Summary The major cause of death from cancer is the relentless growth of metastases that are resistant to conventional therapy. The pathogenesis o f a metastasis is complex and requires that tumor cells complete a sequence of potentially lethal interactions with various host factors. The finding in 1973 that metastasis is a selective process and the finding in 1977 that malignant neoplasms are heterogeneous and contain few preexisting metastatic subpopulations have added a new dimension to our understanding of cancer and its spread. This understanding is now contributing to the design of better therapies against disseminated metastasis. Introduction When T. C. Hsu invited me to contribute to the Roofs section of BioEssuys, I thought that it would be a welcome departure from writing another scientific review. Little did I know how difficult it would be to focus my attention on the events that led me to study the biology of cancer metastasis. I have tried to present my story in an interesting manner while avoiding passing off half-remembered incidents as true stories or enlarging non-events to the status of saga. When I finished military service, my grandmother advised me to choose my vocation with care. She warned me that restless people (she meant me) cannot live one day at a time and also contribute to knowledge (my optimistic goal at that time). She also told me that prior to embarking on a career. T needed to seriously consider whether the efforts would justify the end. I wish that I could state now that my work in the biology and therapy of cancer metastasis had been driven by such a conscious decision. Quite the contrary. My conversation with Grandma convinced me to follow a long-standing desire to minister to sick animals, and T decided to become a veterinarian. In 1963. T graduated from Oklahoma State University with a doctorate in vetcrinary medicine and returned to my hometown, Jerusalem, to open a private practice. I did not find practice particularly challenging so, in 1966, I joined the faculty of the School of Veterinary iMedicine at the University of Pennsylvania in Philadelphia. Soon after my arrival, I was introduced to the late Kobert Brodey, the leading veterinary oncologist in the
United States. Bob Brodey‘s thirst for knowledge was deep, his optimism and enthusiasm were infectious; within one month, I decided that oncology in general and surgical oncology in particular were my calling. It was my privilege to acquire surgical \kills from Bob and to be introduced to the world of clinical research. During those wonderful years, he and I wrote several rcports on the biology of canine mammary cancer and approachcs to therapy. Some of these reports are still relevant today. My contentment with 5urgical oncology was brief, however. as disillusionment set in within two years. Most of my patients (dogs and cats with cancer) were not cured by surgery but, rather, died of metastases. The Dilemma My discussions with many oncologists led me to realize that despite major advances in early diagnosis, surgical tcchniyues, and general patient care. a majority of deaths from cancer are due to the spread of cells from pritnary neoplasms to distant organs where they proliferate. Moreover, since mctastasis frequently occurs prior to diagnosis of a primary tumor, surgical cxcision iq not curative. It became clear to me that continual empiricism in the treatment o€ cancer was unlikely to produce significant improvement and therefore that understanding the mechanisms responsible for canccr metastasis must be a primary goal of cancer research. Only from a better understanding could come the ability to design more effective therapy for different cancers and improvements in the way we dealt with cancer metaytasis. Statcd diffcrently, ‘You cannot fix something if you do not knon how it works.’ Given this frustration with surgical oncology’s role in the treatment of metastasis. I welcomed the suggestion by the Chairman of the Department of Clinical Studies, Kobert Marshak (who would later become the Dean of the Veterinary School at the University of Pennsylvania), to apply to the Graduate School at the University of Pennsylvania and pursue a career in cancer research. My interest in understanding the mechanisms of cancer metastasis led me to seek out Irving Zeidnian, Professor of Pathology at the Univcrsity of Pennsylvania School of Medicine. Subsequent to a long converration, he agreed to accept me as his student. In 196S, I transferred a mere 500 yards to the Department of Pathology at the University of Pennsylvania School or Medicine and entered a new world. The Learning Years In 1968, an international meeling of scientists working in the field of metastaGs could have been held in a small classroom. Metastasis research was not viewed as particularly attractive or even useful. For example, when a leading virologist from The Hebrew University in Jerusalem heard that I planned to study cancer metastasis. he announced. ‘What is there to study?
Either the cancer metastasized or it did not. If it did not, the surgeon will cure the disease, and if it did, it’s too late, so why study its biology?’ 1 have never regretted ignoring this declaration. The Department of Pathology at the University of Pennsylvania Medical School was unique because several of its faculty worked in the area of invasion and metastasis. The Chairman, Dale Rex Coman, had recruited, among many outstanding experimental pathologists, Irving Zeidman, Charles Breedis, Leonard Berwick, Gabriel Gasic, and Peter Nowell (who would become the next chairman). Drs. Coman. Breedis, and Zeidman shared a large laboratory (unheard of today) and started the workday with a morning coffee and discussion period. Dale and Irving were nature lovers, so often the discussion ranged from migratory patterns of birds and seasonal changes in flora and fauna to a recent publication by a colleague, or results of current experiments and their significance. T was embraced by the group, treated as an equal, and taught some principles of successful research. Among them, a question or hypothesis must be based on an observation. Once a question is formulated, one designs appropriate experiments so as to provide as unambiguous answers as possible. In fact, a good experiment may even yield ‘yes’ or ‘no’ answers, although this is rarely achieved in the complex field of cancer biology. A rabbit carcinoma system was one of a few animal models available for the study of cancer metastasis. Using this model, Irving Zeidman had shown that tumor cells can pass through capillary beds of different organs at different rates and that metastases do not necessarily result in the first organ that the blood-borne Cell5 encounter(’). To produce experimental metastases in the lung or liver of rabbits, we had to inject at least one million viable cells into the circulation. This puzzled me. Why was the yield of metastases so low? Did only a few cells survive the injection‘? How could the fate and distribution of tumor cells that enter the circulation be monitored? A thorough review of the relevant literature could have been accomplished in one night(2).Among the few who were working in the field, Bernard and Edwin Fisher reported that chromium-51 can be used to label tumor cells before they are injected into experimental animals(3). I travelled to Pittsburgh to discuss some of the details with Bernie Fisher, whom I admiringly dubbed .the John Wayne of cancer research’ for his gruff but affectionate manner. Since chromium-51 labeled live and dead cells, a better method to study the organ distribution and fate of live tumor cells was required. During one of the early morning coffee sessions in Philadelphia, I decided to concentrate my attention on developing a quantitative analysis of hematogenous metastasis for my Ph.D. thesis. My fellow postdoctoral fellows at Penn were Carlton Stewart and John Kreider. What a trio we made! Carl was working on how mitogens influenced the immune
response and was also interested in the biology of macrophages. Years later 1 would embark on trying to understand this wonderful cell. John taught me the rudiments of tissue culture, a witchcraft never to be practiced during the summer because of constant yeast contamination. This despite his rule that one must rinse his hands in diluted iodine solution before any culture work; one’s hands were permanently stained, but the contamination pcrsisted. However, our salvation soon arrived. John, a Board-certified pathologist, was a consultant to the General Electric Company, involved at that time with exploration of space. While at GE, he became acquainted with the laminar airflow cabinet (used to assemble this and that for space under dustfree conditions) and decided to use it for tissue culture. John published his observation and tissue culturing was revolutionized(4).I take this opportunity to thank him for this incredible contribution. H e is also responsible for another important contribution to my own research. 1 complained that a mouse fibrosarcoma syngeneic to BALB/c mice did not produce visible lung metastases. He recommended that I obtain the B16 melanoma from the Jackson Laboratories because, after intravenous inoculation, this tumor produced visible black tumor colonies in the lung. This 1 have done and, subsequently, the B16 melanoma became the standard tumor system for studies of experimental metastasis. After months of trial and error, I completed a detailed analysis of the distribution and fate of B16 melanoma cells labeled with [12sl]iododeoxyuridine(5). Most cells that cntered the circulation died rapidly, and by 24 h, less than 1YO of the cells survived. In fact, the metastases were produced from less than 0.1 YO of the original inocula. I concluded, in my 1970 paper(’), that a metastasis resulted from only a few surviving cells and could even originate from a single surviving melanoma cell. Sixteen years later, my postdoctoral fellow James Talmadge and I provided definitive evidence that metastases have a clonal origin, and that they can result from the expansion of a single progenitor The fact that the vast majority of tumor cells died in the circulation was not surprising, but it did raise an interesting question. D o metastases result from the fortuitous survival and growth of some cells or from the selective survival and growth of unique subpopulations of cells? The Selective Nature of Cancer Metastasis The conventional wisdom of the time was that metastasis either represented a random process or the inability of the host to prevent cancer spread. To examine this question, I injected mice with syngeneic B16 melanoma cells and harvested lung metastases. Cells isolated from these metastases were injected intravenously into normal mice where they produced lung metastases, repeating the cycle several times. I then compared the metastatic behavior of the cycled cells with that of the wild-type (parent) tumor and
found that the cells isolated from metastases were significantly morc metastatic than the cells from the parental tumor. My excitement led me to write a brief report to Nature, which, after minor revisions, was accepted for publication (to the surprise of my colleagues who predicted that Nature would not publish ‘metastasis data’)(’). In 1W2, Richmond Prehn, who was one of my teachers in thc Department of Pathology at Pennsylvania, invited me to speak at the Gordon Research Conference on Cancer. This was a thrilling and humbling experience. Although I was to discuss what I considered to be a major issue in oncology, it was a mysterious one to most researchers. Moreover. all the data I planned to present were generated by my technician, Marilyn Budmen, and me. I therefore did not have the obligatory slide listing a large number of collaborators to be acknowledged. To remedy this deficiency, Osias Stutman suggested a fictitious collaborator: Morris Shapiro. The presentation was a success, and during the coffee break, Osias Stutman left a cryptic messagc on thc board: ‘Dr. Shapiro, call your office - Josh Fidler appropriated your data.’ Morris Shapiro became a silent hero and stories about him proliferated. Morris was actually instrumental in my meeting my friend, scientific collaborator, and wife. Margaret Kripke. Margaret and I attended an American Cancer Society meeting in 1974. That year’s Morris Shapiro story was the following: Morris’s wife died when he was 65 years old. He decided to get back into circulation and purchased a toupee, elevator shocs. a designer suit, and a wide brimmed hat, and he had his teeth capped and his nose adjusted. While crossing the street, he was run over by a bus. When he reached heaven, he challenged the Almighty: ‘Lord, why did you have to kill me? T was a good man, a loving husband, a devoted father, and I gave to charity. Just because my wife died and I wanted to get back into circulation, you had to kill me?’ The Lord gazed upon Morris and answered, ‘To tell you the truth, Morris, I didn’t recognize you!’ I heard Margaret giggling in the audience and thought that I must meet this immunologist who obviously had a sense of humor. We were married in 1975 and were recruited by Mike Hanna to head independent laboratories in the just established Cancer Biology Program at the NCIFrederick Cancer Research Facility occupying the old biological warfare buildings at Fort Dietrick. My laboratory was designated the Cancer Metastasis Laboratory. To my knowledge. this was the first major government-supported research laboratory dedicated to study the biology and therapy of metastasis.
Tumor Heterogeneity The pioneering days at the NCI-Frederick Cancer Research Center were thrilling. Every day was more exciting than the preceding one. We were converting the old biological warfare facility to a cancer biology program, and our efforts were rewarding. I succeeded
in recruiting several outstanding young scientists, including Douglas Gersten. Ian Hart, Avraham Raz, and Nabil Hanna. At that time, Morris Shapiro retired. In 1976, Douglas Gersten, Marilyn Budmen, and I examined whether tumor cells that were lysed by lymphocytes (under in vitro conditions) differed qualitatively from tumor cells that were not. We succeeded in isolating lymphocyte-resistant variants from the starting populations and concluded that their survival was not random@).Ian Hart then selected for melanoma cells with increased invasive capacity and found that these cells also had increased metastatic capacity in syngeneic mice(9). One obvious criticism of these studies was that the surviving isolatcd tumor cells could have arisen as a result of adaptive rather than selective processes. Although I was keenly aware of this shortcoming, I could not design a definitive experiment to rule this out (or in). Margaret Kripke, who had extensive training in immunology and microbiology, devised a solution: Do a modified Luria and Delbruck fluctuation assay(’’’. After a hasty retreat to the library. T adopted the idea enthusiastically. In 1977, in the first test of the idea, Margaret and I cloned the B16 melanoma and showed that different tumor cell clones, each derived from individual cells isolated from the parent tumor, differed dramatically in their ability to form pulmonary metastases in byngeneic mice. Control subcloning procedures demonstrated that the observed diversity was not a consequence of the cloning procedure. Science published our report(”). Tumor heterogeneity in general and metastatic heterogeneity in particular becamc publishable. To exclude the possibility that the metastatic heterogeneity found in the B16 melanoma might have been introduced as a result of the lengthy in vivo and in vitro cultivation, Margarct and I studied the biological and metastatic heterogeneity in a mouse melanoma induced in a C3H mouse by chronic exposure to UVB radiation and painting with croton oil. One mouse thus treated develo ed a melanoma designated K-1735 (Kripke-l735)(’ 1. The original K-1735 melanoma was established in culture and immediately cloned. We found that the clones differed greatly from each other and from the parent tumor in their ability to produce lung metastases which themselves also varied in size and pigmentation(13).Clearly, tumor heterogeneity was not due to long periods of tissue culture or serial transplantation. That preexisting tumor cell subpopulations proliferating in the same tumor exhibit heterogeneous metastatic potential has since been confirmed in many laboratories (notably, Garth Nicolson’s and George Poste’s). with a wide range of experimental animal tumors of different histories and histological origins. In addition, studies using young nude mice as models for metastasis of human neoplasms have shown that tumor lines and freshly isolated tumors, such as melanoma, colon carcinoma, and renal carcinoma, also contain
