CD63 cancer-associated fibroblasts confer CDK4/6 inhibitor resistance to breast cancer cells by exosomal miR-20.

Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (CDK4/6i) have rapidly received Food and Drug Administration (FDA) approval as a new type of therapy for patients with advanced hormone receptor-positive breast cancer. However, with the widespread application of CDK4/6i, drug resistance has become a new challenge for clinical practice and has greatly limited the treatment effect. Here, the whole microenvironment landscape of ER breast cancer tumors was revealed through single-cell RNA sequencing, and a specific subset of cancer-associated fibroblasts (CD63 CAFs) was identified as highly enriched in CDK4/6i resistant tumor tissues. Then, we found that CD63 CAFs can distinctly promote resistance to CDK4/6i in breast cancer cells and tumor xenografts. In addition, it was discovered that miR-20 is markedly enriched in the CD63 CAFs-derived exosomes, which are used to communicate with ER breast cancer cells, leading to CDK4/6i resistance. Furthermore, exosomal miR-20 could directly target the RB1 mRNA 3'UTR and negatively regulate RB1 expression to decrease CDK4/6i sensitivity in breast cancer cells. Most importantly, we designed and synthesized cRGD-miR-20 sponge nanoparticles and found that they can enhance the therapeutic effect of CDK4/6i in breast cancer. In summary, our findings reveal that CD63 CAFs can promote CDK4/6i resistance via exosomal miR-20, which induces the downregulation of RB1 in breast cancer cells, and suggest that CD63 CAFs may be a novel therapeutic target to enhance CDK4/6i sensitivity.