Necroptosis enhances 'don't eat me' signal and induces macrophage extracellular traps to promote pancreatic cancer liver metastasis.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating cancer with dismal prognosis due to distant metastasis, even in the early stage. Using RNA sequencing and multiplex immunofluorescence, here we find elevated expression of mixed lineage kinase domain-like pseudo-kinase (MLKL) and enhanced necroptosis pathway in PDAC from early liver metastasis T-stage (T1M1) patients comparing with non-metastatic (T1M0) patients. Mechanistically, MLKL-driven necroptosis recruits macrophages, enhances the tumor CD47 'don't eat me' signal, and induces macrophage extracellular traps (MET) formation for CXCL8 activation. CXCL8 further initiates epithelial-mesenchymal transition (EMT) and upregulates ICAM-1 expression to promote endothelial adhesion. METs also degrades extracellular matrix, that eventually supports PDAC liver metastasis. Meanwhile, targeting necroptosis and CD47 reduces liver metastasis in vivo. Our study thus reveals that necroptosis facilitates PDAC metastasis by evading immune surveillance, and also suggest that CD47 blockade, combined with MLKL inhibitor GW806742X, may be a promising neoadjuvant immunotherapy for overcoming the T1M1 dilemma and reviving the opportunity for radical surgery.

Targeting the MCP-GPX4/HMGB1 Axis for Effectively Triggering Immunogenic Ferroptosis in Pancreatic Ductal Adenocarcinoma.

Induction of ferroptosis can inhibit cancer cells in vitro, however, the role of ferroptosis in treatment in vivo is controversial. The immunosuppressive cells activated by the ferroptotic tumor cells can promote the growth of residual tumor cells, hindering the application of ferroptosis stimulation in tumor treatment. In this study, a new strategy is aimed to be identified for effectively triggering immunogenic ferroptosis in pancreatic ductal adenocarcinoma (PDAC) and simultaneously stimulating antitumor immune responses. Toward this, several molecular and biochemical experiments are performed using patient-derived organoid models and a KPC mouse model (LSL-KrasG12D /+, LSL-Trp53R172H/+, Pdx-1-Cre). It is observed that the inhibition of macrophage-capping protein (MCP) suppressed the ubiquitin fold modifier (UFM)ylation of pirin (PIR), a newly identified substrate of UFM1, thereby decreasing the transcription of GPX4, a marker of ferroptosis, and promoting the cytoplasmic transportation of HMGB1, a damage-associated molecular pattern. GPX4 deficiency triggered ferroptosis, and the pre-accumulated cytosolic HMGB1 is released rapidly. This altered release pattern of HMGB1 facilitated the pro-inflammatory M1-like polarization of macrophages. Thus, therapeutic inhibition of MCP yielded dual antitumor effects by stimulating ferroptosis and activating antitumor pro-inflammatory M1-like macrophages. The nanosystem developed for specifically silencing MCP is a promising tool for treating PDAC.

Escherichia coli-Induced cGLIS3-Mediated Stress Granules Activate the NF-κB Pathway to Promote Intrahepatic Cholangiocarcinoma Progression.

Patients with concurrent intrahepatic cholangiocarcinoma (ICC) and hepatolithiasis generally have poor prognoses. Hepatolithiasis is once considered the primary cause of ICC, although recent insights indicate that bacteria in the occurrence of hepatolithiasis can promote the progression of ICC. By constructing in vitro and in vivo ICC models and patient-derived organoids (PDOs), it is shown that Escherichia coli induces the production of a novel RNA, circGLIS3 (cGLIS3), which promotes tumor growth. cGLIS3 binds to hnRNPA1 and G3BP1, resulting in the assembly of stress granules (SGs) and suppression of hnRNPA1 and G3BP1 ubiquitination. Consequently, the IKKα mRNA is blocked in SGs, decreasing the production of IKKα and activating the NF-κB pathway, which finally results in chemoresistance and produces metastatic phenotypes of ICC. This study shows that a combination of Icaritin (ICA) and gemcitabine plus cisplatin (GP) chemotherapy can be a promising treatment strategy for ICC.

