Search

To investigate underlying mechanisms for cancer metastasis and promising therapies in animal models, spontaneous metastasis models can be used to recreate metastasis development. Here, we present three mouse models of spontaneous lung and/or liver metastasis induction. We describe steps for cancer cell preparation, mouse analgesia, and three injection techniques (subcutaneous, intracecal, and intramucosal). We then detail procedures for evaluating metastasis. Most of these models generate metastasis in a time span of 4 weeks in the majority of injected mice. For complete details on the use and execution of this protocol, please refer to Giannou et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Forced metastasis models, those in which the step of intravasation is bypassed, can be used to investigate the mechanisms underlying metastasis and evaluate potential therapeutic targets. Here, we present a protocol for using three forced models of lung and liver metastasis to generate metastasis within 3-4 weeks in approximately 99% of injected mice. We describe steps for cancer cell preparation, mouse analgesia and anesthesia; injecting through intrasplenic, intraportal, and intravenous techniques; and daily evaluation of metastasis. For complete details on the use and execution of this protocol, please refer to Giannou et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Insufficient remnant liver volume (RLV) after the resection of hepatic malignancy could lead to liver failure and mortality. Portal vein ligation (PVL) prior to hepatectomy is subsequently introduced to increase the remnant liver volume and improve the outcome of hepatic malignancy. IL-22 has previously been reported to promote liver regeneration, while facilitating tumor development in the liver via Steap4 upregulation. Here we performed PVL in mouse models to study the role of IL-22 in liver regeneration post-PVL., Methods: Liver weight and volume was measured via magnetic resonance imaging (MRI). Immunohistochemistry for Ki67 and hepatocyte growth factor (HGF) was performed. IL-22 was analyzed by flow cytometry and quantitative polymerase chain reaction (qPCR) was used for acquisition of Il-33, Steap4, Fga, Fgb and Cebpd . To analyze signaling pathways, mice with deletion of STAT3 and a neutralizing antibody for IL-22 were used., Results: The remnant liver weight and volume increased over time after PVL. Additionally, we found that liver regenerative molecules, including Ki67 and HGF, were significantly increased in remnant liver at day 3 post-PVL, as well as IL-22. Administration of IL-22 neutralizing antibody could reduce Ki67 expression after PVL. The upregulation of IL-22 after PVL was mainly derived from innate cells. IL-22 blockade resulted in lower levels of IL-33 and Steap4 in the remnant liver, which was also the case in mice with deletion of STAT3, the main downstream signaling molecule of IL-22, in hepatocytes., Conclusion: IL-22 promotes liver regeneration after PVL. Thus, a combination of IL-22 supplementation and Steap4 blockade could potentially be applied as a novel therapeutic approach to boost liver regeneration without facilitating tumor progression after PVL., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which is responsible for eliciting Coronavirus disease 2019 (COVID-19) still challenges healthcare services worldwide. While many patients only suffer from mild symptoms, patients with some pre-existing medical conditions are at a higher risk for a detrimental course of disease. However, the underlying mechanisms determining disease course are only partially understood. One key factor influencing disease severity is described to be immune-mediated. In this report, we describe a post-mortem analysis of 45 individuals who died from SARS-CoV-2 infection. We could show that although sociodemographic factors and premedical conditions such as obesity and diabetes mellitus reduced survival time in our cohort, they were not associated with changes in the expression of immune-related signature genes at the RNA level in the blood, the gut, or the liver between these different groups. Our data indicate that obesity and diabetes mellitus influence SARS-CoV-2-related mortality, without influencing the extrapulmonary gene expression of immunity-related signature genes at the RNA level., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Samuel Huber reports financial support was provided by 10.13039/501100001659Deutsche Forschungsgemeinschaft. Samuel Huber reports financial support was provided by Ernst 10.13039/501100004038Jung-Stiftung Hamburg. Samuel Huber reports financial support was provided by Stiftung Experimentelle Biomedizin. Fabian Heinrich reports financial support was provided by NATON project within the Netzwerk Universitätsmedizin. Benjamin Ondruschka reports financial support was provided by NATON project within the Netzwerk Universitätsmedizin. Anastasios Giannou reports financial support was provided by Else Kröner Memorial Stipendium. Anastasios Giannou reports financial support was provided by ung Foundation for Science and Research (Ernst Jung Career Development Award 2022). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Differential glycosylation, marked by the presence of truncated O-glycans, is a distinctive feature of epithelial-derived cancers. However, there is a notable gap in research regarding the expression of Tn and STn antigens in esophageal adenocarcinoma (EAC). To address this, we employed commercially available antibodies, previously validated for Tn and STn antigens, to analyze two cohorts of EAC tissues. Initially, large-area tissue sections from formalin-fixed paraffin-embedded (FFPE) EAC and corresponding healthy tissues were subjected to immunohistochemistry (IHC) staining and scoring. Subsequently, we evaluated the RNA expression levels of crucial O-glycosylation related genes- C1GALT1 and C1GALT1C1 -using a quantitative real-time polymerase chain reaction (qRT-PCR). In a comprehensive analysis, a substantial cohort of EAC tissues ( n = 311 for Tn antigen, n = 351 for STn antigen) was investigated and correlated with clinicopathological data. Our findings revealed that Tn and STn antigens are highly expressed (approximately 71% for both) in EAC, with this expression being tumor-specific. Notably, Tn antigen expression correlates significantly with the depth of tumor cell infiltration ( p = 0.026). These antigens emerge as valuable markers and potential therapeutic targets for esophageal adenocarcinoma.

