Metronomic cyclophosphamide eradicates large implanted GL261 gliomas by activating antitumor Cd8+ T-cell responses and immune memory

Cancer chemotherapy using cytotoxic drugs can induce immunogenic tumor cell death; however, dosing regimens and schedules that enable single-agent chemotherapy to induce adaptive immune-dependent ablation of large, established tumors with activation of long-term immune memory have not been identified. Here, we investigate this issue in a syngeneic, implanted GL261 glioma model in immune-competent mice given cyclophosphamide on a 6-day repeating metronomic schedule. Two cycles of metronomic cyclophosphamide treatment induced sustained upregulation of tumor-associated CD8(+) cytotoxic T lymphocyte (CTL) cells, natural killer (NK) cells, macrophages, and other immune cells. Expression of CTL- and NK-cell-shared effectors peaked on Day 6, and then declined by Day 9 after the second cyclophosphamide injection and correlated inversely with the expression of the regulatory T cell (Treg) marker Foxp3. Sustained tumor regression leading to tumor ablation was achieved after several cyclophosphamide treatment cycles. Tumor ablation required CD8(+) T cells, as shown by immunodepletion studies, and was associated with immunity to re-challenge with GL261 glioma cells, but not B16-F10 melanoma or Lewis lung carcinoma cells. Rejection of GL261 tumor re-challenge was associated with elevated CTLs in blood and increased CTL infiltration in tumors, consistent with the induction of long-term, specific CD8(+) T-cell anti-GL261 tumor memory. Co-depletion of CD8(+) T cells and NK cells did not inhibit tumor regression beyond CD8(+) T-cell depletion alone, suggesting that the metronomic cyclophosphamide-activated NK cells function via CD8a(+) T cells. Taken together, these findings provide proof-of-concept that single-agent chemotherapy delivered on an optimized metronomic schedule can eradicate large, established tumors and induce long-term immune memory.