A Novel Trojan Horse Nanotherapy Strategy Targeting the cPKM-STMN1/TGFB1 Axis for Effective Treatment of Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is characterized by its dense fibrotic microenvironment and highly malignant nature, which are associated with chemotherapy resistance and very poor prognosis. Although circRNAs have emerged as important regulators in cancer biology, their role in ICC remains largely unclear. Herein, a circular RNA, cPKM is identified, which is upregulated in ICC and associated with poor prognosis. Silencing cPKM in ICC cells reduces TGFB1 release and stromal fibrosis, inhibits STMN1 expression, and suppresses ICC growth and metastasis, moreover, it also leads to overcoming paclitaxel resistance. This is regulated by the interactions of cPKM with miR-199a-5p or IGF2BP2 and by the ability of cPKM to stabilize STMN1/TGFB1 mRNA. Based on these findings, a Trojan horse nanotherapy strategy with co-loading of siRNA against cPKM (si-cPKM) and paclitaxel (PTX) is developed. The siRNA/PTX co-loaded nanosystem (Trojan horse) efficiently penetrates tumor tissues, releases si-cPKM and paclitaxel (soldiers), promotes paclitaxel sensitization, and suppresses ICC proliferation and metastasis in vivo. Furthermore, it alleviates the fibrosis of ICC tumor stroma and reopens collapsed tumor vessels (opening the gates), thus enhancing the efficacy of the standard chemotherapy regimen (main force). This novel nanotherapy provides a promising new strategy for ICC treatment.

Analgesic efficacy of an opioid-free postoperative pain management strategy versus a conventional opioid-based strategy following open major hepatectomy: an open-label, randomised, controlled, non-inferiority trial.

Background: Convincing clinical evidence regarding completely opioid-free postoperative pain management using erector spinae plane block (ESPB) in patients undergoing open major hepatectomy (OMH) is lacking. Herein, we aimed to compare the postoperative analgesic efficacy of the visualised continuous opioid-free ESPB (VC-ESPB) and conventional intravenous opioid-based postoperative pain management in hepatocellular carcinoma (HCC) patients undergoing OMH. Methods: This open-label, randomised, controlled, non-inferiority trial enrolled patients with HCC undergone open major hepatectomy in Fujian Provincial Hospital and compared the postoperative analgesic efficacy of VC-ESPB (VC-ESPB group) and conventional intravenous opioid-based pain management regimen (conventional group). Patients were randomly assigned (1:1) to VC-ESPB group and conventional group. Patients were not masked to treatment allocation. The VC-ESPB group was treated with intermittent injections of 0.25% ropivacaine (bilateral, 30 mL each side) given every 12 h through catheters placed in the space of erector spinae and an opioid-free intravenous pump (10-mg tropisetron diluted to 100 mL with 0.9% normal saline [NS]) for postoperative pain management. The conventional group did not receive ESPB and was treated with a conventional intravenous opioid-based pump (2.5-µg/kg sufentanil and 10-mg tropisetron diluted to 100 mL with 0.9% NS). Patients in the VC-ESPB group underwent magnetic resonance imaging (MRI) to identify local anaesthetic diffusion after ESPB was performed under ultrasound guidance. The primary outcome was postoperative analgesic efficacy, which was indicated by the cumulative area under the curve (AUC) of the pain visual analogue scale scores (range, 0-10; a higher score indicates more pain) obtained at rest and at movement until 48 h postoperatively after leaving the post-anaesthesia care unit (PACU). Herein, an AUC of 26.5 was set as the noninferiority margin, which needed to be satisfied for both cumulative AUCPACU-48 h at rest and cumulative AUCPACU-48 h at movement. Per protocol participants were included in primary and safety analyses. This trial was registered with ChiCTR.org.cn (ChiCTR1900026583). Findings: Between October 30, 2019, and May 1, 2023, 106 patients were enrolled and randomly assigned to the VC-ESPB group (n = 53) and the conventional group (n = 53). After the dropout (n = 5), a total of 101 patients (VC-ESPB group, n = 50; conventional group, n = 51) were analysed. Both the level of cumulative AUCPACU-48 h (at rest: 160.08 ± 38.00 vs. 164.94 ± 31.00; difference [90% CI], -4.861 [-16.308, 6.585]) and cumulative AUCPACU-48 h (at movement: 209.64 ± 28.98 vs. 212.59 ± 33.11; difference [90% CI], -2.948 [-13.236, 7.339]) were similar between the VC-ESPB and control groups within the first postoperative 48 h. The upper limit of the 90% CIs for the difference in cumulative ACUPACU-48 h at rest and at movement did not reach the upper inferiority margin (26.5). During the first postoperative 48 h, the rate of nonsteroidal anti-inflammatory drug rescue analgesia was similar between the VC-ESPB group and conventional group (n = 16, 32.0% vs. n = 11, 21.6%; P = 0.236). Treatment-related death was not observed in the VC-ESPB group (n = 0, 0%) and conventional group (n = 0, 0%). In VC-ESPB group, local site paralysis (n = 1, 2.0%) was observed in one patient and rash (n = 1, 2.0%) was observed in another patient. One patient in the conventional group was observed with rash preoperatively (n = 1, 2.0%). The VC-ESPB group had significantly lower rates of postoperative nausea (n = 2, 4.0%, vs. n = 9, 17.6%, P = 0.028), vomiting (n = 1, 2.0% vs. n = 8, 15.7%, P = 0.031) and lower incidence of major complications (n = 4, 8.0% vs. n = 6, 11.8%; P = 0.033). Interpretation: This study demonstrates the noninferiority of VC-ESPB when compared with the conventional opioid-based approach for postoperative pain management after OMH, suggesting that it is feasible to achieve opioid-free postoperative pain management for OMH. Funding: The Joint Funds for the Innovation of Science and Technology, Fujian Province, China; the Youth Scientific Research Project of Fujian Provincial Health Commission; the Fujian Research and Training Grants for Young and Middle-aged Leaders in Healthcare; and the Key Clinical Specialty Discipline Construction Program of Fujian, China.

