Longitudinal Proteomics Integrated with Multi‑Omics Reveal Molecular Drivers of Responses to Neoadjuvant Chemotherapy...

Neoadjuvant chemotherapy (NAC) is the standard treatment for locally advanced rectal cancer (LARC), yet its molecular determinants of response remain unclear. We conducted longitudinal 4D-DIA proteomic profiling of pre- and post-treatment specimens from 156 patients, including 31 patients with matched pre- and post-treatment specimens, and integrated with bulk RNA sequencing, single-cell transcriptomics, and functional validation. At baseline, responders were enriched in innate and adaptive immune pathways, whereas non-responders showed proliferative and metabolic programs. Pre-treatment RAC2, elevated in responders, was positively associated with immune activation, oxaliplatin sensitivity, and survival, and was validated in GSE139255 and GSE35640 (all P < 0.05). Moreover, RAC2 demonstrated strong predictive performance (training AUC = 0.867; testing AUC = 0.857). During treatment, NAC induced fibrosis-related remodeling across all groups (all P < 0.05), while responders uniquely preserved CD8⁺ T-cell infiltration (P = 0.031). Responders and RAC2-high tumors exhibited concordant pathway dynamics during NAC. Single-cell analysis further revealed that pre-treatment tumor RAC2, higher in responders, transmitted signals to CD8⁺ T cells while receiving inputs from fibroblasts (P < 0.05). In vitro and in vivo, RAC2 knockdown reduced baseline tumor growth but promoted under oxaliplatin (all P < 0.05). Overall, this study offers proteomic insights into NAC in LARC, supports therapeutic decision-making, and identifies potential targets.