r
su bpopulations of cells with widely differing metastatic properties(''). The implications of neoplastic heterogeneity for cancer treatment are enormous. Cancer is a collection of heterogeneous diseases with each cancer of each tissue or organ displaying different subtypes. Heterogeneity exists among different patients prcsenting with the same disease, e.g., melanoma, arising in different parts of the body. Moreover, genotypic and phenotypic heterogeneity exist among cells populating a single tumor. Stated differently: with the development of better molecular diagnostic tools, we may be able to understand the individual characteristics of each patient's tumor and thus treat each cancer as an individual cancer. So these are the 'Roots' of my research into the biology of metastasis. During the last two decades, research in the field of cancer metastasis has expcrienced a remarkable and welcome renaissance. In part, this is due to the development of new experimental tools that allow analysis of the metastatic phenotype on a cellular, molecular: and biochemical level, and appreciation for the role host factors play in the pathogenesis of metastasis. Today's key issue in metastasis research is not whether neoplasms are heterogeneous - that is substantially settled except for the details. The challenge is to understand its origins and mechanisms. What is clear now is that metastasis is a selective proccss that is regulated by a number of mechanisms. This belief is quite contrary to the once widely accepted view that metastasis represents the ultimate expansion of cellular anarchy. In fact, the realization that, as a whole, the process of metastasis is selective leads me to be optimistic about the prospects for treatment of cancer spread. Belief that certain rules govern metastasis implies that elucidation and understanding of these
rules will lead to bctter therapeutic intervention. These regulatory mechanisms are now being investigated in a large number of laboratories worldwide.
References 1 Z O I D M .I.~(1957). , Melaalasia: ii review of recent advances. Cmcw Re>. 17. 157-161. B. AYD FISHER,E. R. (1967). Recent observations on the concept of . Arch. Patho/. 83, -321-324, 3 FISHFR, E. R. A N D FISHER,B. (1967). The organ distribntion of disseminated "Cr-labeled tumor cell?. Cuizcer Re.
United States. Bob Brodey‘s thirst for knowledge was deep, his optimism and enthusiasm were infectious; within one month, I decided that oncology in general and surgical oncology in particular were my calling. It was my privilege to acquire surgical \kills from Bob and to be introduced to the world of clinical research. During those wonderful years, he and I wrote several rcports on the biology of canine mammary cancer and approachcs to therapy. Some of these reports are still relevant today. My contentment with 5urgical oncology was brief, however. as disillusionment set in within two years. Most of my patients (dogs and cats with cancer) were not cured by surgery but, rather, died of metastases. The Dilemma My discussions with many oncologists led me to realize that despite major advances in early diagnosis, surgical tcchniyues, and general patient care. a majority of deaths from cancer are due to the spread of cells from pritnary neoplasms to distant organs where they proliferate. Moreover, since mctastasis frequently occurs prior to diagnosis of a primary tumor, surgical cxcision iq not curative. It became clear to me that continual empiricism in the treatment o€ cancer was unlikely to produce significant improvement and therefore that understanding the mechanisms responsible for canccr metastasis must be a primary goal of cancer research. Only from a better understanding could come the ability to design more effective therapy for different cancers and improvements in the way we dealt with cancer metaytasis. Statcd diffcrently, ‘You cannot fix something if you do not knon how it works.’ Given this frustration with surgical oncology’s role in the treatment of metastasis. I welcomed the suggestion by the Chairman of the Department of Clinical Studies, Kobert Marshak (who would later become the Dean of the Veterinary School at the University of Pennsylvania), to apply to the Graduate School at the University of Pennsylvania and pursue a career in cancer research. My interest in understanding the mechanisms of cancer metastasis led me to seek out Irving Zeidnian, Professor of Pathology at the Univcrsity of Pennsylvania School of Medicine. Subsequent to a long converration, he agreed to accept me as his student. In 196S, I transferred a mere 500 yards to the Department of Pathology at the University of Pennsylvania School or Medicine and entered a new world. The Learning Years In 1968, an international meeling of scientists working in the field of metastaGs could have been held in a small classroom. Metastasis research was not viewed as particularly attractive or even useful. For example, when a leading virologist from The Hebrew University in Jerusalem heard that I planned to study cancer metastasis. he announced. ‘What is there to study?
