Single-section spatial hypoxia-cytotoxic associations do not consistently reproduce across breast cancer patients

Spatial transcriptomics can reveal localized tumor-immune relationships, but thousands of spots from one tissue section do not provide thousands of biological replicates. We evaluated the distinction between within-section association and patient-level reproducibility using public breast cancer datasets. In a 10x Genomics Visium discovery section containing 3,798 spots, hypoxia-related transcription was inversely associated with cytotoxic gene activity in neighboring spots (Spearman{rho} = -0.202). High-hypoxia spots also had lower neighborhood cytotoxic scores than low-hypoxia spots (rank-biserial effect = -0.286). We then tested the directional association in an independent HER2-positive cohort comprising 36 sections, 13,619 spots, and eight patients. Only 19 of 36 sections and five of eight patients showed negative associations. The median patient-level correlation was -0.043 and did not differ from zero in a one-sided exact Wilcoxon test (P = 0.473). Sensitivity analyses using alternative cytotoxic and hypoxia signatures, neighborhood sizes, and Kendall correlation did not support a consistent inverse patient-level effect. Thus, a strong single-section association did not consistently reproduce across patients. These results caution against interpreting spot-level spatial associations from one section as patient-level biological effects.