Inflammation can induce mutagenic DNA damage to enhance cancer risk and progression. Inflammation also increases fibrosis and stromal stiffening that promotes malignancy, and tissues with higher cancer risk are often stiffer. Despite this connection, how stromal stiffness contributes to inflammatory-mediated DNA damage in tumorigenesis remains unclear. Here, we show that tissue tension engages macrophages to generate lipid peroxidation-induced DNA damage, contributing to mutational burden that may promote malignant progression. We identify that fibrotic breast tumors display higher mutational burdens. Mechanistically, tissue tension increases epithelial STAT3 to drive chemokine-mediated macrophage recruitment. Stiffness promotes reactive oxygen species-induced lipid peroxidation in recruited macrophages, generating aldehydes that damage DNA and enhance progression. Notably, high mammographically dense breast tissues—associated with increased cancer risk—are stiffer and inflamed and display elevated lipid aldehydes and DNA damage. This work links fibrosis and inflammation to tension-mediated cancer initiation and progression.

Tissue stem -progenitor cell frequency has been implicated in tumor risk and progression, but tissue -specific factors linking these associations remain ill-defined. We observed that stiff breast tissue from women with high mammographic density, who exhibit increased lifetime risk for breast cancer, associates with abundant stem -progenitor epithelial cells. Using genetically engineered mouse models of elevated integrin mechanosignaling and collagen density, syngeneic manipulations, and spheroid models, we determined that a stiff matrix and high mechanosignaling increase mammary epithelial stem -progenitor cell frequency and enhance tumor initiation in vivo. Augmented tissue mechanics expand stemness by potentiating extracellular signalrelated kinase (ERK) activity to foster progesterone receptor -dependent RANK signaling. Consistently, we detected elevated phosphorylated ERK and progesterone receptors and increased levels of RANK signaling in stiff breast tissue from women with high mammographic density. The findings link fibrosis and mechanosignaling to stem -progenitor cell frequency and breast cancer risk and causally implicate epidermal growth factor receptor-ERK-dependent hormone signaling in this phenotype.

Tumor progression is accompanied by fibrosis, a condition of excessive extracellular matrix accumulation, which is associated with diminished antitumor immune infiltration. Here we demonstrate that tumor-associated macrophages (TAMs) respond to the stiffened fibrotic tumor microenvironment (TME) by initiating a collagen biosynthesis program directed by transforming growth factor-β. A collateral effect of this programming is an untenable metabolic milieu for productive CD8+ T cell antitumor responses, as collagen-synthesizing macrophages consume environmental arginine, synthesize proline and secrete ornithine that compromises CD8+ T cell function in female breast cancer. Thus, a stiff and fibrotic TME may impede antitumor immunity not only by direct physical exclusion of CD8+ T cells but also through secondary effects of a mechano-metabolic programming of TAMs, which creates an inhospitable metabolic milieu for CD8+ T cells to respond to anticancer immunotherapies.

Intratumor heterogeneity associates with poor patient outcome. Stromal stiffening also accompanies cancer. Whether cancers demonstrate stiffness heterogeneity, and if this is linked to tumor cell heterogeneity remains unclear. We developed a method to measure the stiffness heterogeneity in human breast tumors that quantifies the stromal stiffness each cell experiences and permits visual registration with biomarkers of tumor progression. We present Spatially Transformed Inferential Force Map (STIFMap) which exploits computer vision to precisely automate atomic force microscopy (AFM) indentation combined with a trained convolutional neural network to predict stromal elasticity with micron-resolution using collagen morphological features and ground truth AFM data. We registered high-elasticity regions within human breast tumors colocalizing with markers of mechanical activation and an epithelial-to-mesenchymal transition (EMT). The findings highlight the utility of STIFMap to assess mechanical heterogeneity of human tumors across length scales from single cells to whole tissues and implicates stromal stiffness in tumor cell heterogeneity.

