Multi-model analysis of gallbladder cancer reveals the role of OxLDL-absorbing neutrophils in promoting liver invasion.

BACKGROUND: Gallbladder cancer (GBC) is the most common and lethal malignancy of the biliary tract that lacks effective therapy. In many GBC cases, infiltration into adjacent organs or distant metastasis happened long before the diagnosis, especially the direct liver invasion, which is the most common and unfavorable way of spreading. METHODS: Single-cell RNA sequencing (scRNA-seq), spatial transcriptomics (ST), proteomics, and multiplexed immunohistochemistry (mIHC) were performed on GBC across multiple tumor stages to characterize the tumor microenvironment (TME), focusing specifically on the preferential enrichment of neutrophils in GBC liver invasion (GBC-LI). RESULTS: Multi-model Analysis reveals the immunosuppressive TME of GBC-LI that was characterized by the enrichment of neutrophils at the invasive front. We identified the context-dependent transcriptional states of neutrophils, with the Tumor-Modifying state being associated with oxidized low-density lipoprotein (oxLDL) metabolism. In vitro assays showed that the direct cell-cell contact between GBC cells and neutrophils led to the drastic increase in oxLDL uptake of neutrophils, which was primarily mediated by the elevated OLR1 on neutrophils. The oxLDL-absorbing neutrophils displayed a higher potential to promote tumor invasion while demonstrating lower cancer cytotoxicity. Finally, we identified a neutrophil-promoting niche at the invasive front of GBC-LI that constituted of KRT17+ GBC cells, neutrophils, and surrounding fibroblasts, which may help cultivate the oxLDL-absorbing neutrophils. CONCLUSIONS: Our study reveals the existence of a subset of pro-tumoral neutrophils with a unique ability to absorb oxLDL via OLR1, a phenomenon induced through cell-cell contact with KRT17+ GBC cells in GBC-LI.

A blueprint for tumor-infiltrating B cells across human cancers.

B lymphocytes are essential mediators of humoral immunity and play multiple roles in human cancer. To decode the functions of tumor-infiltrating B cells, we generated a B cell blueprint encompassing single-cell transcriptome, B cell-receptor repertoire, and chromatin accessibility data across 20 different cancer types (477 samples, 269 patients). B cells harbored extraordinary heterogeneity and comprised 15 subsets, which could be grouped into two independent developmental paths (extrafollicular versus germinal center). Tumor types grouped into the extrafollicular pathway were linked with worse clinical outcomes and resistance to immunotherapy. The dysfunctional extrafollicular program was associated with glutamine-derived metabolites through epigenetic-metabolic cross-talk, which promoted a T cell-driven immunosuppressive program. These data suggest an intratumor B cell balance between extrafollicular and germinal-center responses and suggest that humoral immunity could possibly be harnessed for B cell-targeting immunotherapy.

Neutrophil profiling illuminates anti-tumor antigen-presenting potency.

Neutrophils, the most abundant and efficient defenders against pathogens, exert opposing functions across cancer types. However, given their short half-life, it remains challenging to explore how neutrophils adopt specific fates in cancer. Here, we generated and integrated single-cell neutrophil transcriptomes from 17 cancer types (225 samples from 143 patients). Neutrophils exhibited extraordinary complexity, with 10 distinct states including inflammation, angiogenesis, and antigen presentation. Notably, the antigen-presenting program was associated with favorable survival in most cancers and could be evoked by leucine metabolism and subsequent histone H3K27ac modification. These neutrophils could further invoke both (neo)antigen-specific and antigen-independent T cell responses. Neutrophil delivery or a leucine diet fine-tuned the immune balance to enhance anti-PD-1 therapy in various murine cancer models. In summary, these data not only indicate the neutrophil divergence across cancers but also suggest therapeutic opportunities such as antigen-presenting neutrophil delivery.

Single-Cell Profiling of Bone Marrow B Cells and Early B Cell Developmental Disorders Associated With Systemic Lupus Erythematosus.

