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Intrahepatic cholangiocarcinoma (ICC) exhibits very low response rate to immune checkpoint inhibitors (ICIs) and the underlying mechanism is largely unknown. We investigate the tumour immune microenvironment (TIME) of ICCs and the underlying regulatory mechanisms with the aim of developing new target to inhibit tumour growth and improve anti-programmed cell death protein-1 (PD-1) efficacy. Tumour tissues from patients with ICC together with hydrodynamic ICC mouse models were employed to identify the key cell population in TIME of ICCs. Functional analysis and mechanism studies were performed using cell culture, conditional knockout mouse model and hydrodynamic transfection ICC model. The efficacy of single or combined therapy with anti-PD-1 antibody, gene knockout and chemical inhibitor were evaluated in vivo. Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are enriched in advanced ICCs and significantly correlated with N -methylguanosine tRNA methyltransferase METTL1. Using diverse in vivo cancer models, we demonstrate the crucial immunomodulator function of METTL1 in regulation of PMN-MDSC accumulation in TIME and ICC progression. Mechanistically, in human and in mouse are key translational targets of METTL1 that facilitate its function in promoting PMN-MDSC accumulation in TIME and ICC progression in vivo. Co-blockade of METTL1 and its downstream chemokine pathway enhances the anti-PD-1 efficacy in ICC preclinical mouse models. Our data uncover novel mechanisms underlying chemokine regulation and TIME shaping at the layer of messenger RNA translation level and provide new insights for development of efficient cancer immunotherapeutic strategies.

ObjectiveProgrammed death 1 and its ligand 1 (PD-1/PD-L1) immunotherapy is promising for late-stage lung cancer treatment, however, the response rate needs to be improved. Gut microbiota plays a crucial role in immunotherapy sensitisation and Panax ginseng has been shown to possess immunomodulatory potential. In this study, we aimed to investigate whether the combination treatment of ginseng polysaccharides (GPs) and αPD-1 monoclonal antibody (mAb) could sensitise the response by modulating gut microbiota.DesignSyngeneic mouse models were administered GPs and αPD-1 mAb, the sensitising antitumour effects of the combination therapy on gut microbiota were assessed by faecal microbiota transplantation (FMT) and 16S PacBio single-molecule real-time (SMRT) sequencing. To assess the immune-related metabolites, metabolomics analysis of the plasma samples was performed.ResultsWe found GPs increased the antitumour response to αPD-1 mAb by increasing the microbial metabolites valeric acid and decreasing L-kynurenine, as well as the ratio of Kyn/Trp, which contributed to the suppression of regulatory T cells and induction of Teff cells after combination treatment. Besides, the microbial analysis indicated that the abundance of Parabacteroides distasonis and Bacteroides vulgatus was higher in responders to anti-PD-1 blockade than non-responders in the clinic. Furthermore, the combination therapy sensitised the response to PD-1 inhibitor in the mice receiving microbes by FMT from six non-responders by reshaping the gut microbiota from non-responders towards that of responders.ConclusionOur results demonstrate that GPs combined with αPD-1 mAb may be a new strategy to sensitise non-small cell lung cancer patients to anti-PD-1 immunotherapy. The gut microbiota can be used as a novel biomarker to predict the response to anti-PD-1 immunotherapy.

ObjectiveHepatocellular carcinoma (HCC) is heterogeneous, especially in multifocal tumours, which decreases the efficacy of clinical treatments. Understanding tumour heterogeneity is critical when developing novel treatment strategies. However, a comprehensive investigation of tumour heterogeneity in HCC is lacking, and the available evidence regarding tumour heterogeneity has not led to improvements in clinical practice.DesignWe harvested 42 samples from eight HCC patients and evaluated tumour heterogeneity using whole-exome sequencing, RNA sequencing, mass spectrometry-based proteomics and metabolomics, cytometry by time-of-flight, and single-cell analysis. Immunohistochemistry and quantitative polymerase chain reactions were performed to confirm the expression levels of genes. Three independent cohorts were further used to validate the findings.ResultsTumour heterogeneity is considerable with regard to the genomes, transcriptomes, proteomes, and metabolomes of lesions and tumours. The immune status of the HCC microenvironment was relatively less heterogenous. Targeting local immunity could be a suitable intervention with balanced precision and practicability. By clustering immune cells in the HCC microenvironment, we identified three distinctive HCC subtypes with immunocompetent, immunodeficient, and immunosuppressive features. We further revealed the specific metabolic features and cytokine/chemokine expression levels of the different subtypes. Determining the expression levels of CD45 and Foxp3 using immunohistochemistry facilitated the correct classification of HCC patients and the prediction of their prognosis.ConclusionThere is comprehensive intratumoral and intertumoral heterogeneity in all dimensions of HCC. Based on the results, we propose a novel immunophenotypic classification of HCCs that facilitates prognostic prediction and may support decision making with regard to the choice of therapy.

