Epithelial ovarian cancer (EOC) progression is determined by numerous intracellular interactions and the interplay between malignant cells, normal cells, and the tumor acellular microenvironment, formed largely by the extracellular matrix (ECM). The structure and biochemical functioning of various ECM components, along with the activity of agents that regulate ECM remodeling, impact the disease's expansion (adhesion, proliferation, invasion), spread, and response to therapy. It is important to note that the involvement of ECM components and their regulators in the progression of EOC is bidirectional and distinctly depends on a particular tissue context. In certain situations, certain components of the ECM enhance the activity of cancer cells, but in other scenarios, they suppress it. In this review, we summarize the newest knowledge regarding diverse aspects of ECM engagement in EOC pathophysiology and chemotherapy. Moreover, we delineate conditions that exacerbate the pro-cancerous properties of ECM, including diabetes-associated glycation, aging, and cellular senescence. We also explore methods to therapeutically alter the properties of the ECM, which could be beneficial in ovarian cancer prevention and treatment.

Collagen is the most abundant protein in mammals, significantly contributing to cancer progression. Cells express two primary well-conserved collagen receptors, integrins and discoidin domain receptors (DDRs), which bind collagen on distinct sites, suggesting that cancer cells must integrate both signals to decide their fate. The crosstalk between integrins and DDRs mediated by collagen binding produces dynamic, integrated signals that control tumor progression, therapeutic resistance, and cancer cell heterogeneity. This review will discuss the dynamic interplay among collagen, integrins, and DDRs in ECM remodeling during cancer progression and these receptors' crosstalk. In addition, we explored current and future directions for ECM receptor-targeted therapies, including nanotechnologies and precision medicine, to improve therapeutic outcomes by establishing a proper balance between integrins and DDRs in cancer.

The extracellular matrix (ECM) protein Beta-Ig H3 (βigH3, also known as transforming growth factor β induced protein (TGFBI)) is related to poor prognosis in patients with pancreatic ductal adenocarcinoma (PDAC). Proteolytic cleavage of βigH3 has been shown to result in release of the N-terminal fragment covering amino acid 1 to 137, but whether the degradation of βigH3 is associated to prognosis has yet to be determined. In this study we developed an ELISA targeting a collagenase generated fragment of βigH3 (cβigH3) in human serum to use the fragment as a biomarker reflecting degradation of βigH3. We demonstrated that the assay was specific to the cleaved fragment (cβigH3) and confirmed the generation of cβigH3 from degradation of fibroblast generated matrices. Moreover, higher levels of cβigH3 were released upon degradation of matrices produced by TGF-β stimulated pancreatic fibroblast compared to matrices produced by pancreatic fibroblast without TGF-β stimulation, indicating an association of the biomarker with degradation of fibrotic matrix. To evaluate the clinical relevance, we first measured cβigH3 in a cohort of 220 patients with different types of cancer with detectable levels for all 11 cancer types. We then measured the cβigH3 biomarker in pre-treatment serum from a cohort of 469 patients with locally advanced or metastatic PDAC and found that high levels of cβigH3 were associated with longer overall survival independently of age, disease stage, performance status, carbohydrate antigen 19-9 (CA19-9), and the tumor fibrosis biomarker PRO-C3 as compared to patients with high levels of cβigH3 (HR 0.78, 95% CI: 0.0.61-0.98, p = 0.04). In conclusion, cβigH3 reflects proteolytic degradation of βigH3 and shows potential as an independent prognostic biomarker for patients with advanced PDAC.

Collagen type I, a key structural component of the extracellular matrix (ECM), is frequently altered in cancer, with altered fiber organization at the primary tumor site linked to metastasis and poor patient outcomes. Here, we demonstrate that collagen fibers are also altered in metastatic sites such as the omentum of patients with high-grade serous ovarian cancer (HGSOC). Specifically, we observed a significant increase in fiber density, alignment, and width. To determine if the increase in fiber density supports metastasis, we used a semi-interpenetrating methacrylated gelatin (gelMA) network in combination with increasing fibrillar collagen. Cancer cells had significantly increased adhesion as collagen fiber density increased. To determine the responsible mechanisms, we used orthogonal systems to examine 1) the different adhesion peptides exposed in collagen (GFOGER) and gelatin (RGD), and 2) the physical structure of fibers. Cells had minimal response to GFOGER, either alone or in combination with RGD, suggesting that increased adhesion did not result from this collagen-specific interaction. Cell adhesion was significantly higher on electrospun PCL-gelatin fibers compared to flat PCL-gelatin substrates, suggesting that increased cell adhesion resulted from fiber structure. We next investigated the cellular mechanisms involved in increased adhesion on gelMA/coll and found that actin polymerization, but not myosin II contractility, was needed. We further demonstrated that cells on fibrous gels had more robust actin polymerization, and that this resulted in greater adhesion strength. Combined, these results suggest that the increase in collagen fibers with tumor metastasis will support the development of additional metastases. STATEMENT OF SIGNIFICANCE: This work advances the evaluation of the matrisome of the omentum, the most common metastatic site in advanced ovarian cancer by characterizing how collagen fibers change with disease progression. To examine the effect of collagen fibers on metastasis, we utilized a suite of in vitro biomaterials to identify a novel role for collagen fibers in supporting cell adhesion through increased actin dynamics during nascent adhesion formation, which results in increased adhesion strength at later times.

Pancreatic ductal adenocarcinoma (PDA) is an extremely metastatic and lethal disease. In PDA, extracellular matrix (ECM) architectures, known as tumor-associated collagen signatures (TACSs), regulate invasion and metastatic spread in both early dissemination and late-stage disease. As such, TACS has been suggested as a biomarker to aid in pathologic assessment. However, despite its significance, approaches to quantitatively capture these ECM patterns currently require advanced optical systems with signaling processing analysis. Here, we present an expansion of polychromatic polarized microscopy (PPM) with inherent angular information coupled to machine learning and computational pixel-wise analysis of TACS. Using this platform, we are able to accurately capture TACS architectures in hematoxylin and eosin-stained histology sections directly through PPM contrast. Moreover, PPM facilitated identification of transitions to dissemination architectures (ie, transitions from sequestration through expansion to dissemination from both PanINs and throughout PDA). Last, PPM evaluation of architectures in liver metastases, the most common metastatic site for PDA, demonstrates TACS-mediated focal and local invasion as well as identification of unique patterns anchoring aligned fibers into normal-adjacent tumor, suggesting that these patterns may be precursors to metastasis expansion and local spread from micrometastatic lesions. Combined, these findings demonstrate that PPM coupled to computational platforms is a powerful tool for analyzing ECM architecture that can be used to advance cancer microenvironment studies and provide clinically relevant diagnostic information.

