Poster P19 - Single Cell Biomechanical Profiling of the Metastatic Potential of MDA-MB-231 Cells

Introduction: While it is true that solid tumors are often detected due to their increased stiffness and rigidity compared to normal tissues (i.e, detection by palpation), the mechanical properties of tumors can become more complex as they progress [1–3]. During cancer metastasis, attributes of a cancer cell are altered dictating changes in their biomechanical properties and influencing their proliferation, differentiation, and migration. The deviations from mechanical homeostasis are associated with pronounced cell invasiveness and are coupled with re-organization of the extracellular matrix (ECM). The cells will lose their cell-cell and cell-matrix contacts, and undergo increased cell elasticity and deformability, and acquire improved cell motility which helps the cells to metastasize [4–6]. For invasion, cells undergo remodeling in their genetic makeup, where some genes get up-regulated or downregulated. Connexin 43 (Cx43), a gap junction protein with a putative tumor suppressor function, plays a pivotal role in Epithelial to Mesenchymal transition (EMT). Cx43 expression affects cell motility indicators and promotes cytoskeleton reorganization and, ultimately, metastasis when downregulated, corroborating resistance to chemotherapy [7,8]. The lack of universal markers that can predict the metastatic ability of cancer cells necessitates the evaluation of parameters, such as cell adhesion, viscoelasticity, and stiffness, to serve as unique biophysical metrics that can predict cancer cell metastatic potential and elucidate how metastasis and cell mechanics are coupled. These unique biomechanical properties can serve as biomarkers for early cancer detection and can provide a new basis for developing new therapies that can modulate cell mechanical cues for enhanced treatment.

Materials and

Methods: To explore the relationship between cell mechanics and metastatic cell behavior, we quantitatively demonstrate the progression of cancer cell biomechanics across different metastatic states using MDA-MB-231 breast cancer cells with regulated (up- and down-regulated) connexin 43 (Cx43) expression as a model of cells with varying metastatic potential. We utilized fluidic-based single-cell force spectroscopy (SCFS) and Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) to measure the mechanical properties of this cell model, in two different cell states, with varying metastatic potential. We measured, at a single-cell level and in real-time, the adhesion and viscoelastic properties of MDA-MB-231 cells and their response to docetaxel (DTX), a microtubule stabilizing agent that has been shown to disrupt intercellular adhesive forces, impairing cytoskeletal adhesion proteins such as actin and integrins, while also altering cellular morphology, division, and motility [9].

Results, Conclusions, and Discussions: Our data shows that cells with higher metastatic potential (down-regulated Cx43 expression) exhibited a softer, less adherent, and more viscoelastic phenotype, consistent with their metastatic state when in clusters compared to cells at the lower spectrum of metastatic potential (up-regulated Cx43 expression). The Fluidic-based SCFS revealed a 2.6-fold increase in the strength of adhesion and a 1.8-fold increase in elasticity between cells on the lower end of the metastatic spectrum compared to cells on the higher end when the cells are within a cluster. Of particular interest, these mechanical biomarkers are significantly obscured when the cells are in their single cells outside of a cluster confirming the major impact of the cell microenvironment on the metastatic and mechanical transformation of the cells. Additionally, real-time viscoelastic analysis showed a 3-fold elevation in the viscoelastic properties of cell membranes among highly metastatic cells in contrast to their less metastatic counterparts. In fact, cancer cells with higher metastatic potential, when grown in high density in clusters, are more viscoelastic than cells on the opposite end of the metastatic spectrum. We also observed a significant 6-fold increase in cellular stiffening upon treatment with DTX with a rapid and profound response of highly aggressive cells when compared to cells with attenuated metastatic potential. This is illustrated by the timeline of response to treatment and is dose-dependency. Such treatment schemes could render cancer cells less deformable, potentially impeding their ability to invade and metastasize to other tissues. Our study demonstrates a unique quantitative metric that unfolds biomechanical markers for discerning the metastatic state of cancer cells. Our data represents a stepping stone to understanding how mechanical changes in cancer cells may determine how cancer progresses and responds to chemotherapeutic agents and may provide novel modalities to inhibit metastasis.

Acknowledgements (Optional): This research has been funded with support from the Medical Practice Plan (MPP), HITS and National Council for Scientific Research in Lebanon (CNRS-L) grants from the American University of Beirut (AUB).

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