ObjectiveOur goals were to evaluate the antitumour efficacy of Lactobacillus rhamnosus GG (LGG) in combination with immune checkpoint blockade (ICB) immunotherapies on tumour growth and to investigate the underlying mechanisms.DesignWe used murine models of colorectal cancer and melanoma to evaluate whether oral administration of LGG improves the efficacy of ICB therapies. We performed the whole genome shotgun metagenome sequencing of intestinal contents and RNA sequencing of dendritic cells (DCs). In a series of in vitro and in vivo experiments, we further defined the immunological and molecular mechanisms of LGG-mediated antitumour immunity.ResultsWe demonstrate that oral administration of live LGG augmented the antitumour activity of anti-programmed cell death 1 (PD-1) immunotherapy by increasing tumour-infiltrating DCs and T cells. Moreover, the combination treatment shifted the gut microbial community towards enrichment in Lactobacillus murinus and Bacteroides uniformis, that are known to increase DC activation and CD8+tumour recruitment. Mechanistically, treatment with live LGG alone or in combination with anti-PD-1 antibody triggered type I interferon (IFN) production in DCs, enhancing the cross-priming of antitumour CD8+ T cells. In DCs, cyclic GMP-AMP synthase (cGAS)/stimulator of IFN genes (STING) was required for IFN-β induction in response to LGG, as evidenced by the significant decrease in IFN-β levels in cGAS or STING-deficient DCs. LGG induces IFN-β production via the cGAS/STING/TANK binding kinase 1/interferon regulatory factor 7 axis in DCs.ConclusionOur findings have offered valuable insight into the molecular mechanisms of live LGG-mediated antitumour immunity and establish an empirical basis for developing oral administration of live LGG as a combination agent with ICB for cancer therapies.

Cancer is generally regarded as a localised disease, with the well-established role of the tumour microenvironment. However, the realm of cancer goes beyond the tumour microenvironment, and cancer should also be regarded as a systemic and environmental disease. The exposome (ie, the totality of exposures), which encompasses diets, supplements, smoking, alcohol, other lifestyle factors, medications, etc, likely alters the microbiome (inclusive of bacteria, viruses, archaea, fungi, parasites, etc) and immune system in various body sites and influences tumour phenotypes. The systemic metabolic/inflammatory status, which is likely influenced by exposures and intestinal physiological changes, may affect tissue microenvironment of colorectum and any other organs. Germline genomic factors can modify disease phenotypes via gene-by-environment interactions. Although challenges exist, it is crucial to advance not only basic experimental research that can analyse the effects of exposures, microorganisms and microenvironmental components on tumour evolution but also interdisciplinary human population research that can dissect the complex pathogenic roles of the exposome, microbiome and immunome. Metagenomic, metatranscriptomic and metabolomic analyses should be integrated into well-designed population research combined with advanced methodologies of artificial intelligence and molecular pathological epidemiology. Ideally, a prospective cohort study design that enables biospecimen (such as stool) collection before disease detection should be considered to address reverse causation and recall biases. Robust experimental and observational research together can provide insights into dynamic interactions between environmental exposures, microbiota, tumour and immunity during carcinogenesis processes, thereby helping us develop precision prevention and therapeutic strategies to ultimately reduce the cancer burden.

