Single-cell landscape of the ecosystem in early-relapse hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has high relapse and low 5-year survival rates. Single-cell profiling in relapsed HCC may aid in the design of effective anticancer therapies, including immunotherapies. We profiled the transcriptomes of ∼17,000 cells from 18 primary or early-relapse HCC cases. Early-relapse tumors have reduced levels of regulatory T cells, increased dendritic cells (DCs), and increased infiltrated CD8+ T cells, compared with primary tumors, in two independent cohorts. Remarkably, CD8+ T cells in recurrent tumors overexpressed KLRB1 (CD161) and displayed an innate-like low cytotoxic state, with low clonal expansion, unlike the classical exhausted state observed in primary HCC. The enrichment of these cells was associated with a worse prognosis. Differential gene expression and interaction analyses revealed potential immune evasion mechanisms in recurrent tumor cells that dampen DC antigen presentation and recruit innate-like CD8+ T cells. Our comprehensive picture of the HCC ecosystem provides deeper insights into immune evasion mechanisms associated with tumor relapse.

Platelet-mediated circulating tumor cell evasion from natural killer cell killing through immune checkpoint CD155-TIGIT.

BACKGROUND AND AIMS: Circulating tumor cells (CTCs) are precursors of cancer metastasis. However, how CTCs evade immunosurveillance during hematogenous dissemination remains unclear. APPROACH AND RESULTS: We identified CTC-platelet adhesions by single-cell RNA sequencing and multiplex immunofluorescence of blood samples from multiple cancer types. Clinically, CTC-platelet aggregates were associated with significantly shorter progression-free survival and overall survival in patients with HCC. In vitro, ex vivo, and in vivo assays demonstrated direct platelet adhesions gifted cancer cells with an evasive ability from NK cell killing by upregulating inhibitory checkpoint CD155 (PVR cell adhesion molecule), therefore facilitating distant metastasis. Mechanistically, CD155 was transcriptionally regulated by the FAK/JNK/c-Jun cascade in a platelet contact-dependent manner. Further competition assays and cytotoxicity experiments revealed that CD155 on CTCs inhibited NK-cell cytotoxicity only by engaging with immune receptor TIGIT, but not CD96 and DNAM1, another 2 receptors for CD155. Interrupting the CD155-TIGIT interactions with a TIGIT antibody restored NK-cell immunosurveillance on CTCs and markedly attenuated tumor metastasis. CONCLUSIONS: Our results demonstrated CTC evasion from NK-cell-mediated innate immunosurveillance mainly through immune checkpoint CD155-TIGIT, potentially offering an immunotherapeutic strategy for eradicating CTCs.

DNA methylation controlling abscisic acid catabolism responds to light to mediate strawberry fruit ripening.

Phytohormones, epigenetic regulation and environmental factors regulate fruit ripening but their interplay during strawberry fruit ripening remains to be determined. In this study, bagged strawberry fruit exhibited delayed ripening compared with fruit grown in normal light, correlating with reduced abscisic acid (ABA) accumulation. Transcription of the key ABA catabolism gene, ABA 8'-hydroxylase FaCYP707A4, was induced in bagged fruit. With light exclusion whole genome DNA methylation levels were up-regulated, corresponding to a delayed ripening process, while DNA methylation levels in the promoter of FaCYP707A4 were suppressed, correlating with increases in transcript and decreased ABA content. Experiments indicated FaCRY1, a blue light receptor repressed in bagged fruit and FaAGO4, a key protein involved in RNA-directed DNA methylation, could bind to the promoter of FaCYP707A4. The interaction between FaCRY1 and FaAGO4, and an increased enrichment of FaAGO4 directed to the FaCYP707A4 promoter in fruit grown under light suggests FaCRY1 may influence FaAGO4 to modulate the DNA methylation status of the FaCYP707A4 promoter. Furthermore, transient overexpression of FaCRY1, or an increase in FaCRY1 transcription by blue light treatment, increases the methylation level of the FaCYP707A4 promoter, while transient RNA interference of FaCRY1 displayed opposite phenotypes. These findings reveal a mechanism by which DNA methylation influences ABA catabolism, and participates in light-mediated strawberry ripening.

Simulation of future land use/cover change (LUCC) in typical watersheds of arid regions under multiple scenarios.

