MMP14 expression and collagen remodelling support uterine leiomyosarcoma aggressiveness.

Fibrillar collagen deposition, stiffness and downstream signalling support the development of leiomyomas (LMs), common benign mesenchymal tumours of the uterus, and are associated with aggressiveness in multiple carcinomas. Compared with epithelial carcinomas, however, the impact of fibrillar collagens on malignant mesenchymal tumours, including uterine leiomyosarcoma (uLMS), remains elusive. In this study, we analyse the network morphology and density of fibrillar collagens combined with the gene expression within uLMS, LM and normal myometrium (MM). We find that, in contrast to LM, uLMS tumours present low collagen density and increased expression of collagen-remodelling genes, features associated with tumour aggressiveness. Using collagen-based 3D matrices, we show that matrix metalloproteinase-14 (MMP14), a central protein with collagen-remodelling functions that is particularly overexpressed in uLMS, supports uLMS cell proliferation. In addition, we find that, unlike MM and LM cells, uLMS proliferation and migration are less sensitive to changes in collagen substrate stiffness. We demonstrate that uLMS cell growth in low-stiffness substrates is sustained by an enhanced basal yes-associated protein 1 (YAP) activity. Altogether, our results indicate that uLMS cells acquire increased collagen remodelling capabilities and are adapted to grow and migrate in low collagen and soft microenvironments. These results further suggest that matrix remodelling and YAP are potential therapeutic targets for this deadly disease.

Attenuating Epithelial-to-Mesenchymal Transition in Cancer through Angiopoietin-Like 4 Inhibition in a 3D Tumor Microenvironment Model.

Epithelial-to-mesenchymal transition (EMT) plays a crucial role in metastatic cancer progression, and current research, which relies heavily on 2D monolayer cultures, falls short in recapitulating the complexity of a 3D tumor microenvironment. To address this limitation, a transcriptomic meta-analysis is conducted on diverse cancer types undergoing EMT in 2D and 3D cultures. It is found that mechanotransduction is elevated in 3D cultures and is further intensified during EMT, but not during 2D EMT. This analysis reveals a distinct 3D EMT gene signature, characterized by extracellular matrix remodeling coordinated by angiopoietin-like 4 (Angptl4) along with other canonical EMT regulators. Utilizing hydrogel-based 3D matrices with adjustable mechanical forces, 3D cancer cultures are established at varying physiological stiffness levels. A YAP:EGR-1 mediated up-regulation of Angptl4 expression is observed, accompanied by an upregulation of mesenchymal markers, at higher stiffness during cancer EMT. Suppression of Angptl4 using antisense oligonucleotides or anti-cAngptl4 antibodies leads to a dose-dependent abolishment of EMT-mediated chemoresistance and tumor self-organization in 3D, ultimately resulting in diminished metastatic potential and stunted growth of tumor xenografts. This unique programmable 3D cancer cultures simulate stiffness levels in the tumor microenvironment and unveil Angptl4 as a promising therapeutic target to inhibit EMT and impede cancer progression.

The drug efficacy testing in 3D cultures platform identifies effective drugs for ovarian cancer patients.

Most patients with advanced ovarian cancer (OC) relapse and progress despite systemic therapy, pointing to the need for improved and tailored therapy options. Functional precision medicine can help to identify effective therapies for individual patients in a clinically relevant timeframe. Here, we present a scalable functional precision medicine platform: DET3Ct (Drug Efficacy Testing in 3D Cultures), where the response of patient cells to drugs and drug combinations are quantified with live-cell imaging. We demonstrate the delivery of individual drug sensitivity profiles in 20 samples from 16 patients with ovarian cancer in both 2D and 3D culture formats, achieving over 90% success rate in providing results six days after operation. In this cohort all patients received carboplatin. The carboplatin sensitivity scores were significantly different for patients with a progression free interval (PFI) less than or equal to 12 months and those with more than 12 months (p < 0.05). We find that the 3D culture format better retains proliferation and characteristics of the in vivo setting. Using the DET3Ct platform we evaluate 27 tailored combinations with results available 10 days after operation. Notably, carboplatin and A-1331852 (Bcl-xL inhibitor) showed an additive effect in four of eight OC samples tested, while afatinib and A-1331852 led to synergy in five of seven OC models. In conclusion, our 3D DET3Ct platform can rapidly define potential, clinically relevant data on efficacy of existing drugs in OC for precision medicine purposes, as well as provide insights on emerging drugs and drug combinations that warrant testing in clinical trials.

