HKDC1 promotes liver cancer stemness under hypoxia through stabilizing ß-catenin.

BACKGROUND AND AIMS: Hexokinases (HKs), a group of enzymes catalyzing the first step of glycolysis, have been shown to play important roles in liver metabolism and tumorigenesis. Our recent studies identified hexokinase domain containing 1 (HKDC1) as a top candidate associated with liver cancer metastasis. We aimed to compare its cell-type specificity with other HKs upregulated in liver cancer and investigate the molecular mechanisms underlying its involvement in liver cancer metastasis. APPROACH AND RESULTS: We found that, compared to HK1 and HK2, the other 2 commonly upregulated HKs in liver cancer, HKDC1 was most strongly associated with the metastasis potential of tumors and organoids derived from 2 liver cancer mouse models we previously established. RNA in situ hybridization and single-cell RNA-seq analysis revealed that HKDC1 was specifically upregulated in malignant cells in HCC and cholangiocarcinoma patient tumors, whereas HK1 and HK2 were widespread across various tumor microenvironment lineages. An unbiased metabolomic profiling demonstrated that HKDC1 overexpression in HCC cells led to metabolic alterations distinct from those from HK1 and HK2 overexpression, with HKDC1 particularly impacting the tricarboxylic acid cycle. HKDC1 was prometastatic in HCC orthotopic and tail vein injection mouse models. Molecularly, HKDC1 was induced by hypoxia and bound to glycogen synthase kinase 3ß to stabilize ß-catenin, leading to enhanced stemness of HCC cells. CONCLUSIONS: Overall, our findings underscore HKDC1 as a prometastatic HK specifically expressed in the malignant compartment of primary liver tumors, thereby providing a mechanistic basis for targeting this enzyme in advanced liver cancer.

Identification of intracellular bacteria from multiple single-cell RNA-seq platforms using CSI-Microbes.

The study of the tumor microbiome has been garnering increased attention. We developed a computational pipeline (CSI-Microbes) for identifying microbial reads from single-cell RNA sequencing (scRNA-seq) data and for analyzing differential abundance of taxa. Using a series of controlled experiments and analyses, we performed the first systematic evaluation of the efficacy of recovering microbial unique molecular identifiers by multiple scRNA-seq technologies, which identified the newer 10x chemistries (3' v3 and 5') as the best suited approach. We analyzed patient esophageal and colorectal carcinomas and found that reads from distinct genera tend to co-occur in the same host cells, testifying to possible intracellular polymicrobial interactions. Microbial reads are disproportionately abundant within myeloid cells that up-regulate proinflammatory cytokines like IL1Β and CXCL8, while infected tumor cells up-regulate antigen processing and presentation pathways. These results show that myeloid cells with bacteria engulfed are a major source of bacterial RNA within the tumor microenvironment (TME) and may inflame the TME and influence immunotherapy response.

PARP-1 selectively impairs KRAS-driven phenotypic and molecular features in intrahepatic cholangiocarcinoma.

OBJECTIVE: Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer with limited therapeutic options. KRAS mutations are among the most abundant genetic alterations in iCCA associated with poor clinical outcome and treatment response. Recent findings indicate that Poly(ADP-ribose)polymerase1 (PARP-1) is implicated in KRAS-driven cancers, but its exact role in cholangiocarcinogenesis remains undefined. DESIGN: PARP-1 inhibition was performed in patient-derived and established iCCA cells using RNAi, CRISPR/Cas9 and pharmacological inhibition in KRAS-mutant, non-mutant cells. In addition, Parp-1 knockout mice were combined with iCCA induction by hydrodynamic tail vein injection to evaluate an impact on phenotypic and molecular features of Kras-driven and Kras-wildtype iCCA. Clinical implications were confirmed in authentic human iCCA. RESULTS: PARP-1 was significantly enhanced in KRAS-mutant human iCCA. PARP-1-based interventions preferentially impaired cell viability and tumourigenicity in human KRAS-mutant cell lines. Consistently, loss of Parp-1 provoked distinct phenotype in Kras/Tp53-induced versus Akt/Nicd-induced iCCA and abolished Kras-dependent cholangiocarcinogenesis. Transcriptome analyses confirmed preferential impairment of DNA damage response pathways and replicative stress response mediated by CHK1. Consistently, inhibition of CHK1 effectively reversed PARP-1 mediated effects. Finally, Parp-1 depletion induced molecular switch of KRAS-mutant iCCA recapitulating good prognostic human iCCA patients. CONCLUSION: Our findings identify the novel prognostic and therapeutic role of PARP-1 in iCCA patients with activation of oncogenic KRAS signalling.