Classification of left-side hepatolithiasis for laparoscopic middle hepatic vein-guided anatomical hemihepatectomy combined with transhepatic duct lithotomy.

BACKGROUND: Laparoscopic middle hepatic vein-guided anatomical hemihepatectomy combined with transhepatic duct lithotomy (MATL) is an approach that can substantially improve stone clearance rates while reducing the rate of postoperative biliary fistula formation, residual stone rates, and rates of recurrence. In this study, we classified left-side hepatolithiasis cases into four subtypes based upon the diseased stone-containing bile duct, the middle hepatic vein, and the right hepatic duct. We then investigated the risk associated with different subtypes and evaluated the safety and efficacy of the MATL procedure. METHODS: In total, 372 patients who underwent left hemihepatectomy for left intrahepatic bile duct stones were enrolled. Based on the distribution of the stones, the cases could be divided into four types. The risk of surgical treatment was compared for the four types and the safety, short-term efficacy, and long-term efficacy of the MATL procedure in the four types of left intrahepatic bile duct stones were studied. RESULTS: Type II was found to be the most likely to cause intraoperative bleeding while type III was likely to cause biliary tract damage and type IV was associated with the highest stone recurrence rate. The MATL procedure did not increase the risk of surgery and was found to reduce the rate of bile leakage, residual stones, and stone recurrence. CONCLUSION: Left-side hepatolithiasis-associated risk classification is feasible and may represent a viable means of improving the safety and feasibility of the MATL procedure.

Two machine learning-based nomogram to predict risk and prognostic factors for liver metastasis from pancreatic neuroendocrine tumors: a multicenter study.

BACKGROUND: Pancreatic neuroendocrine tumors (PNETs) are one of the most common endocrine tumors, and liver metastasis (LMs) are the most common location of metastasis from PNETS; However, there is no valid nomogram to predict the diagnosis and prognosis of liver metastasis (LMs) from PNETs. Therefore, we aimed to develop a valid predictive model to aid physicians in making better clinical decisions. METHODS: We screened patients in the Surveillance, Epidemiology, and End Results (SEER) database from 2010-2016. Feature selection was performed by machine learning algorithms and then models were constructed. Two nomograms were constructed based on the feature selection algorithm to predict the prognosis and risk of LMs from PNETs. We then used the area under the curve (AUC), receiver operating characteristic (ROC) curve, calibration plot and consistency index (C-index) to evaluate the discrimination and accuracy of the nomograms. Kaplan-Meier (K-M) survival curves and decision curve analysis (DCA) were also used further to validate the clinical efficacy of the nomograms. In the external validation set, the same validation is performed. RESULTS: Of the 1998 patients screened from the SEER database with a pathological diagnosis of PNET, 343 (17.2%) had LMs at the time of diagnosis. The independent risk factors for the occurrence of LMs in PNET patients included histological grade, N stage, surgery, chemotherapy, tumor size and bone metastasis. According to Cox regression analysis, we found that histological subtype, histological grade, surgery, age, and brain metastasis were independent prognostic factors for PNET patients with LMs. Based on these factors, the two nomograms demonstrated good performance in model evaluation. CONCLUSION: We developed two clinically significant predictive models to aid physicians in personalized clinical decision-makings.

Analyzing molecular typing and clinical application of immunogenic cell death-related genes in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is considered one of the most common cancers, characterized by low early detection and high mortality rates, and is a global health challenge. Immunogenic cell death (ICD) is defined as a specific type of regulated cell death (RCD) capable of reshaping the tumor immune microenvironment by releasing danger signals that trigger immune responses, which would contribute to immunotherapy. METHODS: The ICD gene sets were collected from the literature. We collected expression data and clinical information from public databases for the HCC samples in our study. Data processing and mapping were performed using R software to analyze the differences in biological characteristics between different subgroups. The expression of the ICD representative gene in clinical specimens was assessed by immunohistochemistry, and the role of the representative gene in HCC was evaluated by various in vitro assays, including qRT-PCR, colony formation, and CCK8 assay. Lasso-Cox regression was used to screen prognosis-related genes, and an ICD-related risk model (ICDRM) was constructed. To improve the clinical value of ICDRM, Nomograms and calibration curves were created to predict survival probabilities. Finally, the critical gene of ICDRM was further investigated through pan-cancer analysis and single-cell analysis. RESULTS: We identified two ICD clusters that differed significantly in terms of survival, biological function, and immune infiltration. As well as assessing the immune microenvironment of tumors in HCC patients, we demonstrate that ICDRM can differentiate ICD clusters and predict the prognosis and effectiveness of therapy. High-risk subpopulations are characterized by high TMB, suppressed immunity, and poor survival and response to immunotherapy, whereas the opposite is true for low-risk subpopulations. CONCLUSIONS: This study reveals the potential impact of ICDRM on the tumor microenvironment (TME), immune infiltration, and prognosis of HCC patients, but also a potential tool for predicting prognosis.