OBJECTIVE: The peripheral B cell compartment is heavily disturbed in systemic lupus erythematosus (SLE), but whether B cells develop aberrantly in the bone marrow (BM) is largely unknown. METHODS: We performed single-cell RNA/B cell receptor (BCR) sequencing and immune profiling of BM B cells and classified patients with SLE into two groups: early B cell (Pro-B and Pre-B) normal (EBnor) and EB defective/low (EBlo) groups. RESULTS: The SLE-EBlo group exhibited more severe disease activity and proinflammatory status, overaction of type I interferon signaling and metabolic pathways within the B cell compartment, and aberrant BCR repertoires compared with the SLE-EBnor group. Moreover, in one patient with SLE who was initially classified in the SLE-EBlo group, early B cell deficiency and associated abnormalities were largely rectified in a second BM sample at the remission phase. CONCLUSION: In summary, this study suggests that early B cell loss in BM defines a unique pathological state in a subset of patients with SLE that may play an active role in the dysregulated autoimmune responses.

Spatial immunophenotypes predict clinical outcome in intrahepatic cholangiocarcinoma.

Background & Aims: Intrahepatic cholangiocarcinoma (iCCA) is a severe malignant tumour that shows only modest responses to immunotherapy. We aimed to identify the spatial immunophenotypes of iCCA and delineate potential immune escape mechanisms. Method: Multiplex immunohistochemistry (mIHC) was performed to quantitatively evaluate the distribution of 16 immune cell subsets in intratumour, invasive margin and peritumour areas in a cohort of 192 treatment-naïve patients with iCCA. Multiregion unsupervised clustering was used to determine three spatial immunophenotypes, and multiomics analyses were carried out to explore functional differences.Results: iCCA displayed a region-specific distribution of immune cell subsets with abundant CD15+ neutrophil infiltration in intratumour areas. Three spatial immunophenotypes encompassing inflamed (35%), excluded (35%) and ignored (30%) phenotypes were identified. The inflamed phenotype showed characteristics of abundant immune cell infiltration in intratumour areas, increased PD-L1 expression and relatively favourable overall survival. The excluded phenotype with a moderate prognosis was characterized by immune cell infiltration restricted to the invasive margin or peritumour areas and upregulation of activated hepatic stellate cells, extracellular matrix and Notch signalling pathways. The ignored phenotype, with scarce immune cell infiltration across all subregions, was associated with MAPK signalling pathway elevation and a poor prognosis. The excluded and ignored phenotypes, constituting non-inflamed phenotypes, shared features of an increased angiogenesis score, TGF-ß and Wnt-ß catenin pathway upregulation and were enriched for BAP1 mutations and FGFR2 fusions. Conclusion: We identified three spatial immunophenotypes with different overall prognoses in iCCA. Tailored therapies based on the distinct immune evasion mechanisms of the spatial immunophenotypes are needed. Impact and implications: The contribution of immune cell infiltration in the invasive margin and peritumour areas has been proved. We explored the multiregional immune contexture of 192 patients to identify three spatial immunophenotypes in intrahepatic cholangiocarcinoma (iCCA). By integrating genomic and transcriptomic data, phenotype-specific biological behaviours and potential immune escape mechanisms were analysed. Our findings provide a rationale to develop personalized therapies for iCCA.

Elevated Mast Cell Abundance Is Associated with Enrichment of CCR2+ Cytotoxic T Cells and Favorable Prognosis in Lung Adenocarcinoma.

