Exosomes in the tumor microenvironment of cholangiocarcinoma: current status and future perspectives.

Cholangiocarcinoma (CCA) refers to an aggressive malignancy with a high fatality rate and poor prognosis. Globally, the morbidity of CCA is increasing for the past few decades, which has progressed into a disease that gravely endangers human health. Exosomes belong to a class of extracellular vesicles (EVs) with diameters ranging from 40 to 150 nm that can be discharged by all living cells. As communication messengers of the intercellular network, exosomes carry a diverse range of cargoes such as proteins, nucleic acids, lipids, and metabolic substances, which are capable of conveying biological information across different cell types to mediate various physiological activities or pathological changes. Increasing studies have demonstrated that exosomes in the tumor microenvironment participate in regulating tumorigenesis and progression via multiple approaches in the tumor microenvironment. Here, we reviewed the current research progress of exosomes in the context of cancer and particularly highlighted their functions in modulating the development of CCA. Furthermore, the potential values of exosomes as diagnostic and therapeutic targets in CCA were overviewed as well.

Single-cell landscape of immunocytes in patients with extrahepatic cholangiocarcinoma.

BACKGROUND: The intricate landscape of immunocytes in the tumor microenvironment (TME) is fundamental to immunotherapy but notably under-researched in extrahepatic cholangiocarcinoma (ECCA). METHODS: Single-cell RNA sequencing technology was conducted to make an in-depth analysis of immunocytes from matched tumor tissues, paratumor tissues and peripheral blood from ECCA patients. The potential cellular interactions between two cell populations were analyzed with software CellPhoneDB (v2.1.7). RESULTS: We obtained 13526 cells and characterized the transcriptomes and heterogeneity of different clusters and subclusters of immunocytes from ECCA, including CD4+ T cells, CD8+ T cells, B cells and myeloid immunocytes. We observed the rarely described immunocyte subclusters "intermediate" exhausted CD8+ T (CD8+ Tex) cells and "nonclassic" plasmacytes (CD27+ CD138+ CD38-). In addition, we identified potential immunotherapy targets, for example, ACP5, MAGEH1, TNFRSF9 and CCR8 for Tregs and MT1 for CD8+ Tex cells. We also found strong cellular interactions among Treg cells, M2 macrophages and CD8+ Tex cells through ligand-receptor analysis, implying that potential cellular cross-linkage promoted the immunosuppressive nature of the TME. CONCLUSIONS: In a word, our study illuminated the components of the TME and revealed potential cellular interactions at the individual cellular level in ECCA, we aimed to provide a new perspective for further immunological studies and immunotherapy of ECCA.

Prognostic value of periostin in multiple solid cancers: A systematic review with meta-analysis.

Previous studies have shown that the expression of periostin (POSTN) is significantly correlated with prognosis in multiple solid cancers. However, the function of POSTN in tumorigenesis and its relationship with clinical outcomes have not been systematically summarized and analyzed. Thus, a meta-analysis was performed to evaluate the prognostic pertinence of POSTN in solid cancer. We conducted a systematic search in the PubMed, EMBASE, Web of Science, and Cochrane library databases, and a total of 10 studies were used to assess the association of POSTN expression and patients' overall survival (OS) and disease-free survival (DFS). The hazard ratio (HR) or odds ratio (OR) and their corresponding 95% confidence intervals (95% CIs) were further calculated to estimate the association between POSTN and relevant clinical parameters of solid cancer patients. The pooled results indicated that POSTN overexpression was associated with poor OS (HR = 2.35, 95% CI = 1.88-2.93, p < .00001) and DFS (HR = 2.70, 95% CI = 2.00-3.65, p < .00001) in a cohort of 993 patients with cancer. Subsequent analyses showed that the positive expression ratio of POSTN was evidently higher in cancer tissues than in normal tissues (OR = 7.44, 95% CI = 3.66-13.95, p < .00001). In addition, subgroup analysis showed that POSTN was related to microvascular invasion (OR = 5.09, 95% CI = 3.07-8.44, p < .00001), tumor differentiation (OR = 2.03, 95% CI = 1.41-2.91, p = .0001), and lymph node metastasis (OR = 3.05, 95% CI = 2.01-4.64, p < .00001). These data showed that POSTN could be a credible prognostic biomarker and a potential therapeutic target in human solid cancer.

