Abstract A03: Formins stabilize cell-ECM adhesions during invasion onset

The onset of local invasion typically defines the transition from benign to malignant tumors. This pathological definition has clear implications for cell behaviors governing invasion onset, which nonetheless remain mysterious. Thus we asked whether established cytoskeletal regulators contributed specifically to invasion in multicellular ensembles. We investigated roles of actin nucleators from the formin family and the Arp2/3 complex during collective invasion by two cell types: tumor explants from PyMT mice, and acini of Manin-Darby Canine Kidney (MDCK) cells, which initiate collective invasion in response to Hepatocyte Growth Factor (HGF). Contrary to our expectations, inhibition of Arp2/3 did not prevent invasion in either cell type. The pan-formin inhibitor SMIFH2, however, drastically reduced invasive fronts in both cell types. This phenotype was specific to 3D invasion, as formins were dispensable for 2D motility by single cells or cell collectives. We identified Diaphanous 1 (Dia1) and FHOD1 as formin family members required for invasion by MDCK acini. Live imaging of Dia1 knockdown acini revealed that while they formed transient extensions into the collagen, these were too unstable to support invasion. Despite these defects, Dia1 knockdown cells could deform the collagen matrix locally and exerted traction stresses equal to controls. Stiffer, bundled collagen gels potentiated invasion by control acini but could not rescue invasion by Dia1 knockdown acini. We then imaged acini in which GFP-Lifeact and mCherry-Myosin Light Chain were coexpressed as they invaded into labelled collagen. During invasion onset, we observed formation of subcellular actomyosin puncta on minute timescales. These puncta could adhere stably and alter collagen even while the cell continued to change shape and migrate within the acinus. Such adhesions formed rarely and with unstable dynamics in Dia1 knockdown acini. Thus formins may contribute to malignancy through molecular and physical mechanisms distinct from overall cell contractility. Rather, they mediate invasion in 3D contexts where competing cell-cell and cell-ECM interactions make stringent local requirements of adhesion stability. This work provides a bridge between cell motility in 3D and canonical models of 2D migration. Citation Format: Tim Fessenden, Yvonne Beckham, Mathew Perez-Neut, Guillermina Ramirez-San Juan, Margaret L. Gardel. Formins stabilize cell-ECM adhesions during invasion onset. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A03.