The transplantation model provides the opportunity to assess the relevance of a molecule of interest for tumor cell extravasation by using a respective genetically modified donor animal. Here, we present a protocol for orthotopic single-lung transplantation in mice as a tool for lung metastasis studies. We describe steps for animal preparation, lung transplantation, and tumor cell injection. We then detail procedures for the direct comparison of tumor cell spreading between the genetically modified left lung and the naive right lung parenchyma. For complete details on the use and execution of this protocol, please refer to Giannou et al. (2023). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Metastasis is a cancer-related systemic disease and is responsible for the greatest mortality rate among cancer patients. Interestingly, the interaction between the immune system and cancer cells seems to play a key role in metastasis formation in the target organ. However, this complex network is only partially understood. We previously found that IL-22 produced by tissue resident iNKT17 cells promotes cancer cell extravasation, the early step of metastasis. Based on these data, we aimed here to decipher the role of IL-22 in the last step of metastasis formation. We found that IL-22 levels were increased in established metastatic sites in both human and mouse. We also found that Th22 cells were the key source of IL-22 in established metastasis sites, and that deletion of IL-22 in CD4+ T cells was protective in liver metastasis formation. Accordingly, the administration of a murine IL-22 neutralizing antibody in the establishment of metastasis formation significantly reduced the metastatic burden in a mouse model. Mechanistically, IL-22-producing Th22 cells promoted angiogenesis in established metastasis sites. In conclusion, our findings highlight that IL-22 is equally as important in contributing to metastasis formation at late metastatic stages, and thus, identify it as a novel therapeutic target in established metastasis., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Background & Aims: Ulcerative colitis (UC) is characterized by severe inflammation and destruction of the intestinal epithelium, and is associated with specific risk single nucleotide polymorphisms in HLA class II. Given the recently discovered interactions between subsets of HLA-DP molecules and the activating natural killer (NK) cell receptor NKp44, genetic associations of UC and HLA-DP haplotypes and their functional implications were investigated., Methods: HLA-DP haplotype and UC risk association analyses were performed (UC: n = 13,927; control: n = 26,764). Expression levels of HLA-DP on intestinal epithelial cells (IECs) in individuals with and without UC were quantified. Human intestinal 3-dimensional (3D) organoid cocultures with human NK cells were used to determine functional consequences of interactions between HLA-DP and NKp44., Results: These studies identified HLA-DPA1∗01:03-DPB1∗04:01 (HLA-DP401) as a risk haplotype and HLA-DPA1∗01:03-DPB1∗03:01 (HLA-DP301) as a protective haplotype for UC in European populations. HLA-DP expression was significantly higher on IECs of individuals with UC compared with controls. IECs in human intestinal 3D organoids derived from HLA-DP401 pos individuals showed significantly stronger binding of NKp44 compared with HLA-DP301 pos IECs. HLA-DP401 pos IECs in organoids triggered increased degranulation and tumor necrosis factor production by NKp44 + NK cells in cocultures, resulting in enhanced epithelial cell death compared with HLA-DP301 pos organoids. Blocking of HLA-DP401-NKp44 interactions (anti-NKp44) abrogated NK cell activity in cocultures., Conclusions: We identified an UC risk HLA-DP haplotype that engages NKp44 and activates NKp44 + NK cells, mediating damage to intestinal epithelial cells in an HLA-DP haplotype-dependent manner. The molecular interaction between NKp44 and HLA-DP401 in UC can be targeted by therapeutic interventions to reduce NKp44 + NK cell-mediated destruction of the intestinal epithelium in UC., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

An optimized lymph node yield leads to better survival in colon cancer, but extended lymphadenectomy is not associated with survival benefits. Lymphatic mapping shows several colon cancers feature aberrant drainage pathways inducing local recurrence when not resected. Currently, different protocols exist for lymphatic mapping procedures. This meta-analysis assessed which protocol has the best capacity to detect tumor-draining and possibly metastatic lymph nodes. A systematic review was conducted according to PRISMA guidelines, including prospective trials with in vivo tracer application. The risk of bias was evaluated using the QUADAS-2 tool. Traced lymph nodes, total resected lymph nodes, and aberrant drainage detection rate were analyzed. Fifty-eight studies met the inclusion criteria, of which 42 searched for aberrant drainage. While a preoperative tracer injection significantly increased the traced lymph node rates compared to intraoperative tracing (30.1% (15.4, 47.3) vs. 14.1% (11.9, 16.5), p = 0.03), no effect was shown for the tracer used ( p = 0.740) or the application sites comparing submucosal and subserosal injection (22.9% (14.1, 33.1) vs. 14.3% (12.1, 16.8), p = 0.07). Preoperative tracer injection resulted in a significantly higher rate of detected aberrant lymph nodes compared to intraoperative injection (26.3% [95% CI 11.5, 44.0] vs. 2.5% [95% CI 0.8, 4.7], p < 0.001). Analyzing 112 individual patient datasets from eight studies revealed a significant impact on aberrant drainage detection for injection timing, favoring preoperative over intraoperative injection (OR 0.050 [95% CI 0.010-0.176], p < 0.001) while indocyanine green presented itself as the superior tracer (OR 0.127 [95% CI 0.018-0.528], p = 0.012). Optimized lymphatic mapping techniques result in significantly higher detection of aberrant lymphatic drainage patterns and thus enable a personalized approach to reducing local recurrence.

Background: The immune system plays a pivotal role in cancer progression. Interleukin 22 binding protein (IL-22BP), a natural antagonist of the cytokine interleukin 22 (IL-22) has been shown to control the progression of colorectal cancer (CRC). However, the role of IL-22BP in the process of metastasis formation remains unknown., Methods: We used two different murine in vivo metastasis models using the MC38 and LLC cancer cell lines and studied lung and liver metastasis formation after intracaecal or intrasplenic injection of cancer cells. Furthermore, IL22BP expression was measured in a clinical cohort of CRC patients and correlated with metastatic tumor stages., Results: Our data indicate that low levels of IL-22BP are associated with advanced (metastatic) tumor stages in colorectal cancer. Using two different murine in vivo models we show that IL-22BP indeed controls the progression of liver but not lung metastasis in mice., Conclusions: We here demonstrate a crucial role of IL-22BP in controlling metastasis progression. Thus, IL-22 might represent a future therapeutic target against the progression of metastatic CRC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Giannou, Kempski, Zhang, Lücke, Shiri, Zazara, Belios, Machicote, Seeger, Agalioti, Tintelnot, Sagebiel, Tomczak, Bauditz, Bedke, Kocheise, Mercanoglu, Fard-Aghaie, Giorgakis, Lykoudis, Pikouli, Grass, Wahib, Bardenhagen, Brunswig, Heumann, Ghadban, Duprée, Tachezy, Melling, Arck, Stringa, Gentilini, Gondolesi, Nakano, Thomson, Perez, Li, Mann, Izbicki, Gagliani, Maroulis and Huber.)

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This virus does not only lead to pulmonary infection but can also infect other organs such as the gut, the kidney, or the liver. Recent studies confirmed that severe cases of COVID-19 are often associated with liver damage and liver failure, as well as the systemic upregulation of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFα). However, the impact these immune mediators in the liver have on patient survival during SARS-CoV-2 infection is currently unknown. Here, by performing a post-mortem analysis of 45 patients that died from a SARS-CoV-2 infection, we find that an increased expression of TNFA in the liver is associated with elevated mortality. Using publicly available single-cell sequencing datasets, we determined that Kupffer cells and monocytes are the main sources of this TNFα production. Further analysis revealed that TNFα signaling led to the upregulation of pro-inflammatory genes that are associated with an unfavorable outcome. Moreover, high levels of TNFA in the liver were associated with lower levels of interferon alpha and interferon beta. Thus, TNFα signaling in the infected SARS-CoV-2 liver correlates with reduced interferon levels and overall survival time., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Lücke, Nawrocki, Schnell, Meins, Heinrich, Zhang, Bertram, Sabihi, Böttcher, Blankenburg, Pfaff, Notz, Kempski, Reeh, Wolter, Mann, Izbicki, Lütgehetmann, Duprée, Giannou, Ondruschka and Huber.)

During metastasis, cancer cells invade, intravasate, enter the circulation, extravasate, and colonize target organs. Here, we examined the role of interleukin (IL)-22 in metastasis. Immune cell-derived IL-22 acts on epithelial tissues, promoting regeneration and healing upon tissue damage, but it is also associated with malignancy. Il22-deficient mice and mice treated with an IL-22 antibody were protected from colon-cancer-derived liver and lung metastasis formation, while overexpression of IL-22 promoted metastasis. Mechanistically, IL-22 acted on endothelial cells, promoting endothelial permeability and cancer cell transmigration via induction of endothelial aminopeptidase N. Multi-parameter flow cytometry and single-cell sequencing of immune cells isolated during cancer cell extravasation into the liver revealed iNKT17 cells as source of IL-22. iNKT-cell-deficient mice exhibited reduced metastases, which was reversed by injection of wild type, but not Il22-deficient, invariant natural killer T (iNKT) cells. IL-22-producing iNKT cells promoting metastasis were tissue resident, as demonstrated by parabiosis. Thus, IL-22 may present a therapeutic target for prevention of metastasis., Competing Interests: Declaration of interests S.K. declares honoraria from GSK, BMS, Novartis, and TCR2, Inc.; license fees from TCR2, Inc. and Carina Biotech; and research support from TCR2, Inc., Plectonic GmbH, Tabby Therapeutics, and Arcus Biosciences., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

A role of CD4+ T cells during the progression from nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH) has been suggested, but which polarization state of these cells characterizes this progression and the development of fibrosis remain unclear. In addition, a gut-liver axis has been suggested to play a role in NASH, but the role of CD4+ T cells in this axis has just begun to be investigated. Combining single-cell RNA sequencing and multiple-parameter flow cytometry, we provide the first cell atlas to our knowledge focused on liver-infiltrating CD4+ T cells in patients with NAFLD and NASH, showing that NASH is characterized by a population of multicytokine-producing CD4+ T cells. Among these cells, only those with a Th17 polarization state were enriched in patients with advanced fibrosis. In parallel, we observed that Bacteroides appeared to be enriched in the intestine of NASH patients and to correlate with the frequency of multicytokine-producing CD4+ T cells. In short, we deliver a CD4+ T cell atlas of NAFLD and NASH, providing the rationale to target CD4+ T cells with a Th17 polarization state to block fibrosis development. Finally, our data offer an early indication to test whether multicytokine-producing CD4+ T cells are part of the gut-liver axis characterizing NASH.