Efficacy and Safety of Glycosphingolipid SSEA-4 Targeting CAR-T Cells in an Ovarian Carcinoma Model.

Chimeric antigen receptor (CAR) T-cell immunotherapies for solid tumors face critical challenges such as heterogeneous antigen expression. We characterized stage-specific embryonic antigen-4 (SSEA-4) cell-surface glycolipid as a target for CAR T-cell therapy. SSEA-4 is mainly expressed during embryogenesis but is also found in several cancer types making it an attractive tumor-associated antigen. Anti-SSEA-4 CAR-T cells were generated and assessed preclinically in vitro and in vivo for antitumor response and safety. SSEA-4 CAR-T cells effectively eliminated SSEA-4-positive cells in all the tested cancer cell lines, whereas SSEA-4-negative cells lines were not targeted. In vivo efficacy and safety studies using NSG mice and the high-grade serous ovarian cancer cell line OVCAR4 demonstrated a remarkable and specific antitumor response at all the CAR T-cell doses used. At high T-cell doses, CAR T cell-treated mice showed signs of health deterioration after a follow-up period. However, the severity of toxicity was reduced with a delayed onset when lower CAR T-cell doses were used. Our data demonstrate the efficacy of anti-SSEA-4 CAR T-cell therapy; however, safety strategies, such as dose-limiting and/or equipping CAR-T cells with combinatorial antigen recognition should be implemented for its potential clinical translation.

The extracellular matrix - immune microenvironment crosstalk in cancer therapy: Challenges and opportunities.

Targeting the tumour immune microenvironment (TIME) by cancer immunotherapy has led to improved patient outcomes. However, response to these treatments is heterogeneous and cancer-type dependant. The therapeutic activity of classical cancer therapies such as chemotherapy, radiotherapy, and surgical oncology is modulated by alterations of the TIME. A major regulator of immune cell function and resistance to both immune and classical therapies is the extracellular matrix (ECM). Concurrently, cancer therapies reshape the TIME as well as the ECM, causing both pro- and anti-tumour responses. Accordingly, the TIME-ECM crosstalk presents attractive opportunities to improve therapy outcomes. Here, we review the molecular crosstalk between the TIME and the ECM in cancer and its implications in cancer progression and clinical intervention. Additionally, we discuss examples and future directions of ECM and TIME co-targeting in combination with oncological therapies including surgery, chemotherapy, and radiotherapy.

Tumour Suppressor Neuron Navigator 3 and Matrix Metalloproteinase 14 are Co-expressed in Most Melanomas but Downregulated in Thick Tumours.

Melanoma is a highly metastatic tumour originating from neural crest-derived melanocytes. The aim of this study was to analyse the expression of neuron navigator 3 (NAV3) in relation to membrane type-1 matrix metalloproteinase MMP14, a major regulator of invasion, in 40 primary melanomas, 15 benign naevi and 2 melanoma cell lines. NAV3 copy number changes were found in 18/27 (67%) primary melanomas, so that deletions dominated (16/27 of samples, 59%). NAV3 protein was found to be localized at the leading edge of migrating melanoma cells in vitro. Silencing of NAV3 reduced both melanoma cell migration in 2-dimensional conditions, as well as sprouting in 3-dimensional collagen I. NAV3 protein expression correlated with MMP14 in 26/37 (70%) primary melanomas. NAV3 and MMP14 were co-expressed in all tumours with Breslow thickness < 1 mm, in 11/23 of mid-thickness tumours (1-5 mm), but in only 1/6 samples of thick (> 5 mm) melanomas. Altogether, NAV3 number changes are frequent in melanomas, and NAV3 and MMP14, while expressed in all thin melanomas, are often downregulated in thicker tumours, suggesting that the lack of both NAV3 and MMP14 favours melanoma progression.

PROX1 transcription factor controls rhabdomyosarcoma growth, stemness, myogenic properties and therapeutic targets.

