miRNAs in pancreatic cancer progression and metastasis.

Small non-coding RNA or microRNA (miRNA) are critical regulators of eukaryotic cells. Dysregulation of miRNA expression and function has been linked to a variety of diseases including cancer. They play a complex role in cancers, having both tumour suppressor and promoter properties. In addition, a single miRNA can be involved in regulating several mRNAs or many miRNAs can regulate a single mRNA, therefore assessing these roles is essential to a better understanding in cancer initiation and development. Pancreatic cancer is a leading cause of cancer death worldwide, in part due to the lack of diagnostic tools and limited treatment options. The most common form of pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC), is characterised by major genetic mutations that drive cancer initiation and progression. The regulation or interaction of miRNAs with these cancer driving mutations suggests a strong link between the two. Understanding this link between miRNA and PDAC progression may give rise to novel treatments or diagnostic tools. This review summarises the role of miRNAs in PDAC, the downstream signalling pathways that they play a role in, how these are being used and studied as therapeutic targets as well as prognostic/diagnostic tools to improve the clinical outcome of PDAC.

The collagen landscape in cancer: profiling collagens in tumors and in circulation reveals novel markers of cancer-associated fibroblast subtypes.

Cancer-associated fibroblasts (CAFs) deposit and remodel collagens in the tumor stroma, impacting cancer progression and efficacy of interventions. CAFs are the focus of new therapeutics with the aim of normalizing the tumor microenvironment. To do this, a better understanding of CAF heterogeneity and collagen composition in cancer is needed. In this study, we sought to profile the expression of collagens at multiple levels with the goal of identifying cancer biomarkers. We investigated the collagen expression pattern in various cell types and CAF subtypes in a publicly available single-cell RNA sequencing (RNA-seq) dataset of pancreatic ductal adenocarcinoma. Next, we investigated the collagen expression profile in tumor samples across cancer types from The Cancer Genome Atlas (TCGA) database and evaluated if specific patterns of collagen expression were associated with prognosis. Finally, we profiled circulating collagen peptides using a panel of immunoassays to measure collagen fragments in the serum of cancer patients. We found that pancreatic stellate cells and fibroblasts were the primary producers of collagens in the pancreas. COL1A1, COL3A1, COL5A1, COL6A1 were expressed in all CAF subtypes, whereas COL8A1, COL10A1, COL11A1, COL12A1 were specific to myofibroblast CAFs (myCAF) and COL14A1 specific to inflammatory CAFs (iCAF). In TCGA database, myCAF collagens COL10A1 and COL11A1 were elevated across solid tumor types, and multiple associations between high expression and worse survival were found. Finally, circulating collagen biomarkers were elevated in the serum of patients with cancer relative to healthy controls with COL11A1 (myCAF) having the best diagnostic accuracy of the markers measured. In conclusion, CAFs express a noncanonical collagen profile with specific collagen subtypes associated with iCAFs and myCAFs in PDAC. These collagens are deregulated at the cellular, tumor, and systemic levels across different solid tumors and associate with survival. These findings could lead to new discoveries such as novel biomarkers and therapeutic targets. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The role of peroxidasin in solid cancer progression.

Peroxidasin is a heme-containing peroxidase enzyme that plays a vital role in the cross-linking of collagen IV molecules in basement membranes. Collagen IV cross-links are essential for providing structure and mechanical stability throughout tissue development, homeostasis, and wound healing. During cancer progression, the basement membrane is degraded, and proteins typically found in the basement membrane, including peroxidasin and collagen IV, can be found spread throughout the tumour microenvironment where they interact with cancer cells and alter cell behaviour. Whilst peroxidasin is reported to be up-regulated in a number of different cancers, the role that it plays in disease progression and metastasis has only recently begun to be studied. This review highlights the current literature exploring the known roles of peroxidasin in normal tissues and cancer progression, regulators of peroxidasin expression, and the reported relationships between peroxidasin expression and patient outcome in cancer.

Memory of stochastic single-cell apoptotic signaling promotes chemoresistance in neuroblastoma.

