Pancreatic ductal adenocarcinoma (PDA) has a complex tumour microenvironment (TME) that are made up of pancreatic ductal adenocarcinoma cells (PDACs) and pancreatic stellate cells (PSCs). PDACs and PSCs’ interaction allows pancreatic cancer cells to evade the immune system and enhance tumour progression. Hence, identifying and comparing the proteomic profile of PDACs, PSCs and their co-culture will determine which genes are involved in the progression of PDA. PANC10.05 cells (primary PDAC cell line), PSC (hPSC21-S/T) cells and PANC10.05/PSCs co-culture were cultured in 6-well plate for 3 days. Cells collected were lysed to extract the proteins within the cells. The extracted protein lysate then undergoes insolution digestion of proteins. The protein in the samples were analysed and quantified through LCMS/MS before proceeding to data analysis via DEBrowser. The results showed that VIM, KRT8 and KRT18 were differentially expressed proteins (DEPs) detected when compared between PANC10.05, PSCs and their co-culture. In this study, monoculture of PANC10.05 and PSCs were able to express proteins without any interaction between PANC10.05 and PSCs. Hence, PANC10.05-PSCs co-culture is not required for the expression of proteins. The expression of DEPs in PANC10.05 and PSCs are associated in the progression, invasion and metastasis of PDA.

Pancreatic ductal adenocarcinoma (PDAC) is an almost always deadly illness, and early identification is a crucial factor in enhancing survival. The tumour microenvironment (TME), a possible source of biomarkers in PDAC, has a significant impact in cell proliferation, metastasis, and treatment resistance, all of which contribute to a poorer clinical outcome. Metastatic PDAC (SW1990 cell line) and pancreatic stellate cells (PSCs), one of the major cell types that comprise the tumour microenvironment (TME) of pancreatic cancer, foster an immunosuppressive environment around the tumour. The purpose of this investigation was to examine the proteome profiles of conditioned media (CM) from PSCs, SW1990, and PSC+SW1990 co-culture. To establish the probable proteins responsible for the different immunosuppressive efficacy of PSCs, SW1990, and PSC+SW1990 co-culture, the CM containing the secreted proteins from all 3 cell line groups were analysed by Liquid Chromatography with tandem Mass Spectrometry (LC-MS/MS) and then subjected to bioinformatics analysis, including identification of differentially expressed proteins (DEPs), Gene Ontology (GO) terms analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway. TGM2, KRT8, KRT18, KRT19, MYH9, NME1, NME1-NME2, and LDHA were the eight DEPs that were significantly elevated in all three cell line groups comparisons with a false discovery rate (FDR)  0.05 and fold change (FC) ≥ 2. Multiple signalling pathways, including oestrogen signalling, actin cytoskeleton, protein processing in endoplasmic reticulum, vascular smooth muscle contraction, the hypoxia-inducible factor 1 (HIF-1) and the central carbon metabolism, were enriched in the comparison study. The DEPs detected, their GO terns, and the signalling pathways were integrated and matched with literature and proteomic data from prior research in order to better comprehend their association with PSCs and metastatic PDAC. In conclusion, this comparative proteomic research extends 3 the evidence of protein alterations linked with PSCs and metastatic PDAC that should be studied further as possible biomarkers or therapeutic targets

Pancreatic ductal adenocarcinoma cells (PDACs) and pancreatic stellate cells (PSC in a concerted effort protect the tumour microenvironment from immune cells attack. However, most studies have solely focused on their immunosuppressive effects onto T cells, hence, our effort is to study their immunosuppressive effects on B cells viability. Two PDAC cell lines (PANC 10.05 and SW1990) and/or PSC were cultured for 3 days in DMEM : DMEM:f12 (50:50) at 37 oC with 5% CO2 to obtain conditioned media (CM). Collected CM were used to differentiate isolated PBMCs into MDSCs. B cells were isolated from whole blood and treated with CM and on a separate event with isolated MDSCs for 48 hours before proceeding with CellTiter Glo Viability Assay. The cell viability assay showed that CM from PDACs and/or PSCs co-culture are both able to promote B cell proliferation (p<0.05). Similarly, all CM generated MDSCs was also able to promote B cell proliferation but MDSCs untreated by CM is not able to promote B cell proliferation effectively (p < 0.05). These findings indicate that MDSCs’ pro-B cell proliferation activity requires activation by the conditioned media.

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies and its recurrence, delayed diagnosis and resistance to treatments contribute to its poor survival outcomes. Epigenetic modifications such as DNA methylation significantly contribute to tumour heterogeneity, metastasis, and disease progression of PDAC. Additionally, inflammation is one of the major implications of PDAC with the nuclear factor kappa B (NF-κB) being acknowledged as its key mediator. In recent years, multiple evidence have suggested that inhibitor of kappa B kinase alpha (IKKα) plays a central role in the initiation and progression of PDAC through transcriptional activity that enhances the NF-κB-mediated gene expressions. However, there has been lack of evidence correlating the role of IKKα and its epigenetic changes in PDAC. Aim: To determine the epigenetic changes associated with IKKα in a PDAC cell line. To identify gene pathways that are regulated by IKKα in the PDAC cell line. Methodology: Methylome-wide analysis was conducted on two cell lines – the parental PANC1 cell line PANC1_WT and PANC1_33 with the IKKα knockout. Briefly, DNA extracted from both cell lines were subjected to bisulfite conversion and DNA methylation using the Illumina Infinium MethylationEPIC BeadChip array was performed to identify the differentially methylated sites between both cell lines. Pathway enrichment analysis was conducted using the identified differentially methylated regions using EnrichR to gather information on the signaling pathways and genes with altered patterns of DNA methylation in the IKKα knockout cells relative to the parental cell lines. The top 5 most significant hypomethylated and hypermethylated regions were enriched for genes involved in ERBB2 regulation. qRT-PCR was performed to confirm our methylation data and determine the ERBB2 expression in the PDAC cell line. Other confirmatory tests such as in vitro cell assays comprising of cell proliferation and cell migration were also performed to further study the characteristics of the cell lines. Statistical analysis using the EnrichR and Student’s t-test value were performed using the mean and standard deviation values. Findings were deemed statistically significant if p<0.05. Results: Genomic regions that were hypermethylated in the IKKα knockout cells relative to the parental cell lines were significantly enriched for genes involved in ERBB2-related signaling pathways such as the PTK6-ERