Dataset Information

Anti-tumor innate immunity activated by intermittent metronomic cyclophosphamide treatment of 9L brain tumor xenografts is preserved by anti-angiogenic drugs that spare VEGF receptor 2.

ABSTRACT:

Background

Metronomic 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 approach

Mice 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.

Results

Intermittent 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??0.05).

Conclusions

The 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.

SUBMITTER: Doloff JC

PROVIDER: S-EPMC4083145 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Publications

Anti-tumor innate immunity activated by intermittent metronomic cyclophosphamide treatment of 9L brain tumor xenografts is preserved by anti-angiogenic drugs that spare VEGF receptor 2.

Doloff Joshua C JC Chen Chong-Sheng CS Waxman David J DJ

Molecular cancer 20140626

<h4>Background</h4>Metronomic 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.<h4>Methods and approach</h4>Mice 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- ...[more]

PMID: 24965046

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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.

Project description:Cyclophosphamide treatment on a six-day repeating metronomic schedule induces a dramatic, innate immune cell-dependent regression of implanted gliomas. However, little is known about the underlying mechanisms whereby metronomic cyclophosphamide induces innate immune cell mobilization and recruitment, or about the role of DNA damage and cell stress response pathways in eliciting the immune responses linked to tumor regression.Untreated and metronomic cyclophosphamide-treated human U251 glioblastoma xenografts were analyzed on human microarrays at two treatment time points to identify responsive tumor cell-specific factors and their upstream regulators. Mouse microarray analysis across two glioma models (human U251, rat 9L) was used to identify host factors and gene networks that contribute to the observed immune and tumor regression responses.Metronomic cyclophosphamide increased expression of tumor cell-derived DNA damage, cell stress, and cell death genes, which may facilitate innate immune activation. Increased expression of many host (mouse) immune networks was also seen in both tumor models, including complement components, toll-like receptors, interferons, and cytolysis pathways. Key upstream regulators activated by metronomic cyclophosphamide include members of the interferon, toll-like receptor, inflammatory response, and PPAR signaling pathways, whose activation may contribute to anti-tumor immunity. Many upstream regulators inhibited by metronomic cyclophosphamide, including hypoxia-inducible factors and MAP kinases, have glioma-promoting activity; their inhibition may contribute to the therapeutic effectiveness of the six-day repeating metronomic cyclophosphamide schedule.Large numbers of responsive cytokines, chemokines and immune regulatory genes linked to innate immune cell recruitment and tumor regression were identified, as were several immunosuppressive factors that may contribute to the observed escape of some tumors from metronomic CPA-induced, immune-based regression. These factors may include useful biomarkers that facilitate discovery of clinically effective immunogenic metronomic drugs and treatment schedules, and the selection of patients most likely to be responsive to immunogenic drug scheduling.

Dataset Information

Anti-tumor innate immunity activated by intermittent metronomic cyclophosphamide treatment of 9L brain tumor xenografts is preserved by anti-angiogenic drugs that spare VEGF receptor 2.

Background

Methods and approach

Results

Conclusions

Publications

Anti-tumor innate immunity activated by intermittent metronomic cyclophosphamide treatment of 9L brain tumor xenografts is preserved by anti-angiogenic drugs that spare VEGF receptor 2.

Similar Datasets

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