ADAM12-Generated Basigin Ectodomain Binds ß1 Integrin and Enhances the Expression of Cancer-Related Extracellular Matrix Proteins.

Desmoplasia is a common feature of aggressive cancers, driven by a complex interplay of protein production and degradation. Basigin is a type 1 integral membrane receptor secreted in exosomes or released by ectodomain shedding from the cell surface. Given that soluble basigin is increased in the circulation of patients with a poor cancer prognosis, we explored the putative role of the ADAM12-generated basigin ectodomain in cancer progression. We show that recombinant basigin ectodomain binds ß1 integrin and stimulates gelatin degradation and the migration of cancer cells in a matrix metalloproteinase (MMP)- and ß1-integrin-dependent manner. Subsequent in vitro and in vivo experiments demonstrated the altered expression of extracellular matrix proteins, including fibronectin and collagen type 5. Thus, we found increased deposits of collagen type 5 in the stroma of nude mice tumors of the human tumor cell line MCF7 expressing ADAM12-mimicking the desmoplastic response seen in human cancer. Our findings indicate a feedback loop between ADAM12 expression, basigin shedding, TGFß signaling, and extracellular matrix (ECM) remodeling, which could be a mechanism by which ADAM12-generated basigin ectodomain contributes to the regulation of desmoplasia, a key feature in human cancer progression.

Resolution of Acinar Dedifferentiation Regulates Tissue Remodeling in Pancreatic Injury and Cancer Initiation.

BACKGROUND & AIMS: Acinar-to-ductal metaplasia (ADM) is crucial in the development of pancreatic ductal adenocarcinoma. However, our understanding of the induction and resolution of ADM remains limited. We conducted comparative transcriptome analyses to identify conserved mechanisms of ADM in mouse and human. METHODS: We identified Sox4 among the top up-regulated genes. We validated the analysis by RNA in situ hybridization. We performed experiments in mice with acinar-specific deletion of Sox4 (Ptf1a: CreER; Rosa26-LSL-YFPLSL-YFP; Sox4fl/fl) with and without an activating mutation in Kras (KrasLSL-G12D/+). Mice were given caerulein to induce pancreatitis. We performed phenotypic analysis by immunohistochemistry, tissue decellularization, and single-cell RNA sequencing. RESULTS: We demonstrated that Sox4 is reactivated in ADM and pancreatic intraepithelial neoplasias. Contrary to findings in other tissues, Sox4 actually counteracts cellular dedifferentiation and helps maintain tissue homeostasis. Moreover, our investigations unveiled the indispensable role of Sox4 in the specification of mucin-producing cells and tuft-like cells from acinar cells. We identified Sox4-dependent non-cell-autonomous mechanisms regulating the stromal reaction during disease progression. Notably, Sox4-inferred targets are activated upon KRAS inactivation and tumor regression. CONCLUSIONS: Our results indicate that our transcriptome analysis can be used to investigate conserved mechanisms of tissue injury. We demonstrate that Sox4 restrains acinar dedifferentiation and is necessary for the specification of acinar-derived metaplastic cells in pancreatic injury and cancer initiation and is activated upon Kras ablation and tumor regression in mice. By uncovering novel potential strategies to promote tissue homeostasis, our findings offer new avenues for preventing the development of pancreatic ductal adenocarcinoma.

Fibroblast-Derived Lysyl Oxidase Increases Oxidative Phosphorylation and Stemness in Cholangiocarcinoma.

BACKGROUND & AIMS: Metabolic and transcriptional programs respond to extracellular matrix-derived cues in complex environments, such as the tumor microenvironment. Here, we demonstrate how lysyl oxidase (LOX), a known factor in collagen crosslinking, contributes to the development and progression of cholangiocarcinoma (CCA). METHODS: Transcriptomes of 209 human CCA tumors, 143 surrounding tissues, and single-cell data from 30 patients were analyzed. The recombinant protein and a small molecule inhibitor of the LOX activity were used on primary patient-derived CCA cultures to establish the role of LOX in migration, proliferation, colony formation, metabolic fitness, and the LOX interactome. The oncogenic role of LOX was further investigated by RNAscope and in vivo using the AKT/NICD genetically engineered murine CCA model. RESULTS: We traced LOX expression to hepatic stellate cells and specifically hepatic stellate cell-derived inflammatory cancer-associated fibroblasts and found that cancer-associated fibroblast-driven LOX increases oxidative phosphorylation and metabolic fitness of CCA, and regulates mitochondrial function through transcription factor A, mitochondrial. Inhibiting LOX activity in vivo impedes CCA development and progression. Our work highlights that LOX alters tumor microenvironment-directed transcriptional reprogramming of CCA cells by facilitating the expression of the oxidative phosphorylation pathway and by increasing stemness and mobility. CONCLUSIONS: Increased LOX is driven by stromal inflammatory cancer-associated fibroblasts and correlates with diminished survival of patients with CCA. Modulating the LOX activity can serve as a novel tumor microenvironment-directed therapeutic strategy in bile duct pathologies.

