Novel tumor models answering new cancer immunology challenges.
Dr. Untae Kim (1927-2017)
Our colleague and collaborator for many years, Dr. Untae Kim passed away on January 14, 2017. He was 90. He was born and raised in Antung, China and began studying medicine at Seoul National University at the outbreak of the Korean War. His studies were interrupted so he went to meet the U.S. ships landing at Incheon to volunteer his services. Being fluent in multiple languages, he assisted the U.S. Army as an interpreter on the front lines throughout the Korean War. He resumed his medical education after the war and was accepted as an Intern at Brockton Hospital outside of Boston, MA. After completing the residency programs in anatomic pathology and laboratory medicine at the Mallory Institute of Pathology and affiliated hospitals in Boston, MA in 1958, He sought an experience in experimental cancer research trainings under the late professor Jacob Furth at the Dana-Farber Cancer Institute. He worked closely with him for five years in his laboratories in Boston and at Columbia University Cancer Center in New York, learning a great deal about the hormone-dependent and independent tumor problems and developed variously hormone dependent rat mammary tumors. During this period, under the guidance of Dr. Furth, he participated in the Breast Cancer Task Force of the National Cancer Institute. Then, in 1963, he was invited by the Roswell Park Cancer Institute to be an associate chief pathologist, where he also established a research laboratory. In Buffalo, he worked for 34 years, as a fulltime clinical pathologist, and also participated in the Eastern Cooperative Oncology Group (ECOG) and the Cancer-Acute Leukemia Group B (CALGB). At RPCI, he developed and established many highly realistic rat mammary tumor cell lines vis-à-vis metastatic human breast cancer cells (see below). We present the results of some of the rodent model systems he created and in collaboration with us the establishment of the separation and isolation of lymphotrotropic and nonlymphotropic tumor cell subpopulations from the parental tumor. In a subsequent paper, we will explore some of the biology of these two subpopulation and discuss its future potential. In 2011, Dr. Kim was honored as a 50 year emeritus member of the AACR.
Stefan Cohen. March 2017
These are Dr Kim’s notes with his own words. We honor him for his extraordinary research legacy. For us it is a privilege to show these data to the world readers.
Emilio Barbera-Guillem. March 2017
A. Matching Pairs of Isogenic Human Carcinoma Cell Lines with and without Lymphotropic Metastatic Potential
I. Historical Background. Previously, it was first discovered in our laboratory that when spontaneously metastasizing, non-immunogenic, rat mammary tumor cells were transplanted in athymic nude mice, they were non-tumorigenic or rejected, while matching pair of non-metastasizing, immunogenic, syngeneic mammary tumors were readily accepted or highly tumorigenic. These observations completely contradicted the
traditional criteria for malignancy of transformed cells, i.e., “tumorigenic cells in nude mice are considered malignant and non-tumorigenic ones are not. In tissue culture of these cells, again our observations disagreed with or reversed the conventional criteria, i.e., the tumorigenic (non-metastasizing) cells assumed disorganized, “anchorage-independent growth characteristics in vitro, while the non-tumorigenic (lymph node-metastasizing) ones grew more cohesively in anchorage-dependent manner,”
The non-tumorigenic, spontaneously metastasizing cells, dormant in T-cell deficient hosts (nude mice), can be revived by selectively suppressing the T-independent, innate, Natural Killer Cell (NK) mediated anti-tumor immunity with repeated injections of anti-NK antibodies. Such revived tumor cells will grow as long as the NK activity is suppressed. When the resuscitated rat tumor cells were transplanted back into normal syngeneic rats, they promptly manifested their original lymph node metastasizing property. Thus, the lymph node metastasizing, non-tumorigenic cells may be re-defined as NK-sensitive, and the tumorigenic, non-metastasizing cells as NK-resistant, hence we initiated the new paradigm for malignancy for solid tumors contradicting the traditional definition.
