Human Pancreatic Cell Lines Research Articles

Abstract 1530: Tumor cell line-derived xenograft subtype shapes tumor microenvironment composition in BRGSF-HIS mice

Abstract The relevance of preclinical models has vastly improved with mice bearing a human immune system, especially in the context of immunotherapy. BRGSF (Balb/C Rag2−/−, IL2Rγ−/−, SIRPαNOD and Flt3−/−) is a highly immunodeficient mouse featuring reduced murine myeloid cells. BRGSF mice reconstituted with human cord blood CD34+ cells (BRGSF-HIS) develop functional lymphoid and myeloid compartments. This engraftment is stable for over a year (Labarthe et al., 2019) and mice do not develop GvHD. Myeloid compartment can be transiently boosted with exogenous human Flt3L injections. In contrary to other models overexpressing human cytokines to develop human myeloid cells, Flt3L-treated BRGSF-HIS mice do not show side effects. BRGSF-HIS mice are permissive to mouse and human cancer cell lines engraftment and represent a valuable preclinical model to study cancer development and test novel therapeutics. Effect of Flt3L-induced boost on myeloid compartment is seen in a non-small cell lung cancer (A549) tumor model. A single boost prior to tumor cell inoculation associates with increased tumor-infiltrating T-cells (mainly CD8+ T-cells) and myeloid cells, and reduced tumor-infiltrating NK cells in the tumor microenvironment (TME). Human triple negative cancer cell (TNBC) MDA-MB-231 and human pancreatic adenocarcinoma HPAFII cell lines are widely used in cancer research and drug development. We show that upon implantation in BRGSF-HIS mice, in vivo growth of human cell line-derived xenograft (CDX) is CD34+ donor independent and the TME composition varies according to cell line used. As seen in TNBC patients (Zheng et al., 2021); TME of MDA-MB-231 bearing BRGSF-HIS mice is enriched in myeloid cells, mostly CD206+/CD163+ M2 macrophages. CD163-expressing M2 macrophages represent the main tumor-associated macrophages, and are known to promote breast cancer initiation, angiogenesis, invasion, and metastasis by generating an immunosuppressive TME. Interestingly, TME composition evolves over time, with increased T cells frequency in later stage (tumor volume over 1000 mm3 compared to 500 mm3) depicting cancer cell plasticity. Conversely, TME of HPAFII bearing mice is mainly composed of T cells enabling T cells-based therapy. Treatment of BRGSF-HIS mice bearing HPAFII tumor cells with combotherapy based on CD3xTAA + CD28xTAA efficiently reduces tumor growth in vivo compared to vehicle-treated mice or mice injected with CD3xTAA alone. Induced systemic immunomodulation is observed in the TME where increased numbers of both CD4 and CD8 T cells are observed upon combotherapy. Remarkably, while no CD34+ donor effect is observed in CDX growth kinetic, response to treatment appears to be donor dependent with apparition of “weak” and “good” responders. This heterogeneity of response mimics what is observed in clinic and enables further investigations of treatment mode of action and immune escape mechanisms. Citation Format: Perrine Martin-Jeantet, Siham Hedir, Fabiane Sonego, Gaëlle Martin, Yacine Cherifi, Kader Thiam. Tumor cell line-derived xenograft subtype shapes tumor microenvironment composition in BRGSF-HIS 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 1530.

Graphene Nanocomposites in the Treatment of Pancreatic Cancer

The application value of titanium dioxide (TiO2)/graphene nanocomposites in photothermal therapy of pancreatic cancer (PC) was explored. Using scale graphite as raw material, graphene was obtained by Hummer oxidation method and hydrazine hydrate reduction method, and then TiO2/graphene nanocomposites were prepared by ultrasonic heating. Scanning electron microscopy (SEM), X-ray diffraction (XRD), and degraded methyl orange solution were adopted to detect the surface structure, particle size, element morphology, and photocatalytic activity under different composite ratios, different sonication times, and different heating temperatures. Human normal pancreatic ductal epithelial cell line HPDE6-C7 and human metastatic PC cell AsPC-1 were adopted as research models. The cytotoxicity of TiO2/graphene nanocomposites and the killing effect of photothermal therapy based on TiO2/graphene nanocomposites were analyzed by water soluble tetrazolium salt colorimetric assay (WST-1) and methyl thiazolyl tetrazolium salt colorimetric assay (MTT). The results suggested that when the ratio of graphene to TiO2 was 50:1, the ultrasonic time was 100 min, and the heating temperature was 200 °C, TiO2 was better attached to the surface of graphene, the distribution of particles was relatively more uniform, and the concentration of methyl orange was relatively lowest. The XRD pattern showed that the diffraction peak of the doped TiO2/graphene nanocomposite was basically the same as that of the pure TiO2. When the ultrasonic time was 100 min, the diffraction peak intensity in the XRD pattern was the largest. As for AsPC-1 cells, the cell viability was obviously lower than 0.1/1/10/100 μm/mL when the concentration of TiO2/graphene nanocomposites was 500 μm/mL (P <0.05). For HPDE6-C7 cells, when the concentration of TiO2/graphene nanocomposites was 100 and 500 μm/mL, the cell viability was obviously lower than 0.1/1/10 μm/mL (P <0.05), and 500 μm/mL was the lowest. The cell killing rate in group D was clearly higher as against groups A, B, and C (P <0.05). Graphene: The optimal preparation conditions of TiO2/graphene nanocomposites are 50:1, 100 min of ultrasound time, and 200 μC of composite temperature. The photothermal therapy based on TiO2/graphene nanocomposites can effectively kill PC cells, and has a good potential in the field of hyperthermia for pancreatic tumors.

