The entanglement of extracellular matrix molecules and immune checkpoint inhibitors in cancer: a systematic review of the literature.

Introduction: Immune-checkpoint inhibitors (ICIs) have emerged as a core pillar of cancer therapy as single agents or in combination regimens both in adults and children. Unfortunately, ICIs provide a long-lasting therapeutic effect in only one third of the patients. Thus, the search for predictive biomarkers of responsiveness to ICIs remains an urgent clinical need. The efficacy of ICIs treatments is strongly affected not only by the specific characteristics of cancer cells and the levels of immune checkpoint ligands, but also by other components of the tumor microenvironment, among which the extracellular matrix (ECM) is emerging as key player. With the aim to comprehensively describe the relation between ECM and ICIs' efficacy in cancer patients, the present review systematically evaluated the current literature regarding ECM remodeling in association with immunotherapeutic approaches. Methods: This review followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines and was registered at the International Prospective Register of Systematic Reviews (PROSPERO, CRD42022351180). PubMed, Web of Science, and Scopus databases were comprehensively searched from inception to January 2023. Titles, abstracts and full text screening was performed to exclude non eligible articles. The risk of bias was assessed using the QUADAS-2 tool. Results: After employing relevant MeSH and key terms, we identified a total of 5070 studies. Among them, 2540 duplicates, 1521 reviews or commentaries were found and excluded. Following title and abstract screening, the full text was analyzed, and 47 studies meeting the eligibility criteria were retained. The studies included in this systematic review comprehensively recapitulate the latest observations associating changes of the ECM composition following remodeling with the traits of the tumor immune cell infiltration. The present study provides for the first time a broad view of the tight association between ECM molecules and ICIs efficacy in different tumor types, highlighting the importance of ECM-derived proteolytic products as promising liquid biopsy-based biomarkers to predict the efficacy of ICIs. Conclusion: ECM remodeling has an important impact on the immune traits of different tumor types. Increasing evidence pinpoint at ECM-derived molecules as putative biomarkers to identify the patients that would most likely benefit from ICIs treatments. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022351180, identifier CRD42022351180.

Emilin2 fosters vascular stability by promoting pericyte recruitment.

Angiogenesis, the formation of the new blood vessels from pre-existing vasculature, is an essential process occurring under both normal and pathological conditions, such as inflammation and cancer. This complex process is regulated by several cytokines, growth factors and extracellular matrix components modulating endothelial cell and pericyte function. In this study, we discovered that the extracellular matrix glycoprotein Elastin Microfibril Interfacer 2 (Emilin2) plays a prominent role in pericyte physiology. This work was originally prompted by the observations that tumor-associated vessels from Emilin2-/- mice display less pericyte coverage, impaired vascular perfusion, and reduced drug efficacy, suggesting that Emilin2 could promote vessel maturation and stabilization affecting pericyte recruitment. We found that Emilin2 affects different mechanisms engaged in pericyte recruitment and vascular stabilization. First, human primary endothelial cells challenged with recombinant Emilin2 synthesized and released ∼ 2.1 and 1.2 folds more PDGF-BB and HB-EGF, two cytokines known to promote pericyte recruitment. We also discovered that Emilin2, by directly engaging α5ß1 and α6ß1 integrins, highly expressed in pericytes, served as an adhesion substrate and haptotactic stimulus for pericytes. Moreover, Emilin2 evoked increased NCadherin expression via the sphingosine-1-phosphate receptor, leading to enhanced vascular stability by fostering interconnection between endothelial cells and pericytes. Finally, restoring pericyte coverage in melanoma and ovarian tumor vessels developed in Emilin2-/- mice improved drug delivery to the tumors. Collectively, our results implicate Emilin2 as a prominent regulator of pericyte function and suggest that Emilin2 expression could represent a promising maker to predict the clinical outcome of patients with melanoma, ovarian, and potentially other forms of cancer.

Colorectal cancer development is affected by the ECM molecule EMILIN-2 hinging on macrophage polarization via the TLR-4/MyD88 pathway.

