Introduction

Chronic pancreatitis (CP) is a complex inflammatory disorder characterized by progressive destruction of the pancreatic tissue, with fibrotic transformation of the tissue as a major morphological feature. The incidence of the disease is steadily increasing, but no specific therapy is currently available, making the identification of new therapeutic targets a priority. Plasminogen activator inhibitor-1 (SERPINE1/PAI-1) has been implicated in various pathological processes, including fibrosis and inflammation. However, its specific involvement in the pathogenesis of CP is not yet fully understood.

Aims & Methods

We therefore aimed to explore the role of PAI-1 in the development of CP and assess the therapeutic potential of PAI-1 inhibitors as antifibrotic agents to prevent the progression of CP.
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​The PAI-1 expression of the pancreatic tissue was determined in mouse and human pancreatic tissue samples with immunohistochemistry. CP was induced in FVB/N mice with 8X50 µg/kgbw i.p. cerulein injection, given every hour, every 3 days for a total of 5 times. PAI-1 activity was inhibited with a specific inhibitor, which was administered per os at a dose of 10 mg/kgbw for 6 consecutive days after the 3rd cerulein treatment. To assess the extent of inflammation and fibrosis hematoxylin-eosin and Crossman’s trichrome staining were performed, and biochemical assays were used to determine amylase activity and hydroxyproline concentration. PAI-1 expression and location in the pancreas was examined by RT-qPCR and immunofluorescence co-staining.

Results

PAI-1 expression was significantly higher in pancreatic tissue samples obtained from CP patients. After induction of CP in mice, the body weight and the pancreatic weight/body weight ratio significantly reduced, whereas histopathology showed significant pancreatic atrophy and fibrosis. This was also confirmed by the increased hydroxyproline concentration and decreased amylase activity of the pancreatic tissue. The gene expression of SERPINE1 increased in pancreas tissue and fibroblast cell culture in CP group, moreover the elevated PAI-1 signal localization suggests a possible expression role for activated stellate cells. Administration of PAI-1 inhibitor resulted in a decrease in both body weight and pancreatic weight loss rates, additionally the atrophy, tissue fibrosis, hydroxyproline concentrations and SEPRINE1 gene expression were significantly decreased compared to the CP group.

Conclusion

Our results indicate an elevation in PAI-1 expression in CP, with the potential for fibrosis and tissue atrophy reduction by the inhibition of PAI-1. These findings propose PAI-1 as a promising therapeutic target for the treatment of chronic pancreatitis.

Introduction

Chemotherapeutical treatment of pancreatic cancer guided by drug screening of patient-derived tumor organoids (PDTO) is a promising new tool in personalized medicine. However, standard PDTO models do not adequately model the tumor microenvironment, which have been recognized to play a pivotal role in tumor progression, maintenance and therapeutic response. Cancer-associated fibroblasts are dominant mediators of these effects and co-cultures including both PDTOs and CAFs from surgical specimens show that addition CAFs modify resistance of cancer cells to common chemotherapeutic drugs in vitro. However, as previous co-culture models have been established from surgical specimens, they cannot be applied to patients with inoperable disease. To address these shortcomings, we aimed to create a PDTO/CAF co-culture model derived from endoscopic ultrasound-guided fine-needle biopsies (EUS-FNBs) which could be readily used for drug screening purposes.

Aims & Methods

PDTO and CAF cultures were isolated and expanded from EUS-FNBs taken during diagnostic procedures in patients undergoing diagnostic workup for PDAC. Co-cultures were created after thorough characterization and validation of PDTOs and CAFs using next generation sequencing, immunohistochemistry and histopathological evaluation. Interaction between cell types in our co-culture model were evaluated by measuring area of PDTOs in co-cultures vs. standard PDTO cultures.

Results

Validated PDTO and CAF cultures could be expanded from EUS-FNBs with a success rate of 19% and 25%, respectively. In total, eight co-cultures from five different patients were created. A significant increase in growth of PDTOs was observed in 50% of the co-cultures compared to monocultures.

