How the mechanobiology orchestrates the iterative and reciprocal ECM-cell cross-talk that drives microtissue growth.

Controlled tissue growth is essential for multicellular life and requires tight spatiotemporal control over cell proliferation and differentiation until reaching homeostasis. As cells synthesize and remodel extracellular matrix, tissue growth processes can only be understood if the reciprocal feedback between cells and their environment is revealed. Using de novo-grown microtissues, we identified crucial actors of the mechanoregulated events, which iteratively orchestrate a sharp transition from tissue growth to maturation, requiring a myofibroblast-to-fibroblast transition. Cellular decision-making occurs when fibronectin fiber tension switches from highly stretched to relaxed, and it requires the transiently up-regulated appearance of tenascin-C and tissue transglutaminase, matrix metalloprotease activity, as well as a switch from α5ß1 to α2ß1 integrin engagement and epidermal growth factor receptor signaling. As myofibroblasts are associated with wound healing and inflammatory or fibrotic diseases, crucial knowledge needed to advance regenerative strategies or to counter fibrosis and cancer progression has been gained.

Infiltrating CD8+ T cells and M2 macrophages are retained in tumor matrix tracks enriched in low tension fibronectin fibers.

Tracks rich in matrix and cells, as described in several cancer types, have immunosuppressive functions and separate tumor nests and stroma, yet their origin is unknown. Immunostainings of cryosections from mouse breast tumors show that these tracks are bordered by an endothelial-like basement membrane, filled with fibers of collagen adjacent to tenascin-C (TNC) and low-tension fibronectin (Fn) fibers. While present in early-stage tumors and maturing with time, tracks still form under TNC KO conditions, however, host (not tumor cell)-derived TNC is important for track maturation. Tumor infiltrating leukocytes (mostly M2 macrophages and CD8+ T cells) are retained in tracks of early-stage tumors. Following track maturation, retained tumor infiltrating leukocyte (TIL) numbers get reduced and more CD8+ TIL enter the tumor nests in the absence of TNC. As these tracks are enriched with platelets and fibrinogen and have a demarcating endothelial-like basement membrane often adjacent to endothelial cells, this suggests a role of blood vessels in the formation of these tracks. The Fn fiber tension probe FnBPA5 colocalizes with TNC and immune cells in the tracks and shows decreased binding in tracks lacking TNC. Consequently, FnBPA5 can serve as probe for tumor matrix tracks that have immune suppressive properties.

Fibronectin fibers are highly tensed in healthy organs in contrast to tumors and virus-infected lymph nodes.

The extracellular matrix (ECM) acts as reservoir for a plethora of growth factors and cytokines some of which are hypothesized to be regulated by ECM fiber tension. Yet, ECM fiber tension has never been mapped in healthy versus diseased organs. Using our recently developed tension nanoprobe derived from the bacterial adhesin FnBPA5, which preferentially binds to structurally relaxed fibronectin fibers, we discovered here that fibronectin fibers are kept under high tension in selected healthy mouse organs. In contrast, tumor tissues and virus-infected lymph nodes exhibited a significantly higher content of relaxed or proteolytically cleaved fibronectin fibers. This demonstrates for the first time that the tension of ECM fibers is significantly reduced upon pathological tissue transformations. This has wide implications, as the active stretching of fibronectin fibers adjusts critical cellular niche parameters and thereby tunes the reciprocal cell-ECM crosstalk. Mapping the tensional state of fibronectin fibers opens novel and unexpected diagnostic opportunities.