Compositions and methods for the treatment of fibrotic conditions and cancer

Abstract

A method of treating fibrosis and cancer comprising targeting circulating cells is described herein. These circulating cells may be mechanoresponsive immune cells or circulating mesenchymal cells.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF FIBROTIC CONDITIONS AND CANCERCROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 583,522 filed September 18, 2023, the specification of which is incorporated herein in their entirety by reference.FIELD OF THE INVENTION

[0002] The present invention features compositions and methods for the treatment of fibrotic conditions and cancer, specifically through the modulation of one or both of i) circulating cells or ii) myeloid mechanical signaling.BACKGROUND OF THE INVENTION

[0003] Injury in all organs comprises a coordinated process involving resident cells of the injury area as well as cells recruited from the circulation. Many recent studies have examined the importance of tissue resident fibroblast heterogeneity and mechanical signaling in healing and fibrosis. However, tissue repair also involves blood cells, classically part of the inflammatory response, that are recruited from the circulation. Described herein, the present invention identifies mechanoresponsive myeloid subpopulations present in fibrotic and healthy tissue. These subpopulations were then modulated by manipulating mechanical strain in vivo and in vitro and found that specifically targeting myeloid mechanical signaling is sufficient to reduce the pro-fibrotic myeloid subpopulations and instead restore the native, anti-inflammatory subpopulations. These findings reveal a more complicated interplay of circulating cell mechanotransductive signaling during the early and late stages of healing. These findings emphasize the critical importance of mechano-immunomodulation in the healing process and during the development of fibrosis and also identify a new subset of cells that contribute directly to these processes. Collectively, the mechano-immunomodulation of these "early responders" of healing demonstrates that it can trigger a cascade of downstream regenerative healing.

[0004] While the vast majority of circulating cells are immune cells that home to zones of injury during the acute inflammatory phase, rare non-hematopoietic mesenchymal circulating cells (MCC) have been identified in mammalian blood samples over two decades ago. However, since their initial description, the function, long-term fate, and impact of these rare cells on peripheral tissues have been elusive. Here, using single-cell transcriptomics in combination with lineage tracing, the present invention demonstrates that MCC are recruited from the circulation during the chronic remodeling stage of wound healing and persist within scars long-term, up to 6 months after injury. As described herein, a pro-fibrotic function of MCC during healing and fibrosis is revealed and targeted systemic depletion of this rare cell population is demonstrated to reduce pro-fibrotic gene expression within wound fibroblasts, decrease fibrotic collagen architecture, and accelerate wound healing. Thus, targeted inhibition of MCC may lead to novel therapeutic opportunities.BRIEF SUMMARY OF THE INVENTION

[0005] It is an objective of the present invention to provide compositions and methods, etc that allow for treatment of fibrotic conditions and cancer, specifically through the modulation of one or both of i)circulating cells or ii) myeloid mechanical signaling, as specified in the independent claims. Embodiments of the invention are given in the dependent claims. Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.

[0006] The present invention features methods of treating fibrosis and cancer comprising targeting circulating cells. These circulating cells may be mechanoresponsive immune cells or circulating mesenchymal cells.

[0007] The present invention features a method of treating a fibrotic condition or cancer in a subject in need thereof. In some embodiments, the method comprises administering a therapeutic amount of a composition that modulates one or both of i) circulating cells or ii) mechanical signaling in myeloid cells. In other embodiments, the method comprises modulating one or both of i) circulating cells or ii) mechanical signaling in myeloid cells.

[0008] The present invention may also feature a method of treating a fibrotic condition in a subject in need thereof. In some embodiments, the method comprises administering a therapeutic amount of a composition that inhibits one or both of i) circulating cells or ii) mechanical signaling in myeloid cells. In other embodiments, the method comprises inhibiting one or both of i) circulating cells or ii) mechanical signaling in myeloid cells.

[0009] The present invention may feature a method of treating cancer in a subject in need thereof. In some embodiments, the method comprises administering a therapeutic amount of a composition that augments one or both of i) circulating cells or ii) mechanical signaling in myeloid cells. In other embodiments, the method comprises augmenting one or both of i) circulating cells or ii) mechanical signaling in myeloid cells.

[0010] The present invention may further feature a method of increasing wound healing in a subject in need thereof. In some embodiments, the method comprises administering a therapeutic amount of a composition that modulates one or both of i) circulating cells or ii) mechanical signaling in myeloid cells. In other embodiments, the method comprises modulating one or both of i) circulating cells or ii) mechanical signaling in myeloid cells.

[0011] In some embodiments, the circulating cells comprise mesenchymal cells or hematopoietic cells. In some embodiments, the myeloid cells are derived from hematopoietic precursors. The myeloid cells may comprise monocytes, macrophages, neutrophils, dendritic cells, or a combination thereof.

[0012] In some embodiments, the composition comprises small molecules, miRNA, antibodies, peptides, a cell therapy, CRISPR gene editing, or a combination thereof. The small molecule may comprise a small molecule focal adhesion kinase inhibitor (FAKI) or any other inhibitor or activator that modulates mechanical signaling.

[0013] In some embodiments, the composition inhibits one or both of i) circulating cells or ii) myeloid mechanical signaling. In other embodiments, the composition augments one or both of i) circulating cellsor ii) myeloid mechanical signaling. In certain embodiments, modulating one or both of i) circulating cells or ii) mechanical signaling in myeloid cells comprises depleting said cells. Methods to deplete said may comprise but are not limited to plasmaphoresis or a CAR-T cell or other cell type may be used to target and deplete said cells. For example, in FIG. 2 JEDI T cells specifically target, e.g., deplete / destroy, circulating cells expressing GFP. In further embodiments, modulating circulating cells comprises modulating TWEAK (TNF-related weak inducer of apoptosis), MIF (macrophage migration inhibitory factor), CXCL (C-X-C motif chemokines), PTN (pleiotrophin) signaling, THBS1 / THBS2 / THBS4 (Thrombospondins), CD86, CD163, RAC1 , RAC2, MRC1 , EGR1 , SELENOP (Selenoprotein).

[0014] In some embodiments, the fibrotic condition is dermal fibrosis, hypertrophic scarring, fibrosis from the foreign body response to biomedical implants, renal fibrosis, cardiac fibrosis, idiopathic pulmonary fibrosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, musculoskeletal fibrosis, or any other fibrotic condition.

[0015] The present invention may also feature a method of reducing circulating cells at a site of wound healing. In some embodiments, the method may comprise modulating TWEAK (TNF-related weak inducer of apoptosis), MIF (macrophage migration inhibitory factor), CXCL (C-X-C motif chemokines), PTN (pleiotrophin) signaling, THBS1 / THBS2 / THBS4 (Thrombospondins), CD86, CD163, RAC1 , RAC2, MRC1 , EGR1 , SELENOP (Selenoprotein). at the side of wound healing.

[0016] The present invention may further feature a method of treating a fibrotic condition or cancer in a subject in need thereof, the method comprising modulating circulating cells. In some embodiments, the circulating cells comprise mechanoresponsive immune cells. In some embodiments, the circulating cells comprise circulating mesenchymal cells.

[0017] Furthermore, the prior references teach away from the present invention. For example, tissue injury activates a cascade of signaling pathways that recruit various cell types to orchestrate the healing response. However, excessive cell activation and recruitment lead to prolonged inflammation with overproduction and accumulation of extracellular matrix (ECM) proteins, resulting in inert, dysfunctional scar tissue, as observed in pulmonary fibrosis, myocardial infarction, and hypertrophic scar (HTS) formation. Recent examination of fibroblast heterogeneity has resulted in numerous studies focusing on the role of different fibroblast subpopulations during fibrosis and healing. Consequently, most of the literature emphasizes the contribution of resident fibroblasts to scarring across organ systems.

[0018] However, as described herein, tissue repair after injury involves a dynamic interplay not only among resident fibroblast cells but also cells recruited from the circulation. Therefore, the present invention features methods that utilize circulating cells from any origin and / or mechanical signaling in myeloid cells.

[0019] Furthermore, the inventive technical feature of the present invention contributed to a surprising result. For example, circulating fibroblasts are not commonly believed to exist or contribute to fibrosis, making the findings described herein particularly surprising. Additionally, myeloid cells, such asmonocytes and macrophages, are derived from hematopoietic precursors and migrate to injury sites, where they facilitate tissue repair by secreting cytokines to attract tissue-resident fibroblasts. Traditionally, myeloid cells are not thought to respond to mechanical stress or to be highly mechanoresponsive. However, as described herein the Inventors’ unexpectedly identify mechanoresponsive macrophages as key contributors to fibrosis.

[0020] Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0021] The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:

[0022] FIG. 1A shows a schematic showing creation of bone marrow chimeric mice by lethal irradiation of wild-type mice (C57 / BL6) infusion of bone marrow cells from Col1a1-GFP mice. FIG. 1B shows that scar tissue was explanted for analysis 14 days after excisional wounding and one as well as 6 months after ischemic flap creation. FIG. 1C shows masson trichome staining of excisional wound tissue at day 14 (left), circulating Col1a1 expressing cells in excisional wound tissue visualized by co-expression of EGFP using confocal microscopy (middle); rectangle indicates location of magnified image (right). FIG. 1D shows masson trichome staining of ischemic flap tissue at 1 month (left), circulating Col1a1 expressing cells in ischemic flap tissue visualized by co-expression of EGFP (middle), rectangle indicates location of magnified image (right). FIG. 1E shows masson trichome staining of ischemic flap tissue at 6 month (left), circulating Col1a1 expressing cells in ischemic flap tissue visualized by co-expression of EGFP (middle), rectangle indicates location of magnified image (right). FIG. 1F shows relative cell count of EGFP+ cells per DAPI+ cells in excisional wound and ischemic flap tissue. FIG. 1G shows flow cytometry analysis of live, EGFP+ cells in ischemic flap tissue explanted 1 month and FIG. 1H show 6 months after injury. FIG. 11 shows uniform manifold approximation and projection (UMAP) of sorted EGFP+ cells from ischemic flap tissue colored by cell type; Cells colored by cluster (FIG. 1 J); Cells colored by time point (FIG. 1K). FIG 1L shows the expression of marker genes projected onto UMAP embedding. FIG. 1M shows embedding, top right and below: expression of marker genes projected onto spatial embedding.

[0023] FIG. 2A shows unwounded skin of Co / faf-GFP mouse demonstrating GFP expression in dermal fibroblasts. FIG. 2B shows a comparison of mean expression levels of top 5 differentially expressed genes between circulating fibroblasts, resident fibroblasts, and three publicly available bone marrow mesenchymal stromal cell (MSC) datasets. FIG. 2C shows an UpSet plot showing a matrix layout of top 1000 shared across and specific to each cell population. FIG. 2D shows uniform manifold approximation and projection (UMAP) showing subset of MCC and fibrocytes from ischemic flap tissue at one and six months after injury (left) as well as marker gene expression projected onto UMAP embedding (right). FIG. 2E shows a dot plot comparing differentially expressed genes between MCC from ischemic flap tissueexplanted one month vs. six months after injury.

[0024] FIG. 3A shows spatial transcriptomic analysis using 10X Visium in four ischemic flap samples explanted 6 months after injury, top row: hematoxylin & eosin staining, middle row: spatial clusters, bottom row: Anchor-based integration of circulating fibroblast data from single-cell RNA sequencing and Visium. FIG. 3B shows uniform manifold approximation and projection (UMAP) showing integrated data from all Visium tissue sections. FIG. 3C shows UMAP colored by tissue slices. FIG. 3D shows ilntegration of MCC from scRNA-seq projected onto Visium UMAP. FIG. 3E shows expression of marker genes projected onto Visium UMAP.

[0025] FIG 4A shows a schematic showing creation of Col1a1-GFP bone marrow chimeric mice and infusion of JEDI or wild-type (WT) cytotoxic T cells. FIG. 4B shows flow cytometric analysis of excisional wound tissue explanted 18 days after injury and unwounded skin showing relative amounts of live EGFP+ cells. FIG. 4C shows circulating Col1a1 expressing cells in excisional wound tissue and unwounded skin visualized by co-expression of EGFP using confocal microscopy. FIG. 4D shows relative amounts of EGFP+ cells per DAPI+ cells within excisional wound tissue and unwounded skin. FIG. 4E shows analysis of wound area over time. FIG. 4F shows uniform manifold approximation and projection (UMAP) of sorted GFP-negative cells from excisional wound tissue explanted 18 days after injury from mice injected with JEDI or WT T cells, cells colored by cell type. FIG. 4G shows numbers of differentially expressed genes per cell type in mice injected with JEDI vs. WT T cells. FIG. 4H shows a volcano plot showing differentially expressed genes in mice injected with JEDI (right side) vs. WT T cells (left side). FIG. 4I shows UMAP of a subset of resident fibroblasts from excisional wounds. FIG. 4J shows differentially expressed genes in resident fibroblasts from excisional wounds. FIG. 4K, Collagen architecture of wounds explanted at day 7 and 18 after injury in mice injected with JEDI vs. WT T cells and of control wounds (untreated mice), top row: picrosirius red staining, middle row: collagen fiber alignment, bottom row: individual collagen fibers. FIG. 4L shows quantification of the representative images shown in 4K, specifically looking at the alignment and angle variance of the collagen fibers.

[0026] FIG. 5A shows flow cytometric analysis of JEDI T cells and wild-type (WT) cytotoxic T cells immediately post isolation. FIG. 5B shows flow cytometric analysis of live, CD8+ cells from the peripheral blood of WT mice, 4 days after systemic infusion of 4 million JEDI T cells vs. WT cytotoxic T cells. Activated JEDI T cells represent the fraction of CD45.1 and CD44 double positive cells. JEDI T cells highlighted in red. FIG. 5C shows uniform manifold approximation and projection (UMAP) of sorted GFP-negative cells from excisional wound tissue explanted 18 days after injury from mice injected with JEDI or WT T cells, cells colored by group. FIG. 5D, Gene set enrichment analysis (GSEA) using Gene Ontology Biological Product (GO-BP) of resident fibroblasts (GFP-) from excisional wound tissue explanted 18 days after injury from mice injected with JEDI or WT T cells.