Either the cancer metastasized or it did not. If it did not, the surgeon will cure the disease, and if it did, it’s too late, so why study its biology?’ 1 have never regretted ignoring this declaration. The Department of Pathology at the University of Pennsylvania Medical School was unique because several of its faculty worked in the area of invasion and metastasis. The Chairman, Dale Rex Coman, had recruited, among many outstanding experimental pathologists, Irving Zeidman, Charles Breedis, Leonard Berwick, Gabriel Gasic, and Peter Nowell (who would become the next chairman). Drs. Coman. Breedis, and Zeidman shared a large laboratory (unheard of today) and started the workday with a morning coffee and discussion period. Dale and Irving were nature lovers, so often the discussion ranged from migratory patterns of birds and seasonal changes in flora and fauna to a recent publication by a colleague, or results of current experiments and their significance. T was embraced by the group, treated as an equal, and taught some principles of successful research. Among them, a question or hypothesis must be based on an observation. Once a question is formulated, one designs appropriate experiments so as to provide as unambiguous answers as possible. In fact, a good experiment may even yield ‘yes’ or ‘no’ answers, although this is rarely achieved in the complex field of cancer biology. A rabbit carcinoma system was one of a few animal models available for the study of cancer metastasis. Using this model, Irving Zeidman had shown that tumor cells can pass through capillary beds of different organs at different rates and that metastases do not necessarily result in the first organ that the blood-borne Cell5 encounter(’). To produce experimental metastases in the lung or liver of rabbits, we had to inject at least one million viable cells into the circulation. This puzzled me. Why was the yield of metastases so low? Did only a few cells survive the injection‘? How could the fate and distribution of tumor cells that enter the circulation be monitored? A thorough review of the relevant literature could have been accomplished in one night(2).Among the few who were working in the field, Bernard and Edwin Fisher reported that chromium-51 can be used to label tumor cells before they are injected into experimental animals(3). I travelled to Pittsburgh to discuss some of the details with Bernie Fisher, whom I admiringly dubbed .the John Wayne of cancer research’ for his gruff but affectionate manner. Since chromium-51 labeled live and dead cells, a better method to study the organ distribution and fate of live tumor cells was required. During one of the early morning coffee sessions in Philadelphia, I decided to concentrate my attention on developing a quantitative analysis of hematogenous metastasis for my Ph.D. thesis. My fellow postdoctoral fellows at Penn were Carlton Stewart and John Kreider. What a trio we made! Carl was working on how mitogens influenced the immune
response and was also interested in the biology of macrophages. Years later 1 would embark on trying to understand this wonderful cell. John taught me the rudiments of tissue culture, a witchcraft never to be practiced during the summer because of constant yeast contamination. This despite his rule that one must rinse his hands in diluted iodine solution before any culture work; one’s hands were permanently stained, but the contamination pcrsisted. However, our salvation soon arrived. John, a Board-certified pathologist, was a consultant to the General Electric Company, involved at that time with exploration of space. While at GE, he became acquainted with the laminar airflow cabinet (used to assemble this and that for space under dustfree conditions) and decided to use it for tissue culture. John published his observation and tissue culturing was revolutionized(4).I take this opportunity to thank him for this incredible contribution. H e is also responsible for another important contribution to my own research. 