Dataset Information

PD-1-targeted immunotherapy in NASH-triggered liver cancer induces pro-tumorigenic environment through CD8+PD-1+ T-cells

ABSTRACT: Immunotherapy has opened hitherto unknown possibilities to treat cancer. Whereas some cancer types (e.g. melanoma) can be efficiently treated, others lack measurable positive effects (e.g. PDAC). Moreover, stratification of responders/non-responders is only possible in some cancer types (e.g. melanoma). Hepatocellular carcinoma (HCC) has a dismal prognosis, limited treatment options and survival benefit, and represents a potential cancer entity for successful immunotherapy. Here, we investigated NASH-triggered HCC in the context PD-1-targeted immunotherapy. Using flow cytometry, single cell RNA sequencing, immunohistochemistry and mass spectrometric analyses, we found a progressive increase of CD8+PD-1+ effector T-cells with a unique profile of exhaustion and activation markers rising with murine and human NASH severity. Notably, late-stage HCC treatment with PD-1-targeted immunotherapy enhanced hepatic carcinogenesis in mice. Dissecting potential mechanisms of action during tumor-initiation and -progression we analyzed the effects of PD-1-targeted immunotherapy at HCC initiation. PD-1-targeted immunotherapy induced a pro-tumorigenic environment, enhanced necro-inflammation and increased NAFLD-activation score (NAS), leading to increased liver cancer incidence, tumor number and nodule size. In contrast, anti-CD8 or anti-CD8/anti-NK1.1 treatment reduced NAS and abrogated the development of liver cancer, thus identifying CD8+PD-1+ T-cells as drivers of liver cancer in NASH-triggered HCC. Increased apoptotic signaling, STAT3 phosphorylation and hepatic proliferation were detected in intra-tumoral liver tissue upon PD-1-targeted immunotherapy. In line, PD-1-/- mice challenged with a NASH diet displayed early onset of hepatocarcinogenesis, corroborating the pro-tumorigenic role of absent or reduced PD-1. Mechanistically PD-1-targeted immunotherapy mainly affected hepatic abundance of CD8+PD-1+ T-cells, instead of altering the quality of Tox+CXCR6+ expressing CD8+PD-1+TNF+CD39+Gzmb+ T-cells found in NASH livers, leading to an aggressive, pro-tumorigenic liver environment. Single-cell mapping of human NASH-, borderline NASH- or unaffected livers corroborated our preclinical NASH results. Moreover, in human NASH livers a correlation of hepatic CD8+, PD-1+, TNF+ T-cells with fibrosis and NASH severity was observed. Accordingly, HCC patients with NASH etiology display a sharp increase in intra- and peri-tumoral CD8+ PD-1+ T-cells. In a cohort of 65 patients recruited across 6 centers in Germany and Austria, patients with NAFLD/NASH-driven HCC responded worse to PD-1-targeted immunotherapy by Nivolumab or Pembrolizumab compared to non-NAFLD patients. This resulted in significant reduced overall survival, in trends of faster disease progression and reduced progression free survival. Histological analysis of livers derived from HCC patients treated with PD-1-targeted immunotherapy displayed high levels of intra and peri-tumoral CD8+ PD-1+ T-cells and Ki67+ hepatocytes. Taken together, these data indicate that PD-1-targeted immunotherapy induces immune-related adverse effects in NAFLD/NASH-driven HCC through CD8+PD-1+ T-cells. Our data call for stratification of HCC patients subjected to PD-1-targeted immunotherapy, with NAFLD being a negative predictor.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): T Cell

SUBMITTER: Mario Oroshi

LAB HEAD: Felix Meissner

PROVIDER: PXD017236 | Pride | 2021-03-03

REPOSITORIES: Pride

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	20190827_QX8_AnSi_SA_804_01_NA_CD8.raw	Raw
	20190827_QX8_AnSi_SA_804_02_NA_CD8.raw	Raw
	20190827_QX8_AnSi_SA_804_03_NA_CD8.raw	Raw
	20190827_QX8_AnSi_SA_804_04_NA_CD8.raw	Raw
	20190827_QX8_AnSi_SA_804_05_NA_CD8.raw	Raw

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NASH limits anti-tumour surveillance in immunotherapy-treated HCC.

