Cellular composition and the interpretation of candidate RNA readouts of trogocytosis in colorectal cancer

Background: RNA measurements of genes associated with trogocytosis may reflect cellular composition rather than contact-dependent membrane acquisition. We evaluated an eight-gene candidate module in colorectal cancer (CRC) using expression- and lineage-matched random modules, independent compartment analysis and patient-level inference. A separate rare-transcript definition and cancer-model fitness screens provided complementary analyses. Results: In 62 GSE178341 patients, the module correlated with T-cell fraction (ρ=0.767), but this association was typical of 10,000 matched modules (null median 0.766; one-sided empirical P=0.493; equal-tail two-sided P=0.985). A post hoc closest-10% matching sensitivity gave P=0.571. In 23 independent GSE132465 patients, the tumour-wide correlation was 0.735 (permutation P=0.00060) and the epithelial correlation was 0.376 (P=0.0892). The difference in absolute correlations was 0.359, with a 95% confidence interval of −0.041 to 0.759, leaving attenuation uncertain. Among 43,817 leukocytes, 16 CEACAM5-positive candidates arose from four patients; 15 had expression ≤2 transcripts per million. Candidate counts were threshold-sensitive. No survival association was detected for the module in two cohorts. SCD fitness dependency reproduced across CRC organoids and cell lines, whereas adjusted SCD–VPS72 and exploratory ATF3–SMYD2 co-dependencies did not. Exploratory CH25H, ATF3 and FADS2 analyses yielded no Hallmark enrichment meeting the multiple-testing threshold. Conclusions: Matched-module calibration placed the strong association with T-cell abundance within the range expected for comparable immune-lineage genes. Independent compartment analysis was consistent with a contribution from cellular composition, although attenuation remained uncertain. These results identify the comparison conditions needed to interpret the tested RNA readouts. Linking them to membrane transfer requires event-resolved protein measurements.