Duct Cells Research Articles

E2F transcription factors promote tumorigenicity in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with limited treatment options, illustrating an urgent need to identify new drugable targets in PDACs. Using the similarities between tumor development and normal embryonic development, which is accompanied by rapid cell expansion, we aimed to identify and characterize embryonic signaling pathways that were reinitiated during tumor formation and expansion. Here, we report that the transcription factors E2F1 and E2F8 are potential key regulators in PDAC. E2F1 and E2F8 RNA expression is mainly localized in proliferating cells in the developing pancreas and in malignant ductal cells in PDAC. Silencing of E2F1 and E2F8 in PANC-1 pancreatic tumor cells inhibited cell proliferation and impaired cell spreading and migration. Moreover, loss of E2F1 also affected cell viability and apoptosis with E2F expression in PDAC tissues correlating with expression of apoptosis and mitosis pathway genes, suggesting that E2F factors promote cell cycle regulation and tumorigenesis in PDAC cells. Our findings illustrate that E2F1 and E2F8 transcription factors are expressed in pancreatic progenitor and PDAC cells, where they contribute to tumor cell expansion by regulation of cell proliferation, viability, and cell migration making these genes attractive therapeutic targets and potential prognostic markers for pancreatic cancer.

Single-cell transcriptomics reveals a role for pancreatic duct cells as potential mediators of inflammation in diabetes mellitus.

Inflammation of the pancreas contributes to the development of diabetes mellitus. Although it is well-accepted that local inflammation leads to a progressive loss of functional beta cell mass that eventually causes the onset of the disease, the development of islet inflammation remains unclear. Here, we used single-cell RNA sequencing to explore the cell type-specific molecular response of primary human pancreatic cells exposed to an inflammatory environment. We identified a duct subpopulation presenting a unique proinflammatory signature among all pancreatic cell types. Overall, the findings of this study point towards a role for duct cells in the propagation of islet inflammation, and in immune cell recruitment and activation, which are key steps in the pathophysiology of diabetes mellitus.

Highly sensitive ratiometric fluorescent fiber matrices for oxygen sensing with micrometer spatial resolution

AbstractOxygen (O2)-sensing matrices are promising tools for the live monitoring of extracellular O2 consumption levels in long-term cell cultures. In this study, ratiometric O2-sensing membranes were prepared by electrospinning, an easy, low-cost, scalable, and robust method for fabricating nanofibers. Poly(ε-caprolactone) and poly(dimethyl)siloxane polymers were blended with tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) dichloride, which was used as the O2-sensing probe, and rhodamine B isothiocyanate, which was used as the reference dye. The functionalized scaffolds were morphologically characterized by scanning electron microscopy, and their physicochemical profiles were obtained by Fourier transform infrared spectroscopy, thermogravimetric analysis, and water contact angle measurement. The sensing capabilities were investigated by confocal laser scanning microscopy, performing photobleaching, reversibility, and calibration curve studies toward different dissolved O2 (DO) concentrations. Electrospun sensing nanofibers showed a high response to changes in DO concentrations in the physiological-pathological range from 0.5% to 20% and good stability under ratiometric imaging. In addition, the sensing systems were highly biocompatible for cell growth promoting adhesiveness and growth of three cancer cell lines, namely metastatic melanoma cell line SK-MEL2, breast cancer cell line MCF-7, and pancreatic ductal adenocarcinoma cell line Panc-1, thus recreating a suitable biological environment in vitro. These O2-sensing biomaterials can potentially measure alterations in cell metabolism caused by changes in ambient O2 content during drug testing/validation and tissue regeneration processes. Graphic abstract

The pRb/RBL2-E2F1/4-GCN5 axis regulates cancer stem cell formation and G0 phase entry/exit by paracrine mechanisms

The lethality, chemoresistance and metastatic characteristics of cancers are associated with phenotypically plastic cancer stem cells (CSCs). How the non-cell autonomous signalling pathways and cell-autonomous transcriptional machinery orchestrate the stem cell-like characteristics of CSCs is still poorly understood. Here we use a quantitative proteomic approach for identifying secreted proteins of CSCs in pancreatic cancer. We uncover that the cell-autonomous E2F1/4-pRb/RBL2 axis balances non-cell-autonomous signalling in healthy ductal cells but becomes deregulated upon KRAS mutation. E2F1 and E2F4 induce whereas pRb/RBL2 reduce WNT ligand expression (e.g. WNT7A, WNT7B, WNT10A, WNT4) thereby regulating self-renewal, chemoresistance and invasiveness of CSCs in both PDAC and breast cancer, and fibroblast proliferation. Screening for epigenetic enzymes identifies GCN5 as a regulator of CSCs that deposits H3K9ac onto WNT promoters and enhancers. Collectively, paracrine signalling pathways are controlled by the E2F-GCN5-RB axis in diverse cancers and this could be a therapeutic target for eliminating CSCs.