Heterogeneity in Liver Cancer Immune Microenvironment: Emerging Single-Cell and Spatial Perspectives.

Primary liver cancer is a solid malignancy with a high mortality rate. The success of immunotherapy has shown great promise in improving patient care and highlights a crucial need to understand the complexity of the liver tumor immune microenvironment (TIME). Recent advances in single-cell and spatial omics technologies, coupled with the development of systems biology approaches, are rapidly transforming the landscape of tumor immunology. Here we review the cellular landscape of liver TIME from single-cell and spatial perspectives. We also discuss the cellular interaction networks within the tumor cell community in regulating immune responses. We further highlight the challenges and opportunities with implications for biomarker discovery, patient stratification, and combination immunotherapies.

Constructing a Novel Moderately Modulus "Rigid-Flexible" Structure with Synergistic Reinforcement on the Carbon Fiber Surface to Enhance the Mechanical Properties of Carbon Fiber/Epoxy Composites at Elevated Temperatures.

To improve the mechanical performance of carbon fiber (CF)/epoxy composites in high-temperature environments, a moderately modulus gradient modulus interlayer was constructed at the interface phase region of composites. This involved the design of a "rigid-flexible" synergistic reinforcement structure, incorporating rigid nanoparticle GO@CNTs and a flexible polymer polynaphthyl ether nitrile ketone onto the CF surface. Notably, at 180 °C, compared to commercial CF composites, the CF-GO@CNTs-PPENK composites displayed a remarkable improvement in their mechanical characteristics (interfacial shear, interlaminar shear, flexural strength, and modulus), achieving enhancements of 173.0, 91.5, 225.7, and 376.4%, respectively. The principal reason for this the moderately modulus interface phase composed of GO@CNTs-PPENK (where GO and CNTs predominantly consist of carbon atoms with sp2-hybridized orbitals, forming highly stable C-C structures, while PPENK possesses a "twisted non-coplanar" structure), which exhibited resistance to deformation at high temperatures. Moreover, it greatly improved the mechanical interlocking, wettability, and chemical compatibility between CF and the epoxy. It also played a crucial role in balancing and buffering the modulus disparity. The interface failure behavior and reinforcement mechanisms of the CF composites were analyzed. Furthermore, validation of the presence of a moderately modulus gradient interlayer at the interface phase region of CF-GO@CNTs-PPENK composites was performed by using atomic force microscopy. This study has established a theoretical foundation for the development of high-performance CF composites for use in high-temperature fields.

Phosphate-rich cellulose beads for efficient cesium extraction from aqueous solutions: a novel approach for cellulose utilization.

In this work, phosphate-rich cellulose beads (CBPs) were first used for cesium extraction from aqueous solutions. These green, abundant, cheap, and renewable CBPs demonstrated a high adsorption capacity and fast absorption rate. Besides, the CBPs also exhibited excellent stability and recycling performance, as well as good selectivity. This study presents the promising application potential of cellulose for efficient cesium extraction from aqueous media.

Single-Cell Characterization of the Tumor Ecosystem in Liver Cancer.

Tumor heterogeneity along with the complex landscape of the tumor microenvironment create critical challenges for effective liver cancer interventions. Characterizing the tumor ecosystem at the single-cell level may provide insight into the collective behaviors of tumor cells and their interplays with stromal and immune cells. Here we introduce the experimental protocol and computational methods for the single-cell study of liver cancer, which may be essential for a mechanistic understanding of the tumor ecosystem in liver cancer and further pave the way for developing novel therapeutics.

Lineage and ecology define liver tumor evolution in response to treatment.

A tumor ecosystem constantly evolves over time in the face of immune predation or therapeutic intervention, resulting in treatment failure and tumor progression. Here, we present a single-cell transcriptome-based strategy to determine the evolution of longitudinal tumor biopsies from liver cancer patients by measuring cellular lineage and ecology. We construct a lineage and ecological score as joint dynamics of tumor cells and their microenvironments. Tumors may be classified into four main states in the lineage-ecological space, which are associated with clinical outcomes. Analysis of longitudinal samples reveals the evolutionary trajectory of tumors in response to treatment. We validate the lineage-ecology-based scoring system in predicting clinical outcomes using bulk transcriptomic data of additional cohorts of 716 liver cancer patients. Our study provides a framework for monitoring tumor evolution in response to therapeutic intervention.

