Cancer is a heterogeneous systemic disease that is strongly influenced by dynamic interactions with the tumour microenvironment (TME). Despite major advances in understanding spatial and molecular tumour heterogeneity, the temporal dynamics of tumours have received far less attention. Growing evidence has linked circadian clocks to cancer risk, progression, and treatment response, including in breast cancer. However, temporal regulation has yet to be recognized as a cancer hallmark, and its interaction with the TME remains poorly understood. This review examines how circadian rhythms organize breast cancer biology through bidirectional interactions with the TME. Circadian clocks coordinate proliferation, DNA damage responses, metabolism, and immune surveillance. Ageing, chronic stress, and obesity, all of which are established breast cancer risk modifiers, disrupt these rhythms and are reciprocally exacerbated by circadian dysfunction, establishing feed-forward loops that accelerate disease. Within the TME, the extracellular matrix (ECM) plays a central role in mediating this bidirectional control. Stiffened fibrotic stroma dampens epithelial clock amplitude, while circadian rhythms in turn shape collagen turnover and ECM remodelling. These dynamics can foster inflammation, stem cell expansion, and metastatic dissemination, including time-of-day-dependent release of circulating breast tumour cells. Systemically, circadian clocks gate immune cell trafficking, creating predictable windows of immunosurveillance and therapeutic vulnerability. By integrating insights from mechanobiology, metabolism, immune regulation, and ageing, we position circadian timing as a unifying layer that connects cell-intrinsic programmes with the evolving breast TME. Understanding these connections opens new opportunities for chronotherapeutic strategies in which treatment timing is aligned with circadian rhythms to improve outcomes.

The extracellular matrix (ECM) is a critical regulator of cell behavior, with ECM stiffness serving as a key mechanical cue that governs cellular signaling, morphology, and fate. However, experimental investigation of stiffness-dependent cellular responses is often limited by the availability of reproducible, cost-effective, and accessible culture platforms with precisely tunable mechanical properties. This protocol describes polyacrylamide (PA)- and silicone-based methods for fabricating ECM substrates with tunable stiffness. The procedures outline substrate preparation, surface functionalization, and ECM protein conjugation to ensure consistent cell adhesion across a physiologically relevant stiffness range. PA-based substrates are compatible with downstream biochemical assays, including protein and RNA extraction, as well as high-resolution fluorescence imaging. Silicone-based substrates are optimized for total internal reflection fluorescence (TIRF) microscopy, enabling visualization of cell-ECM interactions at the basal membrane. In addition, soft substrates can be adapted to support spheroid cultures positioned within a consistent imaging plane, facilitating consistent image acquisition. These methods provide robust, cost-effective, and reproducible platforms for systematically probing how ECM stiffness regulates cellular processes, advancing mechanistic insight into cell-ECM crosstalk in both physiological and disease contexts.

The CDKN2A locus, which is frequently deleted in pancreatic ductal adenocarcinoma (PDAC), encodes two tumor suppressors, ARF and INK4A, that may influence tumorigenesis through distinct mechanisms. Distinguishing their individual contributions to cancer could help improve the understanding of PDAC pathogenesis and potentially uncover targetable vulnerabilities. Moreover, whereas ARF is known to enhance p53 function, defining its p53-independent activities could elucidate new processes that drive PDAC development. In this study, we sought to understand ARF function in PDAC suppression. Analysis of gene expression and mutational patterns in human PDAC TCGA data indicated that CDKN2AARF and CDKN2AINK4A are commonly both affected by point mutations and/or deletions, suggesting that their combined inactivation contributes to PDAC development. In genetically engineered mouse models, Arf inactivation accelerated KRASG12D-driven PDAC development, both in the presence and absence of Trp53, demonstrating that ARF is a PDAC-suppressor and can act in a p53-independent manner. Transcriptomic analyses of PDACs supported a p53-independent role for ARF, with ARF deficiency promoting extracellular matrix, collagen synthesis/assembly, and epithelial-mesenchymal transition gene expression programs. Accordingly, ARF-deficient PDACs displayed extensive remodeling of the tumor microenvironment (TME), associated with collagen deposition, increased tissue stiffness, and higher fibroblast content-hallmarks of aggressive and treatment-resistant PDAC stroma. Together, this study shows how ARF deficiency associated with CDKN2A inactivation sculpts the PDAC TME in a p53-independent fashion. Given the central role of the TME in PDAC progression and therapeutic resistance, these findings may provide insight critical for improving therapeutic interventions for PDAC. SIGNIFICANCE:ARF deficiency induced by CDKN2AARF alterations promotes remodeling of the pancreatic cancer microenvironment, which could provide a genotype-specific therapeutic vulnerability to improve outcomes of pancreatic cancer patients. See related commentary by Destefanis and Mulvaney, p. 3101.

