Mechanisms of resistance to CDK4/6 inhibitors: Insights from single-cell nucleus sequencing and spatial transcriptomics

Breast cancer incidence is increasing worldwide and represents the second most common malignancy in women after lung cancer. Among its subtypes, luminal B accounts for 10-20% of cases and shows a faster proliferation rate. Also, a poorer prognosis than luminal A, with a 5-year survival rate of only 32% at stage 4. Luminal B tumors are characterized by active estrogen receptor signaling and high expression of proliferation-associated genes such as Ki-67 and E2F , leading to enhanced estrogen-driven cell cycle progression. Consequently, CDK4/6 inhibitors, which block the G1 to S phase transition, have shown favorable therapeutic outcomes. However, in a subset of patients, treatment with CDK4/6 inhibitors induces a luminal-to-basal-like phenotypic conversion, which is associated with poor clinical prognosis. To elucidate the mechanism underlying this phenotype, we performed single-nucleus RNA sequencing and spatial transcriptomic analyses on paired pre- and post-treatment tumor biopsies from three patients who exhibited poor responses to CDK4/6 inhibition. After UMAP projection and batch correction, cell type annotation revealed no major changes in the overall cellular composition, with epithelial/tumor cells remaining dominant. Differential expression analysis showed minimal transcriptional changes in endothelial and fibroblast cells but substantial alterations in tumor/epithelial and immune cell populations. Focusing on immune cells, which displayed the largest number of DEGs, subclustering analysis revealed a marked post-treatment increase in innate lymphoid cells (ILCs), while overall immune cell numbers decreased following treatment. Cell-type-specific DEG analysis further identified substantial transcriptional reprogramming within plasma cells, CD4⁺, and CD8⁺ T cells. In parallel, tumor/epithelial cells exhibited distinct subtype redistribution after CDK4/6 inhibition, which is currently under detailed characterization. Spatial transcriptomic mapping based on nucleus-seq-derived cell type signatures is underway to visualize cell-type distribution and microenvironmental remodeling before and after treatment. Collectively, our integrative analyses aim to uncover the mechanisms of CDK4/6 inhibitor resistance, highlighting the potential roles of ILC expansion, immune remodeling, and epithelial plasticity in luminal-to-basal transition, and to identify biomarkers predictive of therapeutic response.