Gene expression noise is known to promote stochastic drug resistance through the elevated expression of individual genes in rare cancer cells. However, we now demonstrate that chemoresistant neuroblastoma cells emerge at a much higher frequency when the influence of noise is integrated across multiple components of an apoptotic signaling network. Using a JNK activity biosensor with longitudinal high-content and in vivo intravital imaging, we identify a population of stochastic, JNK-impaired, chemoresistant cells that exist because of noise within this signaling network. Furthermore, we reveal that the memory of this initially random state is retained following chemotherapy treatment across a series of in vitro, in vivo, and patient models. Using matched PDX models established at diagnosis and relapse from individual patients, we show that HDAC inhibitor priming cannot erase the memory of this resistant state within relapsed neuroblastomas but improves response in the first-line setting by restoring drug-induced JNK activity within the chemoresistant population of treatment-naïve tumors.

Extracellular matrix profiles determine risk and prognosis of the squamous cell carcinoma subtype of non-small cell lung carcinoma.

BACKGROUND: Squamous cell carcinoma (SqCC) is a subtype of non-small cell lung cancer for which patient prognosis remains poor. The extracellular matrix (ECM) is critical in regulating cell behavior; however, its importance in tumor aggressiveness remains to be comprehensively characterized. METHODS: Multi-omics data of SqCC human tumor specimens was combined to characterize ECM features associated with initiation and recurrence. Penalized logistic regression was used to define a matrix risk signature for SqCC tumors and its performance across a panel of tumor types and in SqCC premalignant lesions was evaluated. Consensus clustering was used to define prognostic matreotypes for SqCC tumors. Matreotype-specific tumor biology was defined by integration of bulk RNAseq with scRNAseq data, cell type deconvolution, analysis of ligand-receptor interactions and enriched biological pathways, and through cross comparison of matreotype expression profiles with aging and idiopathic pulmonary fibrosis lung profiles. RESULTS: This analysis revealed subtype-specific ECM signatures associated with tumor initiation that were predictive of premalignant progression. We identified an ECM-enriched tumor subtype associated with the poorest prognosis. In silico analysis indicates that matrix remodeling programs differentially activate intracellular signaling in tumor and stromal cells to reinforce matrix remodeling associated with resistance and progression. The matrix subtype with the poorest prognosis resembles ECM remodeling in idiopathic pulmonary fibrosis and may represent a field of cancerization associated with elevated cancer risk. CONCLUSIONS: Collectively, this analysis defines matrix-driven features of poor prognosis to inform precision medicine prevention and treatment strategies towards improving SqCC patient outcome.

Bovine and human endometrium-derived hydrogels support organoid culture from healthy and cancerous tissues.

Organoid technology has provided unique insights into human organ development, function, and diseases. Patient-derived organoids are increasingly used for drug screening, modeling rare disorders, designing regenerative therapies, and understanding disease pathogenesis. However, the use of Matrigel to grow organoids represents a major challenge in the clinical translation of organoid technology. Matrigel is a poorly defined mixture of extracellular matrix proteins and growth factors extracted from the Engelbreth-Holm-Swarm mouse tumor. The extracellular matrix is a major driver of multiple cellular processes and differs significantly between tissues as well as in healthy and disease states of the same tissue. Therefore, we envisioned that the extracellular matrix derived from a native healthy tissue would be able to support organoid growth akin to organogenesis in vivo. Here, we have developed hydrogels from decellularized human and bovine endometrium. These hydrogels supported the growth of mouse and human endometrial organoids, which was comparable to Matrigel. Organoids grown in endometrial hydrogels were proteomically more similar to the native tissue than those cultured in Matrigel. Proteomic and Raman microspectroscopy analyses showed that the method of decellularization affects the biochemical composition of hydrogels and, subsequently, their ability to support organoid growth. The amount of laminin in hydrogels correlated with the number and shape of organoids. We also demonstrated the utility of endometrial hydrogels in developing solid scaffolds for supporting high-throughput, cell culture-based applications. In summary, endometrial hydrogels overcome a major limitation of organoid technology and greatly expand the applicability of organoids to understand endometrial biology and associated pathologies.

Temporal profiling of the breast tumour microenvironment reveals collagen XII as a driver of metastasis.

