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ABSTRACTThe expression of viral antigens in chronic hepatitis B virus (HBV) infection drives continuous liver inflammation, one of the main risk factors to develop liver cancer. HBV developed immune-suppressive functions to escape from the host immune system, but their link to liver tumor development is not well understood. Here, we analyzed if and how HBV surface antigen (HBs) expression in combined hepatocellular-cholangiocarcinoma (cHCC/iCCA) cells influences their antigenicity for CD8 T cells. We randomly isolated liver tumor tissues from AlfpCre+-Trp53fl/fl/Alb-HBs+ tg mice and established primary carcinoma cell lines (pCCL) that showed a bilineal (CK7+/HNF4α+) cHCC/iCCA phenotype. These pCCL uniformly expressed HBs (HBshi), and low levels of MHC-I (MHC-Ilo), and were transiently convertible to a high antigenicity (MHC-Ihi) phenotype by IFN-γ treatment. HBshi/pCCL induced HBs/(Kb/S190–197)-specific CD8 T cells and developed slow-growing tumors in subcutaneously transplanted C57Bl/6J (B6) mice. Interestingly, pCCL-ex cells, established from HBshi/pCCL-induced and re-explanted tumors in B6 but not those in immune-deficient Rag1−/− mice showed major alterations, like an MHC-Ihi phenotype, a prominent growth-biased gene expression signature, a significantly decreased HBs expression (HBslo) and a switch to fast-growing tumors in re-transplanted B6 or PD-1−/− hosts with an unlocked PD-1/PD-L1 control system. CD8 T cell-mediated elimination of HBshi/pCCL, together with the attenuation of the negative restraints of HBs in the tumor cells, like ER-stress, reveals a novel mechanism to unleash highly aggressive HBslo/pCCL-ex immune-escape variants. Under certain conditions, HBs-specific CD8 T-cell responses thus potentiate tumor growth, an aspect that should be considered for therapeutic vaccination strategies against chronic HBV infection and liver tumors.

Abstract Background Tumor recurrence is one of the major challenges in clinical management of chordoma. Despite R0-resection, approximately 50% of chordomas recur within ten years after initial surgery. The underlying molecular processes are poorly understood resulting in the lack of associated therapeutic options. This is not least due to the absence of appropriate cell culture models of this orphan disease. Methods The intra-personal progression model cell lines U-CH11 and U-CH11R were compared using array comparative genomic hybridization, expression arrays, RNA-seq, and immunocytochemistry. Cell line origin was confirmed by short tandem repeat analysis. Inter-personal cell culture models (n = 6) were examined to validate whether the new model is representative. Cell viability after HOX/PBX complex inhibition with small peptides was determined by MTS assays. Results Using whole genome microarray analyses, striking differences in gene expression between primary and recurrent chordomas were identified. These expression differences were confirmed in the world’s first intra-personal model of chordoma relapse consisting of cell lines established from a primary (U-CH11) and the corresponding recurrent tumor (U-CH11R). Array comparative genomic hybridization and RNA-sequencing analyses revealed profound genetic similarities between both cell lines pointing to transcriptomic reprogramming as a key mechanism of chordoma progression. Network analysis of the recurrence specific genes highlighted HOX/PBX signaling as a common dysregulated event. Hence, HOX/PBX complexes were used as so far unknown therapeutic targets in recurrent chordomas. Treating chordoma cell lines with the complex formation inhibiting peptide HXR9 induced cFOS mediated apoptosis in all chordoma cell lines tested. This effect was significantly stronger in cell lines established from chordoma relapses. Conclusion Clearly differing gene expression patterns and vulnerabilities to HOX/PBX complex inhibition in highly therapy resistant chordoma relapses were identified using the first intra-personal loco-regional and further inter-personal chordoma progression models. For the first time, HOX/PBX interference was used to induce cell death in chordoma and might serve as the basic concept of an upcoming targeted therapy for chordomas of all progression stages.