The rapid development of the social economy has promoted a continuous increase in the intensity and scale of land use by humans, which has seriously affected the sustainable development of the region. It is important to understand the land use/cover change (LUCC) in the arid region and its future development trends and to make reasonable planning recommendations for the sustainable development of the ecological environment. This study validates the patch-generating land use simulation (PLUS) model in a typical arid region, the Shiyang River Basin (SRB), and analyzes the applicability of the model in arid regions. On this basis, the PLUS model is combined with the scenario analysis method to design four scenarios including no policy intervention, farmland protection, ecological protection and sustainable development to analyze the dynamic changes in past and future land use in the SRB and to make corresponding planning recommendations for the development of each type of land use in the arid region. The results showed that the PLUS model had a better simulation effect in the SRB (its overall accuracy reached 0.97). Coupled models obtain better simulation results than quantitative and spatial models by comparing the mainstream models, with PLUS model that combines CA model and patch generation strategy showing better simulation results in the same category. From 1987 to 2017, the spatial centroid of each LUCC in the SRB moved to varying degrees due to a continuous increase in human activities. The spatial centroid of water bodies had the most obvious change, with a moving speed of 1.49 km/a, while the moving speed of built-up land increased year by year. The spatial centroid of farmland, built-up land and unused land all shifted toward the middle and lower plains, which is a further indication of increased human activity. Due to different government policies, the development trend of land use was also different under different scenarios. However, the four scenarios all showed that the area of built-up land will be increasing exponentially from 2017 to 2037, which would seriously affect the surrounding ecological land and have a negative impact on the local agro-ecological environment. Therefore, we proposed the following planning recommendations: (1) Land leveling work should be carried out on scattered farmland located at high altitudes and with slopes over 25°. Additionally, the land use of low-altitude areas should strictly adhere to basic farmland, increase the diversification of cropping patterns and improve the efficiency of agricultural water. (2) The relationship between ecology, farmland and cities should be reasonably coordinated and the existing idle built-up land should be efficiently used. (3) Forestland and grassland resources should be strictly protected and the ecological redline should be strictly observed. This study can provide new ideas for LUCC modeling and prediction in other parts of the world and provide a strong basis for ecological management and sustainable development in arid areas.

Detection of circulating tumour cells enables early recurrence prediction in hepatocellular carcinoma patients undergoing liver transplantation.

BACKGROUND & AIMS: Liver transplantation (LTx) is one of the most effective treatments for hepatocellular carcinoma (HCC); however, tumour recurrence after LTx often leads to poor outcomes. This study investigated the value of circulating tumour cells (CTCs) as a predictor of recurrence following LTx in patients with HCC. METHODS: This analysis included 193 patients with HCC who underwent LTx at our institute and accepted pre- and post-operative CTC detection; 38 were selected for serial CTC monitoring. The predictive value of CTCs for tumour recurrence in patients with HCC following LTx was evaluated. Single-cell whole genome sequencing was used to characterize CTCs. RESULTS: Overall, the CTC burden decreased after LTx (P < .05). Post-operative CTC count ≥ 1 per 5 mL peripheral blood was identified as a potential biomarker for predicting tumour recurrence after LTx, especially in patients with no detectable CTCs prior to LTx and negative tumour serological biomarkers. The predictive value of post-operative CTC count ≥ 1 per 5 mL blood was retained in patients who did not meet the Milan criteria, University of California San Francisco (UCSF) criteria, or Fudan criteria (all P < .05). Furthermore, post-operative serial CTC detection may be useful in post-surgical surveillance for HCC recurrence. CONCLUSIONS: CTCs may be a useful biomarker to evaluate recurrence risk following LTx in patients with HCC. Evaluation based on CTC detection may enhance the post-transplant management of HCC, and improve the therapeutic efficacy of LTx.

Establishment of a hepatocellular carcinoma patient-derived xenograft platform and its application in biomarker identification.

