Cancer Cells Soften into Metastasis: High Sensitivity Quantitative Characterization of Biomechanical Motility Indicators Across Metastatic Stages

Abstract

Metastasis is the process by which cancer cells spread to other parts of the body. Molecular analysis suggest that metastasis is accompanied by alterations in cellular mechanical properties such as adhesion, viscoelasticity and deformability which regulates cell motility and subsequently disease progression. Quantitative real-time characterization of mobility indicators of cancer cells, such as cell stiffness and detachment can provide much needed insight into the process of cancer cell migration across different metastatic stages. Downregulation of Connexin 43 (Cx43), a gap junction protein, plays a pivotal role in cell motility and promoting cell softness and metastasis, and thus resistance to chemotherapy. In this work, we used MDA-MB-231 epithelial human breast cancer cell line with upregulated and downregulated Cx43 expression as a model for different metastatic stages and Docetaxel (DTX) as a typical microtubule-targeting chemotherapeutic agent. We performed Single Cell Force Spectroscopy (SCFS) using FluidFM to measure the cell-substrate and cell-cell adhesion forces, and Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) to evaluate the cellular viscoelastic properties. Our SFCS results showed that Cx43 upregulation is correlated with increased intercellular adhesion and cell stiffness, with an average cell adhesion force of ~350 nN, while the downregulation of Cx43 resulted in the opposite, with an average adhesion force of ~150 nN. The difference in adhesion forces is observed only in cells present in monolayers when intercellular junctions are present. In addition, our QCM-D data are in line with the adhesion measurements, showing an increase in cellular stiffness upon the upregulation of Cx43 as indicated by the negative energy dissipation shift values averaging around -8 x 10-6, while the downregulation of Cx43 results in a softer and more viscoelastic phenotype with dissipation values as averaging around -35 x 10-6. We also observed increased cell stiffening upon treatment with DTX with a rapid response of cells with downregulated expression of Cx43 compared to other cellular subtypes. Our study is first to provide detailed, quantitative, and real-time biomechanical insight that complements the established standard molecular analysis of cancer mechanical properties across metastatic stages.