BACKGROUND: Cyclin-dependent kinase 4/6 inhibitors (CDK4/6is) in combination with endocrine therapy are the standard treatment for patients with hormone receptor-positive, HER2-negative metastatic breast cancer (mBC). Despite the efficacy of CDK4/6is, intrinsic resistance occurs in approximately one-third of patients, highlighting the need for reliable predictive biomarkers. METHODS: Single-cell RNA sequencing analyzed metastatic tumors from HR+/HER2- mBC patients pre-CDK4/6i treatment at baseline (BL) and/or at disease progression. BL samples were from CDK4/6i responders (median progression-free survival [mPFS] = 25.5 months), while progressors were categorized as early-progressors (EP, mPFS = 3 months) and late-progressors (LP, mPFS = 11 months). Metastatic sites included liver, pleural effusions, ascites, and bone. InferCNV distinguished tumor cells, and functional analysis utilized the Molecular Signatures Database. RESULTS: LP tumors displayed enhanced Myc, EMT, TNF-α, and inflammatory pathways compared to those EP tumors. Samples from BL and LP responders showed increased tumor-infiltrating CD8 CONCLUSION: This study underscores the significance of molecular biomarkers in predicting clinical outcomes to CDK4/6i. Tumor-infiltration CD8