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft-tissue cancer with features of skeletal muscle. Because of poor survival of RMS patients and severe long-term side effects of RMS therapies, alternative RMS therapies are urgently needed. Here we show that the prospero-related homeobox 1 (PROX1) transcription factor is highly expressed in RMS tumors regardless of their cell type of origin. We demonstrate that PROX1 is needed for RMS cell clonogenicity, growth and tumor formation. PROX1 gene silencing repressed several myogenic and tumorigenic transcripts and transformed the RD cell transcriptome to resemble that of benign mesenchymal stem cells. Importantly, we found that fibroblast growth factor receptors (FGFR) mediated the growth effects of PROX1 in RMS. Because of receptor cross-compensation, paralog-specific FGFR inhibition did not mimic the effects of PROX1 silencing, whereas a pan-FGFR inhibitor ablated RMS cell proliferation and induced apoptosis. Our findings uncover the critical role of PROX1 in RMS and offer insights into the mechanisms that regulate RMS development and growth. As FGFR inhibitors have already been tested in clinical phase I/II trials in other cancer types, our findings provide an alternative option for RMS treatment.

An Approach for Systems-Level Understanding of Prostate Cancer from High-Throughput Data Integration to Pathway Modeling and Simulation.

To understand complex diseases, high-throughput data are generated at large and multiple levels. However, extracting meaningful information from large datasets for comprehensive understanding of cell phenotypes and disease pathophysiology remains a major challenge. Despite tremendous advances in understanding molecular mechanisms of cancer and its progression, current knowledge appears discrete and fragmented. In order to render this wealth of data more integrated and thus informative, we have developed a GECIP toolbox to investigate the crosstalk and the responsible genes'/proteins' connectivity of enriched pathways from gene expression data. To implement this toolbox, we used mainly gene expression datasets of prostate cancer, and the three datasets were GSE17951, GSE8218, and GSE1431. The raw samples were processed for normalization, prediction of differentially expressed genes, and the prediction of enriched pathways for the differentially expressed genes. The enriched pathways have been processed for crosstalk degree calculations for which number connections per gene, the frequency of genes in the pathways, sharing frequency, and the connectivity have been used. For network prediction, protein-protein interaction network database FunCoup2.0 was used, and cytoscape software was used for the network visualization. In our results, we found that there were enriched pathways 27, 45, and 22 for GSE17951, GSE8218, and GSE1431, respectively, and 11 pathways in common between all of them. From the crosstalk results, we observe that focal adhesion and PI3K pathways, both experimentally proven central for cellular output upon perturbation of numerous individual/distinct signaling pathways, displayed highest crosstalk degree. Moreover, we also observe that there were more critical pathways which appear to be highly significant, and these pathways are HIF1a, hippo, AMPK, and Ras. In terms of the pathways' components, GSK3B, YWHAE, HIF1A, ATP1A3, and PRKCA are shared between the aforementioned pathways and have higher connectivity with the pathways and the other pathway components. Finally, we conclude that the focal adhesion and PI3K pathways are the most critical pathways, and since for many other pathways, high-rank enrichment did not translate to high crosstalk degree, the global impact of one pathway on others appears distinct from enrichment.

Mechanical Confinement and DDR1 Signaling Synergize to Regulate Collagen-Induced Apoptosis in Rhabdomyosarcoma Cells.

Fibrillar collagens promote cell proliferation, migration, and survival in various epithelial cancers and are generally associated with tumor aggressiveness. However, the impact of fibrillar collagens on soft tissue sarcoma behavior remains poorly understood. Unexpectedly, this study finds that fibrillar collagen-related gene expression is associated with favorable patient prognosis in rhabdomyosarcoma. By developing and using collagen matrices with distinct stiffness and in vivo-like microarchitectures, this study uncovers that the activation of DDR1 has pro-apoptotic and of integrin ß1 pro-survival function, specifically in 3D rhabdomyosarcoma cell cultures. It demonstrates that rhabdomyosarcoma cell-intrinsic or extrinsic matrix remodeling promotes cell survival. Mechanistically, the 3D-specific collagen-induced apoptosis results from a dual DDR1-independent and a synergistic DDR1-dependent TRPV4-mediated response to mechanical confinement. Altogether, these results indicate that dense microfibrillar collagen-rich microenvironments are detrimental to rhabdomyosarcoma cells through an apoptotic response orchestrated by the induction of DDR1 signaling and mechanical confinement. This mechanism helps to explain the preference of rhabdomyosarcoma cells to grow in and metastasize to low fibrillar collagen microenvironments such as the lung.