Tumor Angiocrine Signaling: Novel Targeting Opportunity in Cancer.

The vascular endothelium supplies nutrients and oxygen to different body organs and supports the progression of diseases such as cancer through angiogenesis. Pathological angiogenesis remains a challenge as most patients develop resistance to the approved anti-angiogenic therapies. Therefore, a better understanding of endothelium signaling will support the development of more effective treatments. Over the past two decades, the emerging consensus suggests that the role of endothelial cells in tumor development has gone beyond angiogenesis. Instead, endothelial cells are now considered active participants in the tumor microenvironment, secreting angiocrine factors such as cytokines, growth factors, and chemokines, which instruct their proximate microenvironments. The function of angiocrine signaling is being uncovered in different fields, such as tissue homeostasis, early development, organogenesis, organ regeneration post-injury, and tumorigenesis. In this review, we elucidate the intricate role of angiocrine signaling in cancer progression, including distant metastasis, tumor dormancy, pre-metastatic niche formation, immune evasion, and therapy resistance.

Fibroblast-derived matrix models desmoplastic properties and forms a prognostic signature in cancer progression.

The desmoplastic reaction observed in many cancers is a hallmark of disease progression and prognosis, particularly in breast and pancreatic cancer. Stromal-derived extracellular matrix (ECM) is significantly altered in desmoplasia, and as such plays a critical role in driving cancer progression. Using fibroblast-derived matrices (FDMs), we show that cancer cells have increased growth on cancer associated FDMs, when compared to FDMs derived from non-malignant tissue (normal) fibroblasts. We assess the changes in ECM characteristics from normal to cancer-associated stroma at the primary tumor site. Compositional, structural, and mechanical analyses reveal significant differences, with an increase in abundance of core ECM proteins, coupled with an increase in stiffness and density in cancer-associated FDMs. From compositional changes of FDM, we derived a 36-ECM protein signature, which we show matches in large part with the changes in pancreatic ductal adenocarcinoma (PDAC) tumor and metastases progression. Additionally, this signature also matches at the transcriptomic level in multiple cancer types in patients, prognostic of their survival. Together, our results show relevance of FDMs for cancer modelling and identification of desmoplastic ECM components for further mechanistic studies.

FES null mice demonstrate a reduction in neutrophil dependent pancreatic cancer metastatic burden.

Patients with pancreatic ductal adenocarcinoma (PDAC) have a dismal 5-year survival rate of less than 10%, predominantly due to delayed diagnosis and a lack of effective treatment options. In the PDAC tumor microenvironment (TME), neutrophils are among the immune cell types that are most prevalent and are linked to a poor clinical prognosis. However, treatments that target tumor-associated neutrophils are limited despite recent developments in our understanding of neutrophil function in cancer. The feline sarcoma oncogene (FES) is a nonreceptor tyrosine kinase previously associated with leukemia and hematopoietic homeostasis. Here we describe a newly derived FES null mouse with no distinct phenotype and no defects in hematopoietic homeostasis including neutrophil viability. The immune cell composition and neutrophil population were analyzed with flow cytometry, colony-forming unit (CFU) assay, and a neutrophil viability assay, while the response to PDAC was examined with an in vivo cancer model. In an experimental metastasis model, the FES null model displayed a reduced PDAC hepatic metastatic burden and a reduction in neutrophils granulocytes. Accordingly, our results indicate FES as a potential target for PDAC TME modulation.

Extracting, quantifying, and comparing dynamical and biomechanical properties of living matter through single particle tracking.

A panoply of new tools for tracking single particles and molecules has led to an explosion of experimental data, leading to novel insights into physical properties of living matter governing cellular development and function, health and disease. In this Perspective, we present tools to investigate the dynamics and mechanics of living systems from the molecular to cellular scale via single-particle techniques. In particular, we focus on methods to measure, interpret, and analyse complex data sets that are associated with forces, materials properties, transport, and emergent organisation phenomena within biological and soft-matter systems. Current approaches, challenges, and existing solutions in the associated fields are outlined in order to support the growing community of researchers at the interface of physics and the life sciences. Each section focuses not only on the general physical principles and the potential for understanding living matter, but also on details of practical data extraction and analysis, discussing limitations, interpretation, and comparison across different experimental realisations and theoretical frameworks. Particularly relevant results are introduced as examples. While this Perspective describes living matter from a physical perspective, highlighting experimental and theoretical physics techniques relevant for such systems, it is also meant to serve as a solid starting point for researchers in the life sciences interested in the implementation of biophysical methods.