The tumour stroma, and in particular the extracellular matrix (ECM), is a salient feature of solid tumours that plays a crucial role in shaping their progression. Many desmoplastic tumours including breast cancer involve the significant accumulation of type I collagen. However, recently it has become clear that the precise distribution and organisation of matrix molecules such as collagen I is equally as important in the tumour as their abundance. Cancer-associated fibroblasts (CAFs) coexist within breast cancer tissues and play both pro- and anti-tumourigenic roles through remodelling the ECM. Here, using temporal proteomic profiling of decellularized tumours, we interrogate the evolving matrisome during breast cancer progression. We identify 4 key matrisomal clusters, and pinpoint collagen type XII as a critical component that regulates collagen type I organisation. Through combining our proteomics with single-cell transcriptomics, and genetic manipulation models, we show how CAF-secreted collagen XII alters collagen I organisation to create a pro-invasive microenvironment supporting metastatic dissemination. Finally, we show in patient cohorts that collagen XII may represent an indicator of breast cancer patients at high risk of metastatic relapse.

Pirfenidone Reduces Epithelial-Mesenchymal Transition and Spheroid Formation in Breast Carcinoma through Targeting Cancer-Associated Fibroblasts (CAFs).

The aim of this study was to assess the effects of pirfenidone (PFD) on promoting epithelial-mesenchymal-transition (EMT) and stemness features in breast carcinoma cells through targeting cancer-associated-fibroblasts (CAFs). Using The Cancer Genome Atlas (TCGA) database, we analyzed the association between stromal index, EMT, and stemness-related genes across 1084 breast cancer patients, identifying positive correlation between YAP1, EMT, and stemness genes in samples with a high-stromal index. We monitored carcinoma cell invasion and spheroid formation co-cultured with CAFs in a 3D microfluidic device, followed by exposing carcinoma cells, spheroids, and CAFs with PFD. We depicted a positive association between the high-stromal index and the expression of EMT and stemness genes. High YAP1 expression in samples correlated with more advanced EMT status and stromal index. Additionally, we found that CAFs promoted spheroid formation and induced the expression of YAP1, VIM, and CD44 in spheroids. Treatment with PFD reduced carcinoma cell migration and decreased the expression of these genes at the protein level. The cytokine profiling showed significant depletion of various EMT- and stemness-regulated cytokines, particularly IL8, CCL17, and TNF-beta. These data highlight the potential application of PFD on inhibiting EMT and stemness in carcinoma cells through the targeting of critical cytokines.

Intravital imaging technology guides FAK-mediated priming in pancreatic cancer precision medicine according to Merlin status.

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic, chemoresistant malignancy and is characterized by a dense, desmoplastic stroma that modulates PDAC progression. Here, we visualized transient manipulation of focal adhesion kinase (FAK), which integrates bidirectional cell-environment signaling, using intravital fluorescence lifetime imaging microscopy of the FAK-based Förster resonance energy transfer biosensor in mouse and patient-derived PDAC models. Parallel real-time quantification of the FUCCI cell cycle reporter guided us to improve PDAC response to standard-of-care chemotherapy at primary and secondary sites. Critically, micropatterned pillar plates and stiffness-tunable matrices were used to pinpoint the contribution of environmental cues to chemosensitization, while fluid flow­induced shear stress assessment, patient-derived matrices, and personalized in vivo models allowed us to deconstruct how FAK inhibition can reduce PDAC spread. Last, stratification of PDAC patient samples via Merlin status revealed a patient subset with poor prognosis that are likely to respond to FAK priming before chemotherapy.

Cancer-Associated Fibroblasts in Pancreatic Ductal Adenocarcinoma Determine Response to SLC7A11 Inhibition.