Pfister Dominik D Núñez Nicolás Gonzalo NG Pinyol Roser R Govaere Olivier O Pinter Matthias M Szydlowska Marta M Gupta Revant R Qiu Mengjie M Deczkowska Aleksandra A Weiner Assaf A Müller Florian F Sinha Ankit A Friebel Ekaterina E Engleitner Thomas T Lenggenhager Daniela D Moncsek Anja A Heide Danijela D Stirm Kristin K Kosla Jan J Kotsiliti Eleni E Leone Valentina V Dudek Michael M Yousuf Suhail S Inverso Donato D Singh Indrabahadur I Teijeiro Ana A Castet Florian F Montironi Carla C Haber Philipp K PK Tiniakos Dina D Bedossa Pierre P Cockell Simon S Younes Ramy R Vacca Michele M Marra Fabio F Schattenberg Jörn M JM Allison Michael M Bugianesi Elisabetta E Ratziu Vlad V Pressiani Tiziana T D'Alessio Antonio A Personeni Nicola N Rimassa Lorenza L Daly Ann K AK Scheiner Bernhard B Pomej Katharina K Kirstein Martha M MM Vogel Arndt A Peck-Radosavljevic Markus M Hucke Florian F Finkelmeier Fabian F Waidmann Oliver O Trojan Jörg J Schulze Kornelius K Wege Henning H Koch Sandra S Weinmann Arndt A Bueter Marco M Rössler Fabian F Siebenhüner Alexander A De Dosso Sara S Mallm Jan-Philipp JP Umansky Viktor V Jugold Manfred M Luedde Tom T Schietinger Andrea A Schirmacher Peter P Emu Brinda B Augustin Hellmut G HG Billeter Adrian A Müller-Stich Beat B Kikuchi Hiroto H Duda Dan G DG Kütting Fabian F Waldschmidt Dirk-Thomas DT Ebert Matthias Philip MP Rahbari Nuh N Mei Henrik E HE Schulz Axel Ronald AR Ringelhan Marc M Malek Nisar N Spahn Stephan S Bitzer Michael M Ruiz de Galarreta Marina M Lujambio Amaia A Dufour Jean-Francois JF Marron Thomas U TU Kaseb Ahmed A Kudo Masatoshi M Huang Yi-Hsiang YH Djouder Nabil N Wolter Katharina K Zender Lars L Marche Parice N PN Decaens Thomas T Pinato David J DJ Rad Roland R Mertens Joachim C JC Weber Achim A Unger Kristian K Meissner Felix F Roth Susanne S Jilkova Zuzana Macek ZM Claassen Manfred M Anstee Quentin M QM Amit Ido I Knolle Percy P Becher Burkhard B Llovet Josep M JM Heikenwalder Mathias M

Nature 20210324 7854

Hepatocellular carcinoma (HCC) can have viral or non-viral causes1-5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC ...[more]