Using a method optimized in hepatocellular carcinoma (HCC), we established patient-derived xenograft (PDX) models with an increased take rate (42.2%) and demonstrated that FBS +10% dimethyl sulfoxide exhibited the highest tumor take rate efficacy. Among 254 HCC patients, 103 stably transplantable xenograft lines that could be serially passaged, cryopreserved and revived were established. These lines maintained the diversity of HCC and the essential features of the original specimens at the histological, transcriptome, proteomic and genomic levels. Tumor engraftment was associated with lack of encapsulation, poor tumor differentiation, large size and overexpression of cancer stem cell biomarkers, and was an independent predictor for overall survival and tumor recurrence after resection. To confirm the preclinical value of the PDX model in HCC treatment, several antitumor agents were tested in 16 selected PDX models. The results revealed a high degree of pharmacologic heterogeneity in the cohort, as well as heterogeneity to different agents in the same individual. The sorafenib responses observed between HCC patients and the corresponding PDXs were also consistent. After molecular characterization of the PDX models, we explored the predictive markers for sorafenib response and found that mitogen-activated protein kinase kinase kinase 1 (MAP3K1) might play an important role in sorafenib resistance and sorafenib response is impaired in patients with MAP3K1 downexpression. Our results indicated that PDX models could accurately reproduce patient tumors biology and could aid in the discovery of new treatments to advance in precision medicine.

Clinical Characteristics and Prognostic Factors of Patients with Intrahepatic Cholangiocarcinoma with Fever: A Propensity Score Matching Analysis.

BACKGROUND: Patients with intrahepatic cholangiocarcinoma (ICC) rarely present fever as the initial symptom. We aimed to identify clinical characteristics and prognostic factors for these feverish patients. SUBJECTS, MATERIALS, AND METHODS: This study retrospectively reviewed 31 patients with ICC with fever (≥38.0°C) treated at our hospital between January 2002 and December 2014. A propensity score was used to match patients with and without fever at a ratio of 1:2. RESULTS: Patients with ICC with fever had higher serum γ-glutamyl transferase and carcinoembryonic antigen levels, larger tumors, poorer tumor differentiation, and worse prognosis (all p < .05) than those without fever. This was supported by propensity score matching (PSM) analysis. Univariate and multivariate analyses indicated that microvascular invasion, hilar lymph node metastasis, and temperature ≥ 38.6°C were related to prognosis. Patients with ICC with fever had higher levels of leucocytes, neutrophils, neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR) in peripheral blood before and after PSM analysis. Body temperature positively correlated with leucocytes (r = 0.599, p < .001), neutrophils (r = 0.644, p < .001), NLR (r = 0.681, p < .001), and PLR (r = 0.457, p = .010). CONCLUSION: Patients with ICC with fever ≥38.0°C and ≥38.6°C had poor and extremely poor prognosis, respectively. Radical surgical treatment may improve the prognosis of patients with ICC with fever <38.6°C. However, systemic therapy (e.g., anti-inflammatory and immune therapy) may be preferable to surgery for these patients with fever ≥38.6°C. IMPLICATIONS FOR PRACTICE: Patients with intrahepatic cholangiocarcinoma (ICC) with fever (≥38.0°C) as the initial symptom are extremely rare. Because their symptoms are similar to those of liver abscess, diagnosis is challenging, and most of these patients are already at an advanced stage at the time of diagnosis. Patients with ICC with fever had different clinical characteristics and worse prognosis than those without fever. The prognosis of those with temperature <38.6°C would be improved by timely surgical intervention. Those with fever ≥38.6°C had an extremely dismal outcome, although they all received radical surgical treatment. New therapeutic strategies are needed to improve survival for patients with ICC with temperature ≥38.6°C.

Sphere-forming culture enriches liver cancer stem cells and reveals Stearoyl-CoA desaturase 1 as a potential therapeutic target.

