Effects of CSF on ALL drug sensitivity. (A) NALM-6 cells (B-cell ALL) were treated with asparaginase, cytarabine, dexamethasone, doxorubicin, etoposide, or methotrexate in either CSF or regular tissue culture media. Leukemia cell viability was assessed after 48 hours of drug treatment using fixable viability dye staining and flow cytometry. Error bars represent the mean ± standard deviation (SD) of 3 technical replicates. ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001 by t test. (B-C) NALM-6 (B) and REH (C) cells were treated with methotrexate 220 nM and 180 nM, respectively, or DMSO in regular media or CSF obtained from patients with extraventricular CSF drains (CSF 1-4), normal donors (CSF 5-6), or patients with ALL undergoing routine lumbar punctures during maintenance therapy (CSF 7-10). Leukemia cell viability was assessed after 48 hours of drug treatment using annexin-V and viability dye staining and flow cytometry. Error bars represent the mean ± SD of 3 technical replicates. When comparing methotrexate toxicity in media vs each CSF sample, P < .0001 by analysis of variance with post hoc Dunnett multiple comparisons test. EVD, extraventricular drain; ns, not significant.