Unravelling drug resistant proteotypes through phenotype-resolved proteomics of single-cell derived colonies

Drug resistance in cancer therapy continues to significantly contribute to treatment failure and disease progression, and is linked to intratumoral heterogeneity. Mass spectrometry (MS)-based single-cell proteomics (SCP) provides a unique opportunity to uncover the mechanisms underlying drug-resistant phenotypes; however, current methods lack clonal resolution and are often confounded by cell cycle and cell size differences. Here, we introduce PhenoSCoP, a microscopy-guided discovery proteomics concept for mapping clonal proteomic heterogeneity. By distinguishing between transient and long-lived protein level changes, our approach uncovered hereditary and clone-specific programs associated with chemotherapeutic responses in head and neck squamous cell carcinoma (HNSCC) cells. Combined with fluorescence barcoding and drug treatment assays, we identified pre-existing proteotypes that strongly dominated drug-resistant cell populations. These programs also emerged in HNSCC patient samples and in relapsed tumors after chemoradiotherapy, linking drug-resistant proteotypes to intra- and inter-tissue spatial heterogeneity. In summary, we describe a robust, versatile and phenotype-resolved approach for uncovering single-cell-derived proteotypes associated with the therapeutic responses of distinct tumor cell clones.