Although T cells can exert potent anti-tumor immunity, a subset of T helper (Th) cells producing interleukin-22 (IL-22) in breast and lung tumors is linked to dismal patient outcome. Here, we examined the mechanisms whereby these T cells contribute to disease. In murine models of lung and breast cancer, constitutional and T cell-specific deletion of Il22 reduced metastases without affecting primary tumor growth. Deletion of the IL-22 receptor on cancer cells decreases metastasis to a degree similar to that seen in IL-22-deficient mice. IL-22 induced high expression of CD155, which bound to the activating receptor CD226 on NK cells. Excessive activation led to decreased amounts of CD226 and functionally impaired NK cells, which elevated the metastatic burden. IL-22 signaling was also associated with CD155 expression in human datasets and with poor patient outcomes. Taken together, our findings reveal an immunosuppressive circuit activated by T cell-derived IL-22 that promotes lung metastasis., Competing Interests: Declaration of interests S.K. has received honoraria from TCR2 Inc., Novartis, BMS, and GSK. S.K. and S.E. are inventors of several patents in the field of immuno-oncology. S.K. and S.E. received license fees from TCR2 Inc. and Carina Biotech. S.K. and S.E. received research support from TCR2 Inc., Tabby Therapeutics, Plectonic GmBH, and Arcus Bioscience for work unrelated to the manuscript., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC) ranks among the five most common cancer entities worldwide and leads to hundred-thousands of deaths every year. Despite some groundbreaking therapeutical revelations during the last years, the overall prognosis remains poor. Although the immune system fights malignant transformations with a robust anti-tumor response, certain immune mediators have also been shown to promote cancer development. For example, interleukin (IL)-22 has been associated with HCC progression and worsened prognosis in multiple studies. However, the underlying mechanisms of the pathological role of IL-22-signaling as well as the role of its natural antagonist IL-22 binding protein (IL-22BP) in HCC remain elusive. Here, we corroborate the pathogenic role of IL-22 in HCC by taking advantage of two mouse models. Moreover, we observed a protective role of IL-22BP during liver carcinogenesis. While IL-22 was mainly produced by CD4 + T cells in HCC, IL-22BP was abundantly expressed by neutrophils during liver carcinogenesis. Hepatocytes could be identified as a major target of this pathological IL-22-signaling. Moreover, abrogation of IL-22 signaling in hepatocytes in IL22ra1 flox/flox × Alb Cre+ mice reduced STEAP4 expression-a known oncogene-in HCC in vivo. Likewise, STEAP4 expression correlated with IL22 levels in human HCC samples, but not in healthy liver specimens. In conclusion, these data encourage the development of therapeutical approaches that target the IL-22-IL-22BP axis in HCC.

