IMMU-50. Spatial Immunophenotypic Characterization of Immune Exclusion and Dysfunction via Collagen-Rich Fibrous Septa in Chordomas

Abstract Chordomas are malignant notochordal remnant tumors resistant to standard therapies. Using single-cell RNA-sequencing and spatial transcriptomics, we identified that the chordoma immune landscape is marked by macrophage-rich infiltrates and T-cell dysfunction. Furthermore, we reconstructed a differentiation pathway showing that naïve and memory T-cells in the blood transition into cytotoxic and eventually exhausted T-cells within the tumor, suggesting that immunotherapies may offer benefit despite clinical responses thus far being limited. Profiling chordomas with multiplex immunofluorescence (n=34), we found that immune cells were not randomly distributed but rather accumulated in fibrous septa, and inter-immune cell distances were much closer in fibrous septa versus tumor-resident regions. Using patient-matched (n=2) surgical chordoma specimens obtained before and after nivolumab treatment, a programmed death-1 (PD-1) blocker, we used multiplex imaging to investigate how the tumor microenvironment (TME) shapes immune suppression and therapy resistance through spatial sequestering of immune cells in fibrous septa. All patients had disease progression after PD-1 blockade, and spatial analysis revealed that although there was increased density coverage of CD4+ and CD8+ T-cells around tumor cells, only the proportion of CD8+ T-cells increased minimally from 0.001 to 0.003 (p=0.0010) as a function of nivolumab treatment. Additionally, while the distance between immune cells decreased after treatment, the distance between immune cells and tumor cells increased, reflecting physical confinement of immune cells in fibrous septa. Using treatment-naïve chordoma specimens (n=3), we then characterized the single-cell composition of fibrous septa. Correlating immunofluorescence staining of extracellular matrix (ECM) markers with histological architecture, we identified Type I collagen as a reproducible marker for fibrous septa. Multiplex imaging showed close spatial interactions between fibroblasts and CD8+ T-cells and sequestering of immune cells in collagen-rich tumor stroma. Overall, these data suggest that the intra-tumoral fibrous septa of chordomas are not passive structural elements, but dynamic immune microenvironments shaping T-cell dysfunction.