Metronomic cyclophosphamide activation of anti-tumor immunity: Tumor model, mouse host, and drug schedule dependence of gene responses and their upstream regulators

Abstract

Background: Cyclophosphamide (CPA) can activate immunogenic tumor cell death, which induces immune-based tumor ablation and long-term anti-tumor immunity in a syngeneic C57BL/6 (B6) mouse GL261 glioma model when CPA is given on a 6-day repeating metronomic schedule (CPA/6d). In contrast, we find that two other syngeneic B6 mouse tumors, LLC lung carcinoma and B16F10 melanoma, do not exhibit these drug-induced immune responses despite their intrinsic sensitivity to CPA cytotoxicity. Methods: To elucidate underlying mechanisms, we investigated gene expression and molecular pathway changes associated with the disparate immune responsiveness of these tumors to CPA/6d treatment. Results: Global transcriptome analysis indicated substantial elevation of basal GL261 immune infiltration and strong CPA/6d activation of GL261 immune stimulatory pathways and their upstream regulators, but without preferential depletion of negative immune regulators compared to LLC and B16F10 tumors. In LLC tumors, where CPA/6d treatment is shown to be anti-angiogenic, CPA/6d suppressed VEGFA target genes and down regulated cell adhesion and leukocyte transendothelial migration genes. In GL261 tumors implanted in adaptive immune-deficient scid mice, where CPA/6d-induced GL261 regression is incomplete and late tumor growth rebound can occur, T cell receptor signaling and certain cytokine-cytokine receptor responses seen in B6 mice were deficient. Extending the CPA treatment interval from 6 to 9 days (CPA/9d) - which results in a strong but transient natural killer cell response followed by early tumor growth rebound - induced fewer cytokines and increased expression of drug metabolism genes. Conclusions: These findings elucidate molecular response pathways activated by intermittent metronomic CPA treatment and identify deficiencies that characterize immune-unresponsive tumor models and drug schedules.