Epithelial and Mesenchymal-like Pancreatic Cancer Cells Exhibit Different Stem Cell Phenotypes Associated with Different Metastatic Propensities.

Pancreatic ductal adenocarcinoma (PDAC) is mostly diagnosed at advanced or even metastasized stages, limiting the prognoses of patients. Metastasis requires high tumor cell plasticity, implying phenotypic switching in response to changing environments. Here, epithelial-mesenchymal transition (EMT), being associated with an increase in cancer stem cell (CSC) properties, and its reversion are important. Since it is poorly understood whether different CSC phenotypes exist along the EMT axis and how these impact malignancy-associated properties, we aimed to characterize CSC populations of epithelial and mesenchymal-like PDAC cells. Single-cell cloning revealed CSC (Holoclone) and non-CSC (Paraclone) clones from the PDAC cell lines Panc1 and Panc89. The Panc1 Holoclone cells showed a mesenchymal-like phenotype, dominated by a high expression of the stemness marker Nestin, while the Panc89 Holoclone cells exhibited a SOX2-dominated epithelial phenotype. The Panc89 Holoclone cells showed enhanced cell growth and a self-renewal capacity but slow cluster-like invasion. Contrarily, the Panc1 Holoclone cells showed slower cell growth and self-renewal ability but were highly invasive. Moreover, cell variants differentially responded to chemotherapy. In vivo, the Panc1 and Panc89 cell variants significantly differed regarding the number and size of metastases, as well as organ manifestation, leading to different survival outcomes. Overall, these data support the existence of different CSC phenotypes along the EMT axis in PDAC, manifesting different metastatic propensities.

Liver transplantation for HCC in cirrhosis: Are Milan criteria outdated?

In Germany, organ allocation is based on the MELD-system and lab-MELD is usually low in patients with hepatocellular carcinoma (HCC) in cirrhosis. Higher medical urgency can be achieved by standard exception for HCC (SE-HCC), if Milan criteria (MC) are met. Noteworthy, UNOS T2 reflects MC, but excludes singular lesions < 2 cm. Thus, SE-HCC is awarded to patients with one lesion between 2 and 5 cm or 2 to 3 lesions between 1 and 3 cm. These criteria are static and do not reflect biological properties of HCC.We present a retrospective cohort of 111 patients, who underwent liver transplantation at UKSH, Campus Kiel between 2007 and 2017. No difference was found in overall survival for patient cohorts using Milan, UCSF, up-to-seven, and French-AFP criteria. However, there was a significantly reduced survival, if microvascular invasion was detected in the explanted organ and in patients with HCC-recurrence. The exclusive use of static selection criteria including MC appear to limit the access to liver transplantation.

NF-κB/RelA controlled A20 limits TRAIL-induced apoptosis in pancreatic cancer.

The emergence of resistance to systemic therapies in pancreatic ductal adenocarcinoma (PDAC) is still a major obstacle in clinical practice. Both, constitutive and inducible NF-κB activity are known as key players in this context. To identify differentially expressed and TRAIL resistance mediating NF-κB target genes, TRAIL sensitive and resistant PDAC cell lines were analyzed by transcriptome assays. In this context, A20 was identified as an NF-κB/RelA inducible target gene. Translational PDAC tissue analysis confirmed the correlation of elevated A20 protein expression with activated RelA expression in PDAC patients. In in vitro experiments, an elevated A20 expression is accompanied by a specific resistance toward TRAIL-mediated apoptosis but not to chemotherapeutic-induced cell death. This TRAIL resistance was attributed to A20´s E3-ligase activity-mediating Zink finger domain. Furthermore, the ubiquitin-binding scaffold protein p62 was identified as indispensable for the TRAIL-mediated apoptosis-inducing pathway affected by A20. The results of this study identify A20 as a possible therapeutic target to affect resistance to TRAIL-induced apoptosis in PDAC cells.

Endogenous TRAIL-R4 critically impacts apoptotic and non-apoptotic TRAIL-induced signaling in cancer cells.

Binding of TRAIL to its death domain-containing receptors TRAIL-R1 and TRAIL-R2 can induce cell death and/or pro-inflammatory signaling. The importance of TRAIL and TRAIL-R1/R2 in tumor immune surveillance and cancer biology has meanwhile been well documented. In addition, TRAIL has been shown to preferentially kill tumor cells, raising hope for the development of targeted anti-cancer therapies. Apart from death-inducing receptors, TRAIL also binds to TRAIL-R3 and TRAIL-R4. Whereas TRAIL-R3 is lacking an intracellular domain entirely, TRAIL-R4 contains a truncated death domain but still a signaling-competent intracellular part. It is assumed that these receptors have anti-apoptotic, yet still not well understood regulatory functions. To analyze the significance of the endogenous levels of TRAIL-R4 for TRAIL-induced signaling in cancer cells, we stably knocked down this receptor in Colo357 and MDA-MB-231 cells and analyzed the activation of apoptotic and non-apoptotic pathways in response to treatment with TRAIL. We found that TRAIL-R4 affects a plethora of signaling pathways, partly in an opposite way. While knockdown of TRAIL-R4 in Colo357 strongly increased apoptosis and reduced clonogenic survival, it inhibited cell death and improved clonogenic survival of MDA-MB-231 cells after TRAIL treatment. Furthermore, TRAIL-R4 turned out to be an important regulator of the expression of a variety of anti-apoptotic proteins in MDA-MB-231 cells since TRAIL-R4-KD reduced the cellular levels of FLIPs, XIAP and cIAP2 but upregulated the levels of Bcl-xL. By inhibiting Bcl-xL with Navitoclax, we could finally show that this protein mainly accounts for the acquired resistance of MDA-MB-231 TRAIL-R4-KD cells to TRAIL-induced apoptosis. Analyses of non-apoptotic signaling pathways revealed that in both cell lines TRAIL-R4-KD resulted in a constitutively increased activity of AKT and ERK, while it reduced AKT activity after TRAIL treatment. Furthermore, TRAIL-R4-KD potentiated TRAIL-induced activation of ERK and p38 in Colo357, and NF-κB in MDA-MB-231 cells. Importantly, in both cell lines the activity of AKT, ERK, p38 and NF-κB after TRAIL treatment was higher in TRAIL-R4-KD cells than in respective control cells. Thus, our data provide evidence for the important regulatory functions of endogenous TRAIL-R4 in cancer cells and improve our understanding of the very complex human TRAIL/TRAIL-R system.

Phosphatidylethanol (PEth) for Monitoring Sobriety in Liver Transplant Candidates: Preliminary Results of Differences Between Alcohol-Related and Non-Alcohol-Related Cirrhosis Candidates.

BACKGROUND Monitoring sobriety is mandatory for liver transplant (LT) candidates with alcohol-related cirrhosis in Germany. Prior to listing, abstinence of 6 months is required. However, little is known about biomarker performance in alcohol-related cirrhosis. Routine testing of ethyl glucuronide in urine (uEtG) or hair (hEtG) is prone to manipulation or is unfeasible in anuria. Phosphatidylethanol (PEth) in dried-blood spots is a promising alternative. We compared PEth with routine parameters and self-reports in alcohol-related and non-alcohol-related cirrhosis at our transplant center. MATERIAL AND METHODS All patients received self-report questionnaires (AUDIT & TLFB). Blood, urine and hair samples, as well as PEth dried-blood spots were drawn at baseline. In addition, survival analyses were conducted. RESULTS Out of 66 patients, 53 were listed for LT and 13 were candidates not listed so far. An alcohol-use disorder was found in 25 patients. Positive results for uEtG, hEtG, and PEth were found in 5/65, 9/65, and 34/66 cases, respectively. PEth positivity was found in 52% of patients with alcohol-related cirrhosis, while 53% of patients with other liver diseases were positive. While uEtG, hEtG, and TLFB correlated with higher PEth values, active waiting list status was significantly correlated with negative PEth values. During the mean follow-up of 41.15 months, 23 patients were transplanted (34.9%). None of the biomarkers significantly predicted survival. CONCLUSIONS PEth can importantly assist abstinence monitoring in LT candidates due to its high validity and objectivity. The high percentage of patients with alcohol consumption in the non-alcoholic liver disease cohort underscores the importance of testing all transplant candidates.

Paracrine Interaction of Cholangiocellular Carcinoma with Cancer-Associated Fibroblasts and Schwann Cells Impact Cell Migration.