Cancer is one of the leading causes of death worldwide. When cancer patients are diagnosed with metastasis, meaning that the primary tumor has spread to at least one different site, their life expectancy decreases dramatically. In the past decade, the immune system´s role in fighting cancer and metastasis has been studied extensively. Importantly, immune cells and inflammatory reactions generate potent antitumor responses but also contribute to tumor development. However, the molecular and cellular mechanisms underlying this dichotomic interaction between the immune system and cancer are still poorly understood. Recently, a spotlight has been cast on the distinct subsets of immune cells and their derived cytokines since evidence has implicated their crucial impact on cancer development. T helper 17 cell (T H 17) cells, which express the master transcriptional factor Retinoic acid-receptor-related orphan receptor gamma t, are among these critical cell subsets and are defined by their production of type 3 cytokines, such as IL-17A, IL-17F, and IL-22. Depending on the tumor microenvironment, these cytokines can also be produced by other immune cell sources, such as T cytotoxic 17 cell, innate lymphoid cells, NKT cells, or γδ T cells. To date, a lot of data have been collected describing the divergent functions of IL-17A, IL-17F, and IL-22 in malignancies. In this comprehensive review, we discuss the role of these T H 17- and non-T H 17-derived type 3 cytokines in different tumor entities. Furthermore, we will provide a structured insight into the strict regulation and subsequent downstream mechanisms of these cytokines in cancer and metastasis., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Malignant pleural effusion (MPE) results from the capacity of several human cancers to metastasize to the pleural cavity. No effective treatments are currently available, reflecting our insufficient understanding of the basic mechanisms leading to MPE progression. Here, we found that efferocytosis through the receptor tyrosine kinases AXL and MERTK led to the production of interleukin-10 (IL-10) by four distinct pleural cavity macrophage (Mφ) subpopulations characterized by different metabolic states and cell chemotaxis properties. In turn, IL-10 acts on dendritic cells (DCs) inducing the production of tissue inhibitor of metalloproteinases 1 (TIMP1). Genetic ablation of Axl and Mertk in Mφs or IL-10 receptor in DCs or Timp1 substantially reduced MPE progression. Our results delineate an inflammatory cascade-from the clearance of apoptotic cells by Mφs, to production of IL-10, to induction of TIMP1 in DCs-that facilitates MPE progression. This inflammatory cascade offers a series of therapeutic targets for MPE., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Solitary intestinal lymphoid tissues such as cryptopatches (CPs) and isolated lymphoid follicles (ILFs) constitute steady-state activation hubs containing group 3 innate lymphoid cells (ILC3) that continuously produce interleukin (IL)-22. The outer surface of CPs and ILFs is demarcated by a poorly characterized population of CD11c + cells. Using genome-wide single-cell transcriptional profiling of intestinal mononuclear phagocytes and multidimensional flow cytometry, we found that CP- and ILF-associated CD11c + cells were a transcriptionally distinct subset of intestinal cDCs, which we term CIA-DCs. CIA-DCs required programming by CP- and ILF-resident CCR6 + ILC3 via lymphotoxin-β receptor signaling in cDCs. CIA-DCs differentially expressed genes associated with immunoregulation and were the major cellular source of IL-22 binding protein (IL-22BP) at steady state. Mice lacking CIA-DC-derived IL-22BP exhibited diminished expression of epithelial lipid transporters, reduced lipid resorption, and changes in body fat homeostasis. Our findings provide insight into the design principles of an immunoregulatory checkpoint controlling nutrient absorption., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background & Aims: Unregulated activity of interleukin (IL) 22 promotes intestinal tumorigenesis in mice. IL22 binds the antagonist IL22 subunit alpha 2 (IL22RA2, also called IL22BP). We studied whether alterations in IL22BP contribute to colorectal carcinogenesis in humans and mice., Methods: We obtained tumor and nontumor tissues from patients with colorectal cancer (CRC) and measured levels of cytokines by quantitative polymerase chain reaction, flow cytometry, and immunohistochemistry. We measured levels of Il22bp messenger RNA in colon tissues from wild-type, Tnf -/- , Lta -/- , and Ltb -/- mice. Mice were given azoxymethane and dextran sodium sulfate to induce colitis and associated cancer or intracecal injections of MC38 tumor cells. Some mice were given inhibitors of lymphotoxin beta receptor (LTBR). Intestine tissues were analyzed by single-cell sequencing to identify cell sources of lymphotoxin. We performed immunohistochemistry analysis of colon tissue microarrays from patients with CRC (1475 tissue cores, contained tumor and nontumor tissues) and correlated levels of IL22BP with patient survival times., Results: Levels of IL22BP were decreased in human colorectal tumors, compared with nontumor tissues, and correlated with levels of lymphotoxin. LTBR signaling was required for expression of IL22BP in colon tissues of mice. Wild-type mice given LTBR inhibitors had an increased tumor burden in both models, but LTBR inhibitors did not increase tumor growth in Il22bp -/- mice. Lymphotoxin directly induced expression of IL22BP in cultured human monocyte-derived dendritic cells via activation of nuclear factor κB. Reduced levels of IL22BP in colorectal tumor tissues were associated with shorter survival times of patients with CRC., Conclusions: Lymphotoxin signaling regulates expression of IL22BP in colon; levels of IL22BP are reduced in human colorectal tumors, associated with shorter survival times. LTBR signaling regulates expression of IL22BP in colon tumors in mice and cultured human dendritic cells. Patients with colorectal tumors that express low levels of IL22BP might benefit from treatment with an IL22 antagonist., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Increased expression of osteopontin (secreted phosphoprotein 1, SPP1) is associated with aggressive human lung adenocarcinoma (LADC), but its function remains unknown. Our aim was to determine the role of SPP1 in smoking-induced LADC. We combined mouse models of tobacco carcinogen-induced LADC, of deficiency of endogenous Spp1 alleles, and of adoptive pulmonary macrophage reconstitution to map the expression of SPP1 and its receptors and determine its impact during carcinogenesis. Co-expression of Spp1 and mutant KrasG12C in benign cells was employed to investigate SPP1/KRAS interactions in oncogenesis. Finally, intratracheal adenovirus encoding Cre recombinase was delivered to LSL.KRASG12D mice lacking endogenous or overexpressing transgenic Spp1 alleles. SPP1 was overexpressed in experimental and human LADC and portended poor survival. In response to two different smoke carcinogens, Spp1-deficient mice developed fewer and smaller LADC with decreased cellular survival and angiogenesis. Both lung epithelial- and macrophage-secreted SPP1 drove tumor-associated inflammation, while epithelial SPP1 promoted early tumorigenesis by fostering the survival of KRAS-mutated cells. Finally, loss and overexpression of Spp1 was, respectively, protective and deleterious for mice harboring KRASG12D-driven LADC. Our data support that SPP1 is functionally involved in early stages of airway epithelial carcinogenesis driven by smoking and mutant KRAS and may present an important therapeutic target., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: Reflux promotes esophageal adenocarcinomas (EACs) creating a chronic inflammatory environment. Survival rates are low due to early local recurrences and distant metastasis. Hence, there is a need for new potential treatment options like immunotherapies. However, the inflammatory microenvironment in EACs and its impact on patient outcome remain to be fully understood., Methods: mRNA expression levels of pro- and anti-inflammatory markers in 39 EAC patients without neoadjuvant radio-chemotherapy were measured. Data were confirmed using flow cytometric analysis of freshly resected surgical specimens. Inflammatory alterations in premalignant lesions of Barrett's esophagus were analyzed by immunohistochemistry., Results: Expression levels of IL22 were reduced in EAC, while expression levels of FOXP3, IL10 and CTLA4 were increased. Flow cytometry demonstrated a strong infiltration of CD4 + T cells with a reduction in CD4 + T cells producing IL-22 or IL-17A. We also observed an increase in CD4 + CD127 low FOXP3 + cells producing IL-10. Accumulation of FOXP3 + T cells occurred prior to malignant changes. High expression of IL10 and low expression of IL22 in EAC were associated with reduced overall survival. Moreover, increased expression of IL10, CTLA4 and PD1 in the unaltered esophageal mucosa distant to the EAC was also linked with an unfavorable prognosis., Conclusion: EAC shows an anti-inflammatory environment, which strongly affects patient survival. The microscopically unaltered peritumoral tissue shows a similar anti-inflammatory pattern indicating an immunological field effect, which might contribute to early local recurrences despite radical resection. These data suggest that using checkpoint inhibitors targeting anti-inflammatory T cells would be a promising therapeutic strategy in EAC.

