A pan-cancer single-cell panorama of human natural killer cells.

Natural killer (NK) cells play indispensable roles in innate immune responses against tumor progression. To depict their phenotypic and functional diversities in the tumor microenvironment, we perform integrative single-cell RNA sequencing analyses on NK cells from 716 patients with cancer, covering 24 cancer types. We observed heterogeneity in NK cell composition in a tumor-type-specific manner. Notably, we have identified a group of tumor-associated NK cells that are enriched in tumors, show impaired anti-tumor functions, and are associated with unfavorable prognosis and resistance to immunotherapy. Specific myeloid cell subpopulations, in particular LAMP3+ dendritic cells, appear to mediate the regulation of NK cell anti-tumor immunity. Our study provides insights into NK-cell-based cancer immunity and highlights potential clinical utilities of NK cell subsets as therapeutic targets.

Risk factors of secondary infection/recurrence after ablation for liver cancers: A systemic review and meta-analysis.

Background: This study aimed to systemically explore the risk factors of secondary infection/recurrence after ablation in patients with liver cancer. Methods: Relevant literature in PubMed, EMbase, and Cochrane Library databases were searched with keywords including "liver cancer or carcinoma," "ablation," "infectious or infection or recurrence," and "risk factor or relevant factor or correlative factor or influencing factor." Meta-analyses were performed and forest plots were drawn for risk factors, including the tumor size and location, number of tumor nodules, hepatitis B virus (HBV) DNA levels, serum alpha fetal protein (AFP) levels and serum albumin levels, Child-Pugh Class, and lack of antiviral therapy. A funnel plot was drawn to assess the publication bias. Results: A total of 23 studies were included from the initial 701 potentially relevant articles. Our meta-analyses showed that a large tumor size (odds ratio [OR] = 1.58; 95% confidence interval [CI]: 1.31-1.92); proximity to the colon, large vessels, and large hepatic vein (OR = 4.10; 95% CI: 2.26-7.43); multinodular tumor (OR = 2.10; 95% CI: 1.46-3.03), the higher HBV DNA levels (OR = 1.34; 95% CI: 1.09-0.64); higher serum AFP levels (OR = 1.56; 95% CI: 1.18-2.05), lower serum albumin levels (OR = 1.67; 95% CI: 1.06-2.65); Child-Pugh Class B and Class C (OR = 1.27; 95% CI: 1.05-1.54); and lack of antiviral therapy (OR = 1.75; 95% CI: 0.93-3.28) were associated with an increased risk of post-ablation infection/recurrence in patients with liver cancers. Conclusion: Our results indicated that the tumor size and location, number of tumor nodules, HBV DNA levels, serum AFP levels and serum albumin levels, Child-Pugh Class, and lack of antiviral therapy were the risk factors for post-ablation infection/recurrence in patients with liver cancer. Here, we have provided directions for the clinical prevention of secondary infection/recurrence in patients with liver cancer who underwent ablation therapy.

Dual selective sensor for exosomes in serum using magnetic imprinted polymer isolation sandwiched with aptamer/graphene oxide based FRET fluorescent ignition.

The selective and sensitive detection of cancerous exosomes in serum is critical for early disease diagnosis and improved prognosis. Previous exosome-related research has been limited by a lack of well-understanding in exosomes as well as the challenging background interference of body fluid. Molecularly imprinted polymers (MIPs) and nucleic acid aptamers can be regarded as the two alternatives to antibodies. When using imprinted polymer technology, comprehensive and precise information about the target constituents is not required. In this study, a novel kind of dual selective fluorescent nanosensor for the poorly characterized exosomes was constructed by integrating magnetic MIP selective exosome capture sandwiched with an aptamer/graphene oxide fluorescence resonance energy transfer system (FRET) based selective 'turn-on' exosome labeling heterogeneously. The overall strategy performance was successively evaluated using lysozyme and exosomes as targets. Good linearity and high sensitivity achieved were demonstrated. The LOD of exosomal detection in serum was 2.43 × 106 particles/mL, lower than other immunology based detection methods. The discrimination between serum from breast cancer patients and healthy people was also primarily studied. In conclusion, the developed sensor with outstanding selectivity, high detection sensitivity, simplicity, low cost, and wide applicability for known or unknown targets present significant potential in challenging clinical diagnosis.

