Proteomics and single cell profiling identify keratin driven preexisting immunity influences lung squamous carcinoma neoadjuvant therapy.

Lung squamous cell carcinoma (LUSC) demonstrates heterogeneous responses to neoadjuvant immune checkpoint blockade, necessitating biomarkers for outcome prediction. Here, we identify tumor keratinization as a key determinant of therapeutic resistance. In 470 LUSC patients, elevated serum CYFRA 21-1 correlated with non-complete pathological response (non-CPR), while CK5/6 immunohistochemistry revealed strong association between keratinization and residual tumor burden. Proteomic profiling of 167 treatment-naïve biopsies stratified patients into distinct subtypes based on keratinization levels. KRT_L (low keratinization) exhibited enhanced immune infiltration, a markedly lower residual viable tumor percentage (P < 0.001), and superior survival outcomes (, P = 0.034) compared to the KRT_H (high keratinization). Conversely, KRT_H displayed upregulation of keratin proteins, activation of metabolic pathways, and enhanced cancer stemness features, alongside notable immunosuppression. Single-cell RNA analysis confirmed higher keratinization, metabolism and stemness in non-CPR tumors, with trajectory analysis linking undifferentiated states to keratin overexpression. Through integrated proteomic and single-cell analyses, our findings establish keratinization as a hallmark of immune-cold LUSC microenvironments, mechanistically linking elevated keratin expression with both stemness features and impaired immunotherapy efficacy, proposing keratin-based stratification for personalized therapy.