Textbook outcomes of hepatocellular carcinoma patients with sarcopenia: A multicenter analysis.

BACKGROUND: The impact of sarcopenia on textbook outcome (TO) after hepatectomy in hepatocellular carcinoma (HCC) patients remains unclear. This study aimed to investigate the association between sarcopenia and TO, to clarify its long and short-term prognostic value, and to develop a nomogram model based on sarcopenia and TO for survival prediction. METHODS: Patients who underwent HCC resection between January 2012 and March 2017 in three large hospitals in Fujian were retrospectively recruited and divided into sarcopenia and non-sarcopenia groups based on skeletal muscle index (SMI) values. TO was defined as no 30-day morality, no 30-day readmission, negative margins, no prolonged hospital stay, and no major complications. Multivariate regression was used to screen for clinical factors associated with TO. Nomograms of overall survival (OS) and recurrence-free survival (RFS) after hepatectomy for HCC were developed. RESULTS: A total of 1172 patients were included in the study. The TO rates were 28.74% (121/421 patients) in the sarcopenia group and 43.4% (326/751 patients) in the non-sarcopenia group. The results showed that sarcopenia was an independent predictor of TO (p < 0.001), TO was an independent predictor of perioperative treatment-related sarcopenia (PTRS)(p = 0.002), and TO was an independent predictor of OS and RFS (p < 0.001). Nomogram models based on sarcopenia and TO were generated and accurately predicted OS and RFS at 1, 3, and 5 years. CONCLUSION: Both sarcopenia and TO are independent predictors of OS and RFS after HCC resection. Sarcopenia was an independent predictor of TO. Sarcopenia influenced long-term survival by affecting short-term postoperative outcomes.

Optimization of a laparoscopic procedure for advanced intrahepatic cholangiocarcinoma based on the concept of "waiting time": a preliminary report.

INTRODUCTION: Clinicians increasingly perform laparoscopic surgery for intrahepatic cholangiocarcinoma (ICC). However, this surgery can be difficult in patients with advanced-stage ICC because of the complicated procedures and difficulty in achieving high-quality results. We compared the effects of a three-step optimized procedure with a traditional procedure for patients with advanced-stage ICC. METHODS: Forty-two patients with advanced-stage ICC who received optimized laparoscopic hemihepatectomy with lymph node dissection (LND, optimized group) and 84 propensity score-matched patients who received traditional laparoscopic hemihepatectomy plus LND (traditional group) were analyzed. Surgical quality, disease-free survival (DFS), and overall survival (OS) were compared. RESULTS: The optimized group had a lower surgical bleeding score (P = 0.038) and a higher surgeon satisfaction score (P = 0.001). Blood loss during hepatectomy was less in the optimized group (190 vs. 295 mL, P < 0.001). The optimized group had more harvested LNs (12.0 vs. 8.0, P < 0.001) and more positive LNs (8.0 vs. 5.0, P < 0.001), and a similar rate of adequate LND (88.1% vs. 77.4%, P = 0.149). The optimized group had longer median DFS (9.0 vs. 7.0 months, P = 0.018) and median OS (15.0 vs. 13.0 months, P = 0.046). In addition, the optimized group also had a shorter total operation time (P = 0.001), shorter liver resection time (P = 0.001), shorter LND time (P < 0.001), shorter hospital stay (P < 0.001), and lower incidence of total morbidities (14.3% vs. 36.9%, P = 0.009). CONCLUSIONS: Our optimization of a three-step laparoscopic procedure for advanced ICC was feasible, improved the quality of liver resection and LND, prolonged survival, and led to better intraoperative and postoperative outcomes.

Impact of sarcopenia on the surgical outcomes in patients with hepatolithiasis: A multicenter 10-year experience.