BACKGROUNDS: The role of sphere-forming culture in enriching subpopulations with stem-cell properties in hepatocellular carcinoma (HCC) is unclear. The present study investigates its value in enriching cancer stem cells (CSCs) subpopulations and the mechanism by which HCC CSCs are maintained. METHODS: HCC cell lines and fresh primary tumor cells were cultured in serum-free and ultra-low attachment conditions to allow formation of HCC spheres. In vitro and in vivo experiments were performed to evaluate CSC characteristics. Expression levels of CSC-related genes were assessed by qRT-PCR and the correlation between sphere formation and clinical characteristics was investigated. Finally, gene expression profiling was performed to explore the molecular mechanism underlying HCC CSC maintenance. RESULTS: We found that both cell lines and primary tumor cells formed spheres. HCC spheres possessed the capacity for self-renewal, proliferation, drug resistance, and contained different subpopulations of CSCs. Of interest, 500 sphere-forming Huh7 cells or 200 primary tumor cells could generate tumors in immunodeficient animals. Sphere formation correlated with size, multiple tumors, satellite lesions, and advanced stage. Further investigation identified that the PPARα-SCD1 axis plays an important role in maintenance of the CSC properties of HCC sphere cells by promoting nuclear accumulation of ß-Catenin. Inhibition of SCD1 interfered with sphere formation, down-regulated expression of CSC-related markers, and reduced ß-Catenin nuclear accumulation. CONCLUSIONS: Sphere-forming culture can effectively enrich subpopulations with stem-cell properties, which are maintained through activation of the PPARα-SCD1 axis. Therefore, we suggest that targeting the SCD1-related CSC machinery might provide a novel insight into HCC treatment.

Application of Serum Annexin A3 in Diagnosis, Outcome Prediction and Therapeutic Response Evaluation for Patients with Hepatocellular Carcinoma.

PURPOSE: Annexin A3 (ANXA3) could induce progression of hepatocellular carcinoma (HCC) via promoting stem cell traits of CD133-positive cells. Moreover, serum ANXA3 showed preliminary diagnostic potential, however further validation was required. Meanwhile, the prognostic value of ANXA3 remained elusive. The present study aimed to validate diagnostic performance and further systematically investigate the prognostic value of serum ANXA3. METHODS: Serum ANXA3 of 368 HCC patients was determined by enzyme-linked immunosorbent assay (ELISA); 295 of these patients underwent resection and 73 underwent transcatheter arterial chemoembolization (TACE). Diagnostic performance of ANXA3 was evaluated by receiver operating characteristic (ROC) analysis, and the prognostic value was evaluated by Cox regression and Kaplan-Meier analysis. To evaluate the relationship between serum ANXA3 and circulating CD133 mRNA-positive tumor cells (CD133mRNA+ CTCs), real-time polymerase chain reaction was conducted in 69 patients who underwent resection. RESULTS: Serum ANXA3 provided greater diagnostic performance than α-fetoprotein (area under the curve [AUC] 0.869 vs. 0.782), especially in early diagnosis (AUC 0.852 vs. 0.757) and discriminating HCC from patients at risk (0.832 vs. 0.736). Pretreatment ANXA3 was an independent predictor of tumor recurrence (hazard ratio [HR] 1.87, 95% confidence interval [CI] 1.26-2.76, p = 0.002)/progression (HR 1.88, 95% CI 1.04-3.43, p = 0.038) and survival (resectable: HR 2.26, 95% CI 1.44-3.56, p = 0.001; unresectable: HR 2.08, 95% CI 1.10-4.05, p = 0.025), and retained its performance in low-recurrence-risk subgroups. Specifically, dynamic changes of ANXA3-positive status was associated with worse prognosis. ANXA3 was positively correlated with CD133mRNA+ CTCs (r = 0.601, p < 0.001). In patients with detectable CD133mRNA+ CTC, high ANXA3 was positively associated with a higher risk of recurrence and shorter overall survival. CONCLUSIONS: Serum ANXA3 shows promise as a biomarker for diagnosis, outcome prediction, and therapeutic response evaluation in patients with HCC.

PDXliver: a database of liver cancer patient derived xenograft mouse models.

BACKGROUND: Liver cancer is the second leading cause of cancer-related deaths and characterized by heterogeneity and drug resistance. Patient-derived xenograft (PDX) models have been widely used in cancer research because they reproduce the characteristics of original tumors. However, the current studies of liver cancer PDX mice are scattered and the number of available PDX models are too small to represent the heterogeneity of liver cancer patients. To improve this situation and to complement available PDX models related resources, here we constructed a comprehensive database, PDXliver, to integrate and analyze liver cancer PDX models. DESCRIPTION: Currently, PDXliver contains 116 PDX models from Chinese liver cancer patients, 51 of them were established by the in-house PDX platform and others were curated from the public literatures. These models are annotated with complete information, including clinical characteristics of patients, genome-wide expression profiles, germline variations, somatic mutations and copy number alterations. Analysis of expression subtypes and mutated genes show that PDXliver represents the diversity of human patients. Another feature of PDXliver is storing drug response data of PDX mice, which makes it possible to explore the association between molecular profiles and drug sensitivity. All data can be accessed via the Browse and Search pages. Additionally, two tools are provided to interactively visualize the omics data of selected PDXs or to compare two groups of PDXs. CONCLUSION: As far as we known, PDXliver is the first public database of liver cancer PDX models. We hope that this comprehensive resource will accelerate the utility of PDX models and facilitate liver cancer research. The PDXliver database is freely available online at: http://www.picb.ac.cn/PDXliver/.

