Increased co-expression of PD1 and TIM3 is associated with poor prognosis and immune microenvironment heterogeneity in gallbladder cancer.

BACKGROUND: The effectiveness of immune checkpoint inhibitors in treating gallbladder cancer (GBC) remains unsatisfactory. Recently, several new immune checkpoints have been identified. However, investigations exploring these immune checkpoints in GBC are limited. In this study, we aim to investigate the expression patterns and clinical implications of various immune checkpoints, and further characterize the spatial and quantitative heterogeneity of immune components in GBC. METHODS: We employed single and multiplex immunohistochemistry to evaluate the expression of five immune checkpoint markers and four immune cell markers in the primary tumor core, hepatic invasion margin, and liver metastasis. Subsequently, we analyzed their interrelationships and their prognostic significance. RESULTS: We observed a robust positive correlation between PD1/TIM3 expression in GBC (R = 0.614, P < 0.001). The co-expression of PD1/TIM3 exhibited a synergistic effect in predicting poor prognosis among postoperative GBC patients. Further analysis revealed that the prognostic significance of PD1/TIM3 was prominent in the subgroup with high infiltration of CD8 + T cells (P < 0.001). Multiplex immunohistochemistry reveals that PD1 + TIM3 + FOXP3 + cells constitute a significant proportion of FOXP3 + TILs in GBC tissue. Moreover, the co-high expression of PD1 and TIM3 is positively correlated with the accumulation of CD8 + TILs at the hepatic invasion margin. Lastly, our findings indicated reduced expression levels of immune checkpoints and diminished immune cell infiltration in liver metastases compared to primary tumors. CONCLUSIONS: Increased co-expression of PD1/TIM3 is associated with poor prognosis in GBC patients and is related to the heterogeneity of immune microenvironment between GBC primary tumor and its hepatic invasion margin or liver metastases, which may be a potential target for future immunotherapy of GBC.

Immune Checkpoint Inhibitor-Based Systemic Therapy Shows Remarkable Curative Effect in a Hepatocellular Carcinoma Patient With Intractable Postoperative Recurrence and Metastases: A Case Report and Literature Review.

Hepatocellular carcinoma (HCC) is a systemic disease, and most patients make the diagnosis at an advanced stage. In the past, treatments for recurrence of liver cancer with multiple metastases after surgery was very palliative, The case we present is a primary massive HCC patient with inferior vena cava tumor thrombus. Radical hepatectomy was performed in July 2016. Postoperative follow-up showed that sorafenib (a tyrosine kinase inhibitor TKI, 0.8g qd) failed to stop the progression of the disease. Fourteen months later, the patient gradually developed residual liver recurrence, multiple lung metastases and suspected splenic metastasis. The monotherapy regimen was changed from sorafenib to regorafenib (a TKI,160mg qd), but the disease continued to progress. The systematic treatment regimen was changed to Lenvatinib (a TKI, 8mg qd) plus Pembrolizumab (a immune checkpoint inhibitor ICI, 200mg q3w) in April 2019. Following treatment, partial remission (PR) was achieved. According to the mRECIST standard, the PFS has reached 24 months until March 2021, and the overall postoperative survival is 60 months until July 2021. The case we provide show that immune checkpoint inhibitor (ICI)-based systemic therapy may be an effective rescue treatment choice for HCC patients with intractable postoperative recurrence and metastasis.

