Abstract A59: A rodent in situ glioma allograft model to study bone marrow-derived cell migration

Radiotherapy (RT) induced hypoxia recruits bone marrow-derived cells (BMDCs) to tumors through the upregulation of Stromal Derived Factor-1, a chemokine that binds to receptor CXCR4 present on BMDCs. The infiltration of BMDCs, which includes endothelial progenitors, pericyte progenitors, tumor-associated macrophages, immature monocytes, VEGFR+ hemangiocytes, and CD11b+ myeloid cells has been implicated in supporting tumor establishment by contributing to tumor growth, angiogenesis, vasculogenesis, inflammation, and immune suppression. In this work, we have used a rodent glioma in situ allograft model to study the recruitment/migration of BMDCs post RT. Female albino C57 (8-10 weeks old, Jackson Laboratories) animals underwent +DsRed bone marrow transplant (B6.Cg-Tg(CAG-DsRed*MST)1Nagy/J donor mouse). Following engraftment, glioma cells were cranially implanted with 1x105 glioma cells (GL261-Luc, Perkin Elmer), at an average size of 26.4mm3 indicated by a bioluminescence signal of 5.28x108 photons/sec/cm2/sr, animals underwent 6Gy (RadSource 2000, 250kVp, 25mA) of irradiation limited to a 1cm wide slit centered on the tumor. Bioluminescence (tumor size) and fluorescence imaging (+DsRed BMDCs) was performed prior to and at 1- and 2-weeks post-RT to assess tumor growth and measure the influx of +DsRed BMDCs. In addition, flow cytometry was used to measure the number of +DsRed BMDCs in the tumor and brain. To study the effect of BMDCs in tumor regression, tumors were irradiated at a smaller size (9.3mm3) with 10Gy irradiation. At 1-week post-RT, flow cytometry was used to measure the number of BMDCs (CD45+) in the tumor. In our model 6Gy radiation was able to stall the growth of the tumor. Irradiated tumors did not significantly change in tumor size at either 1- or 2-weeks post irradiation compared to pre-RT size. In addition, irradiated tumors were significantly smaller than untreated tumors (p=0.0008) 1-week post-RT. Using flow cytometry 1-week post irradiation, we observed a significant increase in the number of +DsRed BMDCs in the irradiated tumor (23.5% of cells +DsRed BMDCs) compared to the irradiated brain (6.17% cells +DsRed BMDCs, p=0.03) as well as a significant increase in the tumors (16.6% cells +DsRed BMDCs) compared to the brain (11.1% cells +DsRed BMDCs p=0.02). There was no difference in the number of +DsRed BMDCS in tumors ± irradiation or brains ± irradiation. We found similar results in our tumor regression model using flow cytometry 1-week post-RT; we observed an increase in the number of BMDCs CD45+ in the tumor (~20% of cells CD45+) compared to control tumors (~6% of cells CD45+). Indicating that tumor size at the onset of RT did not effect the migration of BMDCs. Fluorescence imaging did not measure any significant change in the DsRed signal at 1- or 2-weeks post-irradiation. We have successfully shown that post-RT there is an increase in BMDCs migration at 1-week post RT regardless of tumor size at RT. Further experimentation is needed to better elucidate the role of BMDCs in tumor inflammation and immune suppression as well as angiogenesis and vasculogenesis. Our next step is to perform immunohistochemistry on our collected tumor tissue to map out the spatial location of migrated BMDCs within the tumor. Citation Format: Janice A. Zawaski, Tien T. Tang, M Waleed Gaber. A rodent in situ glioma allograft model to study bone marrow-derived cell migration. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A59.