Vasculogenic Mimicry As A Tumor Tissue-Produced Biofilm and an “Apogenetic” ECM Control Element
Andrew J. Maniotis, PhD.
Summary: A consensus became established that anti-angiogenesis would succeed against cancer. It was reasoned that, as normal cells, endothelium couldn’t or wouldn’t “mutate” around blood vessel growth inhibitors, like “highly mutable,” “genetically unstable” tumor cells.
In this as in other contexts, tumor cells are still regarded as supernatural cells. Eventually, the important realization was born and pre-clinically demonstrated that there might be both positive and negative regulators of tumor angiogenesis, or by default, molecularly definable positive and negative regulators of tumor growth, dormancy, and metastasis.
Vasculogenic mimicry (VM) is an exclusively malignant tumor derived extracellular matrix (ECM) phenomenon that functions to perfuse tumors independently of host-made capillaries or blood vessels, and it’s presence in a tumor appears to almost always signal accelerated or enhanced malignancy. From research surveying dozens of human tumor types, the association has been established that cancers harboring VM are typically malignant and are refractory to therapeutic intervention.
Vasculogenic mimicry has been reportedly found in, but not limited to the following contexts: human eye and cutaneous melanoms, breast cancers, astrocytomas, gliomas, glioblastomas, intracranial hemangiopericytomas, renal cell carcinomas, pancreatic cancers, hepatocellular carcinomas, canine breast carcinomas, osteosarcomas, gastric cancers, esophageal squamous cell carcinomas, gallbladder carcinomas, gestational choriocarcinomas, multiple myelomas, nasopharyngeal and laryngeal carcinomas, sebaceous carcinomas, numerous xenograph models in animals, and in the context of so-called cancer stem cells.
Reasons that have been advanced to account for the general failure of cancer therapeutics include the use of immune modified animal models implanted with foreign tumor cells, and the widespread belief that irritants, toxins, viruses, or growth factors are specific cancer inducers.
While these systems and observations have been modestly informative as to some basic mechanisms of tumor growth and rejection phenomenon, the nearly universal use and study of animal models lacking a complete or functional immune system, or animal models that are genetically weakened or modified, to be susceptible to cancers has failed us in the context of therapeutics.
Vaccine-induced tumors in both animals and man, however, provide complete therapeutic models of cancer, to develop better nontoxic methods for cancer reversal, dormancy induction, and rejection. Recent evidence in support of this idea comes from reports of vaccine-induced tumors that arise at vaccine sites and then spread to kill the animal (or human), which are immunologically derived from “self,” circumventing the need for and problems created by weakened hosts that are made to harbor foreign tumor cells.
In light of these new data and conceptual advances, a number of recently published primary reports and reviews regarding malignant cancers support the idea by noted cancer immunologists that the plasticity inherent in tumor vasculogenic mimicry (and perhaps many other cancer-associated phenomena such as redundancy and dormancy) is now being considered as a likely reason that the war on cancer via “proven” approaches have largely failed, and why the often life-ending cytotoxic and immunotoxic drug remedies and radiation have not succeeded. Anti-tumor angiogenesis approaches are now believed even to accelerate certain brain malignancies.
Finally, the study of tissue evolution suggests it is likely that cells and their matrix scaffolds over geological time-periods have exhibited a trend to became increasingly physically associated to form drug-resistant tissue-like aggregations of cells, like microbial biofilms, and matrix-producing multicellular tissues in developing embryos, or they can form tissues at inappropriate times or places, as during malignant drug-resistant tumor formation or fibroses. In all these contexts, a polysaccharide-rich extracellular matrix typically is elaborated with characteristic architectures. A balance of fundamental scaffolding molecules such as fibronectin, that promotes growth, and laminin, that promotes differentiation and typically inhibits new growth, can now be viewed as the positive and negative regulators of these tissue-building processes. Various balances of these molecules appear to not only direct normal development, but also appear to control dormancy, metastasis, drug resistance, tumor rejection, degree of malignancy and in pathological processes such as fibrosis of the lymph nodes in late-staged AIDS, or even perhaps such conditions as terminal alcoholic cirrhosis.
A more comprehensive treatment of this article is attached as a PDF here:
Three brain cancer studies published in in December, 2010, in the journals, Nature and Medical Oncology suggested that malignant tumor phenomena known as vasculogenic mimicry (VM) may be more fundamental and widespread than previously appreciated [1, 2, 3]. These three studies followed a similar report published earlier in 2010 regarding “a new tubular mechanism” of VM found in glioblastoma . These studies provided evidence that a now withdrawn antiangiogenesis drug, and the anti-angiogenesis strategy may have actually promoted or augmented one type of VM pathogenesis, at least in these brain cancer contexts [1, 2, 3].
During the past decade, different hypotheses regarding VM have gained prominence precisely because of a robust scientific methodology and critical peer-review. During 2010 and 2011, there have been two to three papers on average, published per month, that critically explore the basic biology and therapeutic strategies aimed at VM in a variety of tumor contexts, and which have repeatedly confirmed the once controversial finding of VM .
Most scientists would now agree that it is the tumor microenvironment in which the common themes, variations, and problematic issues surrounding VM should be considered. To more fully appreciate the advances, paradoxes, and biological roles of VM, several key historical influences, some widely held beliefs, and the dual and plastic nature of VM should be identified. The need for perfusion to provide gas exchange, nutrients, energy, and clearance is fundamental to all cellular life and tissues. The phenomenon of vasculogenic mimicry has revealed that in the context of malignant cancers, tissue perfusion and metabolism may be solved by cells employing different processes other than those typically associated with normal angiogenesis or vasculogenesis.
For instance, according to Dr. Otto Warburg, in his publication entitled, “On The Origin of Cancer Cells” (Science, 24 February, 1956, Volume 123, Number 3191, pp. 309-314), and from a lecture delivered at Stuttgart on 25 May 1955 before the German Central Committee for Cancer Control and first published in German in Naturwissenschaften (42, 401, 1955) from translation by Dean Burk, Jehu Hunter, and W. H. Everhardy of the U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, Bethesda, Md., and with permission of Naturwissenschaften and with collaboration of Professor Warburg):
“…Goldblatt and Cameron (H. Goldblatt and G. Cameron, J. Exptl. Med. 97, 525, 1953) showed the first experimental induction of cancer by oxygen deficiency, when they exposed heart fibroblasts in tissue culture to intermittent oxygen deficiency for long periods and finally obtained transplantable cancer cells, whereas in control cultures that were maintained without oxygen deficiency, no cancer cells resulted….The driving force of the increase of fermentation, however, is the energy deficiency under which the cells operate after destruction of their respiration, which forces the cells to replace the irretrievably lost respiration energy in some way. They are able to do this by a selective process that makes use of the fermentation of the normal body cells. The more weakly fermenting body cells perish, but the more strongly fermenting ones remain alive, and this selective process continues until the respiratory failure is compensated for energetically by the increase in fermentation. Only then has a cancer cell resulted from the normal body cell.”
To further complicate the potentially key contributions of respiration and fermentation in normal and tumor contexts, new mechanisms of vascular formation are still being discovered and defined. For instance, it is generally acknowledged that in the early vertebrate embryo, blood vessel formation is first accomplished via vasculogenesis, or the spontaneous coalescence of individual cells to form embryonic blood vessels de novo.
In later embryogenesis, the process of developmental angiogenesis (known principally as sprouting angiogenesis) is believed to predominate, with blood vessels arising from pre-existing ones, and also, with such processes as vessel splitting through so-called intussusceptive angiogenesis, and vessel budding . Yet historically, most of these models have been based on studies in developing embryonic contexts or in tissue culture, rather than in adults, including the first known report of angiogenesis that was made when Clark in 1918 observed sprouting angiogenesis in the tail of a developing frog larva . Despite the difficulty of observing formative vascularization processes in adult organisms during physiological events where we think they may occur, it is still generally believed that the formation of vascular tubes is primarily accomplished through classic sprouting or budding angiogenesis (as in wound-healing, various inflammatory diseases, psoriasis, rheumatoid arthritis, cancer, menses, during normal tissue growth or repair like seasonal antler growth among certain ungulates).
However, technological and conceptual advances continue to made, and more evidence is accumulating that neovascular processes such as those that occur in menses, wound healing, (or other contexts) are not directed by classic sprouting angiogenesis, or budding angiogenesis. Instead, it appears that neoangiogenesis may be elaborated by other as yet poorly defined biomechanical mechanisms. Instead of ill-defined soluble factors or tropisms, it now appears as if biomechanical forces such as tension direct the elaboration of functional blood vessels that are passively translocated in groups, are stretched, or are contracted (for an excellent discussion regarding these new biomechanical mechanisms of vascular channel formation in these normal contexts, please see References 6,7).
At least five different types of vascular channel formation at least have been identified in and around tumors that cannot be appropriately described as either classic vasculogenesis, sprouting angiogenesis, or their sub-types, but we will focus on only two types here. These include vasculogenic mimicry of the extravascular matrix type, and vasculogenic mimicry of the tubular type.
In VM of the extravascular matrix type, tubes, pipes, or luminal endothelial cells aren’t seen very frequently, but instead, what is observed is more akin to the spaces between a bunch of packed balloons which are formed by packets of tumor cells, with narrow spaces between them that can function as blood plasma perfusion channels. These tumor-cell packets surrounded by matrix with cells embedded, is an architectural arrangement that was first termed, vascular channels , and recently most descriptively, “the fluid conducting meshwork” .
VM of the tubular type is distinctly different from fluid conducting meshworks , and it occurs frequently In Vitro at low plating densities of malignant cells , and In Vivo in the packed confines of real tumors [1, 2, 3, 4]. Tubular VM is a process whereby tumor cells form “pipes” and cords of cells, or what may be aptly described as lagoons In Vivo (as is presented in Reference 3).
What is the importance about the three recent reports of tubular vasculagenic mimicry in studies 1-3, is that the authors of these reports used the best markers known to be available, in order to define the vessel-lining cells as tumor cells in contact with the luminal surfaces of the channels they observed. In this regard, the technologies they used make it unlikely that either “endothelium dependent,” “endothelium independent,” or intussusceptive growth  can account for their findings as well as tubular vasculogenic mimimcry.
Endothelium-dependent and endothelium-independent growth patterns are self explanatory, as they occur either with or without endothelial cells. Growth of the endometrium during the female menstrual cycle is of the endothelium-dependent kind. The intussusceptive growth pattern, however, may require further definition here to illustrate later why absence of evidence is not evidence of absence. Intussusceptive angiogenesis, or the longitudinal splitting of vessels has been shown to occur during wound healing and tumor vascularization. Intussusceptive growth is initiated by connective tissue cells that wrap around and contract vessels which leads to narrowing and eventually splitting of vessels especially in tissues over-expressing vascular endothelial growth factor (VEGF), and in vascular tumors, like glioblastoma (named endothelium independent intussusception). Yet these type(s) of vascular formation were not elaborated upon in the three reports [1-3]. However, “mosaic vessels,” or “transitional VM,” composed of both tumor cells and endothelial cells were documented, satisfying earlier criticisms that provide evidence of anastomoses between tubular VM, and vessels or lagoons still containing true endothelium .
- The Folkman Legacy:
In 1978, Judah Folkman and Anne Moscona published a paper in Nature  which showed that the shapes of endothelial cells, rather than soluble factors, was at the mechanical basis of the phenomenon of vascular growth and differentiation In Vitro. By experimentally controlling endothelial cell adhesiveness and consequently cell shape, the effect of soluble factors could be shown, or not shown, to effect endothelial cell growth and differentiation, depending on how the shapes of cells were controlled. Thus, their celebrated publication, “Cell Shape in Growth Control” , ushered in a new science of cell shape that could be quantified, and their observations were rapidly and widely replicated in a number of In Vitro systems using modern methods of tissue culturing. Eventually it was demonstrated in these systems, that growth factors, although important stimulants, could not induce growth without specific cell shape constraints.
By establishing this fundamental principle of shape requirement in cell growth and differentiation programs, Folkman and Moscona demonstrated that extracellular matrix “adhesiveness” to fibronectin controlled the switch between endothelial growth or differentiation through shape-dependent cell sensitivity to soluble growth factors. This principle was first established using an elegant approach. They showed they could increase cell spreading at will, and that spreading itself induced increased cellular growth, thymidine labeling, cytoplasmic area, and nuclear size with increasing amounts of fibronectin layered over a substrate of a non-adhesive substance such as polyhema. Conversely, by limiting adhesion by increasing the concentration of adhesion-resistant polyhema, it was demonstrated that cell shape also was a dominant force in differentiation. Other researchers rapidly established that by intentionally increasing or decreasing cell adhesion and spreading by engaging specific adhesion receptors, that they could promote growth or functional differentiation of a number of normal cells including those of the vasculature, breast, liver, and many others.
Further validation of this principle was established by Ingber and his colleagues [see Reference 14: “The Architecture of Life,” [Scientific American for elegant pictures] regarding the necessity of specific cell shapes to respond to soluble growth factors, when they demonstrated that micro-beads coated with concentrations of growth factors many more times than needed to stimulate growth would not effect growth of endothelial cells when they were in a round shape, or not spread well.
Experiments by Ingber and colleagues many years later  employed prepared substrata, that forced cells into various shapes, also demonstrated that cell shape controlled “apoptosis” or “survival” of endothelium and other primary cells. These experiments all established the existence of a so-called “tumor angiogenic switch” governing growth and differentiation in a scientifically measurable and experimentally rigorous context which is now widely acknowledged.
Folkman and Moscona’s landmark “Cell Shape and Growth Control” concept and work defining the angiogenic switch was increasingly characterized and explored in the context of “efficient mechanics.” Efficient mechanics, first described by D’Arcy Thomson were also examined with the rules of biological tensegrity that Donald Ingber and his students painstakingly adapted from Buckminster Fuller’s architectural ideas regarding “geometrically efficient designs” in nature.
Other model systems that were exclusively dependent on cell and tissue mechanics directly and indirectly also supported the principle of matrix control because mechanical activity created by structural factors or arrangements controlled differentiation as well as tissue formation and structure in the context of muscle differentiation [please see: Strohman, R.C., Bayne, E., Spector,D., Obinata,T., Micou-Eastwood, J., Maniotis A. Myogenesis and histogenesis of skeletal muscle on flexible membranes. In Vitro Cell Dev. Biology, Vol 26: pp. 201-208, 1990-photo and description presented in The Age of Regeneration section of this website].
Principles governing matrix control over cell growth, differentiation, and eventually even disease processes such as cancer were then further developed perhaps to their foremost extent, when Dr. Mina Bissell and her collaborators demonstrated how extracellular matrix and cell shape controlled the growth and differentiation not only of normal breast acini (milk glands), but also, she and her collaborators and students showed how cancer cell and tissue properties could be reversed, regardless of any genetic alterations they may harbor [please see: Bissell MJ, Radisky DC, Rizki A, Weaver VM, Petersen OW. The organizing principle: microenvironmental influences in the normal and malignant breast. Differentiation. 2002 Dec;70(9-10):537-46. Review].
As the mystery of cell shape’s role in growth and differentiation control of normal tissues (and certain aspects of cancer) was no longer without an experimental basis. Endothelial growth control, and differentiation each appeared to depend on the architectural constraints of the extracellular matrix, the cytoskeleton, and mechanochemical signaling . These experimental contexts together with these efficient “tensegrity” design rules and constraints, could be exploited not only for anti-cancer strategies, but also in tissue engineering.
During his tenure at Boston’s Children’s Hospital on the Harvard Medical campus, Judah Moses Folkman became a cherished mentor to two generations of students, doctors, colleagues, and researchers. He was regarded by all as the father of tumor anti-angiogenesis. Easily, he had been one of the world’s greatest doctors, judging from how he cared for morbidly ill children and patients with intractable diseases at Boston Children’s Hospital, with his Boston “Vascular Task Force.” As a surgeon, Dr. Folkman at some point in his career noticed that there appeared to be an enrichment of blood vessels both to and surrounding growing tumors, as have surgeons and many pathological surveys subsequently. From the standpoint of establishing and maintaining tumor tissue growth and maintenance as with embryogenesis and normal growth, it seemed to make intuitive sense that a route of oxygenation and clearance is a requisite for the life and expansion of all tissues.
This impression of increased angiogenesis in spontaneously-occurring tumors in humans was strongly reinforced by implanting foreign tumor cells, foreign agents, and tissues into experimental animals with altered immune systems, and into the eye, which lacks lymphatic vessels. Eye implants in particular made it easy to see blood vessel tropisms toward surgically implanted materials or tumor cells in real time. In many contexts, an invariably strong neoangiogenic response would commence toward the foreign cells, substances, or implanted tumors [10-13].
