Multivariate analysis reveals activation-primed fibroblast geometric states in engineered 3D tumor microenvironments

Fibroblasts are a heterogeneous group of cells comprising subpopulations that have been found to be activated in the stromal microenvironment that regulates tumor initiation and growth. The underlying mechanisms of such selective activation of fibroblasts are not understood. We propose that the intrinsic geometric heterogeneity of fibroblasts modulates the nuclear mechanotransduction of signals from the microenvironment, resulting in their selective activation. To test this, we developed an engineered 3D fibroblast tumor coculture system and used high resolution images to quantify multiple cell geometry sensitive nuclear morphological and chromatin organizational features. These features were then mapped to activation levels as measured by the nuclear abundance of transcription cofactor, megakaryoblastic leukemia, and protein levels of its target, αSMA. Importantly, our results indicate the presence of activation-"primed" cell geometries that present higher activation levels, which are further enhanced in the presence of stimuli from cancer cells. Further, we show that by enriching the population of activation-primed cell geometric states by either increasing matrix rigidity or micropatterning primed cell shapes, fibroblast activation levels can be increased. Collectively, our results reveal important cellular geometric states that select for fibroblast activation within the heterogenous tumor microenvironment.