[0027] FIG. 6A shows a schematic creation of Vav-reporter (Vav-R) mice by cross-breeding mTmG and Vavi-Cre mice. FIG. 6B shows a schematic of parabiosis of Vav-R to wild-type (WT) mice to analyze circulating cells (left) and a timeline of ischemic flaps leading to progressive ischemic tissue necrosis over time with resulting skin fibrosis at 1 month post injury (right). FIG. 6C shows masson trichrome staining of ischemic flap tissue explanted at day 3 (D3), 7 (D7), and 1 month (1 M) post injury (left column), circulating hematopoietic cells are identified by enhanced green fluorescent protein (EGFP) expression (middlecolumn), and circulating non-hematopoietic cells are identified by tdTomato expression using confocal microscopy, rectangles show location of magnified images (right column). FIG. 6D and FIG. 6E shows relative quantification of EGFP+ (FIG. 6D) and of tdTomato+ cells per DAPI+ cells in tissue sections across different time points (FIG. 6E). FIG. 6F shows flow cytometric analysis of live, EGFP+ and tdTomato+ cells. FIG. 6G shows sorted tdTomato+ and EGFP+ cells from ischemic flaps harvested at day 7 and 1 month after injury were analyzed by single-cell RNA sequencing and are shown as uniform manifold approximation and projection (UMAP), RFP = red fluorescent protein indicating tdTomato+ cells. FIG. 6H shows expression of EGFP and tdTomato mRNA projected onto UMAP embedding. FIG. 6I shows relative contributions of cell population to total cells per time point. FIG. 6J shows cells colored by cell type. FIG. 6K shows markers of MCC projected onto UMAP embedding.

[0028] FIG. 7A shows uniform manifold approximation and embedding (UMAP) of a subset of fibrocytes and MCC from ischemic flap tissue explanted seven days and one month after injury. GFP = green fluorescent protein, RFP = red fluorescent protein (tdTomato). FIG. 7B shows a violin Plot showing marker expression in fibrocytes and MCC.

[0029] FIG. 8A shows relative contributions of different myeloid sub-populations to total myeloid cells per time point. FIG. 8B shows uniform manifold approximation and projection (UMAP) of myeloid cells with overlaid RNA velocity vectors colored by clusters. Perivasc = perivascular macrophages, proinflam = proinflammatory cells, DC = dendritic cells. FIG. 8C shows, Top: Cells colored by latent time score, bottom: cells colored by time point. FIG. 8D shows expression of cluster markers projected onto UMAP embedding. FIG. 8E shows total cellular interactions between immune cell sub-populations (myeloid and lymphoid) and MCC. FIG. 8F shows a TWEAK signaling network between immune cell subpopulations and MCC. FIG. 8G shows heat maps showing total interaction strength for TWEAK, MIF, CXCL and PTN pathways between myeloid cell subpopulations and MCC. Based on their role in the interaction network groups are classified as sender, receiver, mediator, or influencer. FIG. 8H show schematic showing creation of Vav-reporter (Vav-R) bone marrow chimeric mice and injection of ischemic flaps with TWEAK or phosphate buffered saline (PBS). FIG. 8I shows: Left column: Circulating hematopoietic cells are identified by enhanced green fluorescent protein (EGFP) expression and circulating non-hematopoietic cells are identified by tdTomato expression using confocal microscopy. Rectangles indicate the location of magnified images (right column). FIG. 8J shows Right column: Picrosirius red staining of tissue sections. Center column: Collagen fiber alignment. Left column: Individual collagen fibers as identified by CTFIRE.Top row: ischemic flap tissue explanted 1 month after injury and daily TWEAK injection. Middle row: ischemic flap tissue explanted 1 month after injury and daily PBS injection. Bottom row: unwounded skin. FIG. 8K shows relative quantification of tdTomato+ cells per DAPI+ cells in tissue sections. FIG. 8L shows quantification of collagen fiber alignment.

[0030] FIG. 9A shows relative contributions of lymphoid cell subpopulations to all lymphoid cells per time point. FIG. 9B shows uniform manifold approximation and embedding (UMAP) of lymphoid cell subpopulations. FIG. 9C show: above: Velocity-informed latent time score projected on UMAP embedding, below: cells colored by time point. FIG. 9D shows cluster markers projected on UMAP embedding.

[0031] FIG. 10A, 10B, 10C, 10D, 10E, 10F, 10G, 10H, 101, 10 J, 10K, 10L, and 10M shows establishinga human-like model of HTS. FIG. 10A shows a mechanical strain device with (left) no extension or (right) extension. FIG. 10B shows a mechanical strain device attached to the dorsum of the mouse, creating (left) no strain and (right) extended strain. FIG. 10C and FIG. 10D shows a schematic showing time course of incision on dorsum of mouse, application of device with mechanical strain applied overtime (FIG. 10C), and creation of hypertrophic scar (FIG. 10D). FIG. 10E and FIG 10F shows H&E to quantify inflammatory infiltrate in murine or human unwounded skin and HTS. FIG. 10G and FIG. 10H shows UMAP embedding of HTS and skin colored by cell or sample type. FIG. 101 shows UMAP features plots of cell type defining genes. FIG. 10J shows a number of differentially expressing genes (DEGs) between HTS and skin in each cell type. FIG. 10K and FIG. 10L shows UMAP embedding and differential gene heatmap of myeloid cell subset. FIG. 10M shows a feature and violin plots of top differentiating genes in the myeloid cells. Statistical comparison made using unpaired two-tailed t-tests (*p<0.05, **p<0.01). All data represent mean ± SEM of biological replicates.

[0032] FIG. 11 shows histology and quantification of murine and human unwounded skin and HTS using trichrome staining to quantify collagen density and picrosirius red staining to quantify collagen fiber alignment.

[0033] FIG. 12 shows staining and quantification of COL1 or aSMA (red) in HTS and unwounded (UNW) control skin.

[0034] FIG. 13 shows Plot UMAP embedding of cell type specific markers.

[0035] FIG. 14A, 14B, 14C, 14D, 14E, 14F, 14G, 14H, 141, and 14J show transgenic disruption of myeloid-specific mechanotransduction generates improved healing with reduced inflammation. FIG. 14A shows the mechanical strain device model was applied to WT and genetically modified mice with FAK knocked out (KO) in myeloid (Lyz2+) cells. FIG. 14B shows gross photography and quantification of scars. FIG. 14C and FIG. 14D shows picrosirius red staining of collagen tissue architecture and data analysis with CurveAlign and CT-FIRE algorithms to determine length and alignment of fibers. FIG. 14E and FIG. 14F shows UMAP embedding of all cells showing treatment groups (FIG. 14E) and (FIG. 14F) cell types. FIG. 14G and FIG. 14H shows UMAP embedding of myeloid cells (FIG. 14G) with violin plots of top genes differentiating mechanoresponsive myeloid groups (FIG. 14H). FIG. 141 and FIG. 14J shows UMAP embedding of fibroblasts (FIG. 141) with violin plots of top differentiating genes (FIG. 14J). Statistical comparisons made using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons tests (*p<0.05). All data represent mean ± SEM of biological replicates.

[0036] FIG. 15 shows day 14 picrosirius red images analyzed with CurveAlign and CT-fire collagen architecture algorithms in knockout mice experiments.

[0037] FIG. 16A, 16B, 16C, 16D, 16E, 16F, 16G, 16H, 161, 16 J, 16K, 16L, and 16M shows Identification of circulating, mechanoresponsive myeloid cells which initiate fibrosis. FIG. 16A shows a schematic and gross photography of parabiotic murine models subjected to no strain (control), human-like mechanical strain (generating HTS), and mechanical strain with pharmacological disruption of mechanical signaling using focal adhesion kinase inhibitor (FAKI). FIG. 16B shows FACS results showing percentage of GFP+ circulating cells in wound tissue. FIG. 16C shows a UMAP embedding colored by the treatment group. FIG. 16D shows a UMAP embedding colored by 5 transcriptionally distinct myeloid cell subpopulations with RNA velocity information overlaid. FIG. 16E shows the proportion of eachsubpopulation within each treatment group. FIG. 16F shows a UMAP features plots of differentially expressed myeloid markers. FIG. 16G shows violin plots of differentially expressed myeloid markers. FIG. 16H shows a UMAP embedding colored by differentiation potential of cells generated using CytoTRACE. FIG. 161 shows a partition-based graph abstraction (PAGA) demonstrating subpopulation differentiation directions. FIG. 16J shows RNA velocity analysis plots of unspliced versus spliced constructs for selected genes. FIG. 16K shows confocal imaging of GFP+ cells (green), DAPI+ nuclei (blue), and either EGR1 (red; FIG. 16K) or MRC1 (red; FIG. 16L) expression. FIG. 16M shows quantification of confocal images. Statistical comparisons made using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons tests (*p<0.05). All data represent mean ± SEM of biological replicates.

[0038] FIG. 17A and 17B shows blocking mechanotransduction significantly decreased both scar width and scar collagen content. FIG. 17C shows FACS of GFP+ cells in a wildtype unwounded skin.

[0039] FIG. 18A shows mice subjected to no strain (control), strain (S), strain treated with FAKI (S+FAKI), and unwounded skin (uwd) were analyzed on day 3. FIG. 18B shows histological analysis of scar width. FIG. 18C shows picrosirius red staining of collagen tissue architecture and data analysis with CurveAlign and CT-FIRE algorithms to determine length and alignment of fibers. FIG. 18D and 18E shows immunofluorescent staining of early time point tissue for selenoprotein (SEPP1 , green stain; FIG. 18D) and thrombospondin (green stain; FIG. 18E).

[0040] FIG. 19A and 19B shows confocal imaging and quantification of WT and Lyz2Cre+ / -Brainbow2.1+ / +mouse parabiosis, showing that myeloid cell specific Lyz2 promoter drives stochastic expression of green, red, yellow, or cyan fluorescence (GFP, YFP, RFP, or CFP).

[0041] FIG. 20A shows UMAP embedding showing that both WT and GFP+ cells were captured. FIG. 20B shows UMAP embedding colored by cell type: myeloid cells (monocytes, macrophages, and neutrophils), fibroblasts, keratinocytes, lymphoid cells, and endothelial cells. FIG. 20C shows UMAP embedding colored by group (control, HTS, and FAKI). FIG. 20D shows Fibroblast UMAP colored by group. FIG. 20E shows fibroblast UMAP demonstrating transcriptionally heterogeneous subpopulations. FIG. 20F shows a chart showing that mechanical strain (S) increased the proportion of fibrotic cells and reduced the proportion of regenerative cells when compared to control (C) group cells. FAKI treatment (F) restored the proportions closer to the control group cells. FIG. 20G shows feature plots of cluster defining genes. FIG. 20H shows violin plots of cluster defining genes.

[0042] FIG. 21A, 21B, 21C, 21 D, 21E, 21F, 21G, 21 H, 211, 21J, 21 K, 21L, 21M, 21N, 210, 21P, and 21 Q shows Co-culture of murine fibroblasts and myeloid cells in 3D collagen scaffold confirms mechanoresponsive myeloid response. FIG. 21A shows WT murine dermal fibroblasts and GFP+ murine myeloid cells harvested. FIG. 21 B shows FACS analysis of co-culture collagen scaffolds subjected to strain and strain + FAKI treatment. FIG. 21 C shows Confocal images to analyze the number of GFP+ myeloid cells responding to mechanical disruption. FIG. 21 D shows scRNA-seq UMAP embedding showing population of cells collected from treatment groups. FIG. 21 E shows the Feature plot of eGFP+ expressing myeloid derived cells. FIG. 21 F showsUMAP embedding colored by cell type. FIG. 21 G shows the proportion of myeloid and fibroblast cells in the co-culture groups. FIG. 21 H shows violin plots of myeloid cells. FIG. 211 shows Violin plots of fibroblasts. FIG. 21 J shows FACS quantification showing effects of modulating mechanical strain on macrophage numbers and EGR1 and Thrombospondinlprotein expression. FIG. 21 K shows human fibroblasts and myeloid cells were isolated from plastic surgery procedures and donated blood. FACS analysis of CD45+ myeloid cells and EGR1 + and Thrombospondin1+ myeloid cells within the human co-culture system. FIG. 21 L and FIG. 21 M shows visium spatial transcriptional analysis of human skin and scar from the same patient. FIG. 21 N shows UMAP embedding and cell proportions within each of the spatial clusters between skin and scar (FIG. 210). FIG. 21 P shows feature plots and violin plots of fibrotic and regenerative transcriptional profiles between patient skin and scar (FIG. 21 Q). Statistical comparisons made using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons tests (*p<0.05, **p<0.01, ****p<0.0001). All data represent mean ± SEM of biological replicates.

[0043] FIG. 22A shows WT murine dermal fibroblasts and GFP+ murine myeloid cells harvested. FIG. 22B shows FACS gates of co-culture collagen scaffolds subjected to no strain, strain, and strain + FAKI treatment to quantify the GFP+ cells (FIG. 22C) and EGR1+ and Thrombospondin+ myeloid cells (FIG 22D).

[0044] FIG. 23 shows confocal imaging and quantification showing the expression of thrombospondin and DAPI.

[0045] FIG. 24A shows human dermal fibroblasts and blood derived myeloid cells harvested. FIG. 24B shows FACS gates of co-culture collagen scaffolds subjected to no strain, strain, and strain + FAKI treatment to quantify the fibroblast (Lin-) and macrophage (CD45+) cells, along with (FIG. 24C) EGR1 + and Thrombospondin+ myeloid cells.DETAILED DESCRIPTION OF THE INVENTION

[0046] Disclosed are various peptides, solvents, solutions, carriers, and / or components to be used to prepare compositions to be used within the methods disclosed herein. Also disclosed are the various steps, elements, amounts, routes of administration, symptoms, and / or treatments that are used or observed when performing the disclosed methods, as well as the methods themselves. These and other materials, steps, and / or elements are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed, that while specific reference of each various individual and collective combination and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0047] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed invention belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation. Stated another way, the term "comprising" means "including principally, but not necessarily solely". Furthermore, variations of the word "comprising", such as "comprise" and "comprises", have correspondingly the same meanings. In onerespect, the technology described herein related to the herein described compositions, methods, and respective component(s) thereof, as essential to the invention, yet open to the inclusion of unspecified elements, essential or not ("comprising").