Introduction: Reflux promotes esophageal adenocarcinomas (EAC) creating a chronic inflammatory environment. EAC show an increasing incidence in the Western World and median survival rates are still low. The main reasons for poor prognosis despite new multimodal therapies are diagnosis of EACs at an already advanced stage and distant metastases. Hence, we wanted to investigate the presence of systemic inflammatory interleukins (IL) and their impact on patient prognosis., Material and Methods: Systemic expression levels of pro- and anti-inflammatory markers (IL-2, IL-4, IL-6, IL-10, IL-17A and IL-22) in the sera of 43 EAC patients without neoadjuvant radiochemotherapy were measured by flow cytometric analysis. A correlation to clinicopathological data was performed. Log-rank and Cox regression analysis were used to investigate the impact on patient survival. 43 sera of age and gender matched healthy volunteers were used as controls., Results: Increased systemic IL-6 (p = 0.044) and lower IL-17A (p = 0.002) levels were found in EAC patients as opposed to controls. A correlation of IL-10 levels with an increased T stage was found (p = 0.020). Also, systemic IL-10 levels were highly elevated in patients with distant metastasis (p<0.001). However, only systemic IL-17A levels had an influence on patient survival in multivariate analysis., Conclusion: Systemic IL-6 levels are increased, while IL-17A levels are reduced in EAC patients compared to healthy controls. In addition, circulating IL-10 might help to identify patients with advanced disease and high IL-17A might indicate a limited prognosis., Competing Interests: The authors have declared that no competing interests exist.

Lung adenocarcinoma (LADC) is the leading cause of cancer death worldwide. Nevertheless, syngeneic mouse models of the disease are sparse, and cell lines suitable for transplantable and immunocompetent mouse models of LADC remain unmet needs. We established multiple mouse LADC cell lines by repeatedly exposing two mouse strains (FVB, Balb/c) to the tobacco carcinogens urethane or diethylnitrosamine and by culturing out the resulting lung tumours for prolonged periods of time. Characterization of the resulting cell lines (n = 7) showed that they were immortal and phenotypically stable in vitro, and oncogenic, metastatic and lethal in vivo. The primary tumours that gave rise to the cell lines, as well as secondary tumours generated by transplantation of the cell lines, displayed typical LADC features, such as glandular architecture and mucin and thyroid transcription factor 1 expression. Moreover, these cells exhibited marked molecular similarity with human smokers' LADC, including carcinogen-specific Kras point mutations (KrasQ61R in urethane- and KrasQ61H in diethylnitrosamine-triggered cell lines) and Trp53 deletions and displayed stemness features. Interestingly, all cell lines overexpressed proliferin, a murine prolactin orthologue, which functioned as a lung tumour promoter. Furthermore, prolactin was overexpressed and portended poor prognosis in human LADC. In conclusion, we report the first LADC cell lines derived from mice exposed to tobacco carcinogens. These cells closely resemble human LADC and provide a valuable tool for the functional investigation of the pathobiology of the disease., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In myeloid dendritic cells (DC), deletion of the mechanistic target of rapamycin complex 2 (TORC2) results in an augmented pro-inflammatory phenotype and T cell stimulatory activity; however, the underlying mechanism has not been resolved. Here, we demonstrate that mouse bone marrow-derived TORC2-deficient myeloid DC (TORC2 -/- DC) utilize an altered metabolic program, characterized by enhanced baseline glycolytic function compared to wild-type WT control (Ctrl) DC, increased dependence on glycolytic ATP production, elevated lipid content and higher viability following stimulation with LPS. In addition, TORC2 -/- DC display an increased spare respiratory capacity (SRC) compared to WT Ctrl DC; this metabolic phenotype corresponds with increased mitochondrial mass and mean mitochondrial DNA copy number, and failure of TORC2 -/- DC mitochondria to depolarize following LPS stimulation. Our data suggest that the enhanced metabolic activity of TORC2 -/- DC may be due to compensatory TORC1 pathway activity, namely increased expression of multiple genes upstream of Akt/TORC1 activity, including the integrin alpha IIb, protein tyrosine kinase 2/focal adhesion kinase, IL-7R and Janus kinase 1(JAK1), and the activation of downstream targets of TORC1, including p70S6K, eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) and CD36 (fatty acid translocase). These enhanced TORC1 pathway activities may culminate in increased expression of the nuclear receptor peroxisome proliferator-activated receptor γ (Pparγ) that regulates fatty acid storage, and the transcription factor sterol regulatory element-binding transcription factor 1 (Srebf1). Taken together, our data suggest that TORC2 may function to restrain TORC1-driven metabolic activity and mitochondrial regulation in myeloid DC.

Lung cancer and chronic lung diseases impose major disease burdens worldwide and are caused by inhaled noxious agents including tobacco smoke. The cellular origins of environmental-induced lung tumors and of the dysfunctional airway and alveolar epithelial turnover observed with chronic lung diseases are unknown. To address this, we combined mouse models of genetic labeling and ablation of airway (club) and alveolar cells with exposure to environmental noxious and carcinogenic agents. Club cells are shown to survive KRAS mutations and to form lung tumors after tobacco carcinogen exposure. Increasing numbers of club cells are found in the alveoli with aging and after lung injury, but go undetected since they express alveolar proteins. Ablation of club cells prevents chemical lung tumors and causes alveolar destruction in adult mice. Hence club cells are important in alveolar maintenance and carcinogenesis and may be a therapeutic target against premalignancy and chronic lung disease., Competing Interests: MS, IL, MP, YC, MA, AL, DZ, FR, MV, NK, DW, AG, VA, KA, LK, DT, VK, SZ, IG, AM, VA, RS, GS No competing interests declared, (© 2019, Spella et al.)