Although the Mitogen-activated protein kinase (MAPK) pathway is enriched in cholangiocarcinoma (CCA), treatment with the multityrosine kinase-inhibitor Sorafenib is disappointing. While cancer-associated fibroblasts (CAF) are known to contribute to treatment resistance in CCA, knowledge is lacking for Schwann cells (SC). We investigated the impact of stromal cells on CCA cells and whether this is affected by Sorafenib. Immunohistochemistry revealed elevated expression of CAF and SC markers significantly correlating with reduced tumor-free survival. In co-culture with CAF, CCA cells mostly migrated, which could be diminished by Sorafenib, while in SC co-cultures, SC predominantly migrated towards CCA cells, unaffected by Sorafenib. Moreover, increased secretion of pro-inflammatory cytokines MCP-1, CXCL-1, IL-6 and IL-8 was determined in CAF mono- and co-cultures, which could be reduced by Sorafenib. Corresponding to migration results, an increased expression of phospho-AKT was measured in CAF co-cultured HuCCT-1 cells, although was unaffected by Sorafenib. Intriguingly, CAF co-cultured TFK-1 cells showed increased activation of STAT3, JNK, ERK and AKT pathways, which was partly reduced by Sorafenib. This study indicates that CAF and SC differentially impact CCA cells and Sorafenib partially reverts these stroma-mediated effects. These findings contribute to a better understanding of the paracrine interplay of CAF and SC with CCA cells.

Patients Benefit from Liver Transplantation for Hepatocellular Carcinoma beyond Milan Criteria without Harming the Health Care System.

Liver transplantation (LT) is the only definitive treatment to cure hepatocellular carcinoma (HCC) in cirrhosis. Waiting-list candidates are selected by the model for end-stage liver disease (MELD). However, many indications are not sufficiently represented by labMELD. For HCC, patients are selected by Milan-criteria: Milan-in qualifies for standard exception (SE) and better organ access on the waiting list; while Milan-out patients are restricted to labMELD and might benefit from extended criteria donor (ECD)-grafts. We analyzed a cohort of 102 patients (2011−2020). Patients with labMELD (no SE, Milan-out, n = 56) and matchMELD (SE-HCC, Milan-in, n = 46) were compared. The median overall survival was not significantly different (p = 0.759). No difference was found in time on the waiting list (p = 0.881), donor risk index (p = 0.697) or median costs (p = 0.204, EUR 43,500 (EUR 17,800−185,000) for labMELD and EUR 30,300 (EUR 17,200−395,900) for matchMELD). Costs were triggered by a cut-off labMELD of 12 points. Overall, the deficit increased by EUR 580 per labMELD point. Cost drivers were re-operation (p < 0.001), infection with multiresistant germs (p = 0.020), dialysis (p = 0.017), operation time (p = 0.012) and transfusions (p < 0.001). In conclusion, this study demonstrates that LT for HCC is successful and cost-effective in low labMELD patients independent of Milan-criteria. Therefore, ECD-grafts are favorized in Milan-out HCC patients with low labMELD.

Prognostic significance of high mobility group A2 (HMGA2) in pancreatic ductal adenocarcinoma: malignant functions of cytoplasmic HMGA2 expression.

PURPOSE: HMGA2 has frequently been found in benign as well as malignant tumors and a significant association between HMGA2 overexpression and poor survival in different malignancies was described. In pancreatic ductal adenocarcinoma (PDAC), nuclear HMGA2 expression is associated with tumor dedifferentiation and presence of lymph node metastasis. Nevertheless, the impact of HMGA2 occurrence in other cell compartments is unknown. METHODS: Intracellular distribution of HMGA2 was analyzed in PDAC (n = 106) and peritumoral, non-malignant ducts (n = 28) by immunohistochemistry. Findings were correlated with clinico-pathological data. Additionally, intracellular HMGA2 presence was studied by Western blotting of cytoplasmic and nuclear fractions of cultured cells. RESULTS: HMGA2 was found in the cytoplasm and in the nucleus of cultured cells. In human tumor tissue, HMGA2 was also frequently found in the cytoplasm and the nucleus of tumor cells, however, nuclear staining was generally stronger. Direct comparison from tumor tissue with corresponding non-neoplastic peritumoral tissue revealed significantly stronger expression in tumors (p = 0.003). Of note, the nuclear staining was significantly stronger in lymph node metastatic cell nuclei compared to primary tumor cell nuclei (p = 0.049). Interestingly, cytoplasmic staining positively correlated with lymph vessel (p = 0.004) and venous invasion (p = 0.046). CONCLUSION: HMGA2 is a prognostic marker in PDAC. Firstly, we found a positive correlation for cytoplasmic HMGA2 expression with lympho-vascular invasion and, secondly, we found a significantly stronger nuclear expression of HMGA2 in cancer-positive lymph node nuclei compared to primary tumor cell nuclei. So far, the role of cytoplasmic HMGA2 is nearly unknown, however, our data lend support to the hypothesis that cytoplasmic HMGA2 expression is involved in nodal spread.