PMID: 33762733

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Project description:Hepatocellular carcinoma (HCC) has dismal treatment responses to systemic therapies and is caused by both, viral and non-viral etiologies, including non-alcoholic steatohepatitis (NASH) 1–5. NASH - triggered by high caloric intake and sedentary lifestyle - has become an important driver for HCC development. Immunotherapy has been recently approved for HCC but stratification of responders versus non-responders has remained an unmet need 5–8. Here, we found a progressive accumulation of non-classical activated CD8+PD-1+ T-cells in livers of NASH-affected patients and mice. PD-L1/PD-1-targeted immunotherapy of NASH-induced HCC administered either at cancer-initiation or after cancer-establishment lacked beneficial effects in mice. On the contrary, PD-1-targeted immunotherapy drove hepatic necro-inflammation and increased liver cancer incidence, tumor number, and nodule size. Anti-CD8 or anti-CD8/anti-NK1.1 treatment suppressed liver cancer development, implicating CD8+ T-cells as liver cancer drivers in the context of NASH. In line, PD-1-/- mice challenged with a NASH-inducing diet displayed early-onset of liver cancer. Mechanistically, PD-1-targeted immunotherapy triggered necro-inflammation and a pro-tumorigenic environment in the context of NASH, increasing the abundance of hepatic TOX+CXCR6+ expressing CD8+PD-1+ TNF+ T-cells. Anti-CD8/anti-PD-1 or anti-TNF/anti-PD-1, but not anti-CD4/anti-PD-1 treatment reverted anti-PD1 treatment-related increase of liver inflammation, NASH severity and tumorigenesis. Gene expression profile and increased abundance of murine hepatic CD8+PD-1+ T-cells were corroborated in human CD8+PD-1+ T cells derived from NASH patients. In a meta-analysis of clinical trials testing PD-1-targeting immune checkpoint inhibitors alone (i.e. pembrolizumab) or in combination in 1656 patients with advanced HCC, immunotherapy was not favorable over to control treatment in non-viral- when compared to viral-related HCC. Similarly, in two clinical cohorts tested, patients with NASH-driven HCC had a significantly worse overall survival with PD-1-targeted immunotherapy than HCC patients with other etiologies. Our data identify non-viral etiologies, particularly NASH, as potentially non-responsive in the context of HCC immunotherapy, providing a strong rational basis for patient stratification.

Project description:Background & Aims: Overnutrition is one of the major causes of non-alcoholic fatty liver disease (NAFLD) and its advanced form non-alcoholic steatohepatitis (NASH). Besides the quantity of consumed calories, distinct dietary components are increasingly recognized as important contributor to the pathogenesis of NASH. We aimed to develop and characterize a hitherto missing murine model which resembles both the pathology and nutritional situation of NASH-patients in Western societies. Methods: We developed a NASH-inducing diet (ND) enriched with sucrose, cholesterol and a high concentration of fats rich in saturated fatty acids in a composition which mimics Western food. C57Bl6/N mice were fed with the ND or control chow for 12 weeks. Biochemical, real-time polymerase chain reaction, Western Blot and immunohistochemical analyses were performed to characterize systemic and hepatic changes induced by ND-feeding. Immunohistochemistry was used to assess c-Jun levels and activation in 110 human NAFLD and control liver specimens applying tissue micro array technology. Results: ND-fed mice showed significant body weight gain, impaired glucose tolerance, elevated fasting blood glucose levels as well as decreased adiponectin and increased leptin serum levels compared to control mice. In the liver, ND-feeding led to marked steatosis, enhanced cholesterol levels, distinct signs of oxidative stress, hepatocellular damage, inflammation, activation of hepatic stellate cells, and beginning fibrosis. Transcriptome-wide hepatic gene expression analysis comparing ND-fed mice and control mice indicated main alterations in lipid metabolism and inflammatory processes. Search for over-represented transcription factor target sites among the differentially expressed genes identified AP-1 as the most likely factor to cause the transcriptional changes in ND-livers. Combining differentially expressed gene and protein-protein interaction network analysis identified c-Jun (a component of the AP-1 complex) as hub in the largest connected deregulated sub-network in ND-livers. In accordance, ND-livers revealed c-Jun-phosphorylation and nuclear translocation. Moreover, hepatic c-Jun RNA and protein expression was enhanced in ND-fed compared to control mice. Also NAFLD-patients showed enhanced hepatic c-Jun levels, which correlated with inflammation, and notably, with the degree of hepatic steatosis. Conclusions: The new dietary mouse-model shows important pathological changes also found in human NASH and indicates c-jun/AP-1 activation as critical regulator of hepatic alterations. Abundance of c-jun in NAFLD likely facilitates development and progression of NASH, and thus, c-jun appears as attractive prognostic and therapeutic target of NAFLD progression. 14-weeks old male C57BL/6N mice were fed with either regular diet or a newly designed NASH-inducing diet for 12 weeks. Hepatic gene expression levels were measured thereafter.