G. Davis2, S. A. Northrup1, M. Westcott2, J. H. Stewart1,2 1Duke University Medical Center,Surgical Oncology,Durham, NC, USA 2Wake Forest University School Of Medicine,Winston-Salem, NC, USA
Introduction: Oncolytic virotherapy (OV) using vesicular stomatitis virus (VSV) has become an attractive therapeutic strategy for treating peritoneal surface dissemination of colorectal cancer (PSD of CRC) because it selectively induces apoptosis in malignant cells while sparing non-malignant cells. OV’s also modulate the immune environment to induce a systemic anti-tumor response. The goal of this project was to analyze the immunomodulatory and oncolytic functions of VSV using an established murine model of PSD from CRC. We used an 8-marker flow cytometry panel to characterize the cell types and cytokine environment of the peritoneal cavity in VSV treated and control mice.
Methods: This study utilized an established syngeneic murine model with CT26 cell line coexpressing luciferase and GFP. In this model, 5-6 week old balb/c mice are injected intraperitonealy with 1×106 luciferase/GFP-expressing CT26 cells. On day 4, the mice were imaged utilizing an IVIS system to determine the establishment of tumor. On day 5, mice were injected with either PBS or 1×108 PFU M51R-VSV. Mice were sacrificed at 1, 3, or 7 days after virus/mock treatment, and a peritoneal lavage was performed on each mouse to collect peritoneal exudate cells (PEC) from the cavity. Cells from each sample were processed for flow cytometry, and supernatants from each sample were collected for a multiplex cytokine bead analysis. PECs were immunotyped using an 8-color panel, supernatants were assessed using a Biolegend 13-plex Mouse Inflammation Panel, and all samples were analyzed with a BD Facs Canto.
Results: The results from this study show VSV-mediated alterations in the peritoneal cavity as early 24 hours post virus treatment. At this early time point, M51R-VSV treated animals have significantly fewer CD4+ T cells, CD8a+ T cells, and CD19+ CD11b+ B-1 B Cells. Additionally, M51R treatment altered the cytokine microenvironment; M51R-treated cavities contained less MCP-1 than mock-treated animals. On day 7, M51R-treated mice contained approximately 75% more CD4+ T cells than their PBS-treated cohorts. There were also significant differences in CD11b+ myeloid lineages. These changes in cellular composition were accompanied by marked changes in the local concentration of IL-6 in mock-treated vs M51R-treated animals.
Conclusion: The results from this study suggest that VSV modulates the immune response through a number of key players and facilitates its therapeutic effect by altering the innate response at early time points, in order to produce an anti-tumor adaptive response at later time points. In our model, alterations in early recruitment of T-cells and myeloid cells, along with cytolysis of tumor cells resulted in a robust CD4 helper T cell response and a marked reduction in pro-tumor myeloid lineages 7 days after virus treatment.