Immune phenotypic linkage between colorectal cancer and liver metastasis.

The tumor microenvironment (TME) is connected to immunotherapy responses, but it remains unclear how cancer cells and host tissues differentially influence the immune composition within TME. Here, we performed single-cell analyses for autologous samples from liver metastasized colorectal cancer to disentangle factors shaping TME. By aligning CD45+ cells across different tissues, we classified exhausted CD8+ T cells (Texs) and activated regulatory T cells as M-type, whose phenotypes were associated with the malignancy, while natural killer and mucosal-associated invariant T cells were defined as N-type, whose phenotypes were associated with the niche. T cell receptor sharing between Texs in primary and metastatic tumors implicated the presence of common peripheral non-exhausted precursors. For myeloid cells, a subset of dendritic cells (DC3s) and SPP1+ macrophages were M-type, and the latter were predominant in liver metastasis, indicating its pro-metastasis role. Our analyses bridge immune phenotypes of primary and metastatic tumors, thereby helping to understand the tumor-specific contexture and identify the pro-metastasis components.

Key Candidate Prognostic Biomarkers Correlated with Immune Infiltration in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common subtype of primary liver cancer, which causes ~800,000 deaths annually world-wide. Immune checkpoint inhibitor (ICI) has reformed cancer therapy and achieved unprecedented results in various malignancies, including HCC. However, the response rate of immunotherapy is very low in HCC. Considereing the complicated and unique immune status in liver, we hypothesize that critical molecules will affect prognosis and correlate with immune context in the tumor microenvironment of HCC. METHODS: Using Kaplan-Meier plotter, GEPIA2 and Integrative Molecular Database of Hepatocellular Carcinoma (HCCDB), survival genes and their prognostic value were estimated in HCC. Based on Tumor Immune Estimation Resource (TIMER), association between survival genes and immune infiltration was examined in HCC. FunRich and STRING were used to analyze gene ontology and protein-protein interaction (PPI) Network, qRT-PCR was used to measure mRNA level of candidates; and a Cell Counting Kit-8 was used to measure proliferation of HCC cell line. RESULTS: Using multiple databases, we identified 36 key prognostic genes highly expressed in HCC and associated with poor survival of patients. Meanwhile, the 36 gene signatures correlated with immune infiltration in HCC. Moreover, these genes were significantly associated with exhausted T cells and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in HCC. Among the 36 key genes, SKA3, SGOL2, SPINDOC, TEDC2, TMCO3 and NUP205 were highly expressed in tumor samples compared with adjacent normal tissues in our HCC cohort (n=22). Additionally, proliferation of SMMC7721 cell line was inhibited when it interfered with SiRNA of each gene. CONCLUSION: The 36 genes may serve as potential prognostic biomarkers and molecular targets to ameliorate tumor immune microenvironment (TIME) in HCC and therefore represent a novel avenue for individualized immunotherapy in HCC.

DNASE1L3 as a Prognostic Biomarker Associated with Immune Cell Infiltration in Cancer.