Mast cells constitute indispensable immunoregulatory sentinel cells in the tumor microenvironment. A better understanding of the regulation and functions of mast cells in lung adenocarcinoma (LUAD) could uncover therapeutic approaches to reprogram the immunosuppressive tumor microenvironment. Here, we performed flow cytometry and single-cell RNA sequencing (scRNA-seq) of patient LUAD samples to comprehensively characterize LUAD-infiltrating mast cells. Mast cells exhibited functional heterogeneity and were enriched in LUAD with ground-glass opacity features (gLUAD). The mast cells in gLUAD exhibited proinflammatory and chemotactic properties while those in radiologically solid LUAD (sLUAD) were associated with tumor angiogenesis. Mast cells were an important source of CCL2 and correlated with the recruitment of CCR2+ CTL, a specific subcluster of preexhausted T cells with tissue-resident memory phenotype and enhanced cytotoxicity. Increased infiltration of mast cells and CCR2+ CTLs and their colocalization showed a strong association with favorable prognosis after surgery but were not associated with improved survival after chemotherapy. Collectively, these findings reveal a key role of mast cells in LUAD and their potential cross-talk with CTLs, suggesting that targeting mast cells may be an immunotherapeutic strategy for LUAD. SIGNIFICANCE: Comprehensive characterization of mast cells in lung adenocarcinoma elucidates their heterogeneity and identifies interplay between mast cells and CCR2+ T cells that is associated with a favorable prognosis.

Lactylome analysis suggests lactylation-dependent mechanisms of metabolic adaptation in hepatocellular carcinoma.

Enhanced glycolysis and accumulation of lactate is a common feature in various types of cancer. Intracellular lactate drives a recently described type of posttranslational modification, lysine lactylation (Kla), on core histones. However, the impact of lactylation on biological processes of tumour cells remains largely unknown. Here we show a global lactylome profiling on a prospectively collected hepatitis B virus-related hepatocellular carcinoma (HCC) cohort. Integrative lactylome and proteome analysis of the tumours and adjacent livers identifies 9,275 Kla sites, with 9,256 sites on non-histone proteins, indicating that Kla is a prevalent modification beyond histone proteins and transcriptional regulation. Notably, Kla preferentially affects enzymes involved in metabolic pathways, including the tricarboxylic acid cycle, and carbohydrate, amino acid, fatty acid and nucleotide metabolism. We further verify that lactylation at K28 inhibits the function of adenylate kinase 2, facilitating the proliferation and metastasis of HCC cells. Our study therefore reveals that Kla plays an important role in regulating cellular metabolism and may contribute to HCC progression.

Single-cell profiling reveals mechanisms of uncontrolled inflammation and glycolysis in decidual stromal cell subtypes in recurrent miscarriage.

