Tumor-associated macrophages trigger MAIT cell dysfunction at the HCC invasive margin.

Mucosal-associated invariant T (MAIT) cells represent an abundant innate-like T cell subtype in the human liver. MAIT cells are assigned crucial roles in regulating immunity and inflammation, yet their role in liver cancer remains elusive. Here, we present a MAIT cell-centered profiling of hepatocellular carcinoma (HCC) using scRNA-seq, flow cytometry, and co-detection by indexing (CODEX) imaging of paired patient samples. These analyses highlight the heterogeneity and dysfunctionality of MAIT cells in HCC and their defective capacity to infiltrate liver tumors. Machine-learning tools were used to dissect the spatial cellular interaction network within the MAIT cell neighborhood. Co-localization in the adjacent liver and interaction between niche-occupying CSF1R+PD-L1+ tumor-associated macrophages (TAMs) and MAIT cells was identified as a key regulatory element of MAIT cell dysfunction. Perturbation of this cell-cell interaction in ex vivo co-culture studies using patient samples and murine models reinvigorated MAIT cell cytotoxicity. These studies suggest that aPD-1/aPD-L1 therapies target MAIT cells in HCC patients.

Immunosuppressive CD29+ Treg accumulation in the liver in mice on checkpoint inhibitor therapy.

OBJECTIVE: Liver metastases are often resistant to immune checkpoint inhibitor therapy (ICI) and portend a worse prognosis compared with metastases to other locations. Regulatory T cells (Tregs) are one of several immunosuppressive cells implicated in ICI resistance of liver tumours, but the role played by Tregs residing within the liver surrounding a tumour is unknown. DESIGN: Flow cytometry and single-cell RNA sequencing were used to characterise hepatic Tregs before and after ICI therapy. RESULTS: We found that the murine liver houses a Treg population that, unlike those found in other organs, is both highly proliferative and apoptotic at baseline. On administration of αPD-1, αPD-L1 or αCTLA4, the liver Treg population doubled regardless of the presence of an intrahepatic tumour. Remarkably, this change was not due to the preferential expansion of the subpopulation of Tregs that express PD-1. Instead, a subpopulation of CD29+ (Itgb1, integrin ß1) Tregs, that were highly proliferative at baseline, doubled its size in response to αPD-1. Partial and full depletion of Tregs identified CD29+ Tregs as the prominent niche-filling subpopulation in the liver, and CD29+ Tregs demonstrated enhanced suppression in vitro when derived from the liver but not the spleen. We identified IL2 as a critical modulator of both CD29+ and CD29- hepatic Tregs, but expansion of the liver Treg population with αPD-1 driven by CD29+ Tregs was in part IL2-independent. CONCLUSION: We propose that CD29+ Tregs constitute a unique subpopulation of hepatic Tregs that are primed to respond to ICI agents and mediate resistance.

Metformin treatment rescues CD8+ T-cell response to immune checkpoint inhibitor therapy in mice with NAFLD.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) represents the fastest growing underlying cause of hepatocellular carcinoma (HCC) and has been shown to impact immune effector cell function. The standard of care for the treatment of advanced HCC is immune checkpoint inhibitor (ICI) therapy, yet NASH may negatively affect the efficacy of ICI therapy in HCC. The immunologic mechanisms underlying the impact of NASH on ICI therapy remain unclear. METHODS: Herein, using multiple murine NASH models, we analysed the influence of NASH on the CD8+ T-cell-dependent anti-PD-1 responses against liver cancer. We characterised CD8+ T cells' transcriptomic, functional, and motility changes in mice receiving a normal diet (ND) or a NASH diet. RESULTS: NASH blunted the effect of anti-PD-1 therapy against liver cancers in multiple murine models. NASH caused a proinflammatory phenotypic change of hepatic CD8+ T cells. Transcriptomic analysis revealed changes related to NASH-dependent impairment of hepatic CD8+ T-cell metabolism. In vivo imaging analysis showed reduced motility of intratumoural CD8+ T cells. Metformin treatment rescued the efficacy of anti-PD-1 therapy against liver tumours in NASH. CONCLUSIONS: We discovered that CD8+ T-cell metabolism is critically altered in the context of NASH-related liver cancer, impacting the effectiveness of ICI therapy - a finding which has therapeutic implications in patients with NASH-related liver cancer. LAY SUMMARY: Non-alcoholic steatohepatitis represents the fastest growing cause of hepatocellular carcinoma. It is also associated with reduced efficacy of immunotherapy, which is the standard of care for advanced hepatocellular carcinoma. Herein, we show that non-alcoholic steatohepatitis is associated with impaired motility, metabolic function, and response to anti-PD-1 treatment in hepatic CD8+ T cells, which can be rescued by metformin treatment.

