CX3CR1 Expressing Macrophages Infiltrate the Tumor Microenvironment and Promote Radiation Resistance in a Mouse Model of Lung Cancer

Introduction: Combining radiation and immunotherapy targeting T lymphocytes is extensively studied in lung cancer treatment. Current evidence suggests that only a subset of patients will benefit, while most tumors will eventually develop resistance and progress. Tumor associated macrophages (TAM) are a significant component of the tumor microenvironment (TME) capable of both suppressing (M1) and promoting (M2) tumor growth based on their functional state. Chemokine receptor, CX3CR1, plays an important role in macrophage homeostasis and effector functions, however it’s role in the TME following radiation treatment remains unknown. We hypothesized that macrophages expressing the CX3CR1, play a central role in TME after radiation therapy.

Materials and Methods: Mouse lung cancer model was performed by subcutaneously inoculating Lewis Lung Carcinoma (LLC) expressing luciferase (Luc-2) and mCherry cells in CX3CR1^GFP/GFP reporter mice and CX3CR1^DTR/+ mice. Tumor growth was monitored by bioluminescence Imaging and caliper. TME inflammatory composition was assessed by flow cytometry. Clonogenic assay was used to assess tumor survival after radiation. LLC cell cycle was analyzed by flow cytometry.

Results and discussion: Ten days after tumor irradiation with 8 Gy, irradiated tumors were smaller than non-treated tumors. In-vivo bioluminescent imaging and flow-cytometry demonstrated a significant influx of CX3CR1 expressing cells into the irradiated TME, notably macrophages (F4/80+ CX3CR1+). To establish the direct effect of CX3CR1 expressing macrophages on tumor growth in-vitro, we performed a clonogenic assay, by co-culturing peritoneal macrophages with LLC cells. Eliminating CX3CR1 expressing macrophages from the culture (by negative selection), reduced LLC survival fraction by 25% (P=0.005). Furthermore, co-culture of negative CX3CR1 macrophages with irradiated LLC led to LLC accumulation in the cell cycle S-phase. Finally, to evaluate CX3CR1 depletion effect in-vivo, we injected LLC-mCherry-Luc2 cells subcutaneously into CX3CR1^DTR/+ mice, sensitive to the diphtheria toxin, and C57BL/6J control mice. Two weeks after inoculation, tumors were irradiated with 8Gy, and mice were treated every 3 days with diphtheria toxin, leading to reduction in CX3CR1 expressing cells. Three weeks after radiation, CX3CR1 depleted mice showed reduced tumor growth. Furthermore, flow cytometry analysis showed reduction in pro-tumoral M2 population (F4/80+CD206+), with no difference in T-lymphocyte or programmed cell death-1 expressing cells.

Conclusion: CX3CR1 expressing macrophages invade the TME after radiation therapy and contribute to radiation resistance and lung cancer progression, by promoting tumor survival. Thus, we purpose a novel strategy to improve radiation sensitivity by targeting the CX3CR1 expressing macrophages in the TME.