STUDY QUESTION: Do distinct subpopulations of decidual stromal cells (DSCs) exist and if so, are given subpopulations enriched in recurrent miscarriage (RM)? SUMMARY ANSWER: Three subpopulations of DSCs were identified from which inflammatory DSCs (iDSCs) and glycolytic DSCs (glyDSCs) are significantly enriched in RM, with implicated roles in driving decidual inflammation and immune dysregulation. WHAT IS KNOWN ALREADY: DSCs play crucial roles in establishing and maintaining a successful pregnancy; dysfunction of DSCs has been considered as one of the key reasons for the development of RM. STUDY DESIGN, SIZE, DURATION: We collected 15 early decidual samples from five healthy donors (HDs) and ten RM patients to perform single-cell RNA sequencing (scRNA-seq). A total of 43 RM patients and 37 HDs were enrolled in the validation cohort. PARTICIPANTS/MATERIALS, SETTING, METHODS: Non-immune cells and immune cells of decidual tissues were sorted by flow cytometry to perform scRNA-seq. We used tissue microarrays (TMA) to validate three distinct subpopulations of DSCs. The expression of inflammatory and glycolytic proteins by DSCs was validated by immunohistochemistry (IHC) and multiplex immunohistochemistry (mIHC). Different subsets of decidual NK (dNK) cells and macrophages were also validated by multicolor flow cytometry and mIHC. Cell ligand-receptor and spatial analyses between DSCs and immune cells were analyzed by mIHC. MAIN RESULTS AND THE ROLE OF CHANCE: We classify the DSCs into three subtypes based on scRNA-seq data: myofibroblastic (myDSCs), inflammatory (iDSCs) and glycolytic (glyDSCs), with the latter two being significantly enriched in RM patients. The distribution patterns of DSC subtypes in the RM and HD groups were validated by mIHC. Single-cell analyses indicate that the differentiation of iDSCs and glyDSCs may be coupled with the degrees of hypoxia. Consequently, we propose a pathological model in which a vicious circle is formed and fueled by hypoxic stress, uncontrolled inflammation and aberrant glycolysis. Furthermore, our results show that the inflammatory SPP1+ macrophages and CD18+ dNK cells are preferentially increased in the decidua of RM patients. Cell ligand-receptor and mIHC spatial analyses uncovered close interactions between pathogenic DSCs and inflammatory SPP1+ macrophages and CD18+ NK cells in RM patients. LARGE SCALE DATA: The raw single-cell sequence data reported in this paper were deposited at the National Omics Data Encyclopedia (www.biosino.org), under the accession number OEP002901. LIMITATIONS, REASONS FOR CAUTION: The number of decidual samples for scRNA-seq was limited and in-depth functional studies on DSCs are warranted in future studies. WIDER IMPLICATIONS OF THE FINDINGS: Identification of three DSC subpopulations opens new avenues for further investigation of their roles in RM patients. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Strategic Priority Research Program (No. XDB29030302), Frontier Science Key Research Project (QYZDB-SSW-SMC036), Chinese Academy of Sciences; National Key Research and Development Program of China (2021YFE0200600), National Natural Science Foundation of China (No. 31770960), Shanghai Municipal Science and Technology Major Project (No. 2019SHZDZX02, HS2021SHZX001), and Shanghai Committee of Science and Technology (17411967800). All authors report no conflict of interest.

Single-cell profiling reveals distinct adaptive immune hallmarks in MDA5+ dermatomyositis with therapeutic implications.

Anti-melanoma differentiation-associated gene 5-positive dermatomyositis (MDA5+ DM) is an autoimmune condition associated with rapidly progressive interstitial lung disease and high mortality. The aetiology and pathogenesis of MDA5+ DM are still largely unknown. Here we describe the immune signatures of MDA5+ DM via single-cell RNA sequencing, flow cytometry and multiplex immunohistochemistry in peripheral B and T cells and in affected lung tissue samples from one patient. We find strong peripheral antibody-secreting cell and CD8+ T cell responses as cellular immune hallmarks, and over-stimulated type I interferon signaling and associated metabolic reprogramming as molecular immune signature in MDA5+ DM. High frequency of circulating ISG15+ CD8+ T cells at baseline predicts poor one-year survival in MDA5+ DM patients. In affected lungs, we find profuse immune cells infiltration, which likely contributes to the pro-fibrotic response via type I interferon production. The importance of type I interferons in MDA5+ DM pathology is further emphasized by our observation in a retrospective cohort of MDA5+ DM patients that combined calcineurin and Janus kinase inhibitor therapy show superior efficacy to calcineurin inhibitor monotherapy. In summary, this study reveals key immune-pathogenic features of MDA5+ DM and provides a potential basis for future tailored therapies.

Multi-omics characterization reveals the pathogenesis of liver focal nodular hyperplasia.

The molecular landscape and pathogenesis of focal nodular hyperplasia (FNH) have yet to be elucidated. We performed multi-omics approaches on FNH and paired normal liver tissues from 22 patients, followed by multi-level bioinformatic analyses and experimental validations. Generally, FNH had low mutation burden with low variant allele frequencies, and the mutation frequency significantly correlated with proliferation rate. Although no recurrently deleterious genomic events were found, some putative tumor suppressors or oncogenes were involved. Mutational signatures indicated potential impaired mismatch function and possible poison contact. Integrated analyses unveiled a group of FNH specific endothelial cells that uniquely expressed SOST and probably had strong interaction with fibroblasts through PDGFB/PDGFRB pathway to promote fibrosis. Notably, in one atypical FNH (patient No.11) with pronounced copy number variations, we observed a unique immune module. Most FNH are benign, but molecularly atypical FNH still exist; endothelial cell derived PDGFB probably promotes the fibrogenic process in FNH.