A generalized consensus in the field began to emerge which showed that both primary tumors or their metastases required a recruitment of the host’s blood vessels to oxygenate them, provide nutrients, and provide clearance of wastes to and from the tumor in order for it to grow beyond a few millimeters in diameter. This was contrary to Warburg’s earlier findings and conclusions. Eventually it was generally assumed that all tissues, including tumors, required a blood supply to grow, and new strategies were devised to understand the basic mechanisms of blood vessel stasis, growth, differentiation, and repair, with the aim of exploiting those factors that were believed associated with the elaboration of a host-derived series of blood vessels to and from a growing tumor. These principles, it was believed, would also be useful in the treatment of wound healing, psoriasis, macular degeneration, diabetic retinopathy, Kaposi’s sarcoma, angiosarcomas, hemangiomas of children, vascular anomalies, and in many other contexts, specifically cancer.
While these kinds of hypotheses and advances were specifically being developed, many working scientists and doctors, including Dr. Folkman, were becoming increasingly concerned about the ongoing carnage experienced by cancer patients treated using “proven” toxic remedies or radiation. It was increasingly irrational to assault patients with toxic drugs and deadly radiation. In this context, Folkman’s concept of progress in science he likened to the Wright brothers flying their first airplanes at Kitty Hawk:
“Like the Wright brothers at Kitty Hawk, in the field of cancer we only have flown a few hundred meters,” Folkman would say.
“In the decades ahead, we will construct jets to fly around the earth in a few hours!”
Such perspectives Dr. Folkman held and worked toward.
Dr. Folkman and his vascular task force spent much of their efforts managing young children who had developed hemangiomas, which are typically non-metastasizing tumor-like growths which tend to occur more frequently in low birth-weight children and for unknown reasons as well. Deadly vascular anomalies also were managed, along with diseases such as Graft Versus Host Disease as well as cancers. Hemangiomas, in particular, appeared to be highly angiogenic in the way and abundance they supply themselves with neovasculature. They do not like most malignancies, however, metastasize, and, it is possible that many of Dr. Folkman’s views regarding the immune system and cancer were predicated on these clinical experiences as well as other important morbid syndromes as Kassabach-Merritt syndrome, Kaposi’s sarcoma, and other contexts in which neoangiogenesis appears robust.
In the context of Kaposi’s and the complex role of immunity in cancer, and as is noted in Dr. Robert Gallo’s book, “Virus Hunting—AIDS, Cancer, and the Human Retrovirus: A Story of Scientific Discovery” (New York, Basic Books, 1991, page 267), Gallo consulted Judah Folkman to help find an explanation as to why Kaposi’s sarcoma itself, as one of the first two AIDS-indicator illnesses presenting in a small group of chronically illicit and prescription substance abusing young men, could possibly have anything to do with his “new variant of a known cancer virus” (“HIV”) (as Margart Hekler termed “HIV” before it was named “HIV” by Varmus’s committee at a joint press conference with the sun-glass-wearing Gallo). Although Kaposi’s was a disease that had been described for more than 100 years prior to the AIDS era, it was known that the sarcoma was typically indolent, and contained spindle-shaped cells that had characteristics different than those of endothelial cells, as Dr. Folkman explained to Gallo (Robert Gallo, personal communication). Eventually, after the “tat-transgene” experiments of Jay failed to show any of the toxic “tat” protein within Kaposi lesions, another virus was soon blamed (HHV8), and thus Kaposi’s, as one of the first categories of AIDS-indicator illnesses, was no longer directly or even indirectly due to the molecular signatures associated with “HIV” (Flosie Wong-Staal & Robert C. Gallo. Nature Vol 317, 3 Oct 1985):
“The association of Kaposi’s sarcoma with AIDS deserves special mention. This otherwise extremely rare malignancy occurs predominantly in a restricted group, that is, the homosexuals, and can occur in the absence of any T-cell defect in the patients.”
In fact, Dr. Folkman would often comment regarding the confusing and complex role of the immune system and cancer:
“Although embryos do not have developed immune systems, they do not typically develop cancers, nor do the majority of SCID mice (Severe Combined Immune-Deficient). Unless we place tumor cells in them, they do not, like embryos lacking any immune system, typically develop cancers. The lack or presence of a robust immune system may modulate cancers, but does not appear to cause them to occur.”
The burning question amidst all of these considerations became, could tumor-stimulated blood vessel recruitment to growing tumors be blocked with the same endogenous molecules that are believed to stop new blood vessel growth in non-cancer contexts, such as menses, without harming the patient? Because they weren’t tumor cells believed to “mutate around drugs or drug cocktails,” no resistance to inhibitory compounds should occur in endothelium, it was reasoned.
Not only was cell shape important in the normal context of tissue stasis, growth, and differentiation, but the first antiangiogenic drugs such as the fungus-derived anti-microbial agent, fumigillin (TNP-470 and its later derivitive TNP1470) also were first identified and developed by Ingber and Folkman, and then further developed in collaboration with Takeda Pharmaceuticals and others.
Fumagillin appeared to control the growth of endothelial cells by interfering with their ability to assume the correct shapes for growth (spreading) without killing them, but it had toxic side effects In Vivo.
Regarding the basic physiology of what is known about tumor cell resistance in the realm of drug or radiation exposure, there also was a growing awareness among researchers about a number of reports in the literature whereby the exposure of tumors or tumor cells to either anti-angiogenic agents  or traditional DNA-chain elongation terminating treatments (toxic chemotherapy drugs) had no effect on malignant tumors or even their cells.
Eventually, difficult discussions began to take place during this period regarding the role of the tumor “compartmentalization,” tumor matrix, and the role of tumor stroma in the resistance to drugs and radiation.
Clearly the tissue level of drug effects or resistance was far more problematic than Petri-dish cell or tissue responses, and legions of the world’s foremost workers were invited to give their input as to how the tumor “compartment” In Vivo could be penetrated, or perhaps dissolved.
In subsequent years, and in the context of trying to understand tumor dormancy, Dr. Folkman and his collaborators first showed they could reversibly shrink animal-implanted tumors using highly stable molecules derived from the ECM (Extracellular Matrix), even when they were collected from the urine of mice.
The hypothesized mechanism of tumor reversal in mouse models was likened to be of a similar nature to a phenomenon occasionally seen by surgeons during relatively infrequent surgical observations observed in breast cancer surgery patients who, at a low 5 or so percent incidence, would undergo primary tumor removal, only to present in the clinic several weeks later with lung metastases that had exploded.
The hypothesized reason for this surgical failure was that tumors might employ a mechanism known as “concomitant resistance,” whereby the primary growing tumors both stimulate and repress neoangiogenesis, and cause dormancy of metastases distant from the primary tumor.
The favored explanation at that time for this “concomitant resistance” phenomenon was that tumors must stimulate neoangiogenesis (new blood vessel and capillary growth) close to the primary growing tumor by generating relatively unstable reactive molecules like growth factors, such as VEGF, FGF, NGF, hypoxia-inducing conditions, or with molecules released by reactive cells of the immune system recruited to the site like macrophages, T-cells or neutrophils, or even perhaps because of the acidity tumors tended to produce in their environment. At the same time, as the primary tumors are growing and destroying tissue, the hypothesis of concomitant resistance posited they also may generate stable, degradation-resistant molecules or their molecular sub-fragments. In support of this idea, resistant extracellular matrix-derived molecules, or their cleaved fragments such as a portion of collagen XVIII was shown in in mice bearing large back-inserted tumors to suppress the growth of lung micrometastases, and cause these micromets to lie dormant while the primary tumor continued to grow (endostatin). Especially gratifying was the fact that these stable and postulated long-range acting extracellular matrix molecules or their fragments suppressed tumor growth when added exogenously via injections into tumor-bearing mice harboring metastases. Moreover, endostatin could repeatedly reverse tumor growth when it was administered and withdrawn and then re-administered many times in succession in the same mice.
Other studies also showed that removal of large primary tumors grown on the backs of mice and their subsequent surgical re-attachment could reversibly cause lung metastases to grow or shrink after surgical primary tumor re-implantation or removal.
It was also noted that surgical disturbances, or transforming growth factor beta TGF-b) injections at the metastatic lung site (of Lewis lung carcinomas grown on the backs of mice) caused the lung micrometastases to “wake up” and grow, despite the continued presence of the postulated inhibitory power of the primary tumor, and the stable long-range-acting ECM molecules they hypothetically generated, and which, without disturbance or TGF-B stimulation at the metastatic site, would normally suppress or render dormant the micromets.
Although the hypothesis and strategy that emerged from these studies formulated the most successful pre-clinical models of anti-angiogenesis in mice to date, a number of generations of this idea failed in the context of angiostatin, endostatin, anti-VEGF, thrombin, and other anti-angiogenesis strategies such as avastin in the context of spontaneously-acquired tumors occurring in humans (see Editorial: When a drug fails. New York Times, July, 2010. http://www.nytimes.com/2010/07/26/opinion/26mon1.html, regarding avastin).
However, the important realization was born and demonstrated, that there might be both positive and negative regulators of tumor angiogenesis, or by default, molecularly definable positive and negative regulators of tumor growth, dormancy, and metastasis.
In this context, a paradox was identified, but not pursued, regarding the role of angiogenesis in cancer patients treated with traditional toxic drugs. The well known In Vitro sensitivity of endothelial cells to any and all anti-mitotic, DNA-terminating or disruptive toxic anti-cancer agents that should, in principle, also strongly effect (endothelial) cells In Situ. The endothelium is not only in closest proximity to any intravenous-infused drugs but also experiences or is exposed to the highest doses of infused toxic chemotherapies, and the failure of these toxic compounds to block tumor progression if neoangiogenesis was so important, remained enigmatic
Perhaps all malignant tumors could escape from the constraints of the requirements that normal tissues have of perfusion, clearance, and the need for oxygen and nutrients, for the reasons first advanced by Warburg? Or perhaps a genetically or pharmacologically-crippled immune system was not capable of fostering conditions in which spontaneously acquired tumors thrive, or are rejected?
Conflicting against Warburg’s observations that tumors would predictably form in conditions that lack oxygen, the central tenet of the tumor angiogenesis dogma posited that tumors couldn’t grow (nor could any tissue) in the absence of new blood vessel growth, that endothelial cells which make up new capillaries or blood vessels couldn’t mutate around chemotherapies, and that tumors wouldn’t grow as long as anti-angiogenesis was maintained by whatever means.
Another difficult question or paradox should also have been addressed by those of us studying tumor angiogenesis and tumor anti-angiogenesis. How was it possible that tumors like melanomas or most other solid malignant tumors and their metastases easily invade through matrix, skin, and even bone. At the same time, if tumors depended upon neoangiogenesis to grow or metastasize, how did the (delicate endothelial) cells comprising the delicate tumor-stimulated endothelium-lined capillaries in and around a tumor become exempt from or evade the destructive onslaught of a growing tumor or its metastases? Another way of asking this question is: how are delicate endothelial cells spared or how can they possibly survive and grow along with an expanding tumor amidst the growing and proteolytic tumor inferno whose very existence, growth, and metastasis depended upon an expanding endothelium but at the same time, should destroy that very same endothelium as it does bone, skin, or other tissues, as it grew and metastasized?
In time, it became clear that hypotheses based on anti-angiogenic approaches that blocked vascular growth factors, harmed many patients. The growth factor inhibitors such as antivascular endothelial growth factor blockers caused hemorrhaging, unlike endostatin, and did not even slow down tumor growth. Anti-VEGF and other growth-factors, or growth factor receptor-specific counter-agents, did not quell cancer in a single cancer victim and harmed many. They were also cited as a reason to recently withdraw avastin-a phenomenon also explored in the brain glioblastoma studies previously mentioned (2, 3).
It is now widely acknowledged that none of the endostatin or other anti-angiogenesis trials directed at quelling the growth of host blood vessels in spontaneously acquired human CANCER were successful. It was even reported that the doctors ran out of endostatin early at the Comprehensive Cancer Center in Madison, WI, and perhaps elsewhere. The desperation that cancer creates in the victims and families it visits caused doctors to dose-escalate the endostatin protein in many patients whose tumors were not responsive to the initially calculated and administered Phase I-trial doses.
However, in truly vascular diseases that are associated with the in-growth of genuine new blood vessels such as in macular degeneration, or diabetic retinopathy, Dr. Folkman’s antiangiogenesis hypotheses and approaches now appear to be working, and may constitute a true medical achievement for which he was acknowledged at The Association for Research in Vision and Ophthalmology (ARVO) meeting several years ago before he passed away. Some of the principles his work generated over the years also even may provide reasons to explain, in part, how cyclosporines may contribute to tumor formation in transplant patients, independently of their ability to induce immune system suppression.
- Vasculogenic mimicry:
At The University of Iowa in the context of eye melanomas, a new program had been established that focused on matrix biology of tumor tissue, and the ability of tumor cells to adopt “an interconverted phenotype.” The physicians had a collection of melanoma tissue samples and cells from patients with known outcome going back 35 years. Three-dimensional In Vitro tumor models rich in laminin (Matrigel) were then developed, and access to a continuous stream of human biopsies as well as live human tumor tissue, was available. Conditioned media experiments first suggested that the matrix of tumors, not the soluble factors they made, and their micro-environmental constraints such as laminin and fibronectin, dominated the control of tumor tissue genesis, and its various behaviors.
During this period, the ability to assess the plasticity of aggressive melanoma cells was available, due to collaborations begun in collaboration with the Human Genome Institute, and state of the art In Vitro co-tissue culturing was undertaken. Pathological assessments of hundreds of patient tumors also was accomplished by a talented pathologist named Dr. Robert Folberg, who had studied with Wally Clark, and whose staging methods of melanoma were used almost universally. All these factors made it possible to begin defining the entire gene expression profiles, and their associated phenotypes exhibited by both the interconverting malignant or poorly invasive melanoma cells and tumors.
Most of the cancer community at that time was convinced that tumor anti-angiogenesis was the most rational new approach in cancer biology that had come along in decades because of its lack of toxicity by design (as it would define natural proteins or agents that normally quelled angiogenesis as in menses). Because of its hoped for ability to defeat constantly mutating cancer cells in mouse xenograph models, and because it targeted endothelium rather than angiogenesis-dependent tumor cells by ignoring Warburg’s findings. Thus, we dedicated ourselves at first to study eye melanomas rather than cutaneous melanomas, as this tumor type and its relationship to its blood supply could be studied without the confusion of lymphatic vessels being present, and, because Human eye melanomas are thought to metastasize through a purely hematogenous means (through blood vessels and capillaries only rather than through lymphatics).
Amazingly, however, almost from the beginning of our first experiments, it was found that these eye melanoma tumor cells that begin growing in the uvea of the eye can erect their own vascular channels with their own tumor cells and matrix (through the process our team called vasculogenic mimicry). Poorly invasive melanomas could not.
A principal initial finding was accomplished in co-culture experiments with melanoma and endothelial cells. Negative controls were performed in which endothelial cells were not co-cultured with tumor cells, and the vascular channels formed anyway, but only among the cultures containing the aggressive melanoma cells, but not by poorly invasive ones. The patterns of matrix that formed In Vivo could be recapitulated and shown to be perfused both In Vitro, in animal models, and in living human eye tumors, using laser-scanning ophthalmoscopes obtained at the University of Iowa’s eye clinic.
The September 1999 publication of vasculogenic mimicry was first described as the formation of vascular channels in tumors formed by tumor cells and their extracellular matrix . It was published in The American Journal of Pathology coinciding that same month with the beginning of Dr. Folkman’s first non-toxic and suppposedly mutating-tumor-defeating endostatin anti-angiogenesis trials in humans, that should have, in principle, non-toxically stopped cancer victim’s blood vessels from vascularizing their primary tumors and metastases, based on concomitant resistance findings in mice.
James Watson was interviewed on the eve of the trials by Gina Kolata of the New York Times, and Watson was quoted as saying that Folkman’s endostatin would cure cancer in two years, although Watson later denied it.
Watson, along with everyone else, had been convinced that endothelial cells, being “normal cells,” couldn’t mutate around drugs like “supernatural” tumor cells could, and endostatin had reversibly blocked growth of foreign implanted tumor cells in mice, thus ushering in the principle at least, that it wouldn’t matter if tumor cells wildly mutated, because if one could prohibit the recruitment of new blood vessels using non-toxic, stable, and long-range acting endogenous proteins that inhibited new capillary or blood vessel growth, then tumors couldn’t grow or metastasize.
