Brief Communication: Organ Selectivity for Implantation Survival and Growth of 816 Melanoma. Variant Tumor Lines 1, 2. Isaiah J. Fidler 3 and Garth L. Nicolson ...
Brief Communication: Organ Selectivity for Implantation Survival and Growth of 816 Melanoma Variant Tumor Lines 1, 2 Isaiah J. Fidler 3 and Garth L. Nicolson 4, 5
One important route for spread of malignant tumors is the circulatory system (1-3). After invading local tissue and penetrating the blood stream, tumor cells move into organs and tissues where they may be trapped or arrested in the first capillary bed encountered (4, 5), enter the parenchyma of the organ, or recirculate (3 --6). The presence of tumor cells in circulation, although insufficient by itself to cause neoplasms, seems to be a step toward the establishment of secondary tumor growths. In fact, the recovery of tumor cells from blood has been of little prognostic value for prediction of metastatic disease (3). In several tumor systems, such as melanoma (7), plasmacytoma (8), reticulum cell sarcoma (9), and histiocytoma and others (10), metastases are found only in certain tissues and organs. These findings suggest that this tissue and organ selection is not a result of random distribution, but rather that properties of the circulating tumor cells, the capillary endothelium, and the organ environment may dictate malignant cell distribution, survival, and growth. One system used to study the possible properties of cancer cells and/or their syngeneic host for determination of patterns of arrest, survival, and growth of circulating tumor cells is that of the mouse B16 malignant melanoma variants (11, 12). Several metastatic variant lines were selected from the original melanoma line by repeated iv injection of B16 cells into mice and recovery of tumor colonies from lung growths. The cells from these secondary colonies were put into tissue culture and later injected iv into other normal syngeneic animals to repeat the selection process. The high or low VoL. 57, NO.5, NOVEMBER 1976
survival and in vivo growth properties of these B 16 cells were stable during long-term in vitro culture and were related to the number of selections that occurred in vivo. For example, one in vivo cycle of tumor cells that preferentially formed lung tumor colonies after iv injection yielded a cell variant line (B 16-Fl) of low in vivo survival and growth potential (defined by the number of pulmonary metastases per input cell), but nine more selections in vivo yielded a variant line (B 16-FlO) of high in vivo survival and growth potential. After injection of 125IUDR-labeled BI6-Fl and BI6-FlO cells into tail veins of syngeneic C57BL/6 mice, approximately 70 and 100% of the injected Fl and FlO cells, respectively, were found in the lungs within 2 minutes (11). Although most of the viable cells in the lungs died or left the lungs within 1 day, a few extrapulmonary metastases were found (13). It could be argued that after iv injection, the B 16 cells destined to form pulmonary growths entered the lung capillary bed first, were nonspecifically trapped, and never recirculated. Alternatively, perhaps cells from B 16 lines selected for increased survival in the lungs possessed properties that determined their distribution and subsequent growth independent of the route of cell entry into the circulation. We report here that initial distribution of 125IUDR-labeled BI6-Fl and BI6-FlO cells in the organs varied with the route of tumor cell injection; but within 1 day these differences were small, and the subsequent number of pulmonary metastases did not depend on the arrest of tumor cells in the lungs immediately after injection. MATERIALS AND METHODS
Animals.-Inbred C57BL/6 mice, 8-10 weeks old, were obtained from the Frederick Cancer Research Center Experimental Animal Breeding Facility (Frederick, Md.).
Tumors and culture conditions.-BI6 melanoma lines syngeneic to the C57BL/6 mouse were grown in Falcon tissue culture flasks with Eagle's minimum essential medium supplemented with 10% fetal calf serum (11, 14, 15). In the present studies we used BI6-Fl and BI6-FlO,
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ABBREVIATION USED: 125IUDR=[l25I]5-iodo-2' -deoxyuridine. t Received February 20, 1976; accepted April 16, 1976. 2 Supported by Public Health Service (PHS) contracts NOI C025423 from the Office of the Director and NOI CB33879 from the Tumor Immunology Program within the Division of Cancer Biology and Diagnosis, National Cancer Institute (NCI); by PHS grant CA15l22 (C.L.N.) from NCI; and by American Cancer Society grant BC-2ll, 3 Basic Research Program, Frederick Cancer Research Center, P. O. Box B, Frederick, Md. 2l70L 4 Department of Cancer Biology, The Salk Institute for Biological Studies, San Diego, Calif. 92112, and the Department of Developmental and Cell Biology, University of California, Irvine, Calif. 92664, 5 We thank S. Caines, M. Budmen,J. Smith, and A, Brodginski for technical assistance.
