CDK4/6-Mediated Growth Arrest Enhances Cell Stiffness and Contractility in RB-Proficient Triple-Negative Breast Cancer Cells in 3D Hydrogels.

Abstract

CDK4/6 inhibitors are of increasing interest in cancer therapy, but biomarkers that reliably predict therapeutic effectiveness remain unclear. While the biochemical characterization of CDK4/6 inhibition in cancer cells has been extensively studied, there is a gap in the understanding of the resulting intracellular and extracellular mechanical remodeling after treatment. In this study, we investigated the mechanical implications of cell-cycle inhibitors in breast cancer tumor microenvironments using 3D extracellular matrices. CDK4/6 inhibition in highly invasive retinoblastoma (RB)-proficient triple-negative breast cancer (TNBC) cells results in stable growth arrest and increased cell area. Palbociclib (PD) treatment also results in increased stress fiber formation, focal adhesion formation, and cell stiffness. To evaluate the functional consequences of the intracellular mechanical changes in 3D microenvironments, PD-treated MDA-MB-231 cells were embedded in collagen hydrogels. A significant increase in bulk contractility in the PD-treated cells was observed. Ribociclib treatment of MDA-MB-231 cells resulted in stable growth arrest and increased cell area, cell stiffness, and contractility, similar to PD treatment. PD treatment of an RB-proficient TNBC cell line, SUM159PT, exhibited similar PD sensitivity and increases in cell spreading area and cell stiffness. Furthermore, single-cell contractility was increased and surrounding collagen fibers were significantly more aligned for CDK4/6-inhibited MDA-MB-231 cells, indicating greater extracellular matrix (ECM) remodeling capability. A complementary single-cell computational model recapitulated the experimental trends, supporting a mechanistic link between CDK4/6-mediated mechanical enhancement and ECM remodeling. Taken together, these data suggest that CDK4/6-mediated growth arrest is accompanied by a mechanically enhanced phenotype and greater ECM remodeling. These data suggest that the increase in cell stiffness and contractility may also result in increased tissue stiffness, a marker of tumor aggressiveness. This study motivates further work to better determine whether the mechanical response of cancer cells to cell-cycle inhibitors can serve as a biomarker to predict therapeutic effectiveness.Impact StatementCell-cycle inhibitors are becoming increasingly relevant in cancer treatment. While several clinical trials and Food and Drug Administration approvals have occurred, biomarkers to predict drug effectiveness across different patients remain unclear. This study leverages a 3D extracellular matrix model to investigate the robust mechanical alterations observed in CDK4/6 inhibitor-treated retinoblastoma-proficient triple-negative breast cancer cells. Palbociclib- and ribociclib-treated cancer cells exhibit enhanced cell stiffness and contractility. These findings establish mechanical phenotyping as a tissue engineering-enabled readout of chemotherapeutic drug response and lay a foundation for future studies to evaluate the potential of the readout as a biomarker to predict therapeutic efficacy.