Conclusion

Advanced organoid co-culture models which are able to capture CAF/PDTO interaction can be established from EUS-FNB samples. However, clinical application is hampered by low success rates

References

[1] Tiriac H, Belleau P, Engle DD, et al. Organoid Profiling Identifies Common Responders to Chemotherapy in Pancreatic Cancer. Cancer Discov. 2018;8(9):1112-1129. doi:10.1158/2159-8290.CD-18-0349
[2] Beutel AK, Schütte L, Scheible J, et al. A Prospective Feasibility Trial to Challenge Patient–Derived Pancreatic Cancer Organoids in Predicting Treatment Response. Cancers (Basel). 2021;13(11):2539. doi:10.3390/cancers13112539
[3] Kalluri R. The biology and function of fibroblasts in cancer. Nat Rev Cancer. 2016;16(9):582-598. doi:10.1038/nrc.2016.73
[4] Schuth S, Le Blanc S, Krieger TG, et al. Patient-specific modeling of stroma-mediated chemoresistance of pancreatic cancer using a three-dimensional organoid-fibroblast co-culture system. J Exp Clin Cancer Res. 2022;41(1):312. doi:10.1186/s13046-022-02519-7

Introduction

Tertiary lymphoid structure (TLS) is a pathological structure existing in chronic inflammation or autoimmune diseases by promoting the immunotherapy response. However TLS neogenesis has not been evaluated in primary biliary cholangitis (PBC).

Aims & Methods

To determine the characteristics of liver TLSs neogenesis in patients with PBC and investigate the effect of inhibition of TLSs in a murine model of PBC. We performed a retrospective study of patients with PBC who underwent liver biopsy from April 2015 to November 2022. The cohort was divided into two groups based on TLS neogenesis (TLS- vs TLS+). A comparison of the severity of cholangitis between two groups was performed. Bulk RNA sequencing was performed between the groups of TLS- and TLS+ in liver biopsy. Sphingosine-1-phosphate receptor agonist FTY720 was administrated to suppress TLSs formation in dnTGFβRII mice, in which liver TLSs were developed.

Results

In total, 70 patients were included in the study cohort, of which 50 patients with TLSs formation and 20 patients without TLSs formation. The group of TLS+ showed a higher level of serum liver function compared with TLS- (Table1). The level of IgM and the proportion of liver cirrhosis were higher in the group of TLS+. Transcriptomic analysis in liver biopsy indicated a group of 1,548 significantly upregulated genes and 1,365 downregulated genes comparing the group of TLS+ with TLS-. The KEGG pathway identified liver with TLS expression signatures associated with phospholipase D signaling pathway and B cell receptor signaling pathway. GO assessment of DEGs confirmed the activation of immune system process and leukocyte migration. FTY720 significantly attenuated serum liver function and reduced the levels of inflammation cytokines in dnTGFβRII mice. Notably, TLSs were significantly smaller in FTY720-treated mice, with the levels of TLS-related chemokine and lymphotoxin decreasing significantly. Flow cytometry indicated the percentage of CD19⁺ B cells and CD4⁺ T cells out of the liver CD45⁺population was reduced significantly in FTY720-treated liver.

_{Table1: Clinical and demographic characteristics in patients of tertiary lymphoid structures at the time of biopsy}

	TLS+ (n=50)	TLS-(n=20)	P Value
Female, n (%)	44 (88.0)	19 (95.0)	0.445
Age at liver biopsy	54.5 (48.0-62.5)	50.0 (46.0-55.0)	0.528
ALT (U/L)	46.5 (31.0-79.5)	26.0 (12.0-39.0)	0.018*
AST (U/L)	50.5 (30.0-90.3)	27.0 (22.0-40.0)	0.005*
ALP (U/L)	196.0 (114.3-305.5)	107.0 (72.0-154.0)	0.008*
GGT (U/L)	150.0 (62.5-299.0)	153.0 (61.0-306.0)	0.826
IgG (g/L)	1765.0 (1507.5-2037.5)	1770.0 (1290.0-1890.0)	0.207
IgM (g/L)	342.0 (187.8-507.3)	206.0 (138.0-338.0)	0.013*
Liver cirrhosis, n (%)	14 (28.0)	0	0.007*

Note: Data are presented as a percentage or as the median (25th-75th percentile). Comparisons between the groups were performed by chi-squared tests or nonparametric tests. *P < 0.05.
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; GGT, gamma glutamyl transpeptidase;IgG, immunoglobulin G; IgM, immunoglobulin M.

Conclusion

Our results suggest the potential role of TLSs in building the severity of cholangitis and this understanding may guide therapeutic interventions of PBC.

References

[1]Sato Y, Silina K, van den Broek M, Hirahara K, Yanagita M. The roles of tertiary lymphoid structures in chronic diseases. Nat Rev Nephrol. 2023 : 1-13.
[2]Zhang QY, Ye XP, Zhou Z, et al. Lymphocyte infiltration and thyrocyte destruction are driven by stromal and immune cell components in Hashimoto's thyroiditis. Nat Commun. 2022. 13(1): 775.
[3]Al-Diwani A, Theorell J, Damato V, et al. Cervical lymph nodes and ovarian teratomas as germinal centres in NMDA receptor-antibody encephalitis. Brain. 2022. 145(8): 2742-2754.

Disclosure

Nothing to disclosure