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.

Spatial proximity of tumor-immune interactions predicts patient outcome in hepatocellular carcinoma.

BACKGROUND AND AIMS: The fitness and viability of a tumor ecosystem are influenced by the spatial organization of its cells. We aimed to study the structure, architecture, and cell-cell dynamics of the heterogeneous liver cancer tumor microenvironment using spatially resolved multiplexed imaging. APPROACH AND RESULTS: We performed co-detection by indexing multiplexed immunofluorescence imaging on 68 HCC biopsies from Thai patients [(Thailand Initiative in Genomics and Expression Research for Liver Cancer (TIGER-LC)] as a discovery cohort, and then validated the results in an additional 190 HCC biopsies from Chinese patients [Liver Cancer Institute (LCI)]. We segmented and annotated 117,270 and 465,632 cells from the TIGER-LC and LCI cohorts, respectively. We observed 4 patient groups of TIGER-LC (IC1, IC2, IC3, and IC4) with distinct tumor-immune cellular interaction patterns. In addition, patients from IC2 and IC4 had much better overall survival than those from IC1 and IC3. Noticeably, tumor and CD8 + T-cell interactions were strongly enriched in IC2, the group with the best patient outcomes. The close proximity between the tumor and CD8 + T cells was a strong predictor of patient outcome in both the TIGER-LC and the LCI cohorts. Bulk transcriptomic data from 51 of the 68 HCC cases were used to determine tumor-specific gene expression features of our classified subtypes. Moreover, we observed that the presence of immune spatial neighborhoods in HCC as a measure of overall immune infiltration is linked to better patient prognosis. CONCLUSIONS: Highly multiplexed imaging analysis of liver cancer reveals tumor-immune cellular heterogeneity within spatial contexts, such as tumor and CD8 + T-cell interactions, which may predict patient survival.

Injectable hydrogels embedded with chitosan nanoparticles coated with hyaluronic acid for sequential release of dual drugs.

An effective treatment for some disease, such as the model disease acute retinal necrosis (ARN), requires a combination of different drugs which should be administered at a certain interval. The precise sequential and long-term drug release are the critical questions. In this work, the as-prepared chitosan nanoparticles (CS-NPs) coated with hyaluronic acid (HA) were embedded in the aldehyde ß-cyclodextrin (ACD)/aminated hyaluronic acid (NHA) hydrogels to synthesize injectable hydrogels loaded with dual drugs named DEX-CS-NPs/GCV-Gel and HA-DEX-CS-NPs/GCV-Gel. In the first 24 h and 48 h, the releases of DEX from DEX-CS-NPs/GCV-Gel were 128.5 % and 82.8 % faster than those from HA-DEX-CS-NPs/GCV-Gel, respectively. There was no DEX released from HA-DEX-CS-NPs/GCV-Gel at the first 7 h, which has never been reported before, although some hydrogel systems loaded with different drugs release different drugs simultaneously at different rate which have been well studied. This is a good start of a precise sequence release. The composite hydrogels possessed appropriate rheology, gel time, degradation performance, and ideal cytocompatibility. The injectable hydrogel loaded with dual drugs presenting a precise sequential and long-term release has great potential in the treatment of diseases requiring combinations of drugs being released sequentially at different treating stages.

Recent insights into the pathogenesis and therapeutic targets of chronic liver diseases.

Viral hepatitis, alcohol-associated liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) are the three major causes of chronic liver diseases, which account for approximately 2 million deaths per year worldwide. The current direct-acting antiviral drugs and vaccinations have effectively reduced and ameliorated viral hepatitis infection, but there are still no effective drug treatments for ALD, NAFLD and liver cancer due to the poor understanding of their pathogenesis. To better understand the pathogenesis, the fifth Chinese American Liver Society/Society of Chinese Bioscientists in America Hepatology Division Annual Symposium, which was held virtually on 21-22 October 2022, focused on the topics related to ALD, NAFLD and liver cancer. Here, we briefly highlight the presentations that focus on the current progress in basic and translational research in ALD, NAFLD and liver cancer. The roles of non-coding RNA, autophagy, extrahepatic signalling, macrophages, etc in liver diseases are deliberated, and the application of single-cell RNA sequencing in the study of liver disease is also discussed.