Multimodule characterization of immune subgroups in intrahepatic cholangiocarcinoma reveals distinct therapeutic vulnerabilities.

BACKGROUND: Immune microenvironment is well recognized as a critical regulator across cancer types, despite its complex roles in different disease conditions. Intrahepatic cholangiocarcinoma (iCCA) is characterized by a tumor-reactive milieu, emphasizing a deep insight into its immunogenomic profile to provide prognostic and therapeutic implications. METHODS: We performed genomic, transcriptomic, and proteomic characterization of 255 paired iCCA and adjacent liver tissues. We validated our findings through H&E staining (n=177), multiplex immunostaining (n=188), single-cell RNA sequencing (scRNA-seq) (n=10), in vitro functional studies, and in vivo transposon-based mouse models. RESULTS: Integrated multimodule data identified three immune subgroups with distinct clinical, genetic, and molecular features, designated as IG1 (immune-suppressive, 25.1%), IG2 (immune-exclusion, 42.7%), and IG3 (immune-activated, 32.2%). IG1 was characterized by excessive infiltration of neutrophils and immature dendritic cells (DCs). The hallmark of IG2 was the relatively higher tumor-proliferative activity and tumor purity. IG3 exhibited an enrichment of adaptive immune cells, natural killer cells, and activated DCs. These immune subgroups were significantly associated with prognosis and validated in two independent cohorts. Tumors with KRAS mutations were enriched in IG1 and associated with myeloid inflammation-dominated immunosuppression. Although tumor mutation burden was relatively higher in IG2, loss of heterozygosity in human leucocyte antigen and defects in antigen presentation undermined the recognition of neoantigens, contributing to immune-exclusion behavior. Pathological analysis confirmed that tumor-infiltrating lymphocytes and tertiary lymphoid structures were both predominant in IG3. Hepatitis B virus (HBV)-related samples tended to be under-represented in IG1, and scRNA-seq analyses implied that HBV infection indeed alleviated myeloid inflammation and reinvigorated antitumor immunity. CONCLUSIONS: Our study elucidates that the immunogenomic traits of iCCA are intrinsically heterogeneous among patients, posing great challenge and opportunity for the application of personalized immunotherapy.

Geospatial Immune Heterogeneity Reflects the Diverse Tumor-Immune Interactions in Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) exhibits extensive intratumoral heterogeneity and an extremely high mortality rate. Here, we performed whole-exome sequencing, RNA sequencing, T-cell receptor (TCR) sequencing, and multiplexed immunofluorescence on 207 tumor regions from 45 patients with iCCA. Over half of iCCA displayed intratumoral heterogeneity of immune infiltration, and iCCA were classified into sparsely, heterogeneously, and highly infiltrated subgroups with distinct immunogenomic characteristics. Sparsely infiltrated tumors displayed active copy-number loss of clonal neoantigens, and heterogeneous immune infiltration played an important role in the subclonal evolution across tumor subregions. Highly infiltrated tumors were characterized by extensive immune activation and a similar TCR repertoire across tumor subregions, but counteracted with T-cell exhaustion and pervasive antigen presentation defects. Notably, FGFR2 mutations and fusions correlated with low mutation burden and reduced immune infiltration. Our work delineated the dynamic tumor-immune interactions and developed a robust classification system to divide patients with iCCA into high and low immune evasion groups with different prognoses. SIGNIFICANCE: This study elucidates the impact of spatial immune heterogeneity upon tumor evolution of iCCA and reveals distinct immune evasion mechanisms developed in different immune microenvironments, which can be exploited for the development of personalized immunotherapy strategies. This article is highlighted in the In This Issue feature, p. 2221.