Oxidative stress and histopathological alterations in liver of Cyprinus carpio L. induced by intraperitoneal injection of microcystin-LR.

Microcystins (MCs) are a group of cyclic heptapeptide hepatotoxic peptides produced by cyanobacteria. Microcystins-LR (MC-LR) can inhibit the activities of protein phosphatase type 1 and type 2A (PP1 and PP2A) and induce excessive production of reactive oxygen species (ROS). However, the detailed toxicological mechanism involving oxidative stress in carp (Cyprinus carpio L.) remains largely unclear. In our present study, the effects of sublethal intraperitoneal doses of MC-LR on the oxidative stress and pathological changes in carp liver were investigated. No significant changes of xanthine oxidase were observed, suggesting it might not contribute to over-production of ROS in the liver of fish during 48 h exposure to sublethal intraperitoneal doses of MC-LR. Superoxide dismutase activity in the 50 µg kg(-1) group was significantly induced at 1-24 h. The strongest inhibition of the catalase activity was shown at 48 h after 120 µg kg(-1) MC-LR exposure, with an inhibition rate of 33.7% compared to the control group. In general, a significant depletion of intracellular reduced glutathione was found at 5-12 h after 50 and 120 µg kg(-1) MC-LR exposure, which was mainly due to the conjugation reaction to MC-LR catalyzed by glutathione-S-transferase and its subsequent excretion. Oxidative damages induced by MC-LR were evidenced by the significant elevation in malondialdehyde levels. In addition, a series of histopathological alterations in fish livers were observed, and the most severe hepatic injuries were found at 5-12 h, which could contribute to the efflux of intracellular GSH. Our study further supports the important role of oxidative stress involved in MC-LR induced liver injury in aquatic organisms.

Identifying a novel cuproptosis-related necroptosis gene subtype-related signature for predicting the prognosis, tumor microenvironment, and immunotherapy of hepatocellular carcinoma.

Background: Cuproptosis and necroptosis represent two distinct programmed cell death modalities implicated in neoplastic progression; however, the role of combining cuproptosis and necroptosis in hepatocellular carcinoma (HCC) remains to be elucidated. Methods: A total of 29 cuproptosis-related necroptosis genes (CRNGs) were identified, followed by an extensive analysis of their mutational characteristics, expression patterns, prognostic implications, and associations with the tumor microenvironment (TME). Subsequently, a CRNG subtype-related signature was developed, and its value of prognostic prediction, TME, and therapeutic responses in HCC were thoroughly investigated. Last, quantitative real-time PCR and Western blotting were employed for investigating the signature gene expression in 15 paired clinical tissue samples. Results: Two distinct CRNG subtypes were discerned, demonstrating associations between CRNG expression patterns, clinicopathological attributes, prognosis, and the TME. A CRNG subtype-related prognostic signature, subjected to external validation, was constructed, serving as an independent prognostic factor for HCC patients, indicating poor prognosis for high-risk individuals. Concurrently, the signature's correlations with an immune-suppressive TME, mutational features, stemness properties, immune checkpoint genes, chemoresistance-associated genes, and drug sensitivity were observed, signifying its utility in predicting treatment responses. Subsequently, highly accurate and clinically convenient nomograms were developed, and the signature genes were validated via quantitative real-time PCR and Western blotting, further substantiating the stability and dependability of the CRNG subtype-related prognostic signature. Conclusion: Overall, this investigation presented an extensive panorama of CRNGs and developed the CRNG subtype-related prognostic signature, which holds potential for implementation in personalized treatment strategies and prognostic forecasting for HCC patients.

Identifying the programmed cell death index of hepatocellular carcinoma for prognosis and therapy response improvement by machine learning: a bioinformatics analysis and experimental validation.

Background: Despite advancements in hepatocellular carcinoma (HCC) treatments, the prognosis for patients remains suboptimal. Cumulative evidence suggests that programmed cell death (PCD) exerts crucial functions in HCC. PCD-related genes are potential predictors for prognosis and therapeutic responses. Methods: A systematic analysis of 14 PCD modes was conducted to determine the correlation between PCD and HCC. A novel machine learning-based integrative framework was utilized to construct the PCD Index (PCDI) for prognosis and therapeutic response prediction. A comprehensive analysis of PCDI genes was performed, leveraging data including single-cell sequencing and proteomics. GBA was selected, and its functions were investigated in HCC cell lines by in vitro experiments. Results: Two PCD clusters with different clinical and biological characteristics were identified in HCC. With the computational framework, the PCDI was constructed, demonstrating superior prognostic predictive efficacy and surpassing previously published prognostic models. An efficient clinical nomogram based on PCDI and clinicopathological factors was then developed. PCDI was intimately associated with immunological attributes, and PCDI could efficaciously predict immunotherapy response. Additionally, the PCDI could predict the chemotherapy sensitivity of HCC patients. A multilevel panorama of PCDI genes confirmed its stability and credibility. Finally, the knockdown of GBA could suppress both the proliferative and invasive capacities of HCC cells. Conclusion: This study systematically elucidated the association between PCD and HCC. A robust PCDI was constructed for prognosis and therapy response prediction, which would facilitate clinical management and personalized therapy for HCC.

A Comprehensive Analysis of Pyroptosis-Related lncRNAs Signature Associated With Prognosis and Tumor Immune Microenvironment of Pancreatic Adenocarcinoma.

Background: Globally, pancreatic adenocarcinoma (PAAD) is a common and highly devastating gastrointestinal malignancy that seriously threatens human health. Pyroptosis refers to an emerging form of programmed cell death that has been discovered in recent years, and studies have demonstrated that long non-coding RNA (lncRNA) may act as a moderator in the pyroptosis process of cancer cells. However, relevant explorations about lncRNAs and pyroptosis are still insufficient in PAAD. Therefore, our research is designed to make a comprehensive analysis of the potential values of pyroptosis-related lncRNAs in PAAD. Methods: By integrating the RNA-sequencing, somatic mutation, and copy number variation (CNV) datasets, as well as the clinicopathological features, we established and validated a risk signature based on pyroptosis-related lncRNAs, and comprehensively analyzed its clinical significance and the potential connection with the tumor immune microenvironment (TIME). Consequences: The genetic variation landscape displayed that the somatic mutations were rare while CNV changes were general and mainly concentrated on copy number amplification of these 52 pyroptosis-related genes. Subsequently, a risk signature consisting of 10 lncRNAs (TRAF3IP2-AS1, LINC00519, LINC01133, LINC02251, AC005332.6, AL590787.1, AC090114.2, TRPC7-AS1, MIR223HG, and MIR3142HG) was constructed and patients were divided into different subgroups according to the median risk score; patients with high-risk scores presented worse outcomes compared to those with low-risk scores in the training, testing, and entire cohorts. Furthermore, patients at low-risk scores possessed a higher infiltration abundance of immune cells compared with high-risk patients, which was consistent with the expression levels of lncRNAs between the high/low-risk groups. Drug sensitivity analysis showed that low-risk scores were related to anti-cancer agents like AICAR and Axitinib, whereas high-risk scores were connected with certain drugs such as AUY922. These results demonstrated that our risk signature could be used for prognosis prediction; additionally, it was also related to the TIME that might act as a potential indicator to instruct immunotherapeutic strategies. Conclusion: This work explored the significance of the risk model constructed by pyroptosis-related lncRNAs in prognosis prediction and its internal link with the immune microenvironment of PAAD. The results are expected to assist in the diagnosis, prognostic assessment, and management of patients with PAAD.