Tipping the balance: New insights into stromal regulation of cancer.

Desmoplastic extracellular matrix (ECM) exerts a seemingly paradoxical role in cancer, restricting or promoting progression. Two recent back-to-back Nature reports shed light on this conundrum, revealing the role of different hepatic stellate cell (HSC) populations and different collagen I cleavage states in directing the progression of cancer.

Effects of acute exercise training on tumor outcomes in men with localized prostate cancer: A randomized controlled trial.

Postdiagnosis physical activity is associated with improved cancer outcomes, but biological mechanisms mediating anticancer effects remain unclear. Recent findings suggest that physiological adaptations to acute exercise comprise potential anticancer effects, but these remain poorly explored in clinical settings. The objective of this study was to explore the effects of a single exercise bout on tumor oxygenation and immune cell infiltration in patients with prostate cancer. Thirty patients with localized prostate cancer were randomized (2:1) to either one high-intensity interval training bout or no exercise on the day before radical prostatectomy. Immunohistochemical analyses were performed on prostatic tissue from surgery and assessed for tumor hypoxia, natural killer (NK) cell infiltration, and microvessel density (MVD). Acute systemic response in blood lymphocytes, epinephrine, norepinephrine, IL-6, tumor necrosis factor, cortisol, lactate, and glucose was also evaluated. We did not find between-group differences in tumor hypoxia (Mann-Whitney U test, U = 83.5, p = 0.604) or NK cell infiltration (U = 77.0, p = 0.328). Also, no significant correlation was found between MVD and tumor hypoxia or NK cell infiltration. One exercise bout is likely insufficient to modulate tumor hypoxia or NK cell infiltration. Future studies may elucidate if an accumulation of several exercise bouts can impact these outcomes (NCT03675529, www.clinicaltrials.gov).

Neutrophil granulocytes influence on extracellular matrix in cancer progression.

One in three persons will develop cancer in their lifetime (Siegel RL, Miller KD, Fuchs HE, Jemal A. CA Cancer J Clin 71: 7-33, 2021) and the majority of these patients will die from the spread of cancer throughout their body-a process known as metastasis. Metastasis is strongly regulated by the tumor microenvironment (TME) comprising cellular and noncellular components. In this review, we will focus on the role of neutrophils regulating the extracellular matrix (ECM), enabling ECM remodeling and cancer progression. In particular, we highlight the role of neutrophil-secreted proteases (NSP) and how these promote metastasis.

Matritecture: Mapping the extracellular matrix architecture during health and disease.

All cells in multicellular organisms are housed in the extracellular matrix (ECM), an acellular edifice built up by more than a thousand proteins and glycans. Cells engage in a reciprocal relationship with the ECM; they build, inhabit, maintain, and remodel the ECM, while, in turn, the ECM regulates their behavior. The homeostatic balance of cell-ECM interactions can be lost, due to ageing, irritants or diseases, which results in aberrant cell behavior. The ECM can suppress or promote disease progression, depending on the information relayed to cells. Instructions come in the form of biochemical (e.g., composition), biophysical (e.g., stiffness), and topographical (e.g., structure) cues. While advances have been made in many areas, we only have a very limited grasp of ECM topography. A detailed atlas deciphering the spatiotemporal arrangement of all ECM proteins is lacking. We feel that such an extracellular matrix architecture (matritecture) atlas should be a priority goal for ECM research. In this commentary, we will discuss the need to resolve the spatiotemporal matritecture to identify potential disease triggers and therapeutic targets and present strategies to address this goal. Such a detailed matritecture atlas will not only identify disease-specific ECM structures but may also guide future strategies to restructure disease-related ECM patterns reverting to a normal pattern.

Filopodia rotate and coil by actively generating twist in their actin shaft.

