To determine whether neuroblastomas acquire a sustained drug-resistant phenotype from exposure to chemotherapeutic agents given to patients in vivo, we studied neuroblastoma cell lines established at different points of therapy: six at diagnosis before therapy (DX), six at progressive disease during induction therapy (PD-Ind), and five at relapse after intensive chemoradiotherapy and bone marrow transplantation (PD-BMT). Cells were maintained in the absence of drug selective pressure. Dose-response curves of melphalan, cisplatin, carboplatin, doxorubicin, and etoposide for the cell line panel were determined by measuring cytotoxicity with a 96-well-plate digital imaging microscopy (DIMSCAN) microassay. Drug resistance of cell lines progressively increased with the intensity of therapy delivered in vivo. The greatest resistance was seen in PD-BMT cell lines: IC90 values in PD-BMT cell lines were higher than clinically achievable drug levels by 1–37 times for melphalan, 1–9 times for carboplatin, 25–78 times for cisplatin, 6–719 times for doxorubicin, and 3–52 times for etoposide. Genomic amplification of MYCN did not correlate with resistance. Cross-resistance by Pearson correlation (r ≥ 0.6) was observed between: (a) cisplatin + doxorubicin; (b) carboplatin + cisplatin, etoposide, or melphalan; (c) etoposide + cisplatin, melphalan, or doxorubicin. These data indicate that during therapy, neuroblastomas can acquire resistance to cytotoxic drugs because of the population expansion of tumor cells possessing stable genetic or epigenetic alterations that confer resistance.
Supported in part by a Childrens Hospital Los Angeles Research Institute Research Career Development Fellowship; the Neil Bogart Memorial Laboratories of the T. J. Martell Foundation for Leukemia, Cancer, and AIDS Research; by National Cancer Institute Grants CA60104, CA13539, and CA14089; and by the American Institute for Cancer Research.