The liver is one of the organs most commonly affected by metastasis. The presence of liver metastases has been reported to be responsible for an immunosuppressive microenvironment and diminished immunotherapy efficacy. Herein, we aimed to investigate the role of IL-10 in liver metastasis and to determine how its modulation could affect the efficacy of immunotherapy in vivo. To induce spontaneous or forced liver metastasis in mice, murine cancer cells (MC38) or colon tumor organoids were injected into the cecum or the spleen, respectively. Mice with complete and cell type-specific deletion of IL-10 and IL-10 receptor alpha were used to identify the source and the target of IL-10 during metastasis formation. Programmed death ligand 1 (PD-L1)-deficient mice were used to test the role of this checkpoint. Flow cytometry was applied to characterize the regulation of PD-L1 by IL-10. We found that Il10-deficient mice and mice treated with IL-10 receptor alpha antibodies were protected against liver metastasis formation. Furthermore, by using IL-10 reporter mice, we demonstrated that Foxp3+ regulatory T cells (Tregs) were the major cellular source of IL-10 in liver metastatic sites. Accordingly, deletion of IL-10 in Tregs, but not in myeloid cells, led to reduced liver metastasis. Mechanistically, IL-10 acted on Tregs in an autocrine manner, thereby further amplifying IL-10 production. Furthermore, IL-10 acted on myeloid cells, i.e. monocytes, and induced the upregulation of the immune checkpoint protein PD-L1. Finally, the PD-L1/PD-1 axis attenuated CD8-dependent cytotoxicity against metastatic lesions. Treg-derived IL-10 upregulates PD-L1 expression in monocytes, which in turn reduces CD8+ T-cell infiltration and related antitumor immunity in the context of colorectal cancer-derived liver metastases. These findings provide the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastases. Liver metastasis diminishes the effectiveness of immunotherapy and increases the mortality rate in patients with colorectal cancer. We investigated the role of IL-10 in liver metastasis formation and assessed its impact on the effectiveness of immunotherapy. Our data show that IL-10 is a pro-metastatic factor involved in liver metastasis formation and that it acts as a regulator of PD-L1. This provides the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastasis.

Human pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies in the world and despite great efforts in research types of treatment remain limited. A frequently detected alteration in PDACs is a truncated O-linked N-acetylgalactosamine (GalNAc) glycosylation with expression of the Tn antigen. Changes in O-glycosylation affect posttranslationally modified O-GalNAc proteins resulting in profound cellular alterations. Tn antigen is a tumor associated glycan detected in 75-90 % of PDACs and up to 67 % in its precursor lesions. Since the role of Tn antigen expression in PDAC is insufficiently understood we analyzed the impact of COSMC mediated Tn antigen expression in two human PDAC cell lines on cellular oncogenic properties. Forced expression of Tn antigen on O-glycosylated proteins in pancreatic cancer cells was induced by lentiviral-mediated knockdown of the COSMC chaperone, which prevented O-glycan elongation beyond the initial GalNAcα1- residue on O-linked glycoproteins. Altered O-GalNAc glycosylation was analyzed in human pancreatic cancer cell lines Panc-1 and L3.6pl using Western and Far-Western blot as well as immunocytochemical techniques. To assess the biological implications of COSMC function on oncogenic properties, cell viability assays, scratch assays combined with live cell imaging, migration and apoptosis assays were performed. Lectin based glycoprotein enrichment with subsequent mass spectrometric analysis identified new cancer O-GalNAc modified proteins. Expression of Tn antigen bearing Nucleolin in patient derived PDAC tumor specimens was evaluated and correlated with clinicopathological data. Tn antigen expression was induced on various O-GalNAc glycoproteins in COSMC deficient cell lines compared to the control. Proliferation was reduced (p < 0.001) in COSMC knockdown cells, whereas migration was increased (p < 0.001) and apoptosis was decreased (p = 0.03), highlighting the importance of Tn antigen expression on metastatic and anti-apoptotic behavior of PDAC derived cells. Nucleolin was identified as O-GalNAc modified protein in COSMC deficient PDAC cell lines. Interestingly, immunohistochemical staining and co-localization studies of patient derived PDACs revealed poor survival for patients with strong co-localization of Tn antigen and Nucleolin (p = 0.037). This study substantiates the influence of altered O-glycan (Tn/STn) expression on oncogenic properties in pancreatic cancer and identifies O-GalNAc modified Nucleolin as novel prognostic marker.

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.

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.

Altered O-glycosylation is a key contributor to various pathophysiological processes. Notably, the expression of the Tn antigen is primarily attributed to dysfunction of the chaperone Cosmc, while the overexpression of polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) has also been implicated in numerous diseases. We generated a transgenic mouse model with conditional Cosmc-knockout and simultaneous overexpression of polypeptide N-acetylgalactosaminyltransferase 2 (GalNT2) mediated by the pancreas-specific transcription factor 1a (Ptf1a)-Cre mouse strain to investigate the effect of Tn antigen overexpression on the pancreas in vivo. Histopathological examination of the transgenic pancreas revealed a chronic pancreatitis phenotype with interlobular fibrosis and focal necrosis after only a few weeks as a result of Tn antigen overexpression. In the later stages, there was a progressive loss of pancreatic parenchyma with consecutive exocrine pancreatic insufficiency and malnutrition in the transgenic mice. Flow cytometric analyses have also confirmed that significant infiltration of immune cells occurs in the course of pancreatitis. In the transgenic mouse model presented here, we demonstrated that overexpression of the Tn antigen in the pancreas results in chronic pancreatitis, highlighting the pathophysiological importance of truncated O-glycosylation.

Colorectal carcinomas are a rare but possible complication in an interposed colonic segment used for reconstruction after esophagectomy. We report the case of a patient who underwent colonic interposition surgery in childhood due to esophageal atresia and was diagnosed with colorectal adenocarcinoma of the interposed colon ~57 years later. The patient underwent gastric pull-up after thoraco-abdominal resection of the colonic interposition en bloc with the adjacent remaining esophagus.