Circulating CD14+ HLA-DR-/low myeloid-derived suppressor cells predicted early recurrence of hepatocellular carcinoma after surgery.

AIM: Myeloid-derived suppressor cells (MDSCs) play an important role in tumor progression. The aim of the present study was to investigate the prognostic value of MDSCs for early recurrence of hepatocellular carcinoma (HCC) in patients undergoing curative resection. METHODS: Myeloid-derived suppressor cells were measured by flow cytometry. The correlation between MDSCs and tumor recurrence was analyzed using a cohort of 183 patients who underwent curative resection between February 2014 and July 2015. Prognostic significance was further assessed using Kaplan-Meier survival estimates and log-rank tests. RESULTS: In vivo, CD14+ HLA-DR-/low MDSCs inhibit T cell proliferation and secretion. The frequency of CD14+ HLA-DR-/low MDSCs was significantly higher in HCC patients (3.7 ± 5.3%, n = 183) than in chronic hepatitis patients (1.4 ± 0.6%, n = 25) and healthy controls (1.1 ± 0.5%, n = 50). High frequency of MDSCs was significantly correlated with recurrence (time to recurrence) (P < 0.001) and overall survival (P = 0.034). Patients with HCC in the high MDSC group were prone to more vascular invasion (P = 0.018) and high systemic immune-inflammation index (SII) (P = 0.009) than those in the low MDSC group. Scatter-plot analyses revealed a significant positive correlation between the SII level and the frequency of MDSCs (r = 0.188, P = 0.011). Patients with HCC with a high MDSC frequency and high SII level had significantly shorter time to recurrence (P < 0.001) and overall survival (P = 0.028) than those with a low MDSC frequency and low SII. CONCLUSIONS: An increased frequency of MDSCs was correlated with early recurrence and predicted the prognosis of patients with HCC undergoing curative resection. The HCC patients with high frequency of MDSCs should be provided more advanced management and frequent monitoring.

A polymeric nanoparticle formulation of curcumin in combination with sorafenib synergistically inhibits tumor growth and metastasis in an orthotopic model of human hepatocellular carcinoma.

Curcumin, a yellow polyphenol extracted from the rhizome of turmeric root (Curcuma longa) has potent anti-cancer properties in many types of tumors with ability to reverse multidrug resistance of cancer cells. However, widespread clinical application of this agent in cancer and other diseases has been limited due to its poor aqueous solubility. The recent findings of polymeric nanoparticle formulation of curcumin (NFC) have shown the potential for circumventing the problem of poor solubility, however evidences for NFC's anti-cancer and reverse multidrug resistance properties are lacking. Here we provide models of human hepatocellular carcinoma (HCC), the most common form of primary liver cancer, in vitro and in vivo to evaluate the efficacy of NFC alone and in combination with sorafenib, a kinase inhibitor approved for treatment of HCC. Results showed that NFC not only inhibited the proliferation and invasion of HCC cell lines in vitro, but also drastically suppressed primary tumor growth and lung metastases in vivo. Moreover, in combination with sorafenib, NFC induced HCC cell apoptosis and cell cycle arrest. Mechanistically, NFC and sorafenib synergistically down-regulated the expression of MMP9 via NF-κB/p65 signaling pathway. Furthermore, the combination therapy significantly decreased the population of CD133-positive HCC cells, which have been reported as cancer initiating cells in HCC. Taken together, NanoCurcumin provides an opportunity to expand the clinical repertoire of this agent. Additional studies utilizing a combination of NanoCurcumin and sorafenib in HCC are needed for further clinical development.