The urolithin B nanomicellar delivery system as an efficient selective anticancer compound.

Urolithin B (UB), the antioxidant polyphenol has a protective impact on several organs against oxidative stress. However, its bioactivity is limited by its hydrophobic structure. In the current study, UB was encapsulated into a liposomal structure to improve its bioactivities anticancer, and antimicrobial potential. The UB nano-emulsions (UB-NE) were synthesized and characterized utilizing FESEM, DLS, FTIR, and Zeta-potential analysis. The UB-NMs' selective toxicity was studied by conducting an MTT assay on MCF-7, PANC, AGS, and ASPC1 cells. The AO/PI analysis verified the UB-NMs' cytotoxicity on ASPC1 cell lines and approved the MTT results. Finally, the antibacterial activity of the UB-NMs was studied on both gram-positive (B. subtilis, S. aureus) and gram-negative (E. Coli, P. aeruginosa) bacteria by conducting MIC and MBC analysis. The 68.15nm UB-NMs did not reduce the normal HDF cells' survival. However, they reduced the cancer cells' (PANC and AGS cell lines) survival at high treatment concentrations (> 250µg/mL) compared with normal HDF and cancer MCF-7 cells. Moreover, the IC50 doses of UB-NMs for the ASPC1 and PANC cancer cells were measured at 44.87, and 221.02µg/mL, respectively. The UB-NMs selectively exhibited apoptotic-mediated cytotoxicity on the human pancreatic tumor cell line (ASPC1) by down-regulating BCL2 and NFKB gene expression. Also, the BAX gene expression was up-regulated in the ASPC1-treated cells. Moreover, they exhibited significant anti-bactericidal activity against the E. coli (MIC = 50µg/mL, MBC = 150µg/mL), P. aeruginosa (MIC = 75µg/mL, MBC = 275µg/mL), B. subtilis (MIC = 125µg/mL, MBC = 450µg/mL), and S. aureus (MIC = 50µg/mL, MBC = 200µg/mL) strains. The significant selective cytotoxic impact of the UB-NMs on the human pancreatic tumor cell line makes it an applicable anti-pancreatic cancer compound. Moreover, the antibacterial activity of UB-NMs has the potential to decrease bacterial-mediated pancreatic cancer. However, several bacterial strains and further cancer cell lines are required to verify the UB-NMs' anticancer potential.

Phytochemicals in Pancreatic Cancer Treatment: A Machine Learning Study.

The discovery of new strategies and novel therapeutic agents is crucial to improving the current treatment methods and increasing the efficacy of cancer therapy. Phytochemicals, naturally occurring bioactive constituents derived from plants, have great potential in preventing and treating various diseases, including cancer. This study reviewed 74 literature studies published between 2006 and 2022 that conducted in vitro cytotoxicity and cell apoptosis analyses of the different concentrations of phytochemicals and their combinations with conventional drugs or supplementary phytochemicals on human pancreatic cell lines. From 34 plant-derived phytochemicals on 20 human pancreatic cancer cell lines, a total of 11 input and 2 output variables have been used to construct the data set that contained 2161 different instances. The machine learning approach has been implemented using random forest for regression, whereas association rule mining has been used to determine the effects of individual phytochemicals. The random forest models developed are generally good, indicating that the phytochemical type, its concentration, and the type of cell line are the most important descriptors for predicting the cell viability. However, for predicting cell apoptosis the primary phytochemical type is the most significant descriptor . Among the studied phytochemicals, catechin and indole-3-carbinol were found to be non-cytotoxic at all concentrations irrespective of the treatment time. On the other hand, berbamine and resveratrol were strongly cytotoxic with cell viabilities of less than 40% at a concentration range between 10 and 100 μM and above 100 μM, respectively, which brings them forward as potential therapeutic agents in the treatment of pancreatic cancer.

Development and Assessment of 1,5-Diarylpyrazole/Oxime Hybrids Targeting EGFR and JNK-2 as Antiproliferative Agents: A Comprehensive Study through Synthesis, Molecular Docking, and Evaluation.

New 1,5-diarylpyrazole oxime hybrid derivatives (scaffolds A and B) were designed, synthesized, and then their purity was verified using a variety of spectroscopic methods. A panel of five cancer cell lines known to express EGFR and JNK-2, including human colorectal adenocarcinoma cell line DLD-1, human cervical cancer cell line Hela, human leukemia cell line K562, human pancreatic cell line SUIT-2, and human hepatocellular carcinoma cell line HepG2, were used to biologically evaluate for their in vitro cytotoxicity for all the synthesized compounds 7a-j, 8a-j, 9a-c, and 10a-c. The oxime containing compounds 8a-j and 10a-c were more active as antiproliferative agents than their non-oxime congeners 7a-j and 9a-c. Compounds 8d, 8g, 8i, and 10c inhibited EGFR with IC50 values ranging from 8 to 21 µM when compared with sorafenib. Compound 8i inhibited JNK-2 as effectively as sorafenib, with an IC50 of 1.0 µM. Furthermore, compound 8g showed cell cycle arrest at the G2/M phase in the cell cycle analysis of the Hela cell line, whereas compound 8i showed combined S phase and G2 phase arrest. According to docking studies, oxime hybrid compounds 8d, 8g, 8i, and 10c exhibited binding free energies ranging from -12.98 to 32.30 kcal/mol at the EGFR binding site whereas compounds 8d and 8i had binding free energies ranging from -9.16 to -12.00 kcal/mol at the JNK-2 binding site.

Xanthoxyletin blocks the RANK/RANKL signaling pathway to suppress the growth of human pancreatic cancer cells.

Xanthoxyletin is a vital plant-derived bioactive coumarin. It has been shown to exhibit anticancer effects against different human cancers. Nonetheless, the anticancer effects of xanthoxyletin against human pancreatic cancer cells have not been evaluated. Against this backdrop, the present study was designed to evaluate the anticancer effects of xanthoxyletin in human pancreatic cancer cells and to decipher the underlying molecular mechanisms. The results revealed a significant (p < 0.05) upregulation of receptor activator of NF-kappaB (RANK), receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) in human pancreatic tissues and cell lines at both transcriptional and translational levels. The administration of pancreatic cancer cells with xanthoxyletin diminished the viability of Capan-2 cells in a concentration-dependent manner and led to a significant decline in RANK, RANKL, and OPG expression. Silencing of RANK and xanthoxyletin treatment declined the viability of Capan-2 pancreatic cancer cells via induction of apoptosis. However, pancreatic cancer cells overexpressing RANK could rescue the growth inhibitory effects. Collectively, xanthoxyletin targets the RANK/RANKL signaling pathway in pancreatic cancer cells to induce cell apoptosis and may prove to be an important lead molecule.

Hybrid AI models allow label-free identification and classification of pancreatic tumor repopulating cell population

Human pancreatic cancer cell lines harbor a small population of tumor repopulating cells (TRCs). Soft 3D fibrin gel allows efficient selection and growth of these tumorigenic TRCs. However, rapid and high-throughput identification and classification of pancreatic TRCs remain technically challenging. Here, we developed deep learning (DL) models paired with machine learning (ML) models to readily identify and classify 3D fibrin gel-selected TRCs into sub-types. Using four different human pancreatic cell lines, namely, MIA PaCa-2, PANC-1, CFPAC-1, and HPAF-II, we classified 3 main sub-types to be present within the TRC population. Our best model was an Inception-v3 convolutional neural network (CNN) used as a feature extractor paired with a Support Vector Machine (SVM) classifier with radial basis function (rbf) kernel which obtained a test accuracy of 90%. In addition, we compared this hybrid method of supervised classification with other methods of supervised classifications and showed that our working model outperforms others. With the help of unsupervised machine learning algorithms, we also validated that the pancreatic TRC subpopulation can be clustered into 3 sub-types. Collectively, our robust model can detect and readily classify tumorigenic TRC subpopulation label-free in a high-throughput fashion which can be very beneficial in clinical settings.

Internalization of extracellular vesicles from Lactobacillus johnsonii N6.2 elicit an RNA sensory response in human pancreatic cell lines.

