EMILIN-1 is an extracellular matrix glycoprotein with tumor-suppressive functions. While its loss is implicated in cancer progression, its specific role in the gastric tumor microenvironment and its clinical relevance remain poorly defined. Using in vitro cellular systems and genetically modified mouse models we investigated the consequences of impaired EMILIN-1 function on gastric epithelial transformation, stromal remodeling, and fibroblast reprogramming. Histopathological analysis, gene expression profiling, and functional assays were employed to assess phenotypic changes in epithelial, fibroblastic, and endothelial compartments. We found that gastric cancer (GC) cells downregulate EMILIN-1 in stromal fibroblasts and lymphatic endothelial cells via paracrine signaling, leading to reduced EMILIN-1 deposition and disrupted lymphatic organization. Mechanistically, the interaction between EMILIN-1 and α4β1 integrin, which is absent in GC cells, modulates tumor cell proliferation; loss of this axis allows tumor cells to escape ECM-mediated growth control. Furthermore, EMILIN-1 downregulation reprograms fibroblasts into a pro-tumorigenic phenotype, which enhances GC cell migration and clonogenic potential. EMILIN-1 loss-of-function (E955A) mice showed increased susceptibility to pre-neoplastic lesions, a finding mirrored in human dysplastic tissues where EMILIN-1 was markedly reduced. Our findings establish EMILIN-1 as a master regulator of gastric tissue integrity. Its loss creates a tumor-permissive microenvironment by disrupting epithelial homeostasis, impairing lymphatic structure, and promoting fibroblast activation. The consistent reduction of EMILIN-1 in early human dysplasia highlights its potential as a novel stromal biomarker for early GC risk stratification. Moreover, restoring the EMILIN-1/integrin axis represents a promising therapeutic strategy to re-establish growth control and suppress tumor progression.

Does the homeostatic molecule Multimerin-2 (MMRN2) undergo alterations in preeclampsia (PE) and oocyte donation (OD) pregnancies, potentially contributing to the increased PE risk in OD? Multimerin-2, is profoundly remodeled in pregnancy and decreases in PE and in OD placentas, suggesting a potential pathogenetic role. Assisted reproductive techniques are associated with a higher risk of developing PE, with a two-fold rise in homologous (HOM) and a four-fold rise in heterologous (OD) settings. PE is a systemic vascular disorder characterized by endothelial cells dysfunction, yet its precise mechanisms remain unclear. A key element granting vascular homeostasis is MMRN2, an extracellular molecule specifically expressed by endothelial cells. While its function in pregnancy is not well understood, its regulatory role in vessel efficiency suggests a potential involvement in PE as well. Investigating MMRN2 alterations in PE and OD pregnancies may provide novel insights into disease pathogenesis. This is an observational pilot study conducted over 12 months. Six patient groups were included: 1) OD pregnancies, not diagnosed with PE (OD, n = 10); 2) homologous ART pregnancies, not diagnosed with PE (HOM, n = 7); 3) spontaneous pregnancies diagnosed with PE (PE, n = 7); 4) uneventful spontaneous pregnancies (SP, n = 8); 5) women occurring physiological pregnancies who chose to undergo voluntary abortion within the first gestational trimester (IVG, n = 6); 6) non-pregnant women (CTRL, n = 20). Placental samples were collected after delivery in groups 1-4 and after IVG in group 5, and MMRN2 deposition analyzed through qPCR and immunohistochemistry. Serum samples have been obtained from CTRL women, as reference, and from PE and SP patients at two time points: within the first (11-13th week) and third (34-38th week) trimesters. The concentration of MMRN2 fragments have been evaluated by ELISA. Statistical comparisons were made using ANOVA and student t-test when appropriate. Immunohistochemistry analysis indicated that MMRN2 is deposited within the placental capillary wall during the first trimester and remains stable in SP and HOM pregnancies. By contrast, PE and OD samples showed a consistent reduction of MMRN2. qPCR analyses confirmed this trend, showing a comparable decrease in MMRN2 expression in PE and OD groups compared to SP and HOM patients, respectively, uncovering novel shared molecular features between the two conditions. Additionally, we observed a marked increase in MMRN2 fragments, products of proteolytic cleavage, in first- and third-trimester sera from SP women compared to non-pregnant controls, indicating MMRN2 undergoes remodeling during pregnancy. ELISA analysis of PE samples revealed MMRN2 fragment levels comparable to those from SP in the first trimester, but significantly decreased by the third trimester, suggesting a dysregulated MMRN2 remodeling in PE pregnancies. Given MMRN2 essential role for vessel homeostasis, its absence in the placenta may contribute to vascular dysfunction. Moreover, altered MMRN2 deposition in OD patients suggests a link between the loss of the molecule and the higher risk of developing the disease. Collectively, our findings propose MMRN2 as a promising biomarker for PE and a potential tool for understanding common pathways in PE and OD pregnancies. The small sample size of this pilot study highlights the need for larger cohorts to confirm the observed trends and enhance the reliability of these findings. Additional molecular investigations are required to better characterize the impact of MMRN2 loss in PE and OD in terms of vessel functionality and efficiency. This study highlights excessive MMRN2 remodeling as a potential contributor to vascular dysfunction in PE and provides insight into how OD pregnancies carry a higher risk of developing the disease, supporting the evaluation of MMRN2 as a biomarker and target to improve pregnancy outcomes. No

EMILIN-1 (Elastin Microfibril Interface Located Protein 1) is an extracellular matrix homotrimeric glycoprotein belonging to the EMILIN/Multimerin family, with both structural and regulatory roles, increasingly recognized for its tumor-suppressive functions. Initially identified for its involvement in elastogenesis and vascular homeostasis, EMILIN-1 has gradually emerged as a key player in cancer biology. It exerts its anti-tumor activity through both direct and indirect mechanisms: by regulating tumor cell proliferation and survival and by modulating lymphangiogenesis and the associated inflammatory microenvironment. At the molecular level, EMILIN-1 inhibits pro-oncogenic signaling pathways, such as ERK/AKT and TGF-β, via its selective interaction with α4/α9 integrins. In the tumor microenvironment, it contributes to tissue homeostasis by restraining aberrant lymphatic vessel formation, a process closely linked to tumor dissemination and immune modulation. Notably, EMILIN-1 expression is frequently reduced or its structure altered by proteolytic degradation in advanced cancers, correlating with disease progression and poor prognosis. This review summarizes the current knowledge on EMILIN-1 in cancer, focusing on its dual function as an active extracellular matrix regulator of intercellular signaling. Particular attention is given to its mechanistic role in the control of cell proliferation, underscoring its potential as a novel biomarker and therapeutic target in oncology.

Abstract Background About 50% of cutaneous melanoma (CM) harbors the activating BRAFV600 mutation which exerts most of the oncogenic effects through the MAPK signaling pathway. In the last years, a number of MAPK modulators have been identified, including Spry1. In this context, we have recently demonstrated that knockout of Spry1 (Spry1KO) in BRAFV600-mutant CM led to cell cycle arrest and apoptosis, repressed cell proliferation in vitro, and reduced tumor growth in vivo. Despite these findings, however, the precise molecular mechanism linking Spry1 to BRAFV600-mutant CM remains to be elucidated. Materials and methods Immunoprecipitation coupled to mass spectrometry was employed to gain insight into Spry1 interactome. Spry1 gene was knocked-out using the CRISPR strategy in the BRAF-mutant cell lines. Transmission electron microscopy was used to assess the relationship between Spry1 expression and mitochondrial morphology. By using in vitro and in vivo models, the effects of Spry1KO were investigated through RNA-sequencing, quantitative real-time PCR, Western blot, and immunofluorescence analyses. The Seahorse XF24 assay allowed real-time measurement of cellular metabolism in our model. Angiogenic potential was assessed through in vitro tube formation assays and in vivo CD31 staining. Results Spry1 was mainly located in mitochondria in BRAFV600-mutant CM cells where it interacted with key molecules involved in mitochondrial homeostasis. Spry1 loss resulted in mitochondrial shape alterations and dysfunction, which associated with increased reactive oxygen species production. In agreement, we found that nuclear hypoxia-inducible factor-1 alpha (HIF1α) protein levels were reduced in Spry1KO clones both in vitro and in vivo along with the expression of its glycolysis related genes. Accordingly, Ingenuity Pathway Analysis identified “HIF1α Signaling” as the most significant molecular and cellular function affected by Spry1 silencing, whereas the glycolytic function was significantly impaired in Spry1 depleted BRAFV600-mutant CM cells. In addition, our results indicated that the expression of the vascular endothelial growth factor A was down-regulated following Spry1KO, possibly as a result of mitochondrial dysfunction. Consistently, we observed a substantial impairment of angiogenesis, as assessed by the tube formation assay in vitro and the immunofluorescence staining of CD31 in vivo. Conclusions Altogether, these findings identify Spry1 as a potential regulator of mitochondrial homeostasis, and uncover a previously unrecognized role for Spry1 in regulating nuclear HIF1α expression and angiogenesis in BRAFV600-mutant CM. Significance Spry1KO profoundly impacts on mitochondria homeostasis, while concomitantly impairing HIF1α-dependent glycolysis and reducing angiogenesis in BRAF-mutant CM cells, thus providing a potential therapeutic target to improve BRAFV600-mutant CM treatment.

[This retracts the article DOI: 10.1016/j.mbplus.2020.100029.].

In recent years, the tumor microenvironment has gained recognition as a key regulator of cancer progression. A central component of the tumor microenvironment, the extracellular matrix, undergoes dynamic remodeling during tumor development and plays a crucial role in disease pathogenesis. This review highlights the EMILIN/Multimerin family as a paradigm of the extracellular matrix's diverse and complex functions in cancer. Owing to their intricate domain architecture, these proteins engage in multifaceted interactions, not only directly modulating tumor cell proliferation and migration but also influencing other elements of the tumor microenvironment such as blood, lymphatic vessels, and immune cells. The functional landscape of these interactions is further complicated by proteolytic processing, which can both disrupt native functions and generate bioactive fragments with novel biological activities. Importantly, some of these fragments are detectable in biological fluids, suggesting their potential as predictive or prognostic biomarkers and contributing to the advancement of personalized therapeutic strategies.

Objective: Probe-based confocal laser endomicroscopy (pCLE) is a novel real-time imaging technique that is potentially useful for accurately distinguishing between normal and cancerous tissues. The aim of this study was to describe the pCLE patterns of areas suggestive of tumors and evaluate the ability of the method to differentiate between normal and cancerous tissue during cytoreductive surgery for epithelial ovarian cancer. Methods: In vivo pCLE images and subsequent biopsies were acquired from various anatomical sites including the parietal and visceral peritoneum, ovaries, and omentum. Each endomicroscopic sequence was analyzed by highly experienced investigators using pCLE imaging for cancer diagnosis. Each pCLE sequence was compared with the histology of the corresponding specimens. Results: We enrolled 18 women with International Federation of Gynecology and Obstetrics stage III/IV high-grade serous epithelial ovarian cancer referred for primary or interval debulking surgery. A total of 112 biopsies were obtained for histologic analysis. The pCLE images of normal tissue showed a regular distribution of stromal fibers and consistent cellular architecture, regardless of the anatomical region, with vascularized areas characterized by regular vessels. Conversely, the extravasation of fluorescein, used as a contrast agent, was a distinguishing feature of malignant nodules, which were easily recognized by leakage and are typical of tumor-associated vessels. The leakage often surrounded the dark clusters of neoplastic cells. A substantial agreement between pCLE and histology emerged (k = 0.66), whereas only a fair concordance between the surgeon's intra-operative assessment and histology was found (k = 0.30). Conclusions: Our results suggest that pCLE is a promising intra-operative technique to assist surgeons in accurately detecting peritoneal metastases in patients with advanced epithelial ovarian cancer, enhancing surgical radicality while avoiding unnecessary resection.

Abstract Study question Do stromal alterations occur during placentation upon heterologous assisted reproductive techniques (ART) and are they associated with a higher risk of developing preeclampsia? Summary answer The remodelling of extracellular matrix molecules, key for vascularization and immunity, is altered in pregnancies from oocyte donation and resembles the miss-regulations observed in preeclampsia. What is known already Preeclampsia represents one of the main causes of maternal and perinatal morbidity, affecting 2-8% of spontaneous pregnancies. Its incidence increases two times upon homologous ART and up to four times upon oocyte donation, however the underlying factors are still unknown. Preeclampsia is characterized by high blood pressure and endothelial cell dysfunction, but its pathogenesis is multifactorial relying on vascular, metabolic and immune alterations. All these elements are tightly influenced by the extracellular matrix (ECM) that, during an uneventful pregnancy, is remodelled to support an efficient placentation and immunotolerance. Despite its key functions, little is known regarding ECM remodelling in preeclampsia. Study design, size, duration This is an observational pilot study conducted over a 12 months period. Four groups were included: 1) pregnant women that underwent homologous ART (n = 7; HOM); 2) pregnant women that underwent oocyte donation (n = 10; OD); 3) women that conceived spontaneously affected by PE (n = 7); and 4) uneventful spontaneous pregnancies (n = 8; control group CTRL). Participants/materials, setting, methods For each patient placental samples have been collected upon delivery and processed to perform molecular and proteomic analysis by real time PCR and immunohistochemistry The statistical differences among the groups have been evaluated by the ANOVA and Mann-Whitney tests. Main results and the role of chance We focused our study on specific ECM elements known to modulate angiogenesis and immunity, two key players in preeclampsia occurrence. Our data indicated first that collagen I, collagen IV and Multimerin-2, important components of the vessel wall that contribute to vessel stabilization and efficiency, are significantly decreased in OD compared to HOM group (coll I P = 0.04; coll IV P = 0.043; Multimerin-2 P = 0.036). Next, we analyzed EMILIN-2 and versican, two ECM components exerting not only angiogenic but also immunomodulatory functions. We found that EMILIN-2 is decreased in OD compared to CTRL placentas (P = 0.043), whereas versican undergoes an opposite regulation, being slightly increased in the OD placentas (P = 0.045). We observed an analogous mis-regulation when comparing PE to the CTRL group, suggesting that these stromal alterations may take part in the establishment of a microenvironment more prone to preeclampsia onset and associate with the higher risk related to OD pregnancies. We also found that some features of ECM remodelling seem to be strongly associated with OD. Precisely, our data denoted that the glycoprotein tenascin-C decreased specifically in OD patients, while was comparable between HOM, PE and CTRL groups (P = 0.042). Limitations, reasons for caution The main limitation of this pilot study is the restricted number of patients. Further investigations on a larger sample and prospective cohort of patients might provide more robust data. Wider implications of the findings Our data highlighted for the first time that the placental stroma undergoes an altered remodelling in OD and PE pregnancies. We envision that a deep characterization of ECM will help better understanding of the mechanisms behind the pathogenesis of preeclampsia with possible implications for prediction and prevention. Trial registration number N/A

BackgroundThe contribution of the tumor microenvironment and extracellular matrix to the aggressive biology of Gastric Cancer (GC) has been recently characterized; however, the role of EMILIN-1 in this context is unknown. EMILIN-1 is an essential structural element for the maintenance of lymphatic vessel (LV) integrity and displays anti-proliferative properties as demonstrated in skin and colon cancer. Given the key role of LVs in GC progression, the aim of this study was to investigate the role of EMILIN-1 in GC mouse models.MethodsWe used the syngeneic YTN16 cells which were injected subcutaneously and intraperitoneally in genetically modified EMILIN-1 mice. In alternative, carcinogenesis was induced using N-Methyl-N-nitrosourea (MNU). Mouse-derived samples and human biopsies were analyzed by IHC and IF to the possible correlation between EMILIN-1 expression and LV pattern.ResultsTransgenic mice developed tumors earlier compared to WT animals. 20 days post-injection tumors developed in EMILIN-1 mutant mice were larger and displayed a significant increase of lymphangiogenesis. Treatment of transgenic mice with MNU associated with an increased number of tumors, exacerbated aggressive lesions and higher levels of LV abnormalities. A significant correlation between the levels of EMILIN-1 and podoplanin was detected also in human samples, confirming the results obtained with the pre-clinical models.ConclusionsThis study demonstrates for the first time that loss of EMILIN-1 in GC leads to lymphatic dysfunction and proliferative advantages that sustain tumorigenesis, and assess the use of our animal model as a valuable tool to verify the fate of GC upon loss of EMILIN-1.

Anti-angiogenic therapy is an established method for the treatment of several cancers and vascular-related diseases. Most of the agents employed target the vascular endothelial growth factor A, the major cytokine stimulating angiogenesis. However, the efficacy of these treatments is limited by the onset of drug resistance. Therefore, it is of fundamental importance to better understand the mechanisms that regulate angiogenesis and the microenvironmental cues that play significant role and influence patient treatment and outcome. In this context, here we review the importance of the three basement membrane heparan sulfate proteoglycans (HSPGs), namely perlecan, agrin and collagen XVIII. These HSPGs are abundantly expressed in the vasculature and, due to their complex molecular architecture, they interact with multiple endothelial cell receptors, deeply affecting their function. Under normal conditions, these proteoglycans exert pro-angiogenic functions. However, in pathological conditions such as cancer and inflammation, extracellular matrix remodeling leads to the degradation of these large precursor molecules and the liberation of bioactive processed fragments displaying potent angiostatic activity. These unexpected functions have been demonstrated for the C-terminal fragments of perlecan and collagen XVIII, endorepellin and endostatin. These bioactive fragments can also induce autophagy in vascular endothelial cells which contributes to angiostasis. Overall, basement membrane proteoglycans deeply affect angiogenesis counterbalancing pro-angiogenic signals during tumor progression, and represent possible means to develop new prognostic biomarkers and novel therapeutic approaches for the treatment of solid tumors.