So, along with a reluctant acknowledgment of the possibility that tumor-made perfusion channels might exist with the first publication of VM, an idea that prompted derision from angiogenesis “experts” but never from Dr. Folkman who wrote me a letter of support incorporating VM as another form of tumor vascularization a week after our publication came out, the entire international biomedical research establishment watched Folkman’s and Entremed’s human experiment through a microscope, as the first cancer patients lined up to be treated with endostatin at Dana Farber, MD Anderson, NIH, and The University of Wisconsin Comprehensive Cancer Center. Thus, in the hopeful context of anti-angiogenic therapy of cancers, t he “dubious” concept of VM was greeted by the anti-angiogenesis industry with slightly less than what one might call enthusiastic support for the possibility that malignant tumors may have alternative perfusion mechanisms independent of normal angiogenesis or vasculogenesis, with all that could mean for the success or failure of the new non-toxic Human endostatin, or other antiangiogenic therapies that were then in “the pipeline” [see: In 1st Trials, Cancer Drug Endostatin Disappoint-No Shrinkage Seen In Many Tumors. May 14, 2000, By John Crewdson, Tribune Staff Writer].
As a potential alternative perfusion system independent of host-derived angiogenesis or vasculogenesis, intersusseption, circulating stem cells, vessel co-option, and other postulated mechanisms of host-initiated vascular channel formation, VM was perceived as a threat to the now giant anti-angiogenesis industry. Heavily invested critics did not hesitate to point out shortcomings of the finding, or its interpretation by us, as an alternative perfusion pathway to neoangiogenesis. At the time, there were a few supporters who appreciated VM as a possible extension or elaboration of the belief in the absolute necessity for perfusion in tumor growth, despite Warburg’s work and the work of others showing that tumors thrived at lowered or relatively absent oxygen or clearance.
Yet it was clear to us and many others, however, owing to the electron microscopic studies of melanomas we pursued, that the cells in foremost contact with the plasma channels harbored melanosomes at the ultrastructural level, in addition to the many of the “interconverted markers.”
And it was not then, nor is it now, an implausible concept or reality that tumors erect their own vascular channels when they become malignant and “interconvert” to express both kinds of intermediate filament markers, as well as endothelial markers and others. If indeed the VM “channels” only function or prime function was perfusion, rather than something else entirely different from a hypothesized need for perfusion, why was their topology as looping networks in tumors different than that of pipes? Furthermore, oncologists and cancer researchers have known for decades that differentiated structures such as hair, bone, and other tissues can be found growing in tumors: why not capillaries and blood vessels also, derived from the plastic abilities of tumor cells.
- More about “Tubular Vasculogenic Mimicry:”
What is of importance regarding the newer studies describing tubular vasculogenic mimicry in brain tumors, is the thoroughness and rigor with which the structures in tumors that appear as pipes have now been characterized and defined in tissues using today’s best markers. The reality is that there are indeed tumor cells lining pipes within brain tumors as they are now repeatedly observed with unequivocal tumor cell markers (and not only melanosomes) [see Folberg, R, Maniotis, AJ:APMIS: Vasculogenic mimicry. APMIS, 112:508-525, 2004].
Distinct from fluid conducting meshworks, however, these vasculogenic “pipes” are constructed in such a way that “a variable number (range 20-90% mean 60.7%) of endothelial cells in glioblastoma carry the same genomic alteration as tumor cells,” indicating that a significant portion of the vascular endothelium has a neoplastic origin, and that these same vascular channels contained a subset of tumorigenic cells that produced highly vascularized anaplastic tumours with areas of vasculogenic mimicry in immunocompromised mice. Perhaps most significantly, using double immunohistochemistry analysis of p53 and CD31, archival material of human glioblastomas showed “both remarkable angiogenesis and nuclear accumulation of mutant p53 in 83.3% (20/24) of these tumor cells, with the p53-positive cells facing the lumen of the vessels” . The other glioblastoma study revealed that a subpopulation of endothelial cells within glioblastomas harbor the same somatic mutations identified within tumor cells. This includes amplification of EGRFR and chromosome 7 . This study also demonstrated a stem-cell like CD133+ fraction of cells harboring endothelial cadherin (CD144)-expressing cells that show characteristics of endothelial progenitors capable of maturation into endothelial cells. In addition, via extensive lineage analysis including single cell clonal studies, a subpopulation of the CD133+ stem-like cell fraction was shown to be multipotent and capable of differentiation along tumor and endothelial lineages. Finally, evidence was presented in which “exposure to the clinical anti-angiogenesis agent bevacizumab or to a gamma-secretase inhibitor, as well as knockdown shRN studies demonstrated that blocking VEGFR or silencing VEGFR2 inhibits the maturation of tumor endothelial progenitors into endothelium, but not the differentiation of DC133+ cells into endothelial progenitors, whereas gamma-secretase inhibition of NOTCH1 silencing blocks the transition into endothelial precursors” .
It remains an open question, however, what the prognostic significance of tubular vasculogenic mimicry is, as only the presence of vascular channel-type mimicry (the fluid conducting meshwork) has been associated as an independent variable with poor clinical outcome in numerous surveys.
But why is the ECM so important in cancer, and how could it possibly control all aspects of tumor growth, dormancy, or reversion?
One possibility is that the matrices act as a gas and break in all tissue growth including tumors. If a tumor is simply a tissue growing at the wrong time and place, then the same molecules that control tissue formation in embryogenesis might play a key role in tissues that grow at the wrong time and place.
In support of this idea, since 1935 and 1936, it had been suggested by Waddington and Needham  that cancer develops because of a breakdown of what Waddington termed “individuation fields” that are set up early in development and which normally persist throughout life. Related to this idea regarding the control of neoplasia via “individuation fields,” many investigators have provided evidence that tumor cells themselves exhibit many properties of both embryonic cells and pluripotential stem cells, such as dual expression of intermediate filament systems known as keratins and vimentins.
When tumor cells become highly invasive, they acquire the ability to exhibit an interconverted phenotype. In other words, concomitant with invasiveness and increased motility, and, just as during embryonic development, highly invasive cells (but not poorly invasive ones) will express both vimentin and keratins  similar to embryonic cells before they normally differentiate their intermediate filament systems into either vimentin (if they become mesenchyme), or keratins, (if they become epithelia). Poorly invasive types typically only express one or the other depending on their tissue of origin (vimentin-expression=mesenchymal derived, whereas keratin-expression=epithelium derived).
Most importantly perhaps, it is also known that the invasiveness or malignancy of tumor cells can be modulated by controlling their extracellular environment. Studies of malignant matrix conditioned by aggressive melanomas show that normal melanocytes adopt malignant characteristics, while epigenetic transdifferentiation of normal melanocytes by a metastatic melanoma microenvironment, has been experimentally demonstrated [17, 18].
In fact, such revelations have given rise to diagnostic test kits that demonstrate in every malignant tumor studied, both In Vivo or In Vitro, that it isn’t the silencing or exposure of specific genes that is controlled by the matrix, tissue, or nuclear organization. In fact the entire genome’s sequestration or exposure of hundreds of its genes is directly controlled by the nature of the extracellular matrix, giving rise to a new concept I called mechanogenomics  An exception exists for cervical cells harboring supposed copies of HPV, or hepatocytes harboring so-called supposed mutliple copies of HBV-for reasons that remain controversial that will be briefly elaborated later, and then de-controversialized with a little clear reasoning.
In these experimental contexts of VM, studies of tumor mechanogenomics were then initiated and developed by my group. DNA digestion studies In Situ demonstrated that the entire genome’s exposure of Alu-1 or MSP-1, or DNase-1-sensitive sites is altered when cells become highly invasive. In highly malignant cells, the genome is “sequestered” from DNA-degradating enzymes such as Alu-1, MSP-1, Dnase, or micrococcal nuclease. In poorly invasive, or normal cell precursors of malignant cells, genomes are “exposed” and digest easily. Moreover, these genomic characteristics of highly and poorly invasive or normal cells aren’t localized to specific genes, but are characteristic and stable in genomes over generations (the malignancy-specific distribution of Alu-or MSP-1-sensitive sites) .
It is worth pointing out here our earlier published findings regarding drug-sensitivities of these aggressive cord-forming melanoma cells, because aggressive melanoma cells at low plating densities on matrices rich in laminin such as Matrigel, like endothelial cells, form cords on thin matrix adsorbed to a rigid surface, but cord formation of melanoma cells is not blocked by TNP 1470” while it is completely blocked among endothelial cells (Lab Invest 2002, 82:1031–1043).
Thus it can be shown that the morphological phenotype of many normal and neoplastic cells can be controlled by the ECM, and that such phenotypes are accompanied by nuclease resistant or sequestered genomes if malignant, or nuclease sensitive or exposed genomes if normal or poorly invasive.
But what is the role of genomic instability, or perhaps more importantly, genomic stability in cancer and vasculogenic mimicry?
The phenomenon of genomic instability or genomic invariance (stability) in transformation despite the fact that aneuploidies have always been observed in cancer, has been taken to a high quantitative level with the recent work of Nicholson et al.,  and Klein et al., . In their elegant studies, each and every tumor cell chromosome is followed for generations and compared with the numbers of chromosomes counted in their normal progenitors. The data generated from these studies are presented as genomic diagrams called “karyographs.” These karyographs illustrate better than any genetic method to date, how even extreme aneuploidies are stable, yet they are all different among various tumor-derived karyographed cells that truly are neoplastic. The data in these studies presented as karyographs are convincing, and arguably may someday come to constitute something as cherished as “The Feynman diagrams” known to physics, but in this case, genetics. By contrast, karyographs of precursor normal cells show a flat-line of karyotypic stability as one would expect, without any stably inherited chromosomal numerical and structural alterations or aneuploidies that are detectable using chromosome paints, and/or elevated detection of various oncogene marker copies, casting further doubt on the role of specific oncogenes during the process of true neoplastic transformation, compared to the gll contribution of aneuploidy.
Studies using karyograph analysis have shown that in response to lethal chemotherapy drugs like puromycin, some tumor cells spawn yet other stable aneuploidies as long as the anti-cancer drug is present , and that such adapted drug-resistant tumors will die when the toxic chemical is withdrawn. It is well established as a biological principle, that species become defined as stable ploidies, as we see in the “Feynman-like” karyographs of both normal cells or tumor cells, as shown in the work of Ncholson et al. [also please see the HeLa karyographs from: Duesberg P, Li R, Sachs R, Fabarius A, Upender MB, Hehlmann R. Cancer drug resistance: The central role of the karyotype. Drug Resist Update, 10:51-8, 2007; Karyograph of drug-resistant cells; Duesberg P, Mandrioli D, McCormack A, Nicholson JM. Is carcinogenesis a form of speciation? Cell Cycle 10:2100-14, 2011; Nicholson JM, and Deusberg P. On the karyotypic origin and evolution of cancer cells. Cancer Genetics and Cytogenetics 194: 96-110, 2009].
Although Nicholson et al’s interpretation of their karyographs is that these stable random karyotypes may represent a form of speciation, the phenomenon of aneuploidy cannot causally link aneuploidy with cancer, or prove that aneuploidy “drives” cancer, any more than oncogenes can be said to drive cancers. Speciation itself, once established, is a stable phenomenon, this caution regarding aneuploidy driving the initiation of cancer is emphasized here because with our tests that utilize alu digestion of entire melanoma and breast cancer genomes from tissue, the evidence indicates that there is a global biochemical re-organization at the DNA and protein level as well .
The invariance of stable and clonal tumor aneuploidies and microarrays of tumors has been shown to be real by different groups. It may not be how genomes are specifically packaged differently that drives cancer at all. Changes in ploidy may reflect instead, a more powerful information system being initiated on genome structure by the extracellular matrix environment, through the cytoskeleton and nuclear matrix attachment proteins , because tumors are reversible.
- “Apogenetic control” of gene structure:
The karyoypic forms first described by Galeoti’s, Hertwig’s, and Hanseman’s comparisons of cancer cell and normal cell chromosomes illustrated gross differences in chromatin structure and chromosome numbers in human tissue harboring a tumor. Yet normal cells exhibit aneuploidies during normal development in such creatures as Ascarids, that are hundreds of millions of years old. Aneuploidies also are seen in leaf variegation of corn plants, as was first observed and described by Barbara McClintock, with her “jumping genes” and chromatin bridge fusion cycle. These two well-documented observations indicate that even when entire chromosomes are lost or gained by normal cells, that tumors do not result. In the case of early worm development, as Boveri had observed in the Ascaris’s somatic cell line, or when chromosomes are trapped in the division furrow during the “chromatin-bridge-fusion cycle” in normal corn pant development, as was first observed by McClintock, and which produced leaf variagation in corn plants, aneuploidies do not generate cancers. Thus the presence of aneuploidy in normal differentiation of organisms’ somatic tissues doesn’t necessarily generate cancers.
If cancer is indeed a kind of speciation, there are only precious few cancer syndromes inherited through the germ line lineage (e.g.retinoblastomas).
Many students of chromosomes and karyotypes also now appreciate that it isn’t so important how genomes are packaged and appear at the gross level of chromosomes, but how proteins that inhibit gene activity may be arranged on the chromatin. The seventy million year evolution of primate karyotypes can easily illustrate this hypothesis without controversy: The tree shrew (thought to occupy the branch of evolution at the base of mammalian divergence more than 70 million years ago) has 62 chromosomes: Tarsius, almost as ancient, and one of the oldest known haplorhine (with a lip like ours) primates (which is thought to have given rise to our very early prosimian primate lineage), lives in Borneo, and has 80 chromosomes. However, a similar ancient group of prosimians called slow lorises, have only 5 chromosomes, and all of their chromosomes look like perfect bow ties (are metacentric) including their X and Y chromosomes. Callicebus lugens (not quite as distant to diverge as the prosimians or Tarsius, and which were thought to diverge some 50 million years ago in the New World in South America), have 16 chromosomes, which is less than any other New World primate. Howler monkeys, however, that live in the same jungles of South America as Callicebus with its 16 chromosomes, have 47 chromosomes, or 48 chromosomes like gorillas and chimps, or 49 chromosomes. Squirrel monkeys, another New World primate group, have 44 chromosomes. Old World monkeys, thought to diverge after the New World Monkeys about 30 million years ago, and which currently live in Africa and Asia (like macaques, Papio baboons, and cercopithicus) have as many as 60, 66, and as many as 72 chromosomes. There is almost complete banding homology between baboon and human chromosomes. The apes, including us have 50 (siamangs), 44 (gibbons), 48 (orangutans) 48 (gorillas) 48 (chimps) and 46 (most of us). Primate and human cytogeneticists teach us that some people have 47 chromosomes instead of 46. Some mammals like different groups of muntjaks are morphologically indistinguishable from one another, although they vary in chromosome numbers by as much as 20 chromosomes.
The technique known as chromosome banding shows that the loss of a pair of chromosomes in the human complement of 46 compared to the 48 of chimps, gorillas, and organutans), is probably the result of chromosome fusion resulting in our chromosome 2’s current structure and morphology. A chromosome 5 to chromosome 7 translocation has also occurred in gorillas, that isn’t found among our chromosomes, or those of chimps or orangutans.
Such differences in chromosome numbers among the different groups of primates shows that a chromosomal evolutionary clock does not exist. It is well established that we haven’t really lost or gained many genes during our primate evolution, despite the vast differences in the way our genes have become packaged into chromosomes. There are examples of siabon apes in captivity, whose chomosome numbers are a mixture between the more distant to diverge siamangs, and the more recent to diverge gibbons. These examples would appear to violate the genetic chromosome combination rules that govern the definition of a species, but yet they exist and these siabons are called introgressive hybrids.
Chromosome cytogenetics doesn’t appear to exhibit numerical trends during the evolution of other mammalian groups, reptiles, or amphibians either, and as a peripheral consideration, these different numbers of chromosomes don’t signal more propensity or pre-disposition for the development of cancers in any of these organisms.
- Resistance or refractoriness of malignant cancer cells and tissues to therapeutic intervention is a problem of transdifferentiation and architecture, and not simply mutation:
Tumor cells and tumors aren’t supernatural. In the context of tubular vasculogenic mimimcry, if neoplastic cells can either assume the role of endothelium through complete transdifferentiation into functional endothelium as these recent reports on brain cancers suggest, and can express many of the normal functional markers of endothelia [1,2], or whether they partially transdifferentiate into poor fakers of endothelial characteristics which function as a “poor endothelium,” as in tumor-cell lined lagoons that do not exhibit cells expressing endothelial markers because they are neoplastic without endothelial traits, in all these cases they may be refractory to anti-angiogenesis agents, or traditional toxic agents. In fact this hypothesis is supported by our comparisons between TNP1470-treated melanomas versus endothelial cell cords discussed earlier. Furthermore, tumor cells or tumor cell tissues more so, may become refractory to a plethora of drugs or treatments without genomic instability or mutation playing much of a role, if any. Such tubular mimicry also may represent vessels in transition or anastomoses to real endothelium-lined vessels .
