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Colony Inhibition Mediated by Nonimmune Leukocytes in Vitro and Skin Reactivity in Vivo as Indices of Tumorigenicity of Guinea Pig Cultures Transformed by Chemical Carcinogens

Cytogenetics and Cytology Section, Biology Branch, Carcinogenesis Program, Division of Cancer Cause and Prevention, National Cancer Institute, Bethesda, Maryland 20014

Two short-term quantitative assays that correlate with the tumorigenicity of strain 2 guinea pig fetal cells transformed in culture by chemical carcinogens are described; one measures inhibition of colony growth mediated by nonimmune leukocytes and the other measures skin reactivity in unimmunized syngeneic guinea pigs. Mineral oil-induced peritoneal exudate (PE) cells were obtained from healthy unimmunized syngeneic guinea pigs. The PE cells were cultured for 24 hr and the nonadherent cells with culture medium were incubated with tumorigenic or nontumorigenic target cells in ratios of 1000/1 to 10/1. After 7 to 9 days of incubation in the presence of peritoneal exudate cell culture (PEC), fewer target cell colonies were observed in cultures with tumorigenic than with nontumorigenic cells. The inhibition of tumorigenic cells was dependent on PEC concentration; at 1000/1 PEC/target cell ratio, a reduction of as much as 80% in the number of colonies and a 100% decrease in colony size relative to controls were noted. Inhibitory activity was present primarily in the supernatant fluid of the culture medium of the PE cells. Phytohemagglutinin stimulation of the PE cells increased PEC and peritoneal exudate cell culture medium supernatant (PES) colony-inhibitory activity as much as twofold. The differential colony inhibition activity of PES with or without phytohemagglutinin was stable during storage of PES for 5 months at -35°. In the 2nd assay, 2 or 5 x 10⁶ tumorigenic or nontumorigenic cells were inoculated intradermally into 12- to 16-week-old male unimmunized syngeneic guinea pigs; skin reactivity, in terms of the degree and persistence of induration during the next 4 days, was greater to tumorigenic than to nontumorigenic cells. In both assays, tumor-producing cells, morphologically transformed in culture, or tumor-derived cells, were affected more than early-passage-untreated fetal cells, morphologically nontransformed long-term-cultured cells previously exposed to noncarcinogenic chemicals, or chemical carcinogen-transformed but non-tumor-producing cells. The two assays, particularly the nonimmune leukocyte-mediated colony inhibition with its greater degree of discrimination, provide rapid estimations of the tumorigenic potential of cells transformed in in vitro model systems of chemical carcinogenesis.

Received 10/17/74. Accepted 12/27/74.