Circulating stem cell-like epithelial cell adhesion molecule-positive tumor cells indicate poor prognosis of hepatocellular carcinoma after curative resection.

UNLABELLED: Epithelial cell adhesion molecule-positive (EpCAM+) hepatocellular carcinoma (HCC) cells may constitute a tumor-initiating subpopulation in tumorigenic cell lines and HCC specimens. In the present study, EpCAM+ circulating tumor cells (CTCs) were identified prospectively in HCC patients undergoing curative resection, and the prognostic significance and their stem cell-like characteristics were investigated further. Blood samples from 123 HCC patients were tested prior to resection and 1 month thereafter. CTCs were present in 66.67% of patients, and the cell count measured in 7.5 mL of blood (CTC(7.5) ) ranged between 1 and 34. Fifty-one patients had CTC(7.5) of ≥2 preoperatively, and these patients developed tumor recurrence earlier than those with CTC(7.5) of <2 CTCs (P < 0.001). A preoperative CTC(7.5) of ≥2 was an independent prognostic factor for tumor recurrence (P < 0.001). Its prognostic significance also applied to patients with alpha-fetoprotein (AFP) levels of ≤400 ng/mL or subgroups with low recurrence risk (all P < 0.05). A significant decrease of CTC-positive rates (66.67% to 28.15%, P < 0.05) and CTC(7.5) values (2.60 ± 0.43 to 1.00 ± 0.36, P < 0.05) was observed 1 month after resection. Patients with consistent CTC(7.5) <2 had lower recurrence rates than those with values consistently ≥2 (15.5% versus 87.50%, P < 0.001). EpCAM+ CTCs displayed cancer stem cell biomarkers (CD133 and ABCG2), epithelial-mesenchymal transition, Wnt pathway activation, high tumorigenic potential, and low apoptotic propensity. CONCLUSION: Stem cell-like phenotypes are observed in EpCAM+ CTCs, and a preoperative CTC(7.5) of ≥2 is a novel predictor for tumor recurrence in HCC patients after surgery, especially in patient subgroups with AFP levels of ≤400 ng/mL or low tumor recurrence risk. EpCAM+ CTCs may serve as a real-time parameter for monitoring treatment response and a therapeutic target in HCC recurrence.

Hypoxia inducible factor 2 alpha inhibits hepatocellular carcinoma growth through the transcription factor dimerization partner 3/ E2F transcription factor 1-dependent apoptotic pathway.

UNLABELLED: Hypoxia inducible factors (HIFs) are activated in many tumors and show either promoter or suppressor activity, depending on tumor cell biology and background. However, the role of HIF member HIF-2α remains unclear in hepatocellular carcinoma (HCC). Here, HIF-2α expression was measured in HCC and paired peritumoral tissues by quantitative real-time polymerase chain reaction, western blotting, and immunofluorescence assays, and the clinical significance was explored in 246 HCC patients. In cell culture, HIF-2α levels were up-regulated or down-regulated by use of expression or short hairpin RNA recombinant plasmid, respectively. Cells were analyzed by immunoblotting, chromatin immunoprecipitation coupled with microarray, coimmunoprecipitation, and immunohistochemical staining. In vivo tumor growth was analyzed in nude mice. We found that the average expression of HIF-2α was relatively low in HCC tissues, and the decreased level was associated with lower overall survival (P=0.006). High HIF-2α expression in HCC cells induced higher levels of apoptosis and expression of proapoptotic proteins and inhibited cell and tumor growth. Furthermore, HIF-2α inhibited expression of the novel target gene, transcription factor dimerization partner 3 (TFDP3). TFDP3 protein was found to bind with E2F transcription factor 1 (E2F1) and inhibit its transcriptional activity through both p53-dependent and -independent pathways. Reintroduction of TFDP3 expression reversed HIF-2α-induced apoptosis. CONCLUSIONS: Data gathered from cell lines, tumorigenicity studies, and primary HCC samples demonstrate a negative role of HIF-2α in tumors, which is mediated by the TFDP3/E2F1 pathway. Our study provides evidence supporting a possible tumor-suppressor role for HIF-2α and has uncovered a mechanism that links HIF-2α to a fundamental biological regulator, E2F1.

Integrated multi-omics profiling to dissect the spatiotemporal evolution of metastatic hepatocellular carcinoma.