Two Distinct Immune Cell Signatures Predict the Clinical Outcomes in Patients With Amyopathic Dermatomyositis With Interstitial Lung Disease.

OBJECTIVE: Amyopathic dermatomyositis (ADM) is a heterogeneous and life-threatening autoimmune disease with a high mortality rate. In particular, anti-melanoma differentiation-associated protein 5 antibody-positive patients are at a high risk of developing rapidly progressive interstitial lung disease (RPILD). This study was undertaken to identify immunologic signatures among patients who have ADM with ILD (ADM-ILD) and to discover the biomarkers predicting prognosis. METHODS: The landscape of 42 immune cell phenotypes in the peripheral blood of 82 ADM-ILD patients and 82 age- and sex-matched healthy donors was assessed by multicolor flow cytometry. Patients were stratified using an unsupervised machine learning method (hierarchical clustering analysis) by immune cell subsets. Multiple Wilcoxon's signed rank tests and supervised machine learning methods were performed to identify important immune cell subsets. Kaplan-Meier survival analysis with log rank tests was used to create survival curves. RESULTS: We identified 2 distinct clusters correlating with different disease activities and clinical outcomes in ADM-ILD. Cluster 1 was enriched in the activated CD45RA+HLA-DR+CD8+ T cells with decreased CD56dim natural killer cell proportions and showed a higher prevalence of RPILD and higher mortality. In contrast, the other subgroup, cluster 2 (the nonactivated T cell-dominant cluster), displayed favorable clinical outcomes with high survival rates. Our data also revealed that immunophenotype was an independent risk factor associated with 1-year survival. CONCLUSION: Peripheral immunologic features may have the potential to stratify patients with ADM-ILD according to different disease severity and clinical outcomes, which may have implications for outcome prediction, pathogenesis study, and therapy selection.

Single-cell transcriptomic analysis suggests two molecularly subtypes of intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) is a highly heterogeneous cancer with limited understanding of its classification and tumor microenvironment. Here, by performing single-cell RNA sequencing on 144,878 cells from 14 pairs of iCCA tumors and non-tumor liver tissues, we find that S100P and SPP1 are two markers for iCCA perihilar large duct type (iCCAphl) and peripheral small duct type (iCCApps). S100P + SPP1- iCCAphl has significantly reduced levels of infiltrating CD4+ T cells, CD56+ NK cells, and increased CCL18+ macrophages and PD1+CD8+ T cells compared to S100P-SPP1 + iCCApps. The transcription factor CREB3L1 is identified to regulate the S100P expression and promote tumor cell invasion. S100P-SPP1 + iCCApps has significantly more SPP1+ macrophage infiltration, less aggressiveness and better survival than S100P + SPP1- iCCAphl. Moreover, S100P-SPP1 + iCCApps harbors tumor cells at different status of differentiation, such as ALB + hepatocyte differentiation and ID3+ stemness. Our study extends the understanding of the diversity of tumor cells in iCCA.

Spatiotemporal Immune Landscape of Colorectal Cancer Liver Metastasis at Single-Cell Level.

Liver metastasis, the leading cause of colorectal cancer mortality, exhibits a highly heterogeneous and suppressive immune microenvironment. Here, we sequenced 97 matched samples by using single-cell RNA sequencing and spatial transcriptomics. Strikingly, the metastatic microenvironment underwent remarkable spatial reprogramming of immunosuppressive cells such as MRC1 + CCL18 + M2-like macrophages. We further developed scMetabolism, a computational pipeline for quantifying single-cell metabolism, and observed that those macrophages harbored enhanced metabolic activity. Interestingly, neoadjuvant chemotherapy could block this status and restore the antitumor immune balance in responsive patients, whereas the nonresponsive patients deteriorated into a more suppressive one. Our work described the immune evolution of metastasis and uncovered the black box of how tumors respond to neoadjuvant chemotherapy. SIGNIFICANCE: We present a single-cell and spatial atlas of colorectal liver metastasis and found the highly metabolically activated MRC1 + CCL18 + M2-like macrophages in metastatic sites. Efficient neoadjuvant chemotherapy can slow down such metabolic activation, raising the possibility to target metabolism pathways in metastasis.This article is highlighted in the In This Issue feature, p. 1.

