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Tumors are fibrotic and characterized by abundant, remodeled, and cross-linked collagen that stiffens the extracellular matrix stroma. The stiffened collagenous stroma fosters malignant transformation of the tissue by increasing tumor cell tension to promote focal adhesion formation and potentiate growth factor receptor signaling through kinase. Importantly, collagen cross-linking requires fibronectin (FN). Fibrotic tumors contain abundant FN, and tumor cells frequently up-regulate the FN receptor α5β1 integrin. Using transgenic and xenograft models and tunable two- and three-dimensional substrates, we show that FN-bound α5β1 integrin promotes tension-dependent malignant transformation through engagement of the synergy site that enhances integrin adhesion force. We determined that ligation of the synergy site of FN permits tumor cells to engage a zyxin-stabilized, vinculin-linked scaffold that facilitates nucleation of phosphatidylinositol (3,4,5)-triphosphate at the plasma membrane to enhance phosphoinositide 3-kinase (PI3K)-dependent tumor cell invasion. The data explain why rigid collagen fibrils potentiate PI3K activation to promote malignancy and offer a perspective regarding the consistent up-regulation of α5β1 integrin and FN in many tumors and their correlation with cancer aggression.

The way cells are organized within a tissue dictates how they sense and respond to extracellular signals, as cues are received and interpreted based on expression and organization of receptors, downstream signaling proteins, and transcription factors. Part of this microenvironmental context is the result of forces acting on the cell, including forces from other cells or from the cellular substrate or basement membrane. However, measuring forces exerted on and by cells is difficult, particularly in an in vivo context, and interpreting how forces affect downstream cellular processes poses an even greater challenge. Here, we present a simple method for monitoring and analyzing forces generated from cell collectives. We demonstrate the ability to generate traction force data from human embryonic stem cells grown in large organized epithelial sheets to determine the magnitude and organization of cell-ECM and cell-cell forces within a self-renewing colony. We show that this method can be used to measure forces in a dynamic hESC system and demonstrate the ability to map intracolony protein localization to force organization.

Tumors are stiff and data suggest that the extracellular matrix stiffening that correlates with experimental mammary malignancy drives tumor invasion and metastasis. Nevertheless, the relationship between tissue and extracellular matrix stiffness and human breast cancer progression and aggression remains unclear. We undertook a biophysical and biochemical assessment of stromal-epithelial interactions in noninvasive, invasive and normal adjacent human breast tissue and in breast cancers of increasingly aggressive subtype. Our analysis revealed that human breast cancer transformation is accompanied by an incremental increase in collagen deposition and a progressive linearization and thickening of interstitial collagen. The linearization of collagen was visualized as an overall increase in tissue birefringence and was most striking at the invasive front of the tumor where the stiffness of the stroma and cellular mechanosignaling were the highest. Amongst breast cancer subtypes we found that the stroma at the invasive region of the more aggressive Basal-like and Her2 tumor subtypes was the most heterogeneous and the stiffest when compared to the less aggressive luminal A and B subtypes. Intriguingly, we quantified the greatest number of infiltrating macrophages and the highest level of TGF beta signaling within the cells at the invasive front. We also established that stroma stiffness and the level of cellular TGF beta signaling positively correlated with each other and with the number of infiltrating tumor-activated macrophages, which was highest in the more aggressive tumor subtypes. These findings indicate that human breast cancer progression and aggression, collagen linearization and stromal stiffening are linked and implicate tissue inflammation and TGF beta.

