Pancreatic stent removal with a novel drill dilator.

Video 1Pancreatic stent removal with a novel drill dilator.

Pancreatic RECK inactivation promotes cancer formation, epithelial-mesenchymal transition, and metastasis.

RECK is downregulated in various human cancers; however, how RECK inactivation affects carcinogenesis remains unclear. We addressed this issue in a pancreatic ductal adenocarcinoma (PDAC) mouse model and found that pancreatic Reck deletion dramatically augmented the spontaneous development of PDAC with a mesenchymal phenotype, which was accompanied by increased liver metastases and decreased survival. Lineage tracing revealed that pancreatic Reck deletion induced epithelial-mesenchymal transition (EMT) in PDAC cells, giving rise to inflammatory cancer-associated fibroblast-like cells in mice. Splenic transplantation of Reck-null PDAC cells resulted in numerous liver metastases with a mesenchymal phenotype, whereas reexpression of RECK markedly reduced metastases and changed the PDAC tumor phenotype into an epithelial one. Consistently, low RECK expression correlated with low E-cadherin expression, poor differentiation, metastasis, and poor prognosis in human PDAC. RECK reexpression in the PDAC cells was found to downregulate MMP2 and MMP3, with a concomitant increase in E-cadherin and decrease in EMT-promoting transcription factors. An MMP inhibitor recapitulated the effects of RECK on the expression of E-cadherin and EMT-promoting transcription factors and invasive activity. These results establish the authenticity of RECK as a pancreatic tumor suppressor, provide insights into its underlying mechanisms, and support the idea that RECK could be an important therapeutic effector against human PDAC.

THBS1-producing tumor-infiltrating monocyte-like cells contribute to immunosuppression and metastasis in colorectal cancer.

Mesenchymal activation, characterized by dense stromal infiltration of immune and mesenchymal cells, fuels the aggressiveness of colorectal cancers (CRC), driving progression and metastasis. Targetable molecules in the tumor microenvironment (TME) need to be identified to improve the outcome in CRC patients with this aggressive phenotype. This study reports a positive link between high thrombospondin-1 (THBS1) expression and mesenchymal characteristics, immunosuppression, and unfavorable CRC prognosis. Bone marrow-derived monocyte-like cells recruited by CXCL12 are the primary source of THBS1, which contributes to the development of metastasis by inducing cytotoxic T-cell exhaustion and impairing vascularization. Furthermore, in orthotopically generated CRC models in male mice, THBS1 loss in the TME renders tumors partially sensitive to immune checkpoint inhibitors and anti-cancer drugs. Our study establishes THBS1 as a potential biomarker for identifying mesenchymal CRC and as a critical suppressor of antitumor immunity that contributes to the progression of this malignancy with a poor prognosis.

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.

Simultaneous activation of Kras-Akt and Notch pathways induces extrahepatic biliary cancer via the mTORC1 pathway.

Biliary tract cancer (BTC) has poor prognosis. The Notch receptor is aberrantly expressed in extrahepatic cholangiocarcinoma (eCCA). However, the role of Notch signaling in the initiation and progression of eCCA and gallbladder (GB) cancer remains unknown. Therefore, we investigated the functional role of Notch signaling during tumorigenesis of the extrahepatic bile duct (EHBD) and GB. Activation of Notch signaling and oncogenic Kras resulted in the development of biliary intraepithelial neoplasia (BilINs) in the EHBD and GB, which were premalignant lesions that progressed to adenocarcinoma in mice. The expression of genes involved in the mTORC1 pathway was increased in biliary spheroids from Hnf1b-CreERT2; KrasLSL-G12D ; Rosa26LSL-NotchIC mice and inhibition of the mTORC1 pathway suppressed spheroid growth. Additionally, simultaneous activation of the PI3K-AKT and Notch pathways in EHBD and GB induced biliary cancer development in mice. Consistent with this, we observed a significant correlation between activated NOTCH1 and phosphorylated Ribosomal Protein S6 (p-S6) expression in human eCCA. Furthermore, inhibition of the mTORC1 pathway suppressed the growth of Notch-activated human biliary cancer cells in vitro and in vivo. Mechanistically, the Kras/Notch-Myc axis activated mTORC1 through TSC2 phosphorylation in mutant biliary spheroids. These data indicate that inhibition of the mTORC1 pathway could be an effective treatment strategy for Notch-activated human eCCA. © 2023 The Pathological Society of Great Britain and Ireland.