The breast tumor microenvironment is composed of heterogeneous cell populations, including normal epithelial cells, cancer-associated fibroblasts (CAFs), and tumor cells that lead collective cell invasion. Both leader tumor cells and CAFs are known to play important roles in tumor invasion across the collagen-rich stromal boundary. However, their individual abilities to utilize their cell-intrinsic protrusions and perform force-based collagen remodeling to collectively invade remain unclear. To compare collective invasion phenotypes of leader-like tumor cells and CAFs, we embedded spheroids composed of 4T1 tumor cells or mouse tumor-derived CAF cell lines within 3D collagen gels and analyzed their invasion and collagen deformation. We found that 4T1s undergo greater invasion while generating lower collagen deformation compared with CAFs. Although force-driven collagen deformations are conventionally associated with higher cellular forces and invasion, here 4T1s specifically rely on actin-based protrusions, while CAFs rely on myosin-based contractility for collective invasion. In denser collagen, both cell types slowed their invasion, and selective pharmacological inhibitions show that Arp2/3 is required but myosin-II is dispensable for 4T1 invasion. Furthermore, depletion of CDH3 from 4T1s and DDR2 from CAFs reduces their ability to distinguish between collagen densities. For effective invasion, both cell types reorient and redistribute magnetically prealigned collagen fibers. With heterogeneous cell populations of cocultured CAFs and 4T1s, higher percentage of CAFs impeded invasion while increasing collagen fiber alignment. Overall, our findings demonstrate distinctive mechanisms of collective invasion adopted by 4T1 tumor cells and CAFs, one relying more on protrusions and the other on force-based collagen deformation. These results suggest that individually targeting cellular protrusions or contractility may not be universally applicable for all cell types or collagen densities, and a better cell-type-dependent approach could enhance effectiveness of cancer therapies.

The tumor microenvironment (TME), particularly the extracellular matrix (ECM), plays a crucial role in regulating breast cancer progression. Among ECM components, collagen type I-accounting for over 90% of fibrillar collagen in the human body-is the primary structural component of the tumor ECM. It critically modulates tumor cell behavior, influencing migration, invasion, and therapy resistance. The structural organization of collagen type I fibers can significantly impact clinical outcomes.

This systematic review aimed to assess the association between tumor-stromal collagen type I characteristics and clinical outcomes in breast cancer. A comprehensive search strategy identified studies from major databases, which were appraised using quality assessment tools. Data on collagen quantity, morphology, alignment, and organization were extracted and analyzed to explore their relationship with survival, metastasis, therapy resistance, and clinical characteristics of breast cancer.

Our analysis revealed that increased collagen density-particularly with an organized/aligned fiber orientation-was strongly associated with poor prognosis. Specifically, increased intratumoral collagen quantity was linked to reduced overall survival (HR = 7.84, p = 0.031). Stage III tumors exhibiting elevated collagen uniformity showed higher metastasis rates (p = 0.004), and HER2⁺ tumors with high collagen content correlated with resistance to HER2-targeted therapies (p < 0.05). Furthermore, higher collagen curviness was associated with better outcomes, including a reduced recurrence risk (HR = 0.77, p < 0.001). Subtype-specific trends emerged as ER/PR-negative tumors more frequently exhibited a perpendicular collagen arrangement (p = 0.02), whereas ER/PR-positive tumors showed elevated COL1A1 expression (p < 0.0001). Despite these patterns, the heterogeneity of study methodologies and the complexity of the tumor microenvironment highlight the need for unified frameworks to advance clinical translation.

This review highlights the prognostic significance of tumor-stromal collagen characteristics in breast cancer, suggesting that future research should focus on the molecular mechanisms underlying collagen remodeling and its potential as a cancer biomarker and therapeutic target.

Many biological structures take the form of fibres and filaments, and quantitative analysis of fibre organisation is important for understanding their functions in both normal physiological conditions and disease. In order to visualise these structures, fibres can be fluorescently labelled and imaged, with specialised image analysis methods available for quantifying the degree and strength of fibre alignment. Here we show that fluorescently labelled fibres can display polarised emission, with the strength of this effect varying depending on structure and fluorophore identity. This can bias automated analysis of fibre alignment and mask the true underlying structural organisation. We present a method for quantifying and correcting these polarisation effects without requiring polarisation-resolved microscopy and demonstrate its efficacy when applied to images of fluorescently labelled collagen gels, allowing for more reliable characterisation of fibre microarchitecture.