Steatohepatitis Impairs T-cell-Directed Immunotherapies Against Liver Tumors in Mice.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis causes loss of hepatic CD4+ T cells and promotes tumor growth. The liver is the most common site of distant metastases from a variety of malignancies, many of which respond to immunotherapy. We investigated the effects of steatohepatitis on the efficacy of immunotherapeutic agents against liver tumors in mice. METHODS: Steatohepatitis was induced by feeding C57BL/6NCrl or BALB/c AnNCr mice a methionine and choline-deficient diet or a choline-deficient l-amino acid-defined diet. Mice were given intrahepatic or subcutaneous injections of B16 melanoma and CT26 colon cancer cells, followed by intravenous injections of M30-RNA vaccine (M30) or intraperitoneal injections of an antibody against OX40 (aOX40) on days 3, 7, and 10 after injection of the tumor cells. We measured tumor growth and analyzed immune cells in tumor tissues by flow cytometry. Mice were given N-acetylcysteine to prevent loss of CD4+ T cells from liver. RESULTS: Administration of M30 and aOX40 inhibited growth of tumors from intrahepatic injections of B16 or CT26 cells in mice on regular diet. However, M30 and/or aOX40 did not slow growth of liver tumors from B16 or CT26 cells in mice with diet-induced steatohepatitis (methionine and choline-deficient diet or choline-deficient l-amino acid-defined diet). Steatohepatitis did not affect the ability of M30 to slow growth of subcutaneous B16 tumors. In mice with steatohepatitis given N-acetylcysteine, which prevents loss of CD4+ T cells, M30 and aOX40 were able slow growth of hepatic tumors. Flow cytometry analysis of liver tumors revealed reduced CD4+ T cells and effector memory cells in mice with vs without steatohepatitis. CONCLUSIONS: Steatohepatitis reduces the abilities of immunotherapeutic agents, such as M30 and aOX40, to inhibit tumor liver growth by reducing tumor infiltration by CD4+ T cells and effector memory cells. N-acetylcysteine restores T-cell numbers in tumors and increases the ability of M30 and aOX40 to slow tumor growth in mice.

CD40-mediated immune cell activation enhances response to anti-PD-1 in murine intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: While cholangiocarcinomas (CCAs) commonly express programmed cell death 1 (PD-1) and its ligand (PD-L1), they respond poorly to immune checkpoint inhibitors (ICIs). We aimed to determine whether stimulating antigen-presenting cells, including macrophages and dendritic cells, using a CD40 agonist could improve this response. METHODS: We compared treatment responses in subcutaneous, orthotopic, and 2 plasmid-based murine intrahepatic CCA (iCCA) models. Mice were treated for 4 weeks with weekly IgG control, a CD40 agonistic antibody, anti-PD-1, or the combination of both (anti-CD40/PD-1). Flow cytometric (FACS) analysis of lymphocytes and myeloid cell populations (including activation status) was performed. We used dendritic cell knockout mice, and macrophage, CD4+ and CD8+ T cell depletion models to identify effector cells. Anti-CD40/PD-1 was combined with chemotherapy (gemcitabine/cisplatin) to test for improved therapeutic efficacy. RESULTS: In all 4 models, anti-PD-1 alone was minimally efficacious. Mice exhibited a moderate response to CD40 agonist monotherapy. Combination anti-CD40/PD-1 therapy led to a significantly greater reduction in tumor burden. FACS demonstrated increased number and activation of CD4+ and CD8+ T cells, natural killer cells, and myeloid cells in tumor and non-tumor liver tissue of tumor-bearing mice treated with anti-CD40/PD-1. Depletion of macrophages, dendritic cells, CD4+ T cells, or CD8+ T cells abrogated treatment efficacy. Combining anti-CD40/PD-1 with gemcitabine/cisplatin resulted in a significant survival benefit compared to gemcitabine/cisplatin alone. CONCLUSION: CD40-mediated activation of macrophages and dendritic cells in iCCA significantly enhances response to anti-PD-1 therapy. This regimen may enhance the efficacy of first-line chemotherapy. LAY SUMMARY: Checkpoint inhibition, a common form of immune therapy, is generally ineffective for the treatment of cholangiocarcinoma. These tumors suppress the infiltration and function of surrounding immune cells. Stimulating immune cells such as macrophages and dendritic cells via the CD40 receptor activates downstream immune cells and enhances the response to checkpoint inhibitors.

Laparoscopic liver resection in Caroli disease: A single-centre case series.

BACKGROUND: Liver resection is the treatment of choice for patients with localised Caroli disease. While liver resection was traditionally performed as open procedure, this case series aims to evaluate the safety and efficacy of minimally invasive, laparoscopic liver surgery in these patients. METHODS: A systematic review of electronic case files of patients seen between April 2015 and December 2017 at the Department of Surgery, Charité University Hospital Berlin, was conducted. Patients with Caroli disease in whom laparoscopic liver resection had been performed were identified and analysed in this single-centre case series. RESULTS: Seven patients who underwent laparoscopic liver surgery for Caroli syndrome were identified and presented with a median age of 49 (range = 44-66) years, of which four (57%) were female. Preoperatively, six patients were classified as the American Society of Anaesthesiologists (ASA) 2 and one patient as ASA 3. Two operations were performed as single-incision laparoscopic surgery, whereas the others were done as multi-incision laparoscopic surgery. One patient required a conversion to an open procedure. The length of operation varied between patients, ranging from 128 to 758 min (median = 355). The length of stay in the intensive care unit ranged from 0 to 2 days. Two patients presented with post-operative complications (Clavien-Dindo Grade ≥3a), whereas no patient died. In histopathological analysis, all patients demonstrated characteristic findings of Caroli disease and no cholangiocarcinoma was found. CONCLUSION: These results indicate that minimally invasive, laparoscopic liver surgery is a safe and efficacious treatment option for patients with Caroli disease who require liver resection.

Safety and feasibility of laparoscopic liver resection in patients with a history of abdominal surgeries.

BACKGROUND: Laparoscopic techniques have become the standard approach for most liver resections. Clinical studies providing conclusive evidence which patients benefit most from minimal-invasive surgery remain limited. METHODS: We retrospectively analyzed data of all consecutive cases of laparoscopic liver resection between 2015 and 2018 at our center. We compared patients with and without prior abdominal surgeries with respect to postoperative complications (Clavien-Dindo score), length of operation, length of ICU stay and length of hospitalization in univariate and multivariate analyses. RESULTS: Within the study period 319 patients underwent laparoscopic liver resections, 44% of which had a history of abdominal surgeries. Pre-operative characteristics were similar to patients without prior surgeries. Both groups showed comparable rates of post-operative complications (Clavien-Dindo score ≥3a; 12% in patients without vs. 16% with prior surgeries, p = 0,322). There were no significant differences in length of surgery or length of stay in the ICU or in the hospital. CONCLUSION: Our data suggest that history of prior abdominal surgery is not a risk factor for post-operative complications after laparoscopic liver resection. We conclude that prior abdominal surgery should not be considered a contra-indication for laparoscopic approach in liver resection.

Single-incision laparoscopic surgery portal vein embolisation before extended hepatectomy.

