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.

The Role of C-reactive Protein and Procalcitonin in Predicting the Occurrence of Pancreatic Fistula in Patients who Underwent Laparoscopic Pancreaticoduodenectomy: a Retrospective Study.

The occurrence of postoperative pancreatic fistula following laparoscopic pancreaticoduodenectomy (LPD) is a significant concern, yet there is currently a lack of consensus on reliable predictive methods for this complication. Therefore, the aim of this study was to assess the clinical significance of C-reactive protein (CRP) and procalcitonin (PCT) values and their reliability in early predicting the development of clinically relevant pancreatic fistula (CRPF) following LPD.A retrospective analysis was conducted using data from 120 patients who had LPD between September 2019 and December 2021. Preoperative assessment data, standard patients' demographic and clinicopathological characteristics, intra- and postoperative evaluation, as well as postoperative laboratory values on postoperative days (PODs) 1, 3, and 7, including white blood cells (WBCs), CRP, and PCT, were prospectively recorded on a dedicated database. Two clinicians separately collected and cross-checked all of the data.Among 120 patients [77 men (64%), 43 women (36%], CRPF occurred in 15 patients (11 grade B and 4 grade C fistulas). The incidence rate of CRPF was 12.3%. A comparison of the median values of WBCs, PCT, and CRP across the two groups revealed that the CRPF group had higher values on most PODs than the non-CRPF group. Receiver operating characteristic (ROC) analysis was used to calculate the area under the curve (AUC) and cutoff values. It was discovered that POD 3 has the most accurate and significant values for WBCs, CRP, and PCT. According to the ROC plots, the AUC for WBCs was 0.842, whereas the AUC for PCT was 0.909. As for CRP, the AUC was 0.941 (95% CI 0.899-0.983, p < 0.01) with a cutoff value of 203.45, indicating a sensitivity of 93.3% and specificity of 91.4%.Both CRP and PCT can be used to predict the early onset of CRPF following LPD, with CRP being slightly superior on POD 3.

High mobility group AT-hook 2 promotes tumorigenicity of pancreatic cancer cells via upregulating ANLN.

HMGA2 is associated with the regulation of cellular biological processes in various human disorders and cancer progression, yet little is known about how HMGA2 controls tumorigenesis. This study uncovered the mechanism of HMGA2-mediated regulation of tumorigenicity in pancreatic cancer. We showed that HMGA2 was highly expressed in pancreatic cancer cells and correlated with poor prognosis. HMGA2 expression knockdown inhibited the tumorigenicity of pancreatic cancer cells. Conversely, overexpression of HMGA2 promoted tumorigenicity. Combination of ChIP-Seq, RNA-Seq and dual-luciferase reporter assays revealed HMGA2 could directly regulate ANLN expression. Furthermore, we found ANLN could mediate the HMGA2-induced effects on pancreatic cancer cells. The identification of the regulatory mechanism of HMGA2 and ANLN will provide insights into the progression for human pancreatic cancer.

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.

Overexpression of SOX18 correlates with accelerated cell growth and poor prognosis in human pancreatic ductal adenocarcinoma.

Transcription factor SOX18 has been proved to play a significant role in carcinogenesis. However, no investigation was performed about the expression of SOX18 in pancreatic ductal adenocarcinoma (PDAC). In our work, we found that the PDAC tissues had higher level of SOX18 mRNA and protein expression than matched non-tumor pancreatic tissues and high level of SOX18 protein indicated poor prognosis for PDAC patients. After knockdown of SOX18 gene in PANC-1 and SW1990 cell lines, which showed higher expression level of SOX18 among five PDAC cell lines, the abilities of proliferation, migration and invasion were inhibited and the tumor growth was suppressed in vivo. In addition, the flow cytometry results indicated that down-regulation of SOX18 induced G1/S phase arrest. Furthermore, we found that the expression of cyclin D1, c-myc and MMP-7, three tumorigenesis promoters, was inhabited with downregulation of SOX18. In conclusion, our study reveals that SOX18 plays a significant role in promoting the growth of PDAC, and might serve as a promising target for PDAC therapy.

The decreased N6-methyladenine DNA modification in cancer cells.