[0048] Suitable methods and materials for the practice and / or testing of embodiments of the disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods well known in the art to which the disclosure pertains are described in various general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001 ; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), "Guide to Protein Purification” in Methods in Enzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.), the disclosures of which are incorporated in their entirety herein by reference.

[0049] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In case of conflict, the present specification, including explanations of terms, will control.

[0050] Although methods and materials similar or equivalent to those described herein can be used to practice or test the disclosed technology, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0051] As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, a subject can be a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkey and human). In specific embodiments, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) having a disease, disorder or condition described herein. In another embodiment, the subject is a mammal (e.g., a human) at risk of developing a disease, disorder, or condition described herein. In certain instances, the term patient refers to a human.

[0052] As used herein, the terms "treat," “treating,” or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, with the objective of preventing, reducing, slowing down (lessen), inhibiting, or eliminating an undesired physiological change, symptom, disease, or disorder. For example,the disease may be a fibrotic disorder or cancer. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented or onset delayed. Optionally, the subject or patient may be identified (e.g., diagnosed) as one suffering from the disease or condition prior to administration of the compositions of the invention. Subjects at risk for the disease can be identified by, for example, any or a combination of appropriate diagnostic or prognostic assays known in the art.

[0053] As used herein, “clinical improvement” may refer to a noticeable reduction in the symptoms of a disorder, or cessation thereof.

[0054] The terms “manage,” “managing,” and “management” refer to preventing or slowing the progression, spread or worsening of a disease or disorder, or of one or more symptoms thereof. In certain cases, the beneficial effects that a subject derives from a prophylactic or therapeutic agent do not result in a cure of the disease or disorder.

[0055] The terms “regress,” “regressing,” and “regression” may refer to a decrease in the size of a tumor or in the extent of cancer in the body. In some embodiments, “regression” may refer to a decrease in severity of the disease and / or decrease in the size of a tumor. In some embodiments, regression may generally refer to lighter symptoms without the disease completely disappearing. In certain cases, the beneficial effects that a subject derives from a prophylactic or therapeutic agent do not result in a cure of the disease or disorder. In some embodiments, symptoms of the disease may return.

[0056] The terms “administering” and “administration” refer to methods of providing a pharmaceutical preparation, composition, or formulation to a subject. The compositions described herein can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Such methods are well known to those skilled in the art and include, but are not limited to, administering the compositions orally, intranasally, parenterally (e.g., intravenously and subcutaneously), by intramuscular injection, by intraperitoneal injection, intrathecally, transdermally, extracorporeally, topically or the like.

[0057] Referring now to FIGs. 1A-24C the present invention features methods of treating a fibrotic condition or cancer in a subject in need thereof. In some embodiments, the method comprises a therapeutic amount of a composition that modulates one or both of i) circulating cells of any origin or ii) mechanical signaling in myeloid cells. In other embodiments, the method comprises modulating one or both of i) circulating cells or ii) mechanical signaling in myeloid cells. In some embodiments, the method comprises a therapeutic amount of a composition that modulates one or both of i) circulating cells of any origin (e.g., mesenchymal or hematopoietic origins) or ii) mechanical signaling in myeloid cells. In otherembodiments, the method comprises modulating one or both of i) circulating cells of any origin (mesenchymal or hematopoietic origins) or ii) mechanical signaling in myeloid cells.

[0058] The present invention may also feature a method of treating a fibrotic condition in a subject in need thereof. In some embodiments, the method comprises administering a therapeutic amount of a composition that inhibits one or both of i) circulating cells or ii) myeloid mechanical signaling. In other embodiments, the method comprises inhibiting one or both of i) circulating cells or ii) myeloid mechanical signaling. In certain embodiments, the method comprises administering a therapeutic amount of a composition that augments one or both of i) circulating cells or ii) myeloid mechanical signaling. Alternatively, in certain embodiments, the method comprises augmenting one or both of i) circulating cells or ii) myeloid mechanical signaling.

[0059] The present invention may further feature a method of treating cancer (e.g., solid tumors) in a subject in need thereof. In some embodiments, the method comprises administering a therapeutic amount of a composition that augments one or both of i) circulating cells or ii) myeloid mechanical signaling. In other embodiments, the method comprises augmenting one or both of i) circulating cells or ii) myeloid mechanical signaling. In certain embodiments, the method comprises administering a therapeutic amount of a composition that inhibits one or both of i) circulating cells or ii) myeloid mechanical signaling. Alternatively, in certain embodiments, the method comprises inhibiting one or both of i) circulating cells or ii) myeloid mechanical signaling.

[0060] Without limiting the present invention to any specific theory or mechanism, it is believed that the methods and compositions described herein may be particularly effective for treating solid tumors. As solid tumors grow, they exert mechanical stress on the surrounding tissue, triggering a significant tissue mechanical response that these methods and compositions can target.

[0061] In some embodiments, the circulating cells comprise mesenchymal cells or hematopoietic cells. In some embodiments, the circulating cells are from mesenchymal or hematopoietic origins. In some embodiments, the circulating cells may be mechanoresponsive immune cells or circulating mesenchymal cells. In some embodiments, the myeloid cells are derived from hematopoietic precursors. In some embodiments, the myeloid cells comprise monocytes, macrophages, neutrophils, dendritic cells, or a combination thereof.

[0062] In some embodiments, the composition for modulating circulating cells, mechanical signaling in myeloid cells, or both may include small molecules, drug therapies, cytokines, miRNA, antibodies, peptides, or a combination thereof. In other embodiments, modulation of circulating cells, mechanical signaling in myeloid cells, or both may involve cell therapies, gene editing techniques (such as CRISPR, adenovirus, lentivirus, etc.), or a combination thereof. In certain embodiments, these compositions (e.g., small molecules, drug therapies, etc.) may be used in conjunction with methods for modulating circulating cells, mechanical signaling in myeloid cells, or both, including cell therapies and gene editing techniques. Other known compositions that modulate either circulating cells, mechanical signaling in myeloid cells, or both may be used in accordance with the present invention. In certain embodiments, the composition maycomprise well-known substances in the field. For example, the small molecule may be a focal adhesion kinase inhibitor (FAKI) or any other inhibitor or activator that modulates mechanical signaling (e.g., TWEAK signaling).

[0063] In some embodiments, the composition comprises a focal adhesion kinase inhibitor (FAKI). In other embodiments, the composition comprises an activator of TWEAK.

[0064] In some embodiments, modulating circulating cells or mechanical signaling in myeloid cells, or both, involves depleting these cells. For example, plasmapheresis may be used for cell depletion. Alternatively, in some embodiments, CAR-T cells or other cell types may be employed to target and deplete these cells.

[0065] In some embodiments, modulating circulating cells comprises modulating TWEAK (TNF-related weak inducer of apoptosis), MIF (macrophage migration inhibitory factor), CXCL (C-X-C motif chemokines), PTN (pleiotrophin) signaling, THBS1 / THBS2 / THBS4 (Thrombospondins), CD86, CD163, RAC1 , RAC2, MRC1 , EGR1 , SELENOP (Selenoprotein).

[0066] In some embodiments, the composition described herein inhibits one or both of i) circulating cells or ii) myeloid mechanical signaling. In other embodiments, the compositions described herein augments one or both of i) circulating cells or ii) myeloid mechanical signaling.

[0067] In some embodiments, the methods described herein inhibit one or both of i) circulating cells of any origin or ii) mechanical signaling in myeloid cells. In other embodiments, the methods described herein augment one or both of i) circulating cells of any origin or ii) mechanical signaling in myeloid cells.

[0068] Non-limiting examples of fibrotic conditions may include but are not limited to dermal fibrosis, hypertrophic scarring, fibrosis from the foreign body response to biomedical implants, renal fibrosis, cardiac fibrosis, idiopathic pulmonary fibrosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, musculoskeletal fibrosis, or any other fibrotic condition.

[0069] Additionally, the present invention may feature a method of increasing wound healing in a subject in need thereof. In some embodiments, the method comprising administering a therapeutic amount of a composition that modulates one or both of i) circulating cells or ii) mechanical signaling in myeloid cells. In other embodiments, the method comprises modulating one or both of i) circulating cells or ii) mechanical signaling in myeloid cells.

[0070] The present invention may also feature methods of increasing wound healing in a subject in need thereof. In some embodiments, the method comprises administering a therapeutic amount of a composition that modulates one or both of i) circulating cells or ii) mechanical signaling in myeloid cells. In other embodiments, the method comprises modulating one or both of i) circulating cells or ii) mechanical signaling in myeloid cells. In some embodiments, the present invention may feature a method of increasing wound healing in a subject in need thereof. In some embodiments, the method comprises administering a therapeutic amount of a composition that inhibits one or both of i) circulating cells or ii)mechanical signaling in myeloid cells. In other embodiments, the method comprises inhibiting one or both of i) circulating cells or ii) mechanical signaling in myeloid cells.

[0071] The present invention may also feature a method of reducing circulating cells at a site of wound healing. In some embodiments, the method comprises modulating TWEAK (TNF-related weak inducer of apoptosis), MIF (macrophage migration inhibitory factor), CXCL (C-X-C motif chemokines), PTN (pleiotrophin) signaling, THBS1 / THBS2 / THBS4 (Thrombospondins), CD86, CD163, RAC1 , RAC2, MRC1 , EGR1 , SELENOP (Selenoprotein) at the site of wound healing. In some embodiments, the method comprises inhibiting TWEAK (TNF-related weak inducer of apoptosis), MIF (macrophage migration inhibitory factor), CXCL (C-X-C motif chemokines), PTN (pleiotrophin) signaling, THBS1 / THBS2 / THBS4 (Thrombospondins), CD86, CD163, RAC1 , RAC2, MRC1 , EGR1 , SELENOP (Selenoprotein) at the site of wound healing.

[0072] In certain embodiments, the present invention features a method of treating a fibrotic condition or cancer in a subject in need thereof. The method may comprise modulating circulating cells. In some embodiments, the circulating cells comprise mechanoresponsive immune cells. In other embodiments, the circulating cells comprise circulating mesenchymal cells.

[0073] The present invention may also feature a composition for treating a fibrotic condition or cancer, the composition modulates for one or both of i) circulating cells or ii) mechanical signaling in myeloid cells.

[0074] In some embodiments, the present invention features a composition for use in the method of treating a fibrotic condition or cancer, wherein the composition modulates one or both of i) circulating cells of any origin or ii) mechanical signaling in myeloid cells.

[0075] In some embodiments, the present invention features a composition for use in the method of treating a fibrotic condition, wherein the composition modulates one or both of i) circulating cells of any origin or ii) mechanical signaling in myeloid cells. In other embodiments, the present invention features a composition for use in the method of treating a fibrotic condition, wherein the composition inhibits one or both of i) circulating cells of any origin or ii) mechanical signaling in myeloid cells.

[0076] In some embodiments, the present invention features a composition for use in the method of treating cancer, wherein the composition modulates one or both of i) circulating cells of any origin or ii) mechanical signaling in myeloid cells. In other embodiments, the present invention features a composition for use in the method of treating cancer, wherein the composition augments one or both of i) circulating cells of any origin or ii) mechanical signaling in myeloid cells.EXAMPLE

[0077] The following is a non-limiting example of the present invention. It is to be understood that said example is not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.Circulating mesenchymal cells are recruited during scar remodeling to promote fibrosis afterinjury

[0078] Fibrosis contributes to 45% of all deaths in the United States. In mammals, fibrosis develops as the result of soft tissue injury and can significantly impair organ function. The repair of injured tissue involves a dynamic interplay of different populations of resident cells as well as cells recruited from the circulation. The majority of cells that home to sites of injury are myeloid and lymphoid cells, which are derived from hematopoietic precursors and modulate all stages of wound healing and scar formation. The presence of non-hematopoietic, mesenchymal cells in the circulation has been reported over two decades ago, however, knowledge about these cells is still very limited. Although few studies indicate that these cells may be upregulated in response to injury, their impact on peripheral tissues has not yet been investigated. In addition, how these cells are released into the circulation and whether they home to zones of injury has remained elusive.Non-hematopoietic collagen producing cells home to sites of injury

[0079] To characterize collagen producing cells that are recruited from the circulation and are involved in the development of fibrosis, a transgenic mouse strain (B6.Cg-Tg(Col1a1*3.6-Topaz)2Rowe / J was used, here referred to as Col-GFP), in which all Col1a1 (encoding the pro-alpha(l) chain of collagen-1) expressing cells also express green fluorescent protein (GFP) (FIG. 2A). Wild-type (C57 / BL6) mice were lethally irradiated and rescued by bone marrow transplantation from Col-GFP mice, thus generating Col-GFP bone marrow chimeric mice (Col-GFP-BM) (FIG. 1A). After 3 weeks of bone marrow engraftment, splinted full-thickness excisional wounds or ischemic skin flaps were created on the dorsum of these mice (FIG. 1 B). While mice heal splinted excisional wounds with fibrotic scars over the course of 14-16 days, ischemic skin flaps result in the development of chronic skin fibrosis induced by ischemic tissue necrosis. Therefore, this model allowed for the investigation of the long-term contribution of circulating collagen producing cells to chronic scar remodeling at 6 months after injury (one fifth of the median lifespan of adult mice). Scars were explanted 14 days after excisional wounding and at both 1 and 6 months after creation of ischemic skin flaps (FIG. 1 B-1 E). Histologic analysis using confocal microscopy demonstrated that collagen expressing circulating cells comprised 6-7% of the cells in scar tissue developing from both excisional wounds and ischemic flaps (FIG. 1F). FACS further confirmed these findings, demonstrating similar fractions of GFP+ cells among all live cells isolated from scars at 1 and 6 months (FIG. 1G and 1H). These findings indicate that circulating collagen-expressing cells home to sites of injury to form a niche and contribute to the chronic remodeling stage of healing.