Cells of all domains of life can secrete extracellular vesicles (EV). These secreted vesicles have been indicated as vehicles carrying molecules that facilitate intra- and inter-species interaction. Lactobacillus johnsonii N6.2, a bacterium used in probiotic preparations, has been shown to produce nano-sized EV. In the present work we used L. johnsonii N6.2 EV, concentrated from exosome depleted MRS supernatant, to identify the uptake mechanisms of EV and the impact of the RNA cargo in the EV on the upregulation of the cellular response of βlox5 human pancreatic cells. Using eukaryotic uptake inhibitors, it was found that EV are internalized by the clathrin/dynamin mediated endocytosis pathway. Further co-localization experiments with the endosome markers RAB5, RAB7 and LAMP1 as well as calcein indicated that EV escape the endosome shortly after RAB7 fusion. Using the expression of the 2',5'-oligoadenylate synthetase (OAS) host pathway, previously identified as targeted by L. johnsonii EV, we found that the host cellular response to the EV are dependent on the integrity of the external components of the EV as well as on the RNA cargo. Global transcriptome analysis was performed on EV and the bacterial whole cell. It was found that the RNA transcripts found within the EV largely represent the most abundantly transcribed genes in the bacterial cells such as those associated with protein synthesis and glycolysis. Further analysis showed an enrichment of smaller size transcripts as well as those encoding for membrane bound or extracellular proteins in L. johnsonii's EV.

Antibacterial and anticancer activity (PANC-1) of green synthesized copper oxide nanoparticles from Catharanthus roseus

Copper oxide nanoparticle was biosynthesized using the petals of Catharanthus roseus, and it was found to exhibit anticancer activity in a human pancreatic cell line (PANC-1). The obtained nanoparticles were characterized using XRD, FTIR, FESEM, and TEM techniques. XRD confirms the coexistence of CuO and Cu2O nanoparticles with an average grain size of 15 nm. FTIR spectra possess bands that indicate the formation of copper oxide nanoparticles. FESEM and TEM show spherical shape morphology with an average particle size of 19.6 nm to 32.6 nm. The synthesized copper oxide nanoparticles were tested for antibacterial activity, and the gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa showed a better zone of inhibition than standard streptomycin. The copper oxide nanoparticle was tested for anticancer activity in the PANC-1 line, and the results confirm that cells undergo cell shrinkage in the cytoplasm, which suggested the cytotoxic behavior. The viability of cells was evaluated by an inverted phase contrast microscope followed by the MTT assay method.

760-P: Discovery of GSBR-1290, a Highly Potent, Orally Available, Novel Small Molecule GLP-1 Receptor Agonist

Peptidic glucagon-like peptide 1 receptor agonists (GLP-1RAs) are established treatments for patients with Type 2 Diabetes Mellitus (T2DM) and obesity. However, the need for injection and cold chain storage may limit the utility of most peptidic GLP-1RAs. An oral small molecule GLP-1RA that offers enhanced bioavailability and stability could be more convenient and accessible to patients. Here we report the discovery of GSBR-1290, a highly potent, orally available, novel small molecule GLP-1RA and the characterization of its in vitro and in vivo pharmacology profiles. GSBR-1290 is a small molecule GLP-1RA with high binding affinity to human GLP-1R. GSBR-1290 strongly activated GLP-1R Gαs cAMP pathway without inducing measurable β-arrestin recruitment signaling, which indicates it is a fully biased agonist. The insulin secretion stimulation effect of GSBR-1290 was evaluated in a functional human pancreatic beta cell line, in which GSBR-1290 showed dose dependent induction of insulin secretion. The in vivo efficacy of GSBR-1290 on insulin secretion, glucose control and food intake were evaluated in nonhuman primates (NHPs). In an acute intravenous glucose tolerance test (ivGTT), a single dose of GSBR-1290 strongly induced insulin secretion and glucose clearance. In a repeated dosing study, GSBR-1290 administered orally once daily for 7-day demonstrated robust increase in insulin secretion and glucose clearance in ivGTT and a dose dependent reduction of food intake and body weight. Overall, GSBR-1290 demonstrated potent in vivo efficacy in stimulating insulin secretion, improving glucose tolerance, and reducing food intake and body weight. In conclusion, GSBR-1290 is a highly potent, orally available, fully biased GLP-1RA. Human clinical trials are underway to further evaluate GSBR-1290 as a potential therapy for T2DM and obesity. Disclosure T.Mao: Employee; Structuretx. X.Lin: Employee; Structure Therapeutics. Q.Meng: Employee; Beijing Stonewise Technology, Structure Therapeutics Inc. H.Zhang: Employee; Structuretx. J.J.Zhang: Employee; Structure Therapeutics Inc. S.Shi: Employee; Structure Therapeutics Inc. Z.Guan: Employee; Structure Therapeutics. X.Jiang: Employee; Structure Therapeutics. F.Zhang: Employee; Structure Therapeutics, Eternity BioSciences, Stock/Shareholder; Novo Nordisk. H.Lei: Employee; Structure Therapeutics.

Necroptosis-Related Prognostic Model for Pancreatic Carcinoma Reveals Its Invasion and Metastasis Potential through Hybrid EMT and Immune Escape.