N6-methyladenine (6 mA) is a recently characterized DNA modification in mammalian genomes, although its biological importance remains to be resolved. Using a highly sensitive HPLC/MS/MS approach, here we report regulation of 6 mA modification in mammalian cells. To these aspects, down-regulation of 6 mA modification was first characterized in human cancer cells and tissues, relative to their normal controls. In contrast to the relative stable 5 mC modification, a dramatic decrease of 6 mA modification was found, showing that 6 mA is the most regulated DNA modification in cancers. In addition to the regulation in cancer cells, a hundreds-fold increase of 6 mA modification was found for in vitro cultured human cells, relative to the in vivo cells. This up-regulation was also confirmed with in vitro cultured mouse cells. Taken together, our study revealed distinct 6 mA modification profiles in the cancer and cultured cells. Considering its distinct regulation from that of 5 mC, our study suggests that 6 mA DNA modification may play a crucial role in cell fate transition of mammalian cells.

A Predictive Model Based on the FBXO Family Reveals the Significance of Cyclin F in Hepatocellular Carcinoma.

OBJECTIVE: The F-box protein (FBXO) family plays a key role in the malignant progression of tumors. However, the biological functions and clinical value of the FBXO family in liver cancer remain unclear. Our study comprehensively assessed the clinical value of the FBXO family in hepatocellular carcinoma (HCC) and constructed a novel signature based on the FBXO family to predict prognosis and guide precision immunotherapy. METHODS: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) databases were utilized to investigate the expression characteristics and prognostic value of the FBXO family in HCC. A predictive model based on the FBXO family using TCGA database; and its predictive ability was validated using the ICGC database. Further analyses revealed that this predictive model can independently predict the overall survival (OS) rate of patients with HCC. We further analyzed the association of this predictive model with signaling pathways, clinical pathological features, somatic mutations, and immune therapy responses. Finally, we validated the biological functions of cyclin F (CCNF) through in vitro experiments. RESULTS: A predictive model involving three genes (CCNF, FBXO43, and FBXO45) was constructed, effectively identifying high and low-risk patients with differences in OS, clinicopathological characteristics, somatic mutations, and immune cell infiltration status. Additionally, knock-down of CCNF in HCC cell lines reduced cell proliferation in vitro, suggesting that CCNF may be a potential therapeutic target for HCC. CONCLUSIONS: The predictive model based on the FBXO family can effectively predict OS and the immune therapy response in HCC. Additionally, CCNF is a potential therapeutic target for HCC.

LMNB1/CDKN1A Signaling Regulates the Cell Cycle and Promotes Hepatocellular Carcinoma Progression.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies in the world. Lamin B1 (LMNB1) is a key component of the nuclear skeleton structure. Recent studies have found that LMNB1 is overexpressed in tumor tissues and is associated with the prognosis of patients. However, the underlying mechanism remains unclear in HCC. OBJECTIVE: This study aims to explore the clinical significance and molecular mechanisms of LMNB1 in HCC. METHODS: The expression level of LMNB1 and its clinical values were analyzed with public databases, and the level of LMNB1 in HCC tissues and adjacent normal tissues was confirmed by qRT-PCR and IHC. Functional assays were conducted to explore the impact of LMNB1 knockdown on cell proliferation both in vivo and in vitro. Additionally, Genes and Genomes enrichment analysis, recovery analysis, and ChIP assays were employed to investigate its underlying molecular mechanisms. Finally, we carried out an analysis of the relationship between LMNB1 and immune cell infiltration in HCC. RESULTS: LMNB1 was found to be overexpressed in HCC and correlated with the pathological stage and unfavorable prognosis. Functional assays demonstrated that LMNB1 promotes HCC proliferation both in vitro and in vivo. Further analysis revealed that LMNB1 promotes the progression of HCC by regulating CDKN1A expression. Furthermore, the infiltration of immune cells in HCC tissues suggests a potential correlation between immune infiltration cell markers and the expression of LMNB1. CONCLUSIONS: LMNB1 emerged as a promising therapeutic target and prognostic biomarker for HCC, with its expression showing a correlation with several immune infiltration cell markers.

Prognostic prediction and immune infiltration analysis based on ferroptosis and EMT state in hepatocellular carcinoma.

Background: Ferroptosis is one of the main mechanisms of sorafenib against hepatocellular carcinoma (HCC). Epithelial-mesenchymal transition (EMT) plays an important role in the heterogeneity, tumor metastasis, immunosuppressive microenvironment, and drug resistance of HCC. However, there are few studies looking into the relationship between ferroptosis and EMT and how they may affect the prognosis of HCC collectively. Methods: We downloaded gene expression and clinical data of HCC patients from the Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) databases for prognostic model construction and validation respectively. The Least absolute shrinkage and selection operator (LASSO) Cox regression was used for model construction. The predictive ability of the model was assessed by Kaplan-Meier survival analysis and receiver operating characteristic (ROC) curve. We performed the expression profiles analysis to evaluate the ferroptosis and EMT state. CIBERSORT and single-sample Gene Set Enrichment Analysis (ssGSEA) methods were used for immune infiltration analysis. Results: A total of thirteen crucial genes were identified for ferroptosis-related and EMT-related prognostic model (FEPM) stratifying patients into two risk groups. The high-FEPM group had shorter overall survivals than the low-FEPM group (p<0.0001 in the TCGA cohort and p<0.05 in the ICGC cohort). The FEPM score was proved to be an independent prognostic risk factor (HR>1, p<0.01). Furthermore, the expression profiles analysis suggested that the high-FEPM group appeared to have a more suppressive ferroptosis status and a more active EMT status than the low- FEPM group. Immune infiltration analysis showed that the myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs) were highly enriched in the high-FEPM group. Finally, a nomogram enrolling FEPM score and TNM stage was constructed showing outstanding predictive capacity for the prognosis of patients in the two cohorts. Conclusion: In conclusion, we developed a ferroptosis-related and EMT-related prognostic model, which could help predict overall survival for HCC patients. It might provide a new idea for predicting the response to targeted therapies and immunotherapies in HCC patients.

Inhibition of bone morphogenetic protein receptor 2 suppresses pancreatic ductal adenocarcinoma growth by regulating GRB2/PI3K/AKT axis.

BACKGROUND: Bone morphogenetic protein receptor 2 (BMPR2) is an important transmembrane serine/threonine kinase that involves oncogenic processes in multiple cancers. However, the role of BMPR2 and its regulatory mechanism in pancreatic ductal adenocarcinoma (PDAC) remain unknown. METHODS: We performed a tissue array to explore the expression of BMPR2 in PDAC tissues. The Cell Counting Kit-8 (CCK-8) and colony formation assays were used to measure PDAC cells' proliferation. Proteomics and mass spectrometry technology was applied to analyze the BMPR2-regulating proteins. Flow cytometry was used to analyze the cell cycle distribution of PDAC cells. Orthotopic pancreatic cancer (PC) and patient-derived xenograft (PDX) models were used for in vivo experiments. RESULTS: This study revealed the over-expression of BMPR2 in PDAC tissues and its proliferation-promoting role in PDAC cells. By carrying out protein mass spectrometry technique as well as bioinformatics analysis, we identified that BMPR2 regulated the growth factor receptor-bound protein 2/phosphatidylinositol 3-kinase/protein kinase B (GRB2/PI3K/AKT) signaling pathway, and further in vitro experiments showed that inhibition of BMPR2 resulted in suppressing proliferation and G2/M arrest by inhibiting the GRB2/PI3K/AKT signaling pathway in PDAC cells. The inhibition of BMPR2 by LDN193189 showed similar results in PDAC cells, orthotopic PC, and PDX models, which revealed that inhibition of BMPR2 significantly suppressed tumor growth by suppressing the GRB2/PI3K/AKT axis. CONCLUSIONS: Inhibition of BMPR2 suppresses PDAC growth by regulating the GRB2/PI3K/AKT axis and is a promising PDAC treatment strategy.

Single-cell RNA-seq reveals dynamic change in tumor microenvironment during pancreatic ductal adenocarcinoma malignant progression.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is most aggressive among all gastrointestinal tumors. The complex intra-tumor heterogeneity and special tumor microenvironment in PDAC bring great challenges for developing effective treatment strategies. We aimed to delineate dynamic changes of tumor microenvironment components during PDAC malignant progression utilizing single-cell RNA sequencing. METHODS: A total of 11 samples (4 PDAC I, 4 PDAC II, 3 PDAC III) were used to construct expression matrix. After identifying distinct cell clusters, subcluster analysis for each cluster was performed. New cancer associated fibroblasts (CAFs) subset was validated by weighted gene co-expression network analysis, RNA in situ hybridization and immunofluorescence. FINDINGS: We found that ductal cells were not dominant component while tumor infiltrating immune cells and pancreatic stellate cells gradually accumulated during tumor development. We defined several new Treg and exhausted T cell signature genes, including DUSP4, FANK1 and LAIR2. The analysis of TCGA datasets showed that patients with high expression of DUSP4 had significantly worse prognosis. In addition, we identified a new CAFs subset (complement-secreting CAFs, csCAFs), which specifically expresses complement system components, and constructed csCAFs-related module by weighted gene co-expression network analysis. The csCAFs were located in the tissue stroma adjacent to malignant ductal cells only in early PDAC. INTERPRETATION: We systematically explored PDAC heterogeneity and identified csCAFs as a new CAFs subset special to PDAC, which may be valuable for understanding the crosstalk inside tumor. FUNDING: This study was supported by The Natural Science Foundation of China (NO.81572339, 81672353, 81871954) and the Youth Clinical Research Project of Peking University First Hospital (2018CR28).

Pancreatic stellate cells derived exosomal miR-5703 promotes pancreatic cancer by downregulating CMTM4 and activating PI3K/Akt pathway.

Exosomes play important role in tumor microenvironment, and mediate the crosstalk between pancreatic cancer (PC) cells and matrix components including pancreatic stellate cells (PSCs) to regulate pancreatic cancer progression. Here we isolated primary PSCs from PC patients, and demonstrated that PSC-derived exosomes could be internalized by PC cells to promote cell proliferation. Furthermore, we identified that miR-5703 in the exosomes acted as a driver of cell proliferation and its inhibitor suppressed the function of exosomes to promote PC cell proliferation. miR-5703 directly bound to the 3'UTR of CMTM4 and downregulated its expression. CMTM4 knockdown promoted PC cell proliferation, while overexpression of CMTM4 suppressed PC cell proliferation both in vivo and in vitro. Mechanistically, we revealed that CMTM4 suppressed PI3K/Akt pathway via downregulating PAK4. In conclusion, our results suggest that PSC-derived exosomal miR-5703 could target CMTM4 in PC cells and promote cell proliferation due to PAK4 activated PI3K/Akt pathway.

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.

BRM transcriptionally regulates miR-302a-3p to target SOCS5/STAT3 signaling axis to potentiate pancreatic cancer metastasis.

Brahma (BRM) has recently been documented as a significant predictor of pancreatic cancer (PC) metastasis. This study aimed to further elucidate molecular mechanism by which BRM promotes PC metastasis. We found that silencing BRM reduced PC cell migration and invasion both in vivo and in vitro, accompanied by reduced level of miR-302a-3p. BRM positively regulated the transcription of miR-302a-3p, which acted as a metastasis-promoting miRNA in PC cells. miR-302a-3p directly targeted SOCS5 to boost STAT3 phosphorylation and induce the transcription of STAT3 target genes. Furthermore, miR-302a-3p level was higher in tissue and plasma samples derived from PC patients, and was significantly associated with worse clinical pathological features. In xenograft models, inhibiting miR-302a-3p was synergistically lethal in BRM-silenced PC cells. In conclusion, our results suggest that transcriptional regulation of miR-302a-3p by BRM potentiates PC metastasis by epigenetically suppressing SOCS5 expression and activating STAT3 signaling. These new findings provide potential therapeutic avenues for preventing PC-associated death.

Long non-coding RNA MEG3 functions as a tumour suppressor and has prognostic predictive value in human pancreatic cancer.