Objective: Portal vein embolisation (PVE) represents the standard procedure for augmentation of the contralateral lobe before extended right hepatectomy. However, possible limitations for the percutaneous transhepatic approach exist, for example, large tumours of the right lobe. Here, we present our experiences with single-incision laparoscopic surgery-PVE (SILS-PVE) as an alternative approach for settings where percutaneous routes are technically not feasible. Methods: A small umbilical incision is performed, and a GelPOINT Mini Advanced Access Platform (Santa Margarida, CA, USA) is placed. Staging laparoscopy is performed routinely followed by identification of an appropriate ileal segment, which is subsequently exteriorized through the small umbilical incision. A peripheral mesenteric vein is encircled and cannulated to access right portal vein branches. After sufficient embolisation of the right lobe, the peripheral vein is ligated, the single port is extracted and the umbilical wound is closed. Results: SILS-PVE was successfully applied in 10 patients (median age 60.5 years) between 12/2015 and 03/2018. The technique was indicated due to extensive tumours in the right lobe (n = 8), extensive hydatid cyst (n = 1) and during SILS right hemicolectomy in Stage IV colon cancer (n = 1). Mean operative time was 184 min (range 116-315). Patients were discharged on post-operative day 4 (range 2-9). Augmentation of the future liver remnant volume was assessed by computed tomography-volumetry 3-4 weeks after SILS-PVE and showed a mean relative increase of 64.95%, future remnant liver function showed a mean increase of 120.77%. Conclusion: The proposed SILS-PVE represents a technically simple and safe alternative to standard percutaneous transhepatic approaches. Perioperative risks can be minimised by minimally-invasive surgery, which is of explicit importance in multimodal approaches before major hepatectomy.

Minimally Invasive Liver Surgery in Elderly Patients-A Single-Center Experience.

BACKGROUND: In recent years, laparoscopic liver resection has elicited growing attention as a safe procedure for various forms of hepatic resection. In the context of an aging population, this study aims to evaluate outcomes in elderly patients (>70 y) compared with younger patients (≤70 y). METHODS: All consecutive patients undergoing minimally invasive liver resections between December 2013 and January 2018 at the Department of Surgery, Charité-Universitätsmedizin Berlin, were included in this analysis. Patients' characteristics, such as body mass index, American Society of Anesthesiologists classification, as well as underlying liver disease and function, were examined and the perioperative outcomes of patients aged >70 y (group 1; G1) contrasted with patients aged ≤ 70 y (group 2; G2). RESULTS: Of 250 patients, 67 were >70 y old (G1) and 183 were ≤70 y old (G2). Patients in G1 were characterized by a higher body mass index (27.6 kg/m2versus 24.9 kg/m2; P = 0.004) and impaired physical states (American Society of Anesthesiologists score III/IV; 60% versus 37%; P = 0.002) when compared with group 2. G1 also exhibited higher rates of primary and secondary hepatic malignancies (G1: n = 62; 92.5%; G2: n = 115, 62.8%; P = 0.031) in addition to higher rates of cirrhosis (G1: n = 30, 44.8%; G2: n = 38, 20.8%; P = <0.001). The rate of major complications (Dindo-Clavien grade ≥ III) was similar between both groups (P = 0.58), with no differences regarding resection extent (P = 0.469). No difference was evident with regard to the median intensive care unit (median 1 versus 1 d; range, G1, 0-8 d, G2, 0-23 d; P = 0.1). However, we observed a significant longer hospital stay in G1 of 1 d (median 8 versus 9 d; G1 range: 4-35 d: G2 range: 4-59 d; P = 0.015). CONCLUSIONS: Minimally invasive liver resection is a feasible and safe procedure in elderly patients despite this age group exhibiting a higher rate of primary and secondary malignancy and cirrhosis, as well as an overall more severely compromised physical health when compared with patients under the age of 70 y. Therefore, it stands to reason that patients in poorer general health might particularly benefit from a minimally invasive approach.

Human Stem Cells Promote Liver Regeneration After Partial Hepatectomy in BALB/C Nude Mice.