It is becoming increasingly clear that the refractoriness of malignant tumors against innumerable attempts to cure them therapeutically probably has much more to do with tumor-matrix architecture, and the way cells are juxtaposed to matrices, than does a potential enormous lottery of mutation, genetic determinism, or genetic instability once believed to drive all aspects of cancer. In this context, numerous mutation, oncogene, retrovirus, reverse-transcriptase-telomerase-based models (MORRTT-based models) that accompanied the gene mutation views of cancer, typically regarded the disease as a tumor cell-instigated reshuffling of once believed mutated or over-expressed genes and proteins that evade all chemotherapy cocktails or radiation treatments, every time a cancer victim dies, and as assuredly and as easily as a Las Vegas card sharp shuffles and reshuffles their deck of cards. Modern gene array analyses, gene expression studies, and stable chromosomal changes in tumor cells as shown by Nikolson et al., and by our group with Alu 1 and MSP1 sequestration and exposure of entire genomes of numerous human cancers cells over generations (and even tumors in humans in situ versus their marginal tissues) have shown this hypothesis is not the case [1,2,3,4,22]. Relatively consistent and repeated microarray analyses and gene expression studies that have been performed in a variety of tumor contexts especially do not suggest randomness [4, 22], nor do the Feynman-like karyograms discussed earlier.
- Vasculogenic mimicry, “the fluid conducting meshwork,” “extracellular matrix patterns” or what is being termed “the tumor biofilm” is shown in the 3 figures on the left, showing a primary and malignant melanoma: The figures on the left show three photographs of human melanoma cells first placed into the liver of a mouse (top photo). The melanoma cells then metastasized to the lungs (middle and bottom photos).
Top photo-Liver:—The top photo shows vasculogenic mimicry looping patterns surrounding packages of tumor cells at low magnification in a liver-situated melanoma. The brown pigment is the reaction product of an antibody that recognizes only human fibronectin, so everything that is brown comes from the human tumor cells. Human laminin also stains equally intensively (not shown).
Middle photo-Lung—Single cell metastatic cell:—In this second photo of melanoma metastasizing to the lung, we can observe perhaps no more than one human melanoma cell that came from the liver of the same mouse (above), and which itself is surrounded with a thick layer of human fibronectin. Note the dense brown loop around what appears to be one cell. This thick matrix constitutes the tumor biofilm. When the biofilm is present, cells become impervious to chemotherapy, radiation, or high temperature, and are highly perfused through this matrix. This is why we believe mutation in cancer is irrelevant, as this feature is common in all malignant melanoma and in a plethora of other malignant cancers (see Maniotis et al., 1999).
Bottom photograph-Lung—of an established lung metastasis is a large melanoma colony that metastasized to the lung. Vasculogenic mimicry patterns (think brown wavy lines) can be seen to circle groups of tumor cells, and can be seen surrounding only two individual tumor cells (black arrow). These vasculogenic mimicry patterns have also been called “the fluid conducting meshwork” by other scientists who have validated their existence and their functions in different contexts.Instead, the data suggest that relatively stable aneuploidies persist concomitant with transformation. Consistent with this view, VM may be best appreciated through a consideration of how rigidly invariant processes govern cell metabolism, cell growth, tissue growth, cell and tissue differentiation, tumor dormancy, drug and therapeutic resistance, tumor plasticity, and tissue architecture. Many other processes are probably not controlled genetically, but are controlled, instead, by physiological and mechanical microenvironmental constraints. Their apparent redundancies that solve mechanical and metabolic problems in different ways suggest that a tumor might be simply a tissue growing at the wrong place and time.
- Is vasculogenic mimicry homologous to microbial biofilms?
In Vitro, as cellular density increases to the density of cells found in a tumor, not only do cords and perfusible pipe or tube-like or lagoon-like structures form (tubular VM), as is shown in studies 1-3, 4, but networks of perfusible matrices with tumor cells embedded in them also form (vascular channels or the fluid conducting meshwork). In this form of VM (which has been repeatedly shown to be an independent variable associated with malignancy unlike tubular VM), packets of tumor cells are surrouded by matrix, and these amalgams of matrix and tumor cells direct the routes of plasma perfusion along the juxtapposed surfaces of the packed together tumor-cell groups.
Along these plasma perfusion routes, malignant cells differentiate into a highly elongated and benign-phenotype (spindle A melanoma cells) when in contact with the laminin-rich channel matrices , which together constitute the fluid conducting meshwork. If the fluid conducting meshwork itself had the same constraints as true neo-angiogenesis-derived “pipes,” biomechanical fluid sheer forces would play an important role in this differentiation of the perfusion channel-lining cells, but, the transdifferentiation of the lumenal cells occurs automatically in vitro when malignant cells touch micropatterned laminin matrices, in the absence of any sheer forces , and the cell types lining them are indistinguishable from those seen In Vivo . Their nucleolar size also becomes markedly diminished . The fluid conducting meshworks also have been shown to perfuse tumors from 11-21 times more than pipe-like endothelial cell lined blood vessels . The “patterns” as they are called (the fluid conducting meshwork patterns), appear as back to back loops of PAS in a two-dimentional plane [4, 24-34]. They even form in malignant tumors genetically engineered in Drosophila , and they typically appear as ropes of tumor cells each surrounded by matrix in 3-dimensional reconstructions of human tumors harboring this form of vascular channels. They also have been shown to be highly perfused [4, 36-38], despite their narrow diameters as crawl-spaces between adjacent groups of tumor cells, and sometimes only one or two tumor cell is encircled by fibronectin and laminin-rich matrix. Rare red blood cells are also seen.
So we began to wonder if the matrix-embedded cell amalgams of the extravascular matrix type have other perhaps more important functions other than perfusion, although it is not completely understood how perfusion can occur in tumor cell lined pipes or channels as efficiently as it apparently does in tracer labeling study experiments we and others have conducted on tumors in humans while imaging them with ophthalmascopes. In this context, it has been shown they are perfused within seconds to minutes of tracer-dye labeling, they are filled with plasminogen , they are refractory to anti-angiogenic or highly toxic chemotherapy agents [39, 40] and high temperature, they are not thrombogenic, and they rarely are associated with necrosis. Yet they are not lined by endothelium [1,2, 4] but by transdifferentiated tumor cells that somehow, on their laminin-rich matrices, allow a limited flow of perfusion, without clotting, and they appear predominantly in malignant tumors.
The laminin rich matrices that surround the packets of tumor cells may be a chief reason that thrombosis is resisted, as unpublished experiments show that platelets don’t activate on laminin-rich surfaces, which line the vascular channels. The fluid-conducting meshwork also may provide different efficient causes other than perfusion derived from a variety of observations. Away from the organism, a wealth of In Vitro data have demonstrated that it is the type of extracellular environment (outside the cells), and not the genes, which can be modulated to revert cancer cells to normal cells and tissues [41,42]. The extracellular matrix is not only like the glue on the outside of cells that cement them together: it provides perhaps the most important information of all for cell growth, differentiation, and genome organization and gene expression. This matrix determines how normal cells, cancer cells , or viruses  behave, and only two matrix molecules, laminin, and fibronectin, appear to be required to completely and consistently change the organization and expression of genes reversibly between cancer-like and normal. And much like Dr. Folkman’s positive and negative regulators of angiogenesis, if fibronectin (the matrix binding domain) acts like the “gas” for tumor growth, while laminin appears to dampen growth and act like “a break,” can this relationship usher in a new non-toxic approach to cancer treatment that may even involve dietary precursors that antagonize the production of new VM matrix?
Targeting of this slimy and often dense matrix with antibodies against the gel-binding domain of fibronectin (instead of the RGD domain) in the presence of other matrices such as laminin, reverse transforms or kills growing tumor tissue. The anti-gel-binding domain of the fibronectin molecule for instance, reverse transforms tumor cells and their chromatin structure. Alu and MSP-1 digestion experiments showed that tumor cell DNA, depending on its matrix environment, can be reversed to that seen in normal cells  when fibronectin is targeted by specific antibodies to its gel-binding domain (matrix binding domain rather than the RGD or cell-binding domain). At high concentrations, targeting the gel-binding domain of fibronectin completely disrupts tumors and is non-toxic at high concentrations in mice.
Targeting laminin with specific antibodies to Type I laminin causes tumors to exhibit explosive growth. These matrices are made exclusively by tumor’s cells, and it confers resistance to drugs, to viral infections, and has properties in a way analogous, or perhaps even homologous to the manner by which microbial biofilms exert this effect among the killing agent-resistant microbial colonies that generate them. To determine whether chromatin exposure in T4-2 cells that have been reverted to polarized acini is as reversible as the phenotype of acini transfoming into tumors, tissue-engineered breast acini (milk glands) were gradually released from exposure to 6 mol/L LY294002 and 1 mmol/L dibutyryl-cAMP at day 0, and the preparations were subjected to digestion by AluI for 24 hours followed by staining with ethidium bromide. Note that changes in morphology from an acinus (day 0-normal looking milk duct sphere) to a large disorganized tumorigenic aggregate (day 15), as visualized by DIC and phase microscopy, and is accompanied by increased sequestration of chromatin. The middle row of micrographs shows intermediate sequestration by day seven after release from the reversion. The percentage of digested DNA before and after AluI digestion on revertant T4-2 at day 0, day 7, and day 15 after drug release by flow cytometry is summarized for three experiments is summarized in the table:
Above photographs and flow cytometry chart shows data that demonstrates the reversible manipulation of phenotype and genome sequestration (as determined by flow cytometry measurements of digested DNA quantity).
Day O Row of Photos: Tumorigenic T4-2 cells were grown for 2 weeks on Matrigel and could be reverted to form polarized acini with 6 mol/L LY294002 and 1 mmol/L dibutyryl-cAMP, or antifibronectiin alone, and the DNA becomes digested when these revertants were permeabilized and exposed to Alu I for 24 hours followed by labeling with ethidium bromide. The reversal of both tissue phenotype and DNA sequestration occurred as one would expect, if one changed the malignant phenotype of cancer back to normal tissue. The tissue forms normally, and the protein sequestration of DNA of the genomes of the cells comprising that tissue also is normal. Top row: matched laser-scanning confocal microscopy and DIC for two magnifications). Note in the higher magnification images ( top horizontal row) that only the nucleoli remain undigested in the “normal” nuclei of completely digested T4-2 revertants. These data are representative images of observations obtained from five separate identical experiments. Compare the top row fluorescence staining of DNA with the bottom row of micrographs (Day 15) illustrating the complete resistance of DNA to Alu digestion in nonreverted tumorigenic (in mice) T4-2 cells grown under identical conditions. Middle Row: 7 days, are representative of partial (intermediate quantities of phenotypic and DNA reversion).
- Vaccines induce cancers at the site of injection, which then spread metastatically to kill the vaccinated:
That foreign proteins or vaccine irritants can induce cancers at their injection sites has been increasingly and alarmingly been brought to the attention of the cancer community by organizations such as the American Veterinary Association and other physician groups:
“The American Veterinary Medical Association (AVMA) and Vaccine-Associated Feline Sarcoma Task Force initiated several studies to find out why 160,000 cats each year in the USA develop terminal cancer at their vaccine injection sites. [45 a] The fact that cats can get vaccine-induced cancer has been acknowledged by veterinary bodies around the world, and even the British Government acknowledged it through its Working Group charged with the task of looking into canine and feline vaccines [45 b] following pressure from Canine Health Concern.”
“ A team at Purdue University School of Veterinary Medicine conducted several studies [45 c, d] to determine if vaccines can cause changes in the immune system of dogs that might lead to life-threatening immune-mediated diseases. The vaccinated, but not the non-vaccinated, dogs in the Purdue studies developed autoantibodies to many of their own biochemicals, including fibronectin, laminin, DNA, albumin, cytochrome C, cardiolipin and collagen.
This means that the vaccinated dogs — ”but not the non-vaccinated dogs”–were attacking their own fibronectin, which is involved in tissue repair, cell multiplication and growth, and differentiation between tissues and organs in a living organism,” which may explain why cancer is the biggest killer of dogs over the age of two.
Tumors at vaccine sites in cattle as well as basal cell carcinomas and melanomas also spread to kill both cattle or humans as has been pointed out by Harald zur Hausen during his Nobel lecture (see: Nobel Lecture presentation of zur Hausen exactly 25 minutes into his 2008 Nobel speech-http://nobelprize.org/mediaplayer/index.php?id=1052].
Antibodies against fibronectin and laminin, among other molecular moieties such as collagen, cardiolipin, and DNA may also develop among these vaccinated cattle and humans, but nobody to date has looked into the Human context, as they have in cats, dogs, or cattle, for evidence of tumor tissue developing in the wrong time and place. It should be emphasized here that it is known that such vaccine-induced tumors typically proceed to aggressively spread metastatically from the vaccination site to kill dogs or cats, and yet vaccine induced cancers remain unstudied without any analysis to date of antibodies against these ECM or other molecules (DNA, collagen).
It is in these contexts in which vaccines are causally linked to tumors formed at injection sites where it could be most convincingly demonstrated that one could reverse transform cancer tissue at will, and at the same time revert cancer DNA alu sensitivity back to the sensitivity normally seen among poorly invasive or normal cells and tissues (on genome-wide digestion analyses). One advantage of this approach is that no foreign tumor cells are ever present in the vaccinated companion animal cancer patients, and at least until they are vaccinated, these vaccine site tumor-afflicted animals have “normal” immune systems (not genetically modified to accept foreign tumor cells as are rodents discussed earlier). Another advantage of such an approach would be that if this reverse transformation from vaccine site tumor back to normal tissue could be effected by exogenously added ubiquitous extracellular matrix components such as antifibronectin and/or excess laminin, then the flow of information from DNA to protein would be shown to reversible also In Vivo.
The basis of this In Vivo plasticity was named “dynamic reciprocity” by Dr. Mina Bissell some years ago, to describe her In Vitro work on breast cancer reversion, and to indicate there exists communication between the genome and the matrix that can control the reversal of cancer or its resistance to toxic treatments [see: Poniggia O, Sampayo R, Raffo D, Motter A, Xu R, Bissell MJ, de Kier Joffe, Simian M. The tumor microenvironment modulates tamoxifen resistance in breast cancer resistance: a role for soluble stromal factors and fibronectin through B1 integrin. Breast Cancer Res. Treat. 2O11, Sept. 21].
The companion animal victims of vaccine-induced cancers (our beloved dogs and cats) may come to represent the first model of cancer with a spontaneously-evoked tumor generated from the “host’s” own cells, that would serve as a far better indicator for therapeutic outcomes for new strategies to combat cancer, rather than rodent models, with foreign cells implanted, and initially genetically or pharmacologically crippled immune systems.
A letter of consent from pet owners to try new methods of tumor reversal not involving “proven” toxic remedies currently used in humans is all that would be needed.
- Apparent cancer “evolution” or “speciation” is more easily explained by such processes as vasculogenic mimicry or a “tumor biofilm,” than by mutation or MORRTT:
Is metastatic cancer a disease of the entire organism that manifests first locally in one place? Or does malignant cancer always begin as a local manifestation (the tumor) that then evolves, speciates and then spreads? Such questions signal back to Dr. Folkman’s theorizing regarding such processes as concomitant resistance and tumor dormancy.
During the progression or promotion of cancer, the commonly observed and often encountered phenomenon of dormancy also violates genetic instability and mutation models, and hypotheses that posit that gene or chromosomal mutation(s) or expression changes drive cancer. The reasoning goes as follows: if specific malignancy mutations or gene expression changes are initially required in a normal cell or benign (non-metastasizing) tumor cell in order for it to leave or exit the primary tumor site (imagine such mutations as p53, MMP-9, e-cadherin, etc), then why after they acquire this invasive genotype and phenotype and initially metastasize, don’t then continue to spread once they leave the primary site, or at least begin to grow in their new metastatic site, after already having acquired their malignancy-specific genes or expression changes? Why do the malignant abilities that are now made possible with those specific mutaions or malignacy specific gene expression changes “go to sleep” and become dormant? Is it because these highly invasive genes or gene products aren’t able to breech their new tissue prison of the metastatic site, and have to “wait” until they reshuffle their genomes or change expression patterns, to enable them now to escape from the metastatic site? Or is there a better hypothesis and explanation of dormancy.
