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The liver is the sixth most common site of primary cancer in humans and the fourth leading cause of cancer-related death in the world. Hepatocellular carcinoma (HCC) accounts for 90% of liver cancers. HCC is a prevalent disease with a progression that is modulated by the immune system. Half of the patients with HCC receive systemic therapies, traditionally sorafenib or lenvatinib, as a first-line therapy. In the last few years, immune-checkpoint inhibitors (ICIs) have revolutionized cancer therapy and have gained an increased interest in the treatment of HCC. In 2020, the combination of atezolizumab (anti-programmed death-ligand 1) and bevacizumab (anti-vascular endothelial growth factor) improved overall survival over sorafenib, resulting in Food and Drug Administration (FDA) approval as a first-line treatment for patients with advanced HCC. Despite these major advances, a better molecular and cellular characterization of the tumor microenvironment is still needed because it has a crucial role in the development and progression of HCC. Inflamed (hot) and noninflamed (cold) HCC tumors and genomic signatures have been associated with response to ICIs. However, there are no additional biomarkers to guide clinical decision-making. Other immune-targeting strategies, such as adoptive T-cell transfer, vaccination, and virotherapy, are currently under development. This review provides an overview on the HCC immune microenvironment, different cellular players, current available immunotherapies, and potential immunotherapy modalities.

Simple Summary Polyploidy, a balanced amplification of the genome is a defining feature of the liver. Up to 90% of adult hepatocytes in rodents and around 40% of those in humans are polyploid. The polyploidy of these cells depends on both the DNA content of each nucleus (nuclear ploidy) and the number of nuclei per cell (cellular ploidy). Remarkably, the liver is one of the few mammalian organs that display changes in ploidy content during normal homeostasis, regeneration and pathological conditions. Although polyploid hepatocytes were documented over a century ago, the significance of this original phenomenon in the pathophysiology of the liver remains unclear. In this review, we focused on the mechanisms regulating hepatocyte polyploidization both during liver development and under pathological conditions. We also detailed the effects of polyploidy on liver function and explored the fate and the role of the polyploid state during chronic liver diseases. Abstract Polyploidy, also known as whole-genome amplification, is a condition in which the organism has more than two basic sets of chromosomes. Polyploidy frequently arises during tissue development and repair, and in age-associated diseases, such as cancer. Its consequences are diverse and clearly different between systems. The liver is a particularly fascinating organ in that it can adapt its ploidy to the physiological and pathological context. Polyploid hepatocytes are characterized in terms of the number of nuclei per cell (cellular ploidy; mononucleate/binucleate hepatocytes) and the number of chromosome sets in each nucleus (nuclear ploidy; diploid, tetraploid, octoploid). The advantages and disadvantages of polyploidy in mammals are not fully understood. About 30% of the hepatocytes in the human liver are polyploid. In this review, we explore the mechanisms underlying the development of polyploid cells, our current understanding of the regulation of polyploidization during development and pathophysiology and its consequences for liver function. We will also provide data shedding light on the ways in which polyploid hepatocytes cope with centrosome amplification. Finally, we discuss recent discoveries highlighting the possible roles of liver polyploidy in protecting against tumor formation, or, conversely, contributing to liver tumorigenesis.

La polyploïdie (amplification du génome entier) fait référence à des organismes dont les cellules ont plus de deux jeux complets de chromosomes homologues. La polyploïdie a été observée pour la première fois chez les plantes, il y a plus d'un siècle. Il est dorénavant connu que ce processus se produit chez de nombreux eucaryotes dans diverses circonstances. Chez les mammifères, le développement de cellules polyploïdes peut contribuer à la différenciation des tissus. Il peut donc présenter un gain de fonction. Alternativement, il peut être associé au développement de différentes pathologies comme le cancer. Il existe différents mécanismes qui favorisent la genèse des cellules polyploïdes, dont la fusion cellulaire ou une division cellulaire anormale. Chez les mammifères, la polyploïdie est une des caractéristiques des cellules hépatiques. La polyploïdisation survient en effet principalement au cours du développement du parenchyme hépatique, mais également chez l'adulte, à la suite de différents stress. Des progrès récents ont permis de comprendre les mécanismes de polyploïdisation du tissu hépatique et ses conséquences fonctionnelles dans un contexte physiologique et pathologique.