Metastatic progression depends upon the ability of disseminated tumor cells to evade immune surveillance. MHC molecule expression facilitates T cell recognition and activation to permit the eradication of metastatic tumor cells. We identified nuclear corepressor 2 (NCOR2) as a key epigenetic regulator of MHC class I molecule expression on breast tumor cells. Patients with triple negative breast cancers (TNBC) that expressed high levels of NCOR2 also exhibited reduced metastasis free survival and decreased MHC class I expression, and the metastatic lesions in patients with TNBC had high nuclear NCOR2 and reduced CD8 T cell levels and activity. Genetically and experimentally reducing NCOR2 expression in tumor cells permitted interferon gamma upregulation of MHC class I, and potentiated CD8 T cell activity and induction of apoptosis to repress metastatic progression of disseminated breast cancer cells. These studies provide evidence to support NCOR2 as a targetable epigenetic regulator of metastasis towards which therapies could be developed to reduce patient mortality.

Altered glucose metabolism is an auspicious feature of solid tumors, but is it an intrinsic property of tumor cells or a metabolic adaptation to the tumor microenvironment? Using normal epithelial cells cultured in a physiological culture medium under conditions that mimic the physical properties of healthy or cancerous tissues, we establish multiomics relationships between the biochemical and physical properties of the microenvironment and its impact on biosynthetic outputs of altered glucose metabolism. We find that microenvironmental properties, such as hyperglycemia, can affect the composition and thickness of the epithelial glycocalyx, in part through the activity of mechanosensitive stress responses associated with Heat Shock Factor 1 (HSF1). Because glycocalyx thickness alters immune surveillance of epithelial-origin tumor cells, we examined the relationship between the HSF1-hyperglycemia axis in human breast tumors and validate it as a druggable vulnerability to license natural killer cell lethality against cancer cells.

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.

Transcriptome analysis of PDACs from KTC, KTC;Trp53fl/fl, KTC;Arffl/fl and KTC;Trp53fl/fl; Arffl/fl mice

Tissue fibrosis arises from a critical imbalance between the production and breakdown of extracellular matrix (ECM) components. Whereas current strategies predominantly focus on curbing ECM production, the possibility of promoting ECM degradation to resolve fibrosis remains largely untapped. The role of hepatic stellate cells (HSCs) in ECM degradation is an intriguing area for investigation. We previously demonstrated that inhibiting acid ceramidase (aCDase) increases ceramide in HSCs to ameliorate hepatic fibrosis. Here, we uncover a key signaling pathway that promotes ECM degradation in primary human HSCs, which is dependent upon the activation of protein kinase Cα (PKCα) and the induction of matrix metalloproteinase 1 (MMP-1) through extracellular signal-regulated kinase 1/2 (ERK1/2). Genetic reduction and pharmacological inhibition with a small molecule reduced aCDase activity, leading to increased collagen degradation and hepatic fibrosis resolution in the carbon tetrachloride (CCl4) and fructose, palmitate, cholesterol, and trans-fat (FPC) mouse models. Consistently, ceramide signaling correlated with ECM remodeling and degradation in patients with metabolic dysfunction-associated steatotic liver disease. The findings show that ceramide regulates ECM degradation and establish aCDase as a target for therapeutic regression of fibrosis.

Lysyl oxidases crosslink type I collagen to promote fibrosis and cancer progression in mouse mammary tumor models. Pancreatic ductal adenocarcinomas (PDACs) are highly fibrotic and contain abundant type I collagen with elevated expression of lysyl oxidases. Indeed, inhibition of lysyl oxidases constitute an attractive anti-tumor therapeutic strategy, with several reported preclinical studies demonstrating efficacy at reducing PDAC fibrosis and progression. Yet, lysyl oxidase was first described as an anti-oncogene through its effect of directly suppressing cell transformation by mutant Ras which is present in around 90% of human pancreatic tumors. These prior studies highlight the dual functions, anti-ras and pro-fibrotic, of lysyl oxidases in pancreatic cancer. As a result, clinical trials targeting lysyl oxidase in cancers have demonstrated limited efficacy. Here we examined the effects of perturbation of lysyl oxidase activity or expression using syngeneic orthotopic transplantation models expressing mutant Ras and intravital imaging. Unexpectedly, genetic or pharmacological inhibition of lysyl oxidases increased invasion along collagen fibers and distant metastasis. Furthermore, inhibition of lysyl oxidases promoted focal adhesion kinase (FAK) activity which was required for metastasis. We found that mutant Kras status dictated lysyl oxidase-mediated suppression on FAK signaling in both mouse and human pancreatic cancer cells. These results suggest that the effect of lysyl oxidase on metastasis are dependent on signaling from Ras and FAK. These results strongly caution against inhibiting lysyl oxidases for cancers driven by mutant Ras. Lijuan Sun, Jean Albrengues, John E. Wilkinson, Sarah L. Dallas, Valerie M. Weaver, Mikala Egeblad, Mario A. Shields. Lysyl oxidases suppress pancreatic cancer progression by inhibiting focal adhesion kinase signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 138.

