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

Drug resistance remains a major cause of treatment failure and disease progression and is closely linked to intratumoral heterogeneity. Mass spectrometry (MS)-based single-cell proteomics (SCP) can resolve drug-resistant cell states, but current methods lack clonal and phenotype information and are often confounded by cell-cycle and cell-size effects. Here, we introduce PhenoSCoP, a microscopy-guided discovery proteomics framework that profiles single-cell-derived colonies to map proteomic heterogeneity while preserving clonal origin and image-defined phenotypes. In head and neck squamous cell carcinoma models, PhenoSCoP distinguished heritable clone-intrinsic cisplatin (CP) response programs from adaptive treatment-induced changes, uncovered pre-existing resistant-like proteotypes in treatment-naive populations, and identified an NDUFB11-associated mitochondrial vulnerability in a resistant clone. In patient-derived primary HNSCC cells, CP-tolerant cells resolved into two proteomic states with distinct phenotypes and high or low S100A7/A8/A9 expression. These programs were also detected in HNSCC patient tissues and, in an illustrative matched progression case, increased across metastatic sites. PhenoSCoP therefore provides a phenotype-resolved framework for discovering stable and adaptive proteotypes associated with therapeutic response.