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. 2023 Jun 22;14:3724. doi: 10.1038/s41467-023-39287-7

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — Methods workflow for proteomics experiments of breast cancer cell line (BCCL) conditioned media (a) and formalin-fixed paraffin-embedded (FFPE) tumor samples (b). From hypoxic BCCL secretome experiments, 150 proteins showed hypoxia-increased secretion (Hx). From microdissected FFPE material, 283 proteins showed subtype differences only in the stromal compartment (basal-like (BL) vs. luminal-like (LL) subtypes). The 33-protein hypoxia stromal signature (33P) was generated from the overlapping proteins between the 150 hypoxia-increased proteins (BCCL secretome experiments) and the 283 proteins showing stroma-exclusive subtype differences (microdissected breast cancer patient material) (c). The hypoxia response proteins and 33P signature were validated using bioinformatic analysis and experimental validation (d). The hypoxia response proteins were investigated with bioinformatic analyses (gene ontology analysis (GO), gene set enrichment analysis (GSEA), ingenuity pathway analysis (IPA), network biology analysis using Cytoscape). The 33P signature was explored bioinformatically (GSEA, connectivity map analysis (CMAP), Cibersort, Search-Based Exploration of Expression Compendium (SEEK)) and validated with external clinical validation (METABRIC-Discovery, n = 852; KMplotter merged cohorts) for survival analysis and permutation test, and with extended cell line validation (BCCLs; LL n = 6, BL n = 6). 33P: 33-protein hypoxia stromal signature. BCCL breast cancer cell line. BL basal-like breast cancer subtype. CMAP Connectivity map analysis. ELISA enzyme-linked immunosorbent assay. FFPE formalin-fixed paraffin-embedded tissue. GO gene ontology analysis. GSEA gene set enrichment analysis. Hx hypoxia. IHC immunohistochemistry. LL luminal-like breast cancer subtype. MS mass spectrometry. Nx normoxia. SEEK search-based exploration of expression compendium.