Cancer-associated fibroblasts (CAF) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression through protumor signaling and the generation of fibrosis, the latter of which creates a physical barrier to drugs. CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11 is a cystine transporter that has been identified as a potential therapeutic target in PDAC cells. However, no prior study has evaluated the role of SLC7A11 in PDAC tumor stroma and its prognostic significance. Here we show that high expression of SLC7A11 in human PDAC tumor stroma, but not tumor cells, is independently prognostic of poorer overall survival. Orthogonal approaches showed that PDAC-derived CAFs are highly dependent on SLC7A11 for cystine uptake and glutathione synthesis and that SLC7A11 inhibition significantly decreases CAF proliferation, reduces their resistance to oxidative stress, and inhibits their ability to remodel collagen and support PDAC cell growth. Importantly, specific ablation of SLC7A11 from the tumor compartment of transgenic mouse PDAC tumors did not affect tumor growth, suggesting the stroma can substantially influence PDAC tumor response to SLC7A11 inhibition. In a mouse orthotopic PDAC model utilizing human PDAC cells and CAFs, stable knockdown of SLC7A11 was required in both cell types to reduce tumor growth, metastatic spread, and intratumoral fibrosis, demonstrating the importance of targeting SLC7A11 in both compartments. Finally, treatment with a nanoparticle gene-silencing drug against SLC7A11, developed by our laboratory, reduced PDAC tumor growth, incidence of metastases, CAF activation, and fibrosis in orthotopic PDAC tumors. Overall, these findings identify an important role of SLC7A11 in PDAC-derived CAFs in supporting tumor growth. SIGNIFICANCE: This study demonstrates that SLC7A11 in PDAC stromal cells is important for the tumor-promoting activity of CAFs and validates a clinically translatable nanomedicine for therapeutic SLC7A11 inhibition in PDAC.

In Vitro 3D Models of Tunable Stiffness.

Three-dimensional models of spheroid formation have been routinely used in the cancer field to test the colony forming capacity of malignant cells in an in vitro setting. Use of such a model provides a robust surrogate for in vivo testing, enabling large-scale interrogation into the effect of certain treatment conditions. This adapted protocol describes a high throughput and readily accessible composite alginate hydrogel system for spheroid formation, within a biomechanically tunable three-dimensional environment. This model therefore allows users to examine the effect of certain treatment conditions while cells are embedded within a hydrogel of defined stiffness. This is particularly important in the context of cancer where cells experience a wide range of mechanical properties within their microenvironment, driven by widespread changes in the extracellular matrix composition and architecture.This protocol describes a high-throughput method which results in homogeneous interpenetrating polymer networks of collagen and alginate. We show that this network readily supports single-cell spheroid formation in numerous malignant cell lines (breast cancer, lung cancer, and melanoma) and that these can be robustly analyzed for colony formation measures such as spheroid size, spheroid number, and overall cell viability; therefore, allowing users to undertake high-throughput, in vitro screening against a controlled biomechanical background.

The matrix in cancer.

The extracellular matrix is a fundamental, core component of all tissues and organs, and is essential for the existence of multicellular organisms. From the earliest stages of organism development until death, it regulates and fine-tunes every cellular process in the body. In cancer, the extracellular matrix is altered at the biochemical, biomechanical, architectural and topographical levels, and recent years have seen an exponential increase in the study and recognition of the importance of the matrix in solid tumours. Coupled with the advancement of new technologies to study various elements of the matrix and cell-matrix interactions, we are also beginning to see the deployment of matrix-centric, stromal targeting cancer therapies. This Review touches on many of the facets of matrix biology in solid cancers, including breast, pancreatic and lung cancer, with the aim of highlighting some of the emerging interactions of the matrix and influences that the matrix has on tumour onset, progression and metastatic dissemination, before summarizing the ongoing work in the field aimed at developing therapies to co-target the matrix in cancer and cancer metastasis.

Targeting Lysyl Oxidase Family Meditated Matrix Cross-Linking as an Anti-Stromal Therapy in Solid Tumours.

The lysyl oxidase (LOX) family of enzymes are a major driver in the biogenesis of desmoplastic matrix at the primary tumour and secondary metastatic sites. With the increasing interest in and development of anti-stromal therapies aimed at improving clinical outcomes of cancer patients, the Lox family has emerged as a potentially powerful clinical target. This review examines how lysyl oxidase family dysregulation in solid cancers contributes to disease progression and poor patient outcomes, as well as an evaluation of the preclinical landscape of LOX family targeting therapeutics. We also discuss the suitability of the LOX family as a diagnostic and/or prognostic marker in solid tumours.

