Introduction

The development of pancreatic malignancies is related to metabolic and genetic alterations. Genetic alterations of both progenitor genes and stem cells genes and markers were described in pancreatic neoplastic and inflammatory disorders. In our study we comprehensively asses the expression of mRNA of both stem cells and progenitor pancreatic genes in peripherial blood of patients with pancreatic malignancy in different clinical stages,neuroendocrine tumors, and pancreatic inflammatory disorders (acute(AP) and chronic pancreatitis(CP)), to identify the tumor markers and underlying mechanisms of tumor progression.

Aims & Methods

Our aim was to assess the peripheral blood mononuclear cells (PBMNCs) messenger RNA (mRNA) expression profile of stem cell genes (OCT4, NANOG) and pancreatic progenitor genes (CK19, HES1, INS, PDX1) in selected pancreatic diseases: neoplastic (cancer, neuroendocrine tumors) and inflammatory (acute and chronic pancreatitis).
The 49 patients with pancreatic neoplastic diseases (37 with cancer, 12 with neuroendocrine tumors) and 48 patients with pancreatitis (28 with acute, 23 with chronic) and 34 patients from gender and age-matched control group hospitalized in tertiary center were enrolled into the study. Venous blood was collected and RNA was isolated from PBMNCs. The mRNA expression of 6 stem cell and pancreatic progenitor genes: OCT4 (POU class 5 homeobox 1), NANOG, CK19 (keratin 19), HES1 (hes family bHLH transcription factor 1), INS (insulin), PDX1 (pancreatic and duodenal homeobox 1) was assessed by real-time quantitative PCR. The results were analyzed statistically and their associations with available clinical, biochemical and morphological data (WBC, ERY, PLT, CRP, Ca 19.9, weight, height and BMI) were studied with Kruskall Wallis test, Mann-Whitney U test and Spearman rank correlation coefficient.

Results

We found that the expression of gene characteristic for early stem cells NANOG (p=0.03) and the expression of genes encoding insulin and cytokeratin 19 (INS p=0.02,CK19 p=0.005) was increased in pancreatic cancer patients. However, it was not significantly different in other neoplastic (neuroendocrine) and inflammatory conditions. Moreover, we found that OCT4, HES1, PDX1 expression is significantly decreased in PBMNCs of patients with pancreatitis (both acute and chronic). There were no significant differences in genetic expression between the patients with different stages of pancreatic cancer and patients with acute pancreatitis with different severity (mild vs moderate/severe). What is more, CK19 overexpression correlated with inflammatory markers (both CRP and WBC) in patients with pancreatic cancer.

Conclusion

PBMNCs mRNA expression of selected stem and progenitor genes (NANOG, INS, CK19) may serve as a marker of pancreatic cancer and correlates with coexisting inflammation.

Introduction

Pancreatic solid pseudopapillary neoplasm (SPN) is a rare tumor of low malignant potential that predominantly affects young women and is characterized pathologically by a pseudopapillary structure. Surgical resection is the only curative treatment for SPN, and there are few effective treatment options for patients with metastasis. However, the natural history of SPN, their molecular biological characteristics, and the factors that determine its malignant potential have not been clarified. Recently, organoid culture technology has enabled more specific and precise analyses using only cultured tumor cells. In this study, we aimed to clarify the molecular biological mechanisms that define the origin of SPN, what genetic factors define the malignant transformation of SPN by combining advanced high throughput technology and organoid technology, and examined its correlation with clinical findings.

Aims & Methods

Age, gender, tumor diameter, and presence or absence of malignant pathological findings were extracted from six SPN cases resected from December 2019 to April 2021 at our hospital. In addition, organoids were derived from tumor and normal pancreatic tissues. Gene expression of the organoids and surgical samples were analyzed by RNA-seq.

Results

The median age of the patients was 41 years (IQR: 38-49 years) with a male-to-female ratio of 1:7. The median tumor diameter was 34 mm (IQR: 18-101 mm). Three patients had nerve plexus or vascular invasion, and one of them had liver metastasis. SPN with malignant findings showed significantly increased nucleus diameter (mean 5.8 μm vs. 8.1 μm, p<0.001). The results suggest molecular biological differences associated with malignant transformation. RNA-Seq was performed using normal and tumor surgical tissues, as well as normal and tumor organoids generated from the surgical samples. Comparison of tumor and normal tissue from surgical specimens showed marked changes in gene expression related to cellular localization, and this feature was even more pronounced in tumors with higher grades of malignancy. A similar trend was observed using organoids.

Conclusion

We examined clinical features and performed RNA-seq using surgical tissues and organoids to elucidate the gene expression patterns that characterize SPN. We found that SPN showed a significant enlargement of cell nuclei in high-grade cases, and that there were clear differences in gene expression related to cellular localization and extracellular matrix. Based on the findings of this study, we hope to elucidate the molecular biological mechanisms leading to the development of novel therapies for SPN.