Brg1 controls stemness and metastasis of pancreatic cancer through regulating hypoxia pathway.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. We previously reported that chromatin remodeler Brg1 is essential for acinar cell-derived PDAC formation in mice. However, the functional role of Brg1 in established PDAC and its metastasis remains unknown. Here, we investigated the importance of Brg1 for established PDAC by using a mouse model with a dual recombinase system. We discovered that Brg1 was a critical player for the cell survival and growth of spontaneously developed PDAC in mice. In addition, Brg1 was essential for metastasis of PDAC cells by inhibiting apoptosis in splenic injection and peritoneal dissemination models. Moreover, cancer stem-like property was compromised in PDAC cells by Brg1 ablation. Mechanistically, the hypoxia pathway was downregulated in Brg1-deleted mouse PDAC and BRG1-low human PDAC. Brg1 was essential for HIF-1α to bind to its target genes to augment the hypoxia pathway, which was important for PDAC cells to maintain their stem-like properties and to metastasize to the liver. Human PDAC cells with high BRG1 expression were more susceptible to BRG1 suppression. In conclusion, Brg1 plays a critical role for cell survival, stem-like property and metastasis of PDAC through the regulation of hypoxia pathway, and thus could be a novel therapeutic target for PDAC.

p53 protects against formation of extrahepatic biliary precancerous lesions in the context of oncogenic Kras.

KRAS and TP53 mutations are frequently observed in extrahepatic biliary cancer. Mutations of KRAS and TP53 are independent risk factors for poor prognosis in biliary cancer. However, the exact role of p53 in the development of extrahepatic biliary cancer remains elusive. In this study, we found that simultaneous activation of Kras and inactivation of p53 induces biliary neoplasms that resemble human biliary intraepithelial neoplasia in the extrahepatic bile duct and intracholecystic papillary-tubular neoplasm in the gall bladder in mice. However, inactivation of p53 was not sufficient for the progression of biliary precancerous lesions into invasive cancer in the context of oncogenic Kras within the observation period. This was also the case in the context of additional activation of the Wnt signaling pathway. Thus, p53 protects against formation of extrahepatic biliary precancerous lesions in the context of oncogenic Kras.

Innovative method for the diagnosis of bile duct lesions using a novel tapered-tip sheath system to facilitate biliary biopsies.

BACKGROUND AND AIMS: Pathologic evaluation of bile duct lesions is crucial for a definitive diagnosis and determination of an appropriate therapeutic strategy; however, current methods are limited by several challenges. This study evaluated the impact of a novel tapered-tip sheath system on biliary stricture diagnosis. METHODS: This observational study evaluated 47 consecutive patients who underwent transpapillary biliary stricture biopsy using the novel tapered-tip sheath system from July 2020 to March 2022 compared with 51 historical control subjects undergoing conventional biopsies. Technical success rate, total biopsy time, number of biopsy specimens, adequate tissue sampling rate, adverse events, and diagnostic performance for biliary strictures were assessed. RESULTS: The technical success rate was favorable in both groups, showing no significant difference (97.9% [46 of 47] vs 88.2% [45 of 51], P = .114). However, the total biopsy time was significantly shorter in the novel system group (3.7 vs 7.7 minutes, P < .001). The number of biopsy specimens did not differ between the groups; however, the novel system group had significantly more cases in which ≥3 tissue samples could be obtained (71.7% [33 of 46] vs 51.1% [23 of 45], P = .043), a higher adequate tissue sampling rate (88.2% vs 66.4%, P < .001), and fewer adverse events (6.4% vs 21.6%, P = .043). Although the diagnostic specificity of both groups was 100%, the novel system group had significantly higher diagnostic sensitivity and accuracy (82.1% vs 50% [P = .004] and 84.8% vs 55.5% [P = .005], respectively). CONCLUSIONS: The novel tapered-tip sheath system is a promising tool for biliary stricture diagnosis.

JNK pathway plays a critical role for expansion of human colorectal cancer in the context of BRG1 suppression.

Tumor stem cells (TSCs), capable of self-renewal and continuous production of progeny cells, could be potential therapeutic targets. We have recently reported that chromatin remodeling regulator Brg1 is required for maintenance of murine intestinal TSCs and stemness feature of human colorectal cancer (CRC) cells by inhibiting apoptosis. However, it is still unclear how BRG1 suppression changes the underlying intracellular mechanisms of human CRC cells. We found that Brg1 suppression resulted in upregulation of the JNK signaling pathway in human CRC cells and murine intestinal TSCs. Simultaneous suppression of BRG1 and the JNK pathway, either by pharmacological inhibition or silencing of c-JUN, resulted in even stronger inhibition of the expansion of human CRC cells compared to Brg1 suppression alone. Consistently, high c-JUN expression correlated with worse prognosis for survival in human CRC patients with low BRG1 expression. Therefore, the JNK pathway plays a critical role for expansion and stemness of human CRC cells in the context of BRG1 suppression, and thus a combined blockade of BRG1 and the JNK pathway could be a novel therapeutic approach against human CRC.

