ECM dimensionality tunes actin tension to modulate endoplasmic reticulum function and spheroid phenotypes of mammary epithelial cells.

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.

Alterations in corneal biomechanics underlie early stages of autoimmune-mediated dry eye disease.

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.

Mechanosensitive hormone signaling promotes mammary progenitor expansion and breast cancer risk.

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 signal-related 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-associated macrophages restrict CD8+ T cell function through collagen deposition and metabolic reprogramming of the breast cancer microenvironment.

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-ß. 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.

Programming the Self-Organization of Endothelial Cells into Perfusable Microvasculature.

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 statement Bioprinting 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.

A convolutional neural network STIFMap reveals associations between stromal stiffness and EMT in breast cancer.

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.

Immunosuppressive glycoproteins associate with breast tumor fibrosis and aggression.

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.

Adhesion-mediated mechanosignaling forces mitohormesis.

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.

Pancreatic ductal adenocarcinoma progression is restrained by stromal matrix.

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 PDA 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 PDA 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 PDA and urge caution in clinically deploying stromal depletion strategies.

Raman Spectroscopy for Rapid Evaluation of Surgical Margins during Breast Cancer Lumpectomy.

Failure to precisely distinguish malignant from healthy tissue has severe implications for breast cancer surgical outcomes. Clinical prognoses depend on precisely distinguishing healthy from malignant tissue during surgery. Laser Raman spectroscopy (LRS) has been previously shown to differentiate benign from malignant tissue in real time. However, the cost, assembly effort, and technical expertise needed for construction and implementation of the technique have prohibited widespread adoption. Recently, Raman spectrometers have been developed for non-medical uses and have become commercially available and affordable. Here we demonstrate that this current generation of Raman spectrometers can readily identify cancer in breast surgical specimens. We evaluated two commercially available, portable, near-infrared Raman systems operating at excitation wavelengths of either 785 nm or 1064 nm, collecting a total of 164 Raman spectra from cancerous, benign, and transitional regions of resected breast tissue from six patients undergoing mastectomy. The spectra were classified using standard multivariate statistical techniques. We identified a minimal set of spectral bands sufficient to reliably distinguish between healthy and malignant tissue using either the 1064 nm or 785 nm system. Our results indicate that current generation Raman spectrometers can be used as a rapid diagnostic technique distinguishing benign from malignant tissue during surgery.