RAPID ACQUISITION OF MULTICELLULAR DRUG-RESISTANCE AFTER A SINGLE EXPOSURE OF MAMMARY-TUMOR CELLS TO ANTITUMOR ALKYLATING-AGENTS

Background: Clinical drug resistance is either intrinsic (de novo) or often acquired rapidly in conjunction with chemotherapy. By contrast, the selection of drug-resistant mutant cell lines in monolayer culture systems is usually a more protracted process. Sublines of mouse EMT-6 mammary tumor cells selected for resistance to various alkylating age nts in vivo after serial passage into syngeneic mice manifest their re sistance in vitro only when cultured as three-dimensional multicellula r aggregates or spheroids. Purpose: We examined whether a single expos ure of mouse FMT-6 or human MDA-MB-231 breast cancer cells to alkylati ng agents in vitro is sufficient for the induction of a resistance phe notype, which may be detected by re-applying the drugs to cells grown as three-dimensional aggregates. Methods: Mouse EMT-6 and human MDA-MB -231 breast cancer cells cultured as three-dimensional aggregates were exposed to a single dose of alkylating agent for 1-5 days. Aggregates were dispersed, and cells were plated as monolayer cultures for up to 8 weeks to allow for recovery. Colony-forming ability was assessed af ter a subsequent alkylating-agent exposure of cells cultured as monola yers or three-dimensional aggregates. Results: A single in vitro expos ure to 12.5-mu M cisplatin (CDDP) for 5 days or 25 mu M 4-hydroperoxyc yclophasphamide (4-O2H CTX) for 1 or 3 days without changing the mediu m was sufficient to induce transient but substantial resistance in EMT -6 cells as determined by clonogenic assays. Such resistance was not d etected when monolayer cell cultures were used. The concentration of 4 -O2H-CTX and the length of time the cells remained in three-dimensiona l culture after initial exposure to this drug was associated with the degree of subsequent drug resistance of cells grown as three-dimension al cultures. Furthermore, this acquired resistance after a single drug exposure was accompanied by changes in the three-dimensional architec ture of the cell aggregates, which now formed much more compact multic ellular spheroids. Similarly, a single exposure to 4-O2H-CTX was enoug h to bring about resistance in MDA-MB-231 cells detectable only in thr ee-dimensional cultures, as well as the change in three-dimensional ar chitecture. Conclusions: Rapid acquisition of resistance likely repres ents a physiologic mechanism of adaptation operative at the multicellu lar level rather than a stable genetic change and may be one of the re asons for the rapid development of drug resistance acquired by tumors in vivo. Implications: In vivo drug exposure may result in transient a nd low levels of drug resistance that mag nevertheless be clinically r elevant.