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

ABSTRACT: 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.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.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.These findings elucidate molecular response pathways activated by intermittent metronomic CPA treatment and identify deficiencies that characterize immune-unresponsive tumor models and drug schedules.

SUBMITTER: Wu J

PROVIDER: S-EPMC4982114 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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

Wu Junjie J Jordan Marie M Waxman David J DJ

BMC cancer 20160811

<h4>Background</h4>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.<h4>Met ...[more]

PMID: 27515027

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Project description:BackgroundMetronomic cyclophosphamide given on an intermittent, 6-day repeating schedule, but not on an exposure dose-equivalent daily schedule, activates an anti-tumor innate immune response that leads to major regression of large implanted gliomas, without anti-angiogenesis.Methods and approachMice bearing implanted 9L gliomas were used to investigate the effects of this 6-day repeating, immunogenic cyclophosphamide schedule on myeloid-derived suppressor cells, which are pro-angiogenic and can inhibit anti-tumor immunity, and to elucidate the mechanism whereby the innate immune cell-dependent tumor regression response to metronomic cyclophosphamide treatment is blocked by several anti-angiogenic receptor tyrosine kinase inhibitors.ResultsIntermittent metronomic cyclophosphamide scheduling strongly increased glioma-associated CD11b+ immune cells but not CD11b+Gr1+ myeloid-derived suppressor cells, while bone marrow and spleen reservoirs of the suppressor cells were decreased. The inhibition of immune cell recruitment and tumor regression by anti-angiogenic receptor tyrosine kinase inhibitors, previously observed in several brain tumor models, was recapitulated in the 9L tumor model with the VEGFR2-specific inhibitory monoclonal antibody DC101 (p?<?0.01), implicating VEGFR2 signaling as an essential step in metronomic cyclophosphamide-stimulated immune cell recruitment. In contrast, sorafenib, a multi-receptor tyrosine kinase inhibitor with comparatively weak VEGF receptor phosphorylation inhibitory activity, was strongly anti-angiogenic but did not block metronomic cyclophosphamide-induced innate immunity or tumor regression (p?>?0.05).ConclusionsThe interference by receptor tyrosine kinase inhibitors in the immunogenic actions of intermittent metronomic chemotherapy is not a consequence of anti-angiogenesis per se, as demonstrated in an implanted 9L tumor model. Furthermore, this undesirable interaction with tyrosine kinase inhibitors can be avoided by using anti-angiogenic drugs that spare the VEGFR2 pathway.

Project description:BackgroundTargeting tumor vasculature is a strategy with great promise in the treatment of many cancers. However, anti-angiogenic reagents that target VEGF/VEGFR2 signaling have met with variable results clinically. Among the possible reasons for this may be heterogeneous expression of the target protein.MethodsDouble immunofluorescent staining was performed on formalin-fixed paraffin embedded sections of treated and control SW480 (colorectal) and WM239 (melanoma) xenografts, and tissue microarrays of human colorectal carcinoma and melanoma. Xenografts were developed using RAG1-/- mice by injection with WM239 or SW480 cells and mice were treated with 20 mg/kg/day of cyclophosphamide in their drinking water for up to 18 days. Treated and control tissues were characterized by double immunofluorescence using the mural cell marker ?-SMA and CD31, while the ratio of desmin/CD31 was also determined by western blot. Hypoxia in treated and control tissues were quantified using both western blotting for HIF-1? and immunohistochemistry of CA-IX.ResultsVEGFR2 is heterogeneously expressed in tumor vasculature in both malignant melanoma and colorectal carcinoma. We observed a significant decrease in microvascular density (MVD) in response to low dose metronomic cyclophosphamide chemotherapy in both malignant melanoma (with higher proportion VEGFR2 positive blood vessels; 93%) and colorectal carcinoma (with lower proportion VEGFR2 positive blood vessels; 60%) xenografts. This reduction in MVD occurred in the absence of a significant anti-tumor effect. We also observed less hypoxia in treated melanoma xenografts, despite successful anti-angiogenic blockade, but no change in hypoxia of colorectal xenografts, suggesting that decreases in tumor hypoxia reflect a complex relationship with vascular density. Based on ?-SMA staining and the ratio of desmin to CD31 expression as markers of tumor blood vessel functionality, we found evidence for increased stabilization of colorectal microvessels, but no such change in melanoma vessels.ConclusionsOverall, our study suggests that while heterogeneous expression of VEGFR2 is a feature of human tumors, it may not affect response to low dose metronomic cyclophosphamide treatment and possibly other anti-angiogenic approaches. It remains to be seen whether this heterogeneity is partly responsible for the variable clinical success seen to date with targeted anti-VEGFR2 therapy.

Dataset Information

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

Publications

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

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