Cancer associated fibroblasts (CAFs) play a critically important role in facilitating tumour cell invasion during metastasis. They also modulate local biophysical features of the tumour microenvironment through the formation of fibrotic foci, which have been correlated with breast cancer aggression. However, the impact of the evolving three-dimensional biophysical tumour microenvironment on CAF function remains undefined. Here, by isolating CAFs from primary human triple-negative breast cancer tissue at the time of surgery, we find that their ability to remodel the local microenvironment and invade into a three-dimensional matrix correlates with disease state. We then engineered culture models to systematically deconstruct and recreate mechanical tissue features of early breast cancer fibrotic foci; and demonstrate that invasion is mechanically-activated only in CAFs from patients with no detectable pre-existing metastases, but is independent of mechanical cues in CAFs isolated from patients with later-stage axillary lymph node metastases. By comparing the differential transcriptional response of these cells to microenvironmental tissue stiffness, we identify the aryl hydrocarbon receptor (AhR) as being significantly upregulated in invasive sub-populations of both mechanically-activated and mechanically-insensitive CAFs. Increasing AhR expression in CAFs induced invasion, while suppressing AhR significantly reduced invasion in both mechanically-activated and mechanically-insensitive CAF populations, even on stiffnesses that recapitulate late-stage disease. This work therefore uses mechanobiological analyses to identify AhR as a mediator of CAF invasion, providing a potential stratification marker to identify those patients who might respond to future mechanics-based prophylactic therapies, and provides a targetable mechanism to limit CAF-associated metastatic disease progression in triple-negative breast cancer patients. STATEMENT OF SIGNIFICANCE: By designing a mechanically-tunable tissue-engineered model of fibroblastic foci, and using this to culture patient-derived cancer-associated fibroblasts, we demonstrate that these cells are differentially mechanosensitive, depending on disease stage of the patient. While comparing transcriptomic profiles of patient-derived cells produces too many pathways to screen, identifying the pathways activated by local tissue mechanics that were common across each patient allowed us to identify a specific target to limit fibroblast invasion. This broad discovery strategy may be useful across a variety of biomaterials-based tissue engineered models; and these specific findings suggest (1) a strategy to identify patients who might respond to CAF- or matrix-targeting therapies, and (2) a specific actionable target to limit CAF-associated metastatic disease progression.

The orientation of collagen fibers in relation to malignant epithelium is known to carry prognostic information in a variety of tissues. The data are the strongest for breast and pancreatic ductal adenocarcinoma. However, information inherent in collagen fiber topology in malignant tissues remains untapped in daily surgical pathology practice, largely because collagen fibers within areas of desmoplasia cannot be resolved with standard diagnostic microscopy. The methodologies used to visualize collagen fiber orientation are either of insufficient resolution to consistently capture collagen fiber topology or require resources in time and money that do not fit into the daily surgical pathology workflow. Polychromatic polarization microscopy has the potential to bring collagen topology to the attention of pathologists during their routine work. It has been demonstrated to be equivalent to the gold standard methodology used to research collagen, second harmonic generation. We use polychromatic polarization microscopy to visualize and describe the differences in collagen topology in normal pancreas, chronic pancreatitis, and pancreatic ductal adenocarcinoma with a standard microscope, using hematoxylin and eosin-stained sections. In the process, we propose a lexicon with which to describe the morphologic characteristics of collagen in benign and malignant pancreatic tissues.

Triple-negative breast cancer (TNBC) represents the most aggressive subtype of breast cancer with an extremely dismal prognosis and few treatment options. As a desmoplastic tumor, TNBC tumor cells are girdled by stroma composed of cancer-associated fibroblasts (CAFs) and their secreted stromal components. The rapidly proliferating tumor cells, together with the tumor stroma, exert additional solid tissue pressure on tumor vasculature and surrounding tissues, severely obstructing therapeutic agent from deep intratumoral penetration, and resulting in tumor metastasis and treatment resistance.

Fucoxanthin (FX), a xanthophyll carotenoid abundant in marine algae, has attracted widespread attention as a promising alternative candidate for tumor prevention and treatment. Twist is a pivotal regulator of epithelial to mesenchymal transition, and its depletion has proven to sensitize antitumor drugs, inhibit metastasis, reduce CAFs activation and the following interstitial deposition, and increase tumor perfusion. The nanodrug delivery system co-encapsulating FX and nucleic acid drug Twist siRNA (siTwist) was expected to form a potent anti-TNBC therapeutic cyclical feedback loop.

Herein, our studies constituted a novel self-assembled polymer nanomedicine (siTwist/FX@HES-CH) based on the amino-modified hydroxyethyl starch (HES-NH2) grafted with hydrophobic segment cholesterol (CH). The MTT assay, flow cytometry apoptosis analysis, transwell assay, western blot, and 3D multicellular tumor spheroids growth inhibition assay all showed that siTwist/FX@HES-CH could kill tumor cells and inhibit their metastasis in a synergistic manner. The in vivo anti-TNBC efficacy was demonstrated that siTwist/FX@HES-CH remodeled tumor microenvironment, facilitated interstitial barrier crossing, killed tumor cells synergistically, drastically reduced TNBC orthotopic tumor burden and inhibited lung metastasis.

Systematic studies revealed that this dual-functional nanomedicine that targets both tumor cells and tumor microenvironment significantly alleviates TNBC orthotopic tumor burden and inhibits lung metastasis, establishing a new paradigm for TNBC therapy.

Collagen stroma interactions within the extracellular microenvironment of breast tissue play a significant role in breast cancer, including risk, progression, and outcomes. Hydroxylation of proline (HYP) is a common post-translational modification directly linked to breast cancer survival and progression. Changes in HYP status lead to alterations in epithelial cell signaling, extracellular matrix remodeling, and immune cell recruitment. In the present study, we test the hypothesis that the breast cancer microenvironment presents unique PTMs of collagen, which form bioactive domains at these sites that are associated with spatial histopathological characteristics and influence breast epithelial cell signaling. Mass spectrometry imaging proteomics targeting collagens were paired with comprehensive proteomic methods to identify novel breast cancer-related collagen domains based on spatial localization and regulation in 260 breast tissue samples. As ancestry plays a significant role in breast cancer outcomes, these methods were performed on ancestry diverse breast cancer tissues. Lumpectomies from the Cancer Genome Atlas (TCGA; n=10) reported increased levels of prolyl 4-hydroxylase subunit alpha-3 (P4HA3) accompanied by spatial regulation of fibrillar collagen protein sequences. A concise set of triple negative breast cancer lumpectomies (n=10) showed spatial regulation of specific domain sites from collagen alpha-1(I) chain. Tissue microarrays identified proteomic alterations around post-translationally modified collagen sites in healthy breast (n=81) and patient matched normal adjacent (NAT; n=76) and invasive ductal carcinoma (n=83). A collagen alpha-1(I) chain domain encompassing amino acids 506-514 with site-specific proline hydroxylation reported significant alteration between patient matched normal adjacent tissue and invasive breast cancer. Functional testing of domain 506-514 on breast cancer epithelial cells showed proliferation, chemotaxis and cell signaling response dependent on site localization of proline hydroxylation within domain 506-514 variants. These findings support site localized collagen HYP forms novel bioactive domains that are spatially distributed within the breast cancer microenvironment and may play a role in ancestral traits of breast cancer.

