Cold-Atmospheric Plasma Induces Tumor Cell Death in Preclinical In Vivo and In Vitro Models of Human Cholangiocarcinoma

Through the last decade, cold atmospheric plasma (CAP) has emerged as an innovative therapeutic option for cancer treatment. Recently, we have set up a potentially safe atmospheric pressure plasma jet device that displays antitumoral properties in a preclinical model of cholangiocarcinoma (CCA), a rare and very aggressive cancer emerging from the biliary tree with few efficient treatments. In the present study, we aimed at deciphering the molecular mechanisms underlying the antitumor effects of CAP towards CCA in both an in vivo and in vitro context. In vivo, using subcutaneous xenografts into immunocompromised mice, CAP treatment of CCA induced DNA lesions and tumor cell apoptosis, as evaluated by 8-oxoguanine and cleaved caspase-3 immunohistochemistry, respectively. The analysis of the tumor microenvironment showed changes in markers related to macrophage polarization. In vitro, the incubation of CCA cells with CAP-treated culture media (i.e., plasma-activated media, PAM) led to a dose response decrease in cell survival. At molecular level, CAP treatment induced double-strand DNA breaks, followed by an increased phosphorylation and activation of the cell cycle master regulators CHK1 and p53, leading to cell cycle arrest and cell death by apoptosis. In conclusion, CAP is a novel therapeutic option to consider for CCA in the future

Cancer-associated fibroblasts in cholangiocarcinoma

International audiencePurpose of review: To give a state-of-art knowledge regarding cancer-associated fibroblasts (CAF) in cholangiocarcinoma (CCA) based both on direct evidence and studies on other desmoplastic cancers. High contingency of CAF characterizes CCA, a tumor with a biliary epithelial phenotype that can emerge anywhere in the biliary tree. Current treatments are very limited, the surgical resection being the only effective treatment but restricted to a minority of patients, whereas the remaining patients undergo palliative chemotherapy regimens. In cancer, CAF shape the tumor microenvironment, drive cancer growth and progression, and contribute to drug resistance. All these functions are accomplished through an interplay network between CAF and surrounding cells including tumor and other stromal cells, i.e. immune and endothelial cells.Recent findings: Several studies have pointed out the existence of CAF sub-populations carrying out several and opposite functions, cancer-promoting or cancer-restraining as shown in pancreatic cancer, another prototypic desmoplastic tumor in which heterogeneity of CAF is well demonstrated.Summary: New CAF functions are now emerging in pancreatic and breast cancers like the modulation of immune responses or tumor metabolism, opening new area for treatments

Treatment with mTOR inhibitors after liver transplantation enables a sustained increase in regulatory T-cells while preserving their suppressive capacity.

BACKGROUND: The mammalian targets of rapamycin (mTOR) inhibitors (sirolimus [SRL] and everolimus [EVR]) are used after transplantation for their immunosuppressive activity. Regulatory T-cells (Tregs) play a crucial role in immune tolerance. mTOR inhibitors appear to preserve Tregs, unlike Tacrolimus (Tac). AIM: The aim of this study was to evaluate the number and function of Tregs in liver transplant recipients before and after conversion from Tac to mTOR inhibitors. METHODS: Fifteen patients with stable graft function where converted to SRL (n=5) or EVR (n=10). Tregs (CD4+ CD25+ FoxP3+ CD127low) number and activity were analysed prospectively in blood cells using flow cytometry, and functional assay. RESULTS: Patients of both groups displayed a sustained rise in Treg levels after introduction of mTOR inhibitors (Treg levels at 3 months: 6.45±0.38% of CD4 T-cells, vs. baseline level of 3.61±0.37%, P<0.001; mean fold increase 2.04±0.73). In SRL group, 3-month Treg levels were 6.01±0.53 vs. 3.79±0.39; P=0.037, while in EVR group they were 6.63±0.67 vs. 3.54±0.51; P=0.001. By contrast, no statistical change was observed in an unconverted Tac control group. Tregs also preserved their functional ability to suppress activated T-cells. CONCLUSION: These results suggest that mTOR inhibitors induce a significant increase in Tregs while maintaining suppressive activity after LT

Ex-Vivo Pharmacological Defatting of the Liver: A Review

International audienceThe ongoing organ shortage has forced transplant teams to develop alternate sources of liver grafts. In this setting, ex-situ machine perfusion has rapidly developed as a promising tool to assess viability and improve the function of organs from extended criteria donors, including fatty liver grafts. In particular, normothermic machine perfusion represents a powerful tool to test a liver in full 37 °C metabolism and add pharmacological corrections whenever needed. In this context, many pharmacological agents and therapeutics have been tested to induce liver defatting on normothermic machine perfusion with promising results even on human organs. This systematic review makes a comprehensive synthesis on existing pharmacological therapies for liver defatting, with special focus on normothermic liver machine perfusion as an experimental ex-vivo translational model. View Full-Tex

Early high levels of regulatory T cells and T helper 1 may predict the progression of recurrent hepatitis C after liver transplantation.