Long non-coding RNA (lncRNA) MEG3 has been demonstrated to be a tumour suppressor in many malignancies. However, the functional role of MEG3 in pancreatic cancer (PC) is unclear. In this study, the expression pattern of MEG3 was evaluated in 25 samples of microdissected PC tissues and 8 PC cell lines and was compared to the expression in adjacent non­cancerous tissues and a human pancreatic normal epithelial cell line. Loss of MEG3 expression was observed in both the cancerous tissues and cancer cell lines. Although the absence of expression of MEG3 was not statistically correlated to either histological grade or TNM stage in the 25 cases, the prognosis was significantly worse. MEG3 knockdown enhanced cell proliferation, promoted cell migration and invasion, induced epithelial­mesenchymal transition (EMT), increased the sphere­forming ability and cancer stem cell (CSC) properties, and decreased the chemosensitivity to gemcitabine in vitro. In contrast, forced expression of MEG3 resulted in a reverse effect. In conclusion, MEG3 functions as a tumour suppressor in human PC. The underlying cause of the poor prognosis induced by low levels of MEG3 expression in PC patients might involve EMT induction, enhanced CSC phenotypes and reduced chemoresistance, all of which might be associated with Snail activation.

Long noncoding RNA H19 derived miR-675 regulates cell proliferation by down-regulating E2F-1 in human pancreatic ductal adenocarcinoma.

The long noncoding RNA (lncRNA) H19 has been proven to be overexpressed in human pancreatic ductal adenocarcinoma (PDAC). H19-induced PDAC cell proliferation is cell cycle-dependent by modulating E2F-1. However, the mechanism of how H19 regulates E2F-1 remains unclear. In this study, we investigated the expression of miR-675 in PDAC tumours and cells, the biological function of miR-675 in PDAC cell proliferation and the possible relationship among H19, miR-675 and E2F-1. As a transcript of the first exon of H19, the level of miR-675 was negatively correlated with H19 expression in microdissected PDAC tissues (r=-0.0646, P=0.001). The serum miR-675 expression was significantly down-regulated in patients with PDAC compared to those in healthy individuals. Moreover, an evaluation of five PDAC cases showed that there was a remarkable increase of serum miR-675 levels after resection of the primary tumours. Ectopic overexpression of miR-675 in AsPC-1 and PANC-1 cells decreased cell viability, the colony-forming ability and the percentage of cells in S phase; contrarily, miR-675 knockdown resulted in enhanced cell proliferation. Furthermore, the suppressed cell proliferation caused by H19 knockdown could be rescued by inhibiting miR-675 expression. Additionally, intratumoural injection of either miR-675 agomir or antagomir could significantly affect tumour growth in vivo. Both the bioinformatic prediction and luciferase activity assay confirmed that E2F-1 was a direct target of miR-675. And the decrease of E2F-1 protein expression caused by siH19 could be partially reversed by miR-675 knockdown. We concluded that there might be a H19/miR-675/E2F-1 regulatory loop in cell cycle modulation. Serum miR-675 might serve as a potential biomarker for not only early diagnosis but also outcome prediction in PDAC.

In vivo response of AZ31 alloy as biliary stents: a 6 months evaluation in rabbits.

Mg-based metallic materials have been making continuing progress as vascular stents. However, the research of Mg-based materials as non-vascular stents is still at its primary stage. AZ31 stents hereby were implanted into the common bile duct of rabbits for 6 months. The results revealed an existence of 93.82 ± 1.36% and 30.89 ± 2.46% of the original volume after 1 and 3 month, respectively. Whole blood tests indicated an inflammation decreasing to normal level after 3 month implantation. A benign host response was observed via H&E staining. Nonuniform corrosion at the two ends of the stents was observed and considered the results of flow or local inflammation. Moreover, the application of Mg-based materials for different stenting treatment were reviewed and compared. Esophagus was hypothesized most destructive, whilst blood vessel and bile duct considered similar and less destructive. Trachea and nasal cavity were thought to be mildest.

BRM/SMARCA2 promotes the proliferation and chemoresistance of pancreatic cancer cells by targeting JAK2/STAT3 signaling.