Expression of ZKSCAN3 protein suppresses proliferation, migration, and invasion of pancreatic cancer through autophagy.

Elevated autophagy activity enhances the malignancy of pancreatic cancer (PaCa), and autophagy is recognized as a novel therapeutic target. Zinc finger protein with KRAB and SCAN domains 3 (ZKSCAN3) is a transcription factor that suppresses autophagy, but its association with PaCa is unknown. We analyzed the function of ZKSCAN3 in PaCa and investigated whether autophagy regulation through ZKSCAN3 could become a new therapeutic target for PaCa. Using reverse transcription-quantitative polymerase chain reaction and western blotting, we observed that ZKSCAN3 expression was upregulated in several PaCa cell lines compared with normal pancreatic ductal epithelial cells. Additionally, comparing ZKSCAN3 expression with the prognosis of PaCa patients using web databases, we found that higher ZKSCAN3 expression in PaCa was associated with extended overall survival. Knocking down ZKSCAN3 promoted the proliferation of PaCa cells. Moreover, following ZKSCAN3 knockdown, PaCa cells exhibited significantly enhanced migratory and invasive properties. Conversely, overexpression of ZKSCAN3 significantly suppressed the proliferation, migration and invasion of PaCa cells. Additionally, the knockdown of ZKSCAN3 increased the expression of LC3-II, a marker of autophagy, whereas ZKSCAN3 overexpression decreased LC3-II expression. In a xenograft mouse model, tumors formed by MIA PaCa-2 cells in which ZKSCAN3 was knocked down significantly increased in size compared with the control group. In conclusion, ZKSCAN3 expression was upregulated in several pancreatic cancer cells. Additionally, it was revealed that ZKSCAN3 is negatively correlated with the malignancy of PaCa through autophagy. These results suggest that autophagy regulation via ZKSCAN3 may be a new therapeutic target for PaCa.

Anti-pancreatic cancer activity of cassane diterpenoids isolated from the seeds of Caesalpinia sappan mediated by autophagy activation via ROS/AMPK/mTORC1 pathway

Three undescribed cassane diterpenoids, caesalpanins D–F (1–3), and seven known ones were isolated from the seeds of Caesalpinia sappan. Structures and absolute configurations of 1–3 were elucidated based on the extensive spectroscopic analysis, single-crystal X-ray diffraction analysis, and ECD calculations. Structurally, compound 1 was the first example of 18-norcassane diterpenoid and 2 was a rare 20-norcassane diterpenoid having an unusual five-membered oxygen bridge between C-10/C-18. The anti-proliferative activity of 1, 3, and 4–10 against PANC-1 cells (pancreatic ductal adenocarcinoma cell line) was evaluated, and phanginin H (4) was found to exhibit anti-cancer activity with IC50 value of 18.13 ± 0.63 μM. Compound 4 inhibited PANC-1 cell growth by arresting the cell cycle at G2/M phase via regulation of cyclin-dependent kinases, and the self-renewal and metastasis of PANC-1 cells by suppressing cancer cell stemness. Furthermore, compound 4 induced ROS generation and subsequently activated autophagy, which was demonstrated by the formation of autophagic vacuoles and dynamic change of autophagic flux. The induced ROS accumulation resulted in AMPK activation and subsequently regulation of mTORC1 activity and ULK phosphorylation, indicating that 4 triggered autophagy through ROS/AMPK/mTORC1 pathway. These findings suggested that 4 might potentially be an autophagy inducer for the therapy of pancreatic cancer.

Comprehensive Analysis of Immune Signatures in Primary Biliary Cholangitis and Autoimmune Hepatitis.

