Beyond density: Topography-aware spatial metrics predict immunotherapy outcomes

Background: Accumulating evidence suggests that the spatial organization of tumor-immune ecosystems determines the benefit of checkpoint inhibitors. Therefore, topography-aware metrics that adjust for baseline cell abundance are better suited to quantify spatial immune patterns, such as attraction, repulsion, and clustering, across scales. We hypothesized that such multiscale spatial metrics would outperform immune cell density alone for explaining therapy benefit and survival in advanced non-small cell lung cancer (NSCLC) patients. Methods: We applied a multiplex immunofluorescence pipeline on diagnostic tissue of 54 NSCLC patients treated with checkpoint inhibitors. Annotated cell classes included cancer cells, CD8⁺, CD8⁺FoxP3⁺, M1 and M2 macrophages (-/+PDL1), CD68 - CD163 + cells and stromal non-immune cells. To spatially resolve the immune patterns, we computed neighborhood enrichment (ENR) and radial local enrichment (RLE). ENR was computed over k-nearest neighborhoods (k=5-100) and provided information about the attraction tendency between two cell classes, independent from cell density. RLE was evaluated over 10-50 µm radii and reflected the spatial cell clustering tendency. The spatial metrics were associated with therapy response and overall survival. Results: CD8 ⁺ FoxP3 ⁺ cells stratify risk. Responders demonstrate neighboring of CD8⁺ or CD8⁺PD1⁺ cells to CD8⁺FoxP3⁺ at different scales (ENR k=5-100, p=0.04-0.007), strongest at shorter distances. In the survival analysis, CD8⁺FoxP3⁺ cells were associated with shorter survival when they were closer to PDL1+ cancer cells (ENR: HR 3.7-7.5, p=0.01-0.04). CD8 geometry in stroma tracks response but not survival. Increased clustering of CD8⁺PD1⁺ associated with higher response to therapy (ENR k=10-100, p=0.05- 0.002). Notably, CD8⁺PD1 - cells did not show such associations. This positive impact on therapy response did not translate into a longer overall survival in this cohort. Tumor purity and compactness is associated with shorter survival. Local enrichment of cancer cells within 10-50 µm was associated with poor response and shorter OS, (RLE: HR 2.2-2.5, p=0.03-0.007), pointing to denser, homogeneous tumor nests as a risk factor. Conclusion: Topography-aware spatial metrics capture clinically meaningful organization beyond simple density. These findings supported the cellular context-dependent evaluation of biomarkers. Furthermore our findings also provide in situ evidence for a focused evaluation of a rare, hitherto poorly described subtype of CD8⁺FoxP3⁺ immune cells.