ObjectiveSolid tumours respond poorly to immune checkpoint inhibitor (ICI) therapies. One major therapeutic obstacle is the immunosuppressive tumour microenvironment (TME). Cancer-associated fibroblasts (CAFs) are a key component of the TME and negatively regulate antitumour T-cell response. Here, we aimed to uncover the mechanism underlying CAFs-mediated tumour immune evasion and to develop novel therapeutic strategies targeting CAFs for enhancing ICI efficacy in oesophageal squamous cell carcinoma (OSCC) and colorectal cancer (CRC).DesignAnti-WNT2 monoclonal antibody (mAb) was used to treat immunocompetent C57BL/6 mice bearing subcutaneously grafted mEC25 or CMT93 alone or combined with anti-programmed cell death protein 1 (PD-1), and the antitumour efficiency and immune response were assessed. CAFs-induced suppression of dendritic cell (DC)-differentiation and DC-mediated antitumour immunity were analysed by interfering with CAFs-derived WNT2, either by anti-WNT2 mAb or with short hairpin RNA-mediated knockdown. The molecular mechanism underlying CAFs-induced DC suppression was further explored by RNA-sequencing and western blot analyses.ResultsA negative correlation between WNT2+ CAFs and active CD8+ T cells was detected in primary OSCC tumours. Anti-WNT2 mAb significantly restored antitumour T-cell responses within tumours and enhanced the efficacy of anti-PD-1 by increasing active DC in both mouse OSCC and CRC syngeneic tumour models. Directly interfering with CAFs-derived WNT2 restored DC differentiation and DC-mediated antitumour T-cell responses. Mechanistic analyses further demonstrated that CAFs-secreted WNT2 suppresses the DC-mediated antitumour T-cell response via the SOCS3/p-JAK2/p-STAT3 signalling cascades.ConclusionsCAFs could suppress antitumour immunity through WNT2 secretion. Targeting WNT2 might enhance the ICI efficacy and represent a new anticancer immunotherapy.

ObjectiveIntrahepatic cholangiocarcinoma (ICC) exhibits very low response rate to immune checkpoint inhibitors (ICIs) and the underlying mechanism is largely unknown. We investigate the tumour immune microenvironment (TIME) of ICCs and the underlying regulatory mechanisms with the aim of developing new target to inhibit tumour growth and improve anti-programmed cell death protein-1 (PD-1) efficacy.DesignTumour tissues from patients with ICC together with hydrodynamic ICC mouse models were employed to identify the key cell population in TIME of ICCs. Functional analysis and mechanism studies were performed using cell culture, conditional knockout mouse model and hydrodynamic transfection ICC model. The efficacy of single or combined therapy with anti-PD-1 antibody, gene knockout and chemical inhibitor were evaluated in vivo.ResultsPolymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are enriched in advanced ICCs and significantly correlated with N7-methylguanosine tRNA methyltransferase METTL1. Using diverse in vivo cancer models, we demonstrate the crucial immunomodulator function of METTL1 in regulation of PMN-MDSC accumulation in TIME and ICC progression. Mechanistically, CXCL8 in human and Cxcl5 in mouse are key translational targets of METTL1 that facilitate its function in promoting PMN-MDSC accumulation in TIME and ICC progression in vivo. Co-blockade of METTL1 and its downstream chemokine pathway enhances the anti-PD-1 efficacy in ICC preclinical mouse models.ConclusionsOur data uncover novel mechanisms underlying chemokine regulation and TIME shaping at the layer of messenger RNA translation level and provide new insights for development of efficient cancer immunotherapeutic strategies.

ObjectiveIn this study, we determined whether Helicobacter pylori (H. pylori) infection dampens the efficacy of cancer immunotherapies.DesignUsing mouse models, we evaluated whether immune checkpoint inhibitors or vaccine-based immunotherapies are effective in reducing tumour volumes of H. pylori-infected mice. In humans, we evaluated the correlation between H. pylori seropositivity and the efficacy of the programmed cell death protein 1 (PD-1) blockade therapy in patients with non-small-cell lung cancer (NSCLC).ResultsIn mice engrafted with MC38 colon adenocarcinoma or B16-OVA melanoma cells, the tumour volumes of non-infected mice undergoing anticytotoxic T-lymphocyte-associated protein 4 and/or programmed death ligand 1 or anti-cancer vaccine treatments were significantly smaller than those of infected mice. We observed a decreased number and activation status of tumour-specific CD8+ T cells in the tumours of infected mice treated with cancer immunotherapies independent of the gut microbiome composition. Additionally, by performing an in vitro co-culture assay, we observed that dendritic cells of infected mice promote lower tumour-specific CD8+ T cell proliferation. We performed retrospective human clinical studies in two independent cohorts. In the Dijon cohort, H. pylori seropositivity was found to be associated with a decreased NSCLC patient survival on anti-PD-1 therapy. The survival median for H. pylori seropositive patients was 6.7 months compared with 15.4 months for seronegative patients (p=0.001). Additionally, in the Montreal cohort, H. pylori seropositivity was found to be associated with an apparent decrease of NSCLC patient progression-free survival on anti-PD-1 therapy.ConclusionOur study unveils for the first time that the stomach microbiota affects the response to cancer immunotherapies and that H. pylori serology would be a powerful tool to personalize cancer immunotherapy treatment.