Primary Biliary Cholangitis (PBC) and Autoimmune Hepatitis (AIH) are autoimmune diseases that target hepatocytes and bile duct cells, respectively. Despite their shared autoimmune nature, the differences in immunologic characteristics between them remain largely unexplored. This study seeks to elucidate the unique immunological profiles of PBC and AIH, and to identify key differences. We comprehensively analyzed various T-cell subsets and their receptor expression in a cohort of 45 patients, including 27 PBC and 18 AIH cases. Both diseases exhibited T cell exhaustion and senescence along with a surge in inflammatory cytokines. Significantly increased CD38+HLA-DR+CD8+T cell populations were observed in both diseases. AIH was characterized by an upregulation of CD8+TEMRA, CD4+TEM, and CD4+TEMRA cells, and a concurrent reduction in Treg cells. In contrast, PBC displayed a pronounced presence of Tfh cells and a contraction of CD4-CD8-T cell populations. Correlation analysis revealed that NKP46+NK frequency was closely tied to ALT and AST levels, and TIGIT expression on T cells was associated with GLB level in AIH. In PBC, there is a significant correlation between Tfh cells and ALP levels. Moreover, the identified immune landscapes in both diseases strongly related to disease severity. Through logistic regression analysis, γδ T, TIGIT+Vδ2 T, and Tfh1 cell frequencies emerged as distinct markers capable of differentiating PBC from AIH. In conclusion, our analyses reveal that PBC and AIH share similarities and differences regarding to immune profiles. And γδ T, TIGIT+Vδ2 T, and Tfh1 cell frequencies are potential noninvasive immunological markers that can differentiate PBC from AIH.

Effects of transmembrane serine protease 4 on the survival in patients with pancreatic ductal adenocarcinoma undergoing surgery followed by adjuvant chemotherapy.

The transmembrane serine protease 4 (TMPRSS4) gene is upregulated in various human cancers. However, its biological functions in pancreatic ductal adenocarcinoma remain unclear. We examined the expression of TMPRSS4 in pancreatic ductal adenocarcinoma tissues and its correlation with clinicopathological parameters in patients with pancreatic ductal adenocarcinoma who underwent surgery. The TMPRSS4 expression was immunohistochemically examined in 81 PDAC patients with pancreatic ductal adenocarcinoma. We analyzed the association between the TMPRSS4 expression and clinicopathological factors, the recurrence-free survival (RFS), and the overall survival (OS) and examined the effect of TMPRSS4 expression on cell migration and sensitivity to 5-fluorouracil. The expression rate of TMPRSS4 in the samples was 62.9% (51/81). The TMPRSS4 expression was not correlated with any clinicopathological feature. The five-year overall and recurrence-free survival rates were significantly lower in the TMPRSS4-positive group than in the TMPRSS4-negative group. On a multivariate analysis, TMPRSS4 positivity, poorly differentiated histology, and non-adjuvant chemotherapy predicted a poor OS, while TMPRSS4 positivity and poorly differentiated histology predicted a poor RFS. TMPRSS4-silenced pancreatic ductal adenocarcinoma cells showed higher sensitivity to 5-fluorouracil than did the control siRNA-transfected cells. TMPRSS4 can be considered a prognostic factor and therapeutic target for pancreatic ductal adenocarcinoma.

Multiparametric Characterization of the DSL-6A/C1 Pancreatic Cancer Model in Rats.

The DSL-6A/C1 murine pancreatic ductal adenocarcinoma (PDAC) tumor model was established in Lewis rats and characterized through a comprehensive multiparametric analysis to compare it to other preclinical tumor models and explore potential diagnostic and therapeutical targets. DSL-6A/C1 tumors were histologically analyzed to elucidate PDAC features. The tumor microenvironment was studied for immune cell prevalence. Multiparametric MRI and PET imaging were utilized to characterize tumors, and 68Ga-FAPI-46-targeting cancer-associated fibroblasts (CAFs), were used to validate the histological findings. The histology confirmed typical PDAC characteristics, such as malformed pancreatic ductal malignant cells and CAFs. Distinct immune landscapes were identified, revealing an increased presence of CD8+ T cells and a decreased CD4+ T cell fraction within the tumor microenvironment. PET imaging with 68Ga-FAPI tracers exhibited strong tracer uptake in tumor tissues. The MRI parameters indicated increasing intralesional necrosis over time and elevated contrast media uptake in vital tumor areas. We have demonstrated that the DSL-6A/C1 tumor model, particularly due to its high tumorigenicity, tumor size, and 68Ga-FAPI-46 sensitivity, is a suitable alternative to established small animal models for many forms of preclinical analyses and therapeutic studies of PDAC.