BACKGROUND: The impact of sarcopenia on the surgical outcomes of hepatectomy for hepatolithiasis has not been investigated. The present study elucidated the effect of sarcopenia on short-term outcomes after hemihepatectomy for hepatolithiasis and investigated the benefit of different surgical approaches to hepatectomy in patients with sarcopenia. METHODS: Patients who underwent hemihepatectomy for hepatolithiasis at Fujian Provincial Hospital and 5 other medical centers from 2010 to 2020 were enrolled. The sarcopenic obesity subgroup had sarcopenia coexisting with obesity, and the sarcopenic nonobesity subgroup had sarcopenia without obesity. We analyzed the postoperative outcomes of the sarcopenia group, sarcopenic obesity subgroup and sarcopenic nonobesity subgroup and the corresponding benefits of different surgical approaches. RESULTS: Patients with sarcopenia (n = 481) had worse surgical outcomes than nonsarcopenia, such as longer postoperative hospital duration of stay, longer time to oral intake, longer time to bowel movement, and longer time to off-bed activities. In postoperative short-term outcomes, we also found that sarcopenia had higher rates of major complications, bile leakage, and intensive care unit occupancy than the nonsarcopenic group. Subgroup analysis showed that sarcopenic obesity subgroup (n = 182) had the worst results in intraoperative outcomes and postoperative short-term outcomes. Multivariate analysis identified sarcopenic obesity as a significant risk factor for postoperative hospital duration of stay (hazard ratio = 2.994, P < .001). Furthermore, the sarcopenic obesity and sarcopenic nonobesity (n = 299) subgroups benefited from laparoscopic surgery compared with open surgery, including postoperative recovery and major complications (all P < .05). However, sarcopenic nonobesity subgroup had more significant benefits of laparoscopy than the sarcopenic obesity subgroup. The learning curve for laparoscopic hemihepatectomy for the sarcopenic obesity subgroup had a plateau, and the surgical outcomes of the sarcopenic obesity subgroup were closer to the sarcopenic nonobesity subgroup after the plateau. CONCLUSION: Sarcopenia is associated with more adverse events after hepatectomy and patients with sarcopenic obesity have a higher incidence of adverse events. Patients with sarcopenia could benefit from laparoscopy. Compared with the sarcopenic obesity patients, the sarcopenic nonobesity patients benefited more from laparoscopy. Although the sarcopenic obesity patients had more complications and slower postoperative recovery than the sarcopenic nonobesity patients, laparoscopic also could improve their short-term outcomes, but a longer learning curve was required.

Induced expression of CCL19 promotes the anti-tumor ability of CAR-T cells by increasing their infiltration ability.

Background: Chimeric antigen receptor-engineered T cell (CAR-T) therapy has shown promising potential for anti-cancer treatment. However, for pancreatic ductal adenocarcinoma (PDAC), the lack of infiltrative ability of these CAR-T cells leads to sub-optimal treatment outcome. Methods: Chemokine (C-C motif) ligand 19 (CCL19), the expression of which is regulated by the nuclear factor of activated T cell pathway, was transfected into targeting mesothelin CAR-T cells (mesoCAR-N19) using NFAT regulating element. It was expressed in activated CAR-T cells by OKT3 or mesothelin+ tumor cells but not in inactive cells. The migratory ability of these CAR-T cells was then measured. Subsequently, functional identification of these CAR-T cells was performed in vivo. In addition, the tumor lytic activity and proliferation of the CAR-T cells were measured in vitro. The degree of CAR-T cell infiltration and distribution into the PDAC tumors was examined using the immunohistochemical staining of hCD3 and the detection of CAR gene copy number by quantitative PCR. Finally, the functional assessment of chemokine (C-C motif) receptor 7 knock-out was performed in the CAR-T cells. Results: Through in vitro Transwell assays, it was demonstrated that mesoCAR-N19 can be specifically expressed in CAR-T cells activated by tumor cells compared with conventional mesothelin CAR-T (mesoCAR) cells. We also observed that upregulating the expression of CCL19 can increase the recruitment of additional T cells. In vivo studies subsequently revealed that this highly specific recruitment of T cell infiltration is associated with enhanced tumor-suppressive activities downstream. Conclusion: Induced expression of CCL19 can promote the anti-tumor ability of CAR-T cells by increasing their infiltrative ability. This study potentially uncovered novel method of activating CAR-T cells to enhance their infiltrative capacities, which offers a novel direction for PDAC treatment.

Laparoscopic segment 4b+5 liver resection for stage T3 gallbladder cancer.