Metastatic eye melanomas illustrate this simple point clearly, as do the concomitant resistance models of Folkman’s experience. If vasculogenic mimicry patterns are detected in the primary tumor growing in the eye, and the genetic instability model is evoked to explain this shift from a benign poorly invasive phenotype to a highly invasive and metastatic phenotype, then why don’t these malignant changes in the traveling cells work in the liver as they did in the eye? It has been established that this specific kind of tumor preferentially (90+ percent of the time) first metastatizes to the liver, which is the stereotypic organ to typically first be colonized by metastatic uveal melanomas,
This observation becomes important, because in the initial “management” of eye melanomas, the tumor-bearing eye is completely removed (enucleated), if and when the malignancy is discovered. Presumably, at this point during its evolution, the malignant melanoma, whether it is benign or malignant already, is no longer present anywhere in the organism. Or not? Studies of cohorts of such enucleated patients showed that even after enucleation, recurrance of melanoma in the liver typically occurred in many patients at predictable periods of time after eye removal. Given this information, logically, then, for recurrance years later in the metastatic site of the liver to occur, it must be assumed that at some point, perhaps before the enucleations of the eye happened, and at which time any and all metastatic cells in the primary site were removed along with the enucleation of the entire eye, that some of the uveal melanoma cells must have escaped from the eye before the enucleation, and sat quietly in the liver for 11 or 21 years. What was happening to those fugitive cells sitting in the liver quitely for so many years?
It is clear that they were not dividing, but lay dormant. No primary tumor was present anywhere in the body or eye, so one cannot easily come to any conclusion that a mechanism employing concomitant resistance was at work, whereby the primary tumor secreted long-range-acting metastasis-suppressive molecules (like endostatin), because simply, there was no primary tumor as it had been removed along with the entire eye. If one resorts to explaining this dormancy phenomenon using genomic instability models, then one is then again left with a series of paradoxes, namely, that it is known that the mets (after they later at a distant time explode in the brain, lungs, or elsewhere, are typically clonal progeny derived xerox-like from malignant (metastsizing-capable) cells of the initial primary tumor, and they possess the same genotypes and phenotypes as the metastasis-capable cells of the primary. Moreover, as they sit dormant without being detected as they do not grow while waiting in the liver, there is no or little cell division happening during which time genetic “mistakes” could in principle accumulate, to eventually allow the dormant met to escape the constraint of the liver to invade the liver or lung. Therefore, neither concomitant resistance or genomic instability can account for dormancy behavior of uveal melanoma, or indeed, any dormant metastatic focus’s quiet behavior.
This presents yet a simpler explanation that was, and has been testable. Namely, that there aren’t significant genomic alterations in a metastatic cell that escaped the primary tumor that confer special supernatural properties, and in the case of eye melanomas, there cannot be any concomitant resistance mechanism at work because there has been no primary tumor (or vision disturbances of retina displacement) in the teye of the patient for years, even when the metastasis is detected years later. The most plausible conclusion for these facts, is, that it is the soil or environment of the sleeping cells that ultimately determine their behavior. Hot soup, prescription drugs, bad diet, alcoholism, perhaps a sub-clinical hepatitis infection, emotional stress, or especially, a long-term lack of matrix-promoting walking or exercise must come into play to weaken the constraining field(s) of matrix that lace through the liver to thereby weaken its inhibitory constraint of the sleeping met, allowing it now to escape and resume its unruly behavior in the lung or brain. Thus it is the context, or “soil” if you will, that constitutes the primary information-bearing substance or mechanism, rather than the genes or the cells themselves.
- Las Vegas card sharps continue to dominate cancer research:
Recently, the need for more focused research to explore such subjects as vasculogenic mimicry and the intact immune system was highlighted by well-known cancer immunologists . The principal issues they identified were again unfortunately framed in the context of how cancer cells may mutate around even cocktails of targeted “specific” inhibitors of tumor growth and metastasis, in the same manner as “HIV” is imagined to mutate its tiny non-isolated genome around anti-retroviral drug cocktails, every time a patient dies on these so-called “life-saving” drugs.
Doctors Prendergast and Jaffee, in their Cancer Research-published position statement describe this long entrenched disconnect between “the cancer-cell-centric” and “”oncogene-driven science of cancer,” and the currently accepted views regarding “cancer immunology and the microenvironment,” with the title, “Cancer Immunologists and Cancer Biologists: Why We Didn’t Talk Then but Need to Now.” In their article, they identified what they felt are some of the principal obstacles for progress in the following way:
“In recent years, a wider appreciation of the role of cancer modifier genes in dictating tumor development in different strains of animals has emerged, but this concept has not yet widely penetrated the field. In any case, whereas genetic and cell biological studies of cancer become prominent, biological studies of the role of microenvironment and immune function tended to lag and to offer limited molecular insights into how an inflammatory microenvironment and immune cells could either control or promote cancer. Although advances in recent years have led to a wider appreciation of these processes licensing cancer cell proliferation, survival, and metastasis, the historical reach of the old biases against these areas tend to cause intellectual segregation and separation of differet camps of investigators.”
Along with these insights, Prendergast and Jaffee, in their Cancer Research-published position statement continue to recite the failed litany of the past 100 years of ineffecive and toxic cancer therapeutics, with their tacit assumptions that in effect, we must “kill the enemy,” which they do acknowledge, however, is really part of us, and “an enemy” that in fact is part of our own immunological selves or biological identity:
“Clearly, the goal of cancer therapy is to kill residual tumors that cannot be excised surgically. However, the inherent nature of the cancer cell limits the full effectiveness of therapies that have been developed, or that arguably can be developed. Being of host origin, cancer cells share features of the host that make effective treatment difficult (impossible-my surmise) due to side effects that limit the therapeutic window. (Which is a euphamistic way of stating that if we could poison cancer victims or irradiate them more than we already do, without murdering their immune systems, nervous systems, digestive systems, kidneys or liver, we could conquer cancer once and for all)! Moreover, the plastic nature of tumors makes them remarkably resilient in rebounding from clinical regimens of radiotherapy and chemotherapy that are traditionally used…Making matters worse, the regrown tumor may no longer respond to the previously successful therapy, due to the capacity of tumor cells to evolve resistence under selective pressures applied by cytotoxic agents. Indeed, the concept of selection is central to understanding this disease: development and progression is driven by selection for cells that can survive conditions that would normally be lethal. (Then why are tumor cells thought to be initially more sensitive to radiation and drugs than normal cells which have justified the usage of these therapies since they were first begun)? Resistance to virturally any lethal pressure can be selected by evolution in a cancer cell population because of its genetic plasticity, a key characteristic of cancer cells. As shown in the treatment of other diseases caused by a highly mutable entity (e.g. HIV), successful targeting of tumor cells will require the application of multiple agents that target different survival mechanisms. However, compared with HIV, the genetic space available for the evolution of a cancer cell is far larger, due to the far greater size of the cancer cell genome.”
Although this comparson about the similarities of “HIV” to cancer cell mutability is not supported by the evidence, especially by their likening cancer resistance to a so-called virus that is claimed to cause no less than 58 diseases accept one of its first “AIDS indicator illnesses,” Kaposi’s sarcoma, at the same time, Prendergast and Jaffee in this same article suggest that vasculogenic mimicry may account for the phenomenon of tumor cell resistance in the context of “cell evolution:”
“For example, by depriving tumors of a blood supply antiangiogenic therapies can indirectly kill cancer cells. Resistance to such therapies should be difficult to evolve, as the argument goes, because stromal cells in the tumor environment are not genetically plastic. However, due to their passive nature, such therapies are still prone to circumvention through tumor cell evolution (e.g. vascular mimicry in the case of antiangiogenesis therapies: ref. 1).”
“Reference 1” that they provide in this position statement, is the first report of VM , in which we first characterized the fluid conducting meshwork and tubular VM as entities or processes seen or shared (mimicry) among both normal and tumor cells under the control of the matrix microenvironment, rather than Las Vegas card sharps, oncogenes, viruses, or even DNA. For how many decades to come, will cancer research and hypothesis formulation be held hostage by the terrorism imposed by genome-centric models, or, the imagined power of the mysterious “immune system,” or for that matter, the unproven belief systems erected around the entire subject regarding the roles or associations between viruses and cancer?
- How relevant to human cancer are animal models that employ weakened immune systems?
Despite the fact that many of our medical ethics laws and progress in this area emerged because of human experimentation, the appreciation of the limitation of genetically, pharmacologically, or immunologically weakened models still isn’t widely acknowledged in the research community. Therefore, for the benefit of younger students of cancer who might not be aware of certain advancements in the context of human cancer decades ago, advancements which are typically not disclosed in basic medical classes, a brief mention of the human experiments of Dr. Chester Southam warrant and reinforce a few of Dr. Folkman’s warnings about the immune system and human cancers.
One of the best historical precedents for considering the role of the immune system and cancer occurred when HeLa cells were injected into some 80 or more Ohio prisoners’ forearms by a Dr. Chester Southam. These “human experiments” followed the first hepatitis B vaccine trials on prisoners of that same prison, and they were conducted by Nobelist Dr. Baruch Blumberg and Saul Krugman and others (who would launch the hepatitis B vaccine era and suggest a link between hepatitis B viral infections and hepatocellular carcinoma without an animal model of the disease). In these cancer cell injection studies, Southam failed to induce metastatic cancer in those prisoners. He attempted again to perform the same experiment on 40 old sick Jewish folks at The Jewish Chronic Disease Hospital in Brooklyn, who only spoke Yiddish, and couldn’t understand the purpose of Southam’s experiment if they wanted to. It would become revealed that Southam also would subject more than 300 gynocological surgery patients to his inoculations of live HeLa cancer cells and other cells at Sloan-Kettering. Southam’s experiments on humans were stopped when 3 young Jewish student doctors on service at the Chronic Disease Hospital refused to follow Southam’s orders, and they alerted the hospital’s top administrator.
Why were Henrietta Lack’s cervical cancer cells injected into the arms of 40 non-informed, aged, and quite ill patients at the Jewish Chronic Disease Hospital in Brooklyn, or without their knowledge, into more than 300 women in New York who had undergone gynocological surgeries of various kinds? Because Dr. Southam wanted to know that if such cells as HeLa were accidently needle-stuck into doctors or researchers, whether they might acquire cancer, since doctors and scientists were special soldiers on the front lines. Some of that work was published, but, it made little impression on those doctors or scientists that living malignant cervical cancer cells didn’t cause cancer even when directly injected into a human being’s skin by a tuberculin syringe, or repeatedly injected into some 80 healthy white prisoners, or injected into 40 morbidly sick and aged old persons some of whom already had cancer, or injected into 600+ unsuspecting females who underwent gynocological surgery. All that happened in all persons studied was that small temporary lesions formed at the inoculation sites, that Southam marked with ink so he could relocate the site in most cases. The lesions became rejected and the injection site and the wound healed in every case.
After the student doctors refused to aide in Southam’s experiment at the Jewish Chronic Disease Hospital and reported his experiment to the top administrator, the ethical conundrum of Dr. Chester Southam’s “human experiments” became the focus of the world’s attention. This ethical conundrum led to many of our modern experimentation rules and informed consent laws that are currently in place when human beings are ever used in medical research.
Was it possible, perhaps, that malignant cancer cells didn’t cause cancer by themselves without the proper “soil, ” or was it purely the effects of an immune system rejecting non-self? After he was caught, Dr. Southam was reprimanded and his work ceased for about a year. In 1967 Dr. Southam was subsequently elected vice president of the American Association for Cancer Research, and he became its president the following year (Katz, Experimentation with Human Beings, 63 and 65). Southam believed that he had found a test for human immune suppression, as the old sick Jewish patients took the longest to reject their tumors, and Southam imagined standardizing the injection of live cancer cells into everybody to “know their status” (their cancer potential status), and identify potential cancer patients harboring latent, yet non-morbid-symptom-inducing cancers, by measuring the length of additional time needed for them to reject his injection of HeLa:
Isoantibodies to Human Cancer Cells in Cancer Patients Following Cancer Homotransplants. Tetsuo Itoh and Chester M. Southam. The Journal of Immunology, 1964, 926-936. From the Division of Clinical Chemotherapy, Sloan-Kettering Institute for Cancer Research, New York 21, New York.
Are animal models of cancer initiation, promotion, and metastasis based more upon foreign body-like reactions, or is it due simply to their irritant qualities, that don’t occur in naturally derived human tumor contexts? In real spontaneously forming human cancer, tumor cells derive from “self,” and are not foreign in the first place. Although it could be convincingly argued that using immune modified animal models implanted with foreign tumor cells, irritants, or toxins such as growth factors was informative as to some basic mechanisms of tumor growth, angiogenesis, and tumor dormancy and rejection phenomenon, on the other hand, it could be argued that the use of animal models lacking a complete or functional immune system, or genetically modified animal models selected to be susceptible to cancers following the implantation of foreign cells or substances, is like studying knee-replacement surgery using a snake as an animal model. With crippled immune systems, genetically, pharmacologically, or immunologically modified rodents, like snakes without legs, can only provide a partial glimpse of the dynamics, dormancy, or metastasis of cancer, especially in the context of testing anti-cancer agents for therapeutic efficacy.
Yet for unknown reasons, the severe limitations using genetically or immunologically susceptible cancer animal models are not widely or fully appreciated among many cancer researchers. Perhaps economic-political rather than scientific reasons continue to sanction the use of these limited models?
- Transplant medicine and AIDS casts doubt that immune suppression itself causes cancer:
Regardless of what most of us want to tacitly assume regarding the immune system and cancer, certain research has invalidated any substantial link between general immune suppression and cancer causation. This research shows instead, a link between certain drugs that are used to intentionally suppress the immune system (as in organ transplants), and the initiation of cancers. The long-term use of the antibiotic cyclosporine A, that was used in virtually all human transplant recipients receiving donor organs or bone-marrow grafts was thought to induce cancers through direct long-term suppression of the so-called immune-surveillance mechanisms of the recipient host. However, elegant studies done only during the last decade have shown that it is the cyclosporine itself that may directly induce cancers in organ or bone-marrow recipients, rather than a generalized failure of the organ recipient’s immune-surveillance mechanisms. The so-called immune-surveillance mechanism, importantly, cannot identify cancer cells as non-self, because cancer cells aren’t non-self. Cancer cells derive from our own normal cells. Hojo et al., [please see: Hojo et al., Cyclosporine induces cancer progression by a cell-autonomous mechanism. Nature. Feb 11;397(6719):530-4, 1999]:
Malignancy is a common and dreaded complication following organ transplantation. The high incidence of neoplasm and its aggressive progression, which are associated with immunosuppressive therapy, are thought to be due to the resulting impairment of the organ recipient’s immune-surveillance system. Here we report a mechanism for the heightened malignancy that is independent of host immunity. We show that cyclosporine (cyclosporin A), an immunosuppressant that has had a major impact on improving patient outcome following organ transplantation, phenotypic changes, including invasiveness of non-transformed cells, by a cell-autonomous mechanism. Our studies show that cyclosporine treatment of adenocarcinoma cells results in striking morphological alterations, including membrane ruffling and numerous pseudopodial protrusions, increased cell motility, and anchorage independent (invasive) growth. These changes are prevented by treatment with monoclonal antibodies directed at transforming growth factor-beta (TGF-beta). In vivo, cyclosporine enhances tumour growth in immunodeficient SCID-beige mice; anti-TGF-beta monoclonal antibodies but not control antibodies prevent the cyclosporine-induced increase in the number of metastases. Our findings suggest that immunosuppressants like cyclosporine can promote cancer progression by a direct cellular effect that is independent of its effect on the host’s immune cells, and that cyclosporine-induced TGF-beta production is involved in this.
In a follow-up study, Toshiyuki et al., and other groups working in this field have repeatedly established that it isn’t the impairment of the organ recipient’s immune-surveillance system that induces cancer, but rather, the drugs used to suppress it which are capable of inducing cancers. Co-administration of antagonists to the effects of the drug on cells suppresses cyclosporine’s carcinogenic potential, which left unchecked, frequently causes cancer. As eported by Toshiyuki Tanakaa and Shiro Takaharab [please see Toshiyuki Tanaka and Shiro Takaharab. Nature. 1999 Feb 11;397(6719):530-4. A novel immunosuppressive drug, FTY720, prevents the cancer progression induced by cyclosporine. See also: Cancer Letters, Volume 181, Issue 2, Pages 165-171 (26 July 2002)]:
Cyclosporine A (CsA), the most frequently used immunosuppressive drug, has been reported to induce cancer by a cell-autonomous mechanism. Herein, we report that FTY720, a novel immunosuppressant, prevents CsA-induced alterations in both morphology and cell motility at a low concentration (0.1μM) and induces the CsA-treated cancer cells to undergo apoptosis at a higher concentration (more than 5μM)….We believe that a combination treatment with FTY720 and CsA not only results in a synergistic effect on allografts, but also, reduces the incidence of cancer in transplant patients.