SMYD3 promotes hepatocellular carcinoma progression by methylating S1PR1 promoters.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. SET and MYND domain-containing protein 3 (SMYD3) has been shown to promote the progression of various types of human cancers, including liver cancer; however, the detailed molecular mechanism is still largely unknown. Here, we report that SMYD3 expression in HCC is an independent prognostic factor for survival and promotes the proliferation and migration of HCC cells. We observed that SMYD3 upregulated sphingosine-1-phosphate receptor 1 (S1PR1) promoter activity by methylating histone 3 (H3K4me3). S1PR1 was expressed at high levels in HCC samples, and high S1PR1 expression was associated with shorter survival. S1PR1 expression was also positively correlated with SMYD3 expression in HCC samples. We confirmed that SMYD3 promotes HCC cell growth and migration in vitro and in vivo by upregulating S1PR1 expression. Further investigations revealed that SMYD3 affects critical signaling pathways associated with the progression of HCC through S1PR1. These findings strongly suggest that SMYD3 has a crucial function in HCC progression that is partially mediated by histone methylation at the downstream gene S1PR1, which affects key signaling pathways associated with carcinogenesis and the progression of HCC.

circFOXM1 contributes to sorafenib resistance of hepatocellular carcinoma cells by regulating MECP2 via miR-1324.

As one of the most common malignant tumors, hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths around the world. Emerging studies have indicated that circular RNAs (circRNAs), which play a crucial role in HCC pathogenesis and metastasis, are differentially expressed in HCC. However, the regulatory mechanisms of circRNA on sorafenib resistance of HCC are still unknown. In our study, we identified a novel circRNA, circFOXM1, using RNA sequencing (RNA-seq) that was increased in sorafenib-resistant HCC tissues. Functionally, circFOXM1 significantly inhibited HCC growth and enhanced sorafenib toxicity in vitro. Mechanistically, circFOXM1 acted as a sponge of microRNA (miR)-1324, which is a negative regulator of MECP2, indicating that circFOXM1 downregulation would regulate sorafenib resistance of HCC via releasing more free miR-1324 and suppressing MECP2 expression. Furthermore, miR-1324 overexpression was capable of reversing the circFOXM1-induced malignant phenotypes and elevated expression of MECP2 in HCC cells. circFOXM1 partially contributed to sorafenib resistance of HCC cells through upregulating MECP2 expression by sponging miR-1324.

PROM2 promotes gemcitabine chemoresistance via activating the Akt signaling pathway in pancreatic cancer.

In recent years, the deoxycytidine analogue gemcitabine (2',2',-difluorodeoxycytidine) has become the first-line chemotherapeutic agent for patients with pancreatic cancer. However, due to the intrinsic resistance of pancreatic cancer cells, gemcitabine-based chemotherapy yields limited disease control, with >85% disease progression at 6 months from diagnosis. Therefore, elucidating the mechanisms of chemoresistance is a critical step in improving cancer therapy, especially for the treatment of pancreatic cancer. We show PROM2, a transmembrane glycoprotein, is ubiquitously upregulated in pancreatic cancer cell. We also found higher PROM2 expression is associated with shortened overall and disease-free survival times in patients diagnosed with pancreatic cancer. We provide evidence that PROM2 promotes chemoresistance to gemcitabine both in vivo and in vitro. Mechanistically, we demonstrate that PROM2 could directly interacted with Akt and activates the Akt signaling pathway, which thus inhibiting gemcitabine-induced apoptosis. As further evidence, we show PROM2 expression and Akt phosphorylation both promote gemcitabine chemoresistance, and cause poorer survival in clinical samples with pancreatic cancer. Combining gemcitabine with the Akt inhibitor MK-2206 facilitated significant tumor shrinkage and dramatically elevated the survival status in mice xenografted with pancreatic cancer cells. Our findings not only establish PROM2 as a novel positive regulator of the Akt signaling pathway and a candidate prognostic indicator of gemcitabine response, but also provide a neo-therapeutic approach for patients resistant to gemcitabine treatment.

Effect of lincRNA-p21 targeting HIF-1α on biological functions of liver cancer cells.