Abstract LB316: Endocrine pancreas alteration in pancreatic ductal adenocarcinoma

Abstract Background: The pancreas is central to energy homeostasis, playing essential roles in digestion and regulation of blood glucose. Exocrine pancreas contains large amounts of of hydrolytic enzyme which is responsible for the digestion. Acinar cells, as well as ductal cells, represent the prime components of exocrine pancreas. They are both supposed to be the origin of pancreatic ductal adenocarcinoma(PDAC) and the researches focused on these exocrine cells, as well as other components in pancreatic cancer, are numerous. Islet cells are crucial for glucose homeostasis and their identity are proved to be altered significantly during diabetes by single-cell RNA-sequencing(scRNA-seq). However, the presence or extent of variation of islet cells in PDAC is not established yet. Moreover, due to the low proportion of endocrine cells in pancreas, it is hard to analyze the data of endocrine cells by using published scRNA-seq data of PDAC. As a consequence of this underrepresentation, the function of endocrine cells in PDAC remains unclear. Methods: We used scRNA-seq, multiplex immunohistochemistry (mIHC), high-throughput spatial transcriptomic sequencing (Stereo-seq) to detect the alteration of endocrine pancreas in PDAC of human and mouse model. KPC mice (KrasLSL-G12Dp53LoxPPdx1-CreER) and pancreatic orthotopic transplantation mice were applied to verify the islet cell alteration in multiple stages of PDAC. We also performed islet isolation and purification to enhance the proportion of endocrine cells when preparing single-cell suspension. Cell co-culture experiments were used to determine the interaction between exocrine-endocrine compartments. Results: Through mIHC of islet cells in PDAC tissues and normal pancreas tissues of mice (KPC vs. C57BL/6J) as well as human (PDAC patients vs. healthy donors), we found the decreased proportions of inulin positive (INS+) cells and increased proportions of glucagon positive (GCG+) cells in PDAC tissues. Furthermore, the proportion of pancreatic polypeptide positive (PPY+) cells was significantly elevated in PDAC tissue in both human and murine. Then, we quantified the double-positive cells and found the proportions of INS+GCG+, INS+PPY+ and GCG+PPY+ were markedly increased in PDAC tissue. Additionally, through the experiments in mouse model, we found the islet cells altered gradually within the multiple stage of PDAC(normal-PanIN-PDAC). Finally, it was found that PDAC cells could induce the transdifferentiation of pancreatic α and β cells into GCG+PPY+ and INS+PPY+ double-positive cells, which further promoted PDAC proliferation in a paracrine-dependent manner and formed a reciprocal interaction. Conclusions: Our study systematically maps the alteration of pancreatic endocrine cells in PDAC and elucidates the potential interaction mechanisms between endocrine and exocrine cells during PDAC carcinogenesis. Meanwhile, it is suggested that pancreatic endocrine cells should be considered as an important component of the PDAC microenvironment. Citation Format: Yuan Chen, Xinpeng Yin, Ruiyuan Xu, Rexiati Ruze, Jianlu Song, Chenglin Hu, Chengcheng Wang, Yupei Zhao. Endocrine pancreas alteration in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB316.

Potential role of lipophagy impairment for anticancer effects of glycolysis-suppressed pancreatic ductal adenocarcinoma cells

