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Background: In malignant melanoma, liver metastases significantly reduce survival, even despite highly effective new therapies. Given the increase in metabolic liver diseases such as metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH), this study investigated the impact of liver sinusoidal endothelial cell (LSEC)-specific alterations in MASLD/MASH on hepatic melanoma metastasis., Methods: Mice were fed a choline-deficient L-amino acid-defined (CDAA) diet for ten weeks to induce MASH-associated liver fibrosis, or a CDAA diet or a high fat diet (HFD) for shorter periods of time to induce early steatosis-associated alterations. Liver metastasis formation was assessed using melanoma cell lines B16F10Luc2 and Wt31. LSEC-specific GATA4 knockout mice (Gata4 LSEC-KO/BL ) developing MASH-like liver fibrosis without steatosis via a pathogenic angiocrine switch were included to compare the impact of liver fibrosis versus hepatic steatosis on hepatic melanoma metastasis. Bulk RNA-Seq of isolated LSECs from CDAA-fed and control mice was performed. Levels of adhesion molecules (VCAM1, ICAM1, E-selectin) were monitored, and ICAM1 and VCAM1 antibody therapy was employed., Results: Feeding a CDAA diet, in contrast to a HFD, led to increased metastasis before the development of liver fibrosis. Gata4 LSEC-KO/BL mice characterized by vascular changes ensuing perisinusoidal liver fibrosis without steatosis also exhibited increased metastasis. Early molecular alterations in the hepatic vascular niche, rather than fibrosis or steatosis, correlated with metastasis, as shown by LSEC dedifferentiation and upregulation of endothelial adhesion molecules. The metastatic process in CDAA-fed mice was also dependent on the respective melanoma cell lines used and on the route of their metastatic spread. ICAM1 inhibition, but not VCAM1 inhibition reduced melanoma cell retention., Conclusion: We discovered that the hepatic vascular niche acts as a delicate sensor to even short-term nutritional alterations during the development of MASLD/MASH. The dynamic adaptations to the metabolic challenges of developing MASLD/MASH caused an early shift from the normal hepatic vascular niche to a pre-metastatic vascular niche that promoted hepatic melanoma metastasis in the context of cell-autonomous and acquired melanoma cell features. Altogether, our findings provide a potential avenue for angiotargeted therapies to prevent hepatic melanoma metastasis., Competing Interests: Declarations. Ethics approval and consent to participate: All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Academy of Sciences and the animal ethics committee of Baden Wuerttemberg (Regierungspraesidium Karlsruhe) approved all animal experiments. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Accurate melanoma diagnosis is crucial for patient outcomes and reliability of AI diagnostic tools. We assess interrater variability among eight expert pathologists reviewing histopathological images and clinical metadata of 792 melanoma-suspicious lesions prospectively collected at eight German hospitals. Moreover, we provide access to the largest panel-validated dataset featuring dermoscopic and histopathological images with metadata. Complete agreement is achieved in 53.5% of cases (424/792), and a majority vote ( ≥ five pathologists) in 90.9% (720/792). Considerable discordance is observed for non-invasive melanomas (complete agreement in only 10/73 cases). The expert panel disagrees with the local pathologists' and dermatologists' diagnoses in 14.9% and 33.5% of cases, respectively. This variability highlights the diagnostic challenges of early-stage melanomas and the need to reconsider how ground truth is established in routine care and AI research. Including at least two pathologists or virtual panels may contribute to more consistent diagnostic results., Competing Interests: Competing interests: Prof Utikal reported personal fees from Amgen, Bristol Myers Squibb, GSK, Immunocore, LEO Pharma, Merck Sharp & Dohme, Novartis, Pierre Fabre, Rheacell, Roche, and Sanofi outside the submitted work. Dr Wohlfeil received honoraria from Bristol Myers Squibb, Novartis, and Sun Pharma outside the submitted work. Prof Meier reported grants from Novartis and Roche and others (travel support or/and speaker’s fees or/and advisor’s honoraria) from BMS, MSD, and Pierre Fabre outside the submitted work. Dr Hobelsberger reported clinical trial support and advisor’s honoraria from Almirall, speaker’s honoraria from Almirall, UCB, and AbbVie, and travel support from UCB, Janssen Cilag, Almirall, Novartis, Lilly, LEO Pharma, and AbbVie outside the submitted work. Prof Heinzerling reported other (clinical studies) from BMS, MSD, Pierre Fabre, Replimune, and Sanofi; personal fees from Biomedx, BMS, MSD, Sun, Pierre Fabre, Novartis, and Sanofi; and grants from Therakos outside the submitted work. Prof Haferkamp reported speaker’s fees and advisor’s honoraria from BMS, MSD, Novartis, and Pierre Fabre outside the submitted work. Dr Schlaak reported personal fees from BMS, Novartis, Immunocore, Sun Pharma, MSD, Recordati, and Sanofi Aventis outside the submitted work. Prof Berking reported personal fees from BMS, MSD, InflaRx, Novartis, Sanofi, LEO Pharma, Almirall Hermal, Pierre Fabre, Immunocore, and Delcath outside the submitted work. Dr Erdmann received travel support and speaker’s honoraria from Immunocore, Novartis, Pierre Fabre, and Sanofi outside the submitted work. Dr Sondermann reported grants from Almirall, Novartis, and Medi GmbH; and personal fees from AbbVie, Almirall, BMS, Boehringer Ingelheim, Celgene, Janssen, LEO Pharma, Lilly, Novartis, Pfizer, Sanofi Genzyme, and UCB outside the submitted work. Prof Goebeler reported grants for clinical studies from Argenx, Novartis, Janssen, and Galderma; personal fees from Almirall (consulting), Janssen (advisory board, speaker), GSK (advisory board, speaker), and Lilly (speaker) outside the submitted work. Dr Llamas-Velasco reported fees as an advisory board member, consultant, research support, participation in clinical trials, and honorary for speaking with the following pharmaceutical companies: Abbvie, Almirall, Amgen, Boehringer, BMS, Celgene, Janssen, Leo, Lilly, Kyowa kirin, Novartis and UCB, outside the submitted work. Dr. Kather declares consulting services for Owkin, France; DoMore Diagnostics, Norway; Panakeia, UK; AstraZeneca, UK; Scailyte, Switzerland; Mindpeak, Germany; and MultiplexDx, Slovakia. Furthermore, he holds shares in StratifAI GmbH, Germany, has received a research grant from GSK, and has received honoraria from AstraZeneca, Bayer, Eisai, Janssen, MSD, BMS, Roche, Pfizer, and Fresenius. Dr Gabriela Poch received travel support and speaker’s honoraria from MSD, BMS, Novartis, Sun Pharma, and Amgen outside the submitted work. Dr Brinker reported being the owner of Smart Health Heidelberg GmbH outside the submitted work. The remaining authors declare no competing interests., (© 2025. The Author(s).)

Background: Cancer immunotherapy has revolutionized melanoma treatment, but the high number of non-responders still emphasizes the need for improvement of therapy. One potential avenue for enhancing anti-tumor treatment is through the modulation of coagulation and platelet activity. Both have been found to play an important role in the tumor microenvironment, tumor growth and metastasis. Preclinical studies indicate a beneficial effect, clinical data has been inconsistent., Methods: We examined a cohort of advanced, non-resectable melanoma patients (n = 2419) derived from the German prospective multicenter skin cancer registry ADOReg, who were treated with immune checkpoint inhibitors (ICI). The patients were classified based on whether it was documented that they received platelet aggregation inhibition (PAI) (n = 137) (acetylsalicylic acid (ASA) or clopidogrel), anticoagulation (AC) (n = 185) (direct oral anticoagulation (DOAC), phenprocoumon, heparins) at the start of ICI or no antithrombotic medication (n = 2097) at any point during ICI treatment. The study endpoints were best overall response (BOR), progression-free survival (PFS) and overall survival (OS)., Results: A significantly improved PFS was observed in patients documented to receive ASA (15.1 vs 6.4 months, HR 0.67, 95 % CI: 0.5 to 0.88, p = 0.0047) as well as in patients to receive AC (15.1 vs. 6.4 months, HR 0.7, 95 % CI: 0.53 to 0.91, p = 0.01) compared to patients for whom no antithrombotic medication was documented. Multivariate analysis of OS showed significant risk reduction in patients who received DOAC (HR 0.68, 95 % CI: 0.49 to 0.92, p = 0.0170) or phenprocoumon (HR: 0.44, 95 % CI: 0.19 to 0.85, p = 0.0301)., Conclusion: Our study indicates a positive prognostic effect of anticoagulant and antiplatelet concomitant medication in melanoma patients receiving ICI. Further studies are needed to confrim the cancer-related benefit of adding anticoagulation or platelet inhibition to ICI treatment., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: JK has received honoraria from Bristol-Myers Squibb and Sanofi Genzyme and has received travel support from SUN Pharma and Pierre Fabre, outside the submitted work. GG has received honoraria from Bristol-Myers Squibb and has research funding from Sanofi Genzyme, outside the submitted work. IsH has received honoraria from Bristol-Myers Squibb and Sysmex, outside the submitted work. SU declares research support from Bristol Myers Squibb and Merck Serono; speakers and advisory board honoraria from Bristol Myers Squibb, Merck Sharp & Dohme, Merck Serono, and Novartis; and meeting and travel support from Almirall, Bristol-Myers Squibb, IGEA Clinical Biophysics, Merck Sharp & Dohme, Novartis, Pierre Fabre, and Sun Pharma; outside the submitted work. UL declares research support from Merck Sharp & Dohme; speakers and advisory board honoraria from Sanofi, Regeneron, Sun Pharma, Merck Sharp & Dohme, Allmiral and Novartis; and meeting and travel support from Pierre Fabre, and Sun Pharma; outside the submitted work. CB declares honoraria as speaker and/or advisory board member from Almirall Hermal, Bristol-Myers Squibb, Delcath, Immunocore, InflaRx, Leo Pharma, MSD, Novartis, Pierre Fabre, Regeneron, and Sanofi, outside the submitted work. RG received honoraria for lectures/ advisory boards from Bristol Myers Squibb, Merck Sharp Dohme, Novartis, Merck-Serono, Amgen, Almirall Hermal, Pierre-Fabre, Sun Pharma, Immunocore, 4SC, Delcath, Sanofi/ Regeneron; Meeting support SUN Pharma, Boehringer Ingelheim, PierreFabre; Research support (to institution) Novartis, Sanofi/ Regeneron, Merck Serono, Amgen, SUN Pharma, Kyowa Kirin, Almirall Hermal. DS Honoraria: Roche/Genentech, Novartis, Bristol Myers Squibb, Merck Sharp & Dohme, Immunocore, Merck Serono, Array BioPharma, Pfizer, Pierre Fabre, Philogen, Regeneron, 4SC, Sanofi/Regeneron, NeraCare GmbH, Sun Pharma, InflarxGmbH, Ultimovacs, Sandoz. Consulting or Advisory Role: Roche/Genentech, Novartis, Bristol Myers Squibb, Merck Sharp & Dohme, Merck Serono, 4SC, Pierre Fabre, Sanofi/Regeneron, Nektar. Speakers' Bureau: Bristol Myers Squibb, Merck Sharp & Dohme, Novartis, Pierre Fabre, Sanofi/Regeneron, Merck KGaA. Research Funding: Bristol Myers Squibb (Inst), Novartis (Inst), Roche (Inst), MSD Oncology (Inst), Array BioPharma/Pfizer (Inst). Travel, Accommodations, Expenses: Roche/Genentech, Bristol Myers Squibb, Merck Serono, Novartis, Merck Sharp & Dohme, Pierre Fabre, Sanofi/Regeneron. LZ served as consultant and has received honoraria from BMS, MSD, Novartis, Pierre Fabre, Sanofi, and Sunpharma and travel support from MSD, BMS, Pierre Fabre, Sanofi, Sunpharma and Novartis, outside the submitted work. EL served as consultant and/or has received honoraria from Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis, Pierre-Fabre, Sanofi, Sunpharma, Takeda and travel support from Bristol-Myers Squibb, Pierre- Fabre, Sunpharma and Novartis, outside the submitted work. IvW received honorairia as speaker from Novartis, BMS, MSD, Sanofi, Stemline, Kyowa Kirin and as consultant or advisory from BMS, MSD, Sanofi; travel, accommodations and expenses from Novartis, BMS, MSD, Sanofi, Stemline and Kyowa Kirin. FriM has received travel support or/and speaker’s fees or/and advisor’s honoraria by Novartis, BMS, MSD, Pierre Fabre, Sanofi and Immunocore. SH declares speakers and advisory board honoraria and/or travel support from BMS, MSD, Immunocore, Novartis, Pierre-Fabre, Sanofi, Sun Pharma outside the submitted work. KD received financial support (speaker’s honoraria, advisory boards, travel expense reimbursements or grants) from Abbvie, Bristol-Myers-Squib, Novartis, and Pierre-Fabre. JU is on the advisory board or has received honoraria and travel support from Amgen, Bristol Myers Squibb, GSK, Immunocore, LeoPharma, Merck Sharp and Dohme, Novartis, Pierre Fabre, Roche, Sanofi outside the submitted work. SAW received honoria from Bristol Myers Squibb, Novartis and Sun Pharma, outside the submitted work. CP served as consultant and/or has received honoraria from Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis, Sanofi, Sunpharma, Pierre Fabre, AbbVie, Kyona Kirin and Amgen and received travel support from Amgen, Merck Sharp & Dohme, Bristol-Myers Squibb, Pierre Fabre, Sunpharma and Novartis, outside the submitted work. PT served as consultant and/or received honoraria form Almirall, Bristol Myers Squibb, Biofrontera, Kyowa Kirin, L′Oréal, Merck Sharp & Dohme, Novartis, Pierre-Fabre, Sanofi, 4SC, and travel support from Bristol Myers Squibb outside the submitted work. JeU is on the advisory board or has received honoraria and travel support from Bristol Myers Squibb, Merck Sharp and Dohme, Novartis, Pierre Fabre, Regeneron, Roche, Sanofi and SUN Pharma outside the submitted work. FraM served as consultant and/or has received honoraria from Novartis, Bristol-Myers Squibb, Merck Sharp & Dohme, Pierre Fabre, Sanofi Genzyme, Sun Pharma and travel support from Novartis, Sun Pharma, Pierre Fabre and Merck Sharp & Dohme, outside the submitted work. JW has received honoraria and travel support from Abbvie, Almirall, BMS, Boehringer Ingelheim, Janssen, Infectopharm Leo, Lilly, Mibe, MSD, Novartis, Pfizer and Sanofi. BS received honoraria from SUN Pharma, Allmiral, Novartis; Advisory Board for Pierre Fabre Pharma, Sanofi, Immunocore; travel support from Novartis, Pierre Fabre Pharma; Research Funding from Novartis; all outside the submitted work. FZ declares speakers and advisory board honoraria and/or travel support from BMS, MSD, Roche, Novartis, Pierre-Fabre, Sanofi, Sun Pharma outside the submitted work. AK received honoraria as speaker from InfectoPharm, Paul-Ehrlich-Gesellschaft für Chemotherapie e.V., Almirall Hermal, MSD Sharp & Dohme, Boehringer Ingelheim, Janssen-Cilag. LH declares speaker and advisory board honoraria from BMS, Immunocore, Novartis and Therakos. CL received honoraria for advisory board from BMS, MSD, Merck, Roche, Immunocore, Novartis, Pierre Fabre, Sanofi, Sun Pharma, Almirall Hermal, Kyowa Kirin, Biontech, Boehriner Ingelheim; speakers fee from BMS, MSD, Merck, Roche, Immunocore, Novartis, Pierre Fabre, Sanofi, Sun Pharma, Almirall Hermal, Kyowa Kirin, Biontech, Lilly and travel reimbursement from BMS, MSD, Merck, Roche, Immunocore, Novartis, Pierre Fabre, Sanofi, Sun Pharma, Almirall Hermal, Kyowa Kirin, Biontech, Pfizer. GS has received honoraria from Bristol-Myers Squibb and Kyowa Hakko Kirin Co. Ltd. outside the submitted work. SG declares honoraria for advisory boards, oral presentations and/ or travel expenses from Roche, Novartis, MSD, BMS, Sun Pharma, Klinge Pharma, Kyowa Kirin, Pierre Fabre, Guidepoint Global and UCB and research funds from Novartis and Pierre Fabre outside the submitted work. ND received honoraria from BMS and MSD outside the submitted work. RH and IK are employee of Helios Klinikum Erfurt GmbH. CS reports support from Kyowa Kirin, BMS, Novartis, Roche, SunPharma in context of dermatooncology as well as from Boehringer Ingelheim, Biotest AG and Janssen Cilag in other medical fields. AE is on advisory board or has received honoraria from Agenus, BioInvent, BioNTech, Boeringer Ingelheim GmbH, Brenus, CatalYm, Eurobio. IO Biotech, IQVIA, Merck&Co, MSD, Pfizer, Pierre Fabre, Sairopa BV, SkylineDX BV, Trained ImmunoTherapeutics Discovery and has Equity in IO Biotech, Sairopa BV and SkylineDX BV. MVH received honoraria from MSD, BMS, Roche, Novartis, Sun Pharma, Sanofi, Almirall, Biofrontera, Galderma, Immunocore, InfectoPharm. CG is on the advisory board or has received honoraria from Almirall, Amgen, Beiersdorf, BioNTech, Bristol-Myers Squibb, Immunocore, Janssen, MSD Sharp & Dohme, Novartis, Pierre-Fabre Pharma, Regeneron, Roche, Sanofi Genzyme, SUN Pharma and Sysmex, research funding from Bristol-Myers Squibb, Novartis, Regeneron and Sanofi, outside the submitted work. C.G. is co-founder of Dermagnostix and Dermagnostix R&D. All remaining authors have declared no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study investigated whether adjuvant treatments in stage III cutaneous melanoma (CM) influenced patterns of recurrence. Patients with primary (n = 1033) or relapsed CM (n = 350) who received adjuvant therapies with Nivolumab (N), Pembrolizumab (P), or Dabrafenib and Trametinib (D + T) were extracted from the prospective multicenter real-world skin cancer registry ADOReg. Endpoints were progression-free survival (PFS), distant metastasis-free survival (DMFS), organ-specific DMFS, and overall survival (OS). For primary cases, D + T indicated an improved PFS (1- and 2-year PFS: 90.9%; 82.7%) as compared to P (81.0%, 73.9%; p = .0208), or N (83.8%, 75.2%; p = .0539). BRAF-mutated(mut) CM demonstrated significantly lower PFS (p = .0022) and decreased DMFS (p = .0580) when treated with immune checkpoint inhibitor (ICI) instead of D + T. Besides, NRAS-mut CM tended to perform worse than wt CM upon ICI (PFS: p = .1349; DMFS: p = .0540). OS was similar between the groups. Relapsed cases showed decreased PFS, DMFS, and OS in comparison to primary (all: p < .001), without significant differences between the subgroups. Organ-specific DMFS was significantly altered for primary cases with bone (p = .0367) or brain metastases (p = .0202). In relapsed CM, the frequency of liver (D + T: 1.5%; P: 12%; N: 9%) and LN metastases (D + T: 1.5%; P: 12%; N: 10.2%) was significantly lower with adjuvant D + T than ICI. NRAS-mut CM showed increased recurrence in primary and relapsed cases. These data show that adjuvant D + T is superior to ICI in primary BRAF-mut CM., (© 2024 The Author(s). International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Background: Immunotherapies for malignant melanoma are challenged by the resistance developed in a significant proportion of patients. Myeloid-derived suppressor cells (MDSC), with their ability to inhibit antitumor T-cell responses, are a major contributor to immunosuppression and resistance to immune checkpoint therapies in melanoma. Damage-associated molecular patterns S100A8, S100A9, and HMGB1, acting as toll like receptor 4 (TLR4) and receptor for advanced glycation endproducts (RAGE) ligands, are highly expressed in the tumor microenvironment and drive MDSC activation. However, the role of TLR4 and RAGE signaling in the acquisition of MDSC immunosuppressive properties remains to be better defined. Our study investigates how the signaling via TLR4 and RAGE as well as their ligands S100A9 and HMGB1, shape MDSC-mediated immunosuppression in melanoma., Methods: MDSC were isolated from the peripheral blood of patients with advanced melanoma or generated in vitro from healthy donor-derived monocytes. Monocytes were treated with S100A9 or HMGB1 for 72 hours. The immunosuppressive capacity of treated monocytes was assessed in the inhibition of T-cell proliferation assay in the presence or absence of TLR4 and RAGE inhibitors. Plasma levels of S100A8/9 and HMGB1 were quantified by ELISA. Single-cell RNA sequencing (scRNA-seq) was performed on monocytes from patients with melanoma and healthy donors., Results: We showed that exposure to S100A9 and HMGB1 converted healthy donor-derived monocytes into MDSC through TLR4 signaling. Our scRNA-seq data revealed in patient monocytes enriched inflammatory genes, including S100 and those involved in NF-κB and TLR4 signaling, and a reduced major histocompatibility complex II gene expression. Furthermore, elevated plasma S100A8/9 levels correlated with shorter progression-free survival in patients with melanoma., Conclusions: These findings highlight the critical role of TLR4 and, to a lesser extent, RAGE signaling in the conversion of monocytes into MDSC-like cells, underscore the potential of targeting S100A9 to prevent this conversion, and highlight the prognostic value of S100A8/9 as a plasma biomarker in melanoma., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: This study analyzed the role of Stabilin-1 on hepatic melanoma metastasis in preclinical mouse models., Methods: In Stabilin-1 -/- mice (Stab1 KO), liver colonization of B16F10 luc2 and Wt31 melanoma was investigated. The numbers, morphology, and vascularization of hepatic metastases and the hepatic microenvironment were analyzed by immunofluorescence., Results: While hepatic metastasis of B16F10 luc2 or Wt31 melanoma was unaltered between Stab1 KO and wildtype (Ctrl) mice, metastases of B16F10 luc2 tended to be smaller in Stab1 KO. The endothelial differentiation of both types of liver metastases was similar in Stab1 KO and Ctrl. No differences in initial tumor cell adhesion and retention to the liver vasculature were detected in the B16F10 luc2 model. Analysis of the immune microenvironment revealed a trend towards higher levels of CD45 + Gr-1 + cells in Stab1 KO as compared to Ctrl in the B16F10 luc2 model. Interestingly, significantly higher levels of POSTN were found in the matrix of hepatic metastases of Wt31, while liver metastases of B16F10 luc2 showed a trend towards increased deposition of RELN., Conclusions: Hepatic melanoma metastases show resistance to Stabilin-1 targeting approaches. This suggests that anti-Stab1 therapies should be considered with respect to the tumor entity or target organs.

The treatment of metastatic cutaneous melanoma was fundamentally improved by the discovery and introduction of immune checkpoint inhibitors, such as anti-PD-1 and anti-CTLA-4 antibodies, and targeted therapy with BRAF and MEK inhibition. Unfortunately, many patients suffer a relapse due to resistance mechanisms that in part are mediated by organ-specific metastatic sites. Especially, brain and liver metastases are negative predictive factors for both treatment modalities. There is still high unmet clinical need to prevent and treat spread to these organs. Therefore, experimental research should focus on mechanisms of hepatic melanoma metastasis to better understand this process and to identify therapeutic targets., (© 2023 The Authors. Journal der Deutschen Dermatologischen Gesellschaft published by John Wiley & Sons Ltd on behalf of Deutsche Dermatologische Gesellschaft.)

Background: Liver metastasis is a poor prognostic factor for treatment of advanced cutaneous melanoma with either immunotherapy or targeted therapies. In this study we focused on NRAS mutated melanoma, a cohort with high unmet clinical need., Methods: WT31 melanoma was repeatedly passaged over the liver after intravenous injections five times generating the subline WT31&#95;P5IV. The colonization of target organs, morphology, vascularization and the gene expression profiles of metastases were analyzed., Results: After intravenous injection lung metastasis was significantly decreased and a trend towards increased liver metastasis was detected for WT31&#95;P5IV as compared to parental WT31. Besides, the ratio of lung to liver metastases was significantly smaller. Histology of lung metastases revealed reduced proliferation of WT31&#95;P5IV in relation to WT31 while both size and necrotic areas were unaltered. Liver metastases of both sublines showed no differences in vascularization, proliferation or necrosis. To identify tumor-intrinsic factors that altered the metastatic pattern of WT31&#95;P5IV RNA sequencing was performed and revealed a differential regulation of pathways involved in cell adhesion. Ex vivo fluorescence imaging confirmed that initial tumor cell retention in the lungs was significantly reduced in WT31&#95;P5IV in comparison to WT31., Conclusion: This study demonstrates that tumor-intrinsic properties influencing the metastatic pattern of NRAS mutated melanoma are strongly affected by hepatic passaging and the hematogenous route tumor cells take. It has implications for the clinical setting as such effects might also occur during metastatic spread or disease progression in melanoma patients., (© 2023. The Author(s).)

Background: Hyaluronan receptor LYVE-1 is expressed by liver sinusoidal endothelial cells (LSEC), lymphatic endothelial cells and specialized macrophages. Besides binding to hyaluronan, LYVE-1 can mediate adhesion of leukocytes and cancer cells to endothelial cells. Here, we assessed the impact of LYVE-1 on physiological liver functions and metastasis., Methods: Mice with deficiency of Lyve-1 (Lyve-1-KO) were analyzed using histology, immunofluorescence, microarray analysis, plasma proteomics and flow cytometry. Liver metastasis was studied by intrasplenic/intravenous injection of melanoma (B16F10 luc2, WT31) or colorectal carcinoma (MC38)., Results: Hepatic architecture, liver size, endothelial differentiation and angiocrine functions were unaltered in Lyve-1-KO. Hyaluronan plasma levels were significantly increased in Lyve-1-KO. Besides, plasma proteomics revealed increased carbonic anhydrase-2 and decreased FXIIIA. Furthermore, gene expression analysis of LSEC indicated regulation of immunological pathways. Therefore, liver metastasis of highly and weakly immunogenic tumors, i.e. melanoma and colorectal carcinoma (CRC), was analyzed. Hepatic metastasis of B16F10 luc2 and WT31 melanoma cells, but not MC38 CRC cells, was significantly reduced in Lyve-1-KO mice. In vivo retention assays with B16F10 luc2 cells were unaltered between Lyve-1-KO and control mice. However, in tumor-free Lyve-1-KO livers numbers of hepatic CD4 + , CD8 + and regulatory T cells were increased. In addition, iron deposition was found in F4/80 + liver macrophages known to exert pro-inflammatory effects., Conclusion: Lyve-1 deficiency controlled hepatic metastasis in a tumor cell-specific manner leading to reduced growth of hepatic metastases of melanoma, but not CRC. Anti-tumorigenic effects are likely due to enhancement of the premetastatic hepatic immune microenvironment influencing early liver metastasis formation., (© 2022. The Author(s).)

Background: Cutaneous melanoma exhibits heterogeneous metastatic patterns and prognosis. In this regard, liver metastasis, which is detected in ~ 10-20% of stage 4 patients, came to the fore of melanoma research, as it recently evolved as decisive indicator of treatment resistance to immune checkpoint inhibition., Methods: Hepatic metastases were induced by intrasplenic injection of five different murine melanoma cell lines. The efficiencies of hepatic colonization, morphologic patterns, gene expression profiles and degree of vascularization were analyzed and Sorafenib was applied as anti-angiogenic treatment., Results: WT31 melanoma showed the highest efficiency of hepatic colonization, while intermediate efficiencies were observed for B16F10 and RET, and low efficiencies for D4M and HCmel12. RNAseq-based gene expression profiles of high and intermediate metastatic melanomas in comparison to low metastatic melanomas indicated that this efficiency predominantly associates with gene clusters involved in cell migration and angiogenesis. Indeed, heterogeneous vascularization patterns were found in the five models. Although the degree of vascularization of WT31 and B16F10 metastases differed, both showed a strong response to Sorafenib with a successful abrogation of the vascularization., Conclusion: Our data indicate that molecular heterogeneity of melanomas can be associated with phenotypic and prognostic features of hepatic metastasis paving the way for organ-specific anti-angiogenic therapeutic approaches., (© 2022. The Author(s).)

The Class H scavenger receptors Stabilin-1 (Stab1) and Stabilin-2 (Stab2) are two of the most highly expressed genes in liver sinusoidal endothelial cells (LSECs). While Stab1-deficient (Stab1KO) and Stab2-deficient (Stab2KO) mice are phenotypically unremarkable, Stab1/2-double-deficient (StabDKO) mice exhibit perisinusoidal liver fibrosis, glomerulofibrotic nephropathy and a reduced life expectancy. These conditions are caused by insufficiently scavenged circulating noxious blood factors. The effects of either Stab-single- or double-deficiency on LSEC differentiation and function, however, have not yet been thoroughly investigated. Therefore, we performed comprehensive transcriptomic analyses of primary LSECs from Stab1KO, Stab2KO and StabDKO mice. Microarray analysis revealed dysregulation of pathways and genes involved in established LSEC functions while sinusoidal endothelial marker gene expression was grossly unchanged. 82 genes were significantly altered in Stab1KO, 96 genes in Stab2KO and 238 genes in StabDKO compared with controls; 42 genes were found to be commonly dysregulated in all three groups and all of these genes were downregulated. These commonly downregulated genes (CDGs) were categorized as "potential scavengers," "cell adhesion molecules," "TGF-β/BMP-signaling" or "collagen and extracellular matrix (ECM) components". Among CDGs, Colec10, Lumican and Decorin, were the most strongly down-regulated genes and the corresponding proteins impact on the interaction of LSECs with chemokines, ECM components and carbohydrate structures. Similarly, "chemokine signaling," "cytokine-cytokine receptor interaction" and "ECM-receptor interaction," were the GSEA categories which represented most of the downregulated genes in Stab1KO and Stab2KO LSECs. In summary, our data show that loss of a single Stabilin scavenger receptor - and to a greater extent of both receptors - profoundly alters the transcriptomic repertoire of LSECs. These alterations may affect LSEC-specific functions, especially interactions of LSECs with the ECM and during inflammation as well as clearance of the peripheral blood., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The interaction of tumor cells with organ-specific endothelial cells (EC) is an important step during metastatic progression. Notch signaling in organ-specific niches has been implicated in mediating opposing effects on organotropic metastasis to the lungs and the liver, respectively. In this study, we scrutinized the role of endothelial Notch activation during liver metastasis. To target hepatic EC (HEC), a novel EC subtype-specific Cre driver mouse was generated. Clec4g-Cre tg/wt mice were crossed to Rosa26 N1ICD-IRES-GFP to enhance Notch signaling in HEC (NICD OE-HEC ). In NICD OE-HEC mice, hepatic metastasis of malignant melanoma and colorectal carcinoma was significantly reduced. These mice revealed reduced liver growth and impaired metabolic zonation due to suppression of hepatic angiocrine Wnt signaling. Hepatic metastasis, however, was not controlled by angiocrine Wnt signaling, as deficiency of the Wnt cargo receptor Wls in HEC of Wls HEC-KO mice did not affect hepatic metastasis. In contrast, the hepatic microvasculature in NICD OE-HEC mice revealed a special form of sinusoidal capillarization, with effacement of endothelial zonation functionally paralleled by reduced tumor cell adhesion in vivo . Notably, expression of endothelial adhesion molecule ICAM1 by HEC was significantly reduced. Treatment with an anti-ICAM1 antibody significantly inhibited tumor cell adhesion to HEC in wild-type mice confirming that Notch controls hepatic metastasis via modulation of HEC adhesion molecules. As endothelial Notch activation in the lung has been shown to promote lung metastasis, tumor therapy will require approaches that target Notch in an organ-, cell type-, and context-specific manner. SIGNIFICANCE: Manipulation of Notch signaling in the endothelium has opposing, organ-specific effects on metastasis to the lung and the liver, demonstrating that this pathway should be targeted in a cell- and context-specific fashion., (©2018 American Association for Cancer Research.)

Postnatal liver development is characterized by hepatocyte growth, proliferation, and functional maturation. Notably, canonical Wnt signaling in hepatocytes has been identified as an important regulator of final adult liver size and metabolic liver zonation. The cellular origin of Wnt ligands responsible for homeostatic liver/body weight ratio (LW/BW) remained unclear, which was also attributable to a lack of suitable endothelial Cre driver mice. To comprehensively analyze the effects of hepatic angiocrine Wnt signaling on liver development and metabolic functions, we used endothelial subtype-specific Stab2-Cre driver mice to delete Wls from hepatic endothelial cells (HECs). The resultant Stab2-Cre tg/wt ;Wls fl/fl (Wls-HECKO) mice were viable, but showed a significantly reduced LW/BW. Specifically, ablation of angiocrine Wnt signaling impaired metabolic zonation in the liver, as shown by loss of pericentral, β-catenin-dependent target genes such as glutamine synthase (Glul), RhBg, Axin2, and cytochrome P450 2E1, as well as by extended expression of periportal genes such as arginase 1. Furthermore, endothelial subtype-specific expression of a c-terminally YFP-tagged Wls fusion protein in Wls-HECKO mice (Stab2-Cre tg/wt ;Wls fl/fl ;Rosa26:Wls-YFP fl/wt [Wls-rescue]) restored metabolic liver zonation. Interestingly, lipid metabolism was altered in Wls-HECKO mice exhibiting significantly reduced plasma cholesterol levels, while maintaining normal plasma triglyceride and blood glucose concentrations. On the contrary, zonal expression of Endomucin, LYVE1, and other markers of HEC heterogeneity were not altered in Wls-HECKO livers., Conclusion: Angiocrine Wnt signaling controls liver growth as well as development of metabolic liver zonation in mice, whereas intrahepatic HEC zonation is not affected. (Hepatology 2017)., (© 2017 The Authors. Hepatology published by Wiley Periodicals, Inc. on behalf of American Association for the Study of Liver Diseases.)

Liver sinusoidal endothelial cells (LSEC) represent a unique, organ-specific type of discontinuous endothelial cells. LSEC instruct the hepatic vascular niche by paracrine-acting angiocrine factors. Recently, we have shown that LSEC-specific transcriptional regulator GATA4 induces expression of BMP2 in cultured endothelial cells (EC) in vitro. Furthermore, angiocrine Bmp2 signaling in the liver in vivo was demonstrated to control iron homeostasis. Here, we investigated GATA4-dependent autocrine BMP2 signaling in endothelial cells by gene expression profiling. GATA4 induced a large cluster of inflammatory endothelial response genes in cultured EC, which is similar to previously identified virus-induced and interferon-associated responses. Treating the cells with the BMP2 inhibitor Noggin counter-regulated the GATA4-dependent inflammatory phenotype of EC, indicating that BMP2 is indeed the major driver. In contrast to continuous EC, LSEC were less prone to activation by BMP2. Notably, GATA4-dependent induction of the inflammatory EC response gene cluster was attenuated by over-expression of the LSEC-specific transcriptional modifier LMO3 while hepatocyte activation was fully preserved, indicating conserved BMP2 synthesis. In summary, our data suggest that transcriptional counter-regulation by GATA4 and LMO3 in LSEC prevents autocrine induction of an inflammatory phenotype, while maintaining angiocrine BMP2-mediated cell-cell communication in the liver vascular niche., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Caveolin-1 (Cav-1) is a key organizer of membrane specializations and a scaffold protein that regulates signaling in multiple cell types. We found increased Cav-1 expression in human and murine T cells after allogeneic hematopoietic cell transplantation. Indeed, Cav-1(-/-)donor T cells caused less severe acute graft-versus-host disease (GVHD) and yielded higher numbers of regulatory T cells (Tregs) compared with controls. Depletion of Tregs from the graft abrogated this protective effect. Correspondingly, Treg frequencies increased when Cav-1(-/-)T cells were exposed to transforming growth factor-β/T-cell receptor (TCR)/CD28 activation or alloantigen stimulation in vitro compared with wild-type T cells. Mechanistically, we found that the phosphorylation of Cav-1 is dispensable for the control of T-cell fate by using a nonphosphorylatable Cav-1 (Y14F/Y14F) point-mutation variant. Moreover, the close proximity of lymphocyte-specific protein tyrosine kinase (Lck) to the TCR induced by TCR-activation was reduced in Cav-1(-/-)T cells. Therefore, less TCR/Lck clustering results in suboptimal activation of the downstream signaling events, which correlates with the preferential development into a Treg phenotype. Overall, we report a novel role for Cav-1 in TCR/Lck spatial distribution upon TCR triggering, which controls T-cell fate toward a regulatory phenotype. This alteration translated into a significant increase in the frequency of Tregs and reduced GVHD in vivo., (© 2016 by The American Society of Hematology.)

Acute graft-versus-host disease (GvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation. During the initiation phase of acute GvHD, endogenous danger signals such as ATP are released and inform the innate immune system via activation of the purinergic receptor P2X7 that a noninfectious damage has occurred. A second ATP-activated purinergic receptor involved in inflammatory diseases is P2Y2. In this study, we used P2y2(-/-) mice to test the role of this receptor in GvHD. P2y2(-/-) recipients experienced reduced GvHD-related mortality, IL-6 levels, enterocyte apoptosis, and histopathology scores. Chimeric mice with P2y2 deficiency restricted to hematopoietic tissues survived longer after GvHD induction than did wild-type mice. P2y2 deficiency of the recipient was connected to lower levels of myeloperoxidase in the intestinal tract of mice developing GvHD and a reduced myeloid cell signature. Selective deficiency of P2Y2 in inflammatory monocytes decreased GvHD severity. Mechanistically, P2y2(-/-) inflammatory monocytes displayed defective ERK activation and reactive oxygen species production. Compatible with a role of P2Y2 in human GvHD, the frequency of P2Y2(+) cells in inflamed GvHD lesions correlated with histopathological GvHD severity. Our findings indicate a novel function for P2Y2 in ATP-activated recipient myeloid cells during GvHD, which could be exploited when targeting danger signals to prevent GvHD., (Copyright © 2015 by The American Association of Immunologists, Inc.)