Molecular, cellular and systemic aspects of epithelial ovarian cancer and its tumor microenvironment.

Ovarian cancer encompasses a heterogeneous group of malignancies that involve the ovaries, fallopian tubes and the peritoneal cavity. Despite major advances made within the field of cancer, the majority of patients with ovarian cancer are still being diagnosed at an advanced stage of the disease due to lack of effective screening tools. The overall survival of these patients has, therefore, not substantially improved over the past decades. Most patients undergo debulking surgery and treatment with chemotherapy, but often micrometastases remain and acquire resistance to the therapy, eventually leading to disease recurrence. Here, we summarize the current knowledge in epithelial ovarian cancer development and metastatic progression. For the most common subtypes, we focus further on the properties and functions of the immunosuppressive tumor microenvironment, including the extracellular matrix. Current and future treatment modalities are discussed and finally we provide an overview of the different experimental models used to develop novel therapies.

Chemotherapy as a regulator of extracellular matrix-cell communication: Implications in therapy resistance.

The development of most solid cancers, including pancreatic, breast, lung, liver, and ovarian cancer, involves a desmoplastic reaction: a process of major remodeling of the extracellular matrix (ECM) affecting the ECM composition, mechanics, and microarchitecture. These properties of the ECM influence key cancer cell functions, including treatment resistance. Furthermore, emerging data show that various chemotherapeutic treatments lead to alterations in ECM features and ECM-cell communication. Here, we summarize the current knowledge around the effects of chemotherapy on both the ECM remodeling and ECM-cell signaling and discuss the implications of these alterations on distinct mechanisms of chemoresistance. Additionally, we provide an overview of current therapeutic strategies and ongoing clinical trials utilizing anti-cancer drugs to target the ECM-cell communication and explore the future challenges of these strategies.

FOXP3+ T cells in uterine sarcomas are associated with favorable prognosis, low extracellular matrix expression and reduced YAP activation.

Uterine sarcomas are rare but deadly malignancies without effective treatment. Immunotherapy is a promising new approach to treat these tumors but has shown heterogeneous effects in sarcoma patients. With the goal of identifying key factors for improved patient treatment, we characterized the tumor immune landscape in 58 uterine sarcoma cases with full clinicopathological annotation. Immune cell characterization revealed the overall prevalence of FOXP3+ cells and pro-tumor M2-like macrophages. Hierarchical clustering of patients showed four tumor type-independent immune signatures, where infiltration of FOXP3+ cells and M1-like macrophages associated with favorable prognosis. High CD8+/FOXP3+ ratio in UUS and ESS correlated with poor survival, upregulation of immunosuppressive markers, extracellular matrix (ECM)-related genes and proteins, and YAP activation. This study shows that uterine sarcomas present distinct immune signatures with prognostic value, independent of tumor type, and suggests that targeting the ECM could be beneficial for future treatments.

The expression and prognostic relevance of CDH3 in tongue squamous cell carcinoma.

P-cadherin (CDH3) is a cell-to-cell adhesion molecule that regulates several cellular homeostatic processes in normal tissues. Lack of CDH3 expression is associated with aggressive behavior in oral squamous cell carcinoma (OSCC). Previous studies have shown that CDH3 is downregulated in high-grade OSCC and its reduced expression is predictive for poorer survival. The aim of this study was to evaluate the expression and prognostic relevance of CDH3 in tongue squamous cell carcinoma (TSCC). A retrospective series of 211 TSCC and 50 lymph node samples were stained immunohistochemically with polyclonal antibody (anti-CDH3). CDH3 expression was assessed semi-quantitatively with light microscopy. Fisher's exact test was used to compare patient and tumor characteristics, and the correlations were tested by Spearman correlation. Survival curves were drawn by the Kaplan-Meier method and analyzed by the log-rank test. Univariate and multivariate Cox regression was used to estimate the association between CDH3 expression and survival. CDH3 expression did not affect TSCC patient's disease-specific survival or overall survival. Strong CDH3 expression in the primary tumor predicted poor disease-specific and overall survival in patients with recurrent disease. CDH3 expression in lymph nodes without metastasis was negative in all cases. CDH3 expression was positive in all lymph node metastases with extranodal extension. In contrast to previous report about the prognostic value of CDH3 in OSCC, we were not able to validate the result in TSCC.

