Path to drugging functional clones of luminal breast cancers using in-depth proteomics with spatially resolved mass spectrometry guided by MALDI imaging

Integrating tumor heterogeneity in the drug discovery process is a key challenge to tackle breast cancer resistance. Identifying protein targets for functionally distinct tumor clones is particularly important to tailor therapy to the heterogeneous tumor subpopulations. For this purpose, we performed an unsupervised, label-free, spatially resolved shotgun proteogenomic guided by MALDI mass spectrometry imaging (MSI) on 124 selected tumor clonal areas from early luminal breast cancers, tumor stroma, and breast cancer metastases. 2868 proteins were identified. The main protein classes found in the clonal proteome dataset were enzymes, cytoskeletal proteins, membrane-traffic, translational or scaffold proteins, or transporters. As a comparison, gene-specific transcriptional regulators, chromatin related proteins or transmembrane signal receptor were more abundant in the TCGA dataset. Moreover, 26 mutated proteins have been identified. Similarly, expanding the search to alternative proteins databases retrieved 126 alternative proteins in the clonal proteome dataset. The majority of these alternative proteins were coded mainly from non-coding RNA. To fully understand the molecular information brought by our approach and its relevance to drug target discovery, the clonal proteomic dataset was further compared to the TCGA breast cancer database and two transcriptomic panels, BC360 (nanoString®) and CDx (Foundation One®). We retrieved 139 pathways in the clonal proteome dataset. Only 55% of these pathways were also present in the TCGA dataset, 68% in BC360 and 50% in CDx. Seven of these pathways have been suggested as candidate for drug targeting, 22 have been associated with breast cancer in experimental or clinical reports, the remaining 19 pathways have been understudied in breast cancer. Among the anticancer drugs, 35 drugs matched uniquely with the clonal proteome dataset, with only 7 of them already approved in breast cancer. The number of target and drug interactions with non-anticancer drugs (such as agents targeting the cardiovascular system, metabolism, the musculoskeletal or the nervous systems) was higher in the clonal proteome dataset (540 interactions) compared to TCGA (83 interactions), BC360 (419 interactions), or CDx (172 interactions). Thus, we described the non-redundant knowledge brought by this approach compared to TCGA or transcriptomic panels, the targetable proteins identified in the clonal proteome dataset, and the potential of this approach for drug discovery and repurposing through drug interactions with antineoplastic agents and non-anticancer drugs.SignificanceSpatially resolved mass spectrometry guided by MALDI MS imaging is a precision oncology tool to map and profile breast cancer proteomic clones with the aim of integrating tumor heterogeneity in the target discovery process to develop clone-tailored therapeutic strategies in breast cancer.HighlightsSpatially resolved mass spectrometry guided by MALDI mass spectrometry imaging allows an in-depth proteomic profiling of breast cancer functional clones.This unsupervised and unlabeled technology performed on intact tumors provides a multidimensional analysis of the clonal proteome including conventional proteins, mutated proteins, and alternative proteins.The rich clonal proteomic information generated was not redundant with TCGA or transcriptomic panels, and showed pathways exclusively found in the proteomic analysis.A large proportion of the proteins in the clonal proteome dataset were druggable with both antineoplastic agents and non-anticancer drugs, showing the potential application to drug repurposing.A significant number of the proteins detected had partially or not yet known drug interactions, showing the potential for discovery.