Cell migration occurs throughout development, tissue homeostasis and regeneration, as well as in diseases such as cancer. Cells migrate along two-dimensional (2D) surfaces or interfaces, within microtracks, or in confining three-dimensional (3D) extracellular matrices. Although the basic mechanisms of 2D migration are known, recent studies have elucidated unexpected migration behaviors associated with more complex substrates and have provided insights into their underlying molecular mechanisms. Studies using engineered biomaterials for 3D culture and microfabricated channels to replicate cell confinement observed in vivo have revealed distinct modes of migration. Across these contexts, the mechanical features of the surrounding microenvironment have emerged as major regulators of migration. In this Cell Science at a Glance article and the accompanying poster, we describe physiological contexts wherein 2D and 3D cell migration are essential, report how mechanical properties of the microenvironment regulate individual and collective cell migration, and review the mechanisms mediating these diverse modes of cell migration.

The lamina propria (LP) of the urinary bladder lies between the urothelial mucosa and the muscularis propria. This complex stratum is composed of extracellular matrix, several cell types, and collagen types I and III fibers. LP invasion by urothelial carcinoma (progression from stage Ta to T1) is a determinant of bladder cancer advancement. We attempted to characterize collagen fiber arrangement in the LP. This could enrich our understanding of this important layer and potentially provide clues for sub-staging of the T1 bladder cancer. A total of 24 Masson trichrome-stained images of normal bladder, including 12,530 collagen fibers were quantitatively analyzed using the Dragonfly software. The LP was divided according to fiber orientation into superficial LP (SLP, 15% of the thickness) and the deep LP (DLP, 85% of the thickness). Collagen fiber geometry analysis demonstrated that the SLP fibers are more parallel to the urothelium with an average angle of 260±230 compared to 400±260 in the DLP (p=3.4x10-144), more packed (average distance to the closest fiber of 0.61±0.67 compared to 0.66±0.77, p=0.0001), and their aspect ratio is considerably longer (average of 1.93±0.12 compared to 0.20±0.11, p=2.84x10-8). No difference was found in fiber perimeter or Feret diameter. Thus, we conclude that bladder collagen fibers are arranged in two distinct layers: a dense-ordered SLP and a loose disorder DLP. This indicates that the physical barrier to cancer cell invasion probably lies in the SLP, immediately underneath the urothelium. Once this barrier is breached, the looser and disorganized DLP poses no remarkable obstacle. Thus, we believe that histology-based subdivisions of stage T1 are expected to fail in providing clinically meaningful prognostic information.

Adipose tissue is present in aortic valves (AVs). Valve interstitial cells (VICs) could differentiate into adipogenic lineages. We here characterize whether the presence of adipose tissue in the AV influences inflammation and extracellular matrix (ECM) composition in patients with aortic regurgitation (AR). A total of 144 AVs were analyzed by histological and molecular techniques. We performed discovery studies using Olink Proteomics® technology in 40 AVs (N = 16 without and N = 24 with adipose tissue). In vitro, human white adipocytes (HWAs) or VICs were cultured with adipogenic media and co-cultured with control VICs. Of Avs, 67% presented white-like adipocytes within the spongiosa. Discovery studies revealed increased levels of inflammatory and ECM molecules in AVs containing adipocytes. Interestingly, the presence of adipocytes was associated with greater AV thickness, higher inflammation, and ECM remodeling, which was characterized by increased proinflammatory molecules, collagen, fibronectin, proteoglycans, and metalloproteinases. AV thickness positively correlated with markers of adipose tissue, inflammation, and ECM. In vitro, adipocyte-like VICs expressed higher levels of adipocyte markers, increased cytokines, fibronectin, decorin, and MMP-13. Analyses of supernatants from co-cultured control VICs with HWA or adipocyte-like VICs showed higher expression of inflammatory mediators, collagen type I, proteoglycans, and metalloproteinases. AVs presenting adipocytes were thicker and exhibited changes characterized by increased inflammation accompanied by aberrant expression of collagen, proteoglycans, and metalloproteinases. VICs could differentiate into adipogenic pathway, affect neighbor VICs, and contribute to inflammation, collagen and proteoglycan accumulation, as well as to metalloproteinases secretion. In summary, the presence of adipose tissue in AV could modify its composition, favoring inflammation and remodeling with an impact on AV thickness.

Breast cancer progression is marked by extracellular matrix (ECM) remodeling, including increased stiffness, faster stress relaxation, and elevated collagen levels. In vitro experiments have revealed a role for each of these factors to individually promote malignant behavior, but their combined effects remain unclear. To address this, we developed alginate-collagen hydrogels with independently tunable stiffness, stress relaxation, and collagen density. We show that these combined tumor-mimicking ECM cues reinforced invasive morphologies and promoted spheroid invasion in breast cancer and mammary epithelial cells. High stiffness and low collagen density in slow-relaxing matrices led to the greatest cell migration speed and displacement. RNA-seq revealed Sp1 target gene enrichment in response to both individual and combined ECM cues, with a greater enrichment observed under multiple cues. Notably, high expression of Sp1 target genes upregulated by fast stress relaxation correlated with poor patient survival. Mechanistically, we found that phosphorylated-Sp1 (T453) was increasingly located in the nucleus in stiff and/or fast relaxing matrices, which was regulated by PI3K and ERK1/2 signaling, as well as actomyosin contractility. This study emphasizes how multiple ECM cues in complex microenvironments reinforce malignant traits and supports an emerging role for Sp1 as a mechanoresponsive transcription factor.

Effective therapies for solid tumors, including breast cancers, are hindered by several roadblocks that can be largely attributed to the fibrotic extracellular matrix (ECM). Fibronectin (FN) is a highly upregulated ECM component in the fibrotic tumor stroma and is associated with poor patient prognosis. This study aimed to investigate the therapeutic potential of an anti-fibrotic peptide that specifically targets FN and blocks the fibrillar assembly of FN.

To target FN, we used PEGylated Functional Upstream Domain (PEG-FUD), which binds to the 70 kDa N-terminal region of FN with high affinity, localizes to mammary tumors, and potently inhibits FN assembly in vitro and in vivo. Here, we used the 4T1 tumor model to investigate the efficacy and mechanisms of PEG-FUD to inhibit tumor growth.

Our data demonstrates that PEG-FUD monotherapy reduces tumor growth without systemic toxicity. Analysis of the tumor microenvironment revealed that PEG-FUD effectively inhibited FN matrix assembly within tumors and reduced adhesion-mediated signaling through α5 integrin and FAK leading to enhanced tumor cell death. Notably, signaling through FAK has been associated with resistance mechanisms to doxorubicin (DOX). Therefore, we tested the combination of PEG-FUD and Dox, which significantly reduced tumor growth by 60% compared to vehicle control and 30% compared to Dox monotherapy.

Our findings demonstrate that PEG-FUD significantly modifies the peritumoral ECM of breast cancer, leading to increased tumor cell death, and potentiates the efficacy of conventional breast cancer therapy.

The tumour microenvironment is the "hotbed" of tumour cells, providing abundant extracellular support for growth and metastasis. However, the tumour microenvironment is not static and is constantly remodelled by a variety of cellular components, including tumour cells, through mechanical, biological and chemical means to promote metastasis. Focal adhesion plays an important role in cell-extracellular matrix adhesion. An in-depth exploration of the role of focal adhesion in tumour metastasis, especially their contribution at the biomechanical level, is an important direction of current research. In this review, we first summarize the assembly of focal adhesions and explore their kinetics in tumour cells. Then, we describe in detail the role of focal adhesion in various stages of tumour metastasis, especially its key functions in cell migration, invasion, and matrix remodelling. Finally, we describe the anti-tumour strategies targeting focal adhesion and the current progress in the development of some inhibitors against focal adhesion proteins. In this paper, we summarize for the first time that focal adhesion play a positive feedback role in pro-tumour metastatic matrix remodelling by summarizing the five processes of focal adhesion assembly in a multidimensional way. It is beneficial for researchers to have a deeper understanding of the role of focal adhesion in the biological behaviour of tumour metastasis and the potential of focal adhesion as a therapeutic target, providing new ideas for the prevention and treatment of metastases.

Using a combined top-down (i.e., operator-directed) and bottom-up (i.e., cell-directed) strategy, an Under-oil Open Microfluidic System (UOMS)-based microtumor model is presented for investigating tumor cell migration and anti-metastasis drug test. Compared to the mainstream closed microfluidics-based microtumor models, the UOMS microtumor model features: i) micrometer-scale lateral resolution of surface patterning with open microfluidic design for flexible spatiotemporal sample manipulation (i.e., top-down); ii) self-organized extracellular matrix (ECM) structures and tumor cell-ECM spontaneous remodeling (i.e., bottom-up); and iii) free physical access to the samples on a device with minimized system disturbance. The UOMS microtumor model - allowing a controlled but also self-organized, cell-directed tumor-ECM microenvironment in an open microfluidic configuration - is used to test an anti-metastasis drug (incyclinide, aka CMT-3) with a triple-negative breast cancer cell line (MDA-MB-231). The in vitro results show a suppression of tumor cell migration and ECM remodeling echoing the in vivo mice metastasis results.

Polarized fluorescence microscopy is a valuable tool for measuring molecular orientations in biological samples, but techniques for recovering three-dimensional orientations and positions of fluorescent ensembles are limited. We report a polarized dual-view light-sheet system for determining the diffraction-limited three-dimensional distribution of the orientations and positions of ensembles of fluorescent dipoles that label biological structures. We share a set of visualization, histogram, and profiling tools for interpreting these positions and orientations. We model the distributions based on the polarization-dependent efficiency of excitation and detection of emitted fluorescence, using coarse-grained representations we call orientation distribution functions (ODFs). We apply ODFs to create physics-informed models of image formation with spatio-angular point-spread and transfer functions. We use theory and experiment to conclude that light-sheet tilting is a necessary part of our design for recovering all three-dimensional orientations. We use our system to extend known two-dimensional results to three dimensions in FM1-43-labeled giant unilamellar vesicles, fast-scarlet-labeled cellulose in xylem cells, and phalloidin-labeled actin in U2OS cells. Additionally, we observe phalloidin-labeled actin in mouse fibroblasts grown on grids of labeled nanowires and identify correlations between local actin alignment and global cell-scale orientation, indicating cellular coordination across length scales.

The extracellular matrix (ECM) controls tumour dissemination. We characterise ECM organization in human and mouse tumours, identifying three regions: tumour body, proximal invasive front and distal invasive front. Invasive areas show increased matrix density, fibre thickness, length, and alignment, with unique radial fibre orientation at the distal invasive front correlating with amoeboid invasive features. Using patient samples and murine models, we find that metastases recapitulate ECM features of the primary tumour. Ex vivo culture of murine cancer cells isolated from the different tumour regions reveals a spatial cytoskeletal and transcriptional memory. Several in vitro models recapitulate the in vivo ECM organisation showing that increased matrix induces 3D confinement supporting Rho-ROCK-Myosin II activity, while radial orientation enhances directional invasion. Spatial transcriptomics identifies a mechano-inflammatory program associated with worse prognosis across multiple tumour types. These findings provide mechanistic insights into how ECM organization shapes local invasion and distant metastasis.

In breast cancer (BC), radial alignment of collagen fibers at the tumor-matrix interface facilitates collective invasion of cancer cells into the surrounding stromal matrix, a critical step toward metastasis. Collagen remodeling is driven by proteases and cellular forces, mediated by matrix mechanical plasticity, or irreversible matrix deformation in response to force. However, the specific mechanisms causing collagen radial alignment remain unclear. Here, we study collective invasion of BC tumor spheroids in collagen-rich matrices. Increasing plasticity to BC-relevant ranges facilitates invasion, with increasing stiffness potentiating a transition from single cell to collective invasion. At enhanced plasticity, cells radially align collagen at the tumor-matrix interface prior to invasion. Surprisingly, cells migrate tangentially to the tumor-matrix interface in a swirling-like motion, perpendicular to the direction of alignment. Mechanistically, swirling generates local shear stresses, leading to distally propagating contractile radial stresses due to negative normal stress, an underappreciated property of collagen-rich matrices. These contractile stresses align collagen fibers radially, facilitating collective invasion. The basement membrane (BM), which separates epithelia from stroma in healthy tissues, acts as a mechanical insulator by preventing swirling cells from aligning collagen. Thus, after breaching the BM, swirling of BC cells at the tumor-stroma interface radially aligns collagen to facilitate invasion.