Loss of Arid1a and Pten in Pancreatic Ductal Cells Induces Intraductal Tubulopapillary Neoplasm via the YAP/TAZ Pathway.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) arises from several types of premalignant lesions, including intraductal tubulopapillary neoplasm (ITPN); however, the molecular pathogenesis of ITPN remains unknown. METHODS: We performed studies with Hnf1b-CreERT2; Ptenf/f; Arid1af/f mice to investigate the consequence of genetic deletion of Arid1a in adult pancreatic ductal cells in the context of oncogenic PI3K/Akt pathway activation. RESULTS: Simultaneous deletion of Arid1a and Pten in pancreatic ductal cells resulted in the development of ITPN, which progressed to PDAC, in mice. Simultaneous loss of Arid1a and Pten induced dedifferentiation of pancreatic ductal cells and Yes-associated protein 1/Transcriptional coactivator with PDZ-binding motif (YAP/TAZ) pathway activation. Consistent with the mouse data, TAZ expression was found elevated in human ITPNs and ITPN-derived PDACs but not in human intraductal papillary mucinous neoplasms, indicating that activation of the TAZ pathway is a distinctive feature of ITPN. Furthermore, pharmacological inhibition of the YAP/TAZ pathway suppressed the dedifferentiation of pancreatic ductal cells and development of ITPN in Arid1a and Pten double-knockout mice. CONCLUSION: Concurrent loss of Arid1a and Pten in adult pancreatic ductal cells induced ITPN and ITPN-derived PDAC in mice through aberrant activation of the YAP/TAZ pathway, and inhibition of the YAP/TAZ pathway prevented the development of ITPN. These findings provide novel insights into the pathogenesis of ITPN-derived PDAC and highlight the YAP/TAZ pathway as a potential therapeutic target.

Clinical impact of a novel device delivery system in the diagnosis of bile duct lesions: A single-center experience.

BACKGROUND AND AIM: Pathological evaluation is essential for the diagnosis of biliary tract diseases. However, existing evaluation methods have various challenges in terms of operability and diagnostic performance. The present study aimed to evaluate the feasibility, utility, and safety of a novel device delivery system for bile duct biopsy. METHODS: This study was conducted as a retrospective, descriptive analysis at a single center. Overall, 25 examinations in 14 consecutive patients who underwent transpapillary biopsies for biliary lesions using the novel device delivery system from July to November 2020 were reviewed. Number and time of biopsy, technical success rate, adequate tissue sampling rate, adverse events, and diagnostic performance of bile duct biopsies using the novel device were evaluated. Moreover, negative surgical margins were assessed in patients who underwent surgical resection after mapping biopsy. RESULTS: The median number of biopsy samples was five (range: 2-13), with a median biopsy time of 11.6 min. The technical success rate was 100% (140/140), with an adequate sampling rate of 82.9% (116/140). These rates did not differ depending on the biopsy site or purpose. There were no serious adverse events related to the procedures. The diagnostic sensitivity, specificity, and accuracy of biliary stricture were 90%, 100%, and 92.3%, respectively. Negative surgical margins were confirmed in all patients undergoing surgical resection, including one patient with a surgical procedure changed based on the results of mapping biopsy. CONCLUSIONS: The novel device delivery system has potentials in diagnosing biliary tract diseases and determining appropriate treatment strategies.

Concurrent Activation of Kras and Canonical Wnt Signaling Induces Premalignant Lesions That Progress to Extrahepatic Biliary Cancer in Mice.