According to the five-year data from the Rapamune Maintenance Regimen (RMR) trial, there have been similar findings in the context of squamous cell cancer of the skin induction by cyclosporine in psoriasis patients, and in the context where transplant patients on sirolimus therapy (rapamune-a non-calcineurin inhibitor like cyclosporine) without cyclosporine have a significantly lower risk of developing malignancies than patients who stay on a combination of cyclosporine plus sirolimus.
Also, a number of cancers have been avoided by early withdrawal of cyclosporine including lung, larynx, oropharynx, kidney, gastrointestinal, lymphoma, leukemia, astrocytoma, prostate, breast, cervix, thyroid, glioma, lip sarcoma, and Kaposi’s sarcomas induced by cyclosporine. In the case of Kaposi’s sarcoma, there have been reported cases of reversal upon discontinuation of cyclosporine. This phenomenon of cyclosporine withdrawal and regression or disappearance of the tumour has been reported in 17% patients with mucocutaneous involvement and 16% with visceral involvement as well. The mutagenic toxicity of the drug cyclosporine itself causes cancer: not because of the immune suppression it causes and that it fails to maintain because organs are rejected anyway in time (for kidney transplant patients, in about 5 years and rarely longer than 10 years), but because of “cell-autonomous mechanisms.
- AIDS suggests that generalized immune suppression isn’t the cause of cancers:
The so-called ‘life saving AIDS drug” AZT is also a known carcinogen and has been shown to induce cancers in both animals and man. Like cycloporin, AZT was shown as it was being given to hundreds of thousands of people, that it was a direct cancer inducer that may be distinguished from cancers that become manifested in the context of immune suppression. The Safe Drinking Water and Toxic Enforcement Act in California of 1986 requires that the Governor revise and republish at least once per year the list of chemicals known to the State to cause cancer or reproductive toxicity. The identification number indicated in the following list is the Chemical Abstracts Service (CAS) Registry Number:
Chemical Type of Toxicity CAS No. Date Listed
Zidovudine (AZT) cancer 30516-87-1 December 18, 2009
Also, it should be emphasized here, that skull and cross bones warning always appears on the Sigma Catalogue purchasing information for AZT: Rather than muscular men climbing a mountain with smiles on their faces because they have “HIV,” the following warning is advanced to scientific research instructions that are on the Sigma AZT bottle:
“Toxic by inhalation, in contact with skin and if swallowed. Target organ: Blood Bone Marrow. If you feel unwell, seek medical advice (show the label where possible). Wear suitable protective clothing.”
Regarding these difficult issues, please also see: http://www.oehha.ca.gov/prop65/prop65_list/files/P65single100810.pdf and Pluda et al., who, after a 3 year study conducted on AZT and its role in inducing cancers in a paper entitled: “Development of Non-Hodgkin Lymphoma in a Cohort of Patients with Severe Human Immunodeficiency Virus (HIV) Infection on Long-Term Antiretroviral Therapy.” (Ann Int Med. 1990;113:276-282) claimed that AZT caused cancer: “after starting antiretroviral treatment…the estimated probability of developing lymphoma …by 36 months, [was] 46.4% (CI, 19.6% to 75.5%)…a direct role of therapy itself cannot be totally discounted…Zidovudine can act as a mutagen.”
What is known about the immune system and cancer is somewhat different than the “surveillance” system typically modeled in medical school text books, that supposedly identifies and destroys cancers, which as mentioned, are “self.” It must be kept in mind that tumor cells are part of self, and as self, they may not be completely recognized by an immune system as “non-self.” The AIDS era has fundamentally changed the established views of how a foreign antigen, introduced through vaccines, will or will not protect an organism from future exposure, because as in no other disease syndrome(s), a positive molecular test has indicated impending death, rather than protection and seroconversion.
In fact, there are numerous common themes in the histories, assumptions, and politics surrounding “HPV,” “HBV,” “HBC”, and “HIV” research, medical ractice, and standards of care that are part of a single story that has directly led to the modern vaccines such as gardasil, cervarix, the hepatitis B vaccines, the 64 failed and in some cases abruptly halted “HIV” vaccine AIDSVAX and STEP trials, and, the 170 active or recruiting $500 million dollar/year “HIV” vaccine trials that are currently in that “HIV” vaccine pipeline [please see: http://aidsinfo.nih.gov/vaccines/MainSearch.aspx? strSponsor=Allamp;strRecruiting=on&status=1&strNoRecruiting=on&NRstatus=1].
It is also in this context where we must bravely consider the rationales and evidence underlying what has been described as “Challenger-sized disasters” such as the recent STEP and non-statistically significant Thailand-US-military “HIV” vaccine trails as well as the $500 million dollar a year efforts to push forward the more than 172 current “HIV” vaccines that are in “the pipeline.” The long love affair with mutability, cancer cell evolution, and the “surveillance role of the immune system against cancers,” together with the biological impossibility of the “mutability” of such entities as constituting the mechanism of their resistance, or even as being viable, are in need of serious reappraisal. The maintence of genomes as invariant entitites, as unchanging harbingers of genomic invariance has been a fundamental law of genetics since H.J. Muller discovered that X-rays cause Drosophila chromosomes to break to cause phenotypic alterations (and not necessarily cancers).
In the real context of the efficient causes within the organism, tissue growth, genomic invariance, and mutability are all subsumed, we know now, to environmental constraints. Cells don’t cause cancer: the soil of the host and the interaction of the cancer cells with that soil does. Genomic and pathological invariance are the efficient constraints that allow cells, bacteria, and viruses to maintain their identities over geological time periods, and these observations argue against the view that a lottery is at the basis of everything biological, including pathogenesis. In fact, our knowledge about the truly invariant and evolutionarily conserved mechanisms of biological stasis that are supported by the material and developmental facts we know argue against such genetically protean abilities of either tumor cells, bacteria, or viruses. What appears to be consistent in malignant cancers that are intractable to treatments, and is operative in such syndromes as malignant cancer, profound immune suppression, or resistant viral or bacterial disease, is not the mutability of the so called “seeds” (cancer cells, flu virus, or so-called “HIV,” that was a year after its imagined role in Acquired Immune Deficiences and its rejected role as an instigator of one of the first two original AIDS indicator diseases such as Kaposi’s as Dr. Gallo conceded earlier, and which cannot be found in late staged AIDS patients), but the environments these false markers or tumor cells are found.
Quantitatively speeking, the majority of the genome remains unaltered when a mutation or inersion of chromosomes occurs from time to time. A view of cancer evolution, or viral escape mutants based on such reasoning, is literally ignoring an elephant in closet.
- The fundamental role of the matrix:
From both a physiological and evolutionary point of view, and in cancer at least, the evidence suggests that it may be instead how the fibrous, fluid-conducting VM-like polysaccharide rich matrices control these tissue constraints that develop and which control the genes depending on the various microenvironments that can no longer restrain cells with their matrix components. Thus, in many intractable disease contexts, even in lymph node fibrosis in AIDS or in VM in terminal cancers, it may be better to pursue how the loss of the ECM “breaks” in these matrices confer a capability to cells to interconvert, exit the cell cycle, become metabolically dormant, or how they are non-penetrable to drugs, as is shown so clearly in the fluid conducting meshwork and now in tubular VM seen in brain cancers that are accelerated by anti-angiogenesis treatments [1-3].
The good news is that the supernatural mutability of tumor cells, bacteria, or viruses to mutate or reshuffle their genes like Las Vegas card sharps, may not constitute the principal obstacle to solve. If it is the extracellular matrices that confer resistance in cancer, and the apogenetic information provided by these matrices that function as the chief barriers to targeted therapies, then effective therapies designed to quell cancer need not penetrate into cells or genomes (gene therapy) or even target these entities directly.
Gene- targeted therapies have been shown again and again to be fantasies at best, and as it turns out, the seeming heretical idea of targeting the environment is really quite proximate to our understanding and intervention.
If for no other reason than a growing appreciation that continued efforts or “dreams of gene therapy,” along with ridiculous propositions such as reverse transcriptase being specific from retroviruses when in fact it is found throughout Nature in bacteria and worms according to Varmus, and indeed at the tips of normal chromosomes as a telomerase co-factor, or by the continuing failure of so called “lentiviral vectors” in “gene therapy” that continue to produce no results (even claimed successes in the context of several rare syndromes have severe caveats), we indeed need to talk.
Rather than continuing to imagine and pursue the failed MORRTT theories of genes and onogenes being transduced from cells a billion years ago, as Varmus argued as his case for the origin of oncogenes against Rous’s warnings, with MORRTT’s imagined “hijacking” and perturbation” of normal cellular oncogenes before such genes were infectiously returned to normal cells once more through “cancer virus infections” that never have been demonstrated to cause cancer in humans to date (as Rous warned), and where these cellular “oncogenes” sit quitely today to NOT induce cancers among most individuals (99+%), how does such reasoning explain how these so-called oncogenes reside in natural cancer-free populations and perform normal functions, despite copy number alteration(s) or perceived mutations? The invariance of the cancer syndromes we observe, with all of their stereotypic features which permit pathologists to typify them in the first place, and their genetic “stability” and clonality, and microenvironmental variation depending on location in the body, all probably are simply associated with cancer, rather than drive cancer.
Cancer formation and promotion are most likely driven by the subtle microenvironmental regional variations noted by pathologists regarding the progression and severity of cancer, and may be dependent on its initial and metastatic locations within the organism. In this regard, it it is entirely possible that we have overlooked a simple rule of “tissue building” which has insured stereotypical constraints for tissue formation for eons, in both bacterial biofilm mats that are resistant to all agents that would destroy them, and in tumors that have become malignant, in the lymph nodes of AIDS patients who have been chronically immunologically stressed sometimes for a decade or more via a host of known oxidizing agents, malnutrition, or drugs, and in liver for various auto-immune reasons.
Are there any other cogent explanations as to why “biofilm-like” fibrosis and fibrotic molecules be legion not only in disease states such as malignant tumors, but are now known to be regarded as THE hallmark of advanced AIDS [47, 48, 49]?
Inflammation, it would be argued, as the chief enemy of doctors world-wide in wound healing, asthma, autoimmune diseases, and a plethora of others might constitute one good explanation. But then one would need to explain from an inflammation-damage-centric viewpoint, the stable expression of alternative p53, EGFR, or characteristic chromosomal abnormalities found in the brain cancer studies discussed here (or stereotypic phenotypes such as the aneuploidy of chromosome 7) [1,2], or stereotypic fibrosis of the lymph nodes now assuming a central role in advanced AIDS. Why is fibrosis found only in lymphoid tissue of late-stage AIDS patients? Lymph node fibrosis is not typically discovered in in asymptomatic p24+ gp 160/120+, gp41+, or gp 30/31 positive, p65+, p17+, p40+, p7+ (“gag/pol/env+”) individuals with normal or even abnormal T-cell subsets.
In this regard, and in the context of AIDS, arguments have had to veer off toward the absurd, and many reference labs and physicians are even now questioning the WB as a gold standard. The latest APHL/CDC (an “HIV” testing regulatory body of the government) proposed algorithm omits WB’s altogether. These realizations should come as no surprise to students of cancer or AIDS because “viral loads,” T-cell numbers, and the “mutability” of the disease-associated molecular markers have always been at odds with clinical observations, just as Kaposi’s sarcoma, as one of the first two types of AIDS indicator illnesses was experimentally rejected as being caused by “an AIDS virus.”
Perhaps these are the principal reasons why the presence of fibrosis, the dissolution of the follicular dendritic cell network of the lymph node germinal center, and the progressive loss of antigen-presenting capacity are now being proposed by many on the front lines of AIDS treatment as diagnostic and associated with profound immune deficiency seen in individuals with full-blown AIDS? A large and more recent study in 2006 claimed that “viral load” does not correlate with T-cell numbers, and the rate of progression (when an individual will exhibit symptoms of AIDS) can only be predicted in 4%-6% of HIV-positives studied (out of 2,800):
“A nationwide team of orthodox AIDS researchers led by doctors Benigno Rodriguez and Michael Lederman of Case Western Reserve University in Cleveland are disputing the value of viral load tests-a standard used since 1996 to assess health, predict progression to disease, and grant approval to new AIDS drugs after their study of 2,800 HIV positives concluded viral load measures failed in more than 90% of cases to predict or explain immune status…
”Viral load is only able to predict progression to disease in 4% to 6% of HIV-positives studied, challenging much of the basis for current AIDS science and treatment policy” [Rodriquez B, Sethi AK, Cheruvu VK, et al. Predictive value of plasma HIV RNA level on rate of CD4 T-cell decline in untreated HIV infection.” JAMA 296(12):1498-506, 2006. Cohen J. Study says HIV blood levels don’t predict immune decline. Science 313(5795):1868, 2006].
These conclusions of Rodriquez et al., should be added to the rest of the failed histories of the cancer and AIDS eras, whose histories have been littered with admissions on package inserts such as the following in the case of “HIV:”
“At present there is no recognized standard for establishing the presence or absence of HIV-1 antibody in human blood. (Abbott Laboratories, ELISA HIV Antibody Test Insert)
“The risk of an asymptomatic person with a repeatedly reactive serum developing AIDS or an AIDS-related condition is not known.” (Abbott Laboratories, ELISA HIV Antibody Test Insert)
“The AMPLICOR HIV-1 MONITOR test, is not intended to be used as a screening test for HIV or as a diagnostic test to confirm the presence of HIV infection.” (Roche, Amplicor HIV-1 Monitor Test Kit)
“Do not use this kit as the sole basis of diagnosis of HIV-1 infection.” (Epitope, Inc., Western Blot HIV Antibody Test Insert)
“Clinical studies continue to clarify and refine the interpretation and medical significance of the presence of antibodies to HIV-1.” (Abbott Laboratories, ELISA HIV Antibody Test Insert).
Given the inability of leading hospitals to predict less than 4% of progressive immune suppression syndromes based on the so-called viral load tests or antibody tests, along with the test-kit manufacturer’s caveats regarding their own 33 or more test kits that cannot detect “HIV” and the recent consensus among California physicians to drop altogether any faith in the so-called “gold standard” WESTERN blot tests by leading physicians there in recent years, and given the non-role of genomic instability in cancer causation or promotion, perhaps it is time to provide a token acknowledgement at least or perhaps a passing hat tip to the importance and role of the extracellular matrices and “apogenetic” environments of living cells and tissues?
The evidence in favor of this suggestion strongly points to the idea that when in the appropriate amounts and types, simple tissue-building and tissue maintaining matrices and matrix molecules (as seen in fluid conducting meshworks for instance) direct the formation and structure not only of healthy living tissues, but when they break down or become deranged in diseased tissues or states, intractable diseases can result. In healthy tissue, these matrices are called the extracellular matrix (ECM), and in disease states, such as resistant microbial infections, they are called simply biofilms in which microbes actually, although single-celled organisms, form resistant tissues. In cancer they are referred to variously as “a fluid conducting meshwork,” “vasculogenic mimicry,” “tumor biofilms,“ or “extravascular matrix patterns. ”
But these ideas are not that new. Polysaccharide-and glycoprotein-rich matrices known as microbial biofilms (that cause prokaryotes to form tissue-like structures) have been known to form in the case of bacterial or fungal infections that can evade all drugs from even entering the cell in the first place for more than 200 years, whereas their planktonic counterparts, the current focus and heavily funded obsessions of such fields as microbial resistance are known (and have been known since lenses were first made), sensitive to as much as 10-10,000 times less drug. In support of the fact that polysaccharide residues constitute a chief cause of resistance within cellular communities that are enshrouded with matrices, similar to what we observe in malignant tumors (and their metastases) containing fluid conducting meshworks, it has been long established that even multicellular colonies of micro-organisms that are known as biofilms made of cells suspended in polysaccharaide matrices help perfuse the mats, and when well developed, render the bacterial colonies highly impervious to antibiotics, metals, radiation, as well as extreme changes in pH, oxygen tension, and heat.
The discovery and increasing importance of such phenomena as vasculogenic mimicry, “the fluid conducting meshwork,” “extracellular matrix patterns,” or what is being termed here “the tumor biofilm suggest, at least, that it is possible that resistance among both microorganisms and tumors employ biofilms which provide resistance, such that mutation isn’t really required for either type of biofilm. As reviewed by Harrison et al :
“The Centers for Disease Control and Prevention estimates that up to 70 percent of the human bacterial infections in the Western world are caused by biofilms. This includes diseases such as prostatitis and kidney infections, as well as illnesses associated with implanted medical devices such as artificial joints and catheters and the dental diseases—both tooth decay and gum disease—that arise from dental plaque, a biofilm. In the lungs of cystic fibrosis patients, Pseudomonas aeruginosa frequently forms biofilms that cause potentially lethal pneumonias. There is a long list of biofilm-related ailments, and many scientists.”
