The tumour stroma regulates nearly all stages of carcinogenesis. Stromal heterogeneity in human breast cancers remains poorly understood, limiting the development of stromal-targeted therapies. To further elucidate this, we applied a multi-omics approach to analyse human breast cancer tissues using single-cell RNA sequencing (scRNA-Seq), single-cell phenotyping (CITE-Seq) and spatial transcriptomics.
Single-cell sequencing of ~150,000 cells from 26 early breast cancers revealed novel subpopulations of cancer-associated fibroblasts (CAFs), smooth muscle cells (SMCs) and endothelial cells consistent with cell differentiation states. Profiling healthy tissue controls revealed proportional changes in distinct stromal subpopulations upon disease progression. Using single-cell phenotyping with CITE-Seq, we validated canonical and novel markers of stromal cells, and further report novel immunoregulatory molecules on inflammatory-CAF subclasses. Using orthogonal methods, we showed that these stromal states have distinct morphologies and functional properties in regulating the extracellular matrix. Furthermore, the investigation of gene signatures from inflammatory-CAFs and differentiated-SMCs in independent TNBC patient cohorts revealed strong associations with cytotoxic T-cell dysfunction and exclusion, respectively. Importantly, integration with spatial transcriptomics revealed that inflammatory-CAFs co-localised with cytotoxic T-cells at distinct tissue regions. Such sites also expressed classical immunomodulatory molecules, presenting as strong candidates to further investigate for new immunotherapy approaches.
Our study highlights the potential of large scale single-cell projects to unravel the complexities of the tumour microenvironment and identify novel cell types underlying carcinogenesis. Such insights will guide the next-generation of therapies, which will likely be based upon an integrated understanding of the neoplastic, stromal and immune states that define a tumour and inform treatment response.