Proliferative diabetic retinopathy transcriptomes reveal angiogenesis, anti-angiogenic therapy escape mechanisms, fibrosis and lymphatic involvement.

Proliferative diabetic retinopathy (PDR) is a sight-threatening diabetic complication in urgent need of new therapies. In this study we identify potential molecular mechanisms and target candidates in the pathogenesis of PDR fibrovascular tissue formation. We performed mRNA sequencing of RNA isolated from eleven excised fibrovascular membranes of type 1 diabetic PDR patients and two non-diabetic patients with rhegmatogenous retinal detachment with proliferative vitreoretinopathy. We determined differentially expressed genes between these groups and performed pathway and gene ontology term enrichment analyses to identify potential underlying mechanisms, pathways, and regulators. Multiple pro-angiogenic processes, including VEGFA-dependent and -independent pathways, as well as processes related to lymphatic development, epithelial to mesenchymal transition (EMT), wound healing, inflammation, fibrosis, and extracellular matrix (ECM) composition, were overrepresented in PDR. Overrepresentation of different angiogenic processes may help to explain the transient nature of the benefits that many patients receive from current intravitreal anti-angiogenic therapies, highlighting the importance of combinatorial treatments. Enrichment of genes and pathways related to lymphatic development indicates that targeting lymphatic involvement in PDR progression could have therapeutic relevance. Together with overrepresentation of EMT and fibrosis as well as differential ECM composition, these findings demonstrate the complexity of PDR fibrovascular tissue formation and provide avenues for the development of novel treatments.

WNT2 activation through proximal germline deletion predisposes to small intestinal neuroendocrine tumors and intestinal adenocarcinomas.

Many hereditary cancer syndromes are associated with an increased risk of small and large intestinal adenocarcinomas. However, conditions bearing a high risk to both adenocarcinomas and neuroendocrine tumors are yet to be described. We studied a family with 16 individuals in four generations affected by a wide spectrum of intestinal tumors, including hyperplastic polyps, adenomas, small intestinal neuroendocrine tumors, and colorectal and small intestinal adenocarcinomas. To assess the genetic susceptibility and understand the novel phenotype, we utilized multiple molecular methods, including whole genome sequencing, RNA sequencing, single cell sequencing, RNA in situ hybridization and organoid culture. We detected a heterozygous deletion at the cystic fibrosis locus (7q31.2) perfectly segregating with the intestinal tumor predisposition in the family. The deletion removes a topologically associating domain border between CFTR and WNT2, aberrantly activating WNT2 in the intestinal epithelium. These consequences suggest that the deletion predisposes to small intestinal neuroendocrine tumors and small and large intestinal adenocarcinomas, and reveals the broad tumorigenic effects of aberrant WNT activation in the human intestine.

Co-evolution of matrisome and adaptive adhesion dynamics drives ovarian cancer chemoresistance.

Due to its dynamic nature, the evolution of cancer cell-extracellular matrix (ECM) crosstalk, critically affecting metastasis and treatment resistance, remains elusive. Our results show that platinum-chemotherapy itself enhances resistance by progressively changing the cancer cell-intrinsic adhesion signaling and cell-surrounding ECM. Examining ovarian high-grade serous carcinoma (HGSC) transcriptome and histology, we describe the fibrotic ECM heterogeneity at primary tumors and distinct metastatic sites, prior and after chemotherapy. Using cell models from systematic ECM screen to collagen-based 2D and 3D cultures, we demonstrate that both specific ECM substrates and stiffness increase resistance to platinum-mediated, apoptosis-inducing DNA damage via FAK and ß1 integrin-pMLC-YAP signaling. Among such substrates around metastatic HGSCs, COL6 was upregulated by chemotherapy and enhanced the resistance of relapse, but not treatment-naïve, HGSC organoids. These results identify matrix adhesion as an adaptive response, driving HGSC aggressiveness via co-evolving ECM composition and sensing, suggesting stromal and tumor strategies for ECM pathway targeting.