II. Human Tumor Systems. The above observations made with highly realistic rodent tumors have helped us to extend this view directly to human cancer, and non-tumorigenic cell clones were successfully separated from tumorigenic ones, by xenografting in nude mice, randomly selected human carcinoma cell lines derived from ATCC. So far, we have separated and established three sets of matching pairs of isogenic human tumor cell clones from human breast, prostate and colon carcinoma cell lines.
The method for separation of NK-sensitive cells from NK-resistant ones has been applied for a US Patent #7989001. With this procedure, one should be able to isolate similar isogenic cells from different human carcinomas.
III. R & D Potential. With these matching sets of isogenic human tumor cells, there are many possibilities for the development of new diagnostic and therapeutic tools for identifying specific human cancer cells with metastatic potential, and selectively controlling or even eradicating such cells. Indeed, some of the molecular markers for
these cells with and without lymphotropic metastatic potential have already been identified as “Lymphoid genes and their products (US Patent #6,790,604).
1) Identification of “Metastasis Specific” molecules by subtractive hybridization technique, functional genomics & proteomics, and microarray techniques, to develop molecular profiles of human carcinoma cells with and without lymphotropic metastatic potential, in relation to our earlier discoveries identified in the above patent.
2) Development of monoclonal antibodies (Mab) specific for recognizing human carcinoma cells with lymphotropic metastatic potential, by subtractive immunization procedure. Previously, we successfully developed the Mab (M-N) which specifically recognizes and inhibits the growth of metastasizing rat mammary tumors (US Patent #6,391,301 – “Agent for Identifying and Treating Metastasizing Tumors”). This tried-and-proven-approach seems to be a practical and relatively easy one, and thus we hope to develop similar but human specific monoclonal antibodies with these matching pairs of human breast, prostate and colon carcinoma cells, respectively. Perhaps, with this procedure, we may also be able to pull out Mab against EGFR, ZAP-70, CD54 (I-CAM), CD4, and other activated T-cell associated molecules, that have been shown in the NK-sensitive (metastatic) human carcinoma cells. Hopefully, we should be able to develop new diagnostic and therapeutic tools better than anything available today.
3) Identification of factors in NK-sensitive human carcinoma cells with lymphotropic metastatic potential. It is extraordinary to observe a complete inhibition of growth of highly malignant cancer cells simply by T-independent, NK-mediated innate immunity. Perhaps, some of the answers may come out from the functional genomic and proteomic analyses of these cells. Experimental creation of such immunologic environments in our syngeneic tumor host systems has been attempted with some success.
B. Spontaneously Metastasizing Tumor Models
1. Historical Background. For many years, experimental cancer research on malignant solid tumors was largely performed with animal tumors that either did not metastasize spontaneously, or with those metastasized only to the lung via the blood stream in the form of passive tumor cell embolization. However, unlike common mouse or rat tumors, human malignant solid tumors, particularly, common human carcinomas usually metastasize to regional lymph nodes first, spread further via the lymphatic route,
merge into the venous return, then traverse the pulmonary artery and vein, and ultimately disseminate further to distant organs along the arterial tributaries. Such animal tumors realistic vis-à-vis human carcinomas were not available, until 1970 when we first developed spontaneously metastasizing mammary carcinomas in the highly-inbred strain of W/Fu rats by immunologically modifying the host together with feeding chemical carcinogens. Since then, we have developed many more spontaneously metastasizing tumors, not only with chemical, viral, X-irradiation, and oncogenes combined with immunological manipulation of the host, but also learned how to convert non-metastasizing tumor cells into metastasizing ones by hybridizing the tumor cells with host lymphoid cells, revealing pathogenesis of how the metastatic potential may be acquired during tumor development. These different categories of tumors have been well characterized and their stable cell lines established. Three groups of spontaneously metastasizing rat and mouse tumors are listed.