AbstractToll-like receptor (TLR)-dependent macrophage responses rely on acute increases in oxidative mitochondrial glucose metabolism that epigenetically support rapid proinflammatory transcriptional programming via histone acetylation. Subsequent suppression of oxidative metabolism restrains this metabolic-epigenetic support of proinflammatory gene transcription to enforce tolerance, an immunosuppressed state of innate immune memory. Identifying biology that promotes or counters these metabolic-epigenetic changes will inform therapeutic approaches to influence proinflammatory, antimicrobial, and immunosuppressed myeloid cellular states. Here, we demonstrate that Coenzyme A (CoA) is a “metabolic adjuvant”, as supplying exogenous CoA to macrophages both enhances the magnitude of TLR-driven proinflammatory and antimicrobial responses, and reverse tolerance, via promotion of oxidative metabolism. Extracellular CoA, which we isotopically trace to show its direct uptake by macrophages, works synergistically with tonic TLR signaling, which we demonstrate is a critical regulator of nutrient uptake, metabolism, histone acetylation, and gene expression in macrophages. Together, TLR signaling and exogenous CoA promote mitochondrial glucose oxidation, acetyl-CoA production, and TLR target gene-specific histone acetylation, enhancing metabolic-epigenetic support of proinflammatory transcriptional programming. Exogenous CoA unlocks tumor-associated macrophage (TAM)-dependent TLR agonist anti-tumor activity in anin vivobreast cancer model, and promotes macrophage restriction of the intracellular bacterial pathogenLegionella pneumophila in vitrovia anIrg1-dependent antimicrobial state of CoA-augmented itaconate biosynthesis. Our findings demonstrate direct acquisition of intact extracellular CoA, and the ability of this exogenously supplemented metabolic cofactor to augment a key oxidative metabolic-epigenetic pathway supporting proinflammatory and antimicrobial macrophage phenotypes. This may inform host-targeted metabolic adjuvant therapies to reverse myeloid immunosuppression.

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.

Abstract 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 KRASG12C (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 YAPS127A 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.

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.

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.

The CDKN2A locus is one of the most frequent alterations in pancreatic ductal adenocarcinoma (PDAC). CDKN2A locus encodes two tumor suppressors, ARF and INK4A, which are rarely studied individually, limiting our understanding of their distinct roles in tumorigenesis. In addition, although ARF is an established positive regulator of the p53 tumor suppressor, it also has proposed p53-independent activities. The p53-dependent and p53-independent roles of ARF in the context of PDAC remain unclear. Here, we sought to understand ARF function in PDAC suppression. To this end, we first characterized expression and mutational patterns of the individual transcripts encoding ARF and INK4A using human TCGA data. Because the discrete roles of these transcripts have long been obscured by the shared CDKN2A locus nomenclature, we propose that future studies should analyze them independently and adopt CDKN2A ARF and CDKN2A INK4A as nomenclature that allows for their distinction. Notably, we found that CDKN2A ARF and CDKN2A INK4A are both commonly altered in human PDACs, through point mutation and/or deletion, suggesting that their combined inactivation contributes to human PDAC development. Next, to unequivocally interrogate a role for ARF as a PDAC suppressor, we used genetically-engineered mouse models (GEMMs). We found that Arf inactivation accelerated KRASG12D-driven PDAC development in GEMMs, both in the presence and absence of Trp53, demonstrating that ARF is a PDAC suppressor, and furthermore that it can act in a p53-independent manner. Transcriptomic analyses of PDACs from mice of various genotypes provided support for a p53-independent role for ARF, with ARF deficiency promoting extracellular matrix, collagen synthesis/assembly and epithelial-mesenchymal transition gene expression programs. Accordingly, ARF-deficient PDACs exhibited extensive remodeling of the tumor microenvironment (TME), associated with collagen deposition, increased tissue stiffness, and higher fibroblast content, all of which are characteristic of aggressive and treatment-resistant PDAC stroma. Similarly, transcriptomic analyses of human PDACs supported the notion that reduced ARF expression drives changes in the TME. Taken together, our findings reveal that ARF deficiency sculpts the PDAC TME and does so in a p53-independent fashion. Given the central role of the TME in PDAC progression and therapeutic resistance, our findings offer critical insights that may inform improved therapeutic strategies for PDAC. Moreover, our observations also underscore the importance of distinguishing between the two CDKN2A-encoded tumor suppressors, ARF and INK4A, and of studying both p53-dependent and p53-independent roles for ARF as they may have distinct and complementary roles in PDAC biology. Sofia Ferreira, Brittany M. Flowers, Won-Young Choi, Maria Farina-Morillas, Alberto Gatto, Sohinee Bhattacharyya, Gábor Boross, Ghmkin Hassan, Abigail S. Mulligan, Hannes Vogel, Laura D. Wood, Valerie M. Weaver, Monte M. Winslow, Dmitri Petrov, Mara H. Sherman, Hyo Young. Choi, D. Neil. Hayes, Andrew J. Aguirre, Jose A. Seoane, Laura D. Attardi. Inactivation of CDKN2A ARF promotes p53-independent remodeling of the PDAC tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A030.