“In almost all instances, the biofilm plays a central role in helping microbes survive or spread within the host. That’s because the slimy matrix acts as a shield, protecting pathogenic bacteria from antibodies and white blood cells, the sentinels of the immune system. Biofilms are also notorious for their ability to withstand extraordinarily high concentrations of antibiotics that are otherwise lethal in smaller doses to their planktonic counterparts. In fact, a biofilm can be 10 to 1,000 times less susceptible to an antimicrobial substance than the same organism in suspension.”
“The Dutch microscopist Antonie van Leeuwenhoek first described biofilms in the late 1600s. Using acetic acid, he had tried to kill a biofilm—the dental plaque on his dentures—but noted that only the free swimming cells could be destroyed. Despite the early discovery of microbial communities, microbiology departed from these observations to focus primarily on planktonic bacteria.”
It is possible that whenever in evolution cells have became physically associated to form tissue-like mats, as with microbial biofilms, develop tissues in embryos, or develop tissues at an inappropriate time or place, as during tumor formation, a slimy extracellular matrix is typically elaborated, like the cement used to hold bricks together, which in turn controls the expression characteristics of all cells that form tissues. Mutation is not required.
Tumors harboring malignant cells should be regarded as only one “symptom” of metastatic cancer. In fact, operable and benign tumors should be considered a different disease than metastatic cancers. And there are many kinds of metastatic cancers that resist being classified as a single disease entity or process. Many studies, for instance, have demonstrated the crucial importance and role of the immune system, or infections in stimulating the organism to reject tumors in experimental models, or to fight off naturally-occurring cancers (not cause them to occur). The role of VM and a blood supply, whether induced by angiogenesis, leakage, vessel cooption, circulating stem cells, or by some other process yet to be discovered, all appear to be critical in the process of tumor progression, yet many types of tumors can live in relative anoxic (hypoxic) conditions as Warburg surmised, so perfusion per se (a blood supply) or clearance (removal of wastes) may not be central to the biology and survival of tumors at all, compared with these same requirements for the continued maintenance and survival of normal cells and tissues.
These so-called biofilms in whatever context they form (both prokaryotic and eukaryotic), may be Nature’s simple way of building tissues both at appropriate times and places (bacterial mats and embryos), and at the same time if disturbed, her way of inducing dormancy and complete drug resistance when she builds tissues at inappropriate times and places (tumors, human biofilm microbial infections). These “inappropriate times and places” include the majority of microbial infections that kill patients with implanted medical devices, cystic fibrosis, mycobacterial infections, hospital-acquired pneumonias, TB, etc., and also, our work has suggested that the biofilm (phenomenon) constitutes the chief reason that malignant tumors harboring vasculogenic mimicry patterns (the fluid conducting meshwork, tumor biofilms) kill everyone we find them in. Mutation in none of these contexts is needed, nor, is mutation the reason for drug resistance, metastasis, dormancy, or refractoriness to antibiotics, chemotherapies (take your pick), acids, heat, or radiation (in either the context of bacterial and tumor biofilm-laced tissue).
The presence of a matrix biofilm as vasculogenic mimicry patterns in malignant tumors have immunological implications as well as implications for differentiation and drug resistance. Toll-like receptors have profound implications for fibronectin-receptor targeting of immune cells themselves. By short-circuiting or debulking the tumor biofilm using anti-fibronectin or laminin, one may alleviate the immunological barrier that the biofilm provides. We are currently testing this idea that prokaryotic biofilms are in part composed of laminin and fibronectin-like moieties because cocci bacteria have recently been reported to bind to fibronectin specifically .
Moreover, it is possible that the phenomenon of reverse transformation of tumor cells is in part due to the fact that all cells within the developing vertebrate embryo are forced to behave as normal functioning cells because of an exact magic balance regarding the amounts of laminin and fibronectin, which may constitute Waddington and Needham’s “individuation fields.” These postulated individuation fields produced early in the embryo whereby the high production of laminin in all the tissues functions as a break and differentiation device, while fibronectin typically is elaborated as tissues grow. It acts as “the gas” during tissue building and wound repair. The “breaks” and “gas” are expressed in different amounts in different tumors. Anti-laminin causes tumors to explode in their growth, while anti-gel-binding fibronectin domains cause them to dissolve, shrivel, or die. Similar, if not identical polysaccharides, can be found in growing bacterial biofilms. Our work recently has shown how we can reverse transform breast cancer tissues and melanomas based on these observations and hypotheses, and in fact laminin is a potent murderer of bacterial colonies, regardless of any mutations that they may harbor.
It is my view that most intriguing, urgent, and potentially revolutionary of these initiatives involve biofilms as a natural resistance mechanism in both cancer and in microbiology. In the context of cancer, perhaps a misguided emphasis has been placed upon aneuploidy, sequential deregulation of oncogenes, escape mutants, drug-resistant pumps, and other types of possible tumor and drug resistance mechanisms. There appears to be a number of striking parallels between microbial biofilms and the formation of tumor biofilms, or what we described as vasculogenic mimicry patterns. It is the purpose here to specifically list these parallels and discuss the implications for this important mode of both tumor, and microbial pathogenesis.
The process, architecture, and materials associated with vasculogenic mimicry and the formation of extravascular matrix patterns as “a fluid conducting meshwork” have some of the following similarities:
1. In malignant tumor progression, the data suggest that vasculogenic mimicry (VM) patterns form where a stream of aqueous solution (plasma) flows in and around a tumor. The literature on biofilms says:
“Biofilms are a collection of microorganisms surrounded by the slime they secrete, attached to either an inert or living surface. You are already familiar with some biofilms: the plaque on your teeth, the slippery slime on river stones, and the gel-like film on the inside of a vase which held flowers for a week. Biofilm exists wherever surfaces contact water.”
2. In malignant tumor progression, the data suggest that the aggressive tumor cells make the VM patterns, and that these cells shape the polysaccharide matrix as they form it. The biofilm literature says:
“Biofilms are a collection of microorganisms surrounded by the slime they secrete, attached to either an inert or living surface.”
3. In malignant tumor progression, the evidence shows that VM pattern-forming matrices are molded by subpopulations of initially invasive tumor cells, and that they become populated by cells of different characteristics from the invasive cells as they become non-proliferative and closely associated with the matrix. According to the literature on microbial biofilms:
“Time-dependent response of biofilms is modeled on the principles of viscoelasticity, with states the biofilm dynamic response is defined in terms of compliance, storage and viscosity, a reflection of the organic polymerlike structure. It highlights why antibiotics alone will not reduce the bioburden of the biofilm.”
In recent years, specific extracellular matrix molecules and their breakdown products have been identified that exhibit anti-biotic and anti-fungal properties [52-72]. Since 1989, materials derived from extracellular matrices (ECMs), have been evaluated as scaffolds for surgical reconstruction of damaged or missing tissues.
Attempts to understand the susceptibility of these biological materials to bacterial infections, several of the major ECM molecules also have been evaluated for their anti-microbial activity in our laboratory. For example, ECMs derived from porcine small intestinal submucosa (SIS) and urinary bladder submucosa (UBS) were found to possess antimicrobial activity. ECM extracts, obtained by digesting these acellular matrices in acetic acid, demonstrated antibacterial activity against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Antimicrobial activity was determined using a minimal inhibitory concentration assay. Bacteriostatic activity was detected at protein concentrations of ECM extracts equivalent to 0.77-1.60 mg/mL. The resulting extracts consisted of water-soluble peptides and proteins with molecular weights ranging from 100 kDa and lower molecular weight compounds, as determined by size exclusion liquid chromatography. In preliminary experiments that systematically tested various types and preparations of ECM molecules and their breakdown products, we observed that laminin was among the most abundant and potent ECM molecule possessing anti-microbial activity.
If angiogenesis, mutation, or viruses, cannot explain the refractoriness of vasculogenic mimicry patterns, or the “evolution” of tumor resistance to treatment that occurs after a relatively satisfactory first treatment whereby the tumor appears to go into remission (or dormancy), then what can?
The evidence says it has to do with the condition the “soil” and the physical-chemical context the cells are in. In this regard, it stands to reason that microenvironmental differences dictate how much a cancer can be knocked down initially, or how refractory the tumor cells are to a drug, antibody, or even radiation. The evidence shows that environmental differences of matrix thickness and composition confer resistance, and it could be that cells associated with those regions “resist” as a function of the thickness of the matrix, and because of its high laminin composition.
The way tumor cells have been regarded as having supernatural powers, isn’t based on the scientific evidence. It may owe more to political and historical considerations, as was the fear of HeLa cells wafting about the planet to infect “building 41’s” breast cancer cell collections. It was (and is still) believed by many that these African American’s supernatural tumor cells invaded every culture collection on the planet, because of a failure to appreciate the non-specificity of Stanley Garler’s G6DP marker…that he believed was specific to persons of recent African origin. Yet at the same time, the mistaken belief in effect ruined the Krim-directed Cold-war bolstered race against the Soviets to find the first so-called cancer virus in Humans, that had been launched with the same resources that “put men on the moon” or split the atom, according to Nixon. Thus HeLa cells had “supernatural” properties during this era that erected “The War On Cancer,” but that is a different story too long to be told here (please see: Sacrifice Of The Virgins on this website).
Most experienced investigators, I’ve noticed, including even a few physicians, come to appreciate the power of “the soil” over the seed eventually.
Luc Montagnier, for instance, said in a recent documentary that: “you can catch it (“HIV”) many times and if your immune system is strong you can get rid of it” no problem! This specific interview with the Nobelist, Luc Montagnier, discoverer of “HIV” can be watched at http://www.youtube.com/watch?v=WQoNW7lOnT4l. Here is the important statement in that award-winning documentary:
“…I believe HIV, we can be exposed to HIV many times without being chronically infected. Our immune system will get rid of the virus in a few weeks, if you have a good immune system; and this is also the problem with African people; their nutrition is not very equilibrated, they are in oxidative stress, even if they are not infected with HIV, so their immune system doesn’t work well already, so it is prone, you know, to allow HIV to get in and persist. So there are many ways, not the vaccine, many ways to decrease the transmission, just by simple measures of nutrition, giving anti-oxidants, proper anti-oxidants-hygiene measures, fighting the other infections.”
“If you have a good immune system, then your body can naturally get rid of HIV?”
And yet these statements are only a glimpse of similar thoughts and statements of others, such as Payton Rous, who emphasized “the soil” over “the seed” because of his experimental genius, and as yet another thinker to whom we have bestowed science’s highest Nobel award.
In fact, when you systematically check the primary sources of various Nobel discoverers as they recount their glory, their hard won hypotheses, and listen to them candidly discuss their certainties and caveats, it soon becomes clear that forces other than science and reason are at work in most, if not all, of our higher educational systems, medical schools, and indeed, even in elementary school science and health classes. Thus, apparently, the views of Peyton Rous, Luc Montagnier, and many others are worthy of our time to do some careful investigations and analyses, and some intense questioning, and for many of the reasons they themselves provided, that may not make it through “the peer-review process.”
Yet with these Nobel prize-worthy suggestions, it should be appeciated, that Dr. Montagnier, the Nobel-bequethed heir of “HIV” has also in effect dusted off the Century-old argument of his own country’s biomedical forerunners, Louis Pasteur and Antoine Beuchamp, regarding the supremacy of soil or terrain versus the seed, microbe, or toxin in the development of clinically visible disease.
Thus among a few biomedical researchers Like Dr. Montagnier, at least, it is becoming ever more widely acknowledged even by the Nobelist who is credited with first discovering “The AIDS Virus” that the soil or terrain of the organism, and not the “seeds” or germs (or “HIV”), ultimately determines if and how much illness will result from any seed, imagined or real. It is my contention here, that cancer is no different.
Similarly, the soil or terrain of the host, and the magical balance of the extracellular matrix molecules laminin and fibronectin, determine the growth and malignancy of tumors, as well as block or promote their metastases. Cancer cells do not cause cancer: their environments do.
And because Montagnier is now claiming that “we can be exposed to HIV many times without being chronically infected…because our immune system will get rid of the virus in a few weeks, if you have a good immune system,” then clearly, Montagnier, as should we, side with Antoine Beauchamp’s argument, and not Pasteur’s in the context of “HIV” and AIDS, or supernatural cancer cells and cancer.
Nature conserves her best ideas, and materials such as chromosomes and extracellular matrix to confuse scientists, using forms, patterns, or even materials that mimic others, to accomplish her efficient causes. More than a Century or more of biological, viral, and cancer research illustrates one thing repeatedly: that it is the environment where the most important information systems reside, which “apogenetically” control the genes.
1. Ricci-Vitaiani L, Pallini R, Biffoni M, Todaro M, Invernici G, Centi T, Maira G, Parati EA, Stassi G, Larocca LM, De Maria R. Tumor vascularization via endothelial differentiation of glioblastoma stem-like cells. Nature, Dec. 9; 468 (7325): 824-8, 2010.
2. Wang R, Chadalavada K, Wilshire J, Kowalik U, Hovinga KE, Geber A, Fligelman B, Leversha M, Brennan C, Tabar V. Glioblastoma stem-like cells give rise to tumour endothelium. Nature. 2010 Dec 9;468(7325):829-33; see also Chen Y, Jing Z, Luo C, Zhuang M, Xia J, Chen Z, Wang Y.. Vasculogenic mimicry-potential target for glioblastoma therapy: an in vitro and in vivo study. Med Oncol. Dec 14, 2010; Dong J, Zhang Q, Huang Q, Chen H, Shen Y, Fei X, Zhang T, Diao Y, Wu Z, Qin Z, Lan Q, Gu X. Glioma stem cells involved in tumor tissue remodeling in a xenograft model. J Neurosurg. Aug;113(2):249-60, 2010.
3. Sopufiane El Hallani, Blandine Boisselier, Audrey Rousseau, Carole Colin, Amhed Idbaih, Yannick Marie, Karima Mokhtari, Jean-Leon Thomas, Anne Eichmann, Jean-yves Delattre, Andrew Maniotis, Jarc Sanson. A new alternative mechanism in glioblastoma vascularization: tubular vasculogenic mimicry. Brain. Apr;133(Pt 4):973-82, 2010.
4. Maniotis A., Folberg R., Hess A., Seftor E., Gardner L., Pe’er J., Trent J., Meltzer P., Hendrix M. Vascular channel formation by human uveal melanoma cells in vivo and in vitro: Vasculogenic mimicry. Amer. J. Path.Vol. I55, No 3, pps. 739-752, September, 1999.
5. Ausprunk DH, Folkman J. Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc Res 14(1):53-65, 1977.
6. Kilarski WW, Samolov B, Petersson L, Kvanta A, Gerwins P. Biomechanical regulation of blood vessel growth during tissue vascularization. Nat Med 15(6):657-664, 2009.
7. Witold Waldemar Kilarski, A New Mechanism of Blood Vessel Growth-Hope for New Treatment Strategies. Discovery Medicine, 8(40):23-27, 2009.
8. Clarijs R. Otte-Holler I, Ruiter, de Waal MW. Presence of a fluid-conducting meshwork in xenografted cutaneous and primary human uveal melanoma. Investigative Ophthalmology and Visual Science Vol. 43 No 4, 2002.
9. Folkman J and Moscona A (1978). Role of cell shape in growth control. Nature 273:345–349.
10. Folkman J (1995). Seminars in medicine of the Beth Israel Hospital, Boston: Clinical applications of research on angiogenesis.N Engl J Med 333:1757–1763.
11. Folkman J (1996). New perspectives in clinical oncology from angiogenesis research. Eur J Cancer 32A:2534-2539.
12. Folkman J (2001). Can mosaic tumor vessels facilitate molecular diagnosis of cancer? Proc Natl Acad Sci USA 98:398–400.
13. Folkman J, Watson K, Ingber D, and Hanahan D (1989).Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature 339:58–61.
14. Donald Ingber. “The Architecture of Life.” Scientific American, January, pp48-57, 1998.
15. Waddington CH. Cancer and the theory of organizers. Nature 1935, April 20, 606-608.
16. Hendrix MJC, Seftor EA, Chu W, Trevor KT, Seftor REB. Role of intermediate filaments in migration, invasion and metastasis. Cancer Metastasis Rev 1996;15:507-525,36
17. Abbott DE, Bailey CM, Postovit LM, Seftor EA, Margaryan N, Seftor RE, Hendrix MJ. The epigenetic influence of tumor and embryonic microenvironments: how different are they? Cancer Microenvironment.Dec;1(1):13- 21, 2008.