The Role of the ECM in Lung Cancer Dormancy and Outgrowth.

The dissemination of tumor cells to local and distant sites presents a significant challenge in the clinical management of many solid tumors. These cells may remain dormant for months or years before overt metastases are re-awakened. The components of the extracellular matrix, their posttranslational modifications and their associated factors provide mechanical, physical and chemical cues to these disseminated tumor cells. These cues regulate the proliferative and survival capacity of these cells and lay the foundation for their engraftment and colonization. Crosstalk between tumor cells, stromal and immune cells within primary and secondary sites is fundamental to extracellular matrix remodeling that feeds back to regulate tumor cell dormancy and outgrowth. This review will examine the role of the extracellular matrix and its associated factors in establishing a fertile soil from which individual tumor cells and micrometastases establish primary and secondary tumors. We will focus on the role of the lung extracellular matrix in providing the architectural support for local metastases in lung cancer, and distant metastases in many solid tumors. This review will define how the matrix and matrix associated components are collectively regulated by lung epithelial cells, fibroblasts and resident immune cells to orchestrate tumor dormancy and outgrowth in the lung. Recent advances in targeting these lung-resident tumor cell subpopulations to prevent metastatic disease will be discussed. The development of novel matrix-targeted strategies have the potential to significantly reduce the burden of metastatic disease in lung and other solid tumors and significantly improve patient outcome in these diseases.

Stromal cell diversity associated with immune evasion in human triple-negative breast cancer.

The tumour stroma regulates nearly all stages of carcinogenesis. Stromal heterogeneity in human triple-negative breast cancers (TNBCs) remains poorly understood, limiting the development of stromal-targeted therapies. Single-cell RNA sequencing of five TNBCs revealed two cancer-associated fibroblast (CAF) and two perivascular-like (PVL) subpopulations. CAFs clustered into two states: the first with features of myofibroblasts and the second characterised by high expression of growth factors and immunomodulatory molecules. PVL cells clustered into two states consistent with a differentiated and immature phenotype. We showed that these stromal states have distinct morphologies, spatial relationships and functional properties in regulating the extracellular matrix. Using cell signalling predictions, we provide evidence that stromal-immune crosstalk acts via a diverse array of immunoregulatory molecules. Importantly, the investigation of gene signatures from inflammatory-CAFs and differentiated-PVL cells in independent TNBC patient cohorts revealed strong associations with cytotoxic T-cell dysfunction and exclusion, respectively. Such insights present promising candidates to further investigate for new therapeutic strategies in the treatment of TNBCs.

Plasma polymerized nanoparticles effectively deliver dual siRNA and drug therapy in vivo.

Multifunctional nanocarriers (MNCs) promise to improve therapeutic outcomes by combining multiple classes of molecules into a single nanostructure, enhancing active targeting of therapeutic agents and facilitating new combination therapies. However, nanocarrier platforms currently approved for clinical use can still only carry a single therapeutic agent. The complexity and escalating costs associated with the synthesis of more complex MNCs have been major technological roadblocks in the pathway for clinical translation. Here, we show that plasma polymerized nanoparticles (PPNs), synthesised in reactive gas discharges, can bind and effectively deliver multiple therapeutic cargo in a facile and cost-effective process compatible with up scaled commercial production. Delivery of siRNA against vascular endothelial growth factor (siVEGF) at extremely low concentrations (0.04 nM), significantly reduced VEGF expression in hard-to-transfect cells when compared with commercial platforms carrying higher siRNA doses (6.25 nM). PPNs carrying a combination of siVEGF and standard of care Paclitaxel (PPN-Dual) at reduced doses (< 100 µg/kg) synergistically modulated the microenvironment of orthotopic breast tumors in mice, and significantly reduced tumor growth. We propose PPNs as a new nanomaterial for delivery of therapeutics, which can be easily functionalised in any laboratory setting without the need for additional wet-chemistry and purification steps.

The Mini-Organo: A rapid high-throughput 3D coculture organotypic assay for oncology screening and drug development.