Distribution and density of tertiary lymphoid structures predict clinical outcome in intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: The prognostic value and clinical relevance of tertiary lymphoid structures (TLSs) in intrahepatic cholangiocarcinoma (iCCA) remain unclear. Thus, we aimed to investigate the prognostic value and functional involvement of TLSs in iCCA. METHODS: We retrospectively included 962 patients from 3 cancer centers across China. The TLSs at different anatomic subregions were quantified and correlated with overall survival (OS) by Cox regression and Kaplan-Meier analyses. Multiplex immunohistochemistry (mIHC) was applied to characterize the composition of TLSs in 39 iCCA samples. RESULTS: A quaternary TLS scoring system was established for the intra-tumor region (T score) and peri-tumor region (P score) respectively. T scores positively correlated with favorable prognosis (p <0.001), whereas a high P score signified worse survival (p <0.001). mIHC demonstrated that both T follicular helper and regulatory T cells were significantly increased in intra-tumoral TLSs compared to peri-tumoral counterparts (p <0.05), and regulatory T cell frequencies within intra-tumoral TLSs were positively associated with P score (p <0.05) rather than T score. Collectively, the combination of T and P scores stratified iCCAs into 4 immune classes with distinct prognoses (p <0.001) that differed in the abundance and distribution pattern of TLSs. Patients displaying an immune-active pattern had the lowest risk, with 5-year OS rates of 68.8%, whereas only 3.4% of patients with an immune-excluded pattern survived at 5 years (p <0.001). The C-index of the immune class was statistically higher than the TNM staging system (0.73 vs. 0.63, p <0.001). These results were validated in an internal and 2 external cohorts. CONCLUSIONS: The spatial distribution and abundance of TLSs significantly correlated with prognosis and provided a useful immune classification for iCCA. T follicular helper and regulatory T cells may play a critical role in determining the functional orientation of spatially different TLSs. LAY SUMMARY: Tertiary lymphoid structures (TLSs) are associated with favorable prognosis in a number of cancers. However, their role in intrahepatic cholangiocarcinoma (iCCA) remains unclear. Herein, we comprehensively evaluated the spatial distribution, abundance, and cellular composition of TLSs in iCCA, and revealed the opposite prognostic impacts of TLSs located within or outside the tumor. This difference could be mediated by the different immune cell subsets present within the spatially distinct TLSs. Based on our analysis, we were able to stratify iCCAs into 4 immune subclasses associated with varying prognoses.

Global immune characterization of HBV/HCV-related hepatocellular carcinoma identifies macrophage and T-cell subsets associated with disease progression.

Diverse immune cells in the tumor microenvironment form a complex ecosystem, but our knowledge of their heterogeneity and dynamics within hepatocellular carcinoma (HCC) still remains limited. To assess the plasticity and phenotypes of immune cells within HBV/HCV-related HCC microenvironment at single-cell level, we performed single-cell RNA sequencing on 41,698 immune cells from seven pairs of HBV/HCV-related HCC tumors and non-tumor liver tissues. We combined bio-informatic analyses, flow cytometry, and multiplex immunohistochemistry to assess the heterogeneity of different immune cell subsets in functional characteristics, transcriptional regulation, phenotypic switching, and interactions. We identified 29 immune cell subsets of myeloid cells, NK cells, and lymphocytes with unique transcriptomic profiles in HCC. A highly complex immunological network was shaped by diverse immune cell subsets that can transit among different states and mutually interact. Notably, we identified a subset of M2 macrophage with high expression of CCL18 and transcription factor CREM that was enriched in advanced HCC patients, and potentially participated in tumor progression. We also detected a new subset of activated CD8+ T cells highly expressing XCL1 that correlated with better patient survival rates. Meanwhile, distinct transcriptomic signatures, cytotoxic phenotypes, and evolution trajectory of effector CD8+ T cells from early-stage to advanced HCC were also identified. Our study provides insight into the immune microenvironment in HBV/HCV-related HCC and highlights novel macrophage and T-cell subsets that could be further exploited in future immunotherapy.

