RESUMO
Epithelial-mesenchymal Transition (EMT) is a multi-step process that involves cytoskeletal rearrangement. Here, developing and using an image quantification tool, Statistical Parametrization of Cell Cytoskeleton (SPOCC), we have identified an intermediate EMT state with a specific cytoskeletal signature. We have been able to partition EMT into two steps: (1) initial formation of transverse arcs and dorsal stress fibers and (2) their subsequent conversion to ventral stress fibers with a concurrent alignment of fibers. Using the Orientational Order Parameter (OOP) as a figure of merit, we have been able to track EMT progression in live cells as well as characterize and quantify their cytoskeletal response to drugs. SPOCC has improved throughput and is non-destructive, making it a viable candidate for studying a broad range of biological processes. Further, owing to the increased stiffness (and by inference invasiveness) of the intermediate EMT phenotype compared to mesenchymal cells, our work can be instrumental in aiding the search for future treatment strategies that combat metastasis by specifically targeting the fiber alignment process.
Assuntos
Transição Epitelial-Mesenquimal , Neoplasias Pulmonares , Citoesqueleto , Transição Epitelial-Mesenquimal/fisiologia , Humanos , Neoplasias Pulmonares/genética , Microtúbulos , FenótipoRESUMO
Actin, a primary component of the cell cytoskeleton can have multiple isoforms, each of which can have specific properties uniquely suited for their purpose. These monomers are then bound together to form polymeric filaments utilizing adenosine triphosphate hydrolysis as a source of energy. Proteins, such as Arp2/3, VASP, formin, profilin, and cofilin, serve important roles in the polymerization process. These filaments can further be linked to form stress fibers by proteins called actin-binding proteins, such as α-actinin, myosin, fascin, filamin, zyxin, and epsin. These stress fibers are responsible for mechanotransduction, maintaining cell shape, cell motility, and intracellular cargo transport. Cancer metastasis, specifically epithelial mesenchymal transition (EMT), which is one of the key steps of the process, is accompanied by the formation of thick stress fibers through the Rho-associated protein kinase, MAPK/ERK, and Wnt pathways. Recently, with the advent of "field cancerization," pre-malignant cells have also been demonstrated to possess stress fibers and related cytoskeletal features. Analytical methods ranging from western blot and RNA-sequencing to cryo-EM and fluorescent imaging have been employed to understand the structure and dynamics of actin and related proteins including polymerization/depolymerization. More recent methods involve quantifying properties of the actin cytoskeleton from fluorescent images and utilizing them to study biological processes, such as EMT. These image analysis approaches exploit the fact that filaments have a unique structure (curvilinear) compared to the noise or other artifacts to separate them. Line segments are extracted from these filament images that have assigned lengths and orientations. Coupling such methods with statistical analysis has resulted in development of a new reporter for EMT in lung cancer cells as well as their drug responses.
RESUMO
Cancer immunotherapy focuses on inhibitors of checkpoint proteins, such as programmed death ligand 1 (PD-L1). Unlike RAS-mutated lung cancers, EGFR mutant tumors have a generally low response to immunotherapy. Because treatment outcomes vary by EGFR allele, intrinsic and microenvironmental factors may be involved. Among all non-immunological signaling pathways surveyed in patients' datasets, EGFR signaling is best associated with high PD-L1. Correspondingly, active EGFRs stabilize PD-L1 transcripts and depletion of PD-L1 severely inhibits EGFR-driven tumorigenicity and metastasis in mice. The underlying mechanisms involve the recruitment of phospholipase C-γ1 (PLC-γ1) to a cytoplasmic motif of PD-L1, which enhances PLC-γ1 activation by EGFR. Once stimulated, PLC-γ1 activates calcium flux, Rho GTPases, and protein kinase C, collectively promoting an aggressive phenotype. Anti-PD-L1 antibodies can inhibit these intrinsic functions of PD-L1. Our results portray PD-L1 as a molecular amplifier of EGFR signaling and improve the understanding of the resistance of EGFR+ tumors to immunotherapy.