BACKGROUND: Mesenchymal stem cells (MSCs) have been suggested to augment liver regeneration after surgically and pharmacologically induced liver failure. To further investigate this we processed human bone marrow-derived MSC according to good manufacturing practice (GMP) and tested those cells for their modulatory capacities of metabolic alterations and liver regeneration after partial hepatectomy in BALB/c nude mice. METHODS: Human MSCs were obtained by bone marrow aspiration of healthy donors as in a previously described GMP process. Transgenic GFP-MSCs were administered i.p. 24 h after 70% hepatectomy in BALB/c nude mice, whereas control mice received phosphate-buffered saline. Mice were sacrificed 2, 3, and 5 d after partial hepatectomy. Blood and organs were harvested and metabolic alterations as well as liver regeneration subsequently assessed by liver function tests, multianalyte profiling immunoassays, histology, and immunostaining. RESULTS: Hepatocyte and sinusoidal endothelial cell proliferation were significantly increased after partial hepatectomy in mice receiving MSC compared to control mice (Hepatocyte postoperative day 3, P < 0.01; endothelial cell postoperative day 5, P < 0.05). Hepatocyte fat accumulation correlated inversely with hepatocyte proliferation (r2 = 0.4064, P < 0.01) 2 d after partial hepatectomy, with mice receiving MSC being protected from severe fat accumulation. No GFP-positive cells could be detected in the samples. Serum levels of IL-6, HGF, and IL-10 were significantly decreased at day 3 in mice receiving MSC when compared to control mice (P < 0.05). Relative body weight loss was significantly attenuated after partial hepatectomy in mice receiving MSC (2 d and 3 d, both P < 0.001) with a trend toward a faster relative restoration of liver weight, when compared to control mice. CONCLUSIONS: Human bone marrow-derived MSC attenuate metabolic alterations and improve liver regeneration after partial hepatectomy in BALB/c nude mice. Obtained results using GMP-processed human MSC suggest functional links between fat accumulation and hepatocyte proliferation, without any evidence for cellular homing. This study using GMP-proceeded MSC has important regulatory implications for an urgently needed translation into a clinical trial.

[Laparoscopic Liver Surgery - a Single Centre Series of 250 Consecutive Cases]. / Laparoskopische Leberchirurgie ­ Berliner Zentrumserfahrungen aus 250 konsekutiven Fällen.

BACKGROUND: In spite of convincing results from internationally prominent liver centres, laparoscopic liver surgery is not widely used in Germany. A structured program to develop laparoscopic liver surgery was established in 2015 at the Department of Surgery, Charité Hospital, Berlin, in order to provide support for establishing this approach in German hospitals. METHODS: We now report the results of our centre for 250 consecutive laparoscopic liver resections between 12/2013 and 1/2018. A retrospective analysis was performed with respect to indications, patient characteristics, complexity of the operations and postoperative results. The development of the program was analysed by comparing period 1 (1/2014 - 12/2015) and period 2 (1/2016 - 12/2017). RESULTS: In comparison with period 1 (n = 16, 25% of patients), patients in period 2 (n = 75, 50% of patients) included a significantly greater percentage of patients with a high ASA score (3/4; p = 0.001). Hepatocellular carcinoma was the most frequent indication (n = 76, 30.4%), followed by colorectal liver metastases (n = 63, 25.2%). Malignant tumours increased over the years from 53.8% in period 1 to 75.7% in period 2 (p = 0.001). 72 major (≥ 3 segments) and 178 minor resections (< 3 segments) were performed, with an increase in major resections (n = 12, 17.9%) in period 1 to period 2 (n = 56, 33.1%, p = 0.02). In spite of the significantly higher percentage of complex operations in period 2, the rate of major complications (Dindo-Clavien ≥ III a) in period 2 was 16% (27 patients) was not significantly higher than in period 1, with 11.9% (8 patients, p = 0.432). 67 patients (26.8%) suffered from liver cirrhosis; the postoperative complication rate (Dindo-Clavien ≥ III a) was not significantly different between patients with (12%) and without cirrhosis (15.8%, p = 0.424). CONCLUSION: Aside from appropriate expertise in conventional open liver surgery, minimally invasive surgery and intraoperative sonography, it is necessary to develop a structured program for the reliable implementation of laparoscopic liver surgery in German hospitals. Because of the convincing results, conventional open liver surgery will be largely replaced in Germany by laparoscopic techniques in the coming years.

Health-related quality of life after laparoscopic liver resection.