BACKGROUND: The use of in vitro cell cultures is a powerful tool for obtaining key insights into the behaviour and response of cells to interventions in normal and disease situations. Unlike in vivo settings, in vitro experiments allow a fine-tuned control of a range of microenvironmental elements independently within an isolated setting. The recent expansion in the use of three-dimensional (3D) in vitro assays has created a number of representative tools to study cell behaviour in a more physiologically 3D relevant microenvironment. Complex 3D in vitro models that can recapitulate human tissue biology are essential for understanding the pathophysiology of disease. AIM: The development of the 3D coculture collagen contraction and invasion assay, the "organotypic assay," has been widely adopted as a powerful approach to bridge the gap between standard two-dimensional tissue culture and in vivo mouse models. In the cancer setting, these assays can then be used to dissect how stromal cells, such as cancer-associated fibroblasts (CAFs), drive extracellular matrix (ECM) remodelling to alter cancer cell behaviour and response to intervention. However, to date, many of the published organotypic protocols are low-throughput, time-consuming (up to several weeks), and work-intensive with often limited scalability. Our aim was to develop a fast, high-throughput, scalable 3D organotypic assay for use in oncology screening and drug development. METHODS AND RESULTS: Here, we describe a modified 96-well organotypic assay, the "Mini-Organo," which can be easily completed within 5 days. We demonstrate its application in a wide range of mouse and human cancer biology approaches including evaluation of stromal cell 3D ECM remodelling, 3D cancer cell invasion, and the assessment of efficacy of potential anticancer therapeutic targets. Furthermore, the organotypic assay described is highly amenable to customisation using different cell types under diverse experimental conditions. CONCLUSIONS: The Mini-Organo high-throughput 3D organotypic assay allows the rapid screening of potential cancer therapeutics in human and mouse models in a time-efficient manner.

Shedding new light on RhoA signalling as a drug target in vivo using a novel RhoA-FRET biosensor mouse.

The small GTPase RhoA is a master regulator of signalling in cell-extracellular matrix interactions. RhoA signalling is critical to many cellular processes including migration, mechanotransduction, and is often disrupted in carcinogenesis. Investigating RhoA activity in a native tissue environment is challenging using conventional biochemical methods; we therefore developed a RhoA-FRET biosensor mouse, employing the adaptable nature of intravital imaging to a variety of settings. Mechanotransduction was explored in the context of osteocyte processes embedded in the calvaria responding in a directional manner to compression stress. Further, the migration of neutrophils was examined during in vivo "chemotaxis" in wound response. RhoA activity was tightly regulated during tissue remodelling in mammary gestation, as well as during mammary and pancreatic carcinogenesis. Finally, pharmacological inhibition of RhoA was temporally resolved by the use of optical imaging windows in fully developed pancreatic and mammary tumours in vivo. The RhoA-FRET mouse therefore constitutes a powerful tool to facilitate development of new inhibitors targeting the RhoA signalling axis.

Targeting the lysyl oxidases in tumour desmoplasia.

The extracellular matrix (ECM) is a fundamental component of tissue microenvironments and its dysregulation has been implicated in a number of diseases, in particular cancer. Tumour desmoplasia (fibrosis) accompanies the progression of many solid cancers, and is also often induced as a result of many frontline chemotherapies. This has recently led to an increased interest in targeting the underlying processes. The major structural components of the ECM contributing to desmoplasia are the fibrillar collagens, whose key assembly mechanism is the enzymatic stabilisation of procollagen monomers by the lysyl oxidases. The lysyl oxidase family of copper-dependent amine oxidase enzymes are required for covalent cross-linking of collagen (as well as elastin) molecules into the mature ECM. This key step in the assembly of collagens is of particular interest in the cancer field since it is essential to the tumour desmoplastic response. LOX family members are dysregulated in many cancers and consequently the development of small molecule inhibitors targeting their enzymatic activity has been initiated by many groups. Development of specific small molecule inhibitors however has been hindered by the lack of crystal structures of the active sites, and therefore alternate indirect approaches to target LOX have also been explored. In this review, we introduce the importance of, and assembly steps of the ECM in the tumour desmoplastic response focussing on the role of the lysyl oxidases. We also discuss recent progress in targeting this family of enzymes as a potential therapeutic approach.