Background & Aims: A coordinated stress and regenerative response is important after hepatocyte damage. Here, we investigate the phenotypes that result from genetic abrogation of individual components of the checkpoint kinase 2/transformation-related protein 53 (p53)/cyclin-dependent kinase inhibitor 1A (p21) pathway in a murine model of metabolic liver injury. Methods: Nitisinone was reduced or withdrawn in Fah-/- mice lacking Chk2, p53, or p21, and survival, tumor development, liver injury, and regeneration were analyzed. Partial hepatectomies were performed and mice were challenged with the Fas antibody Jo2. Results: In a model of metabolic liver injury, loss of p53, but not Chk2, impairs the oxidative stress response and aggravates liver damage, indicative of a direct p53-dependent protective effect on hepatocytes. Cell-cycle control during chronic liver injury critically depends on the presence of both p53 and its downstream effector p21. In p53-deficient hepatocytes, unchecked proliferation occurs despite a strong induction of p21, showing a complex interdependency between p21 and p53. The increased regenerative potential in the absence of p53 cannot fully compensate the surplus injury and is not sufficient to promote survival. Despite the distinct phenotypes associated with the loss of individual components of the DNA damage response, gene expression patterns are dominated by the severity of liver injury, but reflect distinct effects of p53 on proliferation and the anti-oxidative stress response. Conclusions: Characteristic phenotypes result from the genetic abrogation of individual components of the DNA damage-response cascade in a liver injury model. The extent to which loss of gene function can be compensated, or affects injury and proliferation, is related to the level at which the cascade is interrupted. Accession numbers of repository for expression microarray data: GSE156983, GSE156263, GSE156852, and GSE156252.

Abstract Trauma is the leading cause of death in individuals under 44 years of age. Thorax trauma (TxT) is strongly associated with trauma‐related death, an unbalanced innate immune response, sepsis, acute respiratory distress syndrome, and multiple organ dysfunction. It is shown that different in vivo traumata, such as TxT or an in vitro polytrauma cytokine cocktail trigger secretion of small extracellular nanovesicles (sEVs) from endothelial cells with pro‐inflammatory cargo. These sEVs transfer transcripts for ICAM‐1, VCAM‐1, E‐selectin, and cytokines to systemically activate the endothelium, facilitate neutrophil‐endothelium interactions, and destabilize barrier integrity. Inhibition of sEV‐release after TxT in mice ameliorates local as well as systemic inflammation, neutrophil infiltration, and distant organ damage in kidneys (acute kidney injury, AKI). Vice versa, injection of TxT‐plasma‐sEVs into healthy animals is sufficient to trigger pulmonary and systemic inflammation as well as AKI. Accordingly, increased sEV concentrations and transfer of similar cargos are observed in polytrauma patients, suggesting a fundamental pathophysiological mechanism.

Abstract Somatic cell reprogramming and tissue repair share relevant factors and molecular programs. Here, Dickkopf‐3 (DKK3) is identified as novel factor for organ regeneration using combined transcription‐factor‐induced reprogramming and RNA‐interference techniques. Loss of Dkk3 enhances the generation of induced pluripotent stem cells but does not affect de novo derivation of embryonic stem cells, three‐germ‐layer differentiation or colony formation capacity of liver and pancreatic organoids. However, DKK3 expression levels in wildtype animals and serum levels in human patients are elevated upon injury. Accordingly, Dkk3‐null mice display less liver damage upon acute and chronic failure mediated by increased proliferation in hepatocytes and LGR5+ liver progenitor cell population, respectively. Similarly, recovery from experimental pancreatitis is accelerated. Regeneration onset occurs in the acinar compartment accompanied by virtually abolished canonical‐Wnt‐signaling in Dkk3‐null animals. This results in reduced expression of the Hedgehog repressor Gli3 and increased Hedgehog‐signaling activity upon Dkk3 loss. Collectively, these data reveal Dkk3 as a key regulator of organ regeneration via a direct, previously unacknowledged link between DKK3, canonical‐Wnt‐, and Hedgehog‐signaling.

Richter syndrome (RS) is defined as the transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, mostly diffuse large B-cell lymphoma (DLBCL). Despite intensive therapy, patients with RS have an unfavorable clinical outcome. The detailed pathobiology of Richter transformation still needs to be elucidated. Here, we report high mRNA and protein levels of CARD9 in the RS cell line U-RT1. Co-immunoprecipitation revealed the assembly of a CBM complex using CARD9 instead of CARD11. CARD9 is known to be an activator of NF-кB signaling in myeloid cells. U-RT1 Western blot analyses showed phosphorylation of IκB as well as IKK, indicating a constitutively active canonical NF-кB pathway. This was further supported by the significant reduction in cell viability and CYLD cleavage products after CARD9 siRNA knockdown. We also showed immunostaining for CARD9 in 53% of cases analyzed in a series of RS tissue specimens, whereas other lymphomas rarely show CARD9 expression. This is the first report on ectopic expression and function of CARD9 in an aggressive B-cell lymphoma. Our findings suggest that CARD9 may contribute to the pathogenesis of RS.