BACKGROUND: Potential benefits of laparoscopic liver resections (LLRs) over open liver resections (OLRs) such as the clinical outcome and health-related quality of life (HRQoL) have not convincingly been investigated, yet. PATIENTS AND METHODS: All patients who had undergone LLR and OLR at our department between 1 June 2014 and 10 October 2016 were identified. HRQoL was assessed using the short form 36 (SF-36). All patients who returned the surveys were then retrospectively analysed with regards to the perioperative outcome. RESULTS: We received 66 eligible questionnaires (50%). The number of major liver resections did not significantly differ between both groups (LLR: 11 [33%], OLR: 16 [48%], P = 0.211).The proportion of patients with two or more co-morbidities (P = 0.044) and liver cirrhosis (P = 0.016), respectively, was significantly higher in the LLR group, when compared to the OLR group (LLR: 11 [33%] vs. 3 of 33 patients [9%], P = 0.016). HRQoL scores were good with no significant differences between both groups. Among these patients, there were significantly more pulmonary complications in the OLR group, and length of hospital stay was longer when compared to the LLR group. CONCLUSIONS: Laparoscopic liver surgery can be performed safely even in multimorbid elderly patients resulting in high HRQoL scores.

Adenosine A2a receptor inhibition increases the anti-tumor efficacy of anti-PD1 treatment in murine hepatobiliary cancers.

Backgrounds & Aims: The efficacy of immune checkpoint inhibitor (ICI) therapy for liver cancer remains limited. As the hypoxic liver environment regulates adenosine signaling, we tested the efficacy of adenosine A2a receptor (A2aR) inhibition in combination with ICI treatment in murine models of liver cancer. Methods: RNA expression related to the adenosine pathway was analyzed from public databases. Peripheral blood mononuclear cells of 13 patients with hepatocellular carcinoma (HCC) were examined by flow cytometry. The following murine cell lines were used: SB-1, RIL175, and Hep55.1c (liver cancer), CT26 (colon cancer), and B16-F10 (melanoma). C57BL/6 and BALB/c mice were used for orthotopic tumor models and were treated with SCH58261, an A2aR inhibitor, in combination with anti-PD1 therapy. Results: RNA expression of ADORA2A in tumor tissues derived from patients with HCC was higher than in tissues from other cancer types. A2aR+ T cells in peripheral blood from patients with HCC were highly proliferative after immunotherapy. Likewise, in an orthotopic murine model, A2aR expression on T cells increased following anti-PD1 treatment, and the expression of A2aR on T cells increased more in tumor-bearing mice compared with tumor-free mice. The combination of SCH58261 and anti-PD1 led to activation of T cells and reductions in tumor size in orthotopic liver cancer models. In contrast, SCH58261 monotherapy was ineffective in orthotopic liver cancer models and the combination was ineffective in the subcutaneous tumor models tested. CD4+ T-cell depletion attenuated the efficacy of the combination therapy. Conclusion: A2aR inhibition and anti-PD1 therapy had a synergistic anti-tumor effect in murine liver cancer models. Impact and implications: Adenosine A2a receptor (A2aR)-expressing T cells in the liver increased in tumor-bearing mice and after anti-PD1 treatment. The combination of an A2aR inhibitor and anti-PD1 treatment had potent anti-tumor effects in two murine models of orthotopic liver cancer. Adenosine A2a receptor blockade promotes immunotherapy efficacy in murine models, highlighting putative clinical benefits for advanced stage liver cancer patients.

The Gut Microbiome Controls Liver Tumors via the Vagus Nerve.

Liver cancer ranks amongst the deadliest cancers. Nerves have emerged as an understudied regulator of tumor progression. The parasympathetic vagus nerve influences systemic immunity via acetylcholine (ACh). Whether cholinergic neuroimmune interactions influence hepatocellular carcinoma (HCC) remains uncertain. Liver denervation via hepatic vagotomy (HV) significantly reduced liver tumor burden, while pharmacological enhancement of parasympathetic tone promoted tumor growth. Cholinergic disruption in Rag1KO mice revealed that cholinergic regulation requires adaptive immunity. Further scRNA-seq and in vitro studies indicated that vagal ACh dampens CD8+ T cell activity via muscarinic ACh receptor (AChR) CHRM3. Depletion of CD8+ T cells abrogated HV outcomes and selective deletion of Chrm3 on CD8 + T cells inhibited liver tumor growth. Beyond tumor-specific outcomes, vagotomy improved cancer-associated fatigue and anxiety-like behavior. As microbiota transplantation from HCC donors was sufficient to impair behavior, we investigated putative microbiota-neuroimmune crosstalk. Tumor, rather than vagotomy, robustly altered fecal bacterial composition, increasing Desulfovibrionales and Clostridial taxa. Strikingly, in tumor-free mice, vagotomy permitted HCC-associated microbiota to activate hepatic CD8+ T cells. These findings reveal that gut bacteria influence behavior and liver anti-tumor immunity via a dynamic and pharmaceutically targetable, vagus-liver axis.

NAFLD indirectly impairs antigen-specific CD8+ T cell immunity against liver cancer in mice.

Non-alcoholic fatty liver disease (NAFLD) has become an important etiology leading to liver cancer. NAFLD alters adaptive T cell immunity and has a profound influence on liver cancer development. However, it is unclear how NAFLD affects tumor antigen-specific T cell response. In this study, we generated a doxycycline-inducible MHC-I and -II antigen-expressing HCC cell line which allowed us to investigate tumor antigen-specific T cell response in two NAFLD mouse models. The system proved to be an effective and efficient way to study tumor antigen-specific T cells. Using this model, it was found that NAFLD impairs antigen-specific CD8+ T cell immunity against HCC. The effect was not due to reduced generation or intrinsic functional changes of tumor antigen-specific CD8+ T cells but caused by accumulated macrophages in the liver environment. The findings suggest that targeting macrophages in NAFLD-driven HCC may improve therapeutic outcomes.

Platelets control liver tumor growth through P2Y12-dependent CD40L release in NAFLD.

Platelets, the often-overlooked component of the immune system, have been shown to promote tumor growth. Non-alcoholic fatty liver disease (NAFLD) is a common disease in the Western world and rising risk for hepatocellular carcinoma (HCC). Unexpectedly, we observed that platelets can inhibit the growth of established HCC in NAFLD mice. Through pharmacological inhibition and genetic depletion of P2Y12 as well as in vivo transfusion of wild-type (WT) or CD40L-/- platelets, we demonstrate that the anti-tumor function of platelets is mediated through P2Y12-dependent CD40L release, which leads to CD8+ T cell activation by the CD40 receptor. Unlike P2Y12 inhibition, blocking platelets with aspirin does not prevent platelet CD40L release nor accelerate HCC in NAFLD mice. Similar findings were observed in liver metastasis models. All together, our study reveals a complex role of platelets in tumor regulation. Anti-platelet treatment without inhibiting CD40L release could be considered for liver cancer patients with NAFLD.

Evaluating the impact of hydroxychloroquine on mouse lymphocyte proliferation and cytokine production in vivo and in vitro.

Immunomodulatory drugs can alter lymphocyte function. Hydroxychloroquine (HCQ) is prescribed for many autoimmune diseases and is under investigation as an anti-tumor autophagy inhibitor. Here, we describe a protocol to evaluate the influence of HCQ on lymphocyte function by measuring the in vitro and ex vivo proliferation and cytokine production. The protocol can provide insights into potential immunomodulatory effects of HCQ and can be used for assessing other medications' effects on lymphocyte functions. For complete details on the use and execution of this protocol, please refer to Wabitsch et al. (2021).

Hydroxychloroquine can impair tumor response to anti-PD1 in subcutaneous mouse models.

Hydroxychloroquine (HCQ) is a well-known anti-inflammatory drug but is also known as an anti-inflammatory drug. Here, we evaluate the influence of HCQ treatment on the effect of anti-PD1 tumor immunotherapy. Anti-PD1 therapy-sensitive tumor lines MC38, CT26, and RIL-175 were used to investigate the impact of HCQ on anti-PD1 therapy efficacy. In vitro assays demonstrated that HCQ directly inhibited tumor cell growth in all the tested tumor cell lines. HCQ treatment impaired both antigen-specific and nonspecific T-cell production of TNFα and IFNγ in vitro and in vivo. Importantly, in all the three tumor models, HCQ treatment significantly impaired the response to anti-PD1 treatment, accompanying diminished in vivo T-cell activation and reduced tumor-infiltrating, antigen-specific CD8+ T cells. This study shows that HCQ treatment can result in immunotherapy failure due to its immunosuppressive effects that offset both increased MHC-I expression by tumor cell and direct cytotoxicity.