[0080] Single cell RNA sequencing (scRNA-seq) performed on sorted GFP+ cells isolated from ischemic skin flaps explanted 1 and 6 months after injury surprisingly revealed that the collagen expressing circulating cells were composed of both hematopoietic (Ptprc expressing: clusters 0, 2, fraction of cluster 3) and non-hematopoietic cells (Ptprc negative, clusters 1 and 4) (FIG 1I-1K). The hematopoietic cells in clusters 0 and 2 showed minimal expression of Col1a1 but strong expression of Adgrel and Cd68 and were thus identified as macrophages (FIG. 1I-1L). A population of cells within cluster 3 weakly expressed macrophage markers and Ptprc, however strongly expressed Col1a1 and Pdgfra and was thus consistentwith the previously described “fibrocytes” (FIG. 1I-1L). Cluster 4 expressed Pecaml being consistent with endothelial cells. Interestingly, cluster 1 neither expressed Ptprc nor macrophage markers, but showed a strong expression of Col1a1, Pdgfra, and stem cell markers such as Cd34 and Ly6a. Thus, these non-hematopoietic cells appeared to be circulating non-hematopoietic mesenchymal cells (MCC, FIG. 1I-1L).

[0081] Next, the study questioned whether MCCs are more closely related to bone marrow MSCs or differentiated wound fibroblasts, comparing their transcriptomic profiles with those of GFP-negative local wound fibroblasts isolated from Col-GFP-BM mice as well as publicly available scRNA-seq datasets of bone marrow MSCs. The results indicated that MCCs show a greater similarity with differentiated wound fibroblasts than with bone marrow MSCs, demonstrated by a shared overexpression of Col1a1, Col3a1, and Postn. In contrast, MSCs demonstrated an overexpression of markers related to osteogenic differentiation such as Ibsp and Malatl (FIG. 2B and 2C).

[0082] When comparing transcriptional profiles between MCC isolated at 1 vs. 6 months after injury, collagen expression was higher at 1 month, whereas expression of transcription factors that have been associated with the development of fibrosis in different organ systems, such as Jun, Myc and Zfp36 were higher expressed at 6 months, suggesting that this rare cell population adopts a pro-fibrotic regulatory function overtime (FIG. 2D and 2E).

[0083] Spatial transcriptomic analysis was performed on scar tissue explanted 6 months after injury using the 10X Visium platform to determine the localization of MCC within the skin. Using anchor-based integration of scRNA-seq data and Visium, MCC formed a niche within the central portion of the scar (FIG. 1M and FIG. 3A-3E), which was consistent with histologic findings (FIG. 1C-1E). Scar areas where MCC projected onto the spatial embedding showed a strong expression of Col1a1, Cd34, and Ly6a confirming scRNA-seq data (FIG. 1M and FIG. 3A-3E).Systemic deletion of MCC promotes regenerative healing

[0084] To characterize the impact of MCC on wound healing and fibrosis, these cells were systemically depleted using a model of T cell mediated killing. Col-GFP-BM chimeric mice were infused with either JEDI (Just EGFP Death Inducing) cytotoxic (CD8+) T cells or wild-type (WT) cytotoxic T cells via retroorbital injection after vaccinating the recipient mice with EGFP to active the JEDI cells. JEDI mice express a T cell receptor specific for EGFP and produce cytotoxic T cells that enable targeted depletion of EGFP expressing cells with very high specificity and efficacy (FIG. 4A, and FIG. 5A). Four days after infusion, activated JEDI cells expressing CD44 as well as CD45.1 were detected in the blood of the recipient Col-GFP-BM mice at a fraction of 1% of all CD8+ T cells, and excisional wounds were created on the backs of these mice (FIG. 4A, FIG. 5B). JEDI cells significantly depleted MCC in scars explanted on day 18 to almost 0%, similar to levels found in unwounded skin (*p<0.05, FIG. 4B-4D). Analysis of wound healing kinetics over time demonstrated that mice that had received JEDI cells healed their wounds by day 15, significantly (*p<0.05) faster than wounds of untreated mice (~21 days) and mice thatwere injected with WT T cells (~19 days). These findings demonstrate that systematic deletion of MCC may improve healing and that MCC may physiologically exacerbate fibrosis throughout the body (FIG. 4E and FIG. 5C).

[0085] To investigate how depletion of MCC from wounds affects gene expression of other cell populations in the wound bed, scRNA-seq of GFP-negative cells were performed from scars explanted 18 days after wounding from Col-GFP-BM mice that had either received JEDI or WT T cells (FIG. 4F and FIG. 5D). The highest number of differentially expressed genes between the two groups was found in resident fibroblasts, indicating that MCCs have the strongest impact on gene expression of fibroblast subpopulations within wounds (FIG. 4G and 4H). Resident fibroblasts from wounds that had been depleted of MCC showed a marked reduction of profibrotic markers, such as En1 (engrailed-1), Dpp4 (encoding CD36, which marks engrailed positive fibroblasts), Sfrp2, Acta2, and collagen encoding markers (Col1a1, Col3a1) compared to controls (FIG. 4I and 4J). Moreover, depletion of MCC induced an upregulation of regenerative markers such as Apoe and Cxcl12 in resident fibroblasts (FIG. 4J). Analysis of signaling pathway expression between the two groups confirmed an overexpression of pathways related to extracellular matrix organization and collagen biosynthesis in resident fibroblasts from controls, which was absent in mice that had been depleted of MCC.

[0086] To confirm the profibrotic role of MCC on the tissue level, the collagen ultrastructure were analyzed of wound tissue from mice injected with either JEDI or WT T cells explanted 7 and 18 days after wounding. Mice depleted of MCC had significantly less aligned and more randomly organized collagen fibers compared to untreated wounds at day 7 (**p<0.01) and day 18 (*p<0.05), indicating that MCC contributes to the development of fibrosis after wounding.Parabiosis and double-transgenic lineage tracing confirms existence of MCC

[0087] While scRNA-seq data from Col-GFP-BM mice indicated that MCC showed by far the highest Col1a1 expression, circulating myeloid cells also demonstrated collagen expression, albeit at much lower levels. Therefore, to confirm the mesenchymal origin of MCC as well as their impact on fibrosis independent from other circulating collagen expressing cells, a second double-transgenic lineage tracing model was employed that would label circulating non-hematopoietic cells independent of Col1a1 expression. A Cre-activated dual fluorescence (tdTomato and GFP) mTmG mouse strain was bred with mice expressing Cre recombinase under the control of the Vav1 promoter. Vav1 is a pan-hematopoietic marker expressed at early embryologic stages of hematopoiesis and enables a ubiquitous and robust differentiation between hematopoietic and non-hematopoietic cells in vivo. By cross-breeding Vav-Cre and mTmG strains, a double transgenic strain (Vav-Cre / mTmG; termed Vav-recombinant, or VavR) was created, in which all hematopoietic cells (Vav1+) were irreversibly labeled with GFP after Cre-mediated excision of the floxed RFP, and non-hematopoietic cells remained tdTomato (RFP = red fluorescent protein) labeled. Importantly, all cells that expressed Vav1 at any point during development were permanently labeled with GFP, eliminating the problem of transient hematopoietic lineage marker expression (FIG. 6A).

[0088] Since whole body irradiation required for BM transplantation models might have an impact on wound healing and fibrosis, a parabiosis model was used to confirm the recruitment of MCC during scar remodeling (FIG. 7A). VavR mice were parabiosed to WT mice and after a common blood circulation had been established, ischemic skin flaps were created on the backs of the WT mice. Flap tissue was explanted for analysis three days, seven days, and one month after injury (FIG. 6B and 6C). tdTomato labeled cells were barely detected within the tissue in the acute and intermediate phases of tissue ischemia (at days 3 and 7). In line with findings from the Col-GFP model, scars explanted during the chronic remodeling stage (at 1 month) revealed a clearly defined cluster of tdTomato+ cells on FACS that comprised about 0.1% of live cells and a 10-times increase in the amount of tdTomato+ cells in tissue sections (FIG. 6C-6F).

[0089] Sorted tdTomato+ and EGFP+ cells isolated seven days and one month after injury were then analyzed using scRNA-seq (FIG. 6G and 6H). Using a custom transcriptome containing the transgene information for both EGFP and tdTomato, the expression of EGFP and tdTomato mRNA were able to be detected and analyzed within single cells (FIG. 6H). Confirming the FACS findings, the majority of all circulating cells were of hematopoietic origin and expressed high levels of both EGFP and Ptprc. A small population of cells clearly clustered separately from the identified myeloid and lymphoid cells on the UMAP embedding and demonstrated a strong expression of tdTomato (FIG. 6H) as well as fibroblast markers, such as Col1a1, Den, Pdgfra, confirming the mesenchymal origin of MCC and their recruitment during the chronic remodeling stage of healing (FIG. 6I-6K).

[0090] MCC demonstrated many transcriptional similarities to fibrocytes. To further differentiate these cells, both MCC and fibrocytes demonstrated similar expression of collagens and stem cell markers such as Cd34, Itgbl and Ly6a (FIG. 6K, FIG. 7B). However, fibrocytes expressed EGFP and the hematopoietic markers Ptprc, Rac2, and Bcl2a1b, which were absent in MCC. These findings indicate that despite similarities in the expression of profibrotic and stem cell markers, distinct populations of MCC and fibrocytes co-exist during scar remodeling (FIG. 7A and 7B).MCC are recruited to sites of injury via TWEAK signaling

[0091] Next, how MCC interacts with other cell types during healing was investigated. Seven distinct subpopulations of myeloid cells were characterized as well as six subpopulations of T cells in ischemic flap tissue explanted seven days and one month after injury (FIG. 8A and 8B, and FIG. 9A-9D). Myeloid subpopulations were differentially characterized by genes such as Arg1 (Arginase-1 , anti-inflammatory), Trem2 (triggering receptor expressed on myeloid cells, pro-angiogenic and anti-fibrotic), Retnla (anti-inflammatory), Lyvel (perivascular macrophages), and Ly6c (monocytes), which have been described in recent studies (FIG. 8C and 8D). Subpopulations also included dendritic cells (DC) and proinflammatory cells characterized by Ccl3 expression. RNA velocity analysis demonstrated that myeloid cells from ischemic flaps were transcriptionally driven from Ly6c+ monocytes, mainly present at day seven, toward Lyvel and Retnla expressing macrophages, found at the one-month time point.

[0092] When analyzing cellular interactions based on expression of ligands and receptors, MCC mainly interacted with myeloid cell populations detected at the one-month time point (FIG. 8E). Analysis of specific signaling pathways underlying the cellular communications showed that the majority of interactions between MCC and myeloid cells were based on TWEAK (TNF-related weak inducer of apoptosis), MIF (macrophage migration inhibitory factor), CXCL (C-X-C motif chemokines), and PTN (pleiotrophin) signaling (FIG. 8F and 8G). TWEAK activates a variety of intracellular signal pathways to promote pro-inflammatory cytokines and dysregulated tissue repair processes to perpetuate and exacerbate the fibrotic process. CXCL chemokines have also been shown to contribute to the recruitment and activation of myeloid cells, while MIF is a critical mediator of inflammation in hematopoietic cells, leading to fibrosis. PTN has been shown to promote organ fibrosis and was found to be overexpressed in hypertrophic scars. MCC mainly received incoming signals via TWEAK and secreted outgoing signals via CXCL, PTN, MIF and TWEAK signaling. These findings indicate that TWEAK secreted from myeloid cells might promote recruitment of MCC during chronic remodeling after injury.

[0093] To confirm the importance of TWEAK to the recruitment of MCC, BM-chimeric mice were created by reconstituting the bone marrow of lethally irradiated WT mice with cells from VAV-R mice (FIG. 8H). After engraftment, ischemic flaps were created on the backs of VAV-R BM-chimeric mice and the flaps were subcutaneously injected with either TWEAK or PBS (control) for 1 month. Mice that had been injected with TWEAK showed significantly more MCC within scar tissue one month after injury (*p<0.05, FIG. 8I and 8K), demonstrating that TWEAK specifically promoted the recruitment of these rare cells during chronic remodeling. Additionally, a significantly higher collagen fiber alignment was observed in mice treated with TWEAK (*p<0.05, FIG. 8J and 8L), indicating that the increased recruitment of MCC subsequently promoted fibrosis in remodeling scars. These findings confirmed that recruitment of MCC during scar remodeling is regulated by TWEAK and that these cells play a pro-fibrotic role during the chronic remodeling stage of healing.

[0094] The recruitment of mesenchymal cells such as fibroblasts during the proliferative stage of wound healing has been believed to occur via local migration from the wound margin triggered by infiltrating immune cells. Here, circulating non-hematopoietic mesenchymal cells (MCC) also home to sites of injury and persist within the tissue long-term has been demonstrated. These cells demonstrate pro-fibrotic features and are only detectable during chronic scar remodeling.

[0095] The data indicate that recruitment of MCC during scar remodeling is mediated by TWEAK secreted from myeloid cells. As myeloid subpopulations transcriptionally evolve during wound repair, changes in their secretome during the remodeling stage might be the cause of the late recruitment of MCC. Since MCC are a rare cell population that is virtually undetectable during the acute inflammatory phase of wound healing, their accumulation within the skin over time might contribute to the higher abundance during remodeling, which appears to reach a plateau around 1 month and remains at stable levels until 6 months after injury.