Comprehensive molecular analyses of metastatic hepatocellular carcinoma (HCC) are lacking. Here, we generate multi-omic profiling of 257 primary and 176 metastatic regions from 182 HCC patients. Primary tumors rich in hypoxia signatures facilitated polyclonal dissemination. Genomic divergence between primary and metastatic HCC is high, and early dissemination is prevalent. The remarkable neoantigen intratumor heterogeneity observed in metastases is associated with decreased T cell reactivity, resulting from disruptions to neoantigen presentation. We identify somatic copy number alterations as highly selected events driving metastasis. Subclones without Wnt mutations show a stronger selective advantage for metastasis than those with Wnt mutations and are characterized by a microenvironment rich in activated fibroblasts favoring a pro-metastatic phenotype. Finally, metastases without Wnt mutations exhibit higher enrichment of immunosuppressive B cells that mediate terminal exhaustion of CD8+ T cells via HLA-E:CD94-NKG2A checkpoint axis. Collectively, our results provide a multi-dimensional dissection of the complex evolutionary process of metastasis.

High expression of Dickkopf-related protein 1 is related to lymphatic metastasis and indicates poor prognosis in intrahepatic cholangiocarcinoma patients after surgery.

BACKGROUND: Dickkopf-related protein 1 (DKK1) has been reported involved in metastasis and invasion in several tumors. This study sought to investigate the prognostic value of DKK1 in intrahepatic cholangiocarcinoma (ICC) and its role in promoting ICC metastasis. METHODS: Tissue microarrays of 138 ICC patient samples were employed to detect DKK1, vascular endothelial growth factor C (VEGF-C), and matrix metalloproteinase 9 (MMP9) expression using immunohistochemistry. The prognostic significances were assessed by Kaplan-Meier survival estimates. DKK1 expression was measured in an ICC cell line (HCCC-9810) and ICC tissues by immunofluorescence assay, quantitative real-time polymerase chain reaction, and western blot. Serum levels of DKK1 from 37 ICC patients were tested by enzyme-linked immunosorbent assay. The role of DKK1 in proliferation, migration, invasion, and gene expression regulation was assessed by DKK1 depletion using small interfering RNA. RESULTS: Multivariate analyses revealed that DKK1 was an unfavorable predictor for overall survival and time to recurrence. The prognostic significance was retained in ICC patients with low recurrence risk (P < .05). DKK1 expression was elevated in an ICC cell line, tumor samples, and patient sera. High levels of DKK1 in ICC tissues correlated with elevated MMP9, VEGF-C, and metastasis of hepatic hilar lymph nodes. DKK1 depletion caused a decrease in cell migration and invasiveness, and down-regulation of MMP9 and VEGF-C expression. CONCLUSIONS: DKK1 is a novel prognostic biomarker for ICC, and it enhances tumor cell invasion and promotes lymph node metastasis of ICC through the induction of MMP9 and VEGF-C. DKK1 may be a potential therapeutic target for ICC.

Dissecting the tumor ecosystem of liver cancers in the single-cell era.

Primary liver cancers (PLCs) are a broad class of malignancies that include HCC, intrahepatic cholangiocarcinoma, and combined hepatocellular and intrahepatic cholangiocarcinoma. PLCs are often associated with a poor prognosis due to their high relapse and low therapeutic response rates. Importantly, PLCs exist within a dynamic and complex tumor ecosystem, which includes malignant, immune, and stromal cells. It is critical to dissect the PLC tumor ecosystem to uncover the underlying mechanisms associated with tumorigenesis, relapse, and treatment resistance to facilitate the discovery of novel therapeutic targets. Single-cell and spatial multi-omics sequencing techniques offer an unprecedented opportunity to elucidate spatiotemporal interactions among heterogeneous cell types within the complex tumor ecosystem. In this review, we describe the latest advances in single-cell and spatial technologies and review their applications with respect to dissecting liver cancer tumor ecosystems.

DNA methylation mediated by RdDM pathway and demethylation affects furanone accumulation through regulation of QUINONE OXIDOREDUCTASE in strawberry.