PD-1/PD-L1 expression profiles within intrahepatic cholangiocarcinoma predict clinical outcome.

OBJECTIVE: Immunotherapy targeting the programmed cell death protein-1 (PD-1)/programmed cell death protein ligand 1 (PD-L1) pathway has been observed to be efficient in several solid tumors. We aim to investigate the prognostic significance of PD-1/PD-L1 expression profile in intrahepatic cholangiocarcinoma (ICC). MATERIALS AND METHODS: We investigated the expression of PD-1, PD-L1, CD8+ T cells, and CD68+ macrophages in paired tumor and adjacent normal tissues from 322 ICC patients using tyramide signal amplification (TSA)-based multiplexed immunohistochemistry. RESULTS: We found that high proportion of tumor-infiltrating CD8+ PD-1High within CD8+ PD-1+ T cells significantly correlated with advanced TNM stage (P = 0.035). ICC patients with high proportion of CD8+ PD-1High in CD8+ PD-1+ had worse postoperative survival than low proportion patients (P = 0.0037), which was an independently prognostic factor for OS (P = 0.025,). The density of CD68+ PD-L1+ significantly and positively correlated with the density of CD8+ PD-1High (P < 0.0001, r = 0.5927). The proportion of CD68+ PD-L1+ within CD68+ ICC was the risk factor for OS and TTR but not an independently factor for prognosis. The CD68+ PD-L1+ macrophages and CD8+ PD-1High T cells may cooperatively play a role in inhibiting anti-tumor immunity. CONCLUSION: CD68+ PD-L1+ macrophages and CD8+ PD-1High T cells predict unfavorable prognosis, which could also bring new progress about immune target therapy in ICC research.

The distribution of immune cells within combined hepatocellular carcinoma and cholangiocarcinoma predicts clinical outcome.

BACKGROUND: This study aimed to investigate the clinical relevance of the immune microenvironment in patients with combined hepatocellular carcinoma and cholangiocarcinoma (cHCC-ICC). PATIENTS AND METHODS: The density of tumor-infiltrating CD3+ , CD8+ , CD163+ , and Foxp3+ immune cells, as well as Programmed cell death 1, Programmed cell death-ligand 1, and Tumor necrosis factor receptor superfamily member 4, was measured in the peritumor liver, tumor invasive margin, and intratumor subregions of 56 cHCC-ICC by immunohistochemistry. The immune index was established to stratify patients. Prognostic significance of immune cell subsets and immune indices was evaluated. RESULTS: The distribution of immune cells was highly heterogeneous among different subregions of cHCC-ICC. As compared with the hepatocellular carcinoma (HCC) component, the lower density of CD8+ T cells and higher intensity of Foxp3+ Tregs and immune checkpoints in the intrahepatic cholangiocarcinoma (ICC) component may indicate a stronger immune evasive ability of ICC. Based on clustering classification or a combination of random forest and lasso-cox, two models of immune indices were established and both were identified as independent prognostic factors for cHCC-ICC patients. The selected immune variables in the immune prognostic models derived from both HCC and ICC subregions, indicating that the prognosis of cHCC-ICC patients was a complex interaction of both components. CONCLUSIONS: The immune contexture was heterogeneous among different subregions of cHCC-ICC patients and contributed differently to patient prognosis. Immune score based on the densities of immune cells might serve as a promising prognostic predictor for cHCC-ICC patients.