In this study, an advanced nanofiber breast cancer in vitro model was developed and systematically characterized including physico-chemical, cell-biological and biophysical parameters. Using electrospinning, the architecture of tumor-associated collagen signatures (TACS5 and TACS6) was mimicked. By employing a rotating cylinder or static plate collector set-up, aligned fibers (TACS5-like structures) and randomly orientated fibers (TACS6-like structures) fibers were produced, respectively. The biocompatibility of these fibers was enhanced by collagen coating, ensuring minimal toxicity and improved cell attachment. Various breast cancer cell lines (MCF7, HCC1954, MDA-MB-468, and MDA-MB-231) were cultured on these fibers to assess epithelial-to-mesenchymal transition (EMT) markers, cellular morphology, and migration. Aligned fibers (TACS5) significantly influenced EMT-related changes, promoting cellular alignment, spindle-shaped morphology and a highly migratory phenotype in mesenchymal and hybrid EMT cells (MDA-MB-468, MDA-MB-231). Conversely, epithelial cells (MCF7, HCC1954) showed limited response, but - under growth factor treatment - started to infiltrate the fibrous scaffold and underwent EMT-like changes, particularly on TACS5-mimicks, emphasizing the interplay of topographical cues and EMT induction. The biophysical analysis revealed a clear correlation between cellular EMT status and cell mechanics, with increased EMT correlating to decreased total cellular stiffness. Cancer cell mechanics, however, were found to be dynamic during biochemical and topographical EMT-induction, exceeding initial stiffness by up to 2-fold. These findings highlight the potential of TACS5-like nanofiber scaffolds in modeling the tumor microenvironment and studying cancer cell behavior and mechanics.

In natural environments, cells move in the presence of multiple physical and chemical guidance cues. Using a model system for such guided cell migration, Dictyostelium discoideum (Dicty), we investigate how chemical and physical signals compete in guiding the motion of cell groups. In Dicty cells, chemical signals can lead to collective streaming behavior, in which cells follow one another head-to-tail and aggregate into clusters of ∼10^{5} cells. We use experiments and numerical simulations to show that streaming and aggregation can be suppressed by the addition of a physical guidance cue of comparable strength to the chemical signals, parallel nanoridges. The bidirectional character of physical guidance by ridges is a determining factor in the suppression of streaming and aggregation. Thus, combining multiple types of guidance cues is a powerful approach to trigger or explain a broad range of collective cell behaviors.

Breast cancer (BC) is characterised by a high level of heterogeneity, which is influenced by the interaction of neoplastic cells with the tumour microenvironment. The diagnostic and prognostic role of the tumour stroma in BC remains to be defined. Differential interference contrast (DIC) microscopy is a label-free imaging technique well suited to visualise weak optical phase objects such as cells and tissue. This study aims to compare stromal collagen fibre characteristics between in situ and invasive breast tumours using DIC microscopy and investigate the prognostic value of collagen parameters in BC. A tissue microarray was generated from 200 cases, comprising ductal carcinoma in situ (DCIS; n = 100) and invasive tumours (n = 100) with an extra 50 (25 invasive BC and 25 DCIS) cases for validation was utilised. Two sections per case were used: one stained with haematoxylin and eosin (H&E) stain for histological review and one unstained for examination using DIC microscopy. Collagen fibre parameters including orientation angle, fibre alignment, fibre density, fibre width, fibre length and fibre straightness were measured. Collagen fibre density was higher in the stroma of invasive BC (161.68 ± 11.2 fibre/µm2) compared to DCIS (p < 0.0001). The collagen fibres were thinner (13.78 ± 1.08 µm), straighter (0.96 ± 0.006, on a scale of 0-1), more disorganised (95.07° ± 11.39°) and less aligned (0.20 ± 0.09, on a 0-1 scale) in the invasive BC compared to DCIS (all p < 0.0001). A model considering these features was developed that could distinguish between DCIS and invasive tumours with 94% accuracy. There were strong correlations between fibre characteristics and clinicopathological parameters in both groups. A statistically significant association between fibre characteristics and patients' outcomes (breast cancer specific survival, and recurrence free survival) was observed in the invasive group but not in DCIS. Although invasive BC and DCIS were both associated with stromal reaction, the structural features of collagen fibres were significantly different in the two disease stages. Analysis of the stroma fibre characteristics in the preoperative core biopsy specimen may help to differentiate pure DCIS from those associated with invasion.

Mechanical properties of the extracellular matrix (ECM) critically regulate a number of important cell functions including growth, differentiation and migration. Type I collagen and glycosaminoglycans (GAGs) are two primary components of ECMs that contribute to mammalian tissue mechanics, with the collagen fiber network sustaining tension, and GAGs withstanding compression. The architecture and stiffness of the collagen network are known to be important for cell-ECM mechanical interactions via cell surface adhesion receptor integrin. In contrast, studies of GAGs in modulating cell-ECM interactions are limited. Here, we present experimental studies on the roles of hyaluronic acid (HA) in single tumor cell traction force generation using a recently developed 3D cell traction force microscopy method. Our work reveals that CD44, a cell surface receptor to HA, is engaged in cell traction force generation in conjunction with β1-integrin. We find that HA significantly modifies the architecture and mechanics of the collagen fiber network, decreasing tumor cells' propensity to remodel the collagen network, attenuating traction force generation, transmission distance, and tumor invasion. Our findings point to a novel role for CD44 in traction force generation, which can be a potential therapeutic target for diseases involving HA rich ECMs such as breast cancer and glioblastoma.