Recently, increasing evidence suggests that DNA methylation plays a crucial role in fruit ripening. However, the role of DNA methylation in regulating specific traits, such as flavor, remains unclear. Here, we report a role of DNA methylation in affecting furanone biosynthesis in strawberry. Strawberry quinone oxidoreductase (FaQR) is a key enzyme in furanone biosynthesis. There are four FaQR homologs in strawberry cultivar 'Yuexin', and one of them, FaQR3, contributes ~50% of FaQR transcripts, indicating a major role of FaQR3 in furanone biosynthesis. Through characterization of levels of DNA methylation and FaQR3 transcript and furanone contents during fruit ripening and after the application of DNA methylation inhibitor, we found that the DNA methylation level of the FaQR3 promoter was negatively correlated with FaQR3 expression and furanone accumulation, suggesting that DNA methylation may be involved in furanone biosynthesis through adjusting FaQR3 expression, and responded to different temperatures consistently. In addition, transient expression of a gene in the RNA-directed DNA methylation (RdDM) pathway, FaAGO4, and enrichment analysis of the 24-nucleotide siRNAs suggested that DNA methylation in the FaQR3 promoter is mediated by the RdDM pathway. Transient RNA interference (RNAi) of FaDML indicated that the demethylation pathway may be involved in regulating furanone accumulation. These findings provide new insights into the role of DNA methylation and demethylation in affecting flavor quality in strawberry during fruit ripening.

Abscisic acid mediated strawberry receptacle ripening involves the interplay of multiple phytohormone signaling networks.

As a canonical non-climacteric fruit, strawberry (Fragaria spp.) ripening is mainly mediated by abscisic acid (ABA), which involves multiple other phytohormone signalings. Many details of these complex associations are not well understood. We present an coexpression network, involving ABA and other phytohormone signalings, based on weighted gene coexpression network analysis of spatiotemporally resolved transcriptome data and phenotypic changes of strawberry receptacles during development and following various treatments. This coexpression network consists of 18,998 transcripts and includes transcripts related to phytohormone signaling pathways, MADS and NAC family transcription factors and biosynthetic pathways associated with fruit quality. Members of eight phytohormone signaling pathways are predicted to participate in ripening and fruit quality attributes mediated by ABA, of which 43 transcripts were screened to consist of the hub phytohormone signalings. In addition to using several genes reported from previous studies to verify the reliability and accuracy of this network, we explored the role of two hub signalings, small auxin up-regulated RNA 1 and 2 in receptacle ripening mediated by ABA, which are also predicted to contribute to fruit quality. These results and publicly accessible datasets provide a valuable resource to elucidate ripening and quality formation mediated by ABA and involves multiple other phytohormone signalings in strawberry receptacle and serve as a model for other non-climacteric fruits.

Characteristics and Clinical Significance of T-Cell Receptor Repertoire in Hepatocellular Carcinoma.

Several studies have demonstrated that the T-cell receptor (TCR) repertoire is associated with prognosis and immune therapy response in several types of cancer. However, the comprehensive features of TCR repertoire in tumor-infiltrating and circulating T cells, as well as its clinical significance of diagnosis in hepatocellular carcinoma (HCC) patients, are still unknown. In this study, we perform paired tumor/peritumoral tissues and peripheral blood samples from 58 patients with HCC and sequenced them with high-throughput TCR to comprehensively analyze the characteristics of TCR and the clinical significance of peripheral TCR sequence. By exploring the abundance and diversity of TCR repertoires, we observe that there was a significantly higher TCR diversity in peripheral blood than in tumoral and peritumoral tissues, while tumoral and peritumoral tissues showed similar TCR diversity. A substantial difference in the usage frequencies of several Vß, Jß genes, and TCRß VJ pairings was found among three types of tissues. Moreover, we reveal that HCC patients have a unique profile of TCR repertoire in peripheral blood in contrast to healthy individuals. We further establish an HCC diagnostic model based on TCRß VJ pairing usage in peripheral blood, which yields a best-fit area under the curve (AUC) of 0.9746 ± 0.0481 (sensitivity = 0.9675 ± 0.0603, specificity = 0.9998 ± 0.0007, average of 100 repeats) in the test set. Our study describes the characteristics of tissue infiltration and circulating T-cell bank in patients with HCC and shows the potential of using circulating TCR sequence as a biomarker for the non-invasive diagnosis of patients with HCC.