ObjectivePatients with increased PD-L1+ host cells in tumours are more potent to benefit from antiprogrammed death-1/programmed death ligand-1 (PD-L1) treatment, but the underlying mechanism is still unclear. We aim to elucidate the nature, regulation and functional relevance of PD-L1+ host cells in hepatocellular carcinoma (HCC).DesignA total of untreated 184 HCC patients was enrolled randomly. C57BL/6 mice are given injection of Hepa1-6 cells to form autologous hepatoma. ELISpot, flow cytometry and real-time PCR are applied to analyse the phenotypic characteristics of PD-L1+ cells isolated directly from HCC specimens paired with blood samples or generated from ex vivo and in vitro culture systems. Immunofluorescence and immunohistochemistry are performed to detect the presence of immune cells on paraffin-embedded and formalin-fixed samples. The underlying regulatory mechanisms of metabolic switching are assessed by both in vitro and in vivo studies.ResultsWe demonstrate that PD-L1+ host macrophages, which constructively represent the major cellular source of PD-L1 in HCC tumours, display an HLA-DRhighCD86high glycolytic phenotype, significantly produce antitumourigenic IL-12p70 and are polarised by intrinsic glycolytic metabolism. Mechanistically, a key glycolytic enzyme PKM2 triggered by hepatoma cell derived fibronectin 1, via a HIF-1α-dependent manner, concurrently controls the antitumourigenic properties and inflammation-mediated PD-L1 expression in glycolytic macrophages. Importantly, although increased PKM2+ glycolytic macrophages predict poor prognosis of patients, blocking PD-L1 on these cells eliminates PD-L1-dominant immunosuppression and liberates intrinsic antitumourigenic properties.ConclusionsSelectively modulating the ‘context’ of glycolytic macrophages in HCC tumours might restore their antitumourigenic properties and provide a precise strategy for anticancer therapy.

ObjectivePancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant desmoplastic stroma composed of cancer-associated fibroblasts (CAF) and interspersed immune cells. A non-canonical CD8+ T-cell subpopulation producing IL-17A (Tc17) promotes autoimmunity and has been identified in tumours. Here, we evaluated the Tc17 role in PDAC.DesignInfiltration of Tc17 cells in PDAC tissue was correlated with patient overall survival and tumour stage. Wild-type (WT) or Il17ra-/- quiescent pancreatic stellate cells (qPSC) were exposed to conditional media obtained from Tc17 cells (Tc17-CM); moreover, co-culture of Tc17-CM-induced inflammatory (i)CAF (Tc17-iCAF) with tumour cells was performed. IL-17A/F-, IL-17RA-, RAG1-deficient and Foxn1nu/nu mice were used to study the Tc17 role in subcutaneous and orthotopic PDAC mouse models.ResultsIncreased abundance of Tc17 cells highly correlated with reduced survival and advanced tumour stage in PDAC. Tc17-CM induced iCAF differentiation as assessed by the expression of iCAF-associated genes via synergism of IL-17A and TNF. Accordingly, IL-17RA controlled the responsiveness of qPSC to Tc17-CM. Pancreatic tumour cells co-cultured with Tc17-iCAF displayed enhanced proliferation and increased expression of genes implicated in proliferation, metabolism and protection from apoptosis. Tc17-iCAF accelerated growth of mouse and human tumours in Rag1-/- and Foxn1nu/nu mice, respectively. Finally, Il17ra-expressed by fibroblasts was required for Tc17-driven tumour growth in vivo.ConclusionsWe identified Tc17 as a novel protumourigenic CD8+ T-cell subtype in PDAC, which accelerated tumour growth via IL-17RA-dependent stroma modification. We described a crosstalk between three cell types, Tc17, fibroblasts and tumour cells, promoting PDAC progression, which resulted in poor prognosis for patients.