Predicting the fate of tumor cells post mechanically priming on soft substrates

Introduction: With roughly two million new diagnoses per year, cancer remains a global health issue. Despite being responsible for most cancer-related deaths, the mechanisms underpinning cancer metastasis remain unclear. Recently, tissue biophysical properties have been shown to be as potent as genetic perturbations in driving cell fate decisions. One such factor is the mechanical phenotype of cancer cells. The mechanical phenotype encompasses the material properties of cells and extracellular matrix (ECM). ECM stiffness, or elastic modulus, is known to play a crucial role in regulating cancer cell migration, proliferation, and differentiation. Here, we aim to recapitulate the in vivo matrix of metastasis sites such as brain and liver using commercially available soft substrates made of polydimethylsiloxane (PDMS) and to investigate the fate of tumor cells post mechanical priming. The mechanical phenotype, differential growth, and survival of mechanically primed cells can be characterized qualitatively and quantitatively, potentially contributing to the prediction of cancer metastasis and development of new therapeutic strategies.

Materials and

Methods: CytoSoft® 6-well plates with elastic modulus of 0.2 kPa used as soft substrate in this study was obtained from Advanced Biomatrix. The wellplate was incubated with media overnight, followed by seeding human tumor cells (A375P) at 10,000 cells/well. The cells were also seeded on glass tissue culture plate as control. The cells were cultured for 10 days before being examined for mechanical phenotypes (i.e., cell deformation and stiffness) using real-time deformability cytometry (RT-DC). qPCR of cells conditioned for 10 days on glass and 0.2 kPa substrates were performed. Cells harvested from glass and soft primed substrates were injected into mice and monitored for up to 51 days. All statistical tests were performed using Prism with the level of significance set at p< 0.05.

Results, Conclusions, and Discussions: The mechanical phenotypes of tumor cells primed on soft substrates were probed using RT-DC, a high-throughput technique to capture and evaluate cell morphology and rheology in a constricted channel. Figure 1(A) shows that cell stiffness decreased for cells primed on 0.2 kPa substrate compared to those on glass. Mechanically primed cells were harvested and injected into mice. Figure 1(C, D) show that the soft primed cells exhibited shorter latency for metastatic outgrowth following tail vein injection into mice as seen in Figure 1(C, D). qPCR also revealed differences in RNA transcription of genes responsible for regulating tumor cell invasion, migration, and proliferation of cells on soft substrates compared to the control.
The findings expand the understanding of how viscoelasticity would affect tumor cell mechanics that would provide some potential biophysical targets for cancer treatment development. Future studies will explore metastatic potential of these mechanically primed cells by testing if there is a) enhanced migration, b) enhanced survival due to environmental pressures and c) immune evasion in vitro using a microfluidic extravasation-mimicking device.

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