[0096] Targeted depletion of MCC during wound healing shifted transcriptomic profiles of resident wound fibroblasts to more regenerative, benign subsets. Therefore, similar to regulatory T cells that only account for a small subset of the total T cell fraction but have crucial regulatory and homeostatic functions, MCC likely represent a master-regulator of fibroblasts within the wound during chronic remodeling. Since T cell mediated depletion of MCC was found to be an effective strategy to reduce fibrosis after injury, The present invention identifies a new cellular target for anti-fibrotic CAR T cell therapeutics.Targeting Circulating Mechanoresponsive Myeloid Cells Reduces Fibrosis

[0097] Tissue injury activates a cascade of signaling pathways to recruit different cell types to orchestrate the healing response. However, excessive cell activation and recruitment leads to prolonged inflammation with overproduction and accumulation of extracellular matrix (ECM) proteins, leading to inert, dysfunctional scar tissue as seen in pulmonary fibrosis, myocardial infarction, and hypertrophic scar (HTS) formation. Recent examination of fibroblast heterogeneity has resulted in a number of studies focused on the role of different fibroblast subpopulations during fibrosis and healing, and the group has previously characterized how mechanical stimuli influences fibroblast heterogeneity and differentiation. Elevated mechanical tissue stresses trigger mesenchymal mechanotransduction pathways to promote fibroblast differentiation into pro-fibrotic subpopulations. Conversely, pharmacologically disrupting fibroblast mechanical signaling promotes pro-regenerative subpopulations that prevent scar formation and instead promote skin regeneration. In the complex cancer stroma, others have also found that disrupting mechanotransduction in the tissue resident cells may either increase or decrease tumor size, depending on the maturity and size of the tumor.

[0098] However, tissue repair after injury involves a dynamic interplay among not only resident cells but also cells recruited from the circulation. Myeloid cells, such as monocytes and macrophages, are derived from hematopoietic precursors and migrate to sites of injury to facilitate tissue repair by secreting cytokines to attract tissue-resident fibroblasts. Although recent advances in novel single-cell sequencing analytics have been used to characterize fibroblast heterogeneity, myeloid cell heterogeneity remains an additional frontier to explore. Recent work has increasingly identified immune cells not only as initiators but also as active participants throughout the course of healing.

[0099] The present example comprehensively investigates how myeloid cells respond to mechanical signals to influence healing, fibrosis, and regeneration. Beneficial and detrimental mechanoresponsive myeloid subpopulations within scars and unwounded skin are first identified. Manipulating mechanical strain in vivo and in vitro reveals that targeting myeloid mechanical signaling is sufficient to reduce pro-fibrotic myeloid subpopulations and restore native, anti-inflammatory myeloid subpopulations. The human relevance of these findings is confirmed using a co-culture system and spatial transcriptomics. Thus, the present invention highlights the importance of myeloid cell mechanotransduction signaling during healing, potentially opening new avenues for therapeutic treatment of fibrosis across various organ systems.Characterization and validation of a human-like hypertrophic scarring model

[0100] Increasing mechanical strain initiates HTS formation in mice. Here, a modified steel palatal expander was attached with rod extender arms to the mouse dorsum using skin adhesive and staples to initiate a uniform and consistent strain profile across an incisional wound (FIG. 10A and 10B). The incision was created , waited 4 days to allow the wound to close, and then mounted the devices to provide human-levels of mechanical strain for 14 days to generate severe HTS (FIG. 10C and 10D). Compared with unwounded skin, these scars demonstrated significantly increased levels of inflammatory infiltrate (FIG. 10E), density of collagen (FIG. 11), and highly aligned collagen (FIG. 11). Utilizing immunofluorescent staining, these scars demonstrated increased collagen production (COL1) and myofibroblast (aSMA+) differentiation (FIG. 12). These histological analyses were compared with human HTS and unwounded skin collected from standard plastic surgery procedures. Human HTS also demonstrated increased inflammatory infiltrate (FIG. 10F), with more densely packed and highly aligned collagen compared with unwounded skin (FIG. 11). These histological analyses confirmed that the murine hypertrophic scarring device model precipitates human-like fibrosis.

[0101] To understand the identity and importance of the cells driving HTS formation, scRNA-seq (FIG. 10G and 10H) was performed from HTS and unwounded skin using the 10x Genomics Chromium platform. Data for individual cells were subjected to Louvain-based clustering and embedded into a two-dimensional UMAP space in a manner blinded to the phenotype of origin. A variety of cell types were captured including myeloid (monocytes, macrophages, and neutrophils), lymphoid, fibroblast, epithelial, endothelial, and neuron cells (FIG. 10G), which were defined using common cell type genes such as Ptprc for immune cells, Lyz2 for myeloid cells, Pdgfra for fibroblasts, Krt5 for epithelial cells, \Avf for endothelial cells (FIG. 101, FIG. 13). Myeloid cells had 800 differentially expressed genes (DEGs; average log fold change > 0.5) between HTS and skin, which was interestingly more than fibroblasts (~500 DEGs) and all other cell types (FIG. 10J). These findings suggested sustained elevation of inflammatory cell activity in HTS and corroborated the previous findings identifying mechanoresponsive macrophages as key drivers of scar formation.

[0102] The changes in transcriptional profiles between unwounded and HTS myeloid cells were interrogated (FIG. 10K). First, human levels of mechanical strain promoted myeloid cells to upregulate inflammatory markers such as Ccl3 and Ccl4 (FIG. 10L). The family of chemokine (C-C motif) ligand (Co / ) genes encodes for macrophage inflammatory proteins (MIP), and all represent chemoattractants secreted by myeloid cells that recruit a variety of other cells during healing. Mechanically activated myeloid cells also upregulated Thbsl the family of Thbs genes encodes for thrombospondin proteins, which facilitate cell binding to ECM, are associated with increased fibrosis, have antiangiogenic functions, and have been linked to macrophage activation and subsequently increased inflammation. Mechanical strain also upregulated Rac2, a hematopoietic-specific Rho-GTPase inflammatory mechanotransduction marker that mediates the recruitment and activation of immune cells.

[0103] In contrast, healthy unwounded skin expressed an upregulation of anti-inflammatory myeloid markers Mrc1, Cd163, and Selenop (FIG. 10L and 10M). Selenop encodes for selenoproteins, which are upstream regulators of anti-inflammatory macrophage processes, ranging across pathogen clearance, cytokine production, and tissue repair and also responsible for modulating canonically M2 anti-inflammatory markers such as Cd163 and Mrc1. Healthy unwounded skin was also characterized by Egr1 (encoding early growth response protein 1) (FIG. 10M), which represses macrophage inflammatory responses to blunt the immune response. Egr1 signaling is promoted in the absence of mechanical signaling, mediated by Akt signaling, and associated specifically with regenerative phenotypes that suppress fibrosis. Taken together, these findings highlight that mechanical strain ablates a healthy population of anti-inflammatory Mrc1 / Selenop / Egr1+ macrophages to instead promote differentiation into pro-inflammatory Ccl+ and pro-fibrotic Thrombospondin+ phenotypes.Targeting myeloid specific mechanical signaling promotes regeneration after injury

[0104] To precisely interrogate the importance of myeloid-specific mechanical signaling, myeloid-specific (Lyz2) Cre mice with ptk2flox / floxmice were bred to create Lyz2-Cre+ / ~Ptk2flox / floxmice (Ptk2 encodes for FAK) and compared their phenotypes to wildtype controls (FIG. 14A). Mechanical strain in wildtype mice (S WT) produced considerable HTS formation, evidenced by hyperpigmentation and significant widening (500% increase) of the scar tissue compared to control (C) mice not subjected to increased mechanical strain (FIG. 14B). Using picrosirius red staining, the collagen ultrastructure of the fibrotic scar tissue was then analyzed using CurveAlign and CT-FIRE mathematical collagen analysis algorithms and found that mechanical strain also promoted significantly longer and more highly aligned collagen fibers (FIG. 14C and 14D; FIG. 15). In contrast, mechanical strain in myeloid-specific knockout (S KO) mice caused significantly reduced scar widths (FIG. 14B) and promoted significantly shorter and less aligned collagen fibers (FIG. 14C and 14D; FIG. 15). These fibers closely mimicked the physiologic, random, basket-weave skin architecture of unwounded skin which is comprised of short, cross-hatched collagen fibers (FIG. 14C and 14D). These findings demonstrate that specifically targeting mechanical signaling in myeloid cells causes a cascade of downstream effects that influence end-stage scar formation and tissue architecture.

[0105] scRNA-seq was then performed to interrogate the transcriptional effects of myeloid-specific mechanical ablation (FIG. 14E) and identified myeloid, fibroblasts, epithelial, lymphoid, neuron, smooth muscle, and endothelial cells in the collected cellular data (FIG. 14F). First, the myeloid cells were interrogated (FIG. 10A-10L), the mechanical strain upregulated fibrotic Thbsl and mechanical signaling markers Rac2 and Piezol while reducing expression of anti-inflammatory Egr1 (FIG. 14G and 14H). The fibroblasts were also interrogated and it was observed that mechanical strain caused a similar downregulation of fibroblast Egr1 expression as well as upregulation of En1+ cells (FIG. 141 and 14J), which encodes for Engrailed. Interestingly, myeloid specific mechanical knockout eliminated expression of these fibrotic and mechanical signaling markers, along with mechanical signaling markers Mapkl and Rockl, while promoting an upregulation of Egr1 that mimicked levels of control mice (FIG. 14H). Most surprisingly, myeloid specific mechanotransduction ablation also affected downstream fibroblasttranscriptional profiles, including an upregulation of Egr1 and downregulation of En1 and mechanical signaling markers such as Rac1, Mapkl, and Rockl (FIG. 14J).

[0106] Overall, these findings demonstrated that manipulating myeloid cell-specific mechanotransduction sufficiently propagated a regenerative response that mitigated scar formation and promoted regeneration of native collagen architecture after injury. Disrupting myeloid cell mechanical signaling restored anti-inflammatory Egr1+ and reduced Thbs1+ transcriptional profiles back to control and unwounded skin levels. Surprisingly, fibroblasts also demonstrated considerable transcriptional changes from myeloid-specific mechanical modulation. Since immune cells infiltrate into the healing wound environment during the early stages of wound repair, these findings demonstrate that immune cells act as the “drivers” of the healing process, dictating the fate and function of all downstream cells.Disruption of mechanotransduction mitigates shifts immune cell recruitment

[0107] The comprehensive understanding of mechanoresponsive myeloid cell subpopulations was pursued using a parabiosis model in conjunction with an HTS model. Parabiotic pairings of WT and GFP+ mice were established, allowing a shared blood circulation to develop over 2 weeks. Subsequently, HTS was initiated so that any injuries to the WT mouse would promote recruitment of GFP+ circulating immune cells (FIG. 16A). In control parabiotic mice (device not extended), a thin scar with a baseline average of 5.4% GFP+ circulating immune cells within the scar (FIG. 16A and 16B). Mice subjected to human-like mechanical strain produced thicker and wider scars and recruited an average of 12.2% GFP+ immune cells within the HTS, an over 250% increase in circulating inflammatory infiltration during healing (FIG. 16A and 16B; FIG. 17A and 17B). In contrast, blocking mechanotransduction using a small molecule focal adhesion kinase inhibitor (FAKI) in mice subjected to human-like mechanical strain led to mitigated scar formation, indicated by a significantly decreased scar widths and scar collagen content (FIG. 16A and 16B; FIG. 17A and 17B). Most importantly, targeting mechanical signaling reduced the number of circulating inflammatory cells back down to baseline levels, with an average of 6.5% circulating immune cells comprising the scars. Mechanical strain promoted scar widening as early as day 3, characterized by significantly elongated and aligned collagen fibers in the early healing tissue, indicative of early HTS formation (FIG. 18A-18C). Pharmacological disruption of early inflammatory cells at day 3 immediately decreased the underlying scar width and promoted shorter, more randomly aligned collagen fibers, similar to unwounded skin (FIG. 17A-17C).

[0108] To further elucidate the importance of these circulating myeloid cells, the parabiosis HTS experiments were then repeated utilizing a wildtype mouse and a Lyz2Cre+ / -Brainbow2.1+ / +mouse, in which the myeloid cell specific Lyz2 promoter drives stochastic expression of green, red, yellow, or cyan fluorescence (GFP, YFP, RFP, or CFP) (FIG. 19A). Wildtype parabiotic mice with mechanically strained incisions demonstrated increased clonal proliferation of circulating Lyz2-positive myeloid cells, corroborating the increased migration and infiltration of myeloid cells within the mechanically strained hypertrophic scar (FIG. 19B). Disruption of mechanical signaling instead reduced and restored myeloid cell recruitment and proliferation back to control baseline levels (FIG. 19B).

[0109] Overall, these findings demonstrate that pharmacological disruption of mechanical signaling specifically altered the recruitment and proliferation of inflammatory cells within the healing scars and that changes in scar widths and collagen fiber architecture could be observed at both early (day 3) and later (day 14) time points.Disrupting mechanotransduction shifts heterogeneous, mechanoresponsive myeloid subpopulations

[0110] To understand the identity and importance of these cells, scRNA-seq was then performed on the sorted cells (FIG. 16A and 16B) from each of the three groups using the 10x Genomics Chromium platform. scRNA-seq of both GFP+ and GFP- cells from all three groups captured 43,274 cells, including myeloid cells (monocytes, macrophages, and neutrophils), fibroblasts, keratinocytes, lymphoid cells, and endothelial cells (FIG. 20A-20C). Most GFP+ circulating cells were classified as immune cells, with fibroblasts and keratinocytes mostly deriving from the local, GFP- tissue.

[0111] Macrophages demonstrated remarkable heterogeneity in the dataset, highlighting the previously unexplored importance of macrophage mechanotransduction during scar formation and tissue regeneration (FIG 16C and 16D). Circulating myeloid cells clustered in 5 transcriptionally distinct subpopulations (FIG. 16D). Human-like mechanical strain directly increased the proportion of fibrotic myeloid cells, which were primarily defined by the monocyte marker Ly6c2 as well as Thbsl (FIG. 16B-16G), corroborating findings in (FIG. 10A-10L and FIG. 11). Mechanical strain also increased Cd86 expression (FIG. 16G), a classical inflammatory marker, and the proportion of inflammatory myeloid cell populations, which were primarily defined by IL6 and Cc / genes (FIG. 16B-16E). Mechanical strain also slightly increased the proportion of migratory myeloid cells, which differentially upregulated the known myeloid migration marker Cd74. All three groups had similar proportions of Mki67+ / Cks1+ myeloid cells, representing myeloid cells undergoing healthy cell cycling and metabolic behaviors.