Indication of Liver Transplantation for Hepatocellular Carcinoma Should Be Reconsidered in Case of Microvascular Invasion and Multilocular Tumor Occurrence.

Liver transplantation (LT) is routinely performed for hepatocellular carcinoma (HCC) in cirrhosis without major vascular invasion. Although the adverse influence of microvascular invasion is recognized, its occurrence does not contraindicate LT. We retrospectively analyzed in our LT cohort the significance of microvascular invasion on survival and demonstrate bridging procedures. At our hospital, 346 patients were diagnosed with HCC, 171 patients were evaluated for LT, and 153 were listed at Eurotransplant during a period of 11 years. Among these, 112 patients received LT and were included in this study. Overall survival after 1, 3 and 5 years was 86.3%, 73.9%, and 67.9%, respectively. Microvascular invasion led to significantly reduced overall (p = 0.030) and disease-free survival (p = 0.002). Five-year disease-free survival with microvascular invasion was 10.5%. Multilocular tumor occurrence with simultaneous microvascular invasion revealed the worst prognosis. In our LT cohort, predominant bridging treatment was transarterial chemoembolization (TACE) and the number of TACE significantly correlated with poorer overall survival after LT (p = 0.028), which was confirmed in multiple Cox regression analysis for overall and disease-free survival (p = 0.015 and p = 0.011). Microvascular tumor invasion is significantly associated with reduced prognosis after LT, which is aggravated by simultaneous occurrence of multiple lesions. Therefore, indication strategies for LT should be reconsidered.

Inferior Vena Cava Constriction After Liver Transplantation Is a Severe Complication Requiring Individually Adapted Treatment: Report of a Single-Center Experience.

BACKGROUND Reports on vena cava occlusion after liver transplantation (LT) are rare, but this finding represents a severe complication in the early postoperative period. In the context of the complex presentation of a patient after LT, symptoms are often misinterpreted and can be subtle. MATERIAL AND METHODS In our cohort of 138 LTs performed between 2014 and 2017 at our University's Transplantation Department, 117 transplantations were valid for further analysis after exclusion of pediatric transplantations and transplants with primary non-function grafts. In 101 cases (73%), patients received a deceased-donor full-size organ. Living-donor LT was performed in 8 patients (6.4%) and 8 patients (6.4%) received a split graft. We report on 6 patients who had inferior vena cava (IVC) occlusion and summarize the treatment choices. RESULTS In our series, patients with positive findings (age 38-70 years) received an orthotopic full-size deceased-donor graft with end-to-end IVC anastomosis. In the subsequent period, imaging revealing IVC occlusion was done on a follow-up basis (n=2), due to dyspnea (n=1), and for progressive ascites (n=2). In 3 cases, a thrombus was found. We give detailed information on our treatment options from interventional treatment to transcardial thrombus removal and anastomosis augmentation. CONCLUSIONS IVC constriction and subsequent thrombosis are severe complications after LT that require individually adapted treatment in specialized centers. Since patients often present with subclinical symptoms, vascular diagnosis should be performed early to detect caval anastomosis pathologies. Despite regular ultrasonography, we favor CT and cavography for subsequent quantification. We also review the literature on IVC occlusion after LT.

Lethal thrombosis of the iliac artery caused by Aspergillus fumigatus after liver transplantation: case report and review of the literature.