The effects of long intergenic non-coding ribonucleic acid (lincRNA)-p21 targeting hypoxia-inducible factor-1α (HIF-1α) on proliferation, apoptosis and migration of liver cancer cells were investigated. MHCC97H liver cancer cells were infected with control lentivirus (control group) and lincRNA-p21 lentivirus (observation group), and control stable cell lines and lincRNA-p21 stable cell lines were screened and obtained by using puromycin. The expression levels of lincRNA-p21 messenger RNA (mRNA) in the control and observation groups were analyzed via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Bioinformatics was used to search for the lincRNA-p21 target. The expression of target gene was analyzed via western blotting, and the proliferation, apoptosis, migration and in vivo tumor formation of MHCC97H cells in the control and observation groups were also analyzed. Compared with that in control group, the lincRNA-p21 mRNA level in observation group was increased significantly (P<0.05). It was found via bioinformatic comparison that HIF-1α was one of the targets of lincRNA-p21. Results of Western blotting revealed that the expression level of HIF-1α protein in cells in observation group was significantly downregulated (P<0.05). Besides, the level of vascular endothelial growth factor (VEGF) protein in cells in control group was obviously higher than that in observation group (P<0.05). Compared with those in control group, the cell proliferation and migration capacities in observation group were markedly reduced, but the apoptosis level was significantly increased (P<0.05). According to the in vivo tumor formation assay, the cell proliferation rate in control group was obviously higher than that in observation group (P<0.05). The number of tumor blood vessels in cells in control group was obviously reduced compared with that in observation group (P<0.05). lincRNA-p21 can significantly downregulate the level of HIF-1α, thus downregulating the expression of VEGF and affecting the cell proliferation, apoptosis and migration.

CCL18 secreted from M2 macrophages promotes migration and invasion via the PI3K/Akt pathway in gallbladder cancer.

PURPOSE: The presence of M2 macrophages within primary tumors has been correlated with a poor prognosis for many types of cancer. However, little is known about the role of M2 macrophages in gallbladder cancer (GBC). METHODS: The number of M2 macrophages in 78 GBC and 16 normal gallbladder tissue samples was assessed by immunohistochemistry. The THP-1 monocyte cell line was differentiated into M2 macrophages and co-cultured with GBC-derived cell lines. The effect of M2 macrophages on promoting GBC cell migration and invasion was analyzed using migration, invasion and scratch wound healing assays. Western blotting and real-time PCR were used to assess the expression of epithelial-mesenchymal transition (EMT) markers and the activation status of the PI3K/Akt signaling pathway in GBC cells co-cultured with THP-1-derived macrophages. RESULTS: The average number of M2 macrophages was found to be significantly higher in GBC tissues than in normal gallbladder tissues. We also found that GBC patients with higher M2 macrophage counts exhibited poorer overall survival rates. Co-culture with M2 macrophages significantly promoted the migration, invasion and EMT of GBC cells. Moreover, we found that CCL18 secreted from M2 macrophages had the same effect on GBC cells as M2 macrophages. Blocking the function of CCL18 with a neutralizing antibody reversed this effect. Finally, we found that M2 macrophages could activate PI3K/Akt signaling in GBC cells, thereby leading to migration, invasion and EMT of these cells. CONCLUSIONS: Our findings contribute to our understanding of the role of chronic inflammation in GBC development and progression, and may offer potential therapeutic targets for GBC.

Safety and efficacy of dehydrated ethanol soaking of the operative field in the treatment of spontaneous hepatocellular carcinoma rupture.

BACKGROUND: The aim of our study was to evaluate the clinical safety and value of ethanol surgical field infiltration (ESFI), combined with distilled water peritoneal lavage (DWPL), after hepatectomy in patients with hepatocellular carcinoma (HCC) rupture. METHODS: Rat liver tissue samples were soaked in dehydrated ethanol for different soaking times, and 18 rats were assigned to three groups that underwent different soaking methods of the hepatectomy cut surface. We retrospectively reviewed 45 patients who underwent hepatectomy for treatment of ruptured HCC. Among these, EFSI combined with DWPL was used in 21 patients (DAW group), with only DWPL used in the other 24 patients (DW group). Clinical outcomes were compared between the two groups. RESULTS: For in vitro experiments, the depth of coagulation degeneration and necrosis increased with the duration of soaking. For in vivo experiments, rats in all three groups survived until postoperative day 7 without significant postoperative complication. In patients, the rate of post-operation complication was comparable between the two groups (P = 0.398), with no between-group differences in liver function levels. The incidence of peritoneal dissemination was significantly higher for DW than DAW group (P = 0.037). Kaplan-Meier test identified dehydrated ethanol treatment as a significant factor of disease-free survival (DFS) (P = 0.036). On univariate analysis, dehydrated ethanol treatment was associated with better prognostic outcomes, although it was not retained as an independent factor of patient outcome. CONCLUSIONS: Dehydrated ethanol soaking of the cut surface of the hepatectomy could potentially lower the risk of metastasis and improve the effect of hepatectomy for ruptured HCC as well as showed potential therapeutic value for intraoperative iatrogenic rupture of HCC.