Cancer incidence and mortality are rapidly growing, with liver cancer being the sixth most diagnosed cancer worldwide and the third leading cause of cancer death in 2020. A number of risk factors have been identified that trigger the progression to hepatocellular carcinoma. In this review, we focus on iron as a potential risk factor for liver carcinogenesis. Molecules involved in the regulation of iron metabolism are often upregulated in cancer cells, in order to provide a supply of this essential trace element for all stages of tumor development, survival, proliferation, and metastasis. Thus, cellular and systemic iron levels must be tightly regulated to prevent or delay liver cancer progression. Disorders associated with dysregulated iron metabolism are characterized with increased susceptibility to hepatocellular carcinoma. This review discusses the association of iron with metabolic disorders such as hereditary hemochromatosis, non-alcoholic fatty liver disease, obesity, and type 2 diabetes, in the background of hepatocellular carcinoma.

Abstract Chordomas are rare tumours of the bone arising along the spine from clivus to sacrum. We compared three chordoma cell lines of the clivus region including the newly established clivus chordoma cell line, U-CH14, with nine chordoma cell lines originating from sacral primaries by morphology, on genomic and expression levels and with patient samples from our chordoma tissue bank. Clinically, chordomas of the clivus were generally smaller in size at presentation and patients with sacral chordomas had more metastases and more often recurrent disease. All chordoma cell lines had a typical physaliphorous morphology and expressed brachyury, S100-protein and cytokeratin. By expression analyses we detected differentially expressed genes in the clivus derived cell lines as compared to the sacral cell lines. Among these were HOXA7, HOXA9, and HOXA10 known to be important for the development of the anterior-posterior body axis. These results were confirmed by qPCR. Immunohistologically, clivus chordomas had no or very low levels of HOXA10 protein while sacral chordomas showed a strong nuclear positivity in all samples analysed. This differential expression of HOX genes in chordomas of the clivus and sacrum suggests an oncofetal mechanism in gene regulation linked to the anatomic site.

Primary liver cancer (PLC) is the sixth most common tumour disease and one of the leading causes of cancer-related death worldwide. The two most common types of PLC are hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA). Diverse subgroups are described and a manifold number of gene mutations are known. Asymptomatic disease progression and limited therapeutic options are the reasons for the high mortality rate in PLC. Up to date, the multikinase inhibitors sorafenib and lenvatinib are the only FDA-approved first-line treatments for advanced HCC. One of the major drawbacks in the preclinical drug development is the lack of suitable model systems. In recent years, 3D organoid cultures were established from several organs and tumour subtypes, thereby opening new avenues in tumour research. 3D organoid cultures are used to describe the tumour diversity, for cancer modelling in a dish and for therapy responsiveness. The establishment of living biobanks and the development of next-generation matrices are promising approaches to overcome drug resistance and to improve the quality of personalised anticancer strategies for patients with PLC. In this review, we summarise the current knowledge of 3D cultures generated from healthy liver and primary liver cancer.

Pluripotency represents a cell state comprising a fine-tuned pattern of transcription factor activity required for embryonic stem cell (ESC) self-renewal. TBX3 is the earliest expressed member of the T-box transcription factor family and is involved in maintenance and induction of pluripotency. Hence, TBX3 is believed to be a key member of the pluripotency circuitry, with loss of TBX3 coinciding with loss of pluripotency. We report a dynamic expression of TBX3 in vitro and in vivo using genetic reporter tools tracking TBX3 expression in mouse ESCs (mESCs). Low TBX3 levels are associated with reduced pluripotency, resembling the more mature epiblast. Notably, TBX3-low cells maintain the intrinsic capability to switch to a TBX3-high state and vice versa. Additionally, we show TBX3 to be dispensable for induction and maintenance of naive pluripotency as well as for germ cell development. These data highlight novel facets of TBX3 action in mESCs.

Liver cancer is one of the most common cancer types worldwide and the fourth leading cause of cancer-related death. Liver carcinoma is distinguished by a high heterogeneity in pathogenesis, histopathology and biological behavior. Dysregulated signaling pathways and various gene mutations are frequent in hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), which represent the two most common types of liver tumors. Both tumor types are characterized by telomere shortening and reactivation of telomerase during carcinogenesis. Continuous cell proliferation, e.g., by oncogenic mutations, can cause extensive telomere shortening in the absence of sufficient telomerase activity, leading to dysfunctional telomeres and genome instability by breakage–fusion–bridge cycles, which induce senescence or apoptosis as a tumor suppressor mechanism. Telomerase reactivation is required to stabilize telomere functionality and for tumor cell survival, representing a genetic risk factor for the development of liver cirrhosis and liver carcinoma. Therefore, telomeres and telomerase could be useful targets in hepatocarcinogenesis. Here, we review similarities and differences between HCC and iCCA in telomere biology.