OBJECTIVES: Deoxyribonuclease 1 like 3 (DNASE1L3) is critically involved in apoptosis and immune response, however, its role in cancer has yet to be deciphered. We aimed to explore the prognostic value of DNASE1L3 across a series of malignancies. METHODS: Based on Oncomine database and Tumor Immune Estimation Resource (TIMER), expression profiling of DNASE1L3 was detailed in malignancies. Using PrognoScan, Kaplan-Meier Plotter, GEPIA2, and bc-GenEcMiner v4.5, prognostic value of DNASE1L3 was estimated in diverse cancers. Based on TIMER, association between DNASEL13 expression and immune infiltration was examined in various cancers. Then, mRNA level of DNASE1L3 in hepatocellular carcinoma (HCC) samples (n=22) and stomach adenocarcinoma (STAD) samples (n=17) was measured with qRT-PCR. Immunohistochemistry was performed to confirm expression of DNASE1L3 in paraffin-embedded tissues of HCC (n=9) and lung adenocarcinoma (n=20). RESULTS: DNASE1L3 was downregulated in multiple cancers, including breast invasive carcinoma (BRCA), cholangiocarcinoma (CHOL), liver hepatocellular carcinoma (LIHC), and lung adenocarcinoma (LUAD). A lower level of DNASE1L3 correlated with poorer prognosis in various cancers, especially in breast, liver, kidney, stomach, lung adenocarcinoma and sarcoma (SARC). Moreover, DNASE1L3 was positively related to immune cell infiltration in many cancers, including BRCA, LIHC, STAD, LUAD, and SARC. DNASE1L3 was significantly associated with CCR7/CCL19 in cancers. DNASE1L3 was downregulated in HCC and STAD tissues as demonstrated by qRT-PCR, as well as in HCC and LUAD samples, as shown by immunohistochemistry. CONCLUSION: DNASE1L3 has potential to serve as a prognostic biomarker in cancer of the breast, kidney, liver, stomach, lung adenocarcinoma and sarcoma. Down-regulation of DNASE1L3 may participate in immune escape via CCR7/CCL19 axis.

In vitro and in vivo studies of biodegradable Zn-Li-Mn alloy staples designed for gastrointestinal anastomosis.

Zn-0.8 wt.% Li-0.1 wt.% Mn wire with the diameter of 0.3 mm was fabricated and further processed into gastrointestinal staple, and its in vitro and in vivo biodegradation behavior and biocompatibility were studied systematically. The experimental Zn-Li-Mn alloy staple could deform from the original U-shape to B-shape without fracture, indicating its good mechanical property. Due to the residual stress concentration caused by anastomosis deformation, the feet and leg arc part of the staple were more prone to degradation. The Zn-Li-Mn alloy staple sustained integrity after immersion in Hanks' solution and simulated gastric fluid (SGF) for 28 days, and the degradation rate in SGF was about 4 times of that in Hanks' solution. Furthermore, Zn-Li-Mn alloy staples were utilized for gastrointestinal anastomosis in pig models, with clinically-used titanium alloy staples as a comparison. No anastomotic leakage and severe inflammation were observed after operation. The Zn-Li-Mn alloy staple maintained mechanical integrity within 8 weeks' implantation. The gastrointestinal tissue healed after 12 weeks, and no obvious side effects were detected during the whole implantation period, demonstrating the good biocompatibility of Zn-Li-Mn alloy staple. Thus, Zn-Li-Mn alloy staple fabricated in this work displayed the promising potential in the gastrointestinal anastomosis.

Laparoscopic Sleeve Gastrectomy Affects Coagulation System of Obese Patients.

BACKGROUND: Laparoscopic sleeve gastrectomy (LSG) is nowadays the most popular bariatric procedure for obesity. However, whether LSG increases the risk of thrombosis remains unclear. The aim of this study was to investigate potential effects of LSG on coagulation system. METHODS: Fifty-five obese patients underwent LSG between 2016 and 2018. The LSG was performed with pneumoperitoneum pressure maintained at 13 mmHg. Venous blood specimens were collected from each patient before surgery, at the end of pneumoperitoneum (i.e., 0 h after surgery), and at 24 h after surgery to determine prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), platelet count (PLT), D-dimer (D-D), red blood cell count (RBC), hematocrit (HCT), plateletcrit (PCT), cholesterol (CHOL), triglyceride (TRIG), and serum calcium (Ca). All patients were examined on the veins of the lower limbs by color Duplex sonography (CDS) before surgery and at 24 h after surgery, respectively. RESULTS: All patients successfully underwent LSG. No severe surgery-related complications were observed during 1-month follow-up after operation. Preoperative BMI was 43.6 ± 8.3 kg/m2. The levels of coagulation factors were within the normal range before surgery, except a relatively higher PLT. The PT and D-D were increased at 0 h and 24 h after surgery (P < 0.05), whereas APTT was decreased (P < 0.05). The postoperative FIB remained similar to the preoperative one (P > 0.05). The CDS identified no thrombus in the veins of the lower limbs, either before surgery or at 24 h after surgery. CONCLUSIONS: LSG may cause postoperative hypercoagulability of patients with obesity.

Abdominal infectious complications associated with the dislocation of intraperitoneal part of drainage tube and poor drainage after major surgeries.

Abdominal drainage, serving as a diagnostic and therapeutic tool, has been widely applied to prevent complications after major abdominal surgical procedures. However, dislocation of intraperitoneal portion of drainage tube and poor drainage after major surgery has never been detailed. In this retrospective study, we determined whether postoperative abdominal infectious complications are attributed to dislocation of intraperitoneal portion of drainage tube. Patients were recruited from the Department of General Surgery at Beijing Shijitan Hospital, Capital Medical University, between June 2015 and June 2018. All of the enrolled patients had undergone different major abdominal surgical procedures with abdominal drainage. According to different fixation methods of the drainage tube, the patients were categorised as follows: group 1 as conventional extra-abdominal fixation where the tubes were fixed on abdominal wall; group 2 as double fixation where the tubes were fixed by both extra-abdominal and intra-abdominal fixation. Among 60 patients (40 in group 1 and 20 in group 2) with suspected postoperative abdominal infection, abdominal computed tomography (CT) was performed to determine the presence of abnormality. Dislocation of drainage tubes, morbidity, treatment, and prognosis were compared between the two groups. None of the patients showed slip knot or drainage tube slipping from the abdomen based on physical examination and CT imaging. Drainage tube was fixed firmly on the abdominal wall. In group 1, 18 (45%) patients developed postoperative complications resulting from abdominal infection where severe dislocation of intraperitoneal portion of drainage tubes was confirmed by CT. Drainage tubes of six cases were significantly dislocated to the anterior abdominal wall from the target area; 7 upper abdominal drainage tubes dislocated to the lower abdomen; and 5 lower abdominal drainage tubes dislocated to the upper abdomen. Common complications included localised peritonitis (n = 4), abdominal abscess (n = 8), and anastomotic leakage (n = 6). Among them, 8 patients were cured by abdominal puncture catheter drainage; 5 underwent secondary operation and 5 were cured by conservative treatment. In group 2, no tube dislocation was identified by CT. Five patients (25%) developed complications, including localised peritonitis (n = 1), abdominal abscess (n = 1), and anastomotic leakage (n = 3). All the five patients were cured by conservative treatment. Postoperative abdominal infection complications can stem from dislocation of intraperitoneal portion of drainage tube and poor drainage after major abdominal surgery. Maintaining the intraperitoneal portion of drainage tube at the proper location, for example, by applying intraabdominal fixation, is paramount to decrease the incidence and severity of postoperative complications.

Landscape and Dynamics of Single Immune Cells in Hepatocellular Carcinoma.

The immune microenvironment of hepatocellular carcinoma (HCC) is poorly characterized. Combining two single-cell RNA sequencing technologies, we produced transcriptomes of CD45+ immune cells for HCC patients from five immune-relevant sites: tumor, adjacent liver, hepatic lymph node (LN), blood, and ascites. A cluster of LAMP3+ dendritic cells (DCs) appeared to be the mature form of conventional DCs and possessed the potential to migrate from tumors to LNs. LAMP3+ DCs also expressed diverse immune-relevant ligands and exhibited potential to regulate multiple subtypes of lymphocytes. Of the macrophages in tumors that exhibited distinct transcriptional states, tumor-associated macrophages (TAMs) were associated with poor prognosis, and we established the inflammatory role of SLC40A1 and GPNMB in these cells. Further, myeloid and lymphoid cells in ascites were predominantly linked to tumor and blood origins, respectively. The dynamic properties of diverse CD45+ cell types revealed by this study add new dimensions to the immune landscape of HCC.

Comprehensive DNA methylation analysis of tissue of origin of plasma cell-free DNA by methylated CpG tandem amplification and sequencing (MCTA-Seq).

BACKGROUND: Comprehensive analysis of the tissue of origin of plasma cell-free DNA (cfDNA) remains insufficient. A genome-scale DNA methylation method for this analysis is of both biological and clinical interest. METHODS: We used the methylated CpG tandem amplification and sequencing (MCTA-Seq), which is a genome-scale DNA methylation method, for analyzing cfDNA. We performed MCTA-Seq to pair plasma cfDNA and white blood cell genomic DNA from 14 healthy individuals for comparative analysis, with eight tissues being analyzed for identifying tissue-specific markers. The relative contributions of multiple tissues to cfDNA were calculated for plasma cfDNA obtained from healthy adults (n = 25), cholelithiasis patients (n = 13), liver cirrhosis patients (n = 17), hepatocellular carcinoma patients (n = 30), and acute pancreatitis patients (n = 8). RESULTS: We identified a total of 146 tissue-specific hypermethylation markers. Simulation analysis showed that MCTA-Seq can accurately measure DNA fractions contributed by multiple tissues to cfDNA. We demonstrated that the liver is the major non-hematopoietic tissue contributing to plasma cfDNA in healthy adults. The method also detected increases in the liver-derived DNA in the blood from patients with liver diseases, which correlate with an increase in the liver enzyme level. Furthermore, the results indicated that blood cells make a major contribution to the elevation of cfDNA levels in acute pancreatitis, liver cirrhosis, and hepatocellular carcinoma patients. Finally, we characterized a novel set of tissue-specific hypermethylation markers for cfDNA detection, which are located within the intragenic regions of tissue-specific highly expressed genes. CONCLUSIONS: We have used MCTA-Seq for simultaneously measuring cfDNA fractions contributed by multiple tissues. Applying this approach to healthy adults and liver and pancreas disease patients revealed the tissue of origin of cfDNA. The approach and the identified markers should facilitate assessing the cfDNA dynamics in a variety of human diseases.

Distinct epigenetic features of tumor-reactive CD8+ T cells in colorectal cancer patients revealed by genome-wide DNA methylation analysis.

BACKGROUND: Tumor-reactive CD8+ tumor-infiltrating lymphocytes (TILs) represent a subtype of T cells that can recognize and destroy tumor specifically. Understanding the regulatory mechanism of tumor-reactive CD8+ T cells has important therapeutic implications. Yet the DNA methylation status of this T cell subtype has not been elucidated. RESULTS: In this study, we segregate tumor-reactive and bystander CD8+ TILs, as well as naïve and effector memory CD8+ T cell subtypes as controls from colorectal cancer patients, to compare their transcriptome and methylome characteristics. Transcriptome profiling confirms previous conclusions that tumor-reactive TILs have an exhausted tissue-resident memory signature. Whole-genome methylation profiling identifies a distinct methylome pattern of tumor-reactive CD8+ T cells, with tumor-reactive markers CD39 and CD103 being specifically demethylated. In addition, dynamic changes are observed during the transition of naïve T cells into tumor-reactive CD8+ T cells. Transcription factor binding motif enrichment analysis identifies several immune-related transcription factors, including three exhaustion-related genes (NR4A1, BATF, and EGR2) and VDR, which potentially play an important regulatory role in tumor-reactive CD8+ T cells. CONCLUSION: Our study supports the involvement of DNA methylation in shaping tumor-reactive and bystander CD8+ TILs, and provides a valuable resource for the development of novel DNA methylation markers and future therapeutics.