Although increased aerobic glycolysis is common in various cancers, pancreatic ductal adenocarcinoma (PDAC) cells can survive a state of glycolysis suppression. We aimed to identify potential therapeutic targets in glycolysis-suppressed PDAC cells. By screening anticancer metabolic compounds, we identified SP-2509, an inhibitor of lysine-specific histone demethylase 1A (LSD1), which dramatically decreased the growth of PDAC PANC-1 cells and showed an anti-tumoral effect in tumor-bearing mice. The growth of glycolysis-suppressed PANC-1 cells was also inhibited by another LSD1 inhibitor, OG-L002. Similarly, the other two PDAC cells (PK-1 and KLM-1) with suppressed glycolysis exhibited anticancer effects against SP-2509. However, the anticancer effects on PDAC cells were unrelated to LSD1. To investigate how PDAC cells survive in a glycolysis-suppressed condition, we conducted proteomic analyses. These results combined with our previous findings suggested that glucose-starvation causes PDAC cells to enhance mitochondrial oxidative phosphorylation. In particular, mitochondrial fatty acid metabolism was identified as a key factor contributing to the survival of PDAC cells under glycolysis suppression. We further demonstrated that SP-2509 and OG-L002 disturbed fatty acid metabolism and induced lipid droplet accumulation through the impairment of lipophagy, but not bulk autophagy. These findings indicate a significant potential association of lipophagy and anticancer effects in glycolysis-suppressed PDAC cells, offering ideas for new therapeutic strategies for PDAC by dual inhibition of glycolysis and fatty acids metabolism.

Abstract LB365: Generation of a novel severely immunodeficient Sprague Dawley rat model for xenograft studies

Abstract The recombinant-activating gene 2 (Rag2) plays a critical role in the recombination of the immunoglobulin and T cell receptor genes during the maturation of T and B cells. Loss of Rag2 protein leads to a lack of functional T and B cells, which eliminates the adaptive immune response. However, Rag2 deficiency alone may not guarantee the success of xenograft implantation, due to NK cells and other innate cells remaining. Il2rg is a key component of several cytokine receptors involved in differentiation and/or function of numerous adaptive and innate immune cells, including NK cells, mast cells, basophils, and dendritic cells. To generate a more severely immunodeficient rat model, both the Rag2 and Il2rg genes were knocked out in Sprague Dawley (SD) rats; this model was termed B-SDG rats. The immune profile and organ weights of thymus and spleen were investigated in B-SDG rats in comparison with Rag2-/- and wild-type SD rats. The spleen weights of both Rag2-/- and B-SDG rats were similar, but significantly reduced compared to wild-type SD rats. Notably, the thymus weights of B-SDG male rats were significantly smaller than those of Rag2-/- males. The immune cell profiles in thymus, spleen and blood were next evaluated by flow cytometry. A complete lack of B and T cells was observed in both Rag2-/- and B-SDG rats. B-SDG rats lacked CD161+ NK cells in the spleen and blood, which were maintained in Rag2-/- rats. Dendritic cells, monocytes, macrophages, and neutrophils were detected in all strains. In vivo xenograft implantation with human lung, bladder, breast, pancreas, acute monocytic and myelomonocytic leukemia tumor cell lines was next evaluated in B-SDG rats, which demonstrated successful engraftment. Furthermore, several efficacy studies were performed in B-SDG rats, including cisplatin chemotherapy on human breast invasive ductal carcinoma cell (HCC1954) and human large cell lung cancer cell (NCI-H460) xenografts, and a HER2-targeting ADC therapy was tested in B-SDG rats harboring human non-small cell lung cancer tumors (NCI-H1975). Taken together, these data confirm that B-SDG rats provide a powerful immunodeficient model for preclinical in vivo evaluation of novel drug candidates. Citation Format: Yanhui Nie, Meiqi Zhang, Wanling Zhong, Jia Feng, Bahetiyaer Huwatibieke, Zhaoxue Yu. Generation of a novel severely immunodeficient Sprague Dawley rat model for xenograft studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB365.

The protein phosphatase-2A subunit PR130 is involved in the formation of cytotoxic protein aggregates in pancreatic ductal adenocarcinoma cells

As a major source of cellular serine and threonine phosphatase activity, protein phosphatase-2A (PP2A) modulates signaling pathways in health and disease. PP2A complexes consist of catalytic, scaffolding, and B-type subunits. Seventeen PP2A B-type subunits direct PP2A complexes to selected substrates. It is ill-defined how PP2A B-type subunits determine the growth and drug responsiveness of tumor cells. Pancreatic ductal adenocarcinoma (PDAC) is a disease with poor prognosis. We analyzed the responses of murine and human mesenchymal and epithelial PDAC cells to the specific PP2A inhibitor phendione. We assessed protein levels by immunoblot and proteomics and cell fate by flow cytometry, confocal microscopy, and genetic manipulation. We show that murine mesenchymal PDAC cells express significantly higher levels of the PP2A B-type subunit PR130 than epithelial PDAC cells. This overexpression of PR130 is associated with a dependency of such metastasis-prone cells on the catalytic activity of PP2A. Phendione induces apoptosis and an accumulation of cytotoxic protein aggregates in murine mesenchymal and human PDAC cells. These processes occur independently of the frequently mutated tumor suppressor p53. Proteomic analyses reveal that phendione upregulates the chaperone HSP70 in mesenchymal PDAC cells. Inhibition of HSP70 promotes phendione-induced apoptosis and phendione promotes a proteasomal degradation of PR130. Genetic elimination of PR130 sensitizes murine and human PDAC cells to phendione-induced apoptosis and protein aggregate formation. These data suggest that the PP2A-PR130 complex dephosphorylates and thereby prevents the aggregation of proteins in tumor cells.Graphical

Two-generation Exposure to a High-fat Diet Alters Duct Cell Morphology and Induces Connexin 32 and 43 Expression in Rat Submandibular Salivary Glands

Two-generation Exposure to a High-fat Diet Alters Duct Cell Morphology and Induces Connexin 32 and 43 Expression in Rat Submandibular Salivary Glands

Role of Wnt5b-Ror1 signaling in the proliferation of pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most refractory cancers with the worst prognosis. Although several molecules are known to be associated with the progression of PDAC, the molecular mechanisms underlying the progression of PDAC remain largely elusive. The Ror-family receptors, Ror1 and Ror2, which act as a receptor(s) for Wnt-family ligands, particularly Wnt5a, are involved in the progression of various types of cancers. Here, we show that higher expression of Ror1 and Wnt5b, but not Ror2, are associated with poorer prognosis of PDAC patients, and that Ror1 and Wnt5b are expressed highly in a type of PDAC cell lines, PANC-1 cells. Knockdown of either Ror1 or Wnt5b in PANC-1 cells inhibited their proliferation significantly in vitro, and knockout of Ror1 in PANC-1 cells resulted in a significant inhibition of tumor growth in vivo. Furthermore, we show that Wnt5b-Ror1 signaling in PANC-1 cells promotes their proliferation in a cell-autonomous manner by modulating our experimental setting in vitro. Collectively, these findings indicate that Wnt5b-Ror1 signaling might play an important role in the progression of some if not all of PDAC by promoting proliferation.

The crucial prognostic signaling pathways of pancreatic ductal adenocarcinoma were identified by single-cell and bulk RNA sequencing data

Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor with poor prognosis and high mortality. Although a large number of studies have explored its potential prognostic markers using traditional RNA sequencing (RNA-Seq) data, they have not achieved good prediction effect. In order to explore the possible prognostic signaling pathways leading to the difference in prognosis, we identified differentially expressed genes from one scRNA-seq cohort and four GEO cohorts, respectively. Then Cox and Lasso regression analysis showed that 12 genes were independent prognostic factors for PDAC. AUC and calibration curve analysis showed that the prognostic model had good discrimination and calibration. Compared with the low-risk group, the high-risk group had a higher proportion of gene mutations than the low-risk group. Immune infiltration analysis revealed differences in macrophages and monocytes between the two groups. Prognosis related genes were mainly distributed in fibroblasts, macrophages and type 2 ducts. The results of cell communication analysis showed that there was a strong communication between cancer-associated fibroblasts (CAF) and type 2 ductal cells, and collagen formation was the main interaction pathway.

Family case of cystic fibrosis

Cystic fibrosis is an autosomal recessively inherited monogenic disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, located on the long arm of chromosome 7 (7q31.2). This mutation results in a disruption in the transport of sodium and chloride ions between intercellular fluid and the epithelial cells of the excretory ducts of the exocrine glands. This paper presents the description of a family case of cystic fibrosis involving two patients who have been under observation since birth and are sisters (patient A – 4 years 10 months, patient B – 1 year 11 months). In both patients, neonatal screening and sequential diagnosis of cystic fibrosis identified the DF508/CFTRdele2.3(21kb) mutation, the most prevalent among the Caucasian population in the Russian Federation. The primary concern in managing both patients is the correction of nutritional status and minimization of exacerbations of chronic bronchopulmonary inflammation. To determine the management strategy for these patients, a consultation was held with Dr. E.I. Kondratyeva, a leading cystic fibrosis expert and professor at the MGSC Institute, who serves as the head of the Scientific and Clinical Department of Cystic Fibrosis and the Department of Genetics of Respiratory System Diseases, an expert of the European Society for Cystic Fibrosis Committee on Diagnostics and Registry, and Deputy Chief for Scientific Work of Research Clinical Institute of Childhood of the Ministry of Health of the Moscow Region. The peculiarity of the presented clinical cases lies in the fact that within one family, with an interval of 3 years, two girls with severe disease were born. Early diagnosis was performed for both, which has allowed for the slowing down of the pathology's progression.

Abstract 1076: Single nucleus RNA-sequencing using normal human salivary glands revealed a potential etiologic relationship between each salivary gland cell and salivary gland malignancies

Abstract Salivary glands are composed of several types of cells, and each cell type is predicted to be involved in the carcinogenesis of different types of cancers including Adenoid cystic carcinoma (ACC), Acinic cell carcinoma (AciCC), salivary duct carcinoma (SDC), myoepithelial carcinoma (MECA) and other histology. In this study, we performed single nucleus RNA-seq on three human salivary gland samples to clarify the gene expression profile of each complex cellular component of the salivary glands and related these expression patterns to expression found in salivary gland cancers (SGC) to infer cell of origin. By single nucleus RNA-seq, total 13,643 salivary gland cells were stratified into four clusters: acinar cells, ductal cells 1, ductal cells 2, and myoepithelial cells/stromal cells, with differentially expressed genes of each group. The localization of each cell group was anatomically validated by IHC of each cluster marker gene, and one group of ductal cells was found to represent intercalated ductal cells labeled with HES1. Gene ontology analysis for this group included “epithelial cell differentiation” confirming the intercalated duct. Furthermore, to elucidate the potential etiologic relationship between normal salivary glands cell type and salivary gland carcinoma histology, we compared the expression of each cluster marker to SGC public RNA-seq data. In comparison between AciCC and ACC, we used normalized RNA-seq data of 54 ACC and 21 AciCC. ACC had significantly less expression of acinar cells markers (STATH, CST2, and LPO) compared to other markers expression(***P = 4 × 10−51), unlike AciCC (P = 0.54), suggesting a possible contribution of acinar cells to the carcinogenesis of AciCC. In another cohort, we compared 16 SDC, and 38 MECA with publicly available RNA-seq data. Remarkably, HES1 and ELF3, each of two different ductal cell markers, were significantly upregulated in SDC consistent with origin of SDC from ductal cells (P=0.001, P=0.0007, respectively). Consistent with bulk RNA-seq results, expression of HES1 in IHC using clinical human SDC and MECA samples was higher in SDC than MECA, suggesting that the expression of the derived cells is involved in salivary gland carcinogenesis. Cell type expressions in specific SGC histology are similar to those found in normal salivary gland populations, indicating a potential etiologic relationship. On the other hand, there was no significant difference in myoepithelial marker ACTA2 expression between SDC and MECA, suggesting that the markers of origin are not always highly expressed, depending on cancer subtypes. Although publicly available SGC RNA-seq data used in this study are limited, the results remarkably reflect the diverse modes of carcinogenesis of SGC. These findings might provide a better understanding of the molecular basis of SGC and treatment strategies for SGC. Citation Format: Takuya Nakagawa, Chanond Nasamran, Jingjing Hu, Prakriti Sen, Theresa Guo, Kathleen Fisch, Joseph Califano. Single nucleus RNA-sequencing using normal human salivary glands revealed a potential etiologic relationship between each salivary gland cell and salivary gland malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1076.

Abstract 5442: Terthighcells: key players in salivary gland homeostasis and regeneration after radiation therapy in adult mice