Extracellular vesicles (EVs) are naturally occurring membrane-bound vesicles secreted by cells. Functionalized with surface-targeting molecules and carrying signalling proteins and nucleic acids as cargo, EVs can rewire pathways and alter biological processes in recipient cells. Tumour-derived EVs have key roles in cancer progression, particularly in metastasis, by promoting tumour cell invasion and the establishment of pre-metastatic niches. An evolving understanding of EVs in cancer highlights a complex intercellular communication network within and beyond the tumour microenvironment that involves cancer cells and non-cancerous cell types, such as fibroblasts and endothelial cells. More recently, EVs have also been recognized for their role in modulating interactions between host and immune cells and in reprogramming the tumour immune microenvironment. In this Review, we discuss EV biogenesis and function in diverse mechanobiological and mechanoimmunological contexts, highlighting how mechanical cues influence EV targeting and activity. The intricate interplay between mechanical forces and EV dynamics contributes to tumour progression and links EVs to key disease hallmarks. This Review discusses how mechanical cues can influence extracellular vesicle targeting and activity.

Glioblastoma (GBM) is an aggressive and highly therapy-resistant brain tumour1,2. Although advanced disease has been intensely investigated, the mechanisms that underpin the earlier, likely more tractable, stages of GBM development remain poorly understood. Here we identify axonal injury as a key driver of GBM progression, which we find is induced in white matter by early tumour cells preferentially expanding in this region. Mechanistically, axonal injury promotes gliomagenesis by triggering Wallerian degeneration, a targetable active programme of axonal death3, which we show increases neuroinflammation and tumour proliferation. Inactivation of SARM1, the key enzyme activated in response to injury that mediates Wallerian degeneration4, was sufficient to break this tumour-promoting feedforward loop, leading to the development of less advanced terminal tumours and prolonged survival in mice. Thus, targeting the tumour-induced injury microenvironment may supress progression from latent to advanced disease, thereby providing a potential strategy for GBM interception and control.

The rapid increase in cell biology techniques, combined with high-throughput technologies and improved computing, has yielded complex data that classical methods struggle to analyze. The question arises, can a machine overcome human performance to identify and predict distinct biological scenarios? Here, we review the mechanotransduction of extracellular matrix mechanical cues and how Artificial Intelligence-based methodologies are or can be used to predict cell states based on morphological and genetic signatures.

Collagen cross-links created by the lysyl oxidase and lysyl hydroxylase families of enzymes are a significant contributing factor to the biomechanical strength and rigidity of tissues, which in turn influence cell signaling and ultimately cell phenotype. In the clinic, the proteolytically liberated N-terminal cross-linked peptide of collagen I (NTX) is used as a biomarker of bone and connective tissue turnover, which is altered in several disease processes. Despite the clinical utility of these collagen breakdown products, the majority of the cross-linked peptide species have not been identified in proteomic datasets. Here we evaluate several parameters for the preparation and identification of these peptides from the collagen I-rich Achilles tendon. Our refined approach involving chemical digestion for protein solubilization coupled with mass spectrometry allows for the identification of the NTX cross-links in a range of modification states. Based on the specificity of the enzymatic cross-linking reaction we utilized follow-up variable modification searches to facilitate identification with a wider range of analytical workflows. We then applied a spectral library approach to identify differences in collagen cross-links in bovine pulmonary hypertension. The presented method offers unique opportunities to understand extracellular matrix remodeling events in development, aging, wound healing, and fibrotic disease that modulate collagen architecture through lysyl-hydroxylase and lysyl-oxidase enzymes.