BACKGROUND: Immune response failure against hepatitis C virus (HCV) has been associated with an increased regulatory T cell (Treg) activity. After liver transplantation (LT), 80% of patients experience an accelerated progression of hepatitis C recurrence. The aim of this work was to assess the involvement of Tregs, T helper (Th) 1, 2 and 17 cells in recurrent hepatitis C. METHODS: Peripheral blood cells obtained before and one month after LT from 22 recipients were analysed. Forty-four key molecules related to Treg, Th1, 2 and 17 responses, were evaluated using qRT-PCR. Liver recipients were classified in two groups according to graft fibrosis evaluated by the METAVIR score on the biopsy performed one year after LT (mild: F ≤ 1, n = 13; severe: F > 1, n = 9). Patients developing a severe recurrence were compared with patients with a mild recurrence. RESULTS: mRNA levels of Treg markers obtained one month after LT were significantly increased in patients with a severe disease course when compared to patients with a mild recurrence. Markers of the Th1 response were elevated in the same group. No differences in the markers determined before LT were observed. CONCLUSION: These findings suggest that Treg, induced by a multifactorial process, which could include a strong Th1 response itself, may play a role in suppressing the early antiviral response, leading to a severe recurrence of hepatitis C

Modulatory effect of rapamycin and tacrolimus on monocyte-derived dendritic cells phenotype and function

International audienceBackground: Immunosuppressive-drugs are needed after solid organ transplantation to prevent allograft rejection but induce severe side effects. Understanding the alloimmune response is critical to modulate it and to achieve graft operational tolerance. The role of regulatory T cells and tolerogenic dendritic cells (Tol-DCs) is undoubtedly essential in tolerance induction. Tacrolimus is considered as the cornerstone of immunosuppression in solid organ transplantation. mTOR inhibitor such as rapamycin are thought to induce tolerance and are used as anticancer drugs in several cancers. The aim of this study was to better understand the effect of these immunosuppressive drugs on the differentiation, maturation and function of human monocyte derived dendritic cells (DCs).Material and methods: DCs were differentiated from monocytes of healthy donors with either rapamycin (Rapa-DCs) or tacrolimus (Tac-DCs). The phenotype was evaluated by flow cytometry analysis. The production of pro- and anti-inflammatory cytokines was assessed by ELISA. The mRNA expression level of IDO and PD-L1 was assessed by RTqPCR. Mixed leukocytes reactions were performed to analyse suppressive activity of DCs.Results: Rapa-DC were characterised by a lower expression of the co-stimulatory molecules and CD83 than control-DCs (CTR-DC) (p < 0.05). In contrast, tacrolimus had no effect on the expression of surface markers compared to CTR-DCs. Rapamycin reduced both IL-12 and IL-10 secretions (p < 0.05). Rapa-DCs had a suppressive effect on CD4+ allogenic T cells compared to CTR-DCs (p < 0.05). However, neither Rapa-DCs nor Tac-DCs favoured the emergence of a CD4+CD25highFoxp3+ population compared to CTR-DCs. Surprisingly, Rapa-DCs had a reduced expression of IDO and PD-L1 compared to Tac-DCs and CTR-DCs.Conclusion: Rapa-DCs exhibit an incomplete phenotypic tolerogenic profile. To our knowledge this is the first paper showing a reduction of expression of pro-tolerogenic enzyme IDO in DCs. Tacrolimus does not change the phenotypical or functional characteristics of moDCs

Influence of everolimus-based treatment on circulating regulatory T cells after liver transplantation: Comparative study with tacrolimus-based therapy.

Liver transplantation remains the only treatment for terminal liver diseases. However, immunosuppressive drugs required for allograft acceptance are toxic and may be responsible for severe side effects. Modulating the immune system to induce tolerance is a promising approach to reduce immunosuppressive regimen. More particularly, promoting natural CD4 CD25 FoxP3 Tregs could be crucial in achieving tolerance. Contrary to calcineurin inhibitors, reports indicate that mTOR inhibitors may have a positive impact on Tregs. Here we present the first randomized prospective clinical study where Tregs levels from liver transplanted patients receiving either tacrolimus or everolimus were monitored for 6 months, starting from the day of transplantation. A total of 30 patients from four centers were monitored. Blood samples were obtained at day 0, day 14, one month, three months and six months post-transplantation. Flow-cytometry immunophenotyping of Tregs (CD4 CD25 CD127 FoxP3) and functional assays with Tregs were performed to assess their immunosuppressive capacity. Levels of Tregs were significantly reduced after one month of standard tacrolimus-based immunosuppressive regimen (p < 0.05). Four months after conversion, levels of Tregs from patients treated with everolimus was significantly higher than patients under tacrolimus (p < 0.02). Functional assays demonstrated that Tregs conserved their capacity to suppress the proliferation of activated PBMC

CD49b, a Major Marker of Regulatory T-Cells Type 1, Predicts the Response to Antiviral Therapy of Recurrent Hepatitis C after Liver Transplantation

The TRANSPEG study was a prospective study to assess the efficacy of antiviral therapy in patients with a recurrent hepatitis C virus (HCV) after liver transplantation. The influence of regulatory T-cells (Tregs) on the response to antiviral therapy was analyzed. Patients were considered as a function of their sustained virological response (SVR) at 18 months after treatment initiation. A transcriptomic analysis was performed to assess Treg markers (Tr1 and FoxP3+) in serum, PBMC, and liver biopsies. 100 patients had been included in the TRANSPEG study. Data from 27 of these patients were available. The results showed that the expression of CD49b (a predominant marker of Tr1) before the introduction of antiviral therapy was significantly associated with SVR. Responders displayed lower serum levels of CD49b than nonresponders (P<0.02). These findings were confirmed in PBMC and liver biopsies even if in a nonsignificant manner for the limited number of samples. The assessment of CD49b levels is thus predictive of the response to antiviral therapy. This data suggests that CD49b may be a marker of the failure of the immune response and antiviral therapy during HCV recurrence. The assessment of CD49b could help to select patients who require earlier and more intensive antiviral therapy

Investigating the performances of cold plasma endoscopy for cholangiocarcinoma local treatment

International audienc

Investigating the performances of cold plasma endoscopy for cholangiocarcinoma local treatment

International audienc