IL-22 is a potent pro-inflammatory cytokine upregulated in psoriasis and in other inflammatory diseases. The function of IL-22 is regulated by the soluble scavenging receptor, IL-22 binding protein (IL-22BP or IL-22RA2). However, the role and regulation of IL-22BP itself in the pathogenesis of inflammatory disease remain unclear. We used the TLR7 agonist Imiquimod (IMQ) to induce a psoriasis-like skin disease in mice and found a strong downregulation of IL-22BP in the affected skin as well as in the lymph nodes of animals treated with IMQ. We also analysed psoriatic skin of patients and compared this to skin of healthy donors. Interestingly, IL-22BP expression was similarly downregulated in skin biopsies of psoriasis patients compared to the skin of healthy donors. Since IL-22BP is expressed foremost in dendritic cells, we characterized its expression in monocyte-derived dendritic cells (MoDC) during maturation. In this way, we found Prostaglandin E2 (PGE 2 ) to be a potent suppressor of IL-22BP expression in vitro. We conclude that regulation of IL-22BP by inflammatory mediators is an important step for the progression of inflammation in the skin and possibly also in other autoimmune diseases.

Malignant pleural effusion (MPE) is a frequent metastatic manifestation of human cancers. While we previously identified KRAS mutations as molecular culprits of MPE formation, the underlying mechanism remained unknown. Here, we determine that non-canonical IKKα-RelB pathway activation of KRAS-mutant tumor cells mediates MPE development and this is fueled by host-provided interleukin IL-1β. Indeed, IKKα is required for the MPE-competence of KRAS-mutant tumor cells by activating non-canonical NF-κB signaling. IL-1β fuels addiction of mutant KRAS to IKKα resulting in increased CXCL1 secretion that fosters MPE-associated inflammation. Importantly, IL-1β-mediated NF-κB induction in KRAS-mutant tumor cells, as well as their resulting MPE-competence, can only be blocked by co-inhibition of both KRAS and IKKα, a strategy that overcomes drug resistance to individual treatments. Hence we show that mutant KRAS facilitates IKKα-mediated responsiveness of tumor cells to host IL-1β, thereby establishing a host-to-tumor signaling circuit that culminates in inflammatory MPE development and drug resistance.

Malignant pleural effusion (MPE) is the lethal consequence of various human cancers metastatic to the pleural cavity. However, the mechanisms responsible for the development of MPE are still obscure. Here we show that mutant KRAS is important for MPE induction in mice. Pleural disseminated, mutant KRAS bearing tumour cells upregulate and systemically release chemokine ligand 2 (CCL2) into the bloodstream to mobilize myeloid cells from the host bone marrow to the pleural space via the spleen. These cells promote MPE formation, as indicated by splenectomy and splenocyte restoration experiments. In addition, KRAS mutations are frequently detected in human MPE and cell lines isolated thereof, but are often lost during automated analyses, as indicated by manual versus automated examination of Sanger sequencing traces. Finally, the novel KRAS inhibitor deltarasin and a monoclonal antibody directed against CCL2 are equally effective against an experimental mouse model of MPE, a result that holds promise for future efficient therapies against the human condition.

Wound healing is a crucial process which protects our body against permanent damage and invasive infectious agents. Upon tissue damage, inflammation is an early event which is orchestrated by a multitude of innate and adaptive immune cell subsets including T H 17 cells. T H 17 cells and T H 17 cell associated cytokines can impact wound healing positively by clearing pathogens and modulating mucosal surfaces and epithelial cells. Injury of the gut mucosa can cause fast expansion of T H 17 cells and their induction from naïve T cells through Interleukin (IL)-6, TGF-β, and IL-1β signaling. T H 17 cells produce various cytokines, such as tumor necrosis factor (TNF)-α, IL-17, and IL-22, which can promote cell survival and proliferation and thus tissue regeneration in several organs including the skin, the intestine, and the liver. However, T H 17 cells are also potentially pathogenic if not tightly controlled. Failure of these control mechanisms can result in chronic inflammatory conditions, such as Inflammatory Bowel Disease (IBD), and can ultimately promote carcinogenesis. Therefore, there are several mechanisms which control T H 17 cells. One control mechanism is the regulation of T H 17 cells via regulatory T cells and IL-10. This mechanism is especially important in the intestine to terminate immune responses and maintain homeostasis. Furthermore, T H 17 cells have the potential to convert from a pro-inflammatory phenotype to an anti-inflammatory phenotype by changing their cytokine profile and acquiring IL-10 production, thereby limiting their own pathological potential. Finally, IL-22, a signature cytokine of T H 17 cells, can be controlled by an endogenous soluble inhibitory receptor, Interleukin 22 binding protein (IL-22BP). During tissue injury, the production of IL-22 by T H 17 cells is upregulated in order to promote tissue regeneration. To limit the regenerative program, which could promote carcinogenesis, IL-22BP is upregulated during the later phase of regeneration in order to terminate the effects of IL-22. This delicate balance secures the beneficial effects of IL-22 and prevents its potential pathogenicity. An important future goal is to understand the precise mechanisms underlying the regulation of T H 17 cells during inflammation, wound healing, and carcinogenesis in order to design targeted therapies for a variety of diseases including infections, cancer, and immune mediated inflammatory disease.