18. Seftor EA, Brown KM, Chin L, Kirschmann DA, Wheaton WW, Protopopov A, Feng B, Balagurunathan Y, Trent JM, Nickoloff BJ, Seftor RE, Hendrix MJ. Epigenetic transdifferentiation of normal melanomcytes by a metastatic melanoma microenvironment. Cancer Res. Nov 15;65(22):10164-9, 2005.
19. Maniotis AJ, Valyi-Nagy K, Karavitis J, Moses J, Boddipali V, Wang Y, Nuñez R, Setty S, Arbieva Z, Bissell MJ, and Folberg R: Chromatin organization measured by Alu- I restriction enzyme changes with malignancy and is regulated by the extracellular matrix and the cytoskeleton. Am. J. Pathol., 166: No. 4 April 2005.
20. Nicholson JM, Deusberg P. On the karyotypic origin and evolution of cancer cells. Cancer Genetics and Cytogenetics 194: 96-110, 2009.
21. Klein A, Li N, Nicholson JM, McCormack AA, Graessmann A, Duesberg P. Transgenic oncogenes induce oncogene-independent cancers with individual karyotypes and phenotypes. Cancer Genet Cytogenet; 200: 79-99, 2010.
22. Folberg R, Arbieva Z, Moses J, Hayee A, Sandal T, Kadkol S, Lin AY, Valyi-Nagy K, Setty S, Leach L, Chevez-Barrios P, Larsen P, Majumdar D, Pe’er J, Maniotis AJ. Tumor cell plasticity in uveal melanoma: microenvironment directed dampening of the invasive and metastatic genotype and phenotype accompanies the generation of vasculogenic mimicry patterns. Am J Pathol. Oct;169(4):1376-89, 2006.
23. Chen X, Ai Z, Rasmussen M, Bajcsy P, Auvil L, Welge M, Leach L, Vangveeravong S, Maniotis AJ, Folberg R. Three-dimensional reconstruction of extravascular matrix patterns and blood vessels in human uveal melanoma tissue: techniques and preliminary findings. Investigative Ophthalmology and Visual Science, 44:2834-2840, 2003.
24.Makitie, T., Summanen, P., Tarkannen, A., and Kivela, T. Microvascular loops and networks as prognostic indicators in choroidal and ciliary body melanomas. J.Nat.Cancer Inst. 91: 359-367, 1999.
25.Pezella F., Manzotti M, Di Bacco A, Giuseppe V, Nicholson AG, Price R, Ratcliffe C, Pastorino U, Harris A, Altman DG, Pilotti S, Veronesi U. Evidence for a novel non-angiogenic pathway in breast-cancer metastasis. Lancet 355: 1787-1788, 2000.
26. Folberg R, Chen X, Boldt HC, Pe’er J, Brown CK, Woolson RF, Maniotis AJ: Microcirculation patterns other than loops and networks in choroidal and ciliary body melanomas. Ophthalmology 98:996-1001, 2001.
27. Michael A. Warso, Andrew Maniotis, Xue Chen, Dibyen Majumdar, Niu K. Patel, Anne Shilkaitis, tapas K. Das Gupta, Robert Folberg. Prognostic Significance of vasculogenic mimicry patterns in primary cutaneous melanoma. Clinical Cancer Research Vol 7, 473-477, March 2001
28. Chen X, Maniotis AJ, Majumdar D, Pe’er J, Folberg R: Uveal melanoma cell staining for CD34 and the assessment of tumor vascularity. Invest Ophthalmol Vis Sci. Aug; 43(8): 2533-9, 2002.
29. E Passalidou, M Trivella, N Singh, M Ferguson, Jhu, A Cesario, P Granone, AG Nicholson, Goldstraw, C Ratcliffe, M Tetlow, I Leigh, AL Harris, KC Gatter, and F Pezzella. Vascular phenotype in angiogenic and nonangiogenic lung non-small cell carcinomas. British Journal of Cancer 86, 244 – 249. 2002
30. Guzman G, Cotler SJ, Lin AY, Maniotis AJ, Folberg R. A pilot study of vasculogenic mimicry immunohistochemical expression in hepatocellular carcinoma. Arch Pathol Lab Med. 2007 Dec;131(12):1776-81.
31. Sun BC; Zhang SW; Zhao XL; Zhang W; Hao XS. Vasculogenic mimicry is associated with poor survival in 37 patients with mesothelial sarcomas and alveolar rhabdomyosarcomas. International Journal of Oncology 25 (6): 1609-1614, 2004.
32. Sun B, Zhang S, Ni C, Zhang D, Zhao X. The clinical significance of vasculogenic mimicry in 337 cases of bidirectional differential malignant tumors. Chin J Clin Oncol 32: 64-67, 2005.
33. Lin AY, Maniotis AJ, Valyi-Nagy K, Majumdar D, Setty S, Kadkol S, Leach L, Pe’er J, Folberg R. Distinguishing fibrovascular septa from vasculogenic mimicry patterns. Arch Pathol Lab Med., Jul;129(7): 884-92, 2005.
34.Baocun Sun, Shiwu Jhang, Denfang Zhang, Jing Du, Hua Guo, Xiulan Zhao, Wei Zhang, Xishan Hao.Vasculogenic mimicry is associated with high tumor grade, invasion and metastasis, and short survival in patients with hepatocellular carcinoma. Oncology reports 16: 693-698, 2006.
35. Potter CJ. Turenchalk G. Xu T. Drosophila in cancer research, an expanding role.Trends In Genetics January, Volume 16 No 1, 2000.
36. Shirakawa K, Tsuda H, Heike Y, Kato K, Asada R, Inomata M, Sasaki H, Kasumi F, Yoshimoto M, Iwanaga T, Konishi F, Terada M, and Wakasugi H. Absence of Endothelial Cells, Central Necrosis, and Fibrosis Are Associated with Aggressive Inflammatory Breast Cancer. Cancer Research 61, 445–451, January 15, 2001.
37. Shirakawa K, Kobayashi H, Heike Y, Kawamoto S, Brechbiel M, Kasumi F, Iwanaga T, Konishi F, Terada M, Wakasugi H. Hemodynamics in Vasculogenic Mimicry and Angiogenesis of Inflammatory Breast Cancer Xenograft. Cancer Research 62, 560–566, January 15, 2002.
38. Kobayashi H, Shirakawa K, Kawamoto S, Saga T, Sato N, Hiraga A, Watanabe I, Heike Y, Togashi K, Konishi J, Brechbiel MW, Wakasugi H. Rapid accumulation and internalization of radiolabeled herceptin in an inflammatory breast cancer xenograft with vasculogenic mimicry predicted by the contrast-enhanced dynamic MRI with the macromolecular contrast agent G6-(1B4M-Gd)(256). Cancer Res. Feb 1;62(3):860-6, 2002.
39. Andrew Maniotis, Xue Chen, Christopher Garcia, Phillip J. DeChristopher, Ding Wu, Jacob Pe’er, Robert Folberg. Control of Melanoma Morphogenesis Endothelial Survival, and Perfusion By Extracellular Matrix. Lab Investigation Vol. 82 No. 8 p.1083-1092, 2002.
40. van der Schaft D., Seftor REB, Seftor E, Hess AR, Bruman LM, Kirschmann DA, Yokayama Y, Briffieoen AW, Hendrix MJC. Effects of angiogenesis inhibitors of vascular network formation by endothelial cells and melanoma cells. Journal of the National Cancer Institute Vol. 96, No. 19, October 6, 2004.
41. Bissell MJ, Radisky DC, Rizki A, Weaver VM, Petersen OW. The organizing principle: microenvironmental influences in the normal and malignant breast. Differentiation. 2002 Dec;70(9-10):537-46. Review.
42. Weaver VM, Petersen OW, Wang F, et al. Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies. J Cell Biol;137:231-245, 1997.
43. Tone Sandal, Klara Valyi-Nagy, Robert Folberg, Mina Bissell, Virginia Spensor, Andrew Maniotis. Epigenetic reversion of breast carcinoma phenotype and DNA sequestration. American Journal Of Pathology, Vol. 170(5):1739-49. May, 2007.
44. Klara Valyi-Nagy, Robert Folberg, Tibor Valyi-Nagy, Andrew J. Maniotis. Susceptibility of Herpes simplex Virus Type I and II, The Role of Tumor Invasiveness, The Extracellular Matrix, and Chromatin Sequestration. Experimental Eye Research, Vol. 84, 9991-10,000, 2007.
45a). http://www.avma.org/vafstf/default.asp.;b) Veterinary Products Committee (VPC) Working Group on Feline and Canine Vaccination, DEFRA, May 2001; c) “Effects of Vaccination on the Endocrine and Immune Systems of Dogs, Phase II”, Purdue University, November 1,1999, at http://www.homestead.com/vonhapsburg/haywardstudyonvaccines.html. D) See http://www.vet.purdue.edu/epi/gdhstudy.htm%5D.
46.George C. Prendergast, Elizabeth M. Jaffee. Cancer Immunologists and Cancer Biologists: Why We Didn’t Talk Then but Need to Now. Cancer Research 67, 3500-3504, April 15, 2007. http://cancerres.aacrjournals.org/cgi/content/full/67/8/3500
47.Pantaleo G, Graziosi C, Demarest JF, Butini L, et al. HIV infection is active and progressive in lymphoid tissue during the clinically latent stage of disease. Nature;362(6418):355-8, 1993b.
48.Evaluation of the pathogenesis of decreasing CD4+ T cell counts in human immunodeficiency virus type 1-infected patients receiving successfully suppressive antiretroviral therapy. J Infect Dis. Jun 1; 199: 1648, 2009
49. Zugar A. The Puzzle of CD4-Cell Depletion Despite Good Viral Suppression. In some patients, CD4-cell counts fail to rise as expected. Could extensive lymph node fibrosis be responsible?”AIDS Clincial Care, June 1, 2009.
50. Harrison et al., American Scientist, Volume 93, pp.508-515, 2005.
51. Schwarz-Linek U, Hook M, Potts JR. Fibronectin-binding proteins of gram-positive cocci. Microbes Infect;8:2291-2298, 2006.
52. Ayda Sarikaya, Rae Record, Ching-Ching Wu, Bob Tullius, Stephen Badylak, Michael Ladisch. Antimicrobial Activity Associated with Extracellular Matrices. Tissue Engineering. February 2002, 8(1): 63-71.
53. Jason Hodde. Naturally Occurring Scaffolds for Soft Tissue Repair and Regeneration. Tissue Engineering. Apr 2002, Vol. 8, No. 2: 295-308.
54. Stephen F. Badylak, Ching Ching Wu, Melissa Bible, Edward McPherson Host protection against deliberate bacterial contamination of an extracellular matrix bioscaffold versus Dacron? mesh in a dog model of orthopedic soft tissue repair. Journal of Biomedical Materials Research. Nov 2003, Vol. 67B, No. 1: 648-654.
55. Volker Musahl, Steven D. Abramowitch, Thomas W. Gilbert, Eiichi Tsuda, James H-C. Wang, Stephen F. Badylak, Savio L-Y. Woo. The use of porcine small intestinal submucosa to enhance the healing of the medial collateral ligament—a functional tissue engineering study in rabbits. Journal of Orthopaedic Research. Feb 2004, Vol. 22, No. 1: 214-220.
56. T Wright Jernigan, Martin A. Croce, Catherine Cagiannos, Daniel H. Shell, Charles R. Handorf, Timothy C. Fabian. Small Intestinal Submucosa for Vascular Reconstruction in the Presence of Gastrointestinal Contamination. Annals of Surgery. Jun 2004, Vol. 239, No. 5: 733-740.
57. Paul D Holtom, Zachary Shinar, Jennifer Benna, Michael J Patzakis. Porcine Small Intestine Submucosa Does Not Show Antimicrobial Properties. Clinical Orthopaedics and Related Research. Nov 2004, Vol. 427: 18-21.
58. Daniel H. Shell, Martin A. Croce, Catherine Cagiannos, T Wright Jernigan, Norma Edwards, Timothy C. Fabian.Comparison of Small-Intestinal Submucosa and Expanded Polytetrafluoroethylene as a Vascular Conduit in the Presence of Gram-Positive Contamination.Annals of Surgery. Jul 2005, Vol. 241, No. 6: 995-1004.
59. Thore Zantop, Thomas W. Gilbert, Mervin C. Yoder, Stephen F. Badylak.Extracellular matrix scaffolds are repopulated by bone marrow-derived cells in a mouse model of achilles tendon reconstruction.Journal of Orthopaedic Research. Jul 2006, Vol. 24, No. 6: 1299-1309.
60. Ellen P. Brennan, Janet Reing, Douglas Chew, Julie M. Myers-Irvin, E.J. Young, Stephen F. Badylak.Antibacterial Activity within Degradation Products of Biological Scaffolds Composed of Extracellular Matrix.Tissue Engineering. Oct 2006, Vol. 12, No. 10: 2949-2955.
61. Ellen P. Brennan, Janet Reing, Douglas Chew, Julie M. Myers-Irvin, E.J. Young, Stephen F. Badylak. Antibacterial Activity within Degradation Products of Biological Scaffolds Composed of Extracellular Matrix. Tissue Engineering. Oct 2006, Vol. 0, No. 0: 060928131519006.
62. Mohammad Reza Seddighi, Dominique J. Griffon, Peter D. Constable, Tomas Martin-Jimenez. Effects of porcine small intestinal submucosa on elution characteristics of gentamicin-impregnated plaster of Paris.American Journal of Veterinary Research. Mar 2007, Vol. 68, No. 2: 171-177.
63. tes Kadioglu, Oner Sanli, Tolga Akman, Ahmet Ersay, Selcuk Guven, Firdovsi Mammadov. Graft Materials in Peyronie’s Disease Surgery: A Comprehensive Review. The Journal of Sexual Medicine. Jun 2007, Vol. 4, No. 3: 581-595.
64. Meher Antia, Gretchen Baneyx, Kristopher E. Kubow, Viola Vogel. Fibronectin in aging extracellular matrix fibrils is progressively unfolded by cells and elicits an enhanced rigidity response. Faraday Discussions. Feb 2008, Vol. 139: 229.
65. Ellen P. Brennan, Xiao-Han Tang, Ann M. Stewart-Akers, Lorraine J. Gudas, Stephen F. Badylak Chemoattractant activity of degradation products of fetal and adult skin extracellular matrix for keratinocyte progenitor cells. Journal of Tissue Engineering and Regenerative Medicine. Jan 2009, Vol. 2, No. 8: 491-498.
66. Janet E. Reing, Li Zhang, Julie Myers-Irvin, Kevin E. Cordero, Donald O. Freytes, Ellen Heber-Katz, Khamilia Bedelbaeva, Donna McIntosh, Abiche Dewilde, Susan J. Braunhut, Stephen F. Badylak. Degradation Products of Extracellular Matrix Affect Cell Migration and Proliferation.Tissue Engineering Part A. Mar 2009, Vol. 15, No. 3: 605-614.
67. Allison J. Beattie, Thomas W. Gilbert, Juan Pablo Guyot, Adolph J. Yates, Stephen F. Badylak. Chemoattraction of Progenitor Cells by Remodeling Extracellular Matrix Scaffold.Tissue Engineering Part A. May 2009, Vol. 15, No. 5: 1119-1125.
68. Jolene E. Valentin, Ann M. Stewart-Akers, Thomas W. Gilbert, Stephen F. Badylak. Macrophage Participation in the Degradation and Remodeling of Extracellular Matrix Scaffold. Tissue Engineering Part A. Jul 2009, Vol. 15, No. 7: 1687-1694.
69. Karen J. Aitken, Darius J. Bägli. The bladder extracellular matrix. Part I: architecture, development and disease. Nature Reviews Urology. Dec 2009, Vol. 6, No. 11: 596-611.
70. Sara S. Sánchezf1, Susana B. Genta, Manuel J. Aybar, Stella M. Honoré, Evelina I. Villecco and Alicia N. Sánchez Riera. Changes in the expression of small intestine extracelluar matrix proteins in streptozotocininduced diabetic rats. Cell Biology International, Volume 24, Issue 12, December 2000, Pages 881-888.
71. Groos S, Reale E, Hünefeld G, Luciano L. Changes in epithelial cell turnover and extracellular matrix in human small intestine after TPN. J Surg Res. 2003 Feb;109(2):74-85.
72. Jean-François Beaulieu, Pierre H. Vachon, and Serge Chartrand. Immunolocalization of extracellular matrix components during organogenesis in the human small intestine JournalAnatomy and Embryology. Volume 183, Number 4 / April, 1991, 10.1007/BF00196837,Pages 363-369.