The construction of three-dimensional (3D) microvascular networks with defined structures remains challenging. Emerging bioprinting strategies provide a means of patterning endothelial cells (ECs) into the geometry of 3D microvascular networks, but the microenvironmental cues necessary to promote their self-organization into cohesive and perfusable microvessels are not well known. To this end, we reconstituted microvessel formation in vitro by patterning thin lines of closely packed ECs fully embedded within a 3D extracellular matrix (ECM) and observed how different microenvironmental parameters influenced EC behaviors and their self-organization into microvessels. We found that the inclusion of fibrillar matrices, such as collagen I, into the ECM positively influenced cell condensation into extended geometries such as cords. We also identified the presence of a high-molecular-weight protein(s) in fetal bovine serum that negatively influenced EC condensation. This component destabilized cord structure by promoting cell protrusions and destabilizing cell-cell adhesions. Endothelial cords cultured in the presence of fibrillar collagen and in the absence of this protein activity were able to polarize, lumenize, incorporate mural cells, and support fluid flow. These optimized conditions allowed for the construction of branched and perfusable microvascular networks directly from patterned cells in as little as 3 days. These findings reveal important design principles for future microvascular engineering efforts based on bioprinting and micropatterning techniques. Impact statementBioprinting is a potential strategy to achieve microvascularization in engineered tissues. However, the controlled self-organization of patterned endothelial cells into perfusable microvasculature remains challenging. We used DNA Programmed Assembly of Cells to create cell-dense, capillary-sized cords of endothelial cells with complete control over their structure. We optimized the matrix and media conditions to promote self-organization and maturation of these endothelial cords into stable and perfusable microvascular networks.

Patient-derived organoids and cellular spheroids recapitulate tissue physiology with remarkable fidelity. We investigated how engagement with a reconstituted basement membrane in three dimensions (3D) supports the polarized, stress resilient tissue phenotype of mammary epithelial spheroids. Cells interacting with reconstituted basement membrane in 3D had reduced levels of total and actin-associated filamin and decreased cortical actin tension that increased plasma membrane protrusions to promote negative plasma membrane curvature and plasma membrane protein associations linked to protein secretion. By contrast, cells engaging a reconstituted basement membrane in 2D had high cortical actin tension that forced filamin unfolding and endoplasmic reticulum (ER) associations. Enhanced filamin-ER interactions increased levels of PKR-like ER kinase effectors and ER-plasma membrane contact sites that compromised calcium homeostasis and diminished cell viability. Consequently, cells with decreased cortical actin tension had reduced ER stress and survived better. Consistently, cortical actin tension in cellular spheroids regulated polarized basement membrane membrane deposition and sensitivity to exogenous stress. The findings implicate cortical actin tension-mediated filamin unfolding in ER function and underscore the importance of tissue mechanics in organoid homeostasis.

Abstract Women with aggressive DCIS are at high risk of progression to invasive breast cancer. Invasive disease is accompanied by extensive collagen remodeling and crosslinking that contributes to tissue fibrosis and stiffness. Fibrotic breast tumors are associated with an immune desert or immune excluded tumor microenvironment (TME) that functions to promote tumor progression. In particular, tumor-associated macrophages (TAMs) promote tumor invasion and metastasis and, drive immune suppression allowing tumors to evade intrinsic anti-tumor immune responses. Here, we show that collagen remodeling and crosslinking and tissue stiffness associate with DCIS progression and recurrence to invasion in patient tissues. The fibrotic and stiff TME promotes DCIS progression in a mouse model in part by inducing a suppressive TAM phenotype characterized by epigenetic and metabolic reprogramming that accompanies an impaired pro-inflammatory gene signature, as seen in tolerant macrophages. TAMs can be polarized by toll-like receptor (TLR) agonists to eliminate tumor cells by producing pro-inflammatory cytokines or by stimulating cytotoxic T cells, though these are poor cancer therapies alone. Thus, we hypothesized to improve the TLR agonists anti-tumor effects, we can reprogram the metabolic pathways associated with macrophage tolerance, and these together may control DCIS progression in fibrotic and stiff TMEs. We show that fibrotic TAMs can be reprogrammed with monophosphoryl lipid A (MPLA) and coenzyme A (CoA) to kill tumor cells. MPLA + CoA injected into the mammary fat pad reduces high risk features of DCIS in this breast cancer mouse model. We show that CoA supplementation alters macrophage metabolism by supporting acetyl-CoA production needed for histone acetylation and pro-inflammatory gene transcription. MPLA + CoA reprograms CD206+ TAMs to inducible NO synthase (iNOS)+ macrophages and activates cytotoxic T cells through macrophage-secreted cytokines. This treatment approach for engaging an anti-tumor immune response represents an alternative treatment to that of current invasive surgery options for women with high-risk DCIS. Citation Format: Mary-Kate Hayward, Greg A. Timblin, Jason J. Northey, Connor Stashko, Alastair J. Ironside, E. Shelley Hwang, Valerie M. Weaver. Reprogramming of fibrotic macrophages to control DCIS progression [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr B003.

