Genomic and Transcriptomic Perturbation of Cancer Cells During Confined Migration and Organ Colonization
Project Investigator: Jan Lammerding (Cornell University)
Collaborator: Franziska Michor (Harvard University, DFCI-PSOC)
In this proposal, we plan to extent our work on cancer cell migration to address the consequences of repetitive physical stress incurred during tumor cell invasion and entry into the blood circulation, which are key steps preceding metastatic organ colonization. We and others have previously shown that confined migration through microfluidic constrictions and transwell plates can result in nuclear envelope herniation and rupture, chromatin fragmentation and reorganization, and DNA damage4-6. However, it remains unclear whether physical cell stress can also result in transcriptomic changes leading to reprogramming of circulating and extravasating cancer cells, and if this response also occurs during the late steps of the metastatic cascade in vivo. The central hypothesis of this proposal is that the repeated mechanical challenge during invasion and intravasation is associated with transcriptomic reprogramming, which allows cells to better survive and/or migrate through mechanically challenging environments. We anticipate that transcriptomic reprogramming is enhanced by the repetitive mechanical forces accumulating during each step of the metastatic cascade, and that physical perturbation acts in addition to established DNA damage to a stress response which supports survival of disseminating tumor cells. We predict that invasion- and intravasation-associated transcriptomic reprogramming results in two outcomes: (i) successful coping with mechanical stress by enhancing pro-survival pathways, e.g. by upscaled stress response and DNA repair pathways including autocrine cytokine production, and/or (ii) mechanical adaptation, e.g. by cytoskeletal adaptation or regulation of intermediate filaments which control nuclear deformability. We anticipate that tumor cells adapt during early steps of the metastatic cascade to cope with mechanical stress, survive and improve invasion abilities, such as cell deformation and improved mechanical stability during shear stress experienced during circulation in the blood stream. These in vivo analyses will complement in vitro studies on tumor cells moving in mechanically defined microfluidic devices and their short- and long-term adaptations in response to mechanical stress performed within the PSOC. The in vitro studies, which focus on mechanically induced changes in chromatin organization and gene expression, are currently carried out as part of a transnetwork project between the Cornell PSOC (PI: Jan Lammerding) and the Chicago PSOC (PI: Vadim Backman). The experiments proposed here will complement the in vitro experiments by providing corresponding data from tumor cells in vivo. Data from both in vitro and in vivo experiments will be available for the modeling work carried out in this project by: Nuclear perturbation including DNA double strand breaks after in vitro migration through constrictions in microfluidic devices (A) and in clonal MV3 melanoma cells isolated after early colonization of the mouse lungs and in vitro passage for 7 days (B). the DFCI-PSOC to identify to what extent mechanical stress associated with tumor cell invasion and intravasation contributes to the “fitness” and evolution of disseminating cancer cells. By analysis of the alterations and changes emerging before and after undergoing the mechanical stress, performing viability assays on clones that have experienced these stresses, and stochastic computational modeling of the evolution of subclones due to phenotypic changes that arise due to these transcriptomic changes, we aim to identify specific adaptations that can be targeted to reduce the ability of tumor cells to withstand these challenges.