BACKGROUND: Colorectal cancer is one of the most frequent and deadly tumors. Among the key regulators of CRC growth and progression, the microenvironment has emerged as a crucial player and as a possible route for the development of new therapeutic opportunities. More specifically, the extracellular matrix acts directly on cancer cells and indirectly affecting the behavior of stromal and inflammatory cells, as well as the bioavailability of growth factors. Among the ECM molecules, EMILIN-2 is frequently down-regulated by methylation in CRC and the purpose of this study was to verify the impact of EMILIN-2 loss in CRC development and its possible value as a prognostic biomarker. METHODS: The AOM/DSS CRC protocol was applied to Emilin-2 null and wild type mice. Tumor development was monitored by endoscopy, the molecular analyses performed by IHC, IF and WB and the immune subpopulations characterized by flow cytometry. Ex vivo cultures of monocyte/macrophages from the murine models were used to verify the molecular pathways. Publicly available datasets were exploited to determine the CRC patients' expression profile; Spearman's correlation analyses and Cox regression were applied to evaluate the association with the inflammatory response; the clinical outcome was predicted by Kaplan-Meier survival curves. Pearson correlation analyses were also applied to a cohort of patients enrolled in our Institute. RESULTS: In preclinical settings, loss of EMILIN-2 associated with an increased number of tumor lesions upon AOM/DSS treatment. In addition, in the early stages of the disease, the Emilin-2 knockout mice displayed a myeloid-derived suppressor cells-rich infiltrate. Instead, in the late stages, lack of EMILIN-2 associated with a decreased number of M1 macrophages, resulting in a higher percentage of the tumor-promoting M2 macrophages. Mechanistically, EMILIN-2 triggered the activation of the Toll-like Receptor 4/MyD88/NF-κB pathway, instrumental for the polarization of macrophages towards the M1 phenotype. Accordingly, dataset and immunofluorescence analyses indicated that low EMILIN-2 expression levels correlated with an increased M2/M1 ratio and with poor CRC patients' prognosis. CONCLUSIONS: These novel results indicate that EMILIN-2 is a key regulator of the tumor-associated inflammatory environment and may represent a promising prognostic biomarker for CRC patients.

Multimerin-2 orchestrates the cross-talk between endothelial cells and pericytes: A mechanism to maintain vascular stability.

Tumor angiogenesis is vital for the growth and development of various solid cancers and as such is a valid and promising therapeutic target. Unfortunately, the use of the currently available anti-angiogenic drugs increases the progression-free survival by only a few months. Conversely, targeting angiogenesis to prompt both vessel reduction and normalization, has been recently viewed as a promising approach to improve therapeutic efficacy. As a double-edged sword, this line of attack may on one side halt tumor growth as a consequence of the reduction of nutrients and oxygen supplied to the tumor cells, and on the other side improve drug delivery and, hence, efficacy. Thus, it is of upmost importance to better characterize the mechanisms regulating vascular stability. In this context, recruitment of pericytes along the blood vessels is crucial to their maturation and stabilization. As the extracellular matrix molecule Multimerin-2 is secreted by endothelial cells and deposited also in juxtaposition between endothelial cells and pericytes, we explored Multimerin-2 role in the cross-talk between the two cell types. We discovered that Multimerin-2 is an adhesion substrate for pericytes. Interestingly, and consistent with the notion that Multimerin-2 is a homeostatic molecule deposited in the later stages of vessel formation, we found that the interaction between endothelial cells and pericytes promoted the expression of Multimerin-2. Furthermore, we found that Multimerin-2 modulated the expression of key cytokines both in endothelial cells and pericytes. Collectively, our findings posit Multimerin-2 as a key molecule in the cross-talk between endothelial cells and pericytes and suggest that the expression of this glycoprotein is required to maintain vascular stability.

The Efficacy of Anti-PD-L1 Treatment in Melanoma Is Associated with the Expression of the ECM Molecule EMILIN2.