Global landscape of 2-hydroxyisobutyrylation in human pancreatic cancer.

As a new type of post-translational modification (PTM), lysine 2-hydroxyisobutyrylation (Khib) was firstly identified in histones and functioned as a regulator of transactivation in mammals. However, the role of Khib proteins remains to be investigated. Here, we firstly identified 10,367 Khib sites on 2,325 modified proteins in seven patients with pancreatic cancer by applying liquid chromatography with tandem mass spectrometry (LC-MS/MS) qualitative proteomics techniques. Among them, 27 Khib-modified sites were identified in histones. Bioinformatics analysis revealed that the Khib-modified proteins were mainly distributed in the cytoplasm and enhanced in metabolic pathways, including glycolysis/gluconeogenesis, the tricarboxylic acid cycle (TCA cycle), and fatty acid degradation. In an overlapping comparison of lysine 2-hydroxyisobutyrylation, succinylation, and acetylation in humans, 105 proteins with 80 sites were modified by all three PTMs, suggesting there may be a complex network among the different modified proteins and sites. Furthermore, MG149, which was identified as a Tip60 inhibitor, significantly decreased the total Khib modification level in pancreatic cancer (PC) and strongly suppressed PC's proliferation, migration, and invasion ability. Overall, our study is the first profiling of lysine 2-hydroxyisobutyrylome and provides a new database for better investigating Khib in PC.

Proteomic and Phosphoproteomic Profiling Reveals the Oncogenic Role of Protein Kinase D Family Kinases in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a lethal malignancy in the hepatobiliary system, with dysregulated protein expression and phosphorylation signaling. However, the protein and phosphorylation signatures of CCAs are little-known. Here, we performed the proteomic and phosphoproteomic profiling of tumors and normal adjacent tissues (NATs) from patients with CCA and predicted eleven PKs high-potentially related to CCA with a comprehensive inference of the functional protein kinases (PKs) (CifPK) pipeline. Besides the two known CCA-associated PKs, we screened the remaining candidates and uncovered five PKs as novel regulators in CCA. Specifically, the protein kinase D (PKD) family members, including PRKD1, PRKD2, and PRKD3, were identified as critical regulators in CCA. Moreover, the pan-inhibitor of the PKD family, 1-naphthyl PP1 (1-NA-PP1), was validated as a potent agent for inhibiting the proliferation, migration, and invasion ability of CCA cells. This study reveals new PKs associated with CCA and suggests PRKD kinases as novel treatment targets for CCA.

Macrophages-aPKCÉ©-CCL5 Feedback Loop Modulates the Progression and Chemoresistance in Cholangiocarcinoma.

BACKGROUND: Recent data indicated that macrophages may mutually interact with cancer cells to promote tumor progression and chemoresistance, but the interaction in cholangiocarcinoma (CCA) is obscure. METHODS: 10x Genomics single-cell sequencing technology was used to identified the role of macrophages in CCA. Then, we measured the expression and prognostic role of macrophage markers and aPKCÉ© in 70 human CCA tissues. Moreover, we constructed monocyte-derived macrophages (MDMs) generated from peripheral blood monocytes (PBMCs) and polarized them into M1/M2 macrophages. A co-culture assay of the human CCA cell lines (TFK-1, EGI-1) and differentiated PBMCs-macrophages was established, and functional studies in vitro and in vivo was performed to explore the interaction between cancer cells and M2 macrophages. Furthermore, we established the cationic liposome-mediated co-delivery of gemcitabine and aPKCÉ©-siRNA and detect the antitumor effects in CCA. RESULTS: M2 macrophage showed tumor-promoting properties in CCA. High levels of aPKCÉ© expression and M2 macrophage infiltration were associated with metastasis and poor prognosis in CCA patients. Moreover, CCA patients with low M2 macrophages infiltration or low aPKCÉ© expression benefited from postoperative gemcitabine-based chemotherapy. Further studies showed that M2 macrophages-derived TGFß1 induced epithelial-mesenchymal transition (EMT) and gemcitabine resistance in CCA cells through aPKCÉ©-mediated NF-κB signaling pathway. Reciprocally, CCL5 was secreted more by CCA cells undergoing aPKCÉ©-induced EMT and consequently modulated macrophage recruitment and polarization. Furthermore, the cationic liposome-mediated co-delivery of GEM and aPKCÉ©-siRNA significantly inhibited macrophages infiltration and CCA progression. CONCLUSION: our study demonstrates the role of Macrophages-aPKCÉ©-CCL5 Feedback Loop in CCA, and proposes a novel therapeutic strategy of aPKCÉ©-siRNA and GEM co-delivered by liposomes for CCA.

