Abstract BACKGROUND Immunotherapies in the treatment of brain metastases remain a major challenge 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. However, whether modulation of TAM/M can enhance CAR T-cell infiltration and efficacy remains unclear. MATERIAL AND 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 carcnioma 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. CSF1R inhibition significantly reduced TAM/M numbers, resulting in higher intratumoral CAR T-cell densities compared to control diet. Increased CAR T-cell infiltration correlated with reduced tumor growth and improved survival. In vivo imaging demonstrated that TAM/M interact with CAR T-cells, which were reduced following CSF1R inhibition, suggesting TAM/M-mediated impairment of CAR T-cell trafficking. CONCLUSION Our findings demonstrate that TAM/M contribute significantly to CAR T-cell suppression in lung cancer brain metastases. Targeting CSF1R on TAM/M improved CAR T-cell trafficking, intratumoral persistence and therapeutic efficacy resulting in a survival benefit. These results pave the way for combinatorial strategies incorporating CSF1R inhibition to further enhance CAR T-cell functionality and improve outcomes in the treatment of brain metastases.
Blobner et al. (Wed,) studied this question.