1 complained that a mouse fibrosarcoma syngeneic to BALB/c mice did not produce visible lung metastases. He recommended that I obtain the B16 melanoma from the Jackson Laboratories because, after intravenous inoculation, this tumor produced visible black tumor colonies in the lung. This 1 have done and, subsequently, the B16 melanoma became the standard tumor system for studies of experimental metastasis. After months of trial and error, I completed a detailed analysis of the distribution and fate of B16 melanoma cells labeled with [12sl]iododeoxyuridine(5). Most cells that cntered the circulation died rapidly, and by 24 h, less than 1YO of the cells survived. In fact, the metastases were produced from less than 0.1 YO of the original inocula. I concluded, in my 1970 paper(’), that a metastasis resulted from only a few surviving cells and could even originate from a single surviving melanoma cell. Sixteen years later, my postdoctoral fellow James Talmadge and I provided definitive evidence that metastases have a clonal origin, and that they can result from the expansion of a single progenitor The fact that the vast majority of tumor cells died in the circulation was not surprising, but it did raise an interesting question. D o metastases result from the fortuitous survival and growth of some cells or from the selective survival and growth of unique subpopulations of cells? The Selective Nature of Cancer Metastasis The conventional wisdom of the time was that metastasis either represented a random process or the inability of the host to prevent cancer spread. To examine this question, I injected mice with syngeneic B16 melanoma cells and harvested lung metastases. Cells isolated from these metastases were injected intravenously into normal mice where they produced lung metastases, repeating the cycle several times. I then compared the metastatic behavior of the cycled cells with that of the wild-type (parent) tumor and
found that the cells isolated from metastases were significantly morc metastatic than the cells from the parental tumor. My excitement led me to write a brief report to Nature, which, after minor revisions, was accepted for publication (to the surprise of my colleagues who predicted that Nature would not publish ‘metastasis data’)(’). In 1W2, Richmond Prehn, who was one of my teachers in thc Department of Pathology at Pennsylvania, invited me to speak at the Gordon Research Conference on Cancer. This was a thrilling and humbling experience. Although I was to discuss what I considered to be a major issue in oncology, it was a mysterious one to most researchers. Moreover. all the data I planned to present were generated by my technician, Marilyn Budmen, and me. I therefore did not have the obligatory slide listing a large number of collaborators to be acknowledged. To remedy this deficiency, Osias Stutman suggested a fictitious collaborator: Morris Shapiro. The presentation was a success, and during the coffee break, Osias Stutman left a cryptic messagc on thc board: ‘Dr. Shapiro, call your office - Josh Fidler appropriated your data.’ Morris Shapiro became a silent hero and stories about him proliferated. Morris was actually instrumental in my meeting my friend, scientific collaborator, and wife. Margaret Kripke. Margaret and I attended an American Cancer Society meeting in 1974. That year’s Morris Shapiro story was the following: Morris’s wife died when he was 65 years old. He decided to get back into circulation and purchased a toupee, elevator shocs. a designer suit, and a wide brimmed hat, and he had his teeth capped and his nose adjusted. While crossing the street, he was run over by a bus. When he reached heaven, he challenged the Almighty: ‘Lord, why did you have to kill me? T was a good man, a loving husband, a devoted father, and I gave to charity. Just because my wife died and I wanted to get back into circulation, you had to kill me?’ The Lord gazed upon Morris and answered, ‘To tell you the truth, Morris, I didn’t recognize you!’ I heard Margaret giggling in the audience and thought that I must meet this immunologist who obviously had a sense of humor. We were married in 1975 and were recruited by Mike Hanna to head independent laboratories in the just established Cancer Biology Program at the NCIFrederick Cancer Research Facility occupying the old biological warfare buildings at Fort Dietrick. My laboratory was designated the Cancer Metastasis Laboratory. To my knowledge. this was the first major government-supported research laboratory dedicated to study the biology and therapy of metastasis.