BACKGROUND: Aspergillus fumigatus infections frequently occur after solid organ transplantation. Yet, a fungal thrombosis after liver transplantation is an exceptional finding. CASE PRESENTATION: We report on a 44-year-old female with an aspergillosis after liver transplantation for autoimmune hepatitis. On postoperative day (pod) 7, seizures occurred and imaging diagnostics revealed an intracranial lesion. Anidulafungin was initiated in suspicion of mycosis and switched to voriconazole on suspicion of an Aspergillus spp. infection. Progression of the cerebral lesion prompted craniotomy (pod 48) and the aspergillosis was verified. The patient was discharged with oral voriconazole therapy. Re-admission was necessary with acute-on-chronic renal failure after a tacrolimus overdose on pod 130. The patient received a pelvic angiography due to a temperature difference in the legs. It showed a complete iliac artery thrombosis which was subsecutively surgically removed. The histopathological examination revealed an Aspergillus fumigatus conglomerate. The patient died on pod 210 due to systemic aspergillosis. CONCLUSION: The acute development of focal neurologic deficits is common in patients with an aspergillosis of the brain. Nevertheless, arterial thrombosis after Aspergillus fumigatus is less frequent and, to the best of our knowledge, its occurrence after liver transplantation has not yet been reported so far. Due to its rarity, we added a review of the literature to this manuscript.

TRAIL-Receptor 4 Modulates γδ T Cell-Cytotoxicity Toward Cancer Cells.

Acquired immune evasion is one of the mechanisms that contributes to the dismal prognosis of cancer. Recently, we observed that different γδ T cell subsets as well as CD8+ αß T cells infiltrate the pancreatic tissue. Interestingly, the abundance of γδ T cells was reported to have a positive prognostic impact on survival of cancer patients. Since γδ T cells utilize TNF-related apoptosis inducing ligand (TRAIL) for killing of tumor cells in addition to granzyme B and perforin, we investigated the role of the TRAIL-/TRAIL-R system in γδ T cell-cytotoxicity toward pancreatic ductal adenocarcinoma (PDAC) and other cancer cells. Coculture of the different cancer cells with γδ T cells resulted in a moderate lysis of tumor cells. The lysis of PDAC Colo357 cells was independent of TRAIL as it was not inhibited by the addition of neutralizing anti-TRAIL antibodies or TRAIL-R2-Fc fusion protein. In accordance, knockdown (KD) of death receptors TRAIL-R1 or TRAIL-R2 in Colo357 cells had no effect on γδ T cell-mediated cytotoxicity. However, KD of decoy receptor TRAIL-R4, which robustly enhanced TRAIL-induced apoptosis, interestingly, almost completely abolished the γδ T cell-mediated lysis of these tumor cells. This effect was associated with a reduced secretion of granzyme B by γδ T cells and enhanced PGE2 production as a result of increased expression level of synthetase cyclooxygenase (COX)-2 by TRAIL-R4-KD cells. In contrast, knockin of TRAIL-R4 decreased COX-2 expression. Importantly, reduced release of granzyme B by γδ T cells cocultured with TRAIL-R4-KD cells was partially reverted by bispecific antibody [HER2xCD3] and led in consequence to enhanced lysis of tumor cells. Likewise, inhibition of COX-1 and/or COX-2 partially enhanced γδ T cell-mediated lysis of TRAIL-R4-KD cells. The combination of bispecific antibody and COX-inhibitor completely restored the lysis of TRAIL-R4-KD cells by γδ T cells. In conclusion, we uncovered an unexpected novel role of TRAIL-R4 in tumor cells. In contrast to its known pro-tumoral, anti-apoptotic function, TRAIL-R4 augments the anti-tumoral cytotoxic activity of γδ T cells.

NF-κB Dependent Chemokine Signaling in Pancreatic Cancer.

Pancreatic cancer is one of the carcinomas with the worst prognoses, as shown by its five-year survival rate of 9%. Although there have been new therapeutic innovations, the effectiveness of these therapies is still limited, resulting in pancreatic ductal adenocarcinoma (PDAC) becoming the second leading cause of cancer-related death in 2020 in the US. In addition to tumor cell intrinsic resistance mechanisms, this disease exhibits a complex stroma consisting of fibroblasts, immune cells, neuronal and vascular cells, along with extracellular matrix, all conferring therapeutic resistance by several mechanisms. The NF-κB pathway is involved in both the tumor cell-intrinsic and microenvironment-mediated therapeutic resistance by regulating the transcription of a plethora of target genes. These genes are involved in nearly all scenarios described as the hallmarks of cancer. In addition to classical regulators of apoptosis, NF-κB regulates the expression of chemokines and their receptors, both in the tumor cells and in cells of the microenvironment. These chemokines mediate autocrine and paracrine loops among tumor cells but also cross-signaling between tumor cells and the stroma. In this review, we will focus on NF-κB-mediated chemokine signaling, with an emphasis on therapy resistance in pancreatic cancer.