Necroptosis, pro-inflammatory programmed necrosis, has been reported to exert momentous roles in pancreatic cancer (PC). Herein, the objective of this study is to construct a necroptosis-related prognostic model for detecting pancreatic cancer. In this study, the intersection between necroptosis-related genes and differentially expressed genes (DEGs) of pancreatic ductal adenocarcinoma (PDAC) was obtained based on GeneCards database, GEO database (GSE28735 and GSE15471), and verified using The Cancer Genome Atlas (TCGA). Next, a prognostic model with Cox and LASSO regression analysis, and divided the patients into high-risk and low-risk groups. Subsequently, the Kaplan-Meier (KM) survival curve and the receiver operating characteristic (ROC) curves were generated to assess the predictive ability of overall survival (OS) of PC patients. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to predict the potential biofunction and possible mechanical pathways. The EMTome database and an immune analysis were applied to further explore underlying mechanism. Finally, clinical samples of PDAC patients were utilized to verify the expression of model genes via immunohistochemistry (IHC), and the normal human pancreatic ductal cell line, hTERT-HPNE as well as human pancreatic ductal carcinoma cell lines, PANC-1 and PL45, were used to identify the levels of model genes by Western blot (WB) and immunofluorescence (IF) in vitro. The results showed that 13 necroptosis-related DEGs (NRDEGs) were screened based on GEO database, and finally four of five prognostic genes, including KRT7, KRT19, IGF2BP3, CXCL5, were further identified by TCGA to successfully construct a prognostic model. Univariate and multivariate Cox analysis ultimately confirmed that this prognostic model has independent prognostic significance, KM curve suggested that the OS of low-risk group was longer than high-risk group, and the area under receiver (AUC) of ROC for 1, 3, 5 years was 0.733, 0.749 and 0.667, respectively. A GO analysis illustrated that model genes may participate in cell-cell junction, cadherin binding, cell adhesion molecule binding, and neutrophil migration and chemotaxis, while KEGG showed involvement in PI3K-Akt signaling pathway, ECMreceptor interaction, IL-17 signaling pathway, TNF signaling pathway, etc. Moreover, our results showed KRT7 and KRT19 were closely related to EMT markers, and EMTome database manifested that KRT7 and KRT19 are highly expressed in both primary and metastatic pancreatic cancer, declaring that model genes promoted invasion and metastasis potential through EMT. In addition, four model genes were positively correlated with Th2, which has been reported to take part in promoting immune escape, while model genes except CXCL5 were negatively correlated with TFH cells, indicating that model genes may participate in immunity. Additionally, IHC results showed that model genes were higher expressed in PC tissues than that in adjacent tumor tissues, and WB and IF also suggested that model genes were more highly expressed in PANC-1 and PL45 than in hTERT-HPNE. Tracing of a necroptosis-related prognostic model for pancreatic carcinoma reveals its invasion and metastasis potential through EMT and immunity. The construction of this model and the possible mechanism of necroptosis in PDAC was preliminarily explored to provide reliable new biomarkers for the early diagnosis, treatment, and prognosis for pancreatic cancer patients.

Abstract 1485: Elucidating the role of VGLL1 in pancreatic cancer tumorigenesis

Abstract Vestigial-like 1 (VGLL1) is a co-transcriptional activator that binds to TEA domain-containing transcription factors (TEADs). Its expression is upregulated in a variety of cancers and is correlated with decreased overall patient survival. In normal tissues, VGLL1 is most highly expressed in placental trophoblast cells, which share attributes of rapid cellular proliferation and invasion with cancer cells. Binding of VGLL1 to TEADs shows structural similarity to co-transcriptional activators in the Hippo pathway, yes-associated protein 1 (YAP) and WW Domain containing transcription regulator 1 (WWTR1/TAZ). YAP/TAZ has been implicated in tumorigenesis in many cancer types, and this structural relationship has provided insights into the functional role of VGLL1. The impact of VGLL1 expression in pancreatic cancer has not been fully elucidated and no VGLL1-targeted therapy currently exists. The aim of this study was to evaluate the function and downstream targets of VGLL1 in pancreatic cancer cells. Ectopic expression of VGLL1 in human pancreatic cell lines PANC1 and SU8686 stimulated cancer cell proliferation and invasion, consistent with findings from other cancer types. ChIP-seq and Nanostring assays were also performed to identify VGLL1 downstream target genes and pathways. Interestingly, analysis of ChIP-seq data from VGLL1-transduced PANC1 cells identified four novel transcription factors with a VGLL1 binding motif, in addition to TEAD4 which has been previously reported. Furthermore, gene array analysis showed approximately 150 differentially expressed genes in mouse pancreatic cancer cells transduced with VGLL1, including genes involved in regulation of cell cycle and apoptosis. Ectopic VGLL1 expression resulted in the increased expression of AP-1, MMP9, and VEGF, genes associated with cancer progression. Furthermore, VGLL1 decreased the expression of cyclin-dependent kinase inhibitors such as p15, p16 and p21 that are associated with reduced cancer cell proliferation. To identify potential regulatory features of VGLL1, we performed mass spectrometry to identify phosphorylation sites and potential binding partners. Our preliminary data suggests that 14-3-3 binds to VGLL1, an intriguing finding since 14-3-3 is also known to regulate YAP/TAZ by sequestering it in the cytoplasm upon being phosphorylated. Our results demonstrate that VGLL1 interacts with specific transcription factors such as TEAD4 in pancreatic cancer cells to modulate the expression of genes expected to promote cell proliferation and invasion. In addition, we have identified a potential regulatory molecule for VGLL1 that plays a critical role in inhibiting other co-transcription activators from translocating into the nucleus. It is hoped that this work will set the stage for the development of VGLL1-targeted therapies with which to treat patients with pancreatic cancer. Citation Format: Heather M. Sonnemann, Barbara Pazdrak, Barbara Nassif, Yimo Sun, Lama Elzohary, Amjad Talukder, Arjun Katailiha, Salah-Eddine Bentebibel, Adi Diab, Gregory Lizee. Elucidating the role of VGLL1 in pancreatic cancer tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1485.