BACKGROUND: There is still controversy over whether to perform laparoscopic surgery for T3 stage gallbladder cancer. In addition, the necessity of segment 4b+5 liver resection for stage T3 gallbladder has not been reported. This article aims to explore the safety, effectiveness, and short-term prognosis of laparoscopic segment 4b+5 liver resection for T3 stage gallbladder cancer. METHODS: This is a retrospective multicenter propensity score-matched study. Disease-free survival, perioperative complications, and intraoperative safety were analyzed to evaluate safety and effectiveness. RESULTS: There was no significant difference in the incidence of intraoperative bleeding, number of lymph nodes obtained, postoperative complications, or disease-free survival (DFS) between the open group (OG) and laparoscopic group (LG) (P > 0.05). The DFS time of the S4b+5 resection group (S4b5) was longer than that of the wedge group (P = 0.016). Cox regression showed that positive margins (HR, 5.32; 95% CI 1.03-27.63; P = 0.047), lymph node metastasis (HR, 2.70; 95% CI 1.31-5.53; P = 0.007), and liver S4b+5 resection (HR, 0.30; 95% CI 0.14-0.66; P = 0.003) were independent risk factors for DFS. The operative time of indocyanine green (ICG) fluorescence-guided liver S4b5 segment resection was shorter than that of traditional laparoscopic S4b+5 resection guided by hepatic veins (P ≤ 0.001). CONCLUSION: Laparoscopic liver S4b+5 resection for T3 stage gallbladder cancer is safe and feasible and can prolong DFS. ICG fluorescence-guided negative staining may reduce the difficulty of the operation.

SRSF3-mediated regulation of N6-methyladenosine modification-related lncRNA ANRIL splicing promotes resistance of pancreatic cancer to gemcitabine.

Serine/arginine-rich splicing factor 3 (SRSF3) regulates mRNA alternative splicing of more than 90% of protein-coding genes, providing an essential source for biological versatility. This study finds that SRSF3 expression is associated with drug resistance and poor prognosis in pancreatic cancer. We also find that SRSF3 regulates ANRIL splicing and m6A modification of ANRIL in pancreatic cancer cells. More importantly, we demonstrate that m6A methylation on lncRNA ANRIL is essential for the splicing. Moreover, our results show that SRSF3 promotes gemcitabine resistance by regulating ANRIL's splicing and ANRIL-208 (one of the ANRIL spliceosomes) can enhance DNA homologous recombination repair (HR) capacity by forming a complex with Ring1b and EZH2. In conclusion, this study establishes a link between SRSF3, m6A modification, lncRNA splicing, and DNA HR in pancreatic cancer and demonstrates that abnormal alternative splicing and m6A modification are closely related to chemotherapy resistance in pancreatic cancer.

Circular RNA circ-MTHFD1L induces HR repair to promote gemcitabine resistance via the miR-615-3p/RPN6 axis in pancreatic ductal adenocarcinoma.

BACKGROUND: Chemoresistance of pancreatic cancer is the main reason for the poor treatment effect of pancreatic cancer patients. Exploring chemotherapy resistance-related genes has been a difficult and hot topic of oncology. Numerous studies implicate the key roles of circular RNAs (circRNAs) in the development of pancreatic cancer. However, the regulation of circRNAs in the process of pancreatic ductal adenocarcinoma (PDAC) chemotherapy resistance is not yet fully clear. METHODS: Based on the cross-analysis of the Gene Expression Omnibus (GEO) database and the data of our center, we explored a new molecule, hsa_circ_0078297 (circ-MTHFD1L), related to chemotherapy resistance. QRT-PCR was used to detect the expression of circRNAs, miRNAs, and mRNAs in human PDAC tissues and their matched normal tissues. The interaction between circ-MTHFD1L and miR-615-3p/RPN6 signal axis was confirmed by a series of experiments such as Dual-luciferase reporter assay, fluorescence in situ hybridization (FISH) RNA immunoprecipitation (RIP) assays. RESULTS: Circ-MTHFD1L was significantly increased in PDAC tissues and cells. And in PDAC patients, the higher the expression level of circ-MTHFD1L, the worse the prognosis. Mechanism analysis showed that circ-MTHFD1L, as an endogenous miR-615-3p sponge, upregulates the expression of RPN6, thereby promoting DNA damage repair and exerting its effect on enhancing gemcitabine chemotherapy resistance. More importantly, we also found that Silencing circ-MTHFD1L combined with olaparib can increase the sensitivity of pancreatic cancer to gemcitabine. CONCLUSION: Circ-MTHFD1L maintains PDAC gemcitabine resistance through the miR-615-3p/RPN6 signal axis. Circ-MTHFD1L may be a molecular marker for the effective treatment of PDAC.

Construction and Validation of Novel Nomograms for Predicting Prognosis of Pancreatic Ductal Adenocarcinoma After Surgery According to Different Primary Cancer Locations.

