Metronomic cylcophosphamide-activated anti-tumor immune responses: dose and schedule dependence in mouse models
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Metronomic cyclophosphamide (CPA) treatment activates robust anti-tumor immunity and induces regression of implanted tumors in mouse models of brain cancer when administered on an intermittent, every 6-day schedule (CPA/6d), but not on a daily low-dose or a maximum-tolerated dose schedule. Five intermittent metronomic CPA schedules were investigated in GL261 gliomas implanted in scid mice. Metronomic CPA treatments spaced 9 or 12 days apart induced extensive tumor regression, however, tumor-infiltrating natural killer cell responses were not sustained, and tumor growth rapidly resumed after treatment day 24. Increasing the CPA dose prolonged the period of tumor regression on the every 9-day schedule, but natural killer cell activation was markedly decreased. Thus, sustained immune and anti-tumor responses were only achieved on the CPA/6d schedule. Furthermore, CPA/6d treatment eradicated GL261 tumors implanted in immunocompetent C57BL/6 mice by activating anti-tumor CD8-T cell responses and immune memory, which provides proof-of-concept that single agent chemotherapy delivered on an optimized metronomic schedule can cure large established cancers. Transcriptomic profiling, KEGG pathway, and upstream regulator analysis were employed to compare CPA/6d-induced gene expression changes between: immune-responsive GL261 tumors and immune-unresponsive Lewis lung carcinoma (LLC) and B16F10 melanoma tumors; between GL261 tumors implanted in immunocompetent mice versus in scid immunodeficient mice; and between GL261 tumors in scid mice treated with CPA every 6-days or every 9-days. CPA-treated LLC tumors were associated with inhibited VEGFA-targeted genes, down-regulated cell adhesion and transendothelial migration genes, and up-regulated drug metabolism pathways. In B16F10 tumors, CPA activated genes in chemokine signaling and antigen processing and presentation pathways, but no NK cell and T cell effector pathways were activated. GL261 tumors in scid mice were deficient in CPA activation of a subset of cytokine and cytokine receptor genes and T cell receptor signaling genes seen in immunocompetent mice. Cytokine gene expression was lower and drug metabolism gene expression was higher in every 9-day CPA-treated tumors versus CPA/6d-treated tumors. Together, these studies elucidate the dose, schedule, and adaptive immune-dependence of CPA-induced anti-tumor immune responses, giving new insight into the molecular signaling events underlying the deficiencies in immune responses seen in intermittent metronomic CPA-unresponsive tumor models.