More than 1000 cells of a spontaneously transformed line of BALB/3T3 cells are required to initiate tumors in half the nude mice inoculated s.c., although the cells clone with an efficiency approaching 100% in culture. The cells of two tumors with prolonged latent periods, initiated by 2 X 10(4) and 5 X 10(3) cells, were chosen for detailed clonal analysis in culture. The cells from the tumors grew very poorly in culture in the first passages, but with increasing speed and efficiency in later

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... ssages. Cells derived directly from the two tumors cloned in agar with an efficiency of 0.01 and 0.002%. The growth rates on plastic of the rare successful clones derived from agar were generally low but extremely varied. Some clones lost the capacity for multiplication in a few passages, while others persisted but fluctuated unpredictably in growth rate in the early passages. The graded increase in growth rate of the uncloned tumor cell populations was probably the result of selection of the more rapidly growing clones. One of the slower-growing clones was subcloned. About half of the subclones grew at a slower rate than the parental clone. These, however, increased progressively in growth rate over six successive weekly passages, suggesting the occurrence of a gradual physiological adaptation. We conclude that selection of fast-growing clones contributes a major part of the gradually improving growth of tumor cell populations in culture, but that a physiological adaptation extending over many cell generations makes a significant contribution. The mechanism in either case is unknown, and indeed there may not be a unique mechanism in the scientifically rigorous sense.