Tissue stem-progenitor cell frequency has been implicated in tumor risk and progression. Tissue-specific factors linking stem-progenitor cell frequency to cancer risk and progression remain ill defined. Using a genetically engineered mouse model that promotes integrin mechanosignaling with syngeneic manipulations, spheroid models, and patient-derived xenografts we determined that a stiff extracellular matrix and high integrin mechanosignaling increase stem-progenitor cell frequency to enhance breast tumor risk and progression. Studies revealed that high integrin-mechanosignaling expands breast epithelial stem-progenitor cell number by potentiating progesterone receptor-dependent RANK signaling. Consistently, we observed that the stiff breast tissue from women with high mammographic density, who exhibit an increased lifetime risk for breast cancer, also have elevated RANK signaling and a high frequency of stem-progenitor epithelial cells. The findings link tissue fibrosis and integrin mechanosignaling to stem-progenitor cell frequency and causally implicate hormone signaling in this phenotype. Accordingly, inhibiting RANK signaling could temper the tumor promoting impact of fibrosis on breast cancer and reduce the elevated breast cancer risk exhibited by women with high mammographic density. Citation Format: Jason J. Northey, Yoshihiro Yui, Mary-Kate Hayward, Connor Stashko, FuiBoon Kai, Janna Mouw, Dhruv Thakar, Jonathon Lakins, Alastair Ironside, Susan Samson, Rita Mukhtar, E. Shelley Hwang, Valerie Weaver. Mechanosensitive hormone signaling promotes mammary progenitor expansion and breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB021.

Tumors feature elevated sialoglycoprotein content. Sialoglycoproteins promote tumor progression and are linked to immune suppression via the sialic acid-Siglec axis. Understanding factors that increase sialoglycoprotein biosynthesis in tumors could identify approaches to improve patient response to immunotherapy. We quantified higher levels of sialoglycoproteins in the fibrotic regions within human breast tumor tissues. Human breast tumor subtypes, which are more fibrotic, similarly featured increased sialoglycoprotein content. Further analysis revealed the breast cancer cells as the primary cell type synthesizing and secreting the tumor tissue sialoglycoproteins and confirmed that the more aggressive, fibrotic breast cancer subtypes expressed the highest levels of sialoglycoprotein biosynthetic genes. The more aggressive breast cancer subtypes also featured greater infiltration of immunosuppressive SIGLEC7, SIGLEC9, and SIGLEC10-pos myeloid cells, indicating that triple-negative breast tumors had higher expression of both immunosuppressive Siglec receptors and their cognate ligands. The findings link sialoglycoprotein biosynthesis and secretion to tumor fibrosis and aggression in human breast tumors. The data suggest targeting of the sialic acid-Siglec axis may comprise an attractive therapeutic target particularly for the more aggressive HER2+ and triple-negative breast cancer subtypes.

The construction of three-dimensional (3D) microvascular networks with defined structures remains challenging. Emerging bioprinting strategies provide a means of patterning endothelial cells (ECs) into the geometry of 3D microvascular networks, but the microenvironmental cues necessary to promote their self-organization into cohesive and perfusable microvessels are unknown. To this end, we reconstituted microvessel formation in vitro by patterning thin lines of closely packed ECs fully embedded within a 3D extracellular matrix (ECM) and observed how different microenvironmental parameters influenced EC behaviors and their self-organization into microvessels. We found that the inclusion of fibrillar matrices, such as collagen I, into the ECM positively influenced cell condensation into extended geometries such as cords. We also identified the presence of a high molecular weight protein(s) in fetal bovine serum (FBS) that negatively influenced EC condensation. This component destabilized cord structure by promoting cell protrusions and destabilizing cell-cell adhesions. Endothelial cords cultured in the presence of fibrillar collagen and the absence of this protein activity were able to polarize, lumenize, incorporate mural cells, and support fluid flow. These optimized conditions allowed for the construction of branched and perfusable microvascular networks directly from patterned cells in as little as three days. These findings reveal important design principles for future microvascular engineering efforts based on bioprinting techniques.

Tumor progression is accompanied by fibrosis, which is associated with diminished anti-tumor immune infiltrate. Here, we demonstrate that tumor infiltrating myeloid cells respond to the stiffened fibrotic tumor microenvironment (TME) by initiating a TGF-beta (TGFβ)-directed, collagen biosynthesis program. A collateral effect of this programming is an untenable metabolic milieu for productive CD8 T cell anti-tumor responses, as collagen-synthesizing macrophages consume environmental arginine, synthesize proline, and secrete ornithine that compromises CD8+ T cell function. Thus, a stiff and fibrotic TME may impede anti-tumor immunity not only by direct physical exclusion of CD8+ T cells, but also via secondary effects of a myeloid mechano-metabolic programming we identified that creates an inhospitable metabolic milieu for CD8+ T cells.

