Integrin β1 cluster stability in the context of cellular mechanosensing and radiosensitivity

Abstract

The cellular interaction with the extracellular matrix (ECM) modulates many key processes such as proliferation, migration, differentiation and survival. In addition, resistance to ionizing radiation has been found to be higher in cells cultured in presence of a 3D matrix, a process, which has been termed cell-adhesion mediated radio-resistance (CAM-RR). These cells are able to properly organize ECM-binding (extracellular matrix) integrins containing a β1 subunit into firm and stable clusters. Upon irradiation, these clusters are hard to break. On the contrary, cells cultured under standard, monolayer-based conditions are unable to keep this clustered status and are therefore radiosensitive. Radioresistance is thus linked to the ability to maintain a well defined organization of integrins in clusters, making integrin distribution a potential drug target for radiosensitization. With the use of the integrin β1 inhibitory antibody AIIB2, a well-known radiosensitizer, it is possible to induce radiosensitivity and in combination with ionizing radiation (IR) to break integrin β1 clusters of 3D cultured cells. In 2D cultured cells the treatment with AIIB2 completely abolished integrin clustering. As integrins are the key mediators of cell adhesion and mechanosensing, they originate the molecular signaling towards chromatin remodeling in response to a cell’s microenvironment. By following the physical link from integrins up to the nucleus with single molecule localization microscopy, it was found that the disintegration of integrin clusters has a direct impact on this nuclear mechanosensor. Collectively, these results show that, in addition to biochemical also mechanobiological cues and in particular nuclear mechanosensing have to be considered as relevant to uncover the molecular events behind adhesion related radiosensitivity. Therein, 2D cultured cells are highly artificial and do not provide the means to investigate mechanobiological aspects. Not only the involvement of ECM-binding integrins in radioresistance of various tumor types makes them an important target in actual cancer studies, they also contribute to drugresistance, metastasis and angiogenesis. So far, the vast majority of high-content screenings (HCS) use flat cultured, highly artificial monolayer-based 2D cells and standard microscopy techniques. The here achieved results prove that 3D cell cultures and single molecule microscopy are powerful tools for preclinical screenings. It would be possible to combine the virtues of microscopy of the nanoscale with the capability of 3D cultured cells to enhance the predictive value of high-content-screenings (HCS)