Spatial proteomic analysis of breast cancer heterogeneity unravels mechanisms of cancer cell phenotypic plasticity

Tumor heterogeneity is the primary driver of drug resistance and relapse. Treatment-resistant clones lead to tumor relapse and metastasis, even years after an initial cancer diagnosis, and variability in the interactions with the immune system can lead to immune evasion and tumor progression. Despite its critical importance, intratumor heterogeneity (ITH) has been studied primarily at the genomic level over the last decade. In contrast, the functional outcomes of heterogeneity and the interplay with the tumor microenvironment have not been fully addressed. In this study, we integrated multi-region spatial mass spectrometry (MS)-based proteomics of 280 tumor regions, exome sequencing, and imaging to investigate spatial proteomic heterogeneity in breast cancer. We identified 8,263 proteins with relatively uniform receptor expression patterns within each region. Using this multi-layered data, our findings reveal increased proteomic heterogeneity with tumor progression, independent of genomic heterogeneity but closely associated with microenvironmental differences. Integration with immune and stromal imaging highlighted a dynamic interplay where low-grade tumors exhibit constrained immune infiltration, and upon progression, macrophages and T-cells infiltrate. However, pathways involving kynurenine and prostaglandins impede anti-tumorigenic activities. Integration with the global protein network provides potential novel targetable mediators of immune evasion in breast cancer, which can serve as the basis for future development of personalized cancer treatments.