BACKGROUND/AIMS: Pancreatic ductal adenocarcinoma (PDAC) can occur in different parts of the pancreas. This study aimed to identify clinicopathological characteristics independently correlated with the prognosis of PDAC of the pancreatic head/uncinate (PHC) or body-tail (PBTC), and to develop novel nomograms for predicting cancer-specific survival (CSS) according to different primary cancer locations. METHODS: 1160 PDAC patients were retrospectively enrolled and assigned to training and test sets with each set divided into PHC and PBTC groups. Comparative analysis of clinicopathologic characteristics, survival analysis, and multivariate analysis were performed. Independent factors were identified and used for constructing nomograms. The performance of the nomograms was validated in the test set. RESULTS: Primary tumor location was an independent risk factor for prognosis of PDAC after surgery. Specially, gender, fasting blood glucose, and preoperative cancer antigen 19-9 were significantly associated with prognosis of PHC, whereas age, body mass index, and lymph nodes were significantly correlated with the prognosis of PBTC. A significant difference in prognosis was found between PHC and PBTC in stage Ia and stage III. Three nomograms were established for predicting the prognosis for PDAC, PHC, and PBTC. Notably, these nomograms were calibrated modestly (c-indexes of 0.690 for PDAC, 0.669 for PHC, and 0.704 for PBTC), presented better accuracy and reliability than the 8th AJCC staging system, and achieved clinical validity. CONCLUSIONS: PHC and PBTC share the differential clinical-pathological characteristics and survival. The nomograms show good performance for predicting prognosis in PHC and PBTC. Therefore, these nomograms hold potential as novel approaches for predicting survival of PHC and PBTC patients after surgery.

Prognostic value and association of sarcopenic obesity and systemic inflammatory indexes in patients with hepatocellular carcinoma following hepatectomy and the establishment of novel predictive nomograms.

BACKGROUND: The specific impacts of sarcopenic obesity (SO) on hepatocellular carcinoma (HCC) and the association between SO and systemic inflammation remain unclear. This study aimed to investigate the prognostic value and association of SO and systemic inflammation with outcomes after hepatectomy for HCC and develop novel nomograms based on SO and inflammatory indexes for survival prediction. METHODS: We retrospectively enrolled 452 patients with HCC who underwent radical hepatectomy between January 2012 and March 2015 in Fujian Provincial Hospital as the training cohort. In addition, 275 patients during the same period were enrolled as the external validation cohort. Patients were classified into different groups according to the presence of sarcopenia and obesity. Different inflammation indexes were evaluated to select the best predictor of overall survival (OS) and recurrence-free survival (RFS). Univariate and multivariate logistic regression were performed to investigate the associations between inflammatory indexes and SO. The inflammatory indexes with the highest predictive values and SO were selected for subgroup analyses to establish a novel classification system: the SOLMR grade. SOLMR grades identified in the multivariate Cox analysis were selected to construct novel nomograms for OS and RFS. RESULTS: SO (P<0.001) was an independent risk factor for OS and RFS. The lymphocyte-monocyte ratio (LMR) had the highest areas under the receiver operating characteristic (ROC) curves (AUCs) for OS (P<0.001) and RFS (P<0.001) and was identified as an independent factor of SO (P=0.001). SO and the LMR were selected to establish the SOLMR grade. Multivariate Cox analysis revealed that SOLMR grade was a significant independent predictor of OS (P<0.001) and RFS (P<0.001). Nomograms based on SOLMR grades were generated and accurately predicted 1-, 3- and 5-year OS and RFS in HCC patients. The C-index of the novel nomograms was higher than those of the other conventional staging systems (P<0.001). CONCLUSIONS: Both SO and the LMR were independent risk factors for OS and RFS in HCC patients after hepatectomy. The LMR was an independent factor of SO. The novel nomograms developed from the SOLMR grading system combining SO with the LMR provide good prognostic estimates of the outcomes of HCC patients.

CLK1/SRSF5 pathway induces aberrant exon skipping of METTL14 and Cyclin L2 and promotes growth and metastasis of pancreatic cancer.