The physical microenvironment plays a crucial role in tumor development, progression, metastasis and treatment. Recently, we proposed four physical hallmarks of cancer, with distinct origins and consequences, to characterize abnormalities in the physical tumor microenvironment: (1) elevated compressive-tensile solid stresses, (2) elevated interstitial fluid pressure and the resulting interstitial fluid flow, (3) altered material properties (for example, increased tissue stiffness) and (4) altered physical micro-architecture. As this emerging field of physical oncology is being advanced by tumor biologists, cell and developmental biologists, engineers, physicists and oncologists, there is a critical need for model systems and measurement tools to mechanistically probe these physical hallmarks. Here, after briefly defining these physical hallmarks, we discuss the tools and model systems available for probing each hallmark in vitro, ex vivo, in vivo and in clinical settings. We finally review the unmet needs for mechanistic probing of the physical hallmarks of tumors and discuss the challenges and unanswered questions associated with each hallmark.

Ductal carcinoma in situ (DCIS) accounts for ~20% of all breast cancer diagnoses but whilst known to be a precursor of invasive breast cancer (IBC), evidence suggests only one in six patients will ever progress. A key challenge is to distinguish between those lesions that will progress and those that will remain indolent. Molecular analyses of neoplastic epithelial cells have not identified consistent differences between lesions that progressed and those that did not, and this has focused attention on the tumour microenvironment (ME).The DCIS ME is unique, complex and dynamic. Myoepithelial cells form the wall of the ductal-lobular tree and exhibit broad tumour suppressor functions. However, in DCIS they acquire phenotypic changes that bestow them with tumour promoter properties, an important evolution since they act as the primary barrier for invasion. Changes in the peri-ductal stromal environment also arise in DCIS, including transformation of fibroblasts into cancer-associated fibroblasts (CAFs). CAFs orchestrate other changes in the stroma, including the physical structure of the extracellular matrix (ECM) through altered protein synthesis, as well as release of a plethora of factors including proteases, cytokines and chemokines that remodel the ECM. CAFs can also modulate the immune ME as well as impact on tumour cell signalling pathways. The heterogeneity of CAFs, including recognition of anti-tumourigenic populations, is becoming evident, as well as heterogeneity of immune cells and the interplay between these and the adipocyte and vascular compartments. Knowledge of the impact of these changes is more advanced in IBC but evidence is starting to accumulate for a role in DCIS. Detailed in vitro, in vivo and tissue studies focusing on the interplay between DCIS epithelial cells and the ME should help to define features that can better predict DCIS behaviour.

Cell collectives, like other motile entities, generate and use forces to move forward. Here, we ask whether environmental configurations alter this proportional force-speed relationship, since aligned extracellular matrix fibers are known to cause directed migration. We show that aligned fibers serve as active conduits for spatial propagation of cellular mechanotransduction through matrix exoskeleton, leading to efficient directed collective cell migration. Epithelial (MCF10A) cell clusters adhered to soft substrates with aligned collagen fibers (AF) migrate faster with much lesser traction forces, compared to random fibers (RF). Fiber alignment causes higher motility waves and transmission of normal stresses deeper into cell monolayer while minimizing shear stresses and increased cell-division based fluidization. By contrast, fiber randomization induces cellular jamming due to breakage in motility waves, disrupted transmission of normal stresses, and heightened shear driven flow. Using a novel motor-clutch model, we explain that such 'force-effective' fast migration phenotype occurs due to rapid stabilization of contractile forces at the migrating front, enabled by higher frictional forces arising from simultaneous compressive loading of parallel fiber-substrate connections. We also model 'haptotaxis' to show that increasing ligand connectivity (but not continuity) increases migration efficiency. According to our model, increased rate of front stabilization via higher resistance to substrate deformation is sufficient to capture 'durotaxis'. Thus, our findings reveal a new paradigm wherein the rate of leading-edge stabilization determines the efficiency of supracellular collective cell migration.

Ductal carcinoma in situ (DCIS) is a noninvasive breast disease that variably progresses to invasive breast cancer (IBC). Given the unpredictability of this progression, most DCIS patients are aggressively managed similar to IBC patients. Undoubtedly, this treatment paradigm places many DCIS patients at risk of overtreatment and its significant consequences. Historically, prognostic modeling has included the assessment of clinicopathological features and genomic markers. Although these provide valuable insights into tumor biology, they remain insufficient to predict which DCIS patients will progress to IBC. Contemporary work has begun to focus on the microenvironment surrounding the ductal cells for molecular patterns that might predict progression. In this review, extracellular microenvironment alterations occurring with the malignant transformation from DCIS to IBC are detailed. Not only do changes in collagen abundance, organization, and localization mediate the transition to IBC, but also the discrete post-translational regulation of collagen fibers is understood to promote invasion. Other extracellular matrix proteins, such as matrix metalloproteases, decorin, and tenascin C, have been characterized for their role in invasive transformation and further demonstrate the prognostic value of the extracellular matrix. Importantly, these extracellular matrix proteins influence immune cells and fibroblasts toward pro-tumorigenic phenotypes. Thus, the progressive changes in the extracellular microenvironment play a key role in invasion and provide promise for prognostic development.

The tumor-associated stroma is an essential compartment in breast cancer, and collagen fiber organization in the stroma has been reported to be correlated with prognosis. In this study, we sought to evaluate collagen fiber characteristics in relation to pathological complete response (pCR) after neoadjuvant chemotherapy (NAC) in breast cancer patients.

A total of 388 breast cancer patients receiving NAC were enrolled. The stroma type was manually assessed on pretreatment hematoxylin and eosin (HE)-stained slides, and the collagen fiber features were quantified by a computer tool. The relationship between syndecan-1 expression and collagen fibers and its correlation with treatment efficacy were detected by immunohistochemistry.