Differential expression of APE1 in hepatocellular carcinoma and the effects on proliferation and apoptosis of cancer cells.

This research aimed to investigate the differential expression of apurinic-apyrimidinic endonuclease 1 (APE1) in hepatocellular carcinoma (HCC) tissues and cells and the effects on proliferation and apoptosis of cancer cells. Immunohistochemical techniques were used to detect the expression of APE1 in 80 cases of HCC and the corresponding paracancerous tissue microarrays; meanwhile, Western blots were used to detect the expression of APE1 in both human HCC BEL-7402, BEL-7405, HCC-9204, Hep3B, HepG2, SMMC-7721 and Huh-7 cells, and normal hepatocyte L-02 cells. The relationship between APE1 expression and clinical pathological characteristics of HCC was statistically analyzed. APE1 shRNA vector was constructed in Hep 3B cells to establish a stably transfected cell line, using Western blots to determine the interference efficiency. Cell proliferation activity was detected with MTT assays, while apoptosis was detected with the Annexin V-FITC/PI double-labeling technique. The expression of APE1 in HCC tissues and cells was significantly up-regulated, and its expression was significantly different from TNM staging and histopathological grading. Down-regulation of APE1 expression significantly reduced the proliferative activity and increased the apoptosis rate of Hep 3B cells. In conclusion, APE1 demonstrates cancer progression potential at the clinical, tissue and cell level. It provides a new idea and theoretical basis for APE1-based clinical diagnosis, prognosis determination and molecular targeted therapy in treatment of HCC.

Programmed Death Receptor 1 (PD1) Knockout and Human Telomerase Reverse Transcriptase (hTERT) Transduction Can Enhance Persistence and Antitumor Efficacy of Cytokine-Induced Killer Cells Against Hepatocellular Carcinoma.

BACKGROUND The weak antitumor efficacy and limited lifespan are the main obstacles that hinder the therapeutic effect of cytokine-induced killer (CIK) cell immunotherapy. In the study, we enhanced the persistence and the antitumor efficacy of CIK cell through PD-1 knockout and hTERT transduction. MATERIAL AND METHODS CIK cells were cultured from patients with hepatocellular carcinoma and PD-1 gene was knocked out through the Cas9 ribonucleoproteins (Cas9 RNPs) electroporation. TIDE assay, T7E1 mismatch cleavage assay, and clone Sanger sequencing were used to detect PD-1 knockout efficiency. The immunophenotype was analyzed by flow cytometry. After PD-1 knockout, the hTERT gene was transduced into PD-1 KO/CIK cells with lentiviral transduction. The hTERT expression and persistence of hTERT/PD-1 KO/CIK cells were evaluated by Western blotting and proliferation curve. The antitumor efficacy was detected by ELISPOT and cytotoxicity assay. The telomere length was measured by the Q-FISH and qPCR method. The karyotype assay was used to analyze the chromosome structural stability. RESULTS The optimal knockout efficiency of PD-1 gene in CIK cells could reach 41.23±0.52%. PD-1 knockout did not affect the immunophenotype of CIK cells. The hTERT transduction enhanced persistence and increased the telomere length. ELISPOT and cytotoxicity assay showed hTERT/PD-1 KO/CIK cells had an enhanced antitumor efficacy. Meanwhile, PD-1 KO/CIK cells transduced with hTERT showed a normal karyotype. CONCLUSIONS PD-1 knockout combined with hTERT transduction could prolong the lifespan and enhance antitumor efficacy of CIK cells against hepatocellular carcinoma cell line.