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ABSTRACT-The fate of bloodborne malignant melanoma cells selected for their enhanced ability to form lung colonies was examined to determine how specific tumor cells are arrested in certain organs during the experimental metastasis process. After murine B16 melanoma variant tumor cell lines with low (B16-F1) or high (B16-F10) survival and growth potential in vivo were administered by iv or intracardiac injections into syngeneic C57BL/6 mice, the quantitative distribution of [ ' 251]5-iodo-2'-deoxyuridine (' 25IUDR)-labeled cells in the organs and subsequent formation of metastatic lung colonies were assessed. The initial distribution of viable tumor cells was dependent on the route of injection: Soon after iv injection, more 125lUDR-labeled B16 cells were localized in the lungs and fewer in the blood and other organs than after intracardiac injection. However, 1 day after the injection, the number of viable tumor cells in the lungs was independent of the route of injection, and at 14 days the quantity of resulting lung tumor colonies was similar. Variant line B16-F10 cells were better. arrested and formed more tumors per input cell than B16.F1, regardless of the injection route. B16-F10 yielded only lung tumor colonies, whereas B16-F1 formed some' extrapulmonary tumor growths. The results suggested that the ultimate fate of circulating tumor cells was not determined solely by nonspecific arrest in the capillary bed of the first organ encountered, and that in vivo selection could produce tumor line variants with organ preferences.-J Natl Cancer Inst 57: 1199-1202, 1976.
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Statistical analysis .-Data were analyzed by Student's two-tailed t-test. RESULTS
The number and location of experimental metastases were determined after injection of 5X 10 4 viable B16 cells into C57BL/6 mice. The average quantity of experimental pulmonary tumor colonies 2 weeks after iv or intracardiac administration of one of the B16 lines was the same (within experimental error) (table 1). However, iv or intracardiac administration of the B 16-FlO line yielded approximately eight times more pulmonary tumors than the B 16-Flline. An interesting result in this experiment was that intracardiac injection of line Fl led to extrapulmonary metastases (liver, adrenal, and mesentary lymph node) in 4 of 8 test animals, whereas we found no extrapulmonary metastases in mice inoculated by intracardiac administration of cells from line FlO (table 1). In other experiments 2 of 4 and 3 of 10 animals exhibited extrapulmonary metastases 2 weeks after intracardiac injections of BI6-Fl cells, whereas BI6-FI0 injections failed to produce any extrapulmonary tumors in similar groups of animals. Histologic examination of several lungs containing melanoma colonies from mice injected by either iv or intracardiac routes did not reveal any differences in the location of the nodules. With either route of administration, tumor colonies were located near the pleura rather than lung parenchyma, as reported earlier in (1, 2, 5). The tissue distribution of 125lUDR-labeled B 16-Fl and BI6-FlO was assessed after iv or intracardiac injection of 1 X 10 5 viable single cells (table 2). Within 2 minutes after iv injection of line Fl, approximately 64% of the cells were found in the lungs; after intracardic administration, about 31% of F 1 cells were in pulmonary tissues at this time. The amount of viable tumor cells in the lungs declined rapidly (11, 13), but by day 7 the number of viable, originally injected tumor cells was approximately the same. Initially, more cells were localized in the liver and spleen after intracardiac injection of BI6-Fl than after iv administration, but within 3 hours the distribution of cells in the extrapulmonary organs was similar. These initial differences are especially significant, since in some mice given intracardiac injections some tumor emboli may have also entered the right side through a perforated septum. Detectable levels of B 16-Fl cells in the circulation were higher after intracardiac injection, I.-Pulmonary metastases in C57BL/6 mice after iu or intracardiac administration of B16-Fl or B16-FIO melanoma cells
TABLE
Pulmonary metastases a Route of injection iv Intracardiac
BI6-Fl
BI6-FI0 b
9±3 (4-13) 10±2 (9-13) c
79±16 (58-100) 83±10 (60-100) d
a Average No. ±SD (range). Measurements were made 14 days after injection of 5 x 104 viable B16 cells, 10 mice/group. b Differences in pulmonary metastases between mice receiving BI6-Fl and BI6-FI0 were highly significant (P