The Hepatic Microenvironment and TRAIL-R2 Impact Outgrowth of Liver Metastases in Pancreatic Cancer after Surgical Resection.

Most patients with pancreatic ductal adenocarcinoma (PDAC) undergoing curative resection relapse within months, often with liver metastases. The hepatic microenvironment determines induction and reversal of dormancy during metastasis. Both tumor growth and metastasis depend on the Tumor necrosis factor (TNF)-related apoptosis-inducing ligand-receptor 2 (TRAIL-R2). This study investigated the interplay of TRAIL-R2 and the hepatic microenvironment in liver metastases formation and the impact of surgical resection. Although TRAIL-R2-knockdown (PancTu-I shTR2) decreased local relapses and number of macroscopic liver metastases after primary tumor resection in an orthotopic PDAC model, the number of micrometastases was increased. Moreover, abdominal surgery induced liver inflammation involving activation of hepatic stellate cells (HSCs) into hepatic myofibroblasts (HMFs). In coculture with HSCs, proliferation of PancTu-I shTR2 cells was significantly lower compared to PancTu-I shCtrl cells, an effect still observed after switching coculture from HSC to HMF, mimicking surgery-mediated liver inflammation and enhancing cell proliferation. CXCL-8/IL-8 blockade diminished HSC-mediated growth inhibition in PancTu-I shTR2 cells, while Vascular Endothelial Growth Factor (VEGF) neutralization decreased HMF-mediated proliferation. Overall, this study points to an important role of TRAIL-R2 in PDAC cells in the interplay with the hepatic microenvironment during metastasis. Resection of primary PDAC seems to induce liver inflammation, which might contribute to outgrowth of liver metastases.

Drug Repositioning Inferred from E2F1-Coregulator Interactions Studies for the Prevention and Treatment of Metastatic Cancers.

Metastasis management remains a long-standing challenge. High abundance of E2F1 triggers tumor progression by developing protein-protein interactions (PPI) with coregulators that enhance its potential to activate a network of prometastatic transcriptional targets. Methods: To identify E2F1-coregulators, we integrated high-throughput Co-immunoprecipitation (IP)/mass spectometry, GST-pull-down assays, and structure modeling. Potential inhibitors of PPI discovered were found by bioinformatics-based pharmacophore modeling, and transcriptome profiling was conducted to screen for coregulated downstream targets. Expression and target gene regulation was validated using qRT-PCR, immunoblotting, chromatin IP, and luciferase assays. Finally, the impact of the E2F1-coregulator complex and its inhibiting drug on metastasis was investigated in vitro in different cancer entities and two mouse metastasis models. Results: We unveiled that E2F1 forms coactivator complexes with metastasis-associated protein 1 (MTA1) which, in turn, is directly upregulated by E2F1. The E2F1:MTA1 complex potentiates hyaluronan synthase 2 (HAS2) expression, increases hyaluronan production and promotes cell motility. Disruption of this prometastatic E2F1:MTA1 interaction reduces hyaluronan synthesis and infiltration of tumor-associated macrophages in the tumor microenvironment, thereby suppressing metastasis. We further demonstrate that E2F1:MTA1 assembly is abrogated by small-molecule, FDA-approved drugs. Treatment of E2F1/MTA1-positive, highly aggressive, circulating melanoma cells and orthotopic pancreatic tumors with argatroban prevents metastasis and cancer relapses in vivo through perturbation of the E2F1:MTA1/HAS2 axis. Conclusion: Our results propose argatroban as an innovative, E2F-coregulator-based, antimetastatic drug. Cancer patients with the infaust E2F1/MTA1/HAS2 signature will likely benefit from drug repositioning.

Liver metastasis of pancreatic cancer: the hepatic microenvironment impacts differentiation and self-renewal capacity of pancreatic ductal epithelial cells.

Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed at advanced stages with the liver as the main site of metastases. The hepatic microenvironment has been shown to determine outgrowth of liver metastases. Cancer stem cells (CSCs) are essential for initiation and maintenance of tumors and acquisition of CSC-properties has been linked to Epithelial-Mesenchymal-Transition. Thus, this study aimed at elucidating whether and how the hepatic microenvironment impacts stemness and differentiation of disseminated pancreatic ductal epithelial cells (PDECs). Culture of premalignant H6c7-kras and malignant Panc1 PDECs together with hepatocytes and hepatic stellate cells (HSC) promoted self-renewal capacity of both PDEC lines. This was indicated by higher colony formation compared to cells cocultured with hepatocytes and hepatic myofibroblasts. Different Panc1 colony types derived from an HSC-enriched coculture were expanded and characterized revealing that holoclones exhibited an enhanced colony formation ability, elevated and exclusive expression of the CSC-marker Nestin and a more pronounced mesenchymal phenotype compared to paraclones. Moreover, Panc1 holoclone cells showed an increased tumorigenic potential in vivo leading to formation of undifferentiated tumors in 7/10 animals, while inoculation of paraclone cells only led to formation of tumors in 2/10 animals being smaller in number and size. Holoclone tumors were characterized by elevated expression of mesenchymal markers, complete loss of E-cadherin expression and high expression of Nestin. Finally, Etanercept-mediated TNF-α blocking partly reversed the mesenchymal CSC-phenotype of Panc1 holoclone cells. Overall, these data provide evidence that the hepatic microenvironment determines stemness and differentiation of PDECs, thereby substantially contributing to liver metastases of PDAC.

Cytoplasmic TRAIL-R1 is a positive prognostic marker in PDAC.

BACKGROUND: The death receptors TRAIL-R1 and TRAIL-R2 are frequently overexpressed in cancer and there is an emerging evidence for their important role in malignant progression, also in the case of pancreatic ductal adenocarcinoma (PDAC). In their canonical localization at the plasma membrane, TRAIL-R1/-R2 may induce cell death and/or pro-inflammatory signaling leading to cell migration, invasion and metastasis. Although, they have repeatedly been found intracellular, in the cytoplasm and in the nucleus, their functions in intracellular locations are still not well understood. Likewise, studies dealing with the prognostic relevance of TRAIL-Rs located in particular cellular compartments are very rare. For PDAC, the correlation of nuclear TRAIL-R2 with worse patients' prognosis has been shown recently. Corresponding data on TRAIL-R1 are not available so far. METHODS: In the present study we analyzed the expression of TRAIL-R1 in 106 PDACs and 28 adjacent, peritumoral non-malignant pancreatic ducts with special emphasis on its cytoplasmic and nuclear localization and correlated the immunohistochemical findings with clinico-pathological patient characteristics. RESULTS: TRAIL-R1 was found in 93.4% of all PDAC samples. Cytoplasmic staining was present with very similar intensity in tumor and normal tissue. In contrast, nuclear TRAIL-R1 staining was significantly stronger in tumor compared to normal tissue (p = 0.006). Interestingly, we found that the number of cells with cytoplasmic TRAIL-R1 staining negatively correlates with tumor grading (p = 0.043). No such correlation could be detected for nuclear TRAIL-R1. Neither, cytoplasmic nor nuclear TRAIL-R1 staining showed a correlation with other clinico-pathological parameter such as pTNM categories. However, Kaplan-Meier analyses revealed significantly prolonged median survival of patients with positive cytoplasmic TRAIL-R1 expression in more than 80% of tumor cells compared to patients with tumors containing a smaller quantity of cells positively stained for cytoplasmic TRAIL-R1 (20 vs. 8 months; p = 0.004). CONCLUSION: Cytoplasmic TRAIL-R1 is a positive prognostic marker for patients with PDAC. Our findings indicate that loss of cytoplasmic TRAIL-R1 results in recurrent disease with more malignant phenotype thus suggesting anti-tumor activities of cytoplasmic TRAIL-R1 in PDAC.

