TMIC-67. Modulating Tumor-Associated Macrophages to Enhance EpCAM-Directed CAR T Cell Efficacy in Lung Cancer Brain Metastases

Abstract BACKGROUND IImmunotherapies for brain metastases face significant hurdles, largely due to the highly immunosuppressive tumor microenvironment (TME). Tumor-associated macrophages and microglia (TAM/M) have emerged as key mediators of immune suppression. Although EpCAM-directed CAR T-cell therapy has shown safety and efficacy in preclinical models, limited intratumoral persistence contributes to tumor recurrence. Whether targeting TAM/M can improve CAR T-cell infiltration and therapeutic potency, however, remains to be elucidated METHODS We utilized our established syngeneic orthotopic cerebral metastasis mouse model, combining a chronic cranial window with repeated intracerebral two-photon laser scanning microscopy to enable single-cell resolution imaging of fluorescent EpCAM/tdTCAR T-cells, TAM/M, and lewis lung carcinoma cells over weeks. EpCAM/tdTCAR T-cells were stereotactically injected adjacent to the tumor site. TAM/M depletion was achieved via CSF1R inhibition using a PLX 3397 diet. To further characterize the spatial organization of cellular and humoral components within the TME, immunofluorescence staining was performed. RESULTS Immunohistochemistry revealed a high intratumoral density of TAM/M, with a substantial proportion exhibiting an M2-like CD206⁺IBA1⁺ phenotype. Oral CSF1R inhibition reduced TAM/M numbers, leading to increased intratumoral CAR T-cell accumulation compared to control diet. Enhanced CAR T-cell infiltration correlated with reduced 2D and 3D tumor growth, ultimately improving survival. In vivo imaging demonstrated direct interactions between TAM/M and CAR T-cells, which ilimiting their intratumoral accumulation. CSF1R inhibition reduced TAM/M and restored CAR T-cell infiltration, indicating TAM/M-mediated impairment of CAR T-cell traffickin CONCLUSION Our findings demonstrate that TAM/M mediate CAR T-cell suppression in brain metastases of lung cancer. CSF1R inhibition reprogrammed TAM/M, enhancing CAR T-cell trafficking, intratumoral persistence, and antitumor efficacy, resulting in prolonged survival. These results support the development of combinatorial strategies incorporating CSF1R blockade to boost CAR T-cell functionality and improve therapeutic outcomes in brain metastases.