Targeting mitochondrial transcription factor A sensitizes pancreatic cancer cell to gemcitabine

BackgroundThe survival of pancreatic cancer cells, particularly cancer stem cells which are responsible for tumor relapse, depends on mitochondrial function. Mitochondrial transcription factor A (TFAM) is critical for the regulation of mitochondrial DNA and thus mitochondrial function. However, the possible involvement of TFAM in pancreatic cancer is unknown. MethodsHuman samples were obtained from pancreatic cancers and their adjacent tissues; human pancreatic cell lines were cultured in RPMI1640 medium. TFAM expressions in pancreatic tissues and cultured cells were determined using immunohistochemistry, ELISA, and reverse transcription polymerase chain reaction (RT-PCR). The effect of TFAM on cell growth, migration, colony formation and apoptosis were evaluated. Mitochondrial biogenesis in pancreatic cancer and normal cells were examined. ResultsThe majority of pancreatic cancer tissues exhibited higher TFAM expression compared to the adjacent counterparts. Consistently, TFAM mRNA and protein levels were higher in pancreatic cancer cell lines than in immortalized normal pancreatic epithelial cells. There was no difference on TFAM level between gemcitabine-sensitive and resistant pancreatic cancer cells. Functional analysis demonstrated that TFAM overexpression activated pancreatic normal and tumor cells whereas TFAM inhibition effectively inhibited the growth of pancreatic cancer cells. TFAM inhibition enhanced gemcitabine's cytotoxicity and suppressed growth, anchorage-independent colony formation and survival of gemcitabine-resistant pancreatic cancer cells. Mechanistic studies showed that TFAM inhibition resulted in remarkable mitochondrial dysfunction and energy crisis followed by oxidative stress. The basal mitochondrial biogenesis level correlated well with TFAM level in pancreatic cancer cells. ConclusionsTFAM played essential roles in pancreatic cancer via regulating mitochondrial functions which highlighted the therapeutic value of inhibiting TFAM to overcome gemcitabine resistance.

Anticancer Activity of Sunitinib Analogues in Human Pancreatic Cancer Cell Cultures under Normoxia and Hypoxia

Pancreatic cancer remains one of the deadliest cancer types. It is usually characterized by high resistance to chemotherapy. However, cancer-targeted drugs, such as sunitinib, recently have shown beneficial effects in pancreatic in vitro and in vivo models. Therefore, we chose to study a series of sunitinib derivatives developed by us, that were proven to be promising compounds for cancer treatment. The aim of our research was to evaluate the anticancer activity of sunitinib derivatives in human pancreatic cancer cell lines MIA PaCa-2 and PANC-1 under normoxia and hypoxia. The effect on cell viability was determined by the MTT assay. The compound effect on cell colony formation and growth was established by clonogenic assay and the activity on cell migration was estimated using a 'wound healing' assay. Six out of 17 tested compounds at 1 µM after 72 h of incubation reduced cell viability by 90% and were more active than sunitinib. Compounds for more detailed experiments were chosen based on their activity and selectivity towards cancer cells compared to fibroblasts. The most promising compound EMAC4001 was 24 and 35 times more active than sunitinib against MIA PaCa-2 cells, and 36 to 47 times more active against the PANC-1 cell line in normoxia and hypoxia. It also inhibited MIA PaCa-2 and PANC-1 cell colony formation. Four tested compounds inhibited MIA PaCa-2 and PANC-1 cell migration under hypoxia, but none was more active than sunitinib. In conclusion, sunitinib derivatives possess anticancer activity in human pancreatic adenocarcinoma MIA PaCa-2 and PANC-1 cell lines, and they are promising for further research.