Long-limb length difference had no effect on outcomes of laparoscopic Roux-en-Y gastric bypass surgery for obese Chinese patients with type 2 diabetes mellitus: A CONSORT compliant article.

Bariatric surgery is effective in treating different components of metabolic syndrome including obesity, type 2 diabetes mellitus (T2DM), and hyperlipidemia. But there is no consensus on the ideal biliopancreatic and Roux limb length. This study aimed to explore the effect of biliopancreatic limb and Roux limb lengths during laparoscopic Roux-en-Y gastric bypass (LRYGB) procedures on weight loss and T2DM control.We studied the clinical records of 58 patients with metabolic syndrome, T2DM, and body mass index (BMI) 32 to 50 kg/m who underwent LRYGB in our hospital. The short limb group (Group A) underwent LRYGB with a limb length of 160 to 200 cm (n = 31) and the long limb group (Group B) underwent LRYGB with a limb length of 210 to 240 cm (n = 27) were compared.The occurrence of acute or chronic internal hernia in Group B was higher than that in Group A (P = .026). Twelve months after surgery, patients from the 2 groups were also observed with reduction in BMI, percent excess weight loss (EWL), preoperative FPG, and HbA1c as compared with these indicators before surgery. However, the differences of these indicators between 2 groups were not significant at the time point of before and 3, 6, 12 months after surgery.LRYGB had significant effects on weight loss and diabetes control in obese T2DM patients. However, there was no significant difference in the short term on weight loss and diabetes control in the patients receiving different limb lengths.

Identifying tumor promoting genomic alterations in tumor-associated fibroblasts via retrovirus-insertional mutagenesis.

Tumor-associated fibroblasts (TAFs) are often essential for solid tumor growth. However, few genetic or epigenetic alterations have been found in TAFs during the progression of solid tumors. Employing a tumor-stromal cell co-injection model, we adapted here retroviral-insertional mutagenesis to stromal cells to identify novel tumor-associated genes in TAFs. We successfully identified 20 gene candidates that might modulate tumor growth if altered in TAFs at genomic level. To validate our finding, the function of one of the candidate genes, tubulin tyrosine ligase (Ttl), was further studied in TAFs from fibrosarcoma, colon, breast and hepatocarcinoma. We demonstrated that down-regulated TTL expression in TAFs indeed promoted tumor growth in mice. Interestingly, decreased expression of TTL in tumor stromal cells also correlated with poor outcome in human colon carcinoma. Thus, the co-injection model of tumor cells with retrovirus-modified fibroblasts proved a valid method to identify tumor-modulating genes in TAFs, allowing for a deeper insight into the role of the stroma for tumor development.

Detection of circulating tumor cells by reverse transcription­quantitative polymerase chain reaction and magnetic activated cell sorting in the peripheral blood of patients with hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies worldwide. Circulating tumor cells (CTCs) are considered a major cause of recurrence and metastasis in cancer; however, the detection of CTCs is challenging owing to their very low numbers in peripheral blood (around 10 CTCs per 1,000,000 erythrocytes). Cancer­testis antigens (CTAs) are specific tumor markers for CTCs. The present study aimed to evaluate the sensitivity and specificity of reverse transcription­quantitative polymerase chain reaction (RT­qPCR) for the detection of nine CTAs as well as placenta­specific antigen 1 (PLAC1) in peripheral blood mononuclear cell (PBMC) samples collected from 51 patients with HCC. The effectiveness of magnetic­activated cell sorting (MACS) for tumor­cell enrichment, through the depletion of CD45+ leukocytes in PBMC samples, was also assessed. Immunocytochemistry along with hematoxylin and eosin staining demonstrated that RT­qPCR achieved an overall positive detection rate for CTAs and PLAC1 of 70.6%; the highest rates were observed for melanoma­associated antigen A3 (MAGEA3), synovial sarcoma X breakpoint 1, MAGEA1, NY­ESO­1, L antigen 1 and PLAC1. MACS­detected intact CTCs in PBMCs were confirmed by H&E staining and morphological assessment; 12 out of 19 (63.2%) patients were identified as positive for CTAs. Screening for these five CTAs and PLAC1 by RT­qPCR may offer a potentially valuable prognostic tool with good sensitivity and specificity in patients with HCC that may be enhanced by MACS.

