Abstract
INTRODUCTION: Our group previously developed a novel adoptive cellular therapy (ACT) platform employing polyclonal populations of tumor-reactive T cells that are efficacious against multiple models of CNS malignancies including high-grade glioma, brainstem glioma, and medulloblastoma. This platform has been safe and feasible in both human and murine systems. One impactful observation was the significant decrease in abundance of immunomodulatory MDSCs and macrophages in the TME after treatment. The objective of this study is to determine the mechanism by which this reduction occurs. Here we have identified that macrophage-derived CCL12 is a major contributor to the migration of MDSCs to gliomas, and that ACT abrogates this axis. METHODS: C57BL/6J mice received orthotopic KR158b-luciferase cells and were treated with ACT as previously published. In either untreated or treated mice, we performed quantification of various immune cells in the TME using flow cytometry, scRNA-sequencing, and spatial transcriptomics (GeoMX Nanostring). Protein expression was obtained using a multiplex protein array and ELISA. Migration assays were performed using trans-well assays in vitro. RESULTS AND CONCLUSION: In conjunction with a significant reduction in MDSC frequency within the TME after ACT, spatial transcriptomics analysis also revealed a reduction in multiple chemokines including CCL12. The reduction of protein expression was confirmed with a multiplex protein array. ScRNA-sequencing then revealed that the CCL12 originates from macrophages within the glioma TME. To determine if MDSCs responded to macrophage-derived CCL12, migration experiments using various cellular components of excised tumor tissues were conducted. This revealed that macrophage-derived CCL12 significantly increased MDSC migration relative to controls. CCL12 is significantly reduced after ACT, leading to the hypothesis that the previously observed decrease in MDSCs at the TME is due to the decrease in macrophages and macrophage-derived CCL12 after treatment. CCL12 represents a novel therapeutic target in the glioma microenvironment.