Integrin expression and activity are altered in tumors, and aberrant integrin signaling promotes malignancy. However, how integrins become altered in tumors remains poorly understood. We discovered that oncogenic activation of MEK signaling induces cell growth and survival, and promotes the malignant phenotype of mammary epithelial cells (MECs) by increasing α5 integrin expression. We determined that MEK activates c-Myc to reduce the transcription of the SWI/SNF chromatin remodeling enzyme Brahma (BRM). Our studies revealed that reduced BRM expression and/or activity drives the malignant behavior of MECs by epigenetically promoting C/EBPβ expression to directly induce α5 integrin transcription. Consistently, we could show that restoring BRM levels normalized the malignant behavior of transformed MECs in culture and in vivo by preventing C/EBPβ-dependent α5 integrin transcription. Our findings identify a novel mechanism whereby oncogenic signaling promotes malignant transformation by regulating transcription of a key chromatin remodeling molecule that regulates integrin-dependent stromal-epithelial interactions.

Stromal-epithelial interactions modulate mammary epithelial cell (MEC) growth and apoptosis by influencing cell adhesion and tissue organization. Perturbations in the mammary stroma and cell adhesion characterize breast tumors and underlie the altered tissue organization, disrupted tissue homeostasis and enhanced survival phenotype of the disease. Apoptosis resistance likely arises during malignant transformation via genetic and epigenetic modification of cell adhesion pathways induced by a changing tissue microenvironment. Acquisition of adhesion-linked survival networks that enhance MEC viability in the absence of basement membrane interactions probably promote malignant transformation, and may render breast tumors sufficiently resistant to exogenous apoptotic stimuli to generate multidrug resistance.

Recent research suggests that uric acid may be nephrotoxic at lower levels than previously recognized and that it may be one mechanism for lead-related nephrotoxicity. Therefore, in understanding mechanisms for lead-related nephrotoxicity, it would be of value to determine whether genetic polymorphisms that are associated with renal outcomes in lead workers and/or modify associations between lead dose and renal function are also associated with uric acid and/or modify associations between lead dose and uric acid. We analyzed data on three such genetic polymorphisms: delta-aminolevulinic acid dehydratase (ALAD), endothelial nitric oxide synthase (eNOS), and the vitamin D receptor (VDR). Mean (+/- SD) tibia, blood, and dimercaptosuccinic acid-chelatable lead levels were 37.2 +/- 40.4 microg/g bone mineral, 32.0+/- 15.0 g/dL, and 0.77+/- 0.86 microg/mg creatinine, respectively, in 798 current and former lead workers. Participants with the eNOSAsp allele had lower mean serum uric acid compared with those with the Glu/Glu genotype. Among older workers (age > or = median of 40.6 years), ALAD genotype modified associations between lead dose and uric acid levels. Higher lead dose was significantly associated with higher uric acid in workers with the ALAD1-1 genotype; associations were in the opposite direction in participants with the variant ALAD1-2 genotype. In contrast, higher tibia lead was associated with higher uric acid in those with the variant VDRB allele; however, modification was dependent on participants with the bb genotype and high tibia lead levels. We conclude that genetic polymorphisms may modify uric acid mediation of lead-related adverse renal effects. [ABSTRACT FROM AUTHOR]

Recent research suggests that both uric acid and lead may be nephrotoxic at lower levels than previously recognized. We analyzed data from 803 current and former lead workers to determine whether lead biomarkers were associated with uric acid and whether previously reported associations between lead dose and renal outcomes were altered after adjustment for uric acid. Outcomes included uric acid, blood urea nitrogen, serum creatinine, measured and calculated creatinine clearances, and urinary N-acetyl-ß-d-glucosaminidase (NAG) and retinol-binding protein. Mean (+/- SD) uric acid, tibia lead, and blood lead levels were 4.8 +/- 1.2 mg/dL, 37.2 +/- 40.4 microg/g bone mineral, and 32.0 +/- 15.0 microg/dL, respectively. None of the lead measures (tibia, blood, and dimercaptosuccinic-acid-chelatable lead) was associated with uric acid, after adjustment for age, sex, body mass index, and alcohol use. However, when we examined effect modification by age on these relations, both blood and tibia lead were significantly associated (ß = 0.0111, p < 0.01 and ß = 0.0036, p = 0.04, respectively) in participants in the oldest age tertile. These associations decreased after adjustment for blood pressure and renal function, although blood lead remained significantly associated with uric acid (ß = 0.0156, p = 0.01) when the population was restricted to the oldest tertile of workers with serum creatinine greater than the median (0.86 mg/dL). Next, in models of renal function in all workers, uric acid was significantly (p < 0.05) associated with all renal outcomes except NAG. Finally, in the oldest tertile of workers, associations between lead dose and NAG were unchanged, but fewer associations between the lead biomarkers and the clinical renal outcomes remained significant (p