Genome-wide profiling of transcription factor activity in primary liver cancer using single-cell ATAC sequencing.

Primary liver cancer (PLC) consists of two main histological subtypes; hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA). The role of transcription factors (TFs) in malignant hepatobiliary lineage commitment between HCC and iCCA remains underexplored. Here, we present genome-wide profiling of transcription regulatory elements of 16 PLC patients using single-cell assay for transposase accessible chromatin sequencing. Single-cell open chromatin profiles reflect the compositional diversity of liver cancer, identifying both malignant and microenvironment component cells. TF motif enrichment levels of 31 TFs strongly discriminate HCC from iCCA tumors. These TFs are members of the nuclear/retinoid receptor, POU, or ETS motif families. POU factors are associated with prognostic features in iCCA. Overall, nuclear receptors, ETS and POU TF motif families delineate transcription regulation between HCC and iCCA tumors, which may be relevant to development and selection of PLC subtype-specific therapeutics.

Biomarkers for immunotherapy of hepatocellular carcinoma.

Immune-checkpoint inhibitors (ICIs) are now widely used for the treatment of patients with advanced-stage hepatocellular carcinoma (HCC). Two different ICI-containing regimens, atezolizumab plus bevacizumab and tremelimumab plus durvalumab, are now approved standard-of-care first-line therapies in this setting. However, and despite substantial improvements in survival outcomes relative to sorafenib, most patients with advanced-stage HCC do not derive durable benefit from these regimens. Advances in genome sequencing including the use of single-cell RNA sequencing (both of tumour material and blood samples), as well as immune cell identification strategies and other techniques such as radiomics and analysis of the microbiota, have created considerable potential for the identification of novel predictive biomarkers enabling the accurate selection of patients who are most likely to derive benefit from ICIs. In this Review, we summarize data on the immunology of HCC and the outcomes in patients receiving ICIs for the treatment of this disease. We then provide an overview of current biomarker use and developments in the past 5 years, including gene signatures, circulating tumour cells, high-dimensional flow cytometry, single-cell RNA sequencing as well as approaches involving the microbiome, radiomics and clinical markers. Novel concepts for further biomarker development in HCC are then discussed including biomarker-driven trials, spatial transcriptomics and integrated 'big data' analysis approaches. These concepts all have the potential to better identify patients who are most likely to benefit from ICIs and to promote the development of new treatment approaches.

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.

An injectable hydrogel based on hyaluronic acid prepared by Schiff base for long-term controlled drug release.

Drug-loaded injectable hydrogels have been studied widely in biomedical technology while the stable long-term controlled drug release and cytotoxicity are challenges. In this work, an injectable hydrogel with good swelling resistance was in situ synthetized using aminated hyaluronic acid (NHA) and aldehyde ß-cyclodextrin (ACD) via Schiff base reaction. The composition, morphology and mechanical property were characterized with FTIR, 13C NMR, SEM and rheology test, respectively. Voriconazole (VCZ) and Endophthalmitis was selected as a model drug and disease, respectively. The drug release, cytotoxicity and antifungal properties were detected in vitro. The results showed a long-term (> 60 days) drug release was realized, the NHA/ACD2/VCZ presented a zero-order release in the later stage. The cytotoxicity of NHA/ACD was detected by live/dead staining assay and Cell Counting Kit-8 (CCK-8). The survival rate of adult retina pigment epithelial cell line-19 (ARPE-19) was over 100 % after 3 d, it indicated a good cytocompatibility. The antifungal experiment presented samples had antifungal property. Biocompatibility in vivo proved NHA/ACD2 had no adverse effects on ocular tissues. Consequently, the injectable hydrogel based on hyaluronic acid prepared by Schiff base reaction provides a new option for long-term controlled drug release in the course of disease treatment from a material perspective.

Graphene Oxide/Styrene-Butadiene Latex Hybrid Aerogel with Improved Mechanical Properties by PEI Grafted GO and CNT.