Abstract Intratumor heterogeneity in breast cancer associates with poor patient outcome. Tissue fibrosis and stromal stiffening accompany breast cancer development and associate with the aggressiveness of human breast cancer subtypes. Whether human breast cancers demonstrate stiffness heterogeneity, and if this is linked to breast tumor aggression remains unclear. To answer these questions, we developed a spatial method to measure the stiffness heterogeneity in human breast tumor tissues that also quantifies the local stromal stiffness each cell experiences and permits correlation with biomarkers of tumor aggression. Here, we present Spatially Transformed Inferential Force Maps (STIFMaps) to predict the elasticity across whole tissue sections with micron-resolution. The method exploits computer vision to precisely automate AFM indentation and then uses a trained convolutional neural network to predict matrix elasticity using collagen morphological features and ground truth AFM data. Because STIFMaps is compatible with biomarker staining we used the approach to register high-elasticity regions within sections of human breast tumors with markers of mechanical activation and an epithelial to mesenchymal transition (EMT) that associated with tumor aggression. The findings herein highlight the utility of STIFMaps for assessing the mechanical heterogeneity of human breast tissues across length scales from single cells to whole tissues. The method also reveals, for the first time, a direct association between stromal stiffness and EMT, thereby implicating stromal stiffness as a driver of human breast cancer aggression.

Mitochondria control eukaryotic cell fate by producing the energy needed to support life and the signals required to execute programed cell death. The biochemical milieu is known to affect mitochondrial function and contribute to the dysfunctional mitochondrial phenotypes implicated in cancer and the morbidities of aging. However, the physical characteristics of the extracellular matrix are also altered in cancerous and aging tissues. Here, we demonstrate that cells sense the physical properties of the extracellular matrix and activate a mitochondrial stress response that adaptively tunes mitochondrial function via solute carrier family 9 member A1-dependent ion exchange and heat shock factor 1-dependent transcription. Overall, our data indicate that adhesion-mediated mechanosignaling may play an unappreciated role in the altered mitochondrial functions observed in aging and cancer.

Autoimmune-mediated dry eye disease is a pathological feature of multiple disorders including Sjögren's syndrome, lupus and rheumatoid arthritis that has a life-long, detrimental impact on vision and overall quality of life. Although late stage disease outcomes such as epithelial barrier dysfunction, reduced corneal innervation and chronic inflammation have been well characterized in both human patients and mouse models, there is little to no understanding of early pathological processes. Moreover, the mechanisms underlying the loss of cornea homeostasis and disease progression are unknown. Here, we utilize the autoimmune regulatory (Aire)-deficient mouse model of autoimmune-mediated dry eye disease in combination with genome wide transcriptomics, high-resolution imaging and atomic force microscopy to reveal a potential extracellular matrix (ECM)-biomechanical-based mechanism driving cellular and morphological changes at early disease onset. Early disease in the Aire-deficient mouse model is associated with a mild reduction in tear production and moderate immune cell infiltration, allowing for interrogation of cellular, molecular and biomechanical changes largely independent of chronic inflammation. Using these tools, we demonstrate for the first time that the emergence of autoimmune-mediated dry eye disease is associated with an alteration in the biomechanical properties of the cornea. We reveal a dramatic disruption of the synthesis and organization of the extracellular matrix as well as degradation of the epithelial basement membrane during early disease. Notably, we provide evidence that the nerve supply to the cornea is severely reduced at early disease stages and that this is independent of basement membrane destruction or significant immune cell infiltration. Furthermore, diseased corneas display spatial heterogeneity in mechanical, structural and compositional changes, with the limbal compartment often exhibiting the opposite response compared to the central cornea. Despite these differences, however, epithelial hyperplasia is apparent in both compartments, possibly driven by increased activation of IL-1R1 and YAP signaling pathways. Thus, we reveal novel perturbations in corneal biomechanics, matrix organization and cell behavior during the early phase of dry eye that may underlie disease development and progression, presenting new potential targets for therapeutic intervention.

Desmoplasia describes the deposition of extensive extracellular matrix and defines primary pancreatic ductal adenocarcinoma (PDA). The acellular component of this stroma has been implicated in PM pathogenesis and is being targeted therapeutically in clinical trials. By analyzing the stromal content of PDA samples from numerous annotated PDA data sets and correlating stromal content with both anatomic site and clinical outcome, we found MA metastases in the liver, the primary cause of mortality to have less stroma, have higher tumor cellularity than primary tumors. Experimentally manipulating stromal matrix with an anti-lysyl oxidase like-2 (anti-LOXL2) antibody in syngeneic orthotopic PDA mouse models significantly decreased matrix content, led to lower tissue stiffness, lower contrast retention on computed tomography, and accelerated tumor growth, resulting in diminished overall survival. These studies suggest an important protective role of stroma in MA and urge caution in clinically deploying stromal depletion strategies.