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 ρ = −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.