All multicellular systems produce and dynamically regulate extracellular matrices (ECMs) that play essential roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small-molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, non-perturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology., Competing Interests: Competing interests: A.F., G.Y., W.K. and K.P. are authors on a patent application filed by the Howard Hughes Medical Institute relating to this work (US Application Serial No. 63/637,164). The remaining authors declare no competing interests., (© 2025. The Author(s).)

Fibrosis accounts for approximately one-third of disease-related deaths globally. Current therapies fail to cure fibrosis, emphasizing the need to identify new antifibrotic approaches. Fibrosis is defined by the excessive accumulation of extracellular matrix (ECM) and resultant stiffening of tissue stroma. This stiffening appropriates actomyosin-mediated mechanical tension within cells to ultimately affect cell fate decisions and function. Recent studies demonstrate that subcellular organelles are physically connected to the actin cytoskeleton and sensitive to mechanoperturbations. These insights highlight mechanisms that may contribute to the chronic organelle stress in many fibrotic diseases, including those of the lung and liver. In this review, we discuss the hypothesis that a stiffened fibrotic ECM corrupts intracellular mechanical tension to compromise organelle homeostasis. We summarize potential therapeutics that could intervene in this mechanical dialog and that may have clinical benefit for resolving pathological organelle stress in fibrosis., Competing Interests: Declaration of interests None declared by authors., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Non-small cell lung cancers (NSCLC) harboring common mutations in EGFR and KRAS characteristically respond transiently to targeted therapies against those mutations, but invariably, tumors recur and progress. Resistance often emerges through mutations in the therapeutic target or activation of alternative signaling pathways. Mechanisms of acute tumor cell resistance to initial EGFR (EGFRi) or KRAS G12C (G12Ci) pathway inhibition remain poorly understood. Our study reveals that acute response to EGFR/RAS/RAF-pathway inhibition is spatial and culture context specific. In vivo, EGFR mutant tumor xenografts shrink by > 90% following acute EGFRi therapy, and residual tumor cells are associated with dense stroma and have increased nuclear YAP. Interestingly, in vitro EGFRi induced cell cycle arrest in NSCLC cells grown in monolayer, while 3D spheroids preferentially die upon inhibitor treatment. We find differential YAP nuclear localization and activity, driven by the distinct culture conditions, as a common resistance mechanism for selective EGFR/KRAS/BRAF pathway therapies. Forced expression of the YAP S127A mutant partially protects cells from EGFR-mediated cell death in spheroid culture. These studies identify YAP activation in monolayer culture as a non-genetic mechanism of acute EGFR/KRAS/BRAF therapy resistance, highlighting that monolayer vs spheroid cell culture systems can model distinct stages of patient cancer progression., Competing Interests: Conflict of interest The authors have declared that no conflict of interest exists.

Development and disease are regulated by the interplay between genetics and the signaling pathways stimulated by morphogens, growth factors, and cytokines. Experimental data highlight the importance of mechanical force in regulating embryonic development, tissue morphogenesis, and malignancy. Force not only sculpts tissue movements to drive embryogenesis and morphogenesis but also modifies the context of biochemical signaling and gene expression to regulate cell and tissue fate. Not surprisingly, experiments have demonstrated that perturbations in cell tension drive malignancy and metastasis by altering biochemical signaling and gene expression through modifications in cytoskeletal tension, transmembrane receptor structure and function, and organelle phenotype that enhance cell growth and survival, alter metabolism, and foster cell migration and invasion. At the tissue level, tumor-associated forces disrupt cell-cell adhesions to perturb tissue organization, compromise vascular integrity to induce hypoxia, and interfere with antitumor immunity to foster metastasis and treatment resistance. Exciting new approaches now exist with which to clarify the relationship between mechanotransduction, biochemical signaling, and gene expression in development and disease. Indeed, gaining insight into these interactions is essential to unravel molecular mechanisms that regulate development and clarify the molecular basis of cancer., (© 2025 Narain et al.; Published by Cold Spring Harbor Laboratory Press.)

