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THE CouxcIr. FOR ToBAcco RE sE ARCH-U.S.A., INC. May 12, 1986 TO: DR. S. C. SOMMERS AND STAFF FROM: D. H. Ford RE: Site visit with Dr. I. Dam.~ov, Hahnemann University Medical Center, Philadelphia, April 23, 1986. Grant No. 1689R1 entitled "Developmentally Pluripotent Human Lung Cancer Stem Cells" GOAL To determine if there is a single prototypical stem cell in human lung bronchial tissue which is pluripotential and from which the major types of human lung cancer cells develop. He feels that the small cells found in undifferentiated tumors may represent such stem cells. RESULTS To date, Dr. Damjanov has successfully established 5 lung carcinoma cell lines: 1 poorly differentiated adenocarcinoma line, 3 small carcinoma cell lines and 1 squamous cell line. All cell lines produce keratin polypeptides and one of the small cell lines has some cells which produce one of the three types of neurofilament proteins. (Since small cell carcinoma is believed by some investigators to be derived from the pulmonary neuroendocrine APUD class of cells and thus may in some way be related to true neurons, one wonders if these neurofilament producing cells might not produce the other two types of filaments if thyroid hormone were to be added to the culture media (thyroid hormone has been clearly established as inducing differentiation in neurons.) This was discussed with Dr. Damjanov. In one study, Dr. Damjanov has cultured small cell carcinoma cells obtained from pleural fluid on laminin coated plates (laminin enhances cell attachment to the plates). These cells have been characterized by electronmicroscopy and appear to be undifferentiated in that they lack the dense core vesicles commonly observed in SCC cells (vesicles containing bombasin and other peptides as well as serotonin). While these might represent a population of undifferentiated stem cells, Dr. Damjanov has been unable to induce them to differentiate into other types of cancer cells. When injected into nude mice, these cells produce only undifferentiated tumors of small cells which lack the characteristic dense core vesicles of human SCC. Dr. Damjanov is currently attempting to monitor differentiation of tumor cells with monoclonal antibodies to cell surface marker proteins which might characterize specific types of lung tumor. He is using standard hybridoma techniques to obtain sufficient amounts of antibody for study. So far he has obtained an antibody which seems to react with basement membrane (BM) matrix proteins of tumors while a second antibody reacts with some of the cells in bronchial glands, peripheral nerve fibers (not the Schwann cell sheaths) as well as with breast, lymphoma, myeloma, 2 of 6 melanomas, 8 SCCs, 2 squamous cell carcinomas, medullary throid carcinoma and carcinoid. This is obviously not an antibody relating to some tumor specific cell surface marker protein.

Site visit - Dr. I. Damjanov Page 2 Another line of investigation concerns the concept that tumor cells (as well as normal cell types) possess a laminin receptor which is necessary for them to bind to the BM, presumably to the laminin in the BM. He has developed antibodies to laminin which prevent tumor cells from attaching to laminin coated surfaces in culture. Question: Could one block the binding of circulating tumor cells (i.e., from SCC) to BM matrix protein by such an antibody and thus prevent metastasis? This line of investigation appears to be prominent in Dr. Damjanov's priorities, possibly because his attempts to define a common stem cell for all types of lung cancer have not been too rewarding. If his hypothesis that antibodies to the laminin of BM could block attachment of cancer cells and subsequent metastasis is confirmed, this could be an attractive goal. Question: how does a circulating cancer cell penetrate the endothelial lining of blood vessels to attain the surface of the BM? Further, could there be a similar relationship with the fibronectin present in the BM? COMMENT Dr. Damjanov's initial goal of detecting and characterizing a common stem cell for all lung cancers appears to be difficult to attain and quite possibly such a cell does not exist. Thus, while he has established five lines of lung carcinoma cells, none appears likely to provide the "ancestor" cell type for which he has been searching. A new direction in his research which has emerged relating to detecting the presence of specific cell surface markers which characterize specific lung tumor types also appears to be unlikely to clearly delineate specific types of tumors at this time, since the early results so far appear non-specific. However, the third line of investigation on the presence of receptors on BM laminin which could be blocked with antibodies to such receptors may hold some promise in preventing metastasis. While Dr. Damjanov's recently developed interest in antibodies to laminin receptors (and perhaps for receptors of fibronectin as well) in the BM appears exciting and worthy of CTR support, we would hesitate to recommend support beyond the original 3-year period requested, unless his results in this line of investigation turn out to be more rewarding than his search for the primordial stem cell or specific cell surface markers which characterize specific types of tumor cell. While he is moving to Jefferson Medical School this summer, this move should not disrupt his program severely. Thus, his success or failure in determining if blocking receptors on laminin prevents metastasis should be evident before a new proposal needs to be considered. DHF/DS