The lungs are frequently affected by cancer metastasis. Although NRAS mutations have been associated with metastatic potential, their exact role in lung homing is incompletely understood. We cross-examined the genotype of various tumor cells with their ability for automatic pulmonary dissemination, modulated NRAS expression using RNA interference and NRAS overexpression, identified NRAS signaling partners by microarray, and validated them using Cxcr1 - and Cxcr2 -deficient mice. Mouse models of spontaneous lung metastasis revealed that mutant or overexpressed NRAS promotes lung colonization by regulating interleukin-8-related chemokine expression, thereby initiating interactions between tumor cells, the pulmonary vasculature, and myeloid cells. Our results support a model where NRAS -mutant, chemokine-expressing circulating tumor cells target the CXCR1-expressing lung vasculature and recruit CXCR2-expressing myeloid cells to initiate metastasis. We further describe a clinically relevant approach to prevent NRAS-driven pulmonary metastasis by inhibiting chemokine signaling. In conclusion, NRAS promotes the colonization of the lungs by various tumor types in mouse models. IL-8-related chemokines, NRAS signaling partners in this process, may constitute an important therapeutic target against pulmonary involvement by cancers of other organs., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Malignant pleural effusion (MPE) is common and difficult to treat. In the vast majority of patients the presence of MPE heralds incurable disease, associated with poor quality of life, morbidity and mortality. Current therapeutic approaches are inefficient and merely offer palliation of associated symptoms. Recent scientific progress has shed light in the biologic processes governing the mechanisms behind the pathobiology of MPE. Pleural based tumors interfere with pleural fluid drainage, as well as the host vasculature and immune system, resulting in decreased fluid absorption and increased pleural fluid production via enhanced plasma extravasation into the pleural space. In order to achieve this feat, pleural based tumors must elicit critical vasoactive events in the pleura, thus forming a favorable microenvironment for tumor dissemination and MPE development. Such properties involve specific transcriptional signaling cascades in addition to secretion of important mediators which attract and activate host cell populations which, in turn, impact tumor cell functions. The dissection of the biologic steps leading to MPE formation provides novel therapeutic targets and recent research findings provide encouraging results towards future therapeutic innovations in MPE management.

The pleural space, a sterile secluded environment in the thoracic cavity, represents an attractive metastatic site for various cancers of lung, breast and gastrointestinal origins. Whereas lung and breast adenocarcinomas could invade the pleural space because of their anatomic proximity, "distant" cancers like ovarian or gastrointestinal tract adenocarcinomas may employ more active mechanisms to the same end. A pleural metastasis is often accompanied by a malignant pleural effusion (MPE), an unfavorable complication that severely restricts the quality of life and expectancy of the cancer patient. MPE is the net "product" of three different processes, namely inflammation, enhanced angiogenesis and vascular leakage. Current efforts are focusing on the identification of cancer cell autocrine (specific mutation spectra and biochemical pathways) and paracrine (cytokine and chemokine signals) characteristics as well as host features (immunological or other) that underlie the MPE phenotype. Herein we examine the pleural histology, cytology and molecular characteristics that make the pleural cavity an attractive metastasis destination for lung adenocarcinoma. Mesothelial and tumor features that may account for the tumor's ability to invade the pleural space are highlighted. Finally, possible therapeutic interventions specifically targeting MPE are discussed.

Mast cells (MCs) have been identified in various tumors; however, the role of these cells in tumorigenesis remains controversial. Here, we quantified MCs in human and murine malignant pleural effusions (MPEs) and evaluated the fate and function of these cells in MPE development. Evaluation of murine MPE-competent lung and colon adenocarcinomas revealed that these tumors actively attract and subsequently degranulate MCs in the pleural space by elaborating CCL2 and osteopontin. MCs were required for effusion development, as MPEs did not form in mice lacking MCs, and pleural infusion of MCs with MPE-incompetent cells promoted MPE formation. Once homed to the pleural space, MCs released tryptase AB1 and IL-1β, which in turn induced pleural vasculature leakiness and triggered NF-κB activation in pleural tumor cells, thereby fostering pleural fluid accumulation and tumor growth. Evaluation of human effusions revealed that MCs are elevated in MPEs compared with benign effusions. Moreover, MC abundance correlated with MPE formation in a human cancer cell-induced effusion model. Treatment of mice with the c-KIT inhibitor imatinib mesylate limited effusion precipitation by mouse and human adenocarcinoma cells. Together, the results of this study indicate that MCs are required for MPE formation and suggest that MC-dependent effusion formation is therapeutically addressable.

Using genetic interventions, we previously determined that C-C motif chemokine ligand 2 (CCL2) promotes malignant pleural effusion (MPE) formation in mice. Here we conducted preclinical studies aimed at assessing the specific therapeutic potential of antibody-mediated CCL2 blockade against MPE. For this, murine MPEs or skin tumors were generated in C57BL/6 mice by intrapleural or subcutaneous delivery of lung (LLC) or colon (MC38) adenocarcinoma cells. Human lung adenocarcinoma cells (A549) were used to induce MPEs in severe combined immunodeficient mice. Intraperitoneal antibodies neutralizing mouse CCL2 and/or CCL12, a murine CCL2 ortholog, were administered at 10 or 50 mg/kg every three days. We found that high doses of CCL2/12 neutralizing antibody treatment (50 mg/kg) were required to limit MPE formation by LLC cells. CCL2 and CCL12 blockade were equally potent inhibitors of MPE development by LLC cells. Combined CCL2 and CCL12 neutralization was also effective against MC38-induced MPE and prolonged the survival of mice in both syngeneic models. Mouse-specific CCL2-blockade limited A549-caused xenogeneic MPE, indicating that host-derived CCL2 also contributes to MPE precipitation in mice. The impact of CCL2/12 antagonism was associated with inhibition of immune and vascular MPE-related phenomena, such as inflammation, new blood vessel assembly and plasma extravasation into the pleural space. We conclude that CCL2 and CCL12 blockade are effective against experimental MPE induced by murine and human adenocarcinoma in mice. These results suggest that CCL2-targeted therapies may hold promise for future use against human MPE.