The hepatic microenvironment essentially determines tumor cell dormancy and metastatic outgrowth of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed when liver metastases already emerged. This study elucidated the impact of hepatic stromal cells on growth behavior of premalignant and malignant pancreatic ductal epithelial cells (PDECs). Liver sections of tumor-bearing KPC mice comprised micrometastases displaying low proliferation located in an unobtrusive hepatic microenvironment whereas macrometastases containing more proliferating cells were surrounded by hepatic myofibroblasts (HMFs). In an age-related syngeneic PDAC mouse model livers with signs of age-related inflammation exhibited significantly more proliferating disseminated tumor cells (DTCs) and micrometastases despite comparable primary tumor growth and DTC numbers. Hepatic stellate cells (HSC), representing a physiologic liver stroma, promoted an IL-8 mediated quiescence-associated phenotype (QAP) of PDECs in coculture. QAP included flattened cell morphology, Ki67-negativity and reduced proliferation, elevated senescence-associated ß galactosidase activity and diminished p-Erk/p-p38-ratio. In contrast, proliferation of PDECs was enhanced by VEGF in the presence of HMF. Switching the micromilieu from HSC to HMF or blocking VEGF reversed QAP in PDECs. This study demonstrates how HSCs induce and maintain a reversible QAP in disseminated PDAC cells, while inflammatory HMFs foster QAP reversal and metastatic outgrowth. Overall, the importance of the hepatic microenvironment in induction and reversal of dormancy during PDAC metastasis is emphasized.

Off-pump tricuspid valved stent implantation: real-time three-dimensional transesophageal echocardiography for guidance and assessment.

In this case report, the value of real-time three-dimensional transesophageal echocardiography (TEE) for guidance and assessment of orthotopic tricuspid valved stent implantation in the beating heart is assessed in four pigs. A full two-dimensional TEE evaluation of standardized parameters was conducted. Three-dimensional TEE provided dependable imaging of anatomical structures of the right side of the heart, the delivery system, and the valved stent, allowing easier orientation and more accurate views for the surgical team. Although visualization of the tricuspid valve in the porcine model is challenging, it contributed highly to the successful implantation of the tricuspid valved stent.

Off-pump tricuspid valved stent implantation: the next step.

OBJECTIVES: Off-pump transcatheter valved stent implantation could be a treatment option for patients suffering from symptomatic tricuspid regurgitation (TR) who are classified as inoperable. In this study, we present our recent short-term results of transventricular tricuspid valved stent implantation and compare different stent types for atrial anchorage. METHODS: Fifteen pigs received a self-expandable valved stent implantation off-pump via a transventricular access. Successfully implanted pigs were observed over a period of 6 h (n = 9), 48 h (n = 1) and 4 weeks (n = 1). Haemodynamic and full transoesophageal echocardiographic (TOE) evaluations were done before, 1 h, 3 h (n = 11; all successfully implanted pigs), and 6 h (n = 9; acute group) after implantation. Nine days postimplantation, one pig received additional angiography, computed tomography (CT) and transthoracic echocardiography (TTE). Post-mortem, gross examination was conducted to analyse the stent position and deformation. In two pigs (48 h and 4 weeks survival) histological staining and immunohistochemistry of surrounding myocardium was performed. RESULTS: The heart rate significantly increased in all pigs postimplantation from 66.8 ± 13.6 to 101.8 ± 24.6 bpm, whereas cardiac output and pressure levels remained unchanged. Orthotopic positioning was reproducibly achieved. TOE showed an efficient reduction of paravalvular leakages from a mean grade of 1.4 1 h postimplantation to a mean of 0.9 at 6 h postimplantation due to a special sealing pouch. The ratio early and late ventricular filling velocities remained constant and the valvular gradient across the valved stent stayed low during the observation period. Angiography, CT and TTE confirmed orthotopic positioning and mild grade of paravalvular leakage after 9 days (n = 1). Only mild TR was observed here. The ventricular part of the stent was deformed to an oval shape in 7 of 14 animals as shown via post-mortem examination. The surrounding tissue after 1 month (n = 1) showed normal morphology, without inflammation or calcification. CONCLUSION: This study shows the feasibility of catheter-based replacement of the tricuspid valve by a valved stent in an off-pump procedure. The successive enhancements in this tricuspid valved stent design lead to a prototype being ready for mid- to long-term evaluations.