BackgroundHepatocellular carcinoma (HCC) is a major global health concern, causing over 700,000 deaths annually [1]. For decades, treatment for advanced HCC was restricted to broad-acting tyrosine kinase inhibitors that conferred limited survival benefits [2]. Recently, immunotherapies have revolutionized treatment of advanced HCC with objective response rates of around 30% [3]. However, with 70% of patients remaining insensitive, there is an urgent need to identify biomarkers of resistance and response and to design novel combination therapies that restore sensitivity in the resistant patients.MethodsTo understand mechanisms of HCC immune evasion, we performed transcriptomic analysis on a collection of immune-escaped murine tumors harboring MYC overexpression and loss of p53 (MYC;p53-/-) [4]. The immune-escaped tumors could be categorized into ”immune-inflamed” and ”immune-desert” through single sample gene set enrichment analysis. We further identified an enrichment signature of Notch signaling in immune-inflamed tumors that was associated with a specific deficit of CD8+ T cells – a result validated in HCC patient samples. To test the role of Notch in immune escape, we generated a novel murine model of HCC based on MYC overexpression, activated Notch1 Intracellular Domain (MYC;NICD1), and customized expression of tumor antigens.ResultsExpression of tumor antigens in MYC;p53-/- mice led to enhanced survival and active immune surveillance/tumor clearance by antigen-specific CD8+ T cells compared to control MYC;p53-/- mice without tumor antigens. However, this survival advantage conferred by tumor antigen expression was abrogated in MYC;NICD1 mice, demonstrating that Notch activation drives immune evasion in HCC in the presence of antigen expression. Mechanistically, Notch activation in the liver led to a reduced number of tumor antigen-specific CD8+ T cells within the tumor microenvironment compared to the MYC;p53-/- mice while the presence of dendritic cells was similar. Adoptive transfer experiments have revealed a potential impairment of CD8+ T cell priming and activation. Further, Notch1-driven tumors were resistant to anti-PD1 monotherapy as well as anti-PDL1/anti-VEGFR2 and anti-PDL1/anti-TGFB1 combination therapies, indicating a multifaceted immune resistance mechanism mediated in part by VEGF and TGFB1. Resistance to anti-PDL1/anti-VEGF combination therapy has been confirmed in HCC patients.ConclusionsTogether, Notch activation promotes a multifaceted immune escape program in HCC. Additionally, it promotes resistance to currently approved immunotherapies, establishing the Notch pathway as a putative biomarker for HCC patient stratification.AcknowledgementsThis study was funded by Damon Runyon-Rachleff Innovator Award and NCI-NIH R37 Award. KEL was supported by 5T32AI078892-12.ReferencesBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018 Nov;68(6):394–424.Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A, Schwartz M, Porta C, Zeuzem S, Bolondi L, Greten TF, Galle PR, Seitz JF, Borbath I, Häussinger D, Giannaris T, Shan M, Moscovici M, Voliotis D, Bruix J; SHARP Investigators Study Group. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med. 2008 Jul 24;359(4):378–90.Finn RS, Qin S, Ikeda M, Galle PR, Ducreux M, Kim TY, Kudo M, Breder V, Merle P, Kaseb AO, Li D, Verret W, Xu DZ, Hernandez S, Liu J, Huang C, Mulla S, Wang Y, Lim HY, Zhu AX, Cheng AL; IMbrave150 Investigators. Atezolizumab plus Bevacizumab in Unresectable Hepatocellular Carcinoma. N Engl J Med. 2020 May 14;382(20):1894–1905.Ruiz de Galarreta M, Bresnahan E, Molina-Sánchez P, Lindblad KE, Maier B, Sia D, Puigvehi M, Miguela V, Casanova-Acebes M, Dhainaut M, Villacorta-Martin C, Singhi AD, Moghe A, von Felden J, Tal Grinspan L, Wang S, Kamphorst AO, Monga SP, Brown BD, Villanueva A, Llovet JM, Merad M, Lujambio A. ß-Catenin Activation Promotes Immune Escape and Resistance to Anti-PD-1 Therapy in Hepatocellular Carcinoma. Cancer Discov. 2019 Aug;9(8):1124–1141.Ethics ApprovalAll mouse experiments were approved by the ISMMS Animal Care and Use Committee (protocol number IACUC-2014–0229).

Mammalian p38α MAPK (Mitogen-Activated Protein Kinase) transduces a variety of extracellular signals that regulate cellular processes, such as inflammation, differentiation, proliferation or apoptosis. In the liver, depending of the physiopathological context, p38α acts as a negative regulator of hepatocyte proliferation as well as a promotor of inflammatory processes. However, its function during an acute injury, in adult liver, remains uncharacterized. In this study, using mice that are deficient in p38α specifically in mature hepatocytes, we unexpectedly found that lack of p38α protected against acute injury induced by CCl4 compound. We demonstrated that the hepatoprotective effect alleviated ROS accumulation and shaped the inflammatory response to promote efficient tissue repair. Mechanistically, we provided strong evidence that Ccl2/Ccl5 chemokines were crucial for a proper hepatoprotective response observed secondary to p38α ablation. Indeed, antibody blockade of Ccl2/Ccl5 was sufficient to abrogate hepatoprotection through a concomitant decrease of both inflammatory cells recruitment and antioxidative response that result ultimately in higher liver damages. Our findings suggest that targeting p38α expression and consequently orientating immune response may represent an attractive approach to favor tissue recovery after acute liver injury.

ObjectivesPolyploidy is a fascinating characteristic of liver parenchyma. Hepatocyte polyploidy depends on the DNA content of each nucleus (nuclear ploidy) and the number of nuclei per cell (cellular ploidy). Which role can be assigned to polyploidy during human hepatocellular carcinoma (HCC) development is still an open question. Here, we investigated whether a specific ploidy spectrum is associated with clinical and molecular features of HCC.DesignPloidy spectra were determined on surgically resected tissues from patients with HCC as well as healthy control tissues. To define ploidy profiles, a quantitative and qualitative in situ imaging approach was used on paraffin tissue liver sections.ResultsWe first demonstrated that polyploid hepatocytes are the major components of human liver parenchyma, polyploidy being mainly cellular (binuclear hepatocytes). Across liver lobules, polyploid hepatocytes do not exhibit a specific zonation pattern. During liver tumorigenesis, cellular ploidy is drastically reduced; binuclear polyploid hepatocytes are barely present in HCC tumours. Remarkably, nuclear ploidy is specifically amplified in HCC tumours. In fact, nuclear ploidy is amplified in HCCs harbouring a low degree of differentiation and TP53 mutations. Finally, our results demonstrated that highly polyploid tumours are associated with a poor prognosis.ConclusionsOur results underline the importance of quantification of cellular and nuclear ploidy spectra during HCC tumorigenesis.