[0112] The relative distribution of unique mRNA transcripts was then compared using CytoTRACE (FIG. 16H). Mechanically stimulated macrophages had the highest number of uniquely expressed mRNA features, demonstrating that strain upregulated transcription of a wide range of unique pro-fibrotic and pro-inflammatory genes compared with both control and mechanically inhibited cells. RNA velocity analysis using the scVelo package to explore the comparative abundance of spliced and unspliced pre-mRNA transcripts was also employed (FIG. 16D and 161). The fibrotic myeloid cluster demonstrated an increased proportion of unspliced Thbsl, demonstrating that these cells were actively creating new Thrombospondin mRNA transcripts at the time of cell capture. Velocity analysis also identified high unspliced mRNA production of Fn1 (FIG. 16J), which encodes for fibronectin, a well-known foundational ECM protein understood to critically drive the formation of fibrotic scar tissue52. Without fibronectin and thrombospondin establishing the initial matrix for subsequent collagen deposition, minimal fibrotic tissue could be formed thereafter.

[0113] In contrast, control and mechanically disrupted circulating myeloid cells shared considerable overlap in transcriptional states. Disruption of mechanical signaling reduced the proportion of Thbs1+ / Ly6c2+ fibrotic cells by approximately 50% and almost eliminated all Ccl+ / ll6+ inflammatory myeloid cells within the healing injury (FIG. 16E). Blocking mechanical signaling also promoted a unique subset of anti-inflammatory, regenerative myeloid cells. Regenerative myeloid cells primarily upregulated anti-inflammatory myeloid markers Mrc1, Cd163, and Selenop and the beneficial myeloid factor Egr1 (FIG. 16F and 16G), matching the quiescent anti-inflammatory properties of unwounded myeloid cells (FIG. 10L and 10M) and myeloid specific mechanical knockout cells (FIG. 11H). Using RNA velocity analysis, regenerative clusters expressed upregulation of unspliced Mrc1 and Sashl (FIG. 16J). Sashl has specifically been recently identified as a marker that downregulates Thbsl and TGF-B, thus reducing fibrotic complications.

[0114] Partition-based graph abstraction (PAGA) was applied and informed by RNA velocity-inferred directionality to quantify the relationship between the myeloid clusters by showing the connectivity of the cellular clusters. PAGA analysis demonstrated two diverging trajectories of myeloid cell differentiation originating from the migratory myeloid clusters and pointing toward either fibrotic or regenerative clusters, either in the presence or absence of mechanical signaling (FIG. 161). Thus, it could be inferred that as myeloid cells migrate into the injured environment, the presence or absence of mechanical signaling could push or pull their transcriptional fates toward either fibrotic or regenerative phenotypes.

[0115] To confirm the importance of myeloid cell signal transduction, immunofluorescent staining was then performed and imaging of tissue sections from the parabiotic murine tissue. Confocal imaging of these tissue sections first confirmed a >300% increase in circulating myeloid cell numbers (GFP+ cells) in mechanically strained (HTS) tissue compared with both control and mechanically disrupted (FAKI) groups (*p<0.05) (FIG. 16K-16M). Furthermore, mechanical disruption significantly increased EGR1 (FIG. 16K, 16M, *p<0.05) and MRC1 protein levels (FIG. 16L, 16M; *p<0.05), confirming protein-level expression of key anti-inflammatory markers found in regenerative myeloid cells within the scRNA-seq dataset. Overall, these findings reveal the previously unexplored importance of myeloid mechanical signaling and heterogeneity during fibrosis and healing. Mechanical signaling drives myeloid cell differentiation and activity within healing wounds into pro-fibrotic and pro-inflammatory phenotypes that express extracellular matrix proteins, such as thrombospondin and fibronectin, as well as inflammatory CCL and IL6 cytokines. Disrupting mechanical signaling specifically shifted myeloid transcriptional states toward regenerative programs primarily driven by M2 markers MRC1 , CD163, and SELENOP, as well as anti-inflammatory and pro-regenerative EGR1 .Mechano-immunomodulation mediates myeloid-fibroblast cross talk

[0116] Within the complicated, multicellular milieu of wound healing, numerous interweaving signals occur between the various cell types. To isolate the effects of how mechanical signaling modulates the interactions between immune cells and fibroblasts during fibrosis and regeneration, a previously established three-dimensional (3D) collagen scaffold culture system was utilized (FIG. 21 A). To mimic thecirculating GFP+ myeloid cells interacting with tissue-resident fibroblasts in the parabiosis experiments, myeloid cells from the bone marrow of GFP+ mice and fibroblasts from wildtype murine dermis were isolated; after in vitro expansion, these cells were seeded together within 3D collagen scaffolds. The scaffolds were either subjected to 10% strain (Strain: S) or 10% strain with FAK inhibition (Strain + FAKI: S+FAKI) to create a physiologically relevant co-culture system of mechanically activated skin. After 48 hours, FACS was used to observe that treatment with mechanical inhibition significantly reduced the number of myeloid cells (FIG. 21 B), confirming the in vivo findings (FIG. 21 C). Disrupting mechanical signaling did not significantly change the absolute number of fibroblasts. This significant reduction in myeloid cell numbers was then further confirmed with confocal imaging and quantification of GFP+ cells in the hydrogels after treatment (FIG. 21 C).

[0117] To examine the molecular drivers behind this behavior, scRNA-seq on the co-cultured cells was then performed. 23,022 cells were captured corresponding to the S and S+FAKI groups (FIG. 21 D), with the eGFP+ cells corresponding to the myeloid cells and eGFP- cells corresponding to the fibroblasts (FIG. 21 E, 21 F). Co-cultured skin exposed to strain were comprised of 48% fibroblast and 52% myeloid cells, and disrupting mechanical signaling instead reduced myeloid proportions to 13.5% (FIG. 21 G). Pharmacological disruption of myeloid mechanical signaling specifically reduced the expression of ECM guiding and scar promoting Fn1 while instead promoting anti-inflammatory and pro-regenerative Mrc1, Selenop, and Egr1 (FIG. 21 H), confirming the in vivo findings from FIG. 10A-12. These shifts in myeloid subpopulations subsequently reduced fibroblast expression of Thbsl, Col1a1, and Col3a1 (FIG. 211), demonstrating a significant decrease in ECM and collagen production (FIG. 12G) and further confirming the findings from FIG. 11 and FIG. 12.

[0118] The scRNA-seq findings were further confirmed by characterizing the role of EGR1 and Thrombospondinl (encoded by Thbsl) protein expressing macrophages using FACS (FIG. 21 J, and FIG. 22). In murine co-culture collagen scaffolds, mechanical disruption significantly reduced the number of macrophages (GFP+ cells) within the collagen scaffolds (FIG. 21J). The remaining myeloid cells demonstrated significantly increased expression of regenerative marker EGR1 and significantly reduced expression of the fibrotic Thrombospondin protein (FIG. 21J). Immunostaining further confirmed the increase in Thrombospondin expression in mechanically strained versus unstrained and pharmacologically disrupted tissues (FIG. 23).Identification and confirmation of mechanoresponsive macrophage subpopulations in human scar and skin

[0119] The co-culture experiments were repeated using human fibroblasts and human myeloid cells. Human skin samples from common plastic surgery procedures (e.g., abdominoplasty, mastectomy) were collected with IRB approval, and human dermal fibroblasts were isolated and cultured. In addition, human blood donated for research purposes was used to obtain human myeloid cells. The human relevance was confirmed by observing that disrupting mechanical signaling significantly decreased the proportion ofhuman macrophages (CD45+) within the co-culture gels and induced regenerative myeloid cells to upregulate EGR1 and downregulate Thrombospondin expression (FIG. 21 K, FIG. 24A-24C).

[0120] Finally, spatial transcriptomics was then performed with the 10X Visium platform on human skin and human scar samples collected from the same patient (FIG. 21 L, 21 M). Utilizing this platform, the transcriptional profiles of “spots” or groups of cells across the histological sections were able to identify 11 distinct transcriptional clusters between the unwounded skin and scar (FIG. 21L-21O). Most importantly, the spatial datasets was then queried for the log-averaged expression of regenerative macrophage markers Mrc1 / Egr1 / Seppi or for the log-averaged expression of fibrotic macrophage markers Thbs1 / Thbs2 / Cd86, which are all markers. Specifically spatial clusters in human skin demonstrated decreased expression of the fibrotic markers and increased expression of the regenerative macrophage markers compared with scar tissue (FIG. 21 Q).

[0121] Overall, these findings comprehensively confirmed the human relevance of the findings. Analyzing human spatial scar samples as well as activating mechanical signaling in human cells created a pro-fibrotic profile primarily dominated by thrombospondin expression. In contrast, analyzing human spatial skin samples as well as disrupting mechanical signaling in human myeloid cells instead promoted a pro-regenerative phenotype dominated by EGR1 expression

[0122] The study indicates that modulating mechanical stress directly affects myeloid cell phenotypes and interactions with other cell types in the complex, multicellular environment of wound healing. This principle has been previously unexplored in the context of fibrosis and regeneration, with most prior studies focusing on fibroblast heterogeneity and transcriptional profiles. To the Inventors knowledge, this is the first study to directly investigate the effects of modulating mechanotransduction on immune cell response at the single-cell level utilizing in vivo and in vitro systems specifically designed to investigate the contribution of circulating mechanoresponsive myeloid cells.

[0123] Healthy skin, which expresses low mechanical signaling pathways, are comprised of a healthy abundance of myeloid Seppi + / Mrc1+ / Egr1+ expression, while scar, which expresses increased mechanical signaling pathways, are comprised of pro-fibrotic Thrombospondin+ / Cd86+ profiles. The presence of these subpopulations were confirmed in both mice and humans and observed that specifically targeting myeloid mechanical signaling shifted myeloid transcriptional programs from fibrotic Thrombospondin+ / Cd86+ phenotypes into regenerative Seppi + / Mrc1+ / Egr1+ transcriptional profiles. This “mechano-immunomodulation” promoted the formation of thinner, more randomly aligned, and less fibrotic tissue that featured a basket-weave collagen architecture similar to that of unwounded skin. These mechano-immunomodulatory myeloid cells were comprehensively identified and characterized in a wide range of in vivo and in vitro tissue engineered models. These findings emphasize the critical importance of mechano-immunomodulation in the healing process and during the development of fibrosis and also identify a new subset of mechanoresponsive myeloid cells that contribute directly to these processes.

[0124] Here, it was demonstrated that disruption of mechanotransduction strongly directs myeloid-lineage cells toward anti-inflammatory and regenerative states. While previous studies pinpointmechanoresponsive fibroblasts as the main contributors to fibrosis through their recruitment of inflammatory cells via cytokine secretion, the findings reveal a more complicated interplay between immune cell and fibroblast mechanotransducive signaling during the stages of healing. Controlling mechanical signaling in myeloid cells may provide a cascading effect that influences all other cell types, including fibroblasts. Furthermore, since inflammatory cells drive the earlier wound healing phases, targeting these cell types may result in more powerful and earlier acting therapeutic approaches. While some more recent studies have suggested a role for immune cells in regulating some fibrotic processes, the findings reveal that myeloid cells may actually serve as the primary cell type of importance. Collectively, mechano-immunomodulation of these “early responders” of healing can trigger a cascade of downstream regenerative healing was demonstrate. Many studies investigating other fibrotic disease states, such as renal fibrosis, idiopathic pulmonary fibrosis non-alcoholic steatohepatitis (NASH), liver cirrhosis, or even cancer, have typically characterized the importance of one cell type in isolation. Based on the findings, future studies should interrogate the important interplay between multiple cell types to potentially improve therapeutic and translational outcomes for a wide range of fibrotic disease states. Since inflammatory cells circulate and home to injury sites during the initial healing phases in all organs, focusing on mechanoresponsive myeloid subpopulations may also pave new avenues for systemic immunomodulatory therapies to target fibrosis and other diseases across other internal organ systems.

[0125] Animals and Animal: All animal work was conducted in accordance with the Administrative Panel on Laboratory Animal Care protocols (APLAC protocols 12080 and 28410) as approved by Stanford University, as well as the IACUC protocols (2021-0828) approved by the University of Arizona. The following transgenic mouse strains (acquired through Jackson Laboratories) were utilized: eGFP (C57BL / 6- Tg(CAGEGFP)10sb / J), FAKfl / fl(B6.129P2(FVB)-Ptfc2fmt IGuanIJ, Stock: 031956), Ly2 (B6.129P2-Lyz2(m,fcre / f7J, Stock: 004781) and B6 (C57BL / 6J, Stock: 000664). Mice were housed at the Stanford University Comparative Medicine Pavilion per Stanford APLAC guidelines, overseen by the Veterinary Service Center (VSC).

[0126] Animal Preparation: Anaesthesia was induced and maintained using 1-3% inhaled isoflurane, at a flow rate of 2 L / min. Anaesthesia adequacy was confirmed through observed loss of hind-limb reflex to nociceptive stimuli. Skin was sterilized with Betadine Surgical Scrub Veterinary (Avrio Health L.P™, Stamford, CT) followed by sterile alcohol prep pads (FisherScientific™, Pittsburgh, PA).

[0127] Hypertrophic Scarring (HTS) Model: Linear incisions approximately 20 mm long were made on the dorsa of the mice and then closed using simple interrupted nylon monofilament 4-0 sutures (Dynarex™, Orangeburg, NY). A loading device, constructed from 22 mm expansion screws and Luhr plate supports, was then placed over each wound on POD 4. The device was secured using surgical staples. Tension across the wound was produced by a 2 mm expansion of the loading device every 2 days, for 14 days total. Other groups of mice had devices attached but underwent no mechanical extension. Samples were collected for histological and transcriptional analysis on day 14.