Pancreatic Cancer Presents Distinct Nanomechanical Properties During Progression.

Cancer progression is closely related to changes in the structure and mechanical properties of the tumor microenvironment (TME). In many solid tumors, including pancreatic cancer, the interplay among the different components of the TME leads to a desmoplastic reaction mainly due to collagen overproduction. Desmoplasia is responsible for the stiffening of the tumor, poses a major barrier to effective drug delivery and has been associated with poor prognosis. The understanding of the involved mechanisms in desmoplasia and the identification of nanomechanical and collagen-based properties that characterize the state of a particular tumor can lead to the development of novel diagnostic and prognostic biomarkers. In this study, in vitro experiments were conducted using two human pancreatic cell lines. Morphological and cytoskeleton characteristics, cells' stiffness and invasive properties were assessed using optical and atomic force microscopy techniques and cell spheroid invasion assay. Subsequently, the two cell lines were used to develop orthotopic pancreatic tumor models. Tissue biopsies were collected at different times of tumor growth for the study of the nanomechanical and collagen-based optical properties of the tissue using Atomic Force Microscopy (AFM) and picrosirius red polarization microscopy, respectively. The results from the in vitro experiments demonstrated that the more invasive cells are softer and present a more elongated shape with more oriented F-actin stress fibers. Furthermore, ex vivo studies of orthotopic tumor biopsies on MIAPaCa-2 and BxPC-3 murine tumor models highlighted that pancreatic cancer presents distinct nanomechanical and collagen-based optical properties relevant to cancer progression. The stiffness spectrums (in terms of Young's modulus values) showed that the higher elasticity distributions were increasing during cancer progression mainly due desmoplasia (collagen overproduction), while a lower elasticity peak was evident -due to cancer cells softening- on both tumor models. Optical microscopy studies highlighted that collagen content increases while collagen fibers tend to form align patterns. Consequently, during cancer progression nanomechanical and collagen-based optical properties alter in relation to changes in collagen content. Therefore, they have the potential to be used as novel biomarkers for assessing and monitoring tumor progression and treatment outcomes.

Aspirin increases the efficacy of gemcitabine in pancreatic cancer by modulating the PI3K/AKT/mTOR signaling pathway and reversing epithelial‑mesenchymal transition.

Gemcitabine is regarded as a standard medication for patients with pancreatic cancer. The aim of the present study was to investigate the impact of aspirin (ASA) on the efficacy of gemcitabine in pancreatic cancer and the potential mechanism. The SW1990 and BxPC-3 human pancreatic cell lines were treated with 2 mmol/l ASA and/or 1 mg/l gemcitabine. The effects of the treatments were tested on the viability, migration and invasion of the cells using MTT, wound healing and Transwell invasion assays. In addition, cell apoptosis was evaluated via flow cytometry with Annexin V-FITC/PI and the western blotting of Bax and Bcl-2. The expression of epithelial-mesenchymal transition (EMT)-associated proteins and activation of the PI3K/AKT/mTOR pathway were also assessed using western blotting. The results reveal that ASA increased the efficacy of gemcitabine in reducing the proliferation, migration and invasion of pancreatic cancer cells and increasing their apoptosis. These effects are associated with inhibition of the PI3K/AKT/mTOR pathway and the reversal of EMT. Thus, the combined use of ASA and gemcitabine is suggested to be a potential therapeutic strategy for patients with pancreatic cancer.

Targeting Interleukin-6/Glycoprotein-130 Signaling by Raloxifene or SC144 Enhances Paclitaxel Efficacy in Pancreatic Cancer

Interleukine-6 plays a key role in the progression and poor survival in pancreatic ductal adenocarcinoma (PDAC). The present study aimed to clarify if targeting the interleukin-6/glycoprotein-130 signaling cascade using the small-molecule gp130 inhibitor SC144 or raloxifene, a non-steroidal selective estrogen receptor modulator, enhances paclitaxel efficacy. MTT/BrdU assays or TUNEL staining were performed to investigate cell viability, proliferation and apoptosis induction in L3.6pl and AsPC-1 human pancreatic cell lines. In vivo, effects were studied in an orthotopic PDAC mouse model. Tumor specimens were analyzed by qPCR, immunohistochemistry and ELISA. Combination of paclitaxel/raloxifene, but not paclitaxel/SC144, enhanced proliferation and viability inhibition and increased apoptosis compared to single treatment in vitro. Synergy score calculations confirmed an additive influence of raloxifene on paclitaxel. In the PDAC mouse model, both combinations of raloxifene/paclitaxel and SC144/paclitaxel reduced tumor weight and volume compared to single-agent therapy or control. Raloxifene/paclitaxel treatment decreased survivin mRNA expression and showed tendencies of increased caspase-3 staining in primary tumors. SC144/paclitaxel reduced interleukin-6 levels in mice's tumors and plasma. In conclusion, raloxifene or SC144 can enhance the anti-tumorigenic effects of paclitaxel, suggesting that paclitaxel doses might also be reduced in combined chemotherapy to lessen paclitaxel side effects.