BACKGROUND: Both aberrant alternative splicing and m6A methylation play complicated roles in the development of pancreatic cancer (PC), while the relationship between these two RNA modifications remains unclear. METHODS: RNA sequencing (RNA-seq) was performed using 15 pairs of pancreatic ductal adenocarcinoma (PDAC) tissues and corresponding normal tissues, and Cdc2-like kinases 1 (CLK1) was identified as a significantly upregulated alternative splicing related gene. Real-time quantitative PCR (qPCR) and western blotting were applied to determine the CLK1 levels. The prognostic value of CLK1 was elucidated by Immunohistochemistry (IHC) analyses in two independent PDAC cohorts. The functional characterizations and mechanistic insights of CLK1 in PDAC growth and metastasis were evaluated with PDAC cell lines and nude mice. SR-like splicing factors5250-Ser (SRSF5250-Ser) was identified as an important target phosphorylation site by phosphorylation mass spectrometry. Through transcriptome sequencing, Methyltransferase-like 14exon10 (METTL14exon10) and Cyclin L2exon6.3 skipping were identified as key alternative splicing events regulated by the CLK1-SRSF5 axis. RIP assays, RNA-pulldown and CLIP-qPCR were performed to confirm molecular interactions and the precise binding sites. The roles of the shift of METTL14exon 10 and Cyclin L2exon6.3 skipping were surveyed. RESULTS: CLK1 expression was significantly increased in PDAC tissues at both the mRNA and protein levels. High CLK1 expression was associated with poor prognosis. Elevated CLK1 expression promoted growth and metastasis of PC cells in vitro and in vivo. Mechanistically, CLK1 enhanced phosphorylation on SRSF5250-Ser, which inhibited METTL14exon10 skipping while promoted Cyclin L2exon6.3 skipping. In addition, aberrant METTL14exon 10 skipping enhanced the N6-methyladenosine modification level and metastasis, while aberrant Cyclin L2exon6.3 promoted proliferation of PDAC cells. CONCLUSIONS: The CLK1/SRSF5 pathway induces aberrant exon skipping of METTL14 and Cyclin L2, which promotes growth and metastasis and regulates m6A methylation of PDAC cells. This study suggests the potential prognostic value and therapeutic targeting of this pathway in PDAC patients.

A new strategy of laparoscopic anatomical hemihepatectomy guided by the middle hepatic vein combined with transhepatic duct lithotomy for complex hemihepatolithiasis: A propensity score matching study.

BACKGROUND: There have been no studies on laparoscopic anatomical hemihepatectomy guided by the middle hepatic vein combined with transhepatic duct lithotripsy for the treatment of complex hemihepatolithiasis. This study aimed to investigate the safety and efficacy of laparoscopic anatomical hemihepatectomy guided by the middle hepatic vein combined with transhepatic duct lithotomy to treat complex hemihepatolithiasis. METHODS: The clinical data for patients who underwent laparoscopic anatomical hemihepatectomy for complex intrahepatic bile duct stones with or without common bile duct stones from January 2016 to June 2020 were prospectively collected. Patients were divided into 2 groups according to surgical approach: laparoscopic anatomical hemihepatectomy guided by the middle hepatic vein (middle hepatic vein group) or laparoscopic anatomical hemihepatectomy not guided by the middle hepatic vein (nonmiddle hepatic vein group). The safety and short-term and long-term efficacy outcomes of the 2 groups were compared with 1:1 propensity score matching. RESULTS: With only a slightly longer operative time (P = .006), the initial and final stone residual rates in the middle hepatic vein group (n = 70) were significantly lower than those in the nonmiddle hepatic vein group (n = 70) (P = .002, P = .009). The bile leakage rate and stone recurrence rate were also significantly lower (P = .001, P = .001). CONCLUSION: Laparoscopic anatomical hemihepatectomy guided by the middle hepatic vein is safe and effective for treating intrahepatic bile duct stones and can decrease the stone residual rate, reduce the bile leakage rate and stone recurrence rate, and accelerate early recovery. However, owing to the complicated technical requirements for surgeons and anesthesiologists, use of the procedure is limited to large and experienced medical centers.

SHCBP1 interacting with EOGT enhances O-GlcNAcylation of NOTCH1 and promotes the development of pancreatic cancer.

O-GlcNAcylation is important in the development and progression of pancreatic ductal adenocarcinoma (PDAC). The glycosyltransferase EGF domain-specific O-linked GlcNAc transferase (EOGT) acts as a key participant in glycosylating NOTCH1. High-throughput sequencing of specimens from 30 advanced PDAC patients identified SHCBP1 and EOGT as factors of poor prognosis. We hypothesized that they could mediate PDAC progression by influencing NOTCH1 O-GlcNAcylation. Thus, 186 PDAC tissue specimens were immunostained for EOGT and SHCBP1. Pancreatic cancer cell lines and nude mouse models were used for in vitro and in vivo experiments. Respectively, The protein expression of EOGT and SHCBP1 was significantly elevated and correlated with worse prognosis in PDAC patients. In vitro, SHCBP1 overexpression promoted pancreatic cancer cell proliferation, migration and invasion, while knocking down SHCBP1 and EOGT inhibited these malignant processes. In vivo data showed that SHCBP1 overexpression promoted xenograft growth and lung metastasis and shortened survival in mice, whereas knocking down either EOGT or SHCBP1 expression suppressed xenograft growth and metastasis and prolonged survival. We further clarified the molecular mechanisms by which EOGT and SHCBP1 enhance the O-GlcNAcylation of NOTCH1, Subsequently promoting the nuclear localization of the Notch intracellular domain (NICD) and inhibiting the transcription of E-cadherin and P21 in pancreatic cancer cells.