Graphene oxide aerogel (GOA) has wide application prospects due to its low density and high porosity. However, the poor mechanical properties and unstable structure of GOA have limited its practical applications. In this study, polyethyleneimide (PEI) was used to graft onto the surface of GO and carbon nanotubes (CNTs) to improve compatibility with polymers. Composite GOA was prepared by adding styrene-butadiene latex (SBL) to the modified GO and CNTs. The synergistic effect of PEI and SBL, resulted in an aerogel with excellent mechanical properties, compressive resistance, and structural stability. When the ratio of SBL to GO and GO to CNTs was 2:1 and 7:3, respectively, the obtained aerogel performance was the best, and the maximum compressive stress was 784.35% higher than that of GOA. The graft of PEI on the surface of GO and CNT could improve the mechanical properties of the aerogel, with greater improvements observed with grafting onto the surface of GO. Compared with GO/CNT/SBL aerogel without PEI grafting, the maximum stress of GO/CNT-PEI/SBL aerogel increased by 5.57%, that of GO-PEI/CNT/SBL aerogel increased by 20.25%, and that of GO-PEI/CNT-PEI/SBL aerogel increased by 28.99%. This work not only provided a possibility for the practical application of aerogel, but also steered the research of GOA in a new direction.

Tumor biology and immune infiltration define primary liver cancer subsets linked to overall survival after immunotherapy.

Primary liver cancer is a rising cause of cancer deaths in the US. Although immunotherapy with immune checkpoint inhibitors induces a potent response in a subset of patients, response rates vary among individuals. Predicting which patients will respond to immune checkpoint inhibitors is of great interest in the field. In a retrospective arm of the National Cancer Institute Cancers of the Liver: Accelerating Research of Immunotherapy by a Transdisciplinary Network (NCI-CLARITY) study, we use archived formalin-fixed, paraffin-embedded samples to profile the transcriptome and genomic alterations among 86 hepatocellular carcinoma and cholangiocarcinoma patients prior to and following immune checkpoint inhibitor treatment. Using supervised and unsupervised approaches, we identify stable molecular subtypes linked to overall survival and distinguished by two axes of aggressive tumor biology and microenvironmental features. Moreover, molecular responses to immune checkpoint inhibitor treatment differ between subtypes. Thus, patients with heterogeneous liver cancer may be stratified by molecular status indicative of treatment response to immune checkpoint inhibitors.

Immunology and immunotherapy of cholangiocarcinoma.

Cholangiocarcinoma is the second most common primary liver cancer. Its incidence is low in the Western world but is rising globally. Surgery, chemotherapy and radiation therapy have been the only treatment options for decades. Progress in our molecular understanding of the disease and the identification of druggable targets, such as IDH1 mutations and FGFR2 fusions, has provided new treatment options. Immunotherapy has emerged as a potent strategy for many different types of cancer and has shown efficacy in combination with chemotherapy for cholangiocarcinoma. In this Review, we discuss findings related to key immunological aspects of cholangiocarcinoma, including the heterogeneous landscape of immune cells within the tumour microenvironment, the immunomodulatory effect of the microbiota and IDH1 mutations, and the association of immune-related signatures and patient outcomes. We introduce findings from preclinical immunotherapy studies, discuss future immune-mediated treatment options, and provide a summary of results from clinical trials testing immune-based approaches in patients with cholangiocarcinoma. This Review provides a thorough survey of our knowledge on immune signatures and immunotherapy in cholangiocarcinoma.

Multiregional single-cell dissection of tumor and immune cells reveals stable lock-and-key features in liver cancer.

Intratumor heterogeneity may result from the evolution of tumor cells and their continuous interactions with the tumor microenvironment which collectively drives tumorigenesis. However, an appearance of cellular and molecular heterogeneity creates a challenge to define molecular features linked to tumor malignancy. Here we perform multiregional single-cell RNA sequencing (scRNA-seq) analysis of seven liver cancer patients (four hepatocellular carcinoma, HCC and three intrahepatic cholangiocarcinoma, iCCA). We identify cellular dynamics of malignant cells and their communication networks with tumor-associated immune cells, which are validated using additional scRNA-seq data of 25 HCC and 12 iCCA patients as a stable fingerprint embedded in a malignant ecosystem representing features of tumor aggressiveness. We further validate the top ligand-receptor interaction pairs (i.e., LGALS9-SLC1A5 and SPP1-PTGER4 between tumor cells and macrophages) associated with unique transcriptome in additional 542 HCC patients. Our study unveils stable molecular networks of malignant ecosystems, which may open a path for therapeutic exploration.