A genome-wide CRISPR screen identifies CALCOCO2 as a regulator of beta cell function influencing type 2 diabetes risk

Identification of the genes and processes mediating genetic association signals for complex diseases represents a major challenge. As many of the genetic signals for type 2 diabetes (T2D) exert their effects through pancreatic islet-cell dysfunction, we performed a genome-wide pooled CRISPR loss-of-function screen in a human pancreatic beta cell line. We assessed the regulation of insulin content as a disease-relevant readout of beta cell function and identified 580 genes influencing this phenotype. Integration with genetic and genomic data provided experimental support for 20 candidate T2D effector transcripts including the autophagy receptor CALCOCO2. Loss of CALCOCO2 was associated with distorted mitochondria, less proinsulin-containing immature granules and accumulation of autophagosomes upon inhibition of late-stage autophagy. Carriers of T2D-associated variants at the CALCOCO2 locus further displayed altered insulin secretion. Our study highlights how cellular screens can augment existing multi-omic efforts to support mechanistic understanding and provide evidence for causal effects at genome-wide association studies loci.

Role of poly(ADP-ribose) polymerase-1 in regulating human islet cell differentiation

Poly(ADP-ribose) polymerase-1 (PARP1), a fundamental DNA repair enzyme, is known to regulate β cell death, replication, and insulin secretion. PARP1 knockout (KO) mice are resistant to diabetes, while PARP1 overactivation contributes to β cell death. Additionally, PARP1 inhibition (PARPi) improves diabetes complications in patients with type-2 diabetes. Despite these beneficial effects, the use of PARP1 modulating agents in diabetes treatment is largely neglected, primarily due to the poorly studied mechanistic action of PARP1 catalytic function in human β cell development. In the present study, we evaluated PARP1 regulatory action in human β cell differentiation using the human pancreatic progenitor cell line, PANC-1. We surveyed islet census and histology from PARP1 wild-type versus KO mice pancreas in a head-to-head comparison with PARP1 regulatory action for in-vitro β cell differentiation following either PARP1 depletion or its pharmacological inhibition in PANC-1-differentiated islet cells. shRNA mediated PARP1 depleted (SiP) and shRNA control (U6) PANC-1 cells were differentiated into islet-like clusters using established protocols. We observed complete abrogation of new β cell formation with absolute PARP1 depletion while its inhibition using the potent inhibitor, PJ34, promoted the endocrine β cell differentiation and maturation. Immunohistochemistry and immunoblotting for key endocrine differentiation players along with β cell maturation markers highlighted the potential regulatory action of PARP1 and augmented β cell differentiation due to direct interaction of unmodified PARP1 protein elicited p38 MAPK phosphorylation and Neurogenin-3 (Ngn3) re-activation. In summary, our study suggests that PARP1 is required for the proper development and differentiation of human islets. Selective inhibition with PARPi can be an advantage in pushing more insulin-producing cells under pathological conditions and delivers a potential for pilot clinical testing for β cell replacement cell therapies for diabetes.

Polyketides isolated from an endophyte Penicillium oxalicum 2021CDF-3 inhibit pancreatic tumor growth.

Fungal secondary metabolites are inherently considered valuable resources for new drugs discovery. To search for novel fungal secondary metabolites with lead compounds potential, a fungal strain Penicillium oxalicum 2021CDF-3, an endophyte of the marine red algae Rhodomela confervoides, was chemically studied. Cultivation of this fungus on solid rice medium yielded 10 structurally diverse metabolites (1–10), including two new polyketides, namely oxalichroman A (1) and oxalihexane A (2). Their structures were determined by detailed analysis of NMR and HRESIMS spectroscopic data. Oxalihexane A (2) was elucidated as a novel polyketide formed by a cyclohexane and cyclohexanone moiety via an ether bond. The stereochemistry of 2 was successfully assigned by NMR and ECD calculations. In the cytotoxic assay, the new compound 2 showed remarkable inhibitory effect on the human pancreatic cancer PATU8988T cell line. Further pharmacological study demonstrated that the expression level of Cyclin D1 was down-regulated by the treatment with 2, which suggested that cell cyclin abnormity was involved in pancreatic tumor cell apoptosis. Moreover, the activation of Wnt5a/Cyclin D1 signaling pathway might be involved in the mechanism of panreatic tumor cell apoptosis induced by 2.