C. Tumor Models for Anti-Angiogenic Therapy
The great pioneering biochemist Otto Warburg had observed that ‘neoplastic cells tend to have well developed anaerobic glycolytic pathway, and hence do not require as much oxygen as normal cells do for energy generation.’ Indeed, several cancer researchers including us, have found that solid tumors in general, perhaps with the exception of hemangiomas, require 30% less oxygen than adjacent normal tissue. When the functional blood-flow was measured by radio-labeled latex particles, and independently by the direct lymph-angiogram taken at 15 min. intervals following intra-tumoral infusion of water-soluble contrast medium, the lymph node metastasizing rat mammary tumors had only 20% as much blood-flow as that of the non-metastasizing ones. The hematogenously metastasizing tumors, on the other hand, had greater vascularity than either the non-metastasizing and lymphogenously metastasizing ones. Therefore, the current stratagem for anti-angiogenic therapy of human cancer may need to be reevaluated.
D. Tumor-Lymphoid Cell Hybrids
Our observations of the expression of lymphoid cell associated molecules on the surface of lymph node metastasizing rodent and human carcinoma cells suggested that perhaps, during the long-intervals between the initial carcinogenic events and the ultimate clinical detection of cancer, there would likely be repeated interactions between neoplastic cells and host immune cells, and such events might cause hybridization between the two, resulting in the acquisition of some of the lymphoid cell characteristics, including their lymphotropic migratory capability by tumor cells. In order to test this supposition, non-metastasizing animal tumor cells of various etiology were fused in vitro with a chemical fusogen, and the fused cells were implanted into normal syngeneic animals and observed for the development of secondary (metastatic) tumor cell colonies.
With the highly realistic tumor models listed in Tables 2 through 6, new innovative treatment modalities may be tested for their specific effectiveness, and perhaps, the current therapeutic agents may be reevaluated.
E. Models for Natural History or Evolution of Mammary & other Cancer Progressing from Hormone Dependence to Autonomy & Acquisition of Metastatic Potential
A rat mammary tumor was induced with a subthreshold dose chemical carcinogen under continuous stimulation of the female W/Fu rats with mammotrophic hormone. Initially, such a tumor (MT-W9), when transplanted in syngeneic rats, grew only in rats similarly stimulated with the hormone. Otherwise it would not grow in normal syngeneic young adult female rats. MT-W9 is well differentiated, milk-secreting adenocarcinoma, and one may regard it minimally transformed neoplasm from the normal organ. When MT-W9 was continuously transplanted in hormone-treated and un-treated rats, after many transplantation generations, a clone of tumor that would grow in normal adult female rats, and it was designated as MT-W9A. It is characterized as estrogen dependent, indicating that it requires physiological levels of female hormones. It is also well- differentiated adenocarcinoma, but grew extremely slowly. Subsequently, again after many generations, another sub-clone of MT-W9A appeared in surgically ovariectomized female rats or adult male rats. It was designated as MT-W9B. Further sub-coning continued, and MT-W9C was isolated: when transplanted, it grew better in normal males than in normal or ovariectomized females. MT-W9C was characterized as androgen-sensitive. Lastly, MT-W9D was isolated, and was characterized as “Fully
Autonomous” or completely independent, for it fast and grew well in either in males, females, ovariectomized females, and castrated males. As MT-W9 progressed it became less and less differentiated and grew faster and faster. However, none of the autonomous or hormone independent tumors gave rise to a metastasizing tumor. On the contrary, spontaneously metastasizing tumors, MT-W449 & MT-W450, emerged from MT-W9A, the estrogen-dependent one which grew extremely slowly. Perhaps, there could have been repeated tumor-host interactions during dormancy. Thus, we have isolated by “in vivo cloning” the entire spectrum of the natural history of mammary tumors, progressing
from hormone dependence to autonomy and acquisition of metastatic potential during its course. These models may be ideal for studying the molecular mechanisms of tumor progression.
Proposed tumor progression: