Human Gallbladder Cancer Research Articles

Establishment and characterization of a novel human gallbladder cancer cell line, GBC-X1

In this study, we successfully established a novel gallbladder cancer cell line, designated as GBC-X1, derived from a primary tumor of a gallbladder cancer patient. By comprehensively analyzing the cell line’s phenotype, molecular characteristics, biomarkers, and histological characteristics, we confirmed that GBC-X1 serves as a valuable model for investigating the pathogenesis of gallbladder cancer and developing therapeutic agents. GBC-X1 has been continuously cultured for one year, with over 60 stable passages. Morphologically, GBC-X1 exhibits typical features of epithelial tumors. The population doubling time of GBC-X1 is 32 h. STR analysis validated a high consistency between GBC-X1 and the patient’s primary tumor. Karyotype analysis revealed an abnormal hypertetraploid karyotype for GBC-X1, characterized by representative karyotypes of 98, XXXX del (4) p (12) del (5) p (21) der (10). Under suspension culture conditions, GBC-X1 efficiently forms tumor balls, while subcutaneous inoculation of GBC-X1 cells into NXG mice leads to xenograft formation with a rate of 80%. Drug sensitivity testing demonstrated that GBC-X1 is resistant to oxaliplatin and sensitive to 5-FU, gemcitabine, and paclitaxel. Immunohistochemistry revealed positive expression of CK7, CK19, E-cadherin, MMP-2, CD44, SOX2, and TP53 in GBC-X1 cells, weak positive expression of Vimentin, and a Ki67 positive rate of 35%. Our research highlights GBC-X1 as a novel gallbladder cancer cell line and emphasizes its potential as an effective experimental model for investigating the pathogenesis of gallbladder cancer and drug development.

Establishment and characterization of the gemcitabine-resistant human gallbladder cancer cell line NOZ GemR.

Patients with gallbladder cancer (GBC) generally receive gemcitabine as the standard treatment; however, its efficacy is often limited owing to the development of resistance. To identify the mechanisms underlying gemcitabine resistance in GBC, a gemcitabine-resistant GBC cell line (NOZ GemR) was established by exposing the parental NOZ cell line to increasing concentrations of gemcitabine. Morphological changes, growth rates, and migratory and invasive capabilities were evaluated. Protein expression was detected using western blotting. The results demonstrated that the IC50 of NOZ and NOZ GemR was 0.011 and 4.464μM, respectively, and that the resistance index ratio was 405.8. In comparison, NOZ GemR cells grew slower and had significantly lower migration and invasion abilities than NOZ cells. There were altered levels of epithelial-mesenchymal transformation markers in NOZ GemR cells, as well as increased levels of the Akt/mTOR pathway protein. The NOZ GemR cell line could be used as an effective in vitro model to improve our understanding of gemcitabine resistance in GBC.

MicroRNA-34a-5p: A pivotal therapeutic target in gallbladder cancer

Gallbladder cancer incidence has been increasing globally, andit remains challenging to expect long prognosis with the current systemic chemotherapy. We identified a novel nucleic acid-mediated therapeutic target against gallbladder cancer by using innovative organoid-based gallbladder cancer models generated from KrasLSL-G12D/+; Trp53f/f mice. Using comprehensive microRNA expression analyses and a bioinformatics approach, we identified significant microRNA-34a-5p downregulation in both murine gallbladder cancer organoids and resected human gallbladder cancer specimens. In three different human gallbladder cancer cell lines, forced microRNA-34a-5p expression inhibited cell proliferation and induced cell-cycle arrest at the G1 phase by suppressing direct target (CDK6) expression. Furthermore, comprehensive RNA sequencing revealed the significant enrichment of gene sets related to the cell-cycle regulators after microRNA-34a-5p expression in gallbladder cancer cells. In a murine xenograft model, locally injected microRNA-34a-5p mimics significantly inhibited gallbladder cancer progression and downregulated CDK6 expression. These results provide a rationale for promising therapeutics against gallbladder cancer by microRNA-34a-5p injection, as well as a strategy to explore therapeutic targets against cancers using organoid-based models, especially for those lacking useful genetically engineered murine models, such as gallbladder cancer.

ELF3 suppresses gallbladder cancer development through downregulation of the EREG/EGFR/mTOR complex 1 signalling pathway.

The prognosis of gallbladder cancer (GBC) remains poor, and a better understanding of GBC molecular mechanisms is important. Genome sequencing of human GBC has demonstrated that loss-of-function mutations of E74-like ETS transcription factor 3 (ELF3) are frequently observed, with ELF3 considered to be a tumour suppressor in GBC. To clarify the underlying molecular mechanisms by which ELF3 suppresses GBC development, we performed in vivo analysis using a combination of autochthonous and allograft mouse models. We first evaluated the clinical significance of ELF3 expression in human GBC tissues and found that low ELF3 expression was associated with advanced clinical stage and deep tumour invasion. For in vivo analysis, we generated Pdx1-Cre; KrasG12D ; Trp53R172H ; Elf3f/f (KPCE) mice and Pdx1-Cre; KrasG12D ; Trp53R172H ; Elf3wt/wt (KPC) mice as a control and analysed their gallbladders histologically. KPCE mice developed larger papillary lesions in the gallbladder than those developed by KPC mice. Organoids established from the gallbladders of KPCE and KPC mice were analysed in vitro. RNA sequencing showed upregulated expression of epiregulin (Ereg) in KPCE organoids, and western blotting revealed that EGFR/mechanical targets of rapamycin complex 1 (mTORC1) were upregulated in KPCE organoids. In addition, ChIP assays on Elf3-overexpressing KPCE organoids showed that ELF3 directly regulated Ereg. Ereg deletion in KPCE organoids (using CRISPR/Cas9) induced EGFR/mTORC1 downregulation, indicating that ELF3 controlled EGFR/mTORC1 activity through regulation of Ereg expression. We also generated allograft mouse models using KPCE and KPC organoids and found that KPCE organoid allograft tumours exhibited poorly differentiated structures with mTORC1 upregulation and mesenchymal phenotype, which were suppressed by Ereg deletion. Furthermore, EGFR/mTORC1 inhibition suppressed cell proliferation and epithelial-mesenchymal transition in KPCE organoids. Our results suggest that ELF3 suppresses GBC development via downregulation of EREG/EGFR/mTORC1 signalling. EGFR/mTORC1 inhibition is a potential therapeutic option for GBC with ELF3 mutation. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Taurodeoxycholic acid-YAP1 upregulates OTX1 in promoting gallbladder cancer malignancy through IFITM3-dependent AKT activation.

Orthodenticle homeobox (OTX1) is reported to be involved in numerous cancers, but the expression level and molecular function of OTX1 in gallbladder cancer (GBC) remain unknown. Here, we found the elevated level of OTX1 associated with poor prognosis in human gallbladder cancer. In vitro and in vivo studies of human gallbladder cancer cell lines demonstrated that overexpression of OTX1 promoted cell proliferation, whereas the downregulation inhibited it. Additionally, we found a tight correlation between the serum level of taurodeoxycholic acid (TDCA) and OTX1 expression. TDCA-induced activation of YAP1 by phosphorylation inhibition contributed to the transcriptional activation of OTX1. Mechanistically, we identified that OTX1 activated AKT signaling pathway by transactivating the expression of IFITM3 and thus promoted the proliferation of GBC cells. Taken together, our results showed that TDCA-YAP1-dependent expression of OTX1 regulated IFITM3 and affected GBC proliferation via the AKT signaling pathway. Our experiments also suggested that OTX1 is a novel therapeutic target for GBC.

Lithocholic acid inhibits gallbladder cancer proliferation through interfering glutaminase-mediated glutamine metabolism

Lithocholic acid (LCA), one of the most common metabolic products of bile acids (BAs), is originally synthesized in the liver, stored in the gallbladder, and released to the intestine, where it assists absorption of lipid-soluble nutrients. LCA has recently emerged as a powerful reagent to inhibit tumorigenesis; however, the anti-tumor activity and molecular mechanisms of LCA in gallbladder cancer (GBC) remain poorly acknowledged. Here, we analyzed serum levels of LCA in human GBC and found that LCA was significantly downregulated in these patients, and reduced LCA levels were associated with poor clinical outcomes. Treatment of xenografts with LCA impeded tumor growth. Furthermore, LCA treatment in GBC cell lines decreased glutaminase (GLS) expression, glutamine (Gln) consumption, and GSH/GSSG and NADPH/NADP+ ratios, leading to cellular ferroptosis. In contrast, GLS overexpression in tumor cells fully restored GBC proliferation and decreased ROS imbalance, thus suppressing ferroptosis. Our findings reveal that LCA functions as a tumor-suppressive factor in GBC by downregulating GLS-mediated glutamine metabolism and subsequently inducing ferroptosis. This study may offer a new therapeutic strategy tailored to improve the treatment of GBC.

HHLA2 promotes tumor progression by long non‑coding RNA H19 in human gallbladder cancer.

Advanced gallbladder cancer (GBC) is one of the most malignant of all types of biliary tract cancers that is associated with poor prognosis and high mortality. Accumulating evidence suggest that the B7 family of proteins serve an essential role in various types of cancers, including GBC. However, the potential function and regulatory mechanism of human endogenous retrovirus-H long terminal repeat-associating protein 2 (HHLA2; also known as B7-H7 or B7H5) in GBC remain poorly understood. In the present study, immunohistochemistry was used to examine the expression pattern of HHLA2 in samples from 89 patients with GBC. The possible association between HHLA2 expression and the clinicopathological parameters, including prognosis, were then assessed. Using lentiviruses, overexpression of HHLA2 plasmid or short-hairpin RNA (shRNA) of HHLA2 were transfected into GBC-SD cells to overexpress or knock down HHLA2 expression, respectively. The effects of HHLA2 overexpression and knockdown on the epithelial-mesenchymal transition (EMT) process on GBC-SD cells were measured by the western blotting and immunofluorescence staining of collagen I, N-cadherin, E-cadherin, vimentin and α-SMA. By contrast, changes in cell proliferation were measured using EdU assay. Cell invasion and migration were assessed using Transwell and wound-healing assays, respectively. In addition, a xenograft mouse model was established to evaluate the tumorigenic ability of the GBC cell line in vivo after stable transfection with lentivirus for HHLA2 overexpression or shRNA for HHLA2 knockdown. The regulatory relationships among TGF-β1, long non-coding RNA (lncRNA) H19 (H19) and HHLA2 were then investigated. The mRNA expression of lncRNA H19 were assessed using reverse transcription-quantitative PCR, whereas the expression levels of HHLA2 were detected by western blotting and immunofluorescence staining. HHLA2 expression was found to gradually increase as the stages of the GBC samples become more advanced. In addition, the expression level of HHLA2 was calculated to be positively associated with the Nevin stage, American Joint Committee on Cancer stage, tumor invasion and regional lymph node metastasis but was negatively associated with the overall patient survival (OS). In vitro experiments demonstrated that overexpression of HHLA2 promoted GBC migration, invasion, proliferation and EMT, whereas in vivo experiments found a promoting role of HHLA2 overexpression on GBC tumor growth. After transfection with lentiviruses encoding the overexpression plasmid of lncRNA H19, GBC migration, invasion, proliferation and EMT were increased. By contrast, knocking down HHLA2 expression suppressed TGF-β1- or lncRNA H19 overexpression-induced GBC migration, invasion, proliferation and EMT. In addition, HHLA2 knockdown significantly reduced the sizes of the GBC tumors in vivo. These results suggest that HHLA2 overexpression can promote GBC progression. Conversely, ablation of HHLA2 expression inhibited both TGF-β1- and lncRNA H19-induced GBC progression, suggesting that HHLA2 is a potential therapeutic target for this disease.

CD47 expression in human gallbladder cancer and its clinicopathologic significance

CD47 expression in human gallbladder cancer and its clinicopathologic significance

Design, synthesis and evaluation of 4,7-disubstituted 8-methoxyquinazoline derivatives as potential cytotoxic agents targeting β-catenin/TCF4 signaling pathway

Overactivation of Wnt/β-catenin signaling by accumulated β-catenin in the nucleus has been shown to play a crucial role in the etiology of cancer. Interaction of β-catenin with Transcription factor 4 (TCF4) is a key step for the activation of Wnt genes in response to upstream signals of the Wnt/β-catenin pathway. Hence, down regulation of Wnt/β-catenin signaling or targeting downstream events by selective β-catenin/TCF4 protein–protein interaction inhibitors could be a potential therapeutic strategy against such cancers. In this study structure-based drug design approach was followed to design novel 4,7-disubstituted 8-methoxyquinazoline-based derivatives which could act as potential cytotoxic agents inhibiting the β-catenin/TCF4 protein–protein interactions. Fifteen compounds possessing 4,7-disubstituted 8-methoxyquinazoline scaffold were synthesized. Cytotoxic potential of the synthesised derivatives were determined against constitutively activated β-catenin/TCF4 signaling pathway cancer cells (HCT116 and HepG2) using the sulforhodamine B assay. The most potent compound (18B) was selected for detailed biological evaluation. Cell morphology, Hoechst 33342 and Annexin V/PI staining were used to detect apoptosis, while inhibition of cell migration was assessed by in vitro wound healing assay against HCT116 and HepG2 cells. Effect on β-catenin/TCF mediated transcriptional activity was assessed by TOPFlash/FOPFlash assay, TCF4 and β-catenin protein expression by immunocytofluorescence, and Wnt target genes (like c-MYC and Cyclin D1) mRNA levels by RT-PCR against HCT116 cells. Cytotoxic potency of the most potential compound (18B) against primary human gallbladder cancer cells was also evaluated. The derivatives showed interactions with active site residues of β-catenin and were capable of hindering the TCF4 binding, thereby disrupting β-catenin/TCF4 interactions. Cytotoxic potencies (IC50) of these derivatives ranged from 5.64 ± 0.68 to 23.18 ± 0.45 μM against HCT116 and HepG2 cells respectively. Compound (18B), the most potent compound among the series, induced apoptosis and inhibited cell migration against HCT116 and HepG2 cells. Mechanistic studies indicated that compound (18B) downregulated β-catenin/TCF4 signaling pathway, β-catenin and TCF4 protein expression, and mRNA levels of c-MYC andCyclin D1 in HCT116 cells and showed cytotoxicity against primary human gallbladder cancer cells with IC50 value of 8.50 ± 1.44 μM. Thus, novel 4,7-disubstituted 8-methoxyquinazoline derivatives were identified as potential cytotoxic agents with potencies comparable to that of imatinib mesylate. Compound (18B) represents a promising lead molecule as anticancer agent against colon, hepatocellular and gallbladder cancers targeting β-catenin/TCF4 signaling pathway.

IL4Rα and IL13Rα1 Are Involved in the Development of Human Gallbladder Cancer.

Background: Gallbladder cancer is commonly associated with inflammation, which indicates that inflammation-related cytokines and cytokine receptors are related to the progression of gallbladder cancers. Interleukin 4 (IL4) is a well-known cytokine that promotes the differentiation of naive helper T cells (Th0) to T helper type 2 cells (Th2). IL13 is a cytokine that is secreted by Th2 cells. IL4 and IL13 are closely related in immune responses. However, the role of IL4Rα and IL13Rα1 signaling pathway has not been fully understood in the development of gallbladder cancer. Methods: In human gallbladder carcinomas, the expression of IL4Rα and IL13Rα1 were evaluated with immunohistochemical staining in tissue microarray tissue sections. After knockdown of IL4Rα or IL13Rα1, cell assays to measure the proliferation and apoptosis and Western blotting analysis were conducted in SNU308 human gallbladder cancer cells. Since Janus kinases2 (JAK2) was considered as one of the down-stream kinases under IL4Rα and IL13Rα1 complex, the same kinds of experiments were performed in SNU308 cells treated with AZD1480, Janus-associated kinases2 (JAK2) inhibitor, to demonstrate the cytotoxic effect of AZD1480 in SNU308 cells. Results: Immunohistochemical expression of IL4Rα was significantly associated with the expression of IL13Rα1 in human carcinoma tissue. In univariate analysis, nuclear expression of IL4Rα, cytoplasmic expression of IL4Rα, nuclear expression of IL13Rα1, and cytoplasmic expression of IL13Rα1 were significantly associated with shorter overall survival and shorter relapse-free survival. Multivariate analysis revealed nuclear expression of IL4Rα as an independent poor prognostic indicator of overall survival and relapse-free survival. Then, we found that knockdown of IL4Rα or IL13Rα1 decreased viability and induced apoptosis in SNU308 cells via activation of FOXO3 and similarly, AZD1480 decreased viability and induced apoptosis in SNU308 cells with dose dependent manner. Conclusions: Taken together, our results suggest that IL4Rα and IL13Rα1 might be involved in the development of human gallbladder cancer cells and IL4Rα and IL13Rα1 complex/JAK2 signaling pathway could be efficient therapeutic targets for gallbladder cancer treatment.

CD73 and PD-L1 as Potential Therapeutic Targets in Gallbladder Cancer.

Gallbladder cancer (GBC) is one of the most common and aggressive biliary tract cancers with a dismal prognosis. Ongoing clinical trials are evaluating a few selected immune checkpoint inhibitors (ICIs) as monotherapy for the treatment of GBC patients. However, only a subset of patients benefits from these treatments. To improve ICI therapy response, molecular mechanisms that confer resistance to immune checkpoint (IC) blockade needs to be explored. Epithelial-to-mesenchymal transition (EMT) program and cancer stem cells (CSCs) have been implicated as key processes that confer ICI treatment resistance. However, in GBC the EMT-CSC-IC axis has not yet been clearly elucidated. This study aims to examine the aberrant expression of ICs associated with CSC and EMT. We successfully enriched CSCs by utilizing a 3-dimensional culture system and established a reversible EMT model with human GBC NOZ cell line. Notably, ICs CD73 and PD-L1 were closely associated with both CSC and EMT phenotypes. Knockdown of CD73 or PD-L1 reduced the proliferative and motile abilities of both adherent monolayers and anchorage-free spheroids. In conclusion, blocking CD73 and PD-L1 offer a promising therapeutic strategy for targeting highly aggressive populations with CSC and EMT phenotype to improve GBC patient prognosis.

Gallbladder Cancer: Current Insights in Genetic Alterations and their Possible Therapeutic Implications

Introduction: Due to the fast progression of molecular technologies such as next-generation sequencing, knowledge of genetic alterations in gallbladder cancer (GBC) is increasing. This systematic review aims to provide an overview of genetic alterations in GBC and their possible therapeutic implications. Method: This systematic review was prospectively registered in the PROSPERO registry (CRD42021265246). The study was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A literature search was performed utilizing Pubmed, EMBASE, Cochrane Library and Web of Science. Only studies identifying genetic alterations in human GBC were included. Results: In total, data was extracted from 62 articles, describing a total of 3,893 GBC samples. Of all frequently (>5% across all studies) detected gene alterations in GBC, mutations in ATM, ERBB2, and PIK3CA, and amplifications in ERBB2 are potentially targetable. High tumor mutational burden (TMB) and microsatellite instability (MSI) were infrequently observed in GBC (1.7% and 3.5%, respectively). For tumors with high TMB or MSI, the highly active immune checkpoint inhibitor Pembrolizumab is approved by the United States Food and Drug Administration (FDA), with promising objective response rates in solid tumors including biliary tract cancer (50% and 41%, respectively). Only nine clinical trials which target frequently detected genetic alterations were identified. Conclusions: In conclusion, ATM mutations, ERBB2 amplifications and mutations, and PIK3CA mutations are potentially targetable alterations which were frequently detected in GBC. Few clinical trials targeting the frequently altered genes in GBC are performed, emphasizing the need for future, multi-center, clinical basket trials.

Gallbladder Cancer: Current Insights in Genetic Alterations and Their Possible Therapeutic Implications.

Simple SummaryKnowledge of genetic alterations in gallbladder cancer (GBC) continues to increase. This systematic review provides an overview of frequently occurring genetic alterations in GBC and describes their possible therapeutic implications. We detected three frequently (>5%) altered genes (ATM, ERBB2 and PIK3CA) for which targeted therapies are available in other cancer types. For solid cancers with microsatellite instability or a high tumor mutational burden pembrolizumab is FDA-approved. Altogether, these five biomarkers might be used in future molecular panels to enable precision medicine for patients with GBC. We found only nine clinical trials evaluating targeted therapies in GBC directed at frequently altered genes (ERBB2, ARID1A, ATM and KRAS). This underlines the challenges to perform such clinical trials in this rare, heterogeneous cancer type and emphasizes the need for multicenter clinical trials.Due to the fast progression in molecular technologies such as next-generation sequencing, knowledge of genetic alterations in gallbladder cancer (GBC) increases. This systematic review provides an overview of frequently occurring genetic alterations occurring in GBC and their possible therapeutic implications. A literature search was performed utilizing PubMed, EMBASE, Cochrane Library, and Web of Science. Only studies reporting genetic alterations in human GBC were included. In total, data were extracted from 62 articles, describing a total of 3893 GBC samples. Frequently detected genetic alterations (>5% in >5 samples across all studies) in GBC for which targeted therapies are available in other cancer types included mutations in ATM, ERBB2, and PIK3CA, and ERBB2 amplifications. High tumor mutational burden (TMB-H) and microsatellite instability (MSI-H) were infrequently observed in GBC (1.7% and 3.5%, respectively). For solid cancers with TMB-H or MSI-H pembrolizumab is FDA-approved and shows an objective response rates of 50% for TMB-H GBC and 41% for MSI-H biliary tract cancer. Only nine clinical trials evaluated targeted therapies in GBC directed at frequently altered genes (ERBB2, ARID1A, ATM, and KRAS). This underlines the challenges to perform such clinical trials in this rare, heterogeneous cancer type and emphasizes the need for multicenter clinical trials.

From the Editor’s desk…

From the Editor’s desk…

Single-cell RNA-sequencing atlas reveals an MDK-dependent immunosuppressive environment in ErbB pathway-mutated gallbladder cancer

Our previous genomic whole-exome sequencing (WES) data identified the key ErbB pathway mutations that play an essential role in regulating the malignancy of gallbladder cancer (GBC). Herein, we tested the hypothesis that individual cellular components of the tumor microenvironment (TME) in GBC function differentially to participate in ErbB pathway mutation-dependent tumor progression. We engaged single-cell RNA-sequencing to reveal transcriptomic heterogeneity and intercellular crosstalk from 13 human GBCs and adjacent normal tissues. In addition, we performed WES analysis to reveal the genomic variations related to tumor malignancy. A variety of bulk RNA-sequencing, immunohistochemical staining, immunofluorescence staining and functional experiments were employed to study the difference between tissues with or without ErbB pathway mutations. We identified 16 cell types from a total of 114,927 cells, in which epithelial cells, M2 macrophages, and regulatory T cells were predominant in tumors with ErbB pathway mutations. Furthermore, epithelial cell subtype 1, 2 and 3 were mainly found in adenocarcinoma and subtype 4 was present in adenosquamous carcinoma. The tumors with ErbB pathway mutations harbored larger populations of epithelial cell subtype 1 and 2, and expressed higher levels of secreted midkine (MDK) than tumors without ErbB pathway mutations. Increased MDK resulted in an interaction with its receptor LRP1, which is expressed by tumor-infiltrating macrophages, and promoted immunosuppressive macrophage differentiation. Moreover, the crosstalk between macrophage-secreted CXCL10 and its receptor CXCR3 on regulatory T cells was induced in GBC with ErbB pathway mutations. Elevated MDK was correlated with poor overall survival in patients with GBC. This study has provided valuable insights into transcriptomic heterogeneity and the global cellular network in the TME, which coordinately functions to promote the progression of GBC with ErbB pathway mutations; thus, unveiling novel cellular and molecular targets for cancer therapy. We employed single-cell RNA-sequencing and functional assays to uncover the transcriptomic heterogeneity and intercellular crosstalk present in gallbladder cancer. We found that ErbB pathway mutations reduced anti-cancer immunity and led to cancer development. ErbB pathway mutations resulted in immunosuppressive macrophage differentiation and regulatory T cell activation, explaining the reduced anti-cancer immunity and worse overall survival observed in patients with these mutations.

Diversity and intratumoral heterogeneity in human gallbladder cancer progression revealed by single-cell RNA sequencing.

BackgroundGallbladder cancer (GC) is a malignant disease characterized with highly cellular heterogeneity and poor prognosis. Determining the intratumoral heterogeneity and microenvironment (TME) can provide novel therapeutic strategies for GC.MethodsWe performed the single‐cell RNA sequencing on the primary and lymph node metastatic gallbladder tumors and the adjacent normal tissues of five patients. The transcriptomic atlas and ligand–receptor‐based intercellular communication networks of the single cells were characterized.ResultsThe transcriptomic landscape of 24,887 single cells was obtained and characterized as 10 cellular clusters, including epithelial, neuroendocrine tumor cells, T&NK cells, B cells, RGS5+ fibroblasts, POSTN+ fibroblasts, PDGFRA+ fibroblasts, endothelial, myeloid cells, and mast cells. Different types of GC harbored distinct epithelial tumor subpopulations, and squamous cell carcinoma could be differentiated from adenocarcinoma cells. Abundant immune cells infiltrated into adenocarcinoma and squamous cell carcinoma, rather than neuroendocrine neoplasms, which showed significant enrichment of stromal cells. CD4+/FOXP3+ T‐reg and CD4+/CXCL13+ T helper cells with higher exhausting biomarkers, as well as a dynamic lineage transition of tumor‐associated macrophages from CCL20hi/CD163lo, CCL20lo/CD163hi to APOE+, were identified in GC tissues, suggesting the immunosuppressive and tumor‐promoting status of immune cells in TME. Two distinct endothelial cells (KDR+ and ACKR1+), which were involved in angiogenesis and lymphangiogenesis, showed remarkable ligand–receptor interactions with primary GC cells and macrophages in gallbladder tumors.ConclusionsThis study reveals a widespread reprogramming across multiple cell populations in GC progression, dissects the cellular heterogeneity and interactions in gallbladder TME, and provides potential therapeutic targets for GC.

Expression and functional characterization of INPP4B in gallbladder cancer patients and gallbladder cancer cells

BackgroundInositol polyphosphate 4-phosphatase type II (INPP4B) is a negative regulator of the PI3K-Akt signalling pathway and plays a contradictory role in different types of cancers. However, the its biological role played by INPP4B in human gallbladder cancer (GBC) has not been elucidated. In this study, we investigated the expression, clinical significance and biological function of INPP4B in GBC patients and cell lines.MethodsThe INPP4B protein expression levels in gallbladder cancer tissues and normal gallbladder tissues were detected by immunohistochemistry, and the clinical significance of INPP4B was analysed. Knockdown and overexpression of INPP4B in GBC-SD and SGC-996 cells followed by cell proliferation, clonogenic, apoptosis detection, scratch wound-healing and transwell assays were used to identify INPP4B function in vitro.ResultsINPP4B was up-regulated in human GBC tissues compared with normal gallbladder tissues and was related to histopathological differentiation (p = 0.026). Here, we observed that INPP4B was highly expressed in high-moderately differentiated tumours compared with low-undifferentiated tumours (p = 0.022). Additionally, we found that INPP4B expression was not associated with overall survival of GBC patients (p = 0.071) and was not an independent prognostic factor. Furthermore, when we stratified the relationship between INPP4B expression and the prognosis of GBC based on histopathological differentiation, we found that INPP4B played a contradictory role in GBC progression depending on the degree of differentiation. In addition, INPP4B knockdown inhibited the proliferation, colony formation, migration and invasion in GBC cells, while INPP4B overexpression had the opposite effects in vitro, which indicates its role as an oncoprotein.ConclusionsThese findings suggested that INPP4B may play a dual role in the prognosis of GBC depending on the degree of differentiation and that INPP4B might act as an oncogene in gallbladder cancer cells.