Landscape of Infiltrating T Cells in Liver Cancer Revealed by Single-Cell Sequencing.

Systematic interrogation of tumor-infiltrating lymphocytes is key to the development of immunotherapies and the prediction of their clinical responses in cancers. Here, we perform deep single-cell RNA sequencing on 5,063 single T cells isolated from peripheral blood, tumor, and adjacent normal tissues from six hepatocellular carcinoma patients. The transcriptional profiles of these individual cells, coupled with assembled T cell receptor (TCR) sequences, enable us to identify 11 T cell subsets based on their molecular and functional properties and delineate their developmental trajectory. Specific subsets such as exhausted CD8+ T cells and Tregs are preferentially enriched and potentially clonally expanded in hepatocellular carcinoma (HCC), and we identified signature genes for each subset. One of the genes, layilin, is upregulated on activated CD8+ T cells and Tregs and represses the CD8+ T cell functions in vitro. This compendium of transcriptome data provides valuable insights and a rich resource for understanding the immune landscape in cancers.

HDM2 Promotes NEDDylation of Hepatitis B Virus HBx To Enhance Its Stability and Function.

Hepatitis B virus (HBV)-encoded X protein (HBx) plays a critical role in HBV-related hepatocarcinoma development. In this study, we demonstrate that HBx is specifically modified by NEDD8. We found that E3 ligase HDM2 promotes NEDDylation of HBx to enhance HBx stability by preventing its ubiquitination-mediated degradation. Consistently, analysis of 160 hepatocellular carcinoma patient specimens indicated that the amount of HDM2 protein correlates with HBx protein level. We identified that HBx K91 and K95 as the key HBx NEDDylation sites and observed that the NEDDylation-deficient HBx has shorter half-life. We generated Huh7 cell lines which ectopically express wild-type and NEDDylation-deficient HBx and found that NEDDylation-deficient HBx showed less chromatin localization and less DDB1 binding. Consistently, the expression of HBx-regulated genes (IL-8, MMP9, and YAP) and HBV transcription (the activity of HBV enhancer and the amount of pgRNA transcribed from cccDNA) were significantly higher in cells expressing wild-type (WT) HBx than that in cells expressing mutant HBx. In addition, HBx-expressing cells proliferated faster than control and mutant HBx-expressing cells. We also showed that the ability of WT HBx-expressing cells to form tumors in nude mice was significantly higher than that of mutant HBx-expressing cells. In conclusion, we revealed that E3 ligase HDM2 promotes NEDDylation of HBx to enhance HBx stability and chromatin localization, which in turn favors HBx-dependent transcriptional regulation, cell proliferation, and HBV-driven tumor growth.IMPORTANCE Hepatitis B virus (HBV) HBx protein plays a critical role in viral replication and hepatocarcinogenesis. However, the regulation of HBx stability is not well understood. We found that HBx is modified by NEDD8 and that the HDM2 E3 ligase promotes HBx NEDDylation to enhance HBx stability by inhibiting its ubiquitination. We provide a new evidence to show the positive correlation between HDM2 and HBx in clinical hepatocellular carcinoma (HCC) samples. We also identified the major NEDDylation sites on HBx. Our studies indicate that the defective NEDDylation of HBx negatively affects its ability to activate the transcription of downstream genes and promote cell proliferation and tumor growth in vivo Taken together, our findings reveal a novel posttranslational modification of HBx by HDM2 which regulates its stability, subcellular localization, and functions. These findings indicate that HDM2 is an important regulator on HBx and a potential diagnosis/therapeutic marker for HBV-associated HCC.