Recurrence of Hepatocellular Carcinoma After Liver Transplantation is Associated with Episodes of Acute Rejections.

PURPOSE: The impact of acute rejection (AR) after liver transplantation (LT) for hepatocellular carcinoma (HCC) on patient outcome is uncertain. This aim of this study is to investigate whether AR is associated with HCC relapse and overall survival. PATIENTS AND METHODS: Patients undergoing LT for HCC between 2001 and 2015 were retrospectively analyzed with regard to histopathological proven AR within the median time until recurrence. Cox's regression analysis was conducted revealing risk factors for HCC recurrence. RESULTS: HCC recurred in 47 of 252 analyzed patients with a median time to recurrence of 20 months. Patients with AR (28.6%) had a significantly higher frequency of recurrence compared to patients without AR (13.0%, p=0.002). Multiple Cox regression analyses identified AR within 20 months to be an independent risk factor for HCC recurrence both as dichotomized (HR=2.91, 95%CI: 1.30-6.53; p=0.009) and as a continuous variable (HR=1.81, 95%CI: 1.28-2.54; p=0.001). HCC recurrence and AR were associated with higher grades of liver fibrosis one year after LT, when compared to patients without AR (p=0.019). CONCLUSION: Our results demonstrate an association of AR with HCC recurrence after LT with implications for intervals of monitoring in tumor surveillance. Graft fibrosis and immune mechanisms are potentially related and causal interactions are worth further investigation.

Anti-PD-1 in Combination With Trametinib Suppresses Tumor Growth and Improves Survival of Intrahepatic Cholangiocarcinoma in Mice.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (iCCA) accounts for a fraction of primary liver cancers but has a 5-year survival rate of only 10%. Immune checkpoint inhibitors are effective in treating many solid cancers, but immune checkpoint inhibitor monotherapy has no clear benefit in iCCA. Mitogen-activated kinase (MEK) inhibitors, such as trametinib, have shown promising results in preclinical studies for iCCA by inhibiting cell proliferation and modifying the tumor microenvironment. This study aimed to show the potential benefit of combining trametinib with anti-programmed cell death protein 1 (PD-1) therapy in different iCCA mouse models. METHODS: Here, we assessed the in vitro cytotoxicity of trametinib in mouse (SB1 and LD-1) and human (EGI-1) cholangiocarcinoma cell lines. We examined the efficacy of single-agent trametinib, anti-PD-1, and a combination of both in subcutaneous, orthotopic, and plasmid-induced iCCA mouse models. Flow cytometry analysis was used to elucidate changes in the tumor immune microenvironment upon treatment. Whole-exome sequencing (WES) was performed on the SB1 tumor cell line to correlate this preclinical model with iCCAs in patients. RESULTS: Trametinib reduced tumor cell growth of SB1, LD-1, and EGI-1 tumor cells in vitro. Trametinib treatment led to up-regulation of major histocompatibility complex (MHC-I) and programmed cell death ligand 1 (PD-L-1) (programmed cell death ligand 1) on tumor cells in vitro. The combination of trametinib and anti-PD-1 reduced tumor burden in several iCCA tumor models and improved survival in SB1 tumor-bearing mice compared with either agent alone. Immunoprofiling of tumor-bearing mice showed an increase of hepatic effector memory CD8+ and CD4+ T cells, as well as an increased degranulation of CD8+ T cells, indicating enhanced cytotoxicity. WES and somatic mutational analysis showed no mutations of KRAS, BRAF, and ERK in SB1 tumor cells, and showed a similar genetic signature of SB1 found in a cohort of patients with iCCA. CONCLUSIONS: Altogether, our study shows that trametinib improves the immunogenicity of tumor cells by up-regulating MHC-I surface expression. The combination with anti-PD-1 results in optimal treatment efficacy for iCCA. WES of SB1 cells suggests that KRAS wild-type iCCAs also respond to this combination therapy.