The pCR rate of patients with collagen-dominant stroma was lower than that of patients with lymphocyte-dominant stroma (19.6% vs. 40.0%, p = 0.001). Patients who achieved pCR had straighter and less dense fibers in pretreatment biopsied tissue than non-pCR patients (p = 0.031, p = 0.044). Additionally, the pCR group had greater syndecans-1 expression on the tumor epithelium than the non-pCR group (p < 0.001), while there was no statistically significant difference in the stroma (p = 0.333). Collagen fiber density was the only factor associated with pCR after correction for other clinicopathological variables in triple-negative breast cancer (TNBC) patients (OR 0.466, 95% CI 0.227-0.956, p = 0.037); patients with lower fiber density had a greater pCR rate (37.5% vs. 12.5%, p = 0.021).

Collagen fiber density was associated with pCR in patients with breast cancer, and it could be a potential candidate for discriminating between responders and nonresponders for TNBC patients receiving NAC.

Glandular epithelia, including mammary gland (MG) and prostate, are composed of luminal and basal cells. During embryonic development, glandular epithelia arise from multipotent stem cells (SCs) that are replaced after birth by unipotent basal and unipotent luminal SCs. Different conditions, such as basal cell transplantation, luminal cell ablation, and oncogene expression can reinduce adult basal SC (BaSCs) multipotency in different glandular epithelia. The mechanisms regulating the reactivation of multipotency are incompletely understood. Here, we have found that Collagen I expression is commonly upregulated in BaSCs across the different multipotent conditions. Increasing collagen concentration or stiffness of the extracellular matrix (ECM) promotes BaSC multipotency in MG and prostate organoids. Single cell RNA-seq of MG organoids in stiff conditions have uncovered the importance of β1 integrin/FAK/AP-1 axis in the regulation of BaSC multipotency. Altogether our study uncovers the key role of Collagen signaling and ECM stiffness in the regulation of multipotency in glandular epithelia.

Collagen is a crucial protein in the extracellular matrix (ECM) essential for preserving tissue architecture and supporting crucial cellular functions like proliferation and differentiation. There are twenty-eight identified types of collagen, which are further divided into different subgroups. This protein plays a critical role in regulating tissue homeostasis. However, in solid tumors, the balance can be disrupted, due to an abundance of collagen in the tumor microenvironment, which significantly affects tumor growth, cell invasion, and metastasis. It is important to investigate the specific types of collagens in cancer ECM and their distinct roles in tumor progression to comprehend their unique contribution to tumor behavior. The diverse pathophysiological functions of different collagen types in cancers illustrate collagen's dual roles, offering potential therapeutic options and serving as prognostic markers.

Cancer, characterized by its immune evasion, active metabolism, and heightened proliferation, comprises both stroma and cells. Although the research has always focused on parenchymal cells, the non-parenchymal components must not be overlooked. Targeting cancer parenchymal cells has proven to be a formidable challenge, yielding limited success on a broad scale. The tumor microenvironment(TME), a critical niche for cancer cell survival, presents a novel way for cancer treatment. Cancer-associated fibroblast (CAF), as a main component of TME, is a dynamically evolving, dual-functioning stromal cell. Furthermore, their biological activities span the entire spectrum of tumor development, metastasis, drug resistance, and prognosis. A thorough understanding of CAFs functions and therapeutic advances holds significant clinical implications. In this review, we underscore the heterogeneity of CAFs by elaborating on their origins, types and function. Most importantly, by elucidating the direct or indirect crosstalk between CAFs and immune cells, the extracellular matrix, and cancer cells, we emphasize the tumorigenicity of CAFs in cancer. Finally, we highlight the challenges encountered in the exploration of CAFs and list targeted therapies for CAF, which have implications for clinical treatment.

Cell migration during many fundamental biological processes including metastasis requires cells to traverse tissue with heterogeneous mechanical cues that direct migration as well as determine force and energy requirements for motility. However, the influence of discrete structural and mechanical cues on migration remains challenging to determine as they are often coupled. Here, we decouple the pro-invasive cues of collagen fiber alignment and tension to study their individual impact on migration. When presented with both cues, cells preferentially travel in the axis of tension against fiber alignment. Computational and experimental data show applying tension perpendicular to alignment increases potential energy stored within collagen fibers, lowering requirements for cell-induced matrix deformation and energy usage during migration compared to motility in the direction of fiber alignment. Energy minimization directs migration trajectory, and tension can facilitate migration against fiber alignment. These findings provide a conceptual understanding of bioenergetics during migration through a fibrous matrix.

Tumor-associated collagen signature (TACS) is an independent prognostic factor for breast cancer. However, it is unclear whether the complete collagen signature, including TACS, the TACS-based collagen microscopic features (TCMF1), and the TACS-based nuclear features (TCMF2), can provide additional prognostic information for the current tumor-node-metastasis (TNM) staging system.

We included 941 patients with breast cancer from three cohorts: the training (n = 355), internal (n = 334), and external validation cohorts (n = 252). TACS and TCMF1 were obtained by multiphoton microscopy (MPM). TCMF2 was extracted on the hematoxylin and eosin images colocated with MPM images. They were linearly combined to establish a complete collagen signature score for reclassifying current TNM staging into stage Ⅰ (II and Ⅲ)/low risk and stage Ⅰ (II and Ⅲ)/high risk.

The low-risk collagen signatures 'downstaged' patients in stage II or Ⅲ, while the high-risk collagen signatures 'upstaged' patients with stage Ⅰ tumors. After incorporating the complete collagen signature into the current TNM staging system, the modified staging system had a higher ability to stratify patients [referent, Ⅰ-new; Ⅱ-new, hazard ratio (HR) 8.655, 6.136, and 4.699 in the training, internal validation, and external validation cohorts, respectively; Ⅲ-new, HR 14.855, 11.201, and 13.245 in the corresponding three cohorts, respectively] than the current TNM staging system (referent, Ⅰ; Ⅱ, HR 1.642, 1.853, and 1.371 in the corresponding three cohorts, respectively; Ⅲ, HR 4.131, 4.283, and 3.711 in the corresponding three cohorts, respectively). Furthermore, the modified staging system showed a higher area under the curve than the current TNM staging system (training cohort: 0.843 versus 0.683; internal validation cohort: 0.792 versus 0.661; and external validation cohort: 0.793 versus 0.646).

The complete collagen signature is an independent predictor of survival outcomes in breast cancer. It adds significant information about the biological behavior of the disease to staging for breast cancer.