J19 - Shear-Induced Deformation and Drug Effects on Triple-Negative Breast Cancer Cells: Biomechanical Properties and Cytoskeletal Structure

Introduction: : This study investigates the biomechanical and structural responses of triple-negative breast cancer (TNBC) cells to chemotherapeutic agents, aiming to inform novel therapeutic strategies for cancer treatment. Using the MDA-MB-231 TNBC cell line, we examined the effects of Paclitaxel (PTX) and Prodigiosin (PGs) drugs through shear assay techniques integrated with Digital Image Correlation (DIC) software to assess viscoelastic properties, including shear strain, displacement over time, and modulus. Structural deformation was concurrently evaluated using scanning electron microscopy (SEM) and fluorescence staining to provide both qualitative and quantitative insights. PTX-treated groups exhibited burst drug release profiles, while PGs-treated cells showed sustained release. Notably, all treated samples demonstrated substantial cellular degradation at the 12-hour mark. Over time, the mechanical response of treated cells indicated a decrease in Young’s modulus and an increase in displacement, signifying compromised cellular integrity. These findings highlight the utilization of biomechanical and structural assessments in evaluating drug efficacy and guiding the development of targeted cancer therapies.

Materials and

Methods: : Cell Culture: MDA-MB-231(GFP+) were cultured in L-15+ media supplemented with 10% FBS, and 1% Penicillin Streptomycin. Cells were incubated at 37oC and 0% CO2. Shear Assay: ~ 10,000 cells were plated into 35 mm petri dishes and cultured for 48 hours. Drug treatments (PTX and PGs) were added at time periods 1-6-12 hours. Constant laminar flow of 0.5 ml/min was applied using a syringe pump for two minutes while simultaneously recording the cells. Videos were converted to TIFF files, which were uploaded to DaVis 10.2. software, where cells were mapped using the strain mapping features. A set of data including displacement, max shear strain, and other values were produced by the software. Shear stress and modulus were calculated and graphed. About 5,000 cells were seeded on 15 mm glass cover slips. Recommended concentration for each drug treatments were added based on corresponding time points 1-6-12 hours. Samples were then fixed using 4% paraformaldehyde, permeabilized and then blocked. Cell actin cytoskeletal structures were stained using Rhodamine Phalloidin and a DAPI nuclear counterstain was done. Cells were imaged using a confocal microscope. About 5,000 cells were seeded on 9 mm glass cover slips. Drugs were added at various time points 1-3-6-12-18 hours. Afterwards, cells were fixed and dehydrated. Samples were placed on a cylindrical column. The nucleus diameter, cell length, perpendicular cell length, nucleus area, and cell body area were measured and compared between treatments groups and timepoints.

Results, Conclusions, and Discussions:: Results produced throughout this experiment were similar to those of fluorescence. At the 1-hour timepoint, only PTX samples showed significant cell body deterioration. At 3 hours, both the PTX and PGs samples show surface dimpling and retraction, PTX samples were flat and two dimensional while the PGS samples showed more raised topography. The 6-hour timepoint is the turning point for both PTX and PGS treatment.

Similar cell deterioration trends were observed in every model except for the controls. Each experiment assessed a different cellular component vital to cancer cell mechanics. Shear assay focused on the viscoelastic properties which included cell displacement, Modulus, and shear strain. Whereas fluorescence focused on the internal actin cytoskeletal structures and SEM focused on the topographical changes of the cell body.

Each study highlighted a key turning point in cell deterioration at 6 hours. The Modulus increased for each of the samples at this time point, indicating a higher stiffness in the cell. For PTX sample, the mechanism of action of the drug explains this behavior since the microtubule structures within the cells are targeted and stabilized. This prevents the cell from undergoing mitosis or migration, essentially keeping the cell stationary. PGs targets pathways within the mitochondria, which specifically govern cell proliferation and apoptosis. However, results from the experiments showed that there is a higher modulus at 6 hours. This could be a result of polymerization of the actin cytoskeletal structure, the process where G-actin monomers assemble into F-actin filaments, generally increases the shear modulus of the actin network. Furthermore, our results showed that there was significant decrease in the modulus after 6 hours as compared to the control cells. Cell deterioration is clearly seen in fluorescence and SEM images. This is attributed to how the drug treatments target specific structures within the cell. At the 6 hour timepoint we found that the effects of PTX are plateauing, while the effects of PGs are increasing in severity. The implication of this study highlights the potential of using biomechanical properties and structural analysis to evaluate cancer cell responses to chemotherapy.

Acknowledgements and/or References (Optional):: The author wish to acknowledge WPI for financial support.