In brain metastasis, cancer cells remain in close contact with the existing vasculature and can use vessels as migratory paths-a process known as vessel co-option. However, the mechanisms regulating this form of migration are poorly understood. Here we use ex vivo brain slices and an organotypic in vitro model for vessel co-option to show that cancer cell invasion along brain vasculature is driven by the difference in stiffness between vessels and the brain parenchyma. Imaging analysis indicated that cells move along the basal surface of vessels by adhering to the basement membrane extracellular matrix. We further show that vessel co-option is enhanced by both the stiffness of brain vasculature, which reinforces focal adhesions through a talin-dependent mechanism, and the softness of the surrounding environment that permits cellular movement. Our work reveals a mechanosensing mechanism that guides cell migration in response to the tissue's intrinsic mechanical heterogeneity, with implications in cancer invasion and metastasis., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The pleiotropic roles of nSMase2-generated ceramide include regulation of intracellular ceramide signaling and exosome biogenesis. We investigated the effects of eliminating nSMase2 on early and advanced PDA, including its influence on the microenvironment. Employing the KPC mouse model of pancreatic cancer, we demonstrate that pancreatic epithelial nSMase2 ablation reduces neoplasia and promotes a PDA subtype switch from aggressive basal-like to classical. nSMase2 elimination prolongs survival of KPC mice, hinders vasculature development, and fosters a robust immune response. nSMase2 loss leads to recruitment of cytotoxic T cells, N1-like neutrophils, and abundant infiltration of anti-tumorigenic macrophages in the pancreatic preneoplastic microenvironment. Mechanistically, we demonstrate that nSMase2-expressing PDA cell small extracellular vesicles (sEVs) reduce survival of KPC mice; PDA cell sEVs generated independently of nSMase2 prolong survival of KPC mice and reprogram macrophages to a proinflammatory phenotype. Collectively, our study highlights previously unappreciated opposing roles for exosomes, based on biogenesis pathway, during PDA progression.