The use of immune checkpoint inhibitors has revolutionized the treatment of melanoma patients, leading to remarkable improvements in the cure. However, to ensure a safe and effective treatment, there is the need to develop markers to identify the patients that would most likely respond to the therapies. The microenvironment is gaining attention in this context, since it can regulate both the immunotherapy efficacyand angiogenesis, which is known to be affected by treatment. Here, we investigated the putative role of the ECM molecule EMILIN-2, a tumor suppressive and pro-angiogenic molecule. We verified that the EMILIN2 expression is variable among melanoma patients and is associated with the response to PD-L1 inhibitors. Consistently, in preclinical settings,the absence of EMILIN-2 is associated with higher PD-L1 expression and increased immunotherapy efficacy. We verified that EMILIN-2 modulates PD-L1 expression in melanoma cells through indirect immune-dependent mechanisms. Notably, upon PD-L1 blockage, Emilin2-/- mice displayed improved intra-tumoral vessel normalization and decreased tumor hypoxia. Finally, we provide evidence indicating that the inclusion of EMILIN2 in a number of gene expression signatures improves their predictive potential, a further indication that the analysis of this molecule may be key for the development of new markers to predict immunotherapy efficacy.

ECM Remodeling in Squamous Cell Carcinoma of the Aerodigestive Tract: Pathways for Cancer Dissemination and Emerging Biomarkers.

Squamous cell carcinomas (SCC) include a number of different types of tumors developing in the skin, in hollow organs, as well as the upper aerodigestive tract (UADT) including the head and neck region and the esophagus which will be dealt with in this review. These tumors are often refractory to current therapeutic approaches with poor patient outcome. The most important prognostic determinant of SCC tumors is the presence of distant metastasis, significantly correlating with low patient survival rates. Rapidly emerging evidence indicate that the extracellular matrix (ECM) composition and remodeling profoundly affect SSC metastatic dissemination. In this review, we will summarize the current knowledge on the role of ECM and its remodeling enzymes in affecting the growth and dissemination of UADT SCC. Taken together, these published evidence suggest that a thorough analysis of the ECM composition in the UADT SCC microenvironment may help disclosing the mechanism of resistance to the treatments and help defining possible targets for clinical intervention.

Prognostic role of visceral fat for overall survival in metastatic colorectal cancer: A pilot study.

BACKGROUND: Body composition, has been established as a risk factor for colorectal cancer diagnosis and disease progression. Aim of this study was to investigate the prognostic role of adiposity, especially visceral fat (VAT), in patients (pts) with metastatic colorectal cancer (MCRC). MATERIAL AND METHODS: A retrospective cohort of 71 MCRC pts treated between 2013 and 2017 was evaluated. VAT was measured as cross-sectional (cm2) area at the L3 level divided by the square of the height (m2). A ROC analysis was performed to define a prognostic threshold according to VAT. RESULTS: Before first-line therapy start, 40 pts (56%) had a body mass index (BMI) > 25 kg/m2. The obtained cut-off value for VAT was 44. Median OS was 30.97 months. At univariate analysis, primary tumor resection (HR 0.40, p = 0.029), VAT>44 (HR 2.85, p = 0.011) and metastasectomy (HR 0.22, p = 0.005) were significantly associated with OS. By multivariate analysis, VAT>44 (HR 2.6; p = 0.020) and metastasectomy were still significantly associated with OS. CONCLUSION: This exploratory study suggests a prognostic role for VAT in MCRC pts, with higher VAT values predicting worse outcome.

Role of Extracellular Matrix in Gastrointestinal Cancer-Associated Angiogenesis.

Gastrointestinal tumors are responsible for more cancer-related fatalities than any other type of tumors, and colorectal and gastric malignancies account for a large part of these diseases. Thus, there is an urgent need to develop new therapeutic approaches to improve the patients' outcome and the tumor microenvironment is a promising arena for the development of such treatments. In fact, the nature of the microenvironment in the different gastrointestinal tracts may significantly influence not only tumor development but also the therapy response. In particular, an important microenvironmental component and a potential therapeutic target is the vasculature. In this context, the extracellular matrix is a key component exerting an active effect in all the hallmarks of cancer, including angiogenesis. Here, we summarized the current knowledge on the role of extracellular matrix in affecting endothelial cell function and intratumoral vascularization in the context of colorectal and gastric cancer. The extracellular matrix acts both directly on endothelial cells and indirectly through its remodeling and the consequent release of growth factors. We envision that a deeper understanding of the role of extracellular matrix and of its remodeling during cancer progression is of chief importance for the development of new, more efficacious, targeted therapies.

Deregulated expression of Elastin Microfibril Interfacer 2 (EMILIN2) in gastric cancer affects tumor growth and angiogenesis.

Gastric cancer is a frequent human tumor and often a lethal disease. Targeted therapy for gastric carcinomas is far behind vis-à-vis other solid tumors, primarily because of the paucity of cancer-driving mutations that could be efficiently and specifically targeted by current therapy. Thus, there is a need to discover actionable pathways/proteins and new diagnostic and prognostic biomarkers. In this study, we explored the role of the extracellular matrix glycoprotein EMILIN2, Elastin Microfibril Interfacer 2, in a cohort of gastric cancer patients. We discovered that EMILIN2 expression was consistently suppressed in gastric cancer and high expression levels of this glycoprotein were linked to abnormal vascular density. Furthermore, we found that EMILIN2 had a dual effect on gastric carcinoma cells: on one hand, it decreased tumor cell proliferation by triggering apoptosis, and on the other hand, it evoked the production of a number of cytokines involved in angiogenesis and inflammation, such as IL-8. Collectively, our findings posit EMILIN2 as an important onco-regulator exerting pleiotropic effects on the gastric cancer microenvironment.

Multimerin-2 maintains vascular stability and permeability.

Multimerin-2 is an extracellular matrix glycoprotein and member of the elastin microfibril interface-located (EMILIN) family of proteins. Multimerin-2 is deposited along blood vessels and we previously demonstrated that it regulates the VEGFA/VEGFR2 signaling axis and angiogenesis. However, its role in modulating vascular homeostasis remains largely unexplored. Here we identified Multimerin-2 as a key molecule required to maintain vascular stability. RNAi knockdown of Multimerin-2 in endothelial cells led to cell-cell junctional instability and increased permeability. Mechanistically cell-cell junction dismantlement occurred through the phosphorylation of VEGFR2 at Tyr951, activation of Src and phosphorylation of VE-cadherin. To provide an in vivo validation for these in vitro effects, we generated Multimerin-2-/- (Mmrn2-/-) mice. Although Mmrn2-/- mice developed normally and displayed no gross abnormalities, endothelial cells displayed cell junctional defects associated with increased levels of VEGFR2 phospho-Tyr949 (the murine counterpart of human Tyr951), impaired pericyte recruitment and increased vascular leakage. Of note, tumor associated vessels were defective in Mmrn2-/- mice, with increased number of small and often collapsed vessels, concurrent with a significant depletion of pericytic coverage. Consequently, the Mmrn2-/- vessels were less perfused and leakier, leading to increased tumor hypoxia. Chemotherapy efficacy was markedly impaired in Mmrn2-/- mice and this was associated with poor drug delivery to the tumor xenografts. Collectively, our findings demonstrate that Multimerin-2 is required for proper vessel homeostasis and stabilization, and unveil the possibility to utilize expression levels of this glycoprotein in predicting chemotherapy efficacy.

Extracellular matrix: the gatekeeper of tumor angiogenesis.

The extracellular matrix is a network of secreted macromolecules that provides a harmonious meshwork for the growth and homeostatic development of organisms. It conveys multiple signaling cascades affecting specific surface receptors that impact cell behavior. During cancer growth, this bioactive meshwork is remodeled and enriched in newly formed blood vessels, which provide nutrients and oxygen to the growing tumor cells. Remodeling of the tumor microenvironment leads to the formation of bioactive fragments that may have a distinct function from their parent molecules, and the balance among these factors directly influence cell viability and metastatic progression. Indeed, the matrix acts as a gatekeeper by regulating the access of cancer cells to nutrients. Here, we will critically evaluate the role of selected matrix constituents in regulating tumor angiogenesis and provide up-to-date information concerning their primary mechanisms of action.

Abrogation of EMILIN1-ß1 integrin interaction promotes experimental colitis and colon carcinogenesis.

Colon cancer is one of the first tumor types where a functional link between inflammation and tumor onset has been described; however, the microenvironmental cues affecting colon cancer progression are poorly understood. Here we demonstrate that the expression of the ECM molecule EMILIN-1 halts the development of AOM-DSS induced tumors. In fact, upon AOM-DSS treatment the Emilin1-/- (E1-/-) mice were characterized by a higher tumor incidence, bigger adenomas and less survival. Similar results were obtained with the E933A EMILIN-1 (E1-E933A) transgenic mouse model, expressing a mutant EMILIN-1 unable to interact with α4/α9ß1 integrins. Interestingly, upon chronic treatment with DSS, E1-/- and E1-E933A mice were characterized by the presence of increased inflammatory infiltrates, higher colitis scores and more severe mucosal injury respect to the wild type (E1+/+) mice. Since alterations of the intestinal lymphatic network are a well-established feature of human inflammatory bowel disease and EMILIN-1 is a key structural element in the maintenance of the integrity of lymphatic vessels, we assessed the lymphatic vasculature in this context. The analyses revealed that both E1-/- and E1-E933A mice displayed a higher density of LYVE-1 positive vessels; however, their functionality was severely compromised after colitis induction. Taken together, these results suggest that the loss of EMILIN-1 expression may cause the reduction of the inflammatory resolution during colon cancer progression due to a decreased lymph flow and impaired inflammatory cell drainage.

Multiplex staining depicts the immune infiltrate in colitis-induced colon cancer model.

Assessment of the host immune response pattern is of increasing importance as highly prognostic and diagnostic, in immune-related diseases and in some types of cancer. Chronic inflammation is a major hallmark in colon cancer formation, but, despite the extent of local inflammatory infiltrate has been demonstrated to be extremely informative, its evaluation is not routinely assessed due to the complexity and limitations of classical immunohistochemistry (IHC). In the last years, technological advance helped in bypassing technical limits, setting up multiplex IHC (mIHC) based on tyramide signal amplification (TSA) method and designing software suited to aid pathologists in cell scoring analysis. Several studies verified the efficacy of this method, but they were restricted to the analysis of human samples. In the era of translational medicine the use of animal models to depict human pathologies, in a more complete and complex approach, is really crucial. Nevertheless, the optimization and validation of this method to species other than human is still poor. We took advantage of Multispectral Imaging System to identify the immunoprofile of Dextran Sulphate Sodium (DSS)-treated mouse colon. We optimized a protocol to sequentially stain formalin fixed paraffin embedded murine colon samples for CD3, CD8a, CD4, and CD4R5B0 antigens. With this approach we obtained a detailed lymphocyte profile, while preserving the morphological tissue context, generally lost with techniques like gene expression profiling or flow cytometry. This study, comparing the results obtained by mIHC with immunophenotyping performed with cytofluorimetric and standard IHC methods validates the potentiality and the applicability of this innovative approach.

The Probe Based Confocal Laser Endomicroscopy (pCLE) in Locally Advanced Gastric Cancer: A Powerful Technique for Real-Time Analysis of Vasculature.

Probe based confocal laser endomicroscopy (pCLE) is an advanced technique which provides imaging of gastrointestinal mucosa at subcellular resolution and, importantly, a valid tool for the evaluation of microvasculature during endoscopic examination. In order to assess intratumoral vascularization and the efficiency of blood flow in locally advanced gastric cancer, we examined 57 patients through pCLE imaging. The vascular alterations in gastric cancer were mainly characterized by leakage and by the presence of tortuous and large size vessels. Defects in blood flow were detected very rarely. No association between the angiogenic score and the gastric tumor site or histological type was observed. Interestingly, no correlation was also found with the tumor grading indicating that the vascular angiogenic anomalies in gastric cancer represent an early pathological event to be observed and detected. The majority of patients displayed unchanged vascular alterations following neoadjuvant chemotherapy and this positively correlated with stable or progressive disease, suggesting that an unaltered angiogenic score could per se be indicative of poor therapeutic efficacy. Different vascular parameters were evaluated by immunofluorescence using bioptic samples and the vessel density did not correlate with clinical staging, site or histologic type. Interestingly, only CD105, Multimerin-2 and GLUT1 were able to discriminate normal from tumoral gastric mucosa. Taken together, these findings indicate that functional and structural angiogenic parameters characteristic of tumor blood network were fully detectable by pCLE. Moreover, the evaluation of tumor vasculature by real-time assessment may provide useful information to achieve tailored therapeutic interventions for gastric cancer patients.

Sleeping beauty genetic screen identifies miR-23b::BTBD7 gene interaction as crucial for colorectal cancer metastasis.

BACKGROUND: Metastatic colorectal cancer (CRC) remains a deadly disease. Identifying locally advanced CRC patients with high risk of developing metastasis and improving outcome of metastatic CRC patients require discovering master regulators of metastasis. In this context, the non-coding part of the human genome is still largely unexplored. METHODS: To interrogate the non-coding part of the human genome and disclose regulators of CRC metastasis, we combined a transposon-based forward genetic screen with a novel in vitro assay, which forces cells to grow deprived of cell-substrate and cell-cell contacts (i.e. forced single cell suspension assay - fSCS). FINDINGS: We proved that fSCS selects CRC cells with mesenchymal and pro-metastatic traits. Moreover, we found that the transposon insertions conferred CRC cells resistance to fSCS and thus metastatic advantage. Among the retrieved transposon insertions, we demonstrated that the one located in the 3'UTR of BTBD7 disrupts miR-23b::BTBD7 interaction and contributes to pro-metastatic traits. In addition, miR-23b and BTBD7 correlate with CRC metastasis both in preclinical experiments and in clinical samples. INTERPRETATION: fSCS is a simple and scalable in vitro assay to investigate pro-metastatic traits and transposon-based genetic screens can interrogate the non-coding part of the human genome (e.g. miRNA::target interactions). Finally, both Btbd7 and miR-23b represent promising prognostic biomarkers and therapeutic targets in CRC. FUND: This work was supported by Marie Curie Actions (CIG n. 303877) and Friuli Venezia Giulia region (Grant Agreement n°245574), Italian Association for Cancer Research (AIRC, MFAG n°13589), Italian Ministry of Health (GR-2010-2319387 and PE-2016-02361040) and 5x1000 to CRO Aviano.

Loss of Multimerin-2 and EMILIN-2 Expression in Gastric Cancer Associate with Altered Angiogenesis.

Gastric cancer is a deadly tumor and a relatively common disease worldwide. Surgical resection and chemotherapy are the main clinical options to treat this type of disease, however the median overall survival rate is limited to one year. Thus, the development of new therapies is a highly necessary clinical need. Angiogenesis is a promising target for this tumor type, however clinical trials with the use of anti-angiogenic drugs have so far not met expectations. Therefore, it is important to better characterize the expression of molecules whose expression levels may impact on the efficacy of the treatments. In this study the characteristics of the gastric tumor associated blood vessels were first assessed by endomicroscopy. Next, we analyzed the expression of Multimerin-2, EMILIN-2 and EMILIN-1, three molecules of the EMI Domain ENdowed (EDEN) protein family. These molecules play important functions in the tumor microenvironment, affecting cancer progression both directly and indirectly impinging on angiogenesis and lymphangiogenesis. All the molecules were highly expressed in the normal mucosa whereas in a number of patients their expression was altered. We consider that better characterizing the gastric tumor microenvironment and the quality of the vasculature may achieve effective patient tailored therapies.

Endorepellin remodels the endothelial transcriptome toward a pro-autophagic and pro-mitophagic gene signature.

Regulation of autophagy by proteolytically cleaved fragments of heparan sulfate proteoglycans is a novel and current research focus in tumor biology. Endorepellin is the C-terminal angiostatic fragment of the heparan sulfate proteoglycan perlecan and induces autophagy in endothelial cells. To further investigate this property, we used NanoString, a digital PCR platform for measuring pre-defined transcripts in biological samples to analyze a custom subset of 95 autophagy-related genes in human umbilical vein endothelial cells treated with ultrapure human recombinant endorepellin. We discovered an endorepellin-evoked pro-autophagic and pro-mitophagic gene expression signatures, which included two coordinately up-regulated mitochondrial-associated genes encoding the E3 ubiquitin protein ligase Parkin and the tumor suppressor mitostatin. Induction of both proteins required the tyrosine kinase activity of vascular endothelial growth factor receptor 2 (VEGFR2). Furthermore, we discovered that endorepellin evoked mitochondrial depolarization in endothelial cells via a specific interaction between its two proximal LG1/2 domains and VEGFR2. We also found that following loss of membrane potential, mitostatin and parkin interact and that mitostatin associates with the established Parkin receptor mitofusin-2. In conclusion, we have identified a critical role for endorepellin in remodeling the autophagic transcriptome and influencing mitochondrial homeostasis.