Tumor Heterogeneity The pioneering days at the NCI-Frederick Cancer Research Center were thrilling. Every day was more exciting than the preceding one. We were converting the old biological warfare facility to a cancer biology program, and our efforts were rewarding. I succeeded
in recruiting several outstanding young scientists, including Douglas Gersten. Ian Hart, Avraham Raz, and Nabil Hanna. At that time, Morris Shapiro retired. In 1976, Douglas Gersten, Marilyn Budmen, and I examined whether tumor cells that were lysed by lymphocytes (under in vitro conditions) differed qualitatively from tumor cells that were not. We succeeded in isolating lymphocyte-resistant variants from the starting populations and concluded that their survival was not random@).Ian Hart then selected for melanoma cells with increased invasive capacity and found that these cells also had increased metastatic capacity in syngeneic mice(9). One obvious criticism of these studies was that the surviving isolatcd tumor cells could have arisen as a result of adaptive rather than selective processes. Although I was keenly aware of this shortcoming, I could not design a definitive experiment to rule this out (or in). Margaret Kripke, who had extensive training in immunology and microbiology, devised a solution: Do a modified Luria and Delbruck fluctuation assay(’’’. After a hasty retreat to the library. T adopted the idea enthusiastically. In 1977, in the first test of the idea, Margaret and I cloned the B16 melanoma and showed that different tumor cell clones, each derived from individual cells isolated from the parent tumor, differed dramatically in their ability to form pulmonary metastases in byngeneic mice. Control subcloning procedures demonstrated that the observed diversity was not a consequence of the cloning procedure. Science published our report(”). Tumor heterogeneity in general and metastatic heterogeneity in particular becamc publishable. To exclude the possibility that the metastatic heterogeneity found in the B16 melanoma might have been introduced as a result of the lengthy in vivo and in vitro cultivation, Margarct and I studied the biological and metastatic heterogeneity in a mouse melanoma induced in a C3H mouse by chronic exposure to UVB radiation and painting with croton oil. One mouse thus treated develo ed a melanoma designated K-1735 (Kripke-l735)(’ 1. The original K-1735 melanoma was established in culture and immediately cloned. We found that the clones differed greatly from each other and from the parent tumor in their ability to produce lung metastases which themselves also varied in size and pigmentation(13).Clearly, tumor heterogeneity was not due to long periods of tissue culture or serial transplantation. That preexisting tumor cell subpopulations proliferating in the same tumor exhibit heterogeneous metastatic potential has since been confirmed in many laboratories (notably, Garth Nicolson’s and George Poste’s). with a wide range of experimental animal tumors of different histories and histological origins. In addition, studies using young nude mice as models for metastasis of human neoplasms have shown that tumor lines and freshly isolated tumors, such as melanoma, colon carcinoma, and renal carcinoma, also contain
r
su bpopulations of cells with widely differing metastatic properties(''). The implications of neoplastic heterogeneity for cancer treatment are enormous. Cancer is a collection of heterogeneous diseases with each cancer of each tissue or organ displaying different subtypes. Heterogeneity exists among different patients prcsenting with the same disease, e.g., melanoma, arising in different parts of the body. Moreover, genotypic and phenotypic heterogeneity exist among cells populating a single tumor. Stated differently: with the development of better molecular diagnostic tools, we may be able to understand the individual characteristics of each patient's tumor and thus treat each cancer as an individual cancer. So these are the 'Roots' of my research into the biology of metastasis. During the last two decades, research in the field of cancer metastasis has expcrienced a remarkable and welcome renaissance. In part, this is due to the development of new experimental tools that allow analysis of the metastatic phenotype on a cellular, molecular: and biochemical level, and appreciation for the role host factors play in the pathogenesis of metastasis. Today's key issue in metastasis research is not whether neoplasms are heterogeneous - that is substantially settled except for the details. The challenge is to understand its origins and mechanisms. What is clear now is that metastasis is a selective proccss that is regulated by a number of mechanisms. This belief is quite contrary to the once widely accepted view that metastasis represents the ultimate expansion of cellular anarchy. In fact, the realization that, as a whole, the process of metastasis is selective leads me to be optimistic about the prospects for treatment of cancer spread. Belief that certain rules govern metastasis implies that elucidation and understanding of these
rules will lead to bctter therapeutic intervention. These regulatory mechanisms are now being investigated in a large number of laboratories worldwide.
References 1 Z O I D M .I.~(1957). , Melaalasia: ii review of recent advances. Cmcw Re>. 17. 157-161. B. AYD FISHER,E. R. (1967). Recent observations on the concept of . Arch. Patho/. 83, -321-324, 3 FISHFR, E. R. A N D FISHER,B. (1967). The organ distribntion of disseminated "Cr-labeled tumor cell?. Cuizcer Re.