A definitive understanding of the interplay between protein binding/migration and membrane curvature evolution is emerging but needs further study. The mechanisms defining such phenomena are critical to intracellular transport and trafficking of proteins. Among trafficking modalities, exosomes have drawn attention in cancer research as these nano-sized naturally occurring vehicles are implicated in intercellular communication in the tumor microenvironment, suppressing anti-tumor immunity and preparing the metastatic niche for progression. A significant question in the field is how the release and composition of tumor exosomes are regulated. In this perspective article, we explore how physical factors such as geometry and tissue mechanics regulate cell cortical tension to influence exosome production by co-opting the biophysics as well as the signaling dynamics of intracellular trafficking pathways and how these exosomes contribute to the suppression of anti-tumor immunity and promote metastasis. We describe a multiscale modeling approach whose impact goes beyond the fundamental investigation of specific cellular processes toward actual clinical translation. Exosomal mechanisms are critical to developing and approving liquid biopsy technologies, poised to transform future non-invasive, longitudinal profiling of evolving tumors and resistance to cancer therapies to bring us one step closer to the promise of personalized medicine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Tissue folds are structural motifs critical to organ function. In the intestine, bending of a flat epithelium into a periodic pattern of folds gives rise to villi, finger-like protrusions that enable nutrient absorption. However, the molecular and mechanical processes driving villus morphogenesis remain unclear. Here, we identify an active mechanical mechanism that simultaneously patterns and folds the intestinal epithelium to initiate villus formation. At the cellular level, we find that PDGFRA+ subepithelial mesenchymal cells generate myosin II-dependent forces sufficient to produce patterned curvature in neighboring tissue interfaces. This symmetry-breaking process requires altered cell and extracellular matrix interactions that are enabled by matrix metalloproteinase-mediated tissue fluidization. Computational models, together with in vitro and in vivo experiments, revealed that these cellular features manifest at the tissue level as differences in interfacial tensions that promote mesenchymal aggregation and interface bending through a process analogous to the active dewetting of a thin liquid film., Competing Interests: Declaration of interests Z.J.G. and C.S.M. hold patents related to the MULTI-seq barcoding method., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Brain metastatic breast cancer is particularly lethal largely due to therapeutic resistance. Almost half of the patients with metastatic HER2-positive breast cancer develop brain metastases, representing a major clinical challenge. We previously described that cancer-associated fibroblasts are an important source of resistance in primary tumors. Here, we report that breast cancer brain metastasis stromal cell interactions in 3D cocultures induce therapeutic resistance to HER2-targeting agents, particularly to the small molecule inhibitor of HER2/EGFR neratinib. We investigated the underlying mechanisms using a synthetic Notch reporter system enabling the sorting of cancer cells that directly interact with stromal cells. We identified mucins and bulky glycoprotein synthesis as top-up-regulated genes and pathways by comparing the gene expression and chromatin profiles of stroma-contact and no-contact cancer cells before and after neratinib treatment. Glycoprotein gene signatures were also enriched in human brain metastases compared to primary tumors. We confirmed increased glycocalyx surrounding cocultures by immunofluorescence and showed that mucinase treatment increased sensitivity to neratinib by enabling a more efficient inhibition of EGFR/HER2 signaling in cancer cells. Overexpression of truncated MUC1 lacking the intracellular domain as a model of increased glycocalyx-induced resistance to neratinib both in cell culture and in experimental brain metastases in immunodeficient mice. Our results highlight the importance of glycoproteins as a resistance mechanism to HER2-targeting therapies in breast cancer brain metastases., Competing Interests: Competing interests statement:K.P. serves on the Scientific Advisory Boards of Novartis, Ideaya Biosciences, and Scorpion Therapeutics, holds equity options in Scorpion Therapeutics and Ideaya Biosciences. L.E.S. is a current employee of Astra-Zeneca. C.R.B. is a cofounder and member of the Scientific Advisory Boards of Palleon Pharmaceuticals, Lycia Therapeutics, InterVenn Bio, OliLux Bio, Enable Biosciences, Redwood Bioscience (a subsidiary of Catalent), GanNA Bio, Firefly Bio, Neuravid and Valora Therapeutics.

All multicellular systems produce and dynamically regulate extracellular matrices (ECM) that play important roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, nonperturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology., Competing Interests: Competing Interests A patent application relating to this work has been filed by the Howard Hughes Medical Institute (internal reference 2024-017-01).

Targeted protein degradation is an emerging strategy for the elimination of classically undruggable proteins. Here, to expand the landscape of targetable substrates, we designed degraders that achieve substrate selectivity via recognition of a discrete peptide and glycan motif and achieve cell-type selectivity via antigen-driven cell-surface binding. We applied this approach to mucins, O-glycosylated proteins that drive cancer progression through biophysical and immunological mechanisms. Engineering of a bacterial mucin-selective protease yielded a variant for fusion to a cancer antigen-binding nanobody. The resulting conjugate selectively degraded mucins on cancer cells, promoted cell death in culture models of mucin-driven growth and survival, and reduced tumor growth in mouse models of breast cancer progression. This work establishes a blueprint for the development of biologics that degrade specific protein glycoforms on target cells., (© 2023. The Author(s).)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