Treatment of postoperative intestinal dysfunction of hirschsprung's disease based on the principle of "anorectal balance".

Purpose: Radical surgery is the most effective treatment for Hirschsprung's disease. However, some children still have symptoms of intestinal dysfunction such as constipation, abdominal distension, and recurrent enterocolitis after operation. The purpose of this study was to evaluate treatment outcomes of postoperative intestinal dysfunction in children with Hirschsprung's disease by using the principle of "anorectal balance". Methods: The clinical data of postoperative intestinal dysfunction in children with Hirschsprung's disease in the single treatment group from July 2019 to July 2021 were retrospectively analyzed. All the enrolled children underwent botulinum toxin injection (2.5 U/kg); 3 to 6 months later, the injection was performed again; the children who had received more than two botulinum toxin injections underwent the internal sphincter myectomy. Anorectal manometry was performed routinely after operation, and abdominal distension and defecation were recorded. Results: A total of thirty children with postoperative intestinal dysfunction underwent radical surgery for Hirschsprung's disease were included in this study. Symptoms of constipation, abdominal distension and enterocolitis were improved after botulinum toxin injections in most children compared to before surgery (P < 0.01). After re-injection of botulinum toxin in twelve children, the frequency of defecation increased, the anal resting pressure decreased, and the clinical symptoms were relieved again (P < 0.05). Eleven children underwent internal sphincter myectomy, and the symptoms of constipation, abdominal distension and enterocolitis were significantly improved after the operation (P < 0.01). Conclusion: Botulinum toxin injection and internal sphincter myectomy based on the principle of "anorectal balance" can effectively reduce the resting pressure of the anus and relieve intestinal dysfunction, and have satisfactory clinical effect.

A Novel Autophagy-Related IncRNAs Signature for Prognostic Prediction and Clinical Value in Patients With Pancreatic Cancer.

Autophagy is an important bioprocess throughout the occurrence and development of cancer. However, the role of autophagy-related lncRNAs in pancreatic cancer (PC) remains obscure. In the study, we identified the autophagy-related lncRNAs (ARlncRNAs) and divided the PC patients from The Cancer Genome Atlas into training and validation set. Firstly, we constructed a signature in the training set by the least absolute shrinkage and selection operator penalized cox regression analysis and the multivariate cox regression analysis. Then, we validated the independent prognostic role of the risk signature in both training and validation set with survival analysis, receiver operating characteristic analysis, and Cox regression. The nomogram was established to demonstrate the predictive power of the signature. Moreover, high risk scores were significantly correlated to worse outcomes and severe clinical characteristics. The Pearson's analysis between risk scores with immune cells infiltration, tumor mutation burden, and the expression level of chemotherapy target molecules indicated that the signature could predict efficacy of immunotherapy and targeted therapy. Next, we constructed an lncRNA-miRNA-mRNA regulatory network and identified several potential small molecule drugs in the Connectivity Map (CMap). What's more, quantitative real-time PCR (qRT-PCR) analysis showed that serum LINC01559 could serve as a diagnostic biomarker. In vitro analysis showed inhibition of LINC01559 suppressed PC cell proliferation, migration, and invasion. Additionally, silencing LINC01559 suppressed gemcitabine-induced autophagy and promoted the sensitivity of PC cells to gemcitabine. In conclusion, we identified a novel ARlncRNAs signature with valuable clinical utility for reliable prognostic prediction and personalized treatment of PC patients. And inhibition of LINC01559 might be a novel strategy to overcome chemoresistance.