Background: Most liver tumors arise on the basis of chronic liver diseases that trigger inflammatory responses. Besides inflammation, subsequent defects in the p53-signaling pathway frequently occurs in liver cancer. In this study, we analyzed the consequences of inflammation and p53 loss in liver carcinogenesis. Methods: We used inducible liver-specific transgenic mouse strains to analyze the consequences of NF-κB/p65 activation mimicking chronic inflammation and subsequent p53 loss. Results: Ikk2ca driven NF-κB/p65 activation in mice results in liver fibrosis, the formation of ectopic lymphoid structures and carcinogenesis independent of p53 expression. Subsequent deletion of Trp53 led to an increased tumor formation, metastasis and a shift in tumor differentiation towards intrahepatic cholangiocarcinoma. In addition, loss of Trp53 in an inflammatory liver resulted in elevated chromosomal instability and indicated a distinct aberration pattern. Conclusions: In conclusion, activation of NF-κB/p65 mimicking chronic inflammation provokes the formation of liver carcinoma. Collateral disruption of Trp53 supports tumor progression and influences tumor differentiation and heterogeneity.

Liver cholestasis is a chronic liver disease and a major health problem worldwide. Cholestasis is characterised by a decrease in bile flow due to impaired secretion by hepatocytes or by obstruction of bile flow through intra- or extrahepatic bile ducts. Thereby cholestasis can induce ductal proliferation, hepatocyte injury and liver fibrosis. Notch signalling promotes the formation and maturation of bile duct structures. Here we investigated the liver regeneration process in the context of cholestasis induced by disruption of the Notch signalling pathway. Liver-specific deletion of recombination signal binding protein for immunoglobulin kappa j region (Rbpj), which represents a key regulator of Notch signalling, induces severe cholestasis through impaired intra-hepatic bile duct (IHBD) maturation, severe necrosis and increased lethality. Deregulation of the biliary compartment and cholestasis are associated with the change of several signalling pathways including a Kyoto Encyclopedia of Genes and Genomes (KEGG) gene set representing the Hippo pathway, further yes-associated protein (YAP) activation and upregulation of SRY (sex determining region Y)-box 9 (SOX9), which is associated with transdifferentiation of hepatocytes. SOX9 upregulation in cholestatic liver injury in vitro is independent of Notch signalling. We could comprehensively address that in vivo Rbpj depletion is followed by YAP activation, which influences the transdifferentiation of hepatocytes and thereby contributing to liver regeneration.

Ion channels are involved in a large variety of cellular processes including stem cell differentiation. Numerous families of ion channels are present in the organism which can be distinguished by means of, for example, ion selectivity, gating mechanism, composition, or cell biological function. To characterize the distinct expression of this group of ion channels we have compared the mRNA expression levels of ion channel genes between human keratinocyte-derived induced pluripotent stem cells (hiPSCs) and their somatic cell source, keratinocytes from plucked human hair. This comparison revealed that 26% of the analyzed probes showed an upregulation of ion channels in hiPSCs while just 6% were downregulated. Additionally, iPSCs express a much higher number of ion channels compared to keratinocytes. Further, to narrow down specificity of ion channel expression in iPS cells we compared their expression patterns with differentiated progeny, namely, neurons and cardiomyocytes derived from iPS cells. To conclude, hiPSCs exhibit a very considerable and diverse ion channel expression pattern. Their detailed analysis could give an insight into their contribution to many cellular processes and even disease mechanisms.

Atrophy of the olfactory epithelium (OE) associated with impaired olfaction and dry nose represents one of the most common phenotypes of human aging. Impairment in regeneration of a functional olfactory epithelium can also occur in response to injury due to infection or nasal surgery. These complications occur more frequently in aged patients. Although age is the most unifying risk factor for atrophic changes and functional decline of the olfactory epithelium, little is known about molecular mechanisms that could influence maintenance and repair of the olfactory epithelium. Here, we analyzed the influence of telomere shortening (a basic mechanism of cellular aging) on homeostasis and regenerative reserve in response to chemical induced injury of the OE in late generation telomere knockout mice (G3 mTerc(-/-)) with short telomeres compared to wild type mice (mTerc(+/+)) with long telomeres. The study revealed no significant influence of telomere shortening on homeostatic maintenance of the OE during mouse aging. In contrast, the regenerative response to chemical induced injury of the OE was significantly impaired in G3 mTerc(-/-) mice compared to mTerc(+/+) mice. Seven days after chemical induced damage, G3 mTerc(-/-) mice exhibited significantly enlarged areas of persisting atrophy compared to mTerc(+/+) mice (p = 0.031). Telomere dysfunction was associated with impairments in cell proliferation in the regenerating epithelium. Deletion of the cell cycle inhibitor, Cdkn1a (p21) rescued defects in OE regeneration in telomere dysfunctional mice. Together, these data indicate that telomere shortening impairs the regenerative capacity of the OE by impairing cell cycle progression in a p21-dependent manner. These findings could be relevant for the impairment in OE function in elderly people.