Single-Cell Definition and Functional Validation of a CTSD-Associated Macrophage State in Colorectal Cancer

Background/Objectives: Tumor-associated macrophage heterogeneity may influence colorectal cancer (CRC) outcomes. We investigated a CTSD-associated macrophage program and its functional relevance. Methods: Single-cell GSE132465 data (23 tumor and 10 normal specimens), TCGA colorectal expression and survival data, and three spatial sections were integrated with Scissor, exploratory survival modeling and macrophage-conditioned medium experiments. We added specimen-level pseudobulk expression analysis, including 10 matched tumor–normal pairs, and 100 repeats of stratified ten-fold cross-validation of Cox models in 571 TCGA cases. Results: Of 45,520 cells, a survival-informed macrophage state was identified. CTSD and SPP1 increased in 10/10 matched tumor macrophage specimens (both paired p = 0.0020), whereas HSPA1A did not show a significant paired increase (p = 0.131). Cross-validated C-indices were 0.550, 0.612 and 0.533 for single-gene CTSD, HSPA1A and SPP1 models, respectively, and 0.604 for the three-gene model. Adding the three genes to age, sex and stage increased the cross-validated C-index from 0.714 to 0.722. Scissor feature selection and downstream survival analyses reused the same outcome, so these results are not independent prognostic validation. CTSD silencing attenuated the ability of newly collected macrophage-conditioned medium to support CRC cell viability, invasion and colony formation. Conclusions: CTSD is a functionally relevant candidate in the experimental macrophage model; clinical prediction and direct extracellular CTSD mechanisms remain unproven.