Filopodia are actin-rich structures, present on the surface of eukaryotic cells. These structures play a pivotal role by allowing cells to explore their environment, generate mechanical forces or perform chemical signaling. Their complex dynamics includes buckling, pulling, length and shape changes. We show that filopodia additionally explore their 3D extracellular space by combining growth and shrinking with axial twisting and buckling. Importantly, the actin core inside filopodia performs a twisting or spinning motion which is observed for a range of cell types spanning from earliest development to highly differentiated tissue cells. Non-equilibrium physical modeling of actin and myosin confirm that twist is an emergent phenomenon of active filaments confined in a narrow channel which is supported by measured traction forces and helical buckles that can be ascribed to accumulation of sufficient twist. These results lead us to conclude that activity induced twisting of the actin shaft is a general mechanism underlying fundamental functions of filopodia.

Fibrotic activity quantified in serum by measurements of type III collagen pro-peptides can be used for prognosis across different solid tumor types.

Due to activation of fibroblast into cancer-associated fibroblasts, there is often an increased deposition of extracellular matrix and fibrillar collagens, e.g. type III collagen, in the tumor microenvironment (TME) that leads to tumor fibrosis (desmoplasia). Tumor fibrosis is closely associated with treatment response and poor prognosis for patients with solid tumors. To assure that the best possible treatment option is provided for patients, there is medical need for identifying patients with high (or low) fibrotic activity in the TME. Measuring unique collagen fragments such as the pro-peptides released into the bloodstream during fibrillar collagen deposition in the TME can provide a non-invasive measure of the fibrotic activity. Based on data from 8 previously published cohorts, this review provides insight into the prognostic value of quantifying tumor fibrosis by measuring the pro-peptide of type III collagen in serum of a total of 1692 patients with different solid tumor types and discusses the importance of tumor fibrosis for understanding prognosis and for potentially guiding future drug development efforts that aim at overcoming the poor outcome associated with a fibrotic TME.

Modeling Metastatic Colonization in a Decellularized Organ Scaffold-Based Perfusion Bioreactor.

Metastatic cancer spread is responsible for most cancer-related deaths. To colonize a new organ, invading cells adapt to, and remodel, the local extracellular matrix (ECM), a network of proteins and proteoglycans underpinning all tissues, and a critical regulator of homeostasis and disease. However, there is a major lack in tools to study cancer cell behavior within native 3D ECM. Here, an in-house designed bioreactor, where mouse organ ECM scaffolds are perfused and populated with cells that are challenged to colonize it, is presented. Using a specialized bioreactor chamber, it is possible to monitor cell behavior microscopically (e.g., proliferation, migration) within the organ scaffold. Cancer cells in this system recapitulate cell signaling observed in vivo and remodel complex native ECM. Moreover, the bioreactors are compatible with co-culturing cell types of different genetic origin comprising the normal and tumor microenvironment. This degree of experimental flexibility in an organ-specific and 3D context, opens new possibilities to study cell-cell and cell-ECM interplay and to model diseases in a controllable organ-specific system ex vivo.

Fiber finding algorithm using stepwise tracing to identify biopolymer fibers in noisy 3D images.

We present a novel fiber finding algorithm (FFA) that will permit researchers to detect and return traces of individual biopolymers. Determining the biophysical properties and structural cues of biopolymers can permit researchers to assess the progression and severity of disease. Confocal microscopy images are a useful method for observing biopolymer structures in three dimensions, but their utility for identifying individual biopolymers is impaired by noise inherent in the acquisition process, including convolution from the point spread function (PSF). The new, iterative FFA we present here 1) measures a microscope's PSF and uses it as a metric for identifying fibers against the background; 2) traces each fiber within a cone angle; and 3) blots out the identified trace before identifying another fiber. Blotting out the identified traces in each iteration allows the FFA to detect and return traces of single fibers accurately and efficiently-even within fiber bundles. We used the FFA to trace unlabeled collagen type I fibers-a biopolymer used to mimic the extracellular matrix in in vitro cancer assays-imaged by confocal reflectance microscopy in three dimensions, enabling quantification of fiber contour length, persistence length, and three-dimensional (3D) mesh size. Based on 3D confocal reflectance microscopy images and the PSF, we traced and measured the fibers to confirm that colder gelation temperatures increased fiber contour length, persistence length, and 3D mesh size-thereby demonstrating the FFA's use in quantifying biopolymers' structural and physical cues from noisy microscope images.

Organ-Specific, Fibroblast-Derived Matrix as a Tool for Studying Breast Cancer Metastasis.