Introduction

Rare early onset cases of diabetes stem from single gene mutations, whereas frequent gene variants can also contribute to multifactorial diabetes, indicating shared disease mechanisms. Our recent study has identified ONECUT1 as a gene implicated in both monogenic and multifactorial diabetes, establishing it as crucial target for investigating fundamental signaling pathways in diabetes pathology [1,2]. However, numerous diabetic characteristics remain unexplained by typical genetic alterations, highlighting the underestimated significance of the non-coding genome. We have identified a neonatal diabetes patient carrying a heterozygous ONECUT1 truncating variant. Remarkably, the patient exhibited a phenotype akin to individuals with homozygous ONECUT1 truncating mutations. Upon further examination, we detected an additional heterozygous deletion in the non-coding genome, leading us to hypothesize that it could impact the regulation of the remaining ONECUT1 allele. Consequently, the dysregulation of the ONECUT1-dependent transcriptional machinery crucial for proper beta-cell development occurs, ultimately leading to the severe diabetic phenotype observed in the patient.

Aims & Methods

Following deletion of specific genomic regions in human embryonic stem cells, we initiated their differentiation into the pancreatic endocrine lineage and meticulously characterized the developmental stages.

Results

Importantly, we successfully replicated the patient’s diabetic phenotype, elucidating deficiencies in both pancreatic development and beta-cell functionality. The differentiation of pancreatic progenitors was notably hindered by the loss of the regulatory region associated with nearly complete ablation of ONECUT1. Moreover, the stem cell-derived islets exhibited a reduced number of beta-cells as well as impaired insulin secretion, even in heterozygous knockouts.

Conclusion

Altogether, our ongoing comprehensive mechanistic characterization provides insights into the precise role of the identified regulatory element in pancreatic beta-cell development and diabetes pathophysiology, emphasizing the significance of the non-coding genome. By unveiling unknown molecular pathomechanisms linked to alterations in regulatory sequences, we contribute to a deeper understanding of the pancreatic transcriptional network and establish the foundation for personalized diabetes treatment.

References

[1] Philippi, A., Heller, S., Costa, I.G. et al. Mutations and variants of ONECUT1 in diabetes. Nat Med 27, 1928–1940 (2021). https://doi.org/10.1038/s41591-021-01502-7
[2] Heller, S., Li, Z., Lin, Q. et al. Transcriptional changes and the role of ONECUT1 in hPSC pancreatic differentiation. Commun Biol 4, 1298 (2021). https://doi.org/10.1038/s42003-021-02818-3

Introduction

The identification and establishment of reliable biomarkers and early diagnostic system for pancreatic ductal adenocarcinoma (PDAC) are urgently needed. Although blood-based liquid biopsy for comprehensive genomic profiling is useful for cancer diagnosis, we have recently shown that driver gene mutations derived from PDAC can be detected at a higher rate in pancreatic juice (PJ) than blood. We have also reported that some RNAs are carried within extracellular vesicles (EVs) and EV-encapsulated RNAs circulating in body fluid are useful as biomarkers for PDAC diagnosis. We recently developed a unique immunoprecipitation method called EViSTEP™ that utilizes a chelating-based reagent and EV surface marker antibodies (CD9/CD63) to isolate EVs with high purity and high yield.

Aims & Methods

In this study, we aimed to evaluate the usefulness of multi-layer liquid biopsy system by DNA mutation and RNA expression in PJ for cancer diagnosis. PJ samples (n=83; 15 benign hepatopancreatobiliary diseases, 33 low-grade (LG) IPMN, 10 high-grade (HG) IPMN and 25 PDAC) were collected during ERCP examination from each patient in a multicenter study. Cell-free (cf) DNA/RNA and EV-derived RNA (EV RNA) were purified from 2 ml of each PJ. Digital PCR was performed for driver gene mutation analysis (4 types of KRAS and 2 types of GNAS), as well as expression analysis of 10 long non-coding RNAs (lncRNA, e.g., HEVEPA, LINC02280) in cfRNA and EV RNA. The diagnostic performance of pancreatic cancer (PDAC+HG-IPMN) was statistically analyzed after normalizing RNA expression with two reference genes (ACTB/B2M).