aPKCι promotes gallbladder cancer tumorigenesis and gemcitabine resistance by competing with Nrf2 for binding to Keap1.

Gallbladder cancer (GBC) is a highly malignant bile duct cancer with poor prognosis characterized by its insensitivity to chemotherapy. Emerging evidence indicates that cytoprotective antioxidation is involved in drug resistance of various cancers; however, the underlying molecular mechanisms remain obscure. Here, we demonstrated that atypical protein kinase Cι (aPKCι) mediated reactive oxygen species (ROS) inhibition in a kinase-independent manner, which played a crucial role in tumorigenesis and chemoresistance. Mechanistically, we found that aPKCι facilitated nuclear factor erythroid 2-related factor 2 (Nrf2) accumulation, nuclear translocation and activated its target genes by competing with Nrf2 for binding to Kelch-like ECH-associated protein 1 (Keap1) through a highly conserved DLL motif. In addition, the aPKCι-Keap1 interaction was required for antioxidant effect, cell growth and gemcitabine resistance in GBC. Importantly, we further confirmed that aPKCι was frequently upregulated and correlated with poor prognosis in patients with GBC. Collectively, our findings suggested that aPKCι positively modulated the Keap1-Nrf2 pathway to enhance GBC growth and gemcitabine resistance, implying that the aPKCι-Keap1-Nrf2 axis may be a potential approach to overcome the drug resistance for the treatment of GBC.

Downregulation of ASPP2 promotes gallbladder cancer metastasis and macrophage recruitment via aPKC-ι/GLI1 pathway.

Gallbladder cancer (GBC) is a highly malignant bile duct cancer with poor prognosis due to early invasion and metastasis. However, the molecular mechanisms through which GBC cells interact with the tumor microenvironment (TME) remain poorly understood. Here, we examined the role of the tumor suppressor apoptosis-stimulating of p53 protein 2 (ASPP2) in regulating GBC invasion and metastasis and macrophage recruitment. The clinicopathological significance of ASPP2 expression was measured by immunohistochemical analysis in 72 patients with GBC. Lentivirus-mediated knockdown or overexpression of ASPP2 was used to investigate the biological functions and molecular mechanisms of ASPP2 in GBC cells. Our data showed that downregulation of ASPP2 in patients with GBC was linked to poor prognosis. Knockdown of ASPP2 induced epithelial-mesenchymal transition (EMT) in GBC cells and influenced the TME. Mechanistically, we further confirmed that ASPP2 affected the expression and protein binding between atypical protein kinase C (aPKC)-ι and glioma-associated oncogene homolog 1 (GLI1). ASPP2 also induced C-C motif chemokine ligand (CCL) 2, CCL5, and tumor necrosis factor-α secretion by cancer cells, thereby promoting macrophage recruitment. The latter also induced EMT-like changes in GBC. Furthermore, ASPP2 deficiency regulated GLI1 transcriptional activity via the noncanonical Hedgehog (Hh) pathway and aPKC-ι/GLI1 signaling loop and promoted GLI1 nuclear localization and binding to the promoters of target genes. Our findings revealed that downregulation of ASPP2 promoted GBC invasion and metastasis through the aPKC-ι/GLI1 pathway and enhanced macrophage recruitment. Thus, ASPP2/aPKC-ι/GLI1 pathway may be a potential therapeutic target for the treatment of GBC.