During the metastatic process, breast cancer cells must come into contact with the extra-cellular matrix (ECM) at every step. The ECM provides both structural support and biochemical cues, and cell-ECM interactions can lead to changes in drug response. Here, we used fibroblast-derived ECM (FDM) to perform high throughput drug screening of 4T1 breast cancer cells on metastatic organ ECM (lung), and we see that drug response differs from treatment on plastic. The FDMs that we can produce from different organs are abundant in and contains a complex mixture of ECM proteins. We also show differences in ECM composition between the primary site and secondary organ sites. Furthermore, we show that global kinase signalling of 4T1 cells on the ECM is relatively unchanged between organs, while changes in signalling compared to plastic are significant. Our study highlights the importance of context when testing drug response in vitro, showing that consideration of the ECM is critically important.

Suppression of tumor-associated neutrophils by lorlatinib attenuates pancreatic cancer growth and improves treatment with immune checkpoint blockade.

Pancreatic ductal adenocarcinoma (PDAC) patients have a 5-year survival rate of only 8% largely due to late diagnosis and insufficient therapeutic options. Neutrophils are among the most abundant immune cell type within the PDAC tumor microenvironment (TME), and are associated with a poor clinical prognosis. However, despite recent advances in understanding neutrophil biology in cancer, therapies targeting tumor-associated neutrophils are lacking. Here, we demonstrate, using pre-clinical mouse models of PDAC, that lorlatinib attenuates PDAC progression by suppressing neutrophil development and mobilization, and by modulating tumor-promoting neutrophil functions within the TME. When combined, lorlatinib also improves the response to anti-PD-1 blockade resulting in more activated CD8 + T cells in PDAC tumors. In summary, this study identifies an effect of lorlatinib in modulating tumor-associated neutrophils, and demonstrates the potential of lorlatinib to treat PDAC.

Decellularization of the Murine Cardiopulmonary Complex.

We present here a decellularization protocol for mouse heart and lungs. It produces structural ECM scaffolds that can be used to analyze ECM topology and composition. It is based on a microsurgical procedure designed to catheterize the trachea and aorta of a euthanized mouse to perfuse the heart and lungs with decellularizing agents. The decellularized cardiopulmonary complex can subsequently be immunostained to reveal the location of structural ECM proteins. The whole procedure can be completed in 4 days. The ECM scaffolds resulting from this protocol are free of dimensional distortions. The absence of cells enables structural examination of ECM structures down to submicron resolution in 3D. This protocol can be applied to healthy and diseased tissue from mice as young as 4-weeks old, including mouse models of fibrosis and cancer, opening the way to determine ECM remodeling associated with cardiopulmonary disease.

Deciphering the temporal heterogeneity of cancer-associated fibroblast subpopulations in breast cancer.

BACKGROUND: Cancer-associated fibroblasts (CAFs) comprise a heterogeneous population of stromal cells within the tumour microenvironment. CAFs exhibit both tumour-promoting and tumour-suppressing functions, making them exciting targets for improving cancer treatments. Careful isolation, identification, and characterisation of CAF heterogeneity is thus necessary for ex vivo validation and future implementation of CAF-targeted strategies in cancer. METHODS: Murine 4T1 (metastatic) and 4T07 (poorly/non-metastatic) orthotopic triple negative breast cancer tumours were collected after 7, 14, or 21 days. The tumours were analysed via flow cytometry for the simultaneous expression of six CAF markers: alpha smooth muscle actin (αSMA), fibroblast activation protein alpha (FAPα), platelet derived growth factor receptor alpha and beta (PDGFRα and PDGFRß), CD26/DPP4 and podoplanin (PDPN). All non-CAFs were excluded from the analysis using a lineage marker cocktail (CD24, CD31, CD45, CD49f, EpCAM, LYVE-1, and TER-119). In total 128 murine tumours and 12 healthy mammary fat pads were analysed. RESULTS: We have developed a multicolour flow cytometry strategy based on exclusion of non-CAFs and successfully employed this to explore the temporal heterogeneity of freshly isolated CAFs in the 4T1 and 4T07 mouse models of triple-negative breast cancer. Analysing 128 murine tumours, we identified 5-6 main CAF populations and numerous minor ones based on the analysis of αSMA, FAPα, PDGFRα, PDGFRß, CD26, and PDPN. All markers showed temporal changes with a distinct switch from primarily PDGFRα+ fibroblasts in healthy mammary tissue to predominantly PDGFRß+ CAFs in tumours. CD26+ CAFs emerged as a large novel subpopulation, only matched by FAPα+ CAFs in abundance. CONCLUSION: We demonstrate that multiple subpopulations of CAFs co-exist in murine triple negative breast cancer, and that the abundance and dynamics for each marker differ depending on tumour type and time. Our results form the foundation needed to isolate and characterise specific CAF populations, and ultimately provide an opportunity to therapeutically target specific CAF subpopulations.