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Use of Lymph in Cell Culture to Model Hormonal and Nutritional Constraints on Tumor Growth in Vivo¹

Department of Molecular Biology and Virus Laboratory, University of California, Berkeley, California 94720

Transformed BALB/3T3 cells, which proliferate without restraint in culture, consistently produce rapidly growing sarcomas when 10⁵ or more cells are inoculated into nude mice but produce sarcomas, of widely varying latent periods and growth rates, or negatives when 10⁴ or fewer cells are inoculated. In an attempt to simulate the in vivo constraints on tumor development, these cells were cultured on plastic surfaces in concentrations of lymph up to 100%. Calf lymph was less effective in supporting multiplication than calf serum at all concentrations up to about 50%. The rate of cell multiplication progressively decreased with increasing concentrations of both fluids above 50%. Nonetheless, rapid multiplication could be achieved even in 100% serum or lymph by supplementing them with the high concentrations of nutrients used in the synthetic medium MCDB 402. Supplementation of cystine and glutamine was essential for the growth-enhancing effects of the other nutrients. When the cells were suspended in agar, lymph was much less effective than serum in promoting colony formation even when both were supplemented with cystine and glutamine, or with all the constituents of the synthetic medium. We conclude that part of the low efficiency of tumor production and reduced growth rate of the transformed cells in mice resulted from a combination of (a) the paucity of growth factors in interstitial fluid, (b) the marked reduction in concentration of essential amino acids encountered by the cells in passing from culture into mice, and (c) the fact that cells multiplying in s.c. space do so without benefit of attachment to a solid substratum. Other factors, such as the growth inhibiting effects of direct contact with quiescent muscle and connective tissue cells, remain to be evaluated.

¹ This work was supported by USPHS Grant CA-15744 from the Division of Extramural Activities, National Cancer Institute, by American Cancer Society Research Development Program Grant RD-231, and by The Council for Tobacco Research Grant 1948.

² To whom requests for reprints should be addressed, at the Department of Molecular Biology, University of California, Wendell M. Stanley Hall, Berkeley, CA 94720.

Received 2/18/87. Revised 6/ 1/87. Accepted 6/17/87.

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