[0128] Parabiosis Model: Parabiotic pairs of mice were created as previously described. In brief,parabiotic pairs consisted of one female wild-type (C57BL / 6) mouse and one female eGFP (C57BL / 6- Tg(CAGEGFP)10sb / J) mouse that were age-matched and housed together. Incisions were made from the base of the elbow joint (olecranon) to the base of the knee joint on corresponding sides of each mouse. Following this, the joints and skin were sutured together. Flow cytometry was used to confirm peripheral blood chimerism two weeks after parabiosis surgery. Following establishment of the systemic circulation, the HTS model was performed as described above.

[0129] Collection and Cryo-sectioning of Human and Murine Tissue Specimens: Human hypertrophic scar (HTS) and unwounded skin samples were obtained under the approved IRB (54225). Human tissue samples were collected during standard plastic surgery procedures, and no patient identifying information was retained with the samples. Both human and mouse specimens were immediately fixed in 4% paraformaldehyde, dehydrated, and cryo-embedded in optimal cutting temperature (OCT) compound for frozen sectioning on a microtome-cryostat.

[0130] Histological analysis of collagen architecture: Masson’s Trichrome staining and Picrosirius Red staining were performed as per manufacturer instructions, and images were captured with a Leica DM5000 B upright microscope. For Picrosirius Red, polarized light microscopy was used to obtain 40x magnification images, and analysis of fiber alignment was performed using CurveAlign, MatFiber, and CT-FIRE.. The degree of the fiber alignment ranges between a quantified value of 0 (completely random) and 1 (completely aligned).

[0131] Immunofluorescent staining: Immunofluorescent staining was performed using primary antibodies targeting Egr1 (ab194357), Mrc1 (ab64693), Thrombospondin 1 (ab267388), and Seppi (PA50112707). The amount of fluorescent area was quantified and normalized using DAPI nuclei staining, using a custom MATLAB image processing code previously published. All images included in this paper are representative images drawn from multiple histologic and immunofluorescent experiments.

[0132] Flow Cytometry Staining: Both human tissue samples and mouse skin wounds were dissected and first washed once in PBS. Tissue was then diced into a fine, even consistency using sharp surgical scissors. Minced skin tissue was then enzymatically digested using a 1 :1 ratio of Collagenase Type IV (ThermoFisher, Cat:17104019) and Collagenase Type II (ThermoFisher, Cat:17101015) at a concentration of 1500U / ml in DMEM (ThermoFisher, Cat: 10569010) for 90 minutes at 37°C. Following enzymatic digestion, FACS buffer was added, and then the solution was filtered through 70 pm and 40 pm cell strainers. Cells were centrifuged at 1500rpm for 5 minutes at 4°C and then the pellet was resuspended in 150 pL of FACS buffer for primary antibody staining. For lineage negative (Lin-) FACS analysis, the following primary antibodies were used: CD45 (ThermoFisher, Cat:48045942), CD31 (ThermoFisher, Cat: 48031942), CD326 (ThermoFisher, Cat: 13932682). Tie2 (ThermoFisher, Cat: 13598782), CD45 (ThermoFisher, Cat: 48045182 or ThermoFisher, Cat: 13045181), CD31 (ThermoFisher, Cat: RM52280 or ThermoFisher, Cat: 13031181), CD324 (ThermoFisher, Cat: 13324982), CD326 (ThermoFisher, Cat: 45592185), Ter119 (ThermoFisher, Cat: 14592182). Additional primary antibody staining of Thrombospondin 1 and EGR1 was then performed. Following further washing, secondary antibodystaining was performed. Following staining, cells were washed again with FACS buffer before DAPI (4',6-diamidino-2-phenylindole) (Biolegend, Cat: 422801) was added to label dead cells. A BD II FACS Aria machine was used for FACS sorting and analysis.

[0133] Single cell barcoding, library preparation, and single cell RNA sequencing (scRNA-seq): Tissue samples were carefully minced into 1 mm2pieces using surgical scissors, with care taken to ensure an even paste-like consistency in order to ensure that a maximum number of cells was captured for scRNA-seq. 30 mL of a 1 mg / mL Liberase (Sigma-Aldrich 5401127001) enzyme solution in PBS was used to fully break down ECM. The cell-digest solutions were then placed in an oven at 37 °C for 2 hours while undergoing constant rotational agitation. A maximum-speed vortex mixer (VWR) was also utilized before, halfway through, and after the 2-hour oven period, to further disrupt tissue clumping and maximize the surface area exposed to the enzymes. This tissue solution was then filtered through a 100 pm Nylon cell filter (Fisher-Scientific 08-771-19), and 20 mL of DMEM + 10% FBS was also passed through the filterto stop the enzymatic action and maximize cell yield. This solution was then centrifuged at 350 x g for 5 min at 4°C, the supernatant was carefully aspirated, and the cell pellet was resuspended in 20 mL of DMEM + 10% FBS. This was then passed through a 70 pm Nylon cell strainer, and 20 mL of FACS buffer was passed through the filter to maximize yield and wash cells.

[0134] This cellular suspension was then formulated to a concentrated solution, which underwent droplet-based microfluidic single cell RNA sequencing (scRNA-seq) at the Stanford Functional Genomics Facility (SFGF) using the 10x Chromium Single Cell platform (Single Cell 3’ v3, 10x Genomics, USA). The cell solution, a reverse transcription master mix, and partitioning oil were all added to a single-cell chip, processed using the Chromium Controller, and then underwent reverse transcription at 53°C for a total of 45min. cDNA was amplified for a total of 12 cycles (BioRad C1000 Touch Thermocycler) and SpriSelect beads (Beckman Coulter, USA) were used to select cDNA size with a 3:5 ratio of SpriSelect reagent volume to sample volume. cDNA was analyzed on an Agilent Bioanalyzer High Sensitivity DNA chip for qualitative control, fragmented for 5min at 32°C, followed by end repair and A-tailing at 65°C for 30min, and then double-sided size selected with SpriSelect. Sequencing adaptors were ligated to the cDNA at 20°C for 15min. cDNA was amplified using a sample-specific index oligo as primer, followed by another round of double-sided size selection. Final libraries were analyzed on an Agilent Bioanalyzer High Sensitivity DNA chip for qualitative control purposes. cDNA libraries were sequenced on a HiSeq 4000 Illumina platform, with a goal of 50,000 reads per cell.

[0135] Data processing, FASTQ generation, and read mapping: Base calls were converted to reads using the Cell Ranger (10X Genomics; version 3.1) implementation of mkfastq. Cell barcodes were examined and filtered based on quality utilizing the following optimized threshold parameters: a minimum of 200 unique transcripts profiled, less than 10,000 total transcripts, and a maximum of 10% of their transcriptome of mitochondrial origin.

[0136] Data normalization and generation of characteristic subpopulation markers: Unique molecular identifiers (UMIs) generated from each cell barcode were created and used for all subsequent analysis.Using a scale factor of 10,000 UMIs per cell, the raw UMI counts were normalized before undergoing natural log transformation (with a pseudocount of 1) through the R package Seurat (version 3.1.1). After identifying highly variable genes, cells were scaled by regression to the fraction of mitochondrial transcripts. The aggregated data was then evaluated by applying uniform manifold approximation and projection (UMAP) analysis for the first 15 principal components. Automated cell annotations were created with the SingleR toolkit (version 3.11) against the ImmGen Murine database. Genes with differential expression were distinguished with Seurat’s native FindMarkers function, using a log fold change threshold of 0.5 and the ROC test to allocate each gene’s predictive power.

[0137] RNA velocity analysis using scVelo: RNA velocity analysis was executed using the dynamical model of the scVelo package. Partition-based graph abstraction (PAGA) was done with the sc.tl.paga function in scVelo. In order to identify the genes with differentially regulated transcriptional dynamics in comparison to all other clusters, a Welch t-test (with a conservatively overestimated variance) was implemented using the sc.tl.rank_velocity_genes function. A ranking of genes by likelihood, grouped by treatment, was created from the dynamical model.

[0138] Murine Peripheral Blood Mononuclear Cell (PBMC) isolation: Murine bone-marrow derived macrophages were isolated and cultured according to previously published protocols. Briefly, C57 / BL6 mice were euthanized, femurs and tibias were harvested, and the bone epiphyses were removed in order to flush out bone marrow using 5mL RPMI. A mortar and pestle were used to crush bone epiphyses and collect bone marrow. ACK (ammonium-chloride-potassium) lysis buffer was used to lyse red blood cells, and cells were strained and washed. A complete medium was used, consisting of RPMI, 10% fetal bovine serum, penicillin (100 U / ml), streptomycin (0.1 mg / ml), 2 mM glutamine, 50 pM 2-mercaptoethanol, and 200 Units (U) I ml murine granulocyte-macrophage colony-stimulating factor (GM-CSF; PeproTech, Frankfurt, Germany). Cells were plated in 10mL of complete medium at a concentration of 2x106cells per 100mm2plate. On Day 3, an additional 10 mL of complete medium were added in each plate. On Day 7, cells were removed from the culture plates and used for experiments.

[0139] Human Peripheral Blood Mononuclear Cell (PBMC) isolation: Whole blood buffy coats were obtained from the Stanford Blood Center (SBC) from anonymous, healthy donors. Peripheral blood mononuclear cells were isolated from whole blood buffy coats by using Ficoll-Paque density gradient centrifugation (1200 x g for 20 minutes), removing the PBMC layer, and washing with PBS. Next, CD14+ monocytes were isolated from the collected PBMCs via magnetic labeling with Miltenyl CD14 microbeads ((Cat # 130-050-201) and magnetic sorting using Miltenyi LS columns (Cat # 130-042-401). At this stage, flow cytometry was used to ensure monocyte purity: positively CD14-labeled fractions were washed in FACS buffer and labeled with anti-CD3 Brilliant Violet 605 (clone:OKT3, BioLegend), anti-CD4 APC / Cyanine 7 (clone: RPA-T4, BioLegend), anti-CD8 PE (clone: SK1 , BioLegend), anti-CD14 FITC (clone: 61 D3, eBiosciences) and GhostDye 510 (TONBO Biosciences). Next, freshly isolated monocytes were resuspended at a concentration of 0.5 x 106cells / mL in a complete medium consisting of RPMI 1640 supplemented with 10% FBS and 2mM L-glutamine. Monocytes were then seeded into 6 well plates at 1 .5 x 106cells per well (1 ,5mL of previous suspension per well). Next, the total volume per well was increasedto 3mL by adding 1.5 mL of complete RPMI media supplemented with 100ng / mL GM-CSF (Recombinant Human GM-CSF Protein R&D Systems 215GM050), bringing the final GM-CSF concentration to 50ng / ml. On Day 3 of culture, half of the media was discarded from each well and replaced with fresh complete media containing 100ng / mL of GM-CSF. On Day 6, cells were polarized into a pro-inflammatory M1 -like phenotype by discarding media and replacing with fresh media containing 50ng / mL IFN-gamma (R&D Systems 285-IF-100).

[0140] Myeloid and fibroblast populated 3D collagen scaffold experiments: Dermal fibroblasts were isolated and cultured from either healthy human skin or mouse skin samples. Both mechanical and enzymatic digestion were used to isolate fibroblasts, and the cells were then cultured under standard conditions until reaching passage. Following previously published protocols, both cell types were incorporated within the scaffolds, creating cell-populated collagen hydrogels with a concentration of 2 x 105cells / mL and 2 mg / mL collagen (PureCol, Advanced Biomatrix 5005). Briefly, collagen scaffolds were shaped in a cruciform arrangement with sponges in the arms and cultured in petri dishes on top of a layer (~0.5cm thick) of cured polydimethylsiloxane (PDMS; Sylgard 184 Silicone Elastomer Kit; Dow Corning). Pins were placed through the hydrogel cruciform arms to hold the scaffolds in place during an initial pre-culture period (24h). After 24 hours, the scaffolds were subjected to either a 10% equibiaxial strain (either with or without FAKI) or no strain for an additional 48-hour time period. The cells experiencing strain were given 10pL of 20 mM FAKI in DMSO in 20mL of culture media, with a final concentration of 10 pM FAKI. Strained but untreated cells were given DMSO in 20mL of culture media. A MATLAB code was used to confirm the 10% equibiaxial strain. After 48 hours, cells were either fixed in 4% PFA (ChemCruz, Cat:281692) for immunohistochemical analysis or harvested for FACS analysis via enzymatic digestion using a 1500U / mL solution of Collagenase Type I (ThermoFisher, Cat:17018029) in DMEM (ThermoFisher, Cat:10569010).

[0141] As used herein, the term “about” refers to plus or minus 10% of the referenced number.

[0142] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting essentially of’ or “consisting of’, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of’ or “consisting of’ is met.

Claims

WHAT IS CLAIMED IS:1 . A method of treating a fibrotic condition or cancer in a subject in need thereof, the method comprising administering a therapeutic amount of a composition that modulates one or both of i) circulating cells of any origin or ii) mechanical signaling in myeloid cells.

2. A method of treating a fibrotic condition or cancer in a subject in need thereof, the method comprising modulating one or both of i) circulating cells of any origin or ii) mechanical signaling in myeloid cells.

3. The method of claim 1 or claim 2, wherein the circulating cells comprise mesenchymal cells or hematopoietic cells.

4. The method of claim 1 or claim 2, wherein the myeloid cells are derived from hematopoietic precursors.

5. The method of claim 4, wherein the myeloid cells comprise monocytes, macrophages, neutrophils, dendritic cells, or a combination thereof.

6. The method of any one of claims 1-5, wherein the composition comprises small molecules, drug therapy, cytokines, miRNA, antibodies, peptides, or a combination thereof.

7. The method of claim 6, wherein the small molecule comprises a small molecule focal adhesion kinase inhibitor (FAKI) or any other inhibitor or activator that modulates mechanical signaling.

8. The method of any one of claims 1-7, wherein modulating one or both of i) circulating cells of any origin or ii) mechanical signaling in myeloid cells comprises utilizing a cell therapy, gene editing, or a combination thereof.

9. The method of any of claims 1-8, wherein the composition inhibits one or both of i) circulating cells or ii) myeloid mechanical signaling.

10. The method of any of claims 1-8, wherein the composition augments one or both of i) circulating cells or ii) myeloid mechanical signaling.