CCL18/PITPNM3 enhances migration, invasion, and EMT through the NF-κB signaling pathway in hepatocellular carcinoma.

Chemokine ligand 18 (CCL18) has been associated with hepatocellular carcinoma (HCC) metastasis. Here, we demonstrated a novel mechanism through which CCL18 enhances cell migration, invasion, and epithelial-mesenchymal transition (EMT) in HCC. (1) Using immunohistochemistry, we analyzed the expression of PITPNM3, a molecule that correlated with CCL18 signaling, in 149 HCC tissue specimens. The results showed that PITPNM3 expression is highly associated with tumor metastasis and differentiation; (2) in vitro experiments showed that CCL18 enhances cell migration, invasion, and EMT in PITPNM3((+)) HCC cells but not in PITPNM3((-)) cells. Silencing of PITPNM3 by short interfering RNA (siRNA) inhibited the induction of cell migration, invasion, and EMT by CCL18; (3) Cell migration, invasion, and EMT induced by CCL18 accompanied with the phosphorylation of IKK and IKBα as well as p65 nuclear translocation in PITPNM3((+)) HCC cells, but not in the cells that PITPNM3 is silenced with siRNA, implying that the activation of NF-κB signaling is involved in the action of CCL18/PITPNM3. These results suggest that CCL18 enhances HCC cell migration, invasion, and EMT through the expression of PITPNM3 and the activation of the NF-κB signaling pathway.

Long-term results of the paraesophagogastric devascularization with or without esophageal transection: which is more suitable for variceal bleeding?

BACKGROUND: It has been reported that the paraesophagogastric devascularization with esophageal transection procedure, also known as the modified Sugiura procedure, was effective in the treatment of variceal bleeding. However, it was not widely accepted by other surgeons because of the high rate of rebleeding, complications, and mortality. To discover the effects of the paraesophagogastric devascularization procedure and the modified Sugiura procedure, we retrospectively analyzed the outcomes of these two procedures. MATERIALS AND METHODS: During January 1990 and December 2009, 278 patients with variceal bleeding underwent devascularization after failed pharmacotherapy and endotherapy. In these 278 patients, 180 underwent paraesophagogastric devascularization without esophageal transection (group I), and the other 98 patients were subjected to the modified Sugiura procedure (group II). RESULTS: Postoperative mortality was 7.2% in group I, and 9.2% in group II (P = 0.563). The postoperative rebleeding rate in the two groups was 2.2 and 3.1%, respectively (P = 0.474). After a mean follow-up of 67.9 ± 37.3 months and 67.4 ± 44.6 months, respectively, esophageal transaction-related morbidity (leak, bleeding, and stricture) was 8.2% (8/98) in group II and 0% (0/180) in group I (P < 0.001). The overall rebleeding rate was 27% (41/152) in group I, and 27.2% (22/81) in group II (P = 0.976). The overall mortality was 28.3% (43/152) in group I, and 28.4% (23/81) in group II (P = 0.986). CONCLUSIONS: In the management of variceal bleeding, paraesophagogastric devascularization without esophageal transection is as effective and safe as devascularization with esophageal transaction, but with less esophageal transection-related morbidity.