Biliary cancer has long been known to carry a poor prognosis, yet the molecular pathogenesis of carcinoma of the extrahepatic biliary system and its precursor lesions remains elusive. Here we investigated the role of Kras and canonical Wnt pathways in the tumorigenesis of the extrahepatic bile duct (EHBD) and gall bladder (GB). In mice, concurrent activation of Kras and Wnt pathways induced biliary neoplasms that resembled human intracholecystic papillary-tubular neoplasm (ICPN) and biliary intraepithelial neoplasia (BilIN), putative precursors to invasive biliary cancer. At a low frequency, these lesions progressed to adenocarcinoma in a xenograft model, establishing them as precancerous lesions. Global gene expression analysis revealed increased expression of genes associated with c-Myc and TGFß pathways in mutant biliary spheroids. Silencing or pharmacologic inhibition of c-Myc suppressed proliferation of mutant biliary spheroids, whereas silencing of Smad4/Tgfbr2 or pharmacologic inhibition of TGFß signaling increased proliferation of mutant biliary spheroids and cancer formation in vivo. Human ICPNs displayed activated Kras and Wnt signals and c-Myc and TGFß pathways. Thus, these data provide direct evidence that concurrent activation of the Kras and canonical Wnt pathways results in formation of ICPN and BilIN, which could develop into biliary cancer. SIGNIFICANCE: This work shows how dysregulation of canonical cell growth pathways drives precursors to biliary cancers and identifies several molecular vulnerabilities as potential therapeutic targets in these precursors to prevent oncogenic progression.

Long-term Outcomes of Stent Placement Inside the Bile Duct for Biliary Strictures After Living Donor Liver Transplantation.

In living donor liver transplantation (LDLT), anastomotic biliary stricture is a serious and refractory complication. In this study, we reviewed the transition of post-LDLT anastomotic biliary strictures and evaluated long-term outcomes of stent placement inside the bile duct, which is referred to as an "inside-stent." Of 805 consecutive adult LDLT recipients in our institution (2000-2018), we reviewed 639 patients with duct-to-duct biliary reconstruction and analyzed chronological changes of post-LDLT biliary strictures. Moreover, we focused on the year 2006 when various surgical modifications were introduced and compared the details of post-LDLT biliary strictures before and after 2006, especially focusing on the long-term outcome of inside-stent placement. The proportion of left lobe grafts had increased from 1.8% before 2005 to 39.3% after 2006 (P < 0.001) to maximize the living donor safety. Overall, post-LDLT anastomotic biliary strictures occurred in 21.3% of the patients with a median follow-up period of 106.1 months, which was decreased from 32.6% before 2005 to 12.8% after 2006 (P < 0.001). Anastomotic biliary strictures were less frequent in patients with left lobe grafts than with right lobe grafts (9.4% versus 25.4%; P < 0.001). The overall technical success rate of inside-stent placement was 82.4%, with an improvement from 75.3% before 2005 up to 95.7% after 2006 (P < 0.01). Furthermore, the stricture resolution rate remained high at approximately 90% throughout the observation period. Increased use of left lobe grafts with several surgical modifications significantly reduced post-LDLT anastomotic biliary strictures, leading to favorable long-term outcomes of inside-stent placements for this condition.

Self-expandable metallic stent placement for malignant biliary stricture using a novel device delivery system.

Video 1Self-expandable metallic stent placement for malignant biliary strictures using a novel device delivery system.

Brg1 is required to maintain colorectal cancer stem cells.

Tumor cells capable of self-renewal and continuous production of progeny cells are called tumor stem cells (TSCs) and are considered to be potential therapeutic targets. However, the mechanisms underlying the survival and function of TSCs are not fully understood. We previously reported that chromatin remodeling regulator Brg1 is essential for intestinal stem cells in mice and Dclk1 is an intestinal TSC marker. In this study, we investigated the role of Brg1 in Dclk1+ intestinal tumor cells for the maintenance of intestinal tumors in mice. Specific ablation of Brg1 in Dclk1+ intestinal tumor cells reduced intestinal tumors in ApcMin mice, and continuous ablation of Brg1 maintained the reduction of intestinal tumors. Lineage tracing in the context of Brg1 ablation in Dclk1+ intestinal tumor cells revealed that Brg1-null Dclk1+ intestinal tumor cells did not give rise to their descendent tumor cells, indicating that Brg1 is essential for the self-renewal of Dclk1+ intestinal tumor cells. Five days after Brg1 ablation, we observed increased apoptosis in Dclk1+ tumor cells. Furthermore, Brg1 was crucial for the stemness of intestinal tumor cells in a spheroid culture system. BRG1 knockdown also impaired cell proliferation and increased apoptosis in human colorectal cancer (CRC) cells. Microarray analysis revealed that apoptosis-related genes were upregulated and stem cell-related genes were downregulated in human CRC cells by BRG1 suppression. Consistently, high BRG1 expression correlated with poor disease-specific survival in human CRC patients. These data indicate that Brg1 plays a crucial role in intestinal TSCs in mice by inhibiting apoptosis and is critical for cell survival and stem cell features in human CRC cells. Thus, BRG1 represents a new therapeutic target for human CRC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.