Results

Logistic regression analysis (objective variables; PDAC+HG IPMN and others) with the KRAS and GNAS variant allele frequency (VAF), and RNA expression as factors was performed. The Area Under Curves (AUC) in Receiver Operating Characteristic (ROC) curve analysis for PDAC+HG IPMN diagnosis were 0.490-0.74 for KRAS mutations, 0.475-0.554 for GNAS mutations, 0.451-0.654 for cfRNA expressions and 0.362-0.639 for EV RNA expressions. Among them, the factors with relatively high AUC were KRASG12V (0.728), KRASG12R (0.687), cfRNA RP11-750H9.5/ACTB(0.654), EV RNA CRNDE/ACTB(0.633). The AUC values of the combinations, [KRASG12V, KRASG12R, cfRNA RP11-750H9.5/ACTB and EV RNA CRNDE/ACTB] and [the aforementioned four factors and CA19-9], were 0.876 and 0.885, respectively in terms of discriminating PDAC+HG IPMN. Moreover, concerning the diagnosis of Stage 0/I PDAC, the AUC values of EV HEVEPA, CA19-9, and the combination [EV HEVEPA and CA19-9] were 0.693, 0.746 and 0.84, respectively, suggesting that EV HEVEPA would be reliable biomarker for early detection of PDAC.

Conclusion

By incorporating cell-free lncRNA and EV lncRNA expression analysis into DNA mutation analysis in PJ samples could be expected to improve the diagnostic performance of pancreatic cancer. Liquid biopsy using PJ would predict the presence of neoplastic lesions with high probability and has a powerful potential for early diagnosis. Establishing the multilayer gene panel using these DNA and RNA has the potential to improve the early diagnostic accuracy for pancreatic cancer, a disease that exhibits significant diversity.

Disclosure

Grants for commissioned/joint research: Hitachi High-Tech Corporation (KT, YM), H.U. Group Research Institute G.K. (KT)

Introduction

Human stem cell-based organoids are well established modeling systems widely used to investigate a range of different diseases. For the pancreas, especially endocrine stem cell-derived islets are used to model and decipher the complex development of diabetes. Although we and others also generated pancreatic duct-like organoids (PDLO) [1-4], engineering the main pancreatic compartment, the acinar cells, is underrepresented in literature [1, 4, 5]. Here, we used our advanced differentiation protocol to generate pancreatic progenitors (PPs) [1]. We enriched for the glycoprotein-2 (GP2) expressing multipotent pancreatic progenitors and generated a finetuned acinar differentiation protocol based on pathway and transcriptome analysis of PPs on single cell level. The optimized protocol robustly produces pure mature and functionally active pancreatic acinar-like organoids which we extensively characterized and validated by high resolution imaging, electron microscopy, transplantation studies and functional assays.

Aims & Methods

Immunofluorescence staining of key acinar markers was performed to comprehensively characterize the stem cell-based acinar organoids. High resolution imaging and transmission electron microscopy revealed the presence of secretory vesicles and digestive enzymes. Functionality was proven by enzyme activity assays. Further maturation of the acinar organoids was monitored by transplantation on an ex vivo scaffold, the porcine urinary bladder [6, 7].

Results

In this study we succeeded in producing functional and mature pancreatic acinar organoids by differentiation of human embryonic stem cells. The enrichment for multipotent pancreatic progenitors was facilitated by a double magnetic activated cell sorting, a straightforward approach to sort for GP2 high expressing multipotent progenitors. Finetuning and modulating relevant pathways identified with a PP single-cell RNA sequencing dataset was based on building trajectories, cell-cell communication patterns and analyzing expression profiles. A comprehensive pipeline for characterization and analysis of the resulting acinar organoids validated their functionality and maturity.

Conclusion

The here generated stem-cell derived pancreatic acinar-like organoids provide a useful human model system to investigate pancreatic exocrine disorders, like pancreatic cancer or pancreatitis. It offers a valuable tool for understanding cancer initiation and progression in an animal-reduced human setup.

References

1. Merz, S., et al., Single-cell profiling of GP2-enriched pancreatic progenitors to simultaneously create acinar, ductal, and endocrine organoids. Theranostics, 2023. 13(6): p. 1949.
2. Breunig, M., et al., Modeling plasticity and dysplasia of pancreatic ductal organoids derived from human pluripotent stem cells. Cell Stem Cell, 2021. 28(6): p. 1105-1124. e19.
3. Breunig, M., et al., Differentiation of human pluripotent stem cells into pancreatic duct-like organoids.STAR protocols, 2021. 2(4): p. 100913.
4. Huang, L., et al., Commitment and oncogene-induced plasticity of human stem cell-derived pancreatic acinar and ductal organoids. Cell Stem Cell, 2021. 28(6): p. 1090-1104. e6.
5. Hohwieler, M., et al., Human pluripotent stem cell-derived acinar/ductal organoids generate human pancreas upon orthotopic transplantation and allow disease modelling. Gut, 2017. 66(3): p. 473-486.
6. Melzer, M.K., et al., Organoids at the PUB: the porcine urinary bladder serves as a pancreatic niche for advanced cancer modeling. Advanced Healthcare Materials, 2022. 11(11): p. 2102345.
7. Melzer, M.K., et al., Protocol to use de-epithelialized porcine urinary bladder as a tissue scaffold for propagation of pancreatic cells. STAR protocols, 2022. 3(4): p. 101869.