Precision modeling of gall bladder cancer patients in mice based on orthotopic implantation of organoid-derived tumor buds

Genetically engineered mice (GEM) are the gold standard for cancer modeling. However, strict recapitulation of stepwise carcinogenesis from a single tumor-initiating epithelial cell among genetically intact cells in adults is not feasible with the currently available techniques using GEM. In previous studies, we partially overcame this challenge by physically isolating organs from adult animals, followed by genetic engineering in organoids and subcutaneous inoculation in nude mice. Despite the establishment of suitable ex vivo carcinogenesis models for diverse tissues, tumor development remained ectopic and occurred under immunodeficient conditions. Further refinement was, therefore, necessary to establish ideal models. Given the poor prognosis and few models owing to the lack of gall bladder (GB)-specific Cre strain, we assumed that the development of authentic models would considerably benefit GB cancer research. Here, we established a novel model using GB organoids with mutant Kras and Trp53 loss generated in vitro by lentiviral Cre transduction and CRISPR/Cas9 gene editing, respectively. Organoid-derived subcutaneous tumor fragments were sutured to the outer surface of the GB in syngeneic mice, which developed orthotopic tumors that resembled human GB cancer in histological and transcriptional features. This model revealed the infiltration of similar subsets of immune cells in both subcutaneous and orthotopic tumors, confirming the appropriate immune environment during carcinogenesis. In addition, we accurately validated the in vivo efficacy of gemcitabine, a common therapeutic agent for GB cancer, in large cohorts. Taken together, this model may serve as a promising avatar of patients with GB cancer in drug discovery and precision medicine.

Kaempferol suppresses proliferation and induces apoptosis and DNA damage in human gallbladder cancer cells through the CDK4/CDK6/cyclin D1 pathway.

Kaempferol has been reported to play an anti-tumor role in various human cancers, while its role in gallbladder cancer (GBC) is unclear. We found that kaempferol significantly inhibited the growth, invasion and migration, meanwhile induced apoptosis through cells arrested at G0/G1 phase of GBC cell lines, including GBC-SD and SGC996 cells in vitro. Kaempferol promoted the release of cytochrome C from the mitochondria to cytoplasm, the activation of c-caspase-3 and c-caspase-9 and increased the expression levels of pro-apoptotic factor Bax, meanwhile decreased the expression levels of anti-apoptotic factor Bcl-2. In addition, the expression levels of CDK4, CDK6 and cyclin D1, which are members of the CDK4/CDK6/cyclin D1 signaling pathway, were also decreased by kaempferol. Moreover, kaempferol could efficiently prevent tumor progression of GBC in the xenograft in vivo. Our results demonstrated that kaempferol suppressed GBC progression through activation of the CDK4/CDK6/cyclin D1 signaling pathway, suggesting that it might be a potential anti-tumor agent for clinical treatment of GBC.

Lenvatinib induces anticancer activity in gallbladder cancer by targeting AKT.

Gallbladder cancer (GBC) is characterized by poor prognosis, early metastasis, and high recurrence rates, which seriously threaten human health. The effect of lenvatinib, a widely used drug in anti-hepatocellular carcinoma in China, on GBC progress, as well as its underlying molecular mechanism, remains unclear. Therefore, the present study investigated the effect of lenvatinib on human GBC GBC-SD and NOZ cells and its underlying mechanisms. A series of experiments, including cell proliferation, clone formation, wound healing, and cell migration and invasion assays, as well as flow cytometry, were performed to investigate the anticancer effect of lenvatinib on GBC. Western blotting was used to detect alterations in protein expression of CKD2, CKD4, cyclin D1, caspase-9, matrix metalloproteinase (MMP)-2, cell migration-inducing protein (CEMIP) and phospho-AKT (p-AKT). In addition, the chemosensitivity of lenvatinib-treated GBC cells to gemcitabine (GEM) and whether the activation of phosphoinositide 3 kinase (PI3K)/AKT contributed to the chemoresistance were determined. Finally, the anticancer effect of lenvatinib in vivo was detected using a xenograft mouse model. These data showed that treatment with lenvatinib inhibited cell proliferation, colony formation ability, migration, induced apoptosis, regulated cell cycle and resulted in decreased resistance to GEM. Treatment with lenvatinib decreased the expression of MMP-2, CEMIP, CDK2, CDK4 and cyclin D1, and increased the expression of cleaved caspase-9, which was mediated by the inactivation of the PI3K/AKT pathway in vitro. In addition, lenvatinib inhibited autophagy in GBC-SD and NOZ cells. Besides, Lenvatinib suppressed GBC cell growth in vivo by targeting p-AKT. In combination, the present data indicated that lenvatinib plays a potential anticancer role in GBC by downregulating the expression of p-AKT.