Abstract Salivary gland homeostasis and regeneration post-radiotherapy depend significantly on stem/progenitor cells. However, the lineage of SMG progenitor cells is not as well defined as in other normal organs, and better understanding of this population would be important for their future clinical application for tissue regeneration. Using a mouse strain expressing regulated CreERT2 recombinase from the endogenous Tert locus, we identify a distinct Terthigh cell population that are located in the ductal region throughout the submandibular gland (SMG), express ASCL3 (another progenitor cell marker) and can give rise to ductal cells during homeostasis. In contrast, Terthigh cells are found to repopulate both ductal and acinar cells at one year of tracing after radiotherapy. Terthigh cells from adult SMG retain self-renewal capacity when subjected in vitro culture, are resistant to radiation cell kill, and display enhanced proliferative activity post-radiation. Similarly, primary human SMG cells with high Tert expression display enhanced cell survival after radiotherapy. RNA sequencing reveals upregulation of the cell cycling and oxidative stress response pathways these cells after radiation. Mechanistically, Tert appears to modulate cell survival and ROS level in SMG spheres following radiation damage. Collectively, these data suggest that Terthigh cells constitute a novel subset of SMG progenitor cells located throughout the ductal region of the gland that can help to repopulate both ductal and acinar cells after RT damage. Citation Format: Li Guan, Vignesh Viswanathan, Sivakamasundari V, Hongbin Cao, Yuyan Jiang, Junfei Zhao, Deana R. Colburg, Patrick T. Neuhoefer, Yu Xu, Eyiwunmi E. Laseinde, Steven Artandi, Quynh-Thu Le. Terthighcells: key players in salivary gland homeostasis and regeneration after radiation therapy in adult mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5442.

Abstract 991: Unraveling the role of nicotinamide adenine dinucleotide precursors in pancreatic cancer biology

Abstract Introduction: Pancreatic cancer has the lowest 5-year survival rates among all cancers because of the challenges with early detection, its aggressive nature, and resistance to chemotherapy; this makes prevention a promising approach for reducing related mortalities. The disease is predominantly diagnosed in individuals over 60, suggesting that age-related physiological decline impacts its onset; therefore, delaying aging hallmarks may delay its onset. Nicotinamide dinucleotide (NAD+) precursors may hold a unique advantage over other geroprotectors because they target multiple aging hallmarks. Heron, we investigated how NAD+ supplementation impacts normal human pancreatic ductal epithelial cells (HPDE) under stress as a surrogate for cancer prevention in vitro. We also examined the paradoxical impact of NAD+ precursor supplementation on pancreatic cancer growth during chemotherapy. Methods: In vitro, we mimicked age-related NAD+ decline in the media. We stressed HPDE cells under these conditions and evaluated the effect of supplementation with the NAD+ precursor, Nicotinamide mononucleotide (NMN). We assessed cell viability using the PicoGreen assay, mitochondrial function, and reactive oxygen species (ROS) using the Seahorse XF Analyzer and the mitoSox kit, respectively, DNA repair and DNA damage using a western blot for Poly ADP ribose (PAR) and gamma H2AX, respectively, and SIRT3 expression was determined using a western blot. To evaluate the impact of NAD+ supplementation on pancreatic cancer cells, we treated pancreatic cancer cells with NMN and oxaliplatin. We then assessed the mitochondrial function, ROS levels, DNA damage, and repair. We implanted MiaPaCa-2 xenografts into athymic nude mice and treated them daily with 300mg/Kg of NMN and biweekly 5mg/kg of Oxaliplatin. Results: Experiments revealed that NMN is not toxic to HPDE cells, even at high concentrations. There was increased cell survival of HPDE cells under stress (nutrient deprivation and alkylating agents) following NMN supplementation. We also noted reduced ROS levels, increased SIRT3 expression, and PARylation. Pancreatic cancer cells were less sensitive to Oxaliplatin following NMN supplementation and showed an increased oxygen consumption rate, increased PARylation, and reduced ROS levels. There was reduced sensitivity to oxaliplatin treatment in the mice with MiaPaCa-2 xenografts treated with NMN. Conclusion: These findings demonstrate a cytoprotective role of NAD+ precursor supplementation in normal pancreatic ductal epithelial cells, with implications for possible cancer prevention. NAD+ supplementation also rescued cancer cells under cytotoxic stress, meaning that geroprotectors like NMN, in conjunction with chemotherapy, may unintentionally promote cancer progression. Future studies will directly test NAD+ precursor supplementation in genetically engineered pancreatic cancer models. Citation Format: Faith Nakazzi, Mehrad Zarei, Hallie J. Graor, Alexander W. Loftus, Omid Hajihassanni, Jordan M. Winter. Unraveling the role of nicotinamide adenine dinucleotide precursors in pancreatic cancer biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 991.