The ablation of the matricellular protein EMILIN2 causes defective vascularization due to impaired EGFR-dependent IL-8 production affecting tumor growth.

EMILIN2 is an extracellular matrix constituent playing an important role in angiogenesis; however, the underlying mechanism is unknown. Here we show that EMILIN2 promotes angiogenesis by directly binding epidermal growth factor receptor (EGFR), which enhances interleukin-8 (IL-8) production. In turn, IL-8 stimulates the proliferation and migration of vascular endothelial cells. Emilin2 null mice were generated and exhibited delayed retinal vascular development, which was rescued by the administration of the IL-8 murine ortholog MIP-2. Next, we assessed tumor growth and tumor-associated angiogenesis in these mice. Tumor cell growth in Emilin2 null mice was impaired as well as the expression of MIP-2. The vascular density of the tumors developed in Emilin2 null mice was prejudiced and vessels perfusion, as well as response to chemotherapy, decreased. Accordingly, human tumors expressing high levels of EMILIN2 were more responsive to chemotherapy. These results point at EMILIN2 as a key microenvironmental cue affecting vessel formation and unveil the possibility to develop new prognostic tools to predict chemotherapy efficacy.

Dissecting the CD93-Multimerin 2 interaction involved in cell adhesion and migration of the activated endothelium.

The glycoprotein CD93 has recently been recognized to play an important role in the regulation of the angiogenic process. Moreover, CD93 is highly expressed in the endothelial cells of tumor blood vessel and faintly expressed in the non-proliferating endothelium. Much evidence suggests that CD93 mediates adhesion in the endothelium. Here we identify Multimerin 2 (MMRN2), a pan-endothelial extracellular matrix protein, as a specific ligand for CD93. We found that CD93 and MMRN2 are co-expressed in the blood vessels of various human tumors. Moreover, disruption of the CD93-MMRN2 interaction reduced endothelial cell adhesion and migration, making the interaction of CD93 with MMRN2 an ideal target to block pathological angiogenesis. Model structures and docking studies served to envisage the region of CD93 and MMRN2 involved in the interaction. Site-directed mutagenesis identified different residue hotspots either directly or indirectly involved in the binding. We propose a molecular model in which the coiled-coil domain of MMRN2 is engaged by F238 of CD93. Altogether, these studies identify the key interaction surfaces of the CD93-MMRN2 complex and provide a framework for exploring how to inhibit angiogenesis by hindering the CD93-MMRN2 interaction.

The angiostatic molecule Multimerin 2 is processed by MMP-9 to allow sprouting angiogenesis.

Angiogenesis is a crucial process occurring under physiological and pathological conditions, including cancer. The development of blood vessels is tightly regulated by a plethora of cytokines, endothelial cell (EC) receptors and extracellular matrix (ECM) components. In this context, we have shown that Multimerin 2 (MMRN2), an ECM molecule specifically secreted by ECs, exerts angiostatic functions by binding VEGFA and other pro-angiogenic cytokines. Here, we demonstrate that during angiogenic stimuli MMRN2 mRNA levels significantly decrease. Furthermore, we provide evidence that MMRN2 is processed by matrix metalloproteinases (MMPs) including MMP-9 and, to a lesser degree, by MMP-2. This proteolytic cleavage correlates with an increased migration of ECs. Accordingly, MMRN2 down-regulation is associated with an increased number of EC pseudopodia at the migrating front and this effect is attenuated using specific MMP-9 inhibitors. The down-modulation of MMRN2 occurs also in the context of tumor-associated angiogenesis. Immunofluorescence performed on tumor sections indicate a broad co-localization of MMP-9 and MMRN2, suggesting that the molecule may be extensively remodeled during tumor angiogenesis. Given the altered expression in tumors and the key role of MMRN2 in blood vessel function, we postulate that analyses of its expression may serve as a marker to predict the efficacy of the treatments. In conclusion, these data further support the role of MMRN2 as a key molecule regulating EC function and sprouting angiogenesis.