BACKGROUND: BRM is one of two evolutionarily conserved catalytic ATPase subunits of SWI/SNF complexes and plays important role in cell proliferation, linage specification and development, cell adhesion, cytokine responses and DNA repair. BRM is often inactivated in various types of cancer indicating its indispensable roles in oncogenesis but the mechanisms remain poorly understood. METHODS: BRM expression in clinical pancreatic cancer samples was examined by immunohistochemistry and the correlation with patient survival was analyzed. shRNAs targeting BRM were used to establish stable BRM knockdown BxPC-3 and T3M4 cell lines. Cell viability was assessed by CCK-8 assay. Cell proliferation was measured by EdU incorporation assay, colony formation assay and Ki67 staining. Cell cycle and apoptosis were examined by flow cytometry. Growth and chemosensitivity of xenografts initiating from BRM-deficient cells were evaluated, and in situ apoptosis was detected by TUNEL assay. The status of JAK-STAT3 signaling was examined by real-time PCR and Western blot analysis. RESULTS: High BRM expression was correlated with worse survival of pancreatic cancer patients. BRM shRNA reduced the proliferation and increased the sensitivity of pancreatic cancer cells to gemcitabine in vivo and in vitro, and these effects are associated with the inhibition of STAT3 phosphorylation and reduced transcription of STAT3 target genes. CONCLUSION: We reveal a novel mechanism by which BRM could activate JAK2/STAT3 pathway to promote pancreatic cancer growth and chemoresistance. These findings may offer potential therapeutic targets for pancreatic cancer patients with excessive BRM expression.

Single-cell triple omics sequencing reveals genetic, epigenetic, and transcriptomic heterogeneity in hepatocellular carcinomas.

Single-cell genome, DNA methylome, and transcriptome sequencing methods have been separately developed. However, to accurately analyze the mechanism by which transcriptome, genome and DNA methylome regulate each other, these omic methods need to be performed in the same single cell. Here we demonstrate a single-cell triple omics sequencing technique, scTrio-seq, that can be used to simultaneously analyze the genomic copy-number variations (CNVs), DNA methylome, and transcriptome of an individual mammalian cell. We show that large-scale CNVs cause proportional changes in RNA expression of genes within the gained or lost genomic regions, whereas these CNVs generally do not affect DNA methylation in these regions. Furthermore, we applied scTrio-seq to 25 single cancer cells derived from a human hepatocellular carcinoma tissue sample. We identified two subpopulations within these cells based on CNVs, DNA methylome, or transcriptome of individual cells. Our work offers a new avenue of dissecting the complex contribution of genomic and epigenomic heterogeneities to the transcriptomic heterogeneity within a population of cells.

Genome-scale detection of hypermethylated CpG islands in circulating cell-free DNA of hepatocellular carcinoma patients.

Despite advances in DNA methylome analyses of cells and tissues, current techniques for genome-scale profiling of DNA methylation in circulating cell-free DNA (ccfDNA) remain limited. Here we describe a methylated CpG tandems amplification and sequencing (MCTA-Seq) method that can detect thousands of hypermethylated CpG islands simultaneously in ccfDNA. This highly sensitive technique can work with genomic DNA as little as 7.5 pg, which is equivalent to 2.5 copies of the haploid genome. We have analyzed a cohort of tissue and plasma samples (n = 151) of hepatocellular carcinoma (HCC) patients and control subjects, identifying dozens of high-performance markers in blood for detecting small HCC (≤ 3 cm). Among these markers, 4 (RGS10, ST8SIA6, RUNX2 and VIM) are mostly specific for cancer detection, while the other 15, classified as a novel set, are already hypermethylated in the normal liver tissues. Two corresponding classifiers have been established, combination of which achieves a sensitivity of 94% with a specificity of 89% for the plasma samples from HCC patients (n = 36) and control subjects including cirrhosis patients (n = 17) and normal individuals (n = 38). Notably, all 15 alpha-fetoprotein-negative HCC patients were successfully identified. Comparison between matched plasma and tissue samples indicates that both the cancer and noncancerous tissues contribute to elevation of the methylation markers in plasma. MCTA-Seq will facilitate the development of ccfDNA methylation biomarkers and contribute to the improvement of cancer detection in a clinical setting.