Cdc42-dependent modulation of rigidity sensing and cell spreading in tumor repopulating cells

Abstract

Recently, a robust mechanical method has been established to isolate a small subpopulation of highly tumorigenic tumor repopulating cells (TRCs) from parental melanoma cells. In order to characterize the molecular and mechanical properties of TRCs, we utilized the tension gauge tether (TGT) single-molecule platform and investigated force requirements during early cell spreading events. TRCs required the peak single molecular tension of around 40 pN through integrins for initial adhesion like the parental control cells, but unlike the control cells, they did not spread and formed very few mature focal adhesions (FAs). Single molecule resolution RNA quantification of three Rho GTPases showed that downregulation of Cdc42, but not Rac1, is responsible for the unusual biophysical features of TRCs and that a threshold level of Cdc42 transcripts per unit cell area is required to initiate cell spreading. Cdc42 overexpression rescued TRC spreading through FA formation and restored the sensitivity to tension cues such that TRCs, like parental control cells, increase cell spreading with increasing single-molecular tension cues. Our single molecule studies identified an unusual biophysical feature of suppressed spreading of TRCs that may enable us to distinguish TRC population from a pool of heterogeneous tumor cell population.

COL2A1 Is a Novel Biomarker of Melanoma Tumor Repopulating Cells

“…Based on this, we seeded single B16F1 melanoma cells in 90 Pa soft 3D fibrin gels. The majority of the cells do not survive the soft 3D microenvironment, which is consistent with published reports [ 14 , 16 , 17 , 18 ]. We tracked and imaged single cells up to 5 days ( Figure 1 b).…”

“…Established cell lines, such as parental B16F1 cells, are traditionally cultured on tissue 2D culture treated rigid dishes. Previous studies, including our own, demonstrated that 3D fibrin gel of 90 Pa stiffness promotes the growth and selection of multicellular TRC spheroids [14,[16][17][18]. Growing evidence also reports similar findings that physical properties of the cellular microenvironment, such as stiffness, have an important role in promoting tumorigenic cancer cell growth [26,27].…”

“…Figure 1 b shows a single tracked cell growing into a multicellular TRC spheroid over a 5-day culture period. These mechanically selected TRCs have been shown to be highly tumorigenic [ 14 , 16 , 17 , 18 ]. However, it remains largely unknown how these tumorigenic TRCs are different from parental B16F1 control cells in terms of gene expression and gene regulatory network.…”

“…Within a few days of culture in soft 3D fibrin gel, 8-10% of the planted cells established multicellular spheroids. These cells within the spheroids were found to be highly tumorigenic and, thus, were aptly named as tumor repopulating cells (TRCs) [14,[16][17][18]. However, very little is known about the specific and reliable biomarkers of TRCs and their molecular and genetic features.…”

DeepScratch: Single-cell based topological metrics of scratch wound assays

“…Both CDC42 and CDH5 knockdowns significantly reduce the spatial coordination of cell density, reflecting the importance of such coordination in collective cell migration. However, the depletion of these genes affects different mechanisms; the disruption of cell adhesion through CDH5 knockdown reduces cellular tension, while CDC42 knockdown affects cell response to microenvironmental change [35] , [36] . By considering multiple LCD-based measures we can discriminate among the different effects of these perturbations ( Fig.…”