11. The method of any of claims 1-10, wherein modulating one or both of i) circulating cells or ii) mechanical signaling in myeloid cells comprises depleting said cells.

12. The method of claim 11 , wherein plasmaphoresis is used to deplete the cells or wherein a CAR-T cell or other cell type is used to target and deplete said cells.

13. The method of any of claims 1-12, wherein modulating circulating cells comprises modulating TWEAK (TNF-related weak inducer of apoptosis), MIF (macrophage migration inhibitory factor), CXCL (C-X-C motif chemokines), PTN (pleiotrophin) signaling, THBS1 / THBS2 / THBS4 (Thrombospondins), CD86, CD163, RAC1 , RAC2, MRC1 , EGR1 , SELENOP (Selenoprotein).

14. The method of any one of claims 1-13, wherein the fibrotic condition is dermal fibrosis, hypertrophic scarring, fibrosis from the foreign body response to biomedical implants, renal fibrosis, cardiac fibrosis, idiopathic pulmonary fibrosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, musculoskeletal fibrosis, or any other fibrotic condition.

15. A method of treating a fibrotic condition in a subject in need thereof, the method comprising administering a therapeutic amount of a composition that inhibits one or both of i) circulating cells or ii) myeloid mechanical signaling.

16. A method of treating a fibrotic condition in a subject in need thereof, the method comprising inhibiting one or both of i) circulating cells or ii) myeloid mechanical signaling.

17. The method of claim 15 or claim 16, wherein the circulating cells comprise mesenchymal cells or hematopoietic cells.

18. The method of claim 15 or claim 16, wherein the myeloid cells are derived from hematopoietic precursors.

19. The method of claim 18, wherein the myeloid cells comprise monocytes, macrophages, neutrophils, dendritic cells, or a combination thereof.

20. The method of any one of claims 15-19, wherein the composition comprises small molecules, drug therapy, cytokines, miRNA, antibodies, peptides, or a combination thereof.

21. The method of claim 20, wherein the small molecule comprises a small molecule focal adhesion kinase inhibitor (FAKI) or any other inhibitor or activator that modulates mechanical signaling.

22. The method of any one of claims 15-21 , wherein inhibiting one or both of i) circulating cells of any origin or ii) mechanical signaling in myeloid cells comprises utilizing a cell therapy, gene editing, or a combination thereof.

23. The method of any of claims 15-21 , wherein inhibiting one or both of i) circulating cells or ii) mechanical signaling in myeloid cells comprises depleting said cells.

24. The method of claim 23, wherein plasmaphoresis is used to deplete the cells or wherein a CAR-T cell or other cell type is used to target and deplete said cells.

25. The method of any of claims 15-24, wherein inhibiting circulating cells comprises modulating TWEAK (TNF-related weak inducer of apoptosis), MIF (macrophage migration inhibitory factor), CXCL (C-X-C motif chemokines), PTN (pleiotrophin) signaling, THBS1 / THBS2 / THBS4 (Thrombospondins), CD86, CD163, RAC1 , RAC2, MRC1 , EGR1 , SELENOP (Selenoprotein).

26. The method of any one of claims 15-25, wherein the fibrotic condition is dermal fibrosis, hypertrophic scarring, fibrosis from the foreign body response to biomedical implants, renal fibrosis, cardiac fibrosis, idiopathic pulmonary fibrosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, musculoskeletal fibrosis, or any other fibrotic condition.

27. A method of treating cancer in a subject in need thereof, the method comprising administering a therapeutic amount of a composition that augments one or both of i) circulating cells or ii) myeloid mechanical signaling.

28. A method of treating cancer in a subject in need thereof, the method comprising administering augmenting one or both of i) circulating cells or ii) myeloid mechanical signaling.

29. The method of claim 27 or claim 28, wherein the circulating cells comprise mesenchymal cells or hematopoietic cells.

30. The method of claim 27 or claim 28, wherein the myeloid cells are derived from hematopoietic precursors.

31. The method of claim 30, wherein the myeloid cells comprise monocytes, macrophages, neutrophils, dendritic cells, or a combination thereof.

32. The method of any one of claims 27-31 , wherein the composition comprises small molecules, drug therapy, cytokines, miRNA, antibodies, peptides, or a combination thereof.

33. The method of claim 32, wherein the small molecule comprises a small molecule focal adhesion kinase inhibitor (FAKI) or any other inhibitor or activator that modulates mechanical signaling.

34. The method of any one of claims 27-33, wherein augmenting one or both of i) circulating cells of any origin or ii) mechanical signaling in myeloid cells comprises utilizing a cell therapy, gene editing, or a combination thereof.

35. The method of any of claims 27-34, wherein augmenting the circulating cells comprises modulating TWEAK (TNF-related weak inducer of apoptosis), MIF (macrophage migration inhibitory factor), CXCL (C-X-C motif chemokines), PTN (pleiotrophin) signaling, THBS1 / THBS2 / THBS4 (Thrombospondins), CD86, CD163, RAC1 , RAC2, MRC1 , EGR1 , SELENOP (Selenoprotein)36. A method of increasing wound healing in a subject in need thereof, the method comprising administering a therapeutic amount of a composition that modulates one or both of i) circulating cells or ii) mechanical signaling in myeloid cells.

37. A method of increasing wound healing in a subject in need thereof, the method comprising modulating one or both of i) circulating cells or ii) mechanical signaling in myeloid cells.

38. The method of claim 36 or claim 37, wherein the circulating cells comprise mesenchymal cells or hematopoietic cells.

39. The method of claim 36 or claim 37, wherein the myeloid cells are derived from hematopoietic precursors.

40. The method of claim 39, wherein the myeloid cells comprise monocytes, macrophages, neutrophils, dendritic cells, or a combination thereof.

41. The method of any one of claims 36-40, wherein the composition comprises small molecules, drug therapy, cytokines, miRNA, antibodies, peptides, or a combination thereof.

42. The method of claim 41 , wherein the small molecule comprises a small molecule focal adhesion kinase inhibitor (FAKI) or any other inhibitor or activator that modulates mechanical signaling.

43. The method of any one of claims 36-42, wherein modulating one or both of i) circulating cells of any origin or ii) mechanical signaling in myeloid cells comprises utilizing a cell therapy, gene editing, or a combination thereof.

44. The method of any of claims 36-43, wherein the composition inhibits one or both of i) circulating cells or ii) myeloid mechanical signaling.

45. The method of any of claims 36-43, wherein the composition augments one or both of i) circulating cells or ii) myeloid mechanical signaling.

46. The method of any of claims 36-45, wherein modulating one or both of i) circulating cells or ii) mechanical signaling in myeloid cells comprises depleting said cells.

47. The method of claim 46, wherein plasmaphoresis is used to deplete the cells or wherein a CAR-T cell or other cell type is used to target and deplete said cells.

48. The method of any of claims 36-47, wherein modulating circulating cells comprises modulating TWEAK (TNF-related weak inducer of apoptosis), MIF (macrophage migration inhibitory factor), CXCL (C-X-C motif chemokines), PTN (pleiotrophin) signaling, THBS1 / THBS2 / THBS4 (Thrombospondins), CD86, CD163, RAC1 , RAC2, MRC1 , EGR1 , SELENOP (Selenoprotein).

49. A method of reducing circulating cells at a site of wound healing, the method comprising modulating TWEAK (TNF-related weak inducer of apoptosis), MIF (macrophage migration inhibitory factor), CXCL (C-X-C motif chemokines), PTN (pleiotrophin) signaling, THBS1 / THBS2 / THBS4 (Thrombospondins), CD86, CD163, RAC1 , RAC2, MRC1 , EGR1 , SELENOP (Selenoprotein) at the site of wound healing.

50. A method of treating a fibrotic condition or cancer in a subject in need thereof, the method comprising modulating circulating cells.

51. The method of claim 50, wherein the circulating cells comprise mechanoresponsive immune cells.

52. The method of claim 50 or claim 51 , wherein the circulating cells comprise circulating mesenchymal cells.

53. A composition for use in the method of treating a fibrotic condition or cancer, wherein the composition modulates one or both of i) circulating cells of any origin or ii) mechanical signaling in myeloid cells.

54. The composition of claim 53, wherein the circulating cells comprise mesenchymal cells or hematopoietic cells.

55. The composition of claim 53, wherein the myeloid cells are derived from hematopoietic precursors.

56. The composition of claim 55, wherein the myeloid cells comprise monocytes, macrophages, neutrophils, dendritic cells, or a combination thereof.

57. The composition of any one of claims 53-56, wherein the composition comprises small molecules, drug therapy, cytokines, miRNA, antibodies, peptides, or a combination thereof.

58. The composition of claim 57, wherein the small molecule comprises a small molecule focal adhesion kinase inhibitor (FAKI) or any other inhibitor or activator that modulates mechanical signaling.

59. The composition of any one of claims 53-56, wherein modulating one or both of i) circulating cells of any origin or ii) mechanical signaling in myeloid cells comprises utilizing a cell therapy, gene editing, or a combination thereof.

60. The composition of any of claims 53-59, wherein the composition inhibits one or both of i) circulating cells or ii) myeloid mechanical signaling.

61. The composition of any of claims 53-59, wherein the composition augments one or both of i) circulating cells or ii) myeloid mechanical signaling.

62. The composition of any of claims 53-61 , wherein modulating one or both of i) circulating cells or ii) mechanical signaling in myeloid cells comprises depleting said cells.

63. The composition of claim 62, wherein plasmaphoresis is used to deplete the cells or wherein a CAR-T cell or other cell type is used to target and deplete said cells.

64. The composition of any of claims 53-63, wherein modulating circulating cells comprises modulating TWEAK (TNF-related weak inducer of apoptosis), MIF (macrophage migration inhibitory factor), CXCL (C-X-C motif chemokines), PTN (pleiotrophin) signaling, THBS1 / THBS2 / THBS4 (Thrombospondins), CD86, CD163, RAC1 , RAC2, MRC1 , EGR1 ,SELENOP (Selenoprotein).

65. The composition of any one of claims 53-64, wherein the fibrotic condition is dermal fibrosis, hypertrophic scarring, fibrosis from the foreign body response to biomedical implants, renal fibrosis, cardiac fibrosis, idiopathic pulmonary fibrosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, musculoskeletal fibrosis, or any other fibrotic condition.

66. A composition for use in the method of treating a fibrotic condition, wherein the composition inhibits one or both of i) circulating cells of any origin or ii) mechanical signaling in myeloid cells.

67. The composition of claim 66 wherein the circulating cells comprise mesenchymal cells or hematopoietic cells.

68. The composition of claim 66, wherein the myeloid cells are derived from hematopoietic precursors.

69. The composition of claim 68, wherein the myeloid cells comprise monocytes, macrophages, neutrophils, dendritic cells, or a combination thereof.

70. The composition of any one of claims 66-69, wherein the composition comprises small molecules, drug therapy, cytokines, miRNA, antibodies, peptides, or a combination thereof.

71. The composition of claim 70, wherein the small molecule comprises a small molecule focal adhesion kinase inhibitor (FAKI) or any other inhibitor or activator that modulates mechanical signaling.

72. The composition of any one of claims 66-71 , wherein inhibiting one or both of i) circulating cells of any origin or ii) mechanical signaling in myeloid cells comprises utilizing a cell therapy, gene editing, or a combination thereof.

73. The composition of any of claims 66-72, wherein inhibiting one or both of i) circulating cells or ii) mechanical signaling in myeloid cells comprises depleting said cells.

74. The composition of claim 73, wherein plasmaphoresis is used to deplete the cells or wherein a CAR-T cell or other cell type is used to target and deplete said cells.

75. The composition of any of claims 66-74, wherein inhibiting circulating cells comprises modulating TWEAK (TNF-related weak inducer of apoptosis), MIF (macrophage migration inhibitory factor), CXCL (C-X-C motif chemokines), PTN (pleiotrophin) signaling, THBS1 / THBS2 / THBS4 (Thrombospondins), CD86, CD163, RAC1 , RAC2, MRC1 , EGR1 , SELENOP (Selenoprotein).

76. The composition of any one of claims 66-75, wherein the fibrotic condition is dermal fibrosis, hypertrophic scarring, fibrosis from the foreign body response to biomedical implants, renal fibrosis, cardiac fibrosis, idiopathic pulmonary fibrosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, musculoskeletal fibrosis, or any other fibrotic condition.

77. A composition for use in the method of treating cancer, wherein the composition augments one or both of i) circulating cells of any origin or ii) mechanical signaling in myeloid cells.

78. The composition of claim 77, wherein the circulating cells comprise mesenchymal cells or hematopoietic cells.

79. The composition of claim 77, wherein the myeloid cells are derived from hematopoietic precursors.

80. The composition of claim 79, wherein the myeloid cells comprise monocytes, macrophages,neutrophils, dendritic cells, or a combination thereof.

81. The composition of any one of claims 77-80, wherein the composition comprises small molecules, drug therapy, cytokines, miRNA, antibodies, peptides, or a combination thereof.

82. The composition of claim 81 , wherein the small molecule comprises a small molecule focal adhesion kinase inhibitor (FAKI) or any other inhibitor or activator that modulates mechanical signaling.

83. The composition of any one of claims 77-82, wherein augmenting one or both of i) circulating cells of any origin or ii) mechanical signaling in myeloid cells comprises utilizing a cell therapy, gene editing, or a combination thereof.

84. The composition of any of claims 77-83, wherein augmenting one or both of i) circulating cells or ii) mechanical signaling in myeloid cells comprises depleting said cells.

85. The composition of claim 84, wherein plasmaphoresis is used to deplete the cells or wherein a CAR-T cell or other cell type is used to target and deplete said cells.

86. The composition of any of claims 77-85, wherein augmenting circulating cells comprises modulating TWEAK (TNF-related weak inducer of apoptosis), MIF (macrophage migration inhibitory factor), CXCL (C-X-C motif chemokines), PTN (pleiotrophin) signaling, THBS1 / THBS2 / THBS4 (Thrombospondins), CD86, CD163, RAC1 , RAC2, MRC1 , EGR1 , SELENOP (Selenoprotein).

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