Abstract 5124: Tumor and immune cell infiltration are enhanced by irradiation of normal tissues in immunocompromised mice
Authors: Rafat Marjan , Vilalta Marta , Aguilera Todd A. , Giaccia Amato J. , Graves Edward E. 
Published: July 2016

Breast cancer recurrence remains high in triple-negative cases despite aggressive surgical, radiological, and chemotherapeutic intervention. Recent studies suggest that circulating tumor cell re-seeding of primary tumors may facilitate recurrence. However, the tumor microenvironment's role in recurrence is not well understood. We hypothesize that the irradiated tumor stroma and microenvironment may influence tumor cell migration. In this study, we characterize the effects of normal tissue irradiation on tumor and immune cell migration to evaluate how tumor-stromal interactions modulate recurrence after therapy. This work represents the first step toward elucidating how stromal tissue radiation response contributes to tumor and immune cell recruitment.

Mouse embryonic fibroblasts (MEF) were irradiated to 20 Gy with a cesium source. Supernatant was collected after 2 or 7 d incubation to be used as a chemoattractant in a transwell assay to investigate the induction of 4T1 murine or MDA-MB-231 human mammary carcinoma cell invasion. An orthotopic breast cancer model was used to evaluate the effect of radiation on tumor cell migration to normal tissues. Nude mice were inoculated with luciferase labeled 4T1 or MDA-MB-231 cells in the mammary fat pad (MFP), and BALB/c mice with depleted CD4 and CD8+ T cells were inoculated with 4T1 MFP tumors. The contralateral normal MFP was irradiated to a dose of 20 Gy with a 250 kVp cabinet x-ray machine when tumors were palpable. Cell migration was monitored with bioluminescence imaging (BLI) 10 d after irradiation. Irradiated and control tissues were evaluated using immunohistochemistry (IHC). Tissue sections were stained with F4/80 to determine the extent of macrophage infiltration. Flow cytometry was also performed on dissociated irradiated and control tissues to characterize immune cell populations. Of particular interest were CD11b+F4/80+ macrophages and CD11b+GR1+ myeloid-derived suppressor cells (MDSCs).

Radiation enhanced tumor cell migration to normal tissues both in vitro and in vivo. 4T1 and MDA-MB-231 cells exhibited an increase in invasion (p&lt;0.05) in vitro when exposed to irradiated MEF media. Ex vivo BLI analysis revealed that normal tissue irradiation attracted tumor cells to the MFP and surrounding tissues, including the peritoneum and muscle (p&lt;0.001), in nude and BALB/c mice with depleted CD4 and CD8+ T cells. An increase in macrophage infiltration was found in irradiated tissue sections. Flow cytometry confirmed increases in macrophage and MDSC infiltration in irradiated areas.

This study establishes that normal tissue radiation response may play a role in modulating tumor and immune cell migration after radiation. The increase in macrophage and MDSC infiltration after irradiation indicates the immune contribution in tumor cell migration. These results suggest that the tumor stroma may facilitate tumor cell invasion and tumor regrowth following radiotherapy.

Citation Format: Marjan Rafat, Marta Vilalta, Todd A. Aguilera, Amato J. Giaccia, Edward E. Graves. Tumor and immune cell infiltration are enhanced by irradiation of normal tissues in immunocompromised mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5124.