MCAM expression facilitates melanoma-endothelial interactions and promotes metastatic disease progression

MCAM expression facilitates melanoma-endothelial interactions and promotes metastatic disease progression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article MCAM expression facilitates melanoma-endothelial interactions and promotes metastatic disease progression Andreas Dominik Braun, Miriam Mengoni, Thomas Tüting, Evelyn Gaffal This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4183647/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Invasive growth and metastatic dissemination represent the primary cause of death in cancer patients. In order to successfully detach from the primary tumor and establish metastases in distant tissues, cancer cells need to dynamically rewire their cell adhesion machinery. Here we revisit the potential association of MCAM, a member of the immunoglobulin superfamily that was initially identified as a melanoma antigen, with disease progression. Using immunohistochemical stainings and bioinformatic analyses of published datasets, we find similar MCAM expression levels in primary and metastatic human melanomas. In additional bioinformatic analyses, we show that MCAM is highly expressed in fetal melanocytes and subsequently downregulated during melanocyte maturation. Bioinformatic inference of cellular communication networks reveals that melanoma cells with high MCAM expression more actively engage in signaling crosstalk with endothelial cells. Experimental investigations demonstrate that disruption of MCAM in melanoma cells inhibits their migration on endothelial cell surfaces in vitro and decreases their ability to develop spontaneous lung metastases in vivo. Taken together, our results could not confirm the notion that MCAM expression represents a useful biomarker for disease progression, but provide evidence that MCAM expression might represent part of a reactivated embryonal transcriptional program that facilitates melanoma-endothelial cell interactions during metastatic progression. Biological sciences/Cancer/Skin cancer/Melanoma Biological sciences/Cancer/Cancer microenvironment Biological sciences/Cancer/Metastases Biological sciences/Cell biology/Cell migration Biological sciences/Cell biology/Cell adhesion Melanoma migration metastasis cell adhesion MCAM Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Invasive growth and metastatic dissemination are the primary cause of death in patients with cancer (Gerstberger et al. 2023 ). In order to detach from the primary tumor and successfully invade distant tissues to form metastases, cancer cells need to rewire their cell adhesion machinery (Hamidi and Ivaska 2018 ). As a prototype of this process, cells from many cancer types have been shown to downregulate the adhesion molecule E-cadherin, and upregulate the related molecule N-cadherin in a process termed epithelial to mesenchymal transition (Dongre and Weinberg 2019 ). In addition to cadherins, also members from the integrin- and immunoglobulin superfamily have been implicated in the migration of cancer cells (Janiszewska et al. 2020 ). As a prominent example of a cell adhesion molecule of the immunoglobulin superfamily, the melanoma cell adhesion molecule MCAM has been described to drive tumor progression in multiple cancer types (Wang and Yan 2013 ). Initially, MCAM was described as a tumor antigen expressed in primary melanomas but not benign melanocytic nevi (Lehmann et al. 1987 ). Subsequent studies reported an increase of MCAM expression in melanoma cells during metastatic disease progression (Lehmann et al. 1989 ). The specificity of MCAM in malignant lesions has been challenged by observations identifying abundant MCAM expression also in benign melanocytic nevi (Shih et al. 1994 ). First experimental evidence for a tumor promoting role of MCAM was obtained through transgenic overexpression of MCAM in the melanoma cell line SB-2, which resulted in an accelerated tumor growth of MCAM overexpressing cells after transplantation in mice (Xie et al. 1997 ). Furthermore, MCAM has been described to mediate adhesion between melanoma and endothelial cells in vitro (Shih et al. 1997 ). How MCAM promotes tumor invasion and metastatic spread is currently not fully understood. In our current work, we combine immunohistopathologic analyses of 69 advanced human primary melanomas and 42 melanoma metastases, bioinformatic analyses of published transcriptome datasets, and experimental studies in a mouse melanoma model to address the function of MCAM in melanoma progression. Materials and methods Cell culture HCmel12 mouse melanoma cells were derived from the Hgf-Cdk4 mouse melanoma model as previously described (Bald et al. 2014 ). Cells were cultivated in Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies) supplemented with 10% fetal calf serum (Biochrome), 2 mM L-glutamine (Life Technologies), 10 mM non-essential amino acids (Life Technologies), 1 mM HEPES (Life Technologies), 20 µM β-mercaptoethanol (Sigma), 100 IU/ml penicillin and 100 µg/ml streptomycin (Invitrogen). Cultivation was performed in a humidified incubator at 5% CO2. Cells were screened for contamination with mycoplasma with no detection of mycoplasma. Patient material 69 primary melanomas and 42 melanoma metastases were obtained during routine patient care at the Department for Dermatology of the University Hospital Magdeburg. Sample processing and hematoxylin & eosin staining was performed using standard histopathologic procedures. Immunohistochemistry was performed using an automated Ventana BenchMark with anti-MCAM (1:200, Epitomics, catalog #AC-0052), anti-SOX10 (Master diagnóstica, catalog #MAD-000656QD) and anti-CD31 antibodies (Cell Marque, catalog #131M-98). Usage of the routinely acquired material for research was approved by the ethics committee of the Otto-von-Guericke University Magdeburg (approval number 162/20), and informed consent obtained from patients. All experiments were performed in accordance with local ethical and legal regulations. CRISPR/Cas9 Knockout of MCAM To create MCAM knockout cells, 5 x 10 5 HCmel12 melanoma cells were seeded into a 12-well plate and transfected with 1.6 µg MCAM sgRNA-plasmid and 0.4 µg pRP-TagGFP2 plasmid using the FuGene HD transfection system (Promega) according to manufacturer’s instructions. Control cells were transfected with an empty CRISPR vector without containing sgRNA. As the sgRNA backbone, the plasmid px330 (addgene Plasmid #42230) was used. Fluorescently labeled single cells were sorted using a FACSAria III cell sorter (BD) into 96-well plates. Genomic DNA from monoclones was isolated with the NucleoSpin Tissue kit (Macherey-Nagel) according to manufacturer’s protocol. The MCAM knockout target region was amplified via PCR, and subsequently sequenced on a MiSeq Sequencer (Illumina) in single-end mode for 300 cycles. Successful frameshift mutations were identified using cris.py (Connelly and Pruett-Miller 2019 ). Immunoblot Proteins from HCmel12 cells were isolated using the M-PER extraction reagent (Fermentas) supplemented with protease inhibitors (Thermo Scientific). Protein concentrations were quantified using the Pierce BCA protein assay kit (Thermo Scientific) and measurement on a microplate reader at 562 nm (Tecan Group). Subsequently, 10 µg of protein was mixed with Roti Load loading buffer (Roth) and separated by SDS-PAGE. Proteins were transferred to a PVDF-membrane with 0.45 µm pore size (GE Healthcare) by wet blotting. Membranes were blocked with 5% skim milk for 1 h and stained using the anti-MCAM antibody (1:2000, Thermo Scientific, catalog #14-1469-82, RRID AB_1210462) overnight at 4°C for the primary antibody, and the anti-mouse IgG HRP (1:3000, Cell Signaling, catalog #7076) for 1 h at room temperature. As loading control, a HRP-conjugated anti-b-actin antibody was used (1:5000, Santa Cruz, catalog #sc-47778 HRP). Detection was performed using the SignalFire ECL reagent (Cell Signaling) and the chemiluminescence acquired using an Octuplus QPLEX imager (NH DyeAgnostics). In vitro melanoma-endothelial co-culture migration assay Mouse endothelial cells (bEND) were plated at a density of 10 5 cells in a µ-Slide 8-well chambered coverslip (Ibidi) and incubated for 16h at 37° and 5% CO2. Next, 10 4 TagGFP2-labeled HCmel12 CRISPR control or HCmel12 MCAM knockout cells were carefully seeded on top of the attached endothelial cells. Migration was followed using a fully automated Leica TCS SP8 confocal microscope equipped with a climate chamber (37°C, 5% CO2 with humidity). Images were acquired every 5 minutes for 12 h using a 10x objective. For each well, 3 representative viewing fields were captured using the track-and-mark feature. Migration distance and velocity of individual cells were quantified from image stacks using ImageJ with the TrackMate plugin (Tinevez et al. 2017 ). Cells with a track duration < 30 min and track length < 50 mm were removed from further analysis. Tumor transplantation experiments C57BL/6J mice were acquired from Janvier or were taken from own breeding. 2x10 5 HCmel12 CRISPR control cells or a mixture of three HCmel12 MCAM knockout clones in equal proportion were transplanted intracutaneously into the right flank using a 30G needle (BD). Tumor growth monitoring was performed three times per week using a vernier caliper and recorded as mean diameter. Mice were euthanized when tumors exceeded 20 mm in diameter or when signs of illness in accordance with local ethical regulations were observed. Macroscopic counting of lung metastases was conducted by inspection. Mice were age- and sex-matched, and randomly assigned to experimental groups at the start of each experiment. All experiments were conducted using groups of six mice and repeated independently at least twice. Experiments were performed in accordance with local ethical and legal regulations with the approval of the responsible authorities (Landesverwaltungsamt Saxony-Anhalt, Germany, approval number: 42502-2-1393 Uni MD). Bioinformatic analysis of published datasets TCGA bulk RNA sequencing data for cutaneous melanoma samples were downloaded from cBioPortal as rsem normalized count matrix (Akbani et al. 2015 ; Cerami et al. 2012 ). For the survival analysis, MCAM expression in samples was binarized using the mean as threshold. Clinical data including overall survival was also obtained from cBioPortal. The raw count matrix of single-cell RNA sequencing data from human melanocytes was obtained from GEO (accession number GSE151091) (Belote et al. 2021 ) and loaded into scanpy (Wolf et al. 2018 ). Data was preprocessed by filtering cells with < 50.000 unique reds, 20%. Genes detected in < 3 cells were removed from further analysis. Doublets detection was performed using scrublet (Wolock et al. 2019 ), and detected doublets removed. Reads were normalized using the size factor as calculated using scran (L. Lun et al. 2016 ). Mouse single cell RNA sequencing data from the NRAS/Ink4a model was obtained from the Marine group as published ( https://drive.google.com/drive/folders/1poq4Lo5AxVp0WpG1EMgIjIeDR4q98zcA ) (Karras et al. 2022 ). The cell type annotation was used as published, with subsequent annotation of malignant cells as MCMA high for cells with MCAM expression greater than the cohort median + standard deviation. The interaction analysis was performed using CellChatv2 (Jin et al. 2023 ) as outlined in the documentation using standard parameters. Bulk RNA sequencing data from mouse melanocytes was obtained from GEO (accession number GSE140193) (Marie et al. 2020 ). For the heatmap, genes with a spearman correlation coefficient > 0.5 with age in the human melanocyte dataset were shown. Results MCAM protein and mRNA expression do not increase significantly during disease progression from primary to metastatic melanomas. In initial studies, we reassessed the value of MCAM as a potential marker for melanoma progression (Figure 1a). We stained sections of advanced primary melanomas with a vertical tumor thickness ≥ 1 mm that were derived from 69 patients (Table 1) for MCAM, the melanoma marker Sox10, and the endothelial marker CD31 using immunohistochemistry. As previously reported, MCAM was not expressed on epidermal melanocytes, but on many melanoma cells with high intra- and intertumoral heterogeneity (Figure 1b). In addition, endothelial cells constitutively expressed MCAM (Figure 1b). We quantified the expression of MCAM in tumor cells, taking into account both the stain area and intensity (Supplementary Figure 1a). In contrast to previous reports (Lehmann et al. 1989; Shih et al. 1994), we did not observe a significant correlation between MCAM staining and vertical tumor thickness in our cohort (Supplementary Figure 1b). We also stained sections of melanoma skin and lymph node metastases derived from 42 patients and found MCAM expression to be slightly increased compared to primary melanomas (Figure 1c). An analysis of the subset of matched primary and metastatic melanomas derived from 14 patients did not show a clear increase of MCAM staining during disease progression (Figure 1d). Table 1: Characteristics of the primary melanoma cohort Characteristic Total (n=69) Age 1 71 (59-78) Sex: male 43 (62.3%) Sex: female 26 (37.7%) Vertical tumor thickness 1 4.3 (2.8-7.0) Location: Head/neck 14 (20.3%) Location: Trunk 24 (34.8%) Location: Upper extremity 10 (14.5%) Location: Lower extremity 21 (30.4%) Type: SSM 19 (27.5%) Type: Nodular 30 (43.5%) Type: ALM 12 (17.4%) Type: LMM 1 (1.4%) Type: Other 7 (10.1%) Sentinel positive 18 (26.1%) Sentinel negative 38 (55.1%) Sentinel not performed 13 (18.8%) BRAF positive 16 (23.2%) BRAF negative 13 (18.8%) BRAF not tested 40 (58.0%) Stage: IA 1 (1.4%) Stage: IB 6 (8.7%) Stage: IIA 8 (11.6%) Stage: IIB 9 (13.0%) Stage: IIC 12 (17.4%) Stage: IIIA 1 (1.4%) Stage: IIIB 3 (4.3%) Stage: IIIC 24 (34.8%) Stage: IIID 1 (1.4%) Stage: IV 4 (5.8%) 1 Values in parantheses indicate the interquartile range. Next, we evaluated the expression of MCAM in bulk RNA sequencing data of the “Cancer Genome Atlas” project (Akbani et al. 2015). This bioinformatic analysis did not reveal differences of MCAM mRNA expression levels between primary and metastatic melanomas (Figure 1e). Moreover, we stratified patients into cohorts with MCAM mRNA expression above and below the mean. Patients from these cohorts did not differ significantly in their melanoma-specific survival (Figure 1f). In summary, these results do not support the notion that MCAM expression can serve as a marker for melanoma disease progression. MCAM is expressed on fetal melanocyte precursors and downregulated during maturation. Metastatic dissemination of melanoma cells has been associated with the ability to closely interact with endothelial cells and to acquire dedifferentiated and stem-like cell states (Bald et al. 2014; Karras et al. 2022). In this process, melanoma cells are thought to reactivate the migratory abilities of their neural crest precursors. We therefore hypothesized that the upregulation of MCAM on melanoma cells might reflect an embryonal transcriptional program. We addressed this hypothesis in bioinformatic analyses of a scRNA sequencing dataset obtained from human melanocytes at different developmental stages (Belote et al. 2021). Whereas MCAM mRNA expression was strongly expressed in many fetal melanocytes, we detected only few neonatal and hardly any adult melanocytes with high MCAM mRNA levels (Figure 2a-b). Next, we calculated spearman rank correlation coefficients for each gene across melanocyte developmental stages. Interestingly, MCAM was the only cell adhesion molecule that showed a significant inverse correlation with melanocyte maturation (Figure 2c). In another bulk RNA sequencing dataset from mouse melanocytes sampled at different developmental stages (Marie et al. 2020), MCAM expression was also downregulated during melanocyte maturation, indicating an evolutionary conserved role of MCAM during melanocyte maturation (Figure 2c). Bioinformatic analyses support a functional role for MCAM in melanoma cell – endothelial cell interactions. During embryonal development, neural crest cells have been demonstrated to interact with endothelial cells (George et al. 2016). Due to the high expression of MCAM on both melanoma as well as endothelial cells, we hypothesized that MCAM might also promote the interaction of melanoma and endothelial cells. In order to test this hypothesis, we analyzed a scRNA sequencing dataset of mouse melanomas from the Nras/Ink4a model (Karras et al. 2022). As expected, we detected high expression levels of MCAM in melanoma cells, endothelial cells and pericytes (Figure 3a-b). We next used CellChat to computationally infer cellular interaction networks (Jin et al. 2023). In this analysis, we were able to detect a tight interaction network between melanoma cells, endothelial cells, pericytes and cancer-associated fibroblasts (Figure 3c). Interestingly, this interaction was stronger in melanoma cells with high MCAM expression compared to melanoma cells with low MCAM expression (Figure 3d), suggesting that MCAM promotes the interaction of melanoma cells to endothelial cells and pericytes. MCAM-deficient HCmel12 mouse melanoma cells seeded onto endothelial cell monolayers in vitro show reduced motility. To experimentally confirm the hypothesis that MCAM promotes the interaction of melanoma and endothelial cells, we generated MCAM knockout mouse melanoma cells (Figure 4a). For this, we used the cell line HCmel12, which has been shown to readily migrate on endothelial cells in vitro (Bald et al. 2014). Disruption of the MCAM gene was confirmed via next generation sequencing, and MCAM knockout validated on protein level via immunoblot (Figure 4b-c). We then followed migration of HCmel12 MCAM knockout and HCmel12 CRISPR control cells on endothelial cell monolayers in vitro using time-lapse video microscopy (Figure 4d). In agreement with our hypothesis, we observed a significant reduction of migration distance and velocity of HCmel12 MCAM knockout cells compared to HCmel12 CRISPR control cells (Figure 4e). MCAM-deficient HCmel12 mouse melanoma cells transplanted into the skin in vivo show reduced numbers of spontaneous lung metastasis. The angiotropic growth of melanoma cells has been reported to promote melanoma metastatic spread (Bald et al. 2014). Because the knockout of MCAM disrupted the migration of melanoma cells on endothelial cells, we hypothesized that MCAM knockout might also reduce metastatic dissemination of melanoma cells. To experimentally test this hypothesis, we intradermally transplanted HCmel12 CRISPR control and HCmel12 MCAM knockout cells in syngeneic BL6 mice (Figure 5a). Mice transplanted with MCAM knockout cells showed a longer survival (Figure 5b-c) compared to HCmel12 CRISPR control cells. Whereas mice bearing HCmel12 CRISPR control melanomas frequently developed macroscopically visible lung metastases, no lung metastases were observed in mice transplanted with HCmel12 MCAM knockout cells (Figure 5d). Also in immunocompromised NOD/SCID mice, HCmel12 MCAM knockout cells developed significantly fewer lung metastases compared to their MCAM proficient HCmel12 CRISPR control counterparts (Figure 5e-h). These results demonstrate that MCAM promotes metastatic dissemination of melanoma cells. Discussion MCAM has previously been described as a marker of melanoma disease progression and metastasis (Lehmann et al. 1989 ; Shih et al. 1994 ). Here, we profiled MCAM protein expression using immunohistochemistry and MCAM mRNA expression using bioinformatic analyses of published transcriptome datasets. In our work, we found no support for MCAM as a biomarker for melanoma progression. In our bioinformatic analyses, we find abundant expression of MCAM in developmental melanocyte precursors, suggesting that the expression of MCAM in melanoma cells might reflect the reacquisition of stem-like phenotypes of their embryonal precursors. Interestingly, recent reports also identified MCAM expression as a marker of migrating developmental neural crest cell phenotypes (Soldatov et al. 2019 ), further supporting a role for MCAM in melanocyte development and migration. Bioinformatic inference of cellular communication networks revealed that melanoma cells with high MCAM expression more actively engage in signalling crosstalk with endothelial cells. The close interaction of melanoma cells with vessels, a process known as angiotropism, has previously been identified as a driver of melanoma metastasis (Bald et al. 2014 ; Braun et al. 2020 ; Lugassy et al. 2022 ). In addition to securing the supply of oxygen and nutrients, melanoma-endothelial interactions have been demonstrated to promote stem-like, migratory melanoma cell phenotypes (Karras et al. 2022 ; Nowosad et al. 2023 ). Our observations that a CRISPR-Cas9 mediated disruption of the MCAM gene in melanoma cells impairs their ability to migrate on endothelial cell surfaces in vitro and decreases their ability to develop spontaneous lung metastases in vivo functionally support the notion that MCAM mediates melanoma-endothelial interactions and thereby facilitates metastatic progression. MCAM on melanoma cells could interact with molecules such as Laminin-411 that are highly expressed in vessel walls and have been described as ligands for MCAM (Flanagan et al. 2012 ). MCAM has also been described as a co-receptor for several growth factor receptors such as VEGFR2 or PDGFR-β, and may thereby promote cell growth and simulate tumor vascularization (Chen et al. 2018 ; Jiang et al. 2012 ). However, the exact molecular mechanisms how MCAM facilitates melanoma-endothelial interactions remain to be determined in future work. Declarations Data Availability Statement All data generated or analyzed during this study are included in this published article and its supplementary information files. Conflicts of Interest Statement The authors have no conflicts of interest to declare. Acknowledgements We thank A. Lässig, S. Simon, and I. Döring for their expert assistance with histopathologic workup of human melanoma samples, and M. Zenker and D. Schanze for sequencing of genetically engineered cells. Funding A.D.B. and M.M. were funded by the Else Kröner Forschungskolleg Magdeburg (grant numbers 2017_Kolleg.07; TP3 and TP4). E.G. received funding from the Deutsche Forschungsgemeinschaft (grant number FOR2372). Ethics Statement All animal experiments were conducted in accordance with the ARRIVE guidelines and in compliance with federal and international guidelines with the approval of the responsible authorities (Landesverwaltungsamt Saxony-Anhalt, Germany, approval number: 42502-2-1393 Uni MD). The use of the routinely acquired patient-derived melanoma material for research was approved by the ethics committee of the Otto-von-Guericke University Magdeburg (approval number 162/20). Author Contribution Statement A.D.B. and M.M. performed experiments and analyzed data. M.M. and A.D.B. collected clinical data. A.D.B. performed bioinformatics analyses. A.D.B. and T.T. evaluated histopathological sections. A.D.B., T.T. and E.G. designed experiments. A.D.B., M.M., T.T. and E.G. contributed intellectual input and helped to interpret data. E.G. supervised the research project. 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Science. 2019;364(6444):eaas9536 Tinevez J-Y, Perry N, Schindelin J, Hoopes GM, Reynolds GD, Laplantine E, et al. TrackMate: An open and extensible platform for single-particle tracking. Methods. 2017;115:80–90 Wang Z, Yan X. CD146, a multi-functional molecule beyond adhesion. Cancer Lett. 2013;330(2):150–62 Wolf FA, Angerer P, Theis FJ. SCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 2018;19(1):15 Wolock SL, Lopez R, Klein AM. Scrublet: Computational Identification of Cell Doublets in Single-Cell Transcriptomic Data. Cell Syst. 2019;8(4):281-291.e9 Xie S, Luca M, Huang S, Gutman M, Reich R, Johnson JP, et al. Expression of MCAM/MUC18 by Human Melanoma Cells Leads to Increased Tumor Growth and Metastasis. Cancer Res. 1997;57(11):2295–303 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4183647","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":291530694,"identity":"311bea12-0be7-468b-9f37-fc1f6e9c609a","order_by":0,"name":"Andreas Dominik Braun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIiWNgGAWjYHACAySyAogPQIQZG4jTcoZkLYxtRGjhn9287cOHmjoGc+nDz6QL522T4zvAfEzyaxuDbD8OLRJ3jhXPnHHsMINlX5qZ9Mxtt40lD7ClScu2MRjPxGXNjRxjZh62AwwGZxjMpHm33U7ccIDH2FiyjQHIwK5DHqTlz786oBb2b9K8c27Xw7Xsx6HFAKSFsY0ZqIUHaEvD7QSDAzyGDz+CbMHhLsMbacWMvX2HeSx7eIqteY7dNpx5mC3xMcM5CeMZOGyRu5G8meHHtzo5cx72jbd5am7L8x1vPnDwR5mNbD8u70MBD4LJDEQ8DBL41WMAxh8kahgFo2AUjIJhDQBoSFq47pCaTgAAAABJRU5ErkJggg==","orcid":"","institution":"University Hospital Magdeburg","correspondingAuthor":true,"prefix":"","firstName":"Andreas","middleName":"Dominik","lastName":"Braun","suffix":""},{"id":291530695,"identity":"66f141b8-b276-4209-be57-1b6abbeeb5b9","order_by":1,"name":"Miriam Mengoni","email":"","orcid":"","institution":"University Hospital Magdeburg","correspondingAuthor":false,"prefix":"","firstName":"Miriam","middleName":"","lastName":"Mengoni","suffix":""},{"id":291530696,"identity":"6d918289-0d90-4169-8b4e-5e8ea0ed3ff7","order_by":2,"name":"Thomas Tüting","email":"","orcid":"","institution":"University Hospital Magdeburg","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Tüting","suffix":""},{"id":291530697,"identity":"4a248ecf-f6b4-4fcf-a893-86b4b762ff82","order_by":3,"name":"Evelyn Gaffal","email":"","orcid":"","institution":"University Hospital Magdeburg","correspondingAuthor":false,"prefix":"","firstName":"Evelyn","middleName":"","lastName":"Gaffal","suffix":""}],"badges":[],"createdAt":"2024-03-28 16:44:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4183647/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4183647/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54787238,"identity":"a443aaae-7f50-450e-9bc4-64ea8a790cca","added_by":"auto","created_at":"2024-04-16 19:00:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":712856,"visible":true,"origin":"","legend":"\u003cp\u003eMCAM protein and mRNA expression do not increase significantly during disease progression from primary to metastatic melanomas.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. Conceptual outline and overview of the current work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e. Representative immunohistochemistry stainings of vessels, intraepidermal melanocytes and melanomas. Scale bar indicates 100 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e. Distribution of MCAM stain grades in 69 primary melanomas and 42 melanoma metastases. * = p\u0026lt;0.05, Mann-Whitney-U test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e. Comparison of MCAM stain grades from matched primary and metastatic melanomas in 14 patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e. mRNA expression of MCAM in primary and metastatic melanoma samples from the TCGA SKCM cohort. The bars indicate the mean. Samples with MCAM expression ≥ mean are marked in red. ns=not significant, unpaired two-sided t-test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e. Kaplan-Meier curves of melanoma-specific survival for TCGA SKCM cohort stratified by MCAM expression. ns = not significant, log-rank test.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4183647/v1/f02a654d1189ca8a1a9d71d0.png"},{"id":54787217,"identity":"c3496968-718f-4a1a-b32d-cbb3f1c4be8b","added_by":"auto","created_at":"2024-04-16 19:00:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":448465,"visible":true,"origin":"","legend":"\u003cp\u003eMCAM is expressed on fetal melanocyte precursors and downregulated during maturation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. Dimensionality reduction and visualization of single-cell transcriptomes from melanocytes from different developmental stages using UMAP. Data was obtained from the GEO (GSE151091).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e. MCAM expression in single cells grouped by developmental stage. *** = p\u0026lt;0.001, Kruskal-Wallis test with post-hoc Mann-Whitney-U test and Bonferroni-correction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e. Heatmap of genes correlated developmental stage in human melanocytes. Shown are all genes with a spearman correlation coefficient \u0026gt; 0.5. The same genes are also shown for mouse melanocytes obtained at different ages (GSE140193).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4183647/v1/a05b4076536b6cdfbb822d50.png"},{"id":54787219,"identity":"a00a737f-efbc-4a4e-b8cd-abd6b49a07cb","added_by":"auto","created_at":"2024-04-16 19:00:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":271906,"visible":true,"origin":"","legend":"\u003cp\u003eBioinformatic analyses suggest a functional role for MCAM in melanoma cell – endothelial cell interactions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. Dimensionality reduction and visualization of single-cell transcriptomes from melanocytes from the Nras/Ink4a mouse melanoma model using UMAP. Data was obtained from the Marine group as published (Karras et al. 2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e. MCAM expression in single cells grouped by cell type.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e. Computationally inferred interaction network between cell types performed with CellChatv2. The width of the lines indicates the signaling strength, the diameter of the dots indicates the group sizes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e. Incoming and outgoing signal strength from computationally inferred interactions for each cell type.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4183647/v1/73cfcace3214bd3aca2693ca.png"},{"id":54787237,"identity":"69fe30c5-6181-4301-bede-a2008f962477","added_by":"auto","created_at":"2024-04-16 19:00:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":314608,"visible":true,"origin":"","legend":"\u003cp\u003eMCAM-deficient HCmel12 mouse melanoma cells show reduced migratory activity on endothelial cell monolayers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. Chromosomal position of Mcam-gene locus in mice. Red triangles indicate the guide RNA targeting sites.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e. Next generation sequencing of HCmel12 MCAM CRISPR knockout poly- and monoclones.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e. Immunoblot of three HCmel12 MCAM CRISPR knockout monoclones.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e. Representative images of in-vitro melanoma-endothelial co-culture migration assay. Images are shown at 10x magnification. Colors indicate individual tracks as constructed by TrackMate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e. Mean track distance and velocity for individual melanoma cells. *** = p\u0026lt;0.001, Mann-Whitney-U test.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4183647/v1/48596ed73ae4e34e41dd1ceb.png"},{"id":54787235,"identity":"b1c51aa4-09ec-4d15-9002-eea3e4a7004c","added_by":"auto","created_at":"2024-04-16 19:00:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":119280,"visible":true,"origin":"","legend":"\u003cp\u003eMCAM-deficient HCmel12 mouse melanoma cells transplanted into the skin in vivo show reduced numbers of spontaneous lung metastasis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. Experimental protocol for the analysis of tumor growth and spontaneous lung metastasis of HCmel12 CRISPR control and HCmel12 MCAM knockout cells in C57BL/6 mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e., \u003cstrong\u003ec\u003c/strong\u003e. Tumor growth (b) and Kaplan-Meier survival curves (c) of C57BL/6 mice bearing established tumors. * = p\u0026lt;0.05, log-rank test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e. Number of spontaneous macroscopic lung metastases in C57BL/6 mice. * = p\u0026lt;0.05, Mann-Whitney-U test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e. Experimental protocol for the analysis of tumor growth and spontaneous lung metastasis of HCmel12 CRISPR control and HCmel12 MCAM knockout cells in NOD/SCID mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e., \u003cstrong\u003eg\u003c/strong\u003e. Tumor growth (f) and Kaplan-Meier survival curves (g) of NOD/SCID mice bearing established tumors. ns = not significant, log-rank test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh\u003c/strong\u003e. Number of spontaneous macroscopic lung metastases in NOD/SCID mice. * = p\u0026lt;0.05, Mann-Whitney-U test.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4183647/v1/898c258da8029bd77d6a67ae.png"},{"id":58291452,"identity":"0b5408f9-377d-410d-9207-6cc9bd1a351a","added_by":"auto","created_at":"2024-06-13 13:44:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2081067,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4183647/v1/8822887c-03ee-41dd-b18c-c7fff4780a60.pdf"},{"id":54787236,"identity":"7e8dd7f0-5b34-4c14-9c57-eab863219778","added_by":"auto","created_at":"2024-04-16 19:00:45","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11921788,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4183647/v1/c8747121c72549f0e3ceca81.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"MCAM expression facilitates melanoma-endothelial interactions and promotes metastatic disease progression","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInvasive growth and metastatic dissemination are the primary cause of death in patients with cancer (Gerstberger et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In order to detach from the primary tumor and successfully invade distant tissues to form metastases, cancer cells need to rewire their cell adhesion machinery (Hamidi and Ivaska \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). As a prototype of this process, cells from many cancer types have been shown to downregulate the adhesion molecule E-cadherin, and upregulate the related molecule N-cadherin in a process termed epithelial to mesenchymal transition (Dongre and Weinberg \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In addition to cadherins, also members from the integrin- and immunoglobulin superfamily have been implicated in the migration of cancer cells (Janiszewska et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs a prominent example of a cell adhesion molecule of the immunoglobulin superfamily, the melanoma cell adhesion molecule MCAM has been described to drive tumor progression in multiple cancer types (Wang and Yan \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Initially, MCAM was described as a tumor antigen expressed in primary melanomas but not benign melanocytic nevi (Lehmann et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Subsequent studies reported an increase of MCAM expression in melanoma cells during metastatic disease progression (Lehmann et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). The specificity of MCAM in malignant lesions has been challenged by observations identifying abundant MCAM expression also in benign melanocytic nevi (Shih et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1994\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFirst experimental evidence for a tumor promoting role of MCAM was obtained through transgenic overexpression of MCAM in the melanoma cell line SB-2, which resulted in an accelerated tumor growth of MCAM overexpressing cells after transplantation in mice (Xie et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Furthermore, MCAM has been described to mediate adhesion between melanoma and endothelial cells in vitro (Shih et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). How MCAM promotes tumor invasion and metastatic spread is currently not fully understood.\u003c/p\u003e \u003cp\u003eIn our current work, we combine immunohistopathologic analyses of 69 advanced human primary melanomas and 42 melanoma metastases, bioinformatic analyses of published transcriptome datasets, and experimental studies in a mouse melanoma model to address the function of MCAM in melanoma progression.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHCmel12 mouse melanoma cells were derived from the Hgf-Cdk4 mouse melanoma model as previously described (Bald et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Cells were cultivated in Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies) supplemented with 10% fetal calf serum (Biochrome), 2 mM L-glutamine (Life Technologies), 10 mM non-essential amino acids (Life Technologies), 1 mM HEPES (Life Technologies), 20 \u0026micro;M β-mercaptoethanol (Sigma), 100 IU/ml penicillin and 100 \u0026micro;g/ml streptomycin (Invitrogen). Cultivation was performed in a humidified incubator at 5% CO2. Cells were screened for contamination with mycoplasma with no detection of mycoplasma.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePatient material\u003c/h2\u003e \u003cp\u003e69 primary melanomas and 42 melanoma metastases were obtained during routine patient care at the Department for Dermatology of the University Hospital Magdeburg. Sample processing and hematoxylin \u0026amp; eosin staining was performed using standard histopathologic procedures. Immunohistochemistry was performed using an automated Ventana BenchMark with anti-MCAM (1:200, Epitomics, catalog #AC-0052), anti-SOX10 (Master diagn\u0026oacute;stica, catalog #MAD-000656QD) and anti-CD31 antibodies (Cell Marque, catalog #131M-98). Usage of the routinely acquired material for research was approved by the ethics committee of the Otto-von-Guericke University Magdeburg (approval number 162/20), and informed consent obtained from patients. All experiments were performed in accordance with local ethical and legal regulations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCRISPR/Cas9 Knockout of MCAM\u003c/h2\u003e \u003cp\u003eTo create MCAM knockout cells, 5 x 10\u003csup\u003e5\u003c/sup\u003e HCmel12 melanoma cells were seeded into a 12-well plate and transfected with 1.6 \u0026micro;g MCAM sgRNA-plasmid and 0.4 \u0026micro;g pRP-TagGFP2 plasmid using the FuGene HD transfection system (Promega) according to manufacturer\u0026rsquo;s instructions. Control cells were transfected with an empty CRISPR vector without containing sgRNA. As the sgRNA backbone, the plasmid px330 (addgene Plasmid #42230) was used. Fluorescently labeled single cells were sorted using a FACSAria III cell sorter (BD) into 96-well plates. Genomic DNA from monoclones was isolated with the NucleoSpin Tissue kit (Macherey-Nagel) according to manufacturer\u0026rsquo;s protocol. The MCAM knockout target region was amplified via PCR, and subsequently sequenced on a MiSeq Sequencer (Illumina) in single-end mode for 300 cycles. Successful frameshift mutations were identified using cris.py (Connelly and Pruett-Miller \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblot\u003c/h2\u003e \u003cp\u003eProteins from HCmel12 cells were isolated using the M-PER extraction reagent (Fermentas) supplemented with protease inhibitors (Thermo Scientific). Protein concentrations were quantified using the Pierce BCA protein assay kit (Thermo Scientific) and measurement on a microplate reader at 562 nm (Tecan Group). Subsequently, 10 \u0026micro;g of protein was mixed with Roti Load loading buffer (Roth) and separated by SDS-PAGE. Proteins were transferred to a PVDF-membrane with 0.45 \u0026micro;m pore size (GE Healthcare) by wet blotting. Membranes were blocked with 5% skim milk for 1 h and stained using the anti-MCAM antibody (1:2000, Thermo Scientific, catalog #14-1469-82, RRID AB_1210462) overnight at 4\u0026deg;C for the primary antibody, and the anti-mouse IgG HRP (1:3000, Cell Signaling, catalog #7076) for 1 h at room temperature. As loading control, a HRP-conjugated anti-b-actin antibody was used (1:5000, Santa Cruz, catalog #sc-47778 HRP). Detection was performed using the SignalFire ECL reagent (Cell Signaling) and the chemiluminescence acquired using an Octuplus QPLEX imager (NH DyeAgnostics).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro melanoma-endothelial co-culture migration assay\u003c/h2\u003e \u003cp\u003eMouse endothelial cells (bEND) were plated at a density of 10\u003csup\u003e5\u003c/sup\u003e cells in a \u0026micro;-Slide 8-well chambered coverslip (Ibidi) and incubated for 16h at 37\u0026deg; and 5% CO2. Next, 10\u003csup\u003e4\u003c/sup\u003e TagGFP2-labeled HCmel12 CRISPR control or HCmel12 MCAM knockout cells were carefully seeded on top of the attached endothelial cells. Migration was followed using a fully automated Leica TCS SP8 confocal microscope equipped with a climate chamber (37\u0026deg;C, 5% CO2 with humidity). Images were acquired every 5 minutes for 12 h using a 10x objective. For each well, 3 representative viewing fields were captured using the track-and-mark feature. Migration distance and velocity of individual cells were quantified from image stacks using ImageJ with the TrackMate plugin (Tinevez et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Cells with a track duration\u0026thinsp;\u0026lt;\u0026thinsp;30 min and track length\u0026thinsp;\u0026lt;\u0026thinsp;50 mm were removed from further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTumor transplantation experiments\u003c/h2\u003e \u003cp\u003eC57BL/6J mice were acquired from Janvier or were taken from own breeding. 2x10\u003csup\u003e5\u003c/sup\u003e HCmel12 CRISPR control cells or a mixture of three HCmel12 MCAM knockout clones in equal proportion were transplanted intracutaneously into the right flank using a 30G needle (BD). Tumor growth monitoring was performed three times per week using a vernier caliper and recorded as mean diameter. Mice were euthanized when tumors exceeded 20 mm in diameter or when signs of illness in accordance with local ethical regulations were observed. Macroscopic counting of lung metastases was conducted by inspection. Mice were age- and sex-matched, and randomly assigned to experimental groups at the start of each experiment. All experiments were conducted using groups of six mice and repeated independently at least twice. Experiments were performed in accordance with local ethical and legal regulations with the approval of the responsible authorities (Landesverwaltungsamt Saxony-Anhalt, Germany, approval number: 42502-2-1393 Uni MD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatic analysis of published datasets\u003c/h2\u003e \u003cp\u003eTCGA bulk RNA sequencing data for cutaneous melanoma samples were downloaded from cBioPortal as rsem normalized count matrix (Akbani et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Cerami et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). For the survival analysis, MCAM expression in samples was binarized using the mean as threshold. Clinical data including overall survival was also obtained from cBioPortal. The raw count matrix of single-cell RNA sequencing data from human melanocytes was obtained from GEO (accession number GSE151091) (Belote et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and loaded into scanpy (Wolf et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Data was preprocessed by filtering cells with \u0026lt;\u0026thinsp;50.000 unique reds, \u0026lt; 500 genes and a fraction of ERCC spike reads\u0026thinsp;\u0026gt;\u0026thinsp;20%. Genes detected in \u0026lt;\u0026thinsp;3 cells were removed from further analysis. Doublets detection was performed using scrublet (Wolock et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and detected doublets removed. Reads were normalized using the size factor as calculated using scran (L. Lun et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Mouse single cell RNA sequencing data from the NRAS/Ink4a model was obtained from the Marine group as published (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://drive.google.com/drive/folders/1poq4Lo5AxVp0WpG1EMgIjIeDR4q98zcA\u003c/span\u003e\u003cspan address=\"https://drive.google.com/drive/folders/1poq4Lo5AxVp0WpG1EMgIjIeDR4q98zcA\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Karras et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The cell type annotation was used as published, with subsequent annotation of malignant cells as MCMA high for cells with MCAM expression greater than the cohort median\u0026thinsp;+\u0026thinsp;standard deviation. The interaction analysis was performed using CellChatv2 (Jin et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) as outlined in the documentation using standard parameters. Bulk RNA sequencing data from mouse melanocytes was obtained from GEO (accession number GSE140193) (Marie et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For the heatmap, genes with a spearman correlation coefficient\u0026thinsp;\u0026gt;\u0026thinsp;0.5 with age in the human melanocyte dataset were shown.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eMCAM protein and mRNA expression do not increase significantly during disease progression from primary to metastatic melanomas.\u003c/p\u003e\n\u003cp\u003eIn initial studies, we reassessed the value of MCAM as a potential marker for melanoma progression (Figure 1a). We stained sections of advanced primary melanomas with a vertical tumor thickness \u0026ge; 1 mm that were derived from 69 patients (Table 1) for MCAM, the melanoma marker Sox10, and the endothelial marker CD31 using immunohistochemistry. As previously reported, MCAM was not expressed on epidermal melanocytes, but on many melanoma cells with high intra- and intertumoral heterogeneity (Figure 1b). In addition, endothelial cells constitutively expressed MCAM (Figure 1b). We quantified the expression of MCAM in tumor cells, taking into account both the stain area and intensity (Supplementary Figure 1a). In contrast to previous reports (Lehmann et al. 1989; Shih et al. 1994), we did not observe a significant correlation between MCAM staining and vertical tumor thickness in our cohort (Supplementary Figure 1b). We also stained sections of melanoma skin and lymph node metastases derived from 42 patients and found MCAM expression to be slightly increased compared to primary melanomas (Figure 1c). An analysis of the subset of matched primary and metastatic melanomas derived from 14 patients did not show a clear increase of MCAM staining during disease progression (Figure 1d).\u003c/p\u003e\n\u003cp\u003eTable 1: Characteristics of the primary melanoma cohort\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"368\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eCharacteristic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003eTotal (n=69)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eAge\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e71 (59-78)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eSex: male\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e43 (62.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eSex: female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e26 (37.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eVertical tumor thickness\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e4.3 (2.8-7.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eLocation: Head/neck\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e14 (20.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eLocation: Trunk\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e24 (34.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eLocation: Upper extremity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e10 (14.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eLocation: Lower extremity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e21 (30.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eType: SSM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e19 (27.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eType: Nodular\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e30 (43.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eType: ALM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e12 (17.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eType: LMM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e1 (1.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eType: Other\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e7 (10.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eSentinel positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e18 (26.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eSentinel negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e38 (55.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eSentinel not performed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e13 (18.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eBRAF positive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e16 (23.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eBRAF negative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e13 (18.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eBRAF not tested\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e40 (58.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eStage: IA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e1 (1.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eStage: IB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e6 (8.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eStage: IIA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e8 (11.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eStage: IIB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e9 (13.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eStage: IIC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e12 (17.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eStage: IIIA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e1 (1.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eStage: IIIB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e3 (4.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eStage: IIIC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e24 (34.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eStage: IIID\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e1 (1.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"62.22826086956522%\" valign=\"top\"\u003e\n \u003cp\u003eStage: IV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.77173913043478%\" valign=\"top\"\u003e\n \u003cp\u003e4 (5.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Values in parantheses indicate the interquartile range.\u003c/p\u003e\n\u003cp\u003eNext, we evaluated the expression of MCAM in bulk RNA sequencing data of the \u0026ldquo;Cancer Genome Atlas\u0026rdquo; project (Akbani et al. 2015). This bioinformatic analysis did not reveal differences of MCAM mRNA expression levels between primary and metastatic melanomas (Figure 1e). Moreover, we stratified patients into cohorts with MCAM mRNA expression above and below the mean. Patients from these cohorts did not differ significantly in their melanoma-specific survival (Figure 1f). In summary, these results do not support the notion that MCAM expression can serve as a marker for melanoma disease progression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMCAM is expressed on fetal melanocyte precursors and downregulated during maturation.\u003c/p\u003e\n\u003cp\u003eMetastatic dissemination of melanoma cells has been associated with the ability to closely interact with endothelial cells and to acquire dedifferentiated and stem-like cell states (Bald et al. 2014; Karras et al. 2022). In this process, melanoma cells are thought to reactivate the migratory abilities of their neural crest precursors. We therefore hypothesized that the upregulation of MCAM on melanoma cells might reflect an embryonal transcriptional program. We addressed this hypothesis in bioinformatic analyses of a scRNA sequencing dataset obtained from human melanocytes at different developmental stages (Belote et al. 2021). Whereas MCAM mRNA expression was strongly expressed in many fetal melanocytes, we detected only few neonatal and hardly any adult melanocytes with high MCAM mRNA levels (Figure 2a-b). Next, we calculated spearman rank correlation coefficients for each gene across melanocyte developmental stages. Interestingly, MCAM was the only cell adhesion molecule that showed a significant inverse correlation with melanocyte maturation (Figure 2c). In another bulk RNA sequencing dataset from mouse melanocytes sampled at different developmental stages (Marie et al. 2020), MCAM expression was also downregulated during melanocyte maturation, indicating an evolutionary conserved role of MCAM during melanocyte maturation (Figure 2c).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBioinformatic analyses support a functional role for MCAM in melanoma cell \u0026ndash; endothelial cell interactions.\u003c/p\u003e\n\u003cp\u003eDuring embryonal development, neural crest cells have been demonstrated to interact with endothelial cells (George et al. 2016). Due to the high expression of MCAM on both melanoma as well as endothelial cells, we hypothesized that MCAM might also promote the interaction of melanoma and endothelial cells. In order to test this hypothesis, we analyzed a scRNA sequencing dataset of mouse melanomas from the Nras/Ink4a model (Karras et al. 2022). As expected, we detected high expression levels of MCAM in melanoma cells, endothelial cells and pericytes (Figure 3a-b). We next used CellChat to computationally infer cellular interaction networks (Jin et al. 2023). In this analysis, we were able to detect a tight interaction network between melanoma cells, endothelial cells, pericytes and cancer-associated fibroblasts (Figure 3c). Interestingly, this interaction was stronger in melanoma cells with high MCAM expression compared to melanoma cells with low MCAM expression (Figure 3d), suggesting that MCAM promotes the interaction of melanoma cells to endothelial cells and pericytes.\u003c/p\u003e\n\u003cp\u003eMCAM-deficient HCmel12 mouse melanoma cells seeded onto endothelial cell monolayers \u003cem\u003ein vitro\u003c/em\u003e show reduced motility.\u003c/p\u003e\n\u003cp\u003eTo experimentally confirm the hypothesis that MCAM promotes the interaction of melanoma and endothelial cells, we generated MCAM knockout mouse melanoma cells (Figure 4a). For this, we used the cell line HCmel12, which has been shown to readily migrate on endothelial cells \u003cem\u003ein vitro\u003c/em\u003e (Bald et al. 2014). Disruption of the MCAM gene was confirmed via next generation sequencing, and MCAM knockout validated on protein level via immunoblot (Figure 4b-c). We then followed migration of HCmel12 MCAM knockout and HCmel12 CRISPR control cells on endothelial cell monolayers \u003cem\u003ein vitro\u003c/em\u003e using time-lapse video microscopy (Figure 4d). In agreement with our hypothesis, we observed a significant reduction of migration distance and velocity of HCmel12 MCAM knockout cells compared to HCmel12 CRISPR control cells (Figure 4e).\u003c/p\u003e\n\u003cp\u003eMCAM-deficient HCmel12 mouse melanoma cells transplanted into the skin \u003cem\u003ein vivo\u003c/em\u003e show reduced numbers of spontaneous lung metastasis.\u003c/p\u003e\n\u003cp\u003eThe angiotropic growth of melanoma cells has been reported to promote melanoma metastatic spread (Bald et al. 2014). Because the knockout of MCAM disrupted the migration of melanoma cells on endothelial cells, we hypothesized that MCAM knockout might also reduce metastatic dissemination of melanoma cells. To experimentally test this hypothesis, we intradermally transplanted HCmel12 CRISPR control and HCmel12 MCAM knockout cells in syngeneic BL6 mice (Figure 5a). Mice transplanted with MCAM knockout cells showed a longer survival (Figure 5b-c) compared to HCmel12 CRISPR control cells. Whereas mice bearing HCmel12 CRISPR control melanomas frequently developed macroscopically visible lung metastases, no lung metastases were observed in mice transplanted with HCmel12 MCAM knockout cells (Figure 5d). Also in immunocompromised NOD/SCID mice, HCmel12 MCAM knockout cells developed significantly fewer lung metastases compared to their MCAM proficient HCmel12 CRISPR control counterparts (Figure 5e-h). These results demonstrate that MCAM promotes metastatic dissemination of melanoma cells.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMCAM has previously been described as a marker of melanoma disease progression and metastasis (Lehmann et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Shih et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Here, we profiled MCAM protein expression using immunohistochemistry and MCAM mRNA expression using bioinformatic analyses of published transcriptome datasets. In our work, we found no support for MCAM as a biomarker for melanoma progression.\u003c/p\u003e \u003cp\u003eIn our bioinformatic analyses, we find abundant expression of MCAM in developmental melanocyte precursors, suggesting that the expression of MCAM in melanoma cells might reflect the reacquisition of stem-like phenotypes of their embryonal precursors. Interestingly, recent reports also identified MCAM expression as a marker of migrating developmental neural crest cell phenotypes (Soldatov et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), further supporting a role for MCAM in melanocyte development and migration. Bioinformatic inference of cellular communication networks revealed that melanoma cells with high MCAM expression more actively engage in signalling crosstalk with endothelial cells. The close interaction of melanoma cells with vessels, a process known as angiotropism, has previously been identified as a driver of melanoma metastasis (Bald et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Braun et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lugassy et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In addition to securing the supply of oxygen and nutrients, melanoma-endothelial interactions have been demonstrated to promote stem-like, migratory melanoma cell phenotypes (Karras et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Nowosad et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur observations that a CRISPR-Cas9 mediated disruption of the MCAM gene in melanoma cells impairs their ability to migrate on endothelial cell surfaces in vitro and decreases their ability to develop spontaneous lung metastases in vivo functionally support the notion that MCAM mediates melanoma-endothelial interactions and thereby facilitates metastatic progression. MCAM on melanoma cells could interact with molecules such as Laminin-411 that are highly expressed in vessel walls and have been described as ligands for MCAM (Flanagan et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). MCAM has also been described as a co-receptor for several growth factor receptors such as VEGFR2 or PDGFR-β, and may thereby promote cell growth and simulate tumor vascularization (Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Jiang et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, the exact molecular mechanisms how MCAM facilitates melanoma-endothelial interactions remain to be determined in future work.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eData Availability Statement\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003eConflicts of Interest Statement\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe thank A. Lässig, S. Simon, and I. Döring for their expert assistance with histopathologic workup of human melanoma samples, and M. Zenker and D. Schanze for sequencing of genetically engineered cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eA.D.B. and M.M. were funded by the Else Kröner Forschungskolleg Magdeburg (grant numbers 2017_Kolleg.07; TP3 and TP4). E.G. received funding from the Deutsche Forschungsgemeinschaft (grant number FOR2372).\u003c/p\u003e\n\u003cp\u003eEthics Statement\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with the ARRIVE guidelines and in compliance with federal and international guidelines with the approval of the responsible authorities (Landesverwaltungsamt Saxony-Anhalt, Germany, approval number: 42502-2-1393 Uni MD). The use of the routinely acquired patient-derived melanoma material for research was approved by the ethics committee of the Otto-von-Guericke University Magdeburg (approval number 162/20).\u003c/p\u003e\n\u003cp\u003eAuthor Contribution Statement\u003c/p\u003e\n\u003cp\u003eA.D.B. and M.M. performed experiments and analyzed data. M.M. and A.D.B. collected clinical data. A.D.B. performed bioinformatics analyses. A.D.B. and T.T. evaluated histopathological sections. A.D.B., T.T. and E.G. designed experiments. A.D.B., M.M., T.T. and E.G. contributed intellectual input and helped to interpret data. E.G. supervised the research project. A.D.B., M.M., T.T. and E.G. wrote and reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAkbani R, Akdemir KC, Aksoy BA, Albert M, Ally A, Amin SB, et al. Genomic Classification of Cutaneous Melanoma. Cell. 2015;161(7):1681\u0026ndash;96\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBald T, Quast T, Landsberg J, Rogava M, Glodde N, Lopez-Ramos D, et al. Ultraviolet-radiation-induced inflammation promotes angiotropism and metastasis in melanoma. Nature. 2014;507(7490):109\u0026ndash;13\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBelote RL, Le D, Maynard A, Lang UE, Sinclair A, Lohman BK, et al. Human melanocyte development and melanoma dedifferentiation at single-cell resolution. Nat. Cell Biol. 2021;\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBraun AD, Mengoni M, Bonifatius S, T\u0026uuml;ting T, Gaffal E. 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Cancer Res. 1997;57(11):2295\u0026ndash;303\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Melanoma, migration, metastasis, cell adhesion, MCAM","lastPublishedDoi":"10.21203/rs.3.rs-4183647/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4183647/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInvasive growth and metastatic dissemination represent the primary cause of death in cancer patients. In order to successfully detach from the primary tumor and establish metastases in distant tissues, cancer cells need to dynamically rewire their cell adhesion machinery. Here we revisit the potential association of MCAM, a member of the immunoglobulin superfamily that was initially identified as a melanoma antigen, with disease progression. Using immunohistochemical stainings and bioinformatic analyses of published datasets, we find similar MCAM expression levels in primary and metastatic human melanomas. In additional bioinformatic analyses, we show that MCAM is highly expressed in fetal melanocytes and subsequently downregulated during melanocyte maturation. Bioinformatic inference of cellular communication networks reveals that melanoma cells with high MCAM expression more actively engage in signaling crosstalk with endothelial cells. Experimental investigations demonstrate that disruption of MCAM in melanoma cells inhibits their migration on endothelial cell surfaces in vitro and decreases their ability to develop spontaneous lung metastases in vivo. Taken together, our results could not confirm the notion that MCAM expression represents a useful biomarker for disease progression, but provide evidence that MCAM expression might represent part of a reactivated embryonal transcriptional program that facilitates melanoma-endothelial cell interactions during metastatic progression.\u003c/p\u003e","manuscriptTitle":"MCAM expression facilitates melanoma-endothelial interactions and promotes metastatic disease progression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-16 19:00:14","doi":"10.21203/rs.3.rs-4183647/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7b6d0e98-ff41-4603-9365-2852628fc734","owner":[],"postedDate":"April 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":30711192,"name":"Biological sciences/Cancer/Skin cancer/Melanoma"},{"id":30711193,"name":"Biological sciences/Cancer/Cancer microenvironment"},{"id":30711194,"name":"Biological sciences/Cancer/Metastases"},{"id":30711195,"name":"Biological sciences/Cell biology/Cell migration"},{"id":30711196,"name":"Biological sciences/Cell biology/Cell adhesion"}],"tags":[],"updatedAt":"2024-06-13T13:36:14+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-16 19:00:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4183647","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4183647","identity":"rs-4183647","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}