CD109-GP130 interaction drives glioblastoma stem cell plasticity and chemoresistance through STAT3 activity.

Glioma stem cells (GSCs) drive propagation and therapeutic resistance of glioblastomas, the most aggressive diffuse brain tumors. However, the molecular mechanisms that maintain the stemness and promote therapy resistance remain poorly understood. Here we report CD109/STAT3 axis as crucial for the maintenance of stemness and tumorigenicity of GSCs and as a mediator of chemoresistance. Mechanistically, CD109 physically interacts with glycoprotein 130 to promote activation of the IL-6/STAT3 pathway in GSCs. Genetic depletion of CD109 abolished the stemness and self-renewal of GSCs and impaired tumorigenicity. Loss of stemness was accompanied with a phenotypic shift of GSCs to more differentiated astrocytic-like cells. Importantly, genetic or pharmacologic targeting of CD109/STAT3 axis sensitized the GSCs to chemotherapy, suggesting that targeting CD109/STAT3 axis has potential to overcome therapy resistance in glioblastoma.

Mesothelin-Specific CAR T Cells Target Ovarian Cancer.

New therapeutic options for patients with ovarian cancer are urgently needed. Therefore, we evaluated the efficacy of two second-generation mesothelin (MSLN)-directed CAR T cells in orthotopic mouse models of ovarian cancer. Treatment with CAR T cells expressing an MSLN CAR construct including the CD28 domain (M28z) significantly prolonged survival, but no persistent tumor control was observed. Despite lower response rates, MSLN-4-1BB (MBBz) CAR T cells induced long-term remission in some SKOV3-bearing mice. Tumor-infiltrating M28z and MBBz CAR T cells upregulated PD-1 and LAG3 in an antigen-dependent manner while MSLN+ tumor cells expressed the corresponding ligands (PD-L1 and HLA-DR), demonstrating that coinhibitory pathways impede CAR T-cell persistence in the ovarian tumor microenvironment. Furthermore, profiling plasma soluble factors identified a cluster of M28z- and MBBz-treated mice characterized by elevated T-cell secreted factors that had increased survival, higher CD8+ T-cell tumor infiltration, less exhausted CAR T-cell phenotypes, and increased HLA-DR expression by tumor cells. Altogether, our study demonstrates the therapeutic potential of MSLN-CAR T cells to treat ovarian cancer. SIGNIFICANCE: These findings demonstrate that MSLN-directed CAR T cells can provide antitumor immunity against ovarian cancer.

CD11c-CD8 Spatial Cross Presentation: A Novel Approach to Link Immune Surveillance and Patient Survival in Soft Tissue Sarcoma.

Checkpoint inhibitors are slowly being introduced in the care of specific sarcoma subtypes such as undifferentiated pleomorphic sarcoma, alveolar soft part sarcoma, and angiosarcoma even though formal indication is lacking. Proper biomarkers to unravel potential immune reactivity in the tumor microenvironment are therefore expected to be highly warranted. In this study, intratumoral spatial cross presentation was investigated as a novel concept where immune cell composition in the tumor microenvironment was suggested to act as a proxy for immune surveillance. Double immunohistochemistry revealed a prognostic role of direct spatial interactions between CD11c+ antigen-presenting cells (APCs) and CD8+ cells in contrast to each marker alone in a soft tissue sarcoma (STS) cohort of 177 patients from the Karolinska University Hospital (MFS p = 0.048, OS p = 0.025). The survival benefit was verified in multivariable analysis (MFS p = 0.012, OS p = 0.004). Transcriptomics performed in the TCGA sarcoma cohort confirmed the prognostic value of combining CD11c with CD8 (259 patients, p = 0.005), irrespective of FOXP3 levels and in a CD274 (PD-LI)-rich tumor microenvironment. Altogether, this study presents a histopathological approach to link immune surveillance and patient survival in STS. Notably, spatial cross presentation as a prognostic marker is distinct from therapy response-predictive biomarkers such as immune checkpoint molecules of the PD-L1/PD1 pathway.