Title: Mechanical intercellular communication via matrix-borne cell force transmission during vascular network formation Problem or question being addressed: The ability of cells to communicate and coordinate their activity is crucial to the development and homeostatic function of all tissues. Intercellular communication through receptor-ligand engagement at the cell-cell interface or via diffusive soluble factors has been extensively studied (1). In addition to these well established means of biochemically mediated intercellular signaling, a more recent body of evidence has shown that cells also communicate via cell-generated forces transmitted to neighboring cells through the extracellular matrix (ECM), which we term mechanical intercellular communication (MIC) (2, 3). The dynamic and reciprocal nature of generating and sensing mechanical signals, however, makes MIC difficult to investigate. Specifically, we lack an understanding of the cellular machinery required for cells to sense and respond to tensile forces originating from neighboring cells. Further, how tissue-relevant matrix properties mediate the transmission of cell-generated forces has not been established. A deeper understanding of long-range MIC in the context of vasculogenic assembly could prove invaluable for the informed design of vascularized biomaterials (4). Occurring naturally during embryonic development and adult neovascularization, vasculogenesis involves the directed assembly of dispersed, individual ECs into an interconnected network of capillary-like structures and requires cellular communication and coordination over length-scales far larger than a cell. If better understood, control over this process presents a promising approach to engineer microvasculature to support parenchymal cells, a major challenge in the field of tissue engineering and regenerative medicine (5). Rationale of your approach: Several computational models suggest that fibrous matrices are optimal for transmitting forces over large distances (i.e., greater than one cell body away) due to their nonlinear elastic behavior and the potential for strain-induced alignment of ECM fibers (6). Our lab has previously developed synthetic matrices of electrospun dextran-based hydrogel fibers with user-defined architecture and mechanical properties (7, 8). Here, we combined this biomaterial system with a microfabrication-based cell-patterning method to precisely pattern individual ECs within fiber matrices that uniquely allows us to monitor and study MIC at the single-cell level (Figure 1). Figure 1. (A) Schematic depicting microfabrication-based patterning approach to isolate individual ECs at the center of suspended fibrous matrices. Representative confocal fluorescent image of patterned EC (cyan), rhodamine-labeled fibers (magenta), and fluorescent beads embedded in matrix fibers (white) (scale bar, 100 μm). (B) F-actin heat map of patterned ECs with histograms of average patterning error in x- and y-directions (n = 91 cells) (scale bar, 100 μm). Details of suggested approach: In this work, we developed a microfabrication-based cell patterning method to precisely pattern individual ECs within suspended matrices of DexMA fibers that have defined biophysical properties. To visualize cell-generated forces and MIC between ECs, we combined this patterning technique with timelapse confocal microscopy. ECs were patterned as cell pairs at a defined distance of 200 μm away from each other in low stiffness matrices that support cell force-mediated matrix deformations as well as in high stiffness matrices that do not support such deformations (Figure 2A). Strikingly, in low stiffness matrices, ECs frequently exhibited rapid, directional migration towards each other leading to cell-cell contact (Figure 2B-C, Movie 1). However, this phenomenon was not observed in high stiffness matrices that do not support matrix deformations. Figure 2. (A) Schematic of cell pair patterning in which two ECs are patterned 200 μm apart on a suspended DexMA fibrous matrix. (B) Representative image of initial matrix fiber morphology in non-aligned and aligned conditions. Temporally color-coded overlays capturing EC morphology and migration over a 12 hour time course after cell attachment as a function of matrix alignment and stiffness (scale bars, 100 μm). (C) Quantification of the percent of interacting and non-interacting cells over a 12 hour time course. Movie 1. Representative confocal fluorescence 2-hour timelapse movie of a pair of lifeAct-GFP expressing ECs (cyan) and rhodamine-labeled DexMA fibers (magenta) patterned in low stiffness, cell-deformable matrices. After confirming that matrix deformations can act as mechanical signals that mediate communication between neighboring ECs, we also aimed to characterize the molecular machinery underlying this observation. We combined our cell patterning technique with pharmacological inhibition studies to identify a role for both focal adhesion kinase (FAK) signaling as well as the mechanosensitive ion channel TRPV4 in the process of sending, receiving, and responding to mechanical signals. How it will affect the broader field: MIC involves the generation, transmission, and receipt of cell-generated forces conveyed through the ECM, which we posit to be an important but understudied means of intercellular communication. MIC is likely critical to tissue development given the ubiquity of cell-generated forces and dynamic changes to the ECM in the growing embryo. The previous notion that cells divide, migrate, and assemble in a static ECM during embryonic development has been widely discredited (9). In contrast, collective motion, spatial coordination, and multi-scale ECM deformations are essential for the morphogenetic transitions required for organogenesis. This work establishes an in vitro system to study MIC and yield new mechanistic findings that could underly cellular organization during development as well as inform the design of biomaterials that facilitate the self-assembly of functional vascular networks for tissue engineering and regenerative medicine applications. References: 1. B. A. Yang, T. M. Westerhof, K. Sabin, S. D. Merajver, C. A. Aguilar, Engineered Tools to Study Intercellular Communication. Adv. Sci. 8, 1–20 (2021). 2. L. Sapir, S. Tzlil, Talking over the extracellular matrix: How do cells communicate mechanically? Semin. Cell Dev. Biol. 71 (2017). 3. F. Alisafaei, X. Chen, T. Leahy, P. Janmey, V. B. Shenoy, Long-range mechanical signaling in biological systems. Soft Matter (2020). 4. W. Risau, V. Lemmon, Changes in the vascular extracellular matrix during embryonic vasculogenesis and angiogenesis. Dev. Biol. 125, 441–450 (1988). 5. H.-H. G. Song, R. T. Rumma, C. K. Ozaki, E. R. Edelman, C. S. Chen, Vascular tissue engineering : Progress, challenges, and clinical promise. Cell Stem Cell. 22, 340–354 (2018). 6. X. Ma, M. E. Schickel, M. D. Stevenson, A. L. Sarang-Sieminski, K. J. Gooch, S. N. Ghadiali, R. T. Hart, Fibers in the extracellular matrix enable long-range stress transmission between cells. Biophys. J. 104, 1410–1418 (2013). 7. B. M. Baker, B. Trappmann, W. Y. Wang, M. S. Sakar, I. L. Kim, V. B. Shenoy, J. A. Burdick, C. S. Chen, Cell-mediated fibre recruitment drives extracellular matrix mechanosensing in engineered fibrillar microenvironments. Nat. Mater. 14, 1262–1268 (2015). 8. C. D. Davidson, W. Y. Wang, I. Zaimi, D. K. P. Jayco, B. M. Baker, Cell force-mediated matrix reorganization underlies multicellular network assembly. Sci. Rep. 9 (2019). 9. R. Loganathan, B. J. Rongish, C. M. Smith, M. B. Filla, A. Czirok, B. Bénazéraf, C. D. Little, Extracellular matrix motion and early morphogenesis. Development. 143, 2056–2065 (2016).