In this chapter, we aim to bridge basic molecular and cellular principles surrounding membrane curvature generation with rewiring of cellular signals in cancer through multiscale models. We describe a general framework that integrates signaling with other cellular functions like trafficking, cell-cell and cell-matrix adhesion, and motility. The guiding question in our approach is: how does a physical change in cell membrane configuration caused by external stimuli (including those by the extracellular microenvironment) alter trafficking, signaling and subsequent cell fate? We answer this question by constructing a modeling framework based on stochastic spatial continuum models of cell membrane deformations. We apply this framework to explore the link between trafficking, signaling in the tumor microenvironment, and cell fate. At each stage, we aim to connect the results of our predictions with cellular experiments., (Copyright © 2024. Published by Elsevier Inc.)

Multiple respiratory hazards have been identified in the cannabis cultivation and production industry, in which occupational asthma and work-related exacerbation of preexisting asthma have been reported. An employee working in a Massachusetts cannabis cultivation and processing facility experienced progressively worsening work-associated respiratory symptoms, which culminated in a fatal asthma attack in January 2022. This report represents findings of an Occupational Safety and Health Administration inspection, which included a worksite exposure assessment, coworker and next-of-kin interviews, medical record reviews, and collaboration with the Massachusetts Department of Public Health. Respiratory tract or skin symptoms were reported by four of 10 coworkers with similar job duties. Prevention is best achieved through a multifaceted approach, including controlling asthmagen exposures, such as cannabis dust, providing worker training, and conducting medical monitoring for occupational allergy. Evaluation of workers with new-onset or worsening asthma is essential, along with prompt diagnosis and medical management, which might include cessation of work and workers' compensation when relation to work exposures is identified. It is important to recognize that work in cannabis production is potentially causative., Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. Jeremy T. Hua reports support from the Reuben M. Cherniack fellowship award at National Jewish Health. Wigdan Farah reports support from the Mayo Clinic. Trapper J. Braegger and Anne Hart report support from the Occupational Safety and Health Administration. James R. Laing reports support from the U.S. Department of Labor’s Bureau of Labor Statistics for documentation of workplace fatalities for the Census of Fatal Occupational Injuries, unrelated to the current work. No other potential conflicts of interest were disclosed.

Efforts to identify anti-cancer therapeutics and understand tumor-immune interactions are built with in vitro models that do not match the microenvironmental characteristics of human tissues. Using in vitro models which mimic the physical properties of healthy or cancerous tissues and a physiologically relevant culture medium, we demonstrate that the chemical and physical properties of the microenvironment regulate the composition and topology of the glycocalyx. Remarkably, we find that cancer and age-related changes in the physical properties of the microenvironment are sufficient to adjust immune surveillance via the topology of the glycocalyx, a previously unknown phenomenon observable only with a physiologically relevant culture medium., Competing Interests: Competing Interests J.R.C. is an inventor on an issued patent for Human Plasma-Like Medium (HPLM) assigned to the Whitehead Institute (Application number: PCT/US2017/061377. Patent number: 11453858. Issue date: 09/27/2022)

The role of morphogenetic forces in cell fate specification is an area of intense interest. Our prior studies suggested that the development of high cell-cell tension in human embryonic stem cells (hESC) colonies permits the Src-mediated phosphorylation of junctional β-catenin that accelerates its release to potentiate Wnt-dependent signaling critical for initiating mesoderm specification. Using an ectopically expressed nonphosphorylatable mutant of β-catenin (Y654F), we now provide direct evidence that impeding tension-dependent Src-mediated β-catenin phosphorylation impedes the expression of Brachyury (T) and the epithelial-to-mesenchymal transition (EMT) necessary for mesoderm specification. Addition of exogenous Wnt3a or inhibiting GSK3β activity rescued mesoderm expression, emphasizing the importance of force dependent Wnt signaling in regulating mechanomorphogenesis. Our work provides a framework for understanding tension-dependent β-catenin/Wnt signaling in the self-organization of tissues during developmental processes including gastrulation., Competing Interests: Declaration of interests The authors declare no competing interests.

Pre-metastatic niche formation is a critical step during the metastatic spread of cancer. One way by which primary tumors prime host cells at future metastatic sites is through the shedding of tumor-derived microparticles as a consequence of vascular sheer flow. However, it remains unclear how the uptake of such particles by resident immune cells affects their phenotype and function. Here, we show that ingestion of tumor-derived microparticles by macrophages induces a rapid metabolic and phenotypic switch that is characterized by enhanced mitochondrial mass and function, increased oxidative phosphorylation, and upregulation of adhesion molecules, resulting in reduced motility in the early metastatic lung. This reprogramming event is dependent on signaling through the mTORC1, but not the mTORC2, pathway and is induced by uptake of tumor-derived microparticles. Together, these data support a mechanism by which uptake of tumor-derived microparticles induces reprogramming of macrophages to shape their fate and function in the early metastatic lung., Competing Interests: Declaration of interests M.F.K. is a founder and shareholder in Foundery Innovations that prosecutes and develops novel immunotherapeutics, respectively., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2023. The Author(s).)

Tissue fibrosis and extracellular matrix (ECM) stiffening promote tumour progression. The mechanisms by which ECM regulates its contacting cells have been extensively studied. However, how stiffness influences intercellular communications in the microenvironment for tumour progression remains unknown. Here we report that stiff ECM stimulates the release of exosomes from cancer cells. We delineate a molecular pathway that links stiff ECM to activation of Akt, which in turn promotes GTP loading to Rab8 that drives exosome secretion. We further show that exosomes generated from cells grown on stiff ECM effectively promote tumour growth. Proteomic analysis revealed that the Notch signalling pathway is activated in cells treated with exosomes derived from tumour cells grown on stiff ECM, consistent with our gene expression analysis of liver tissues from patients. Our study reveals a molecular mechanism that regulates exosome secretion and provides insight into how mechanical properties of the ECM control the tumour microenvironment for tumour growth., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Breast cancer progression is accompanied by profound extracellular matrix (ECM) remodeling. A greater abundance of aligned fibrillar collagen is characteristic of invasive and aggressive breast cancers and has been associated with elevated activity of collagen crosslinking enzymes, such as lysyl oxidase (LOX) and lysyl hydroxylases (LH) and the formation of more mature collagen matrix crosslinks. Aligned collagen fibers can facilitate metastatic dissemination of tumor cells, and LOX inhibitors have been used to inhibit tumor progression and metastasis in experimental models. Thus, a better understanding of how matrix crosslinking alters tumor cell phenotypes, and behaviors would improve our ability to effectively treat aggressive metastatic breast cancer. Herein described is an experimental approach to glycate and crosslink a collagen-I/basement membrane extract ECM to study the impact of ECM crosslinking on mammary tumor progression in vivo. Moreover, glycation of collagen by sugars to form advanced glycation end products naturally occurs during aging, extending the potential relevance of this approach to research on mechanisms of aging involved in disease progression., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Matrix stiffness and dimensionality have been shown to be major determinants of cell behavior. However, a workflow for examining nanometer-scale responses of the associated molecular machinery is not available. Here, we describe a comprehensive, quantitative workflow that permits the analysis of cells responding to mechanical and dimensionality cues in their native state at nanometer scale by cryogenic electron tomography. Using this approach, we quantified distinct cytoskeletal nanoarchitectures and vesicle phenotypes induced in human mammary epithelial cells in response to stiffness and dimensionality of reconstituted basement membrane. Our workflow closely recapitulates the microenvironment associated with acinar morphogenesis and identified distinct differences in situ at nanometer scale. Using drug treatment, we showed that molecular events and nanometer-scale rearrangements triggered by engagement of apical cell receptors with reconstituted basement membrane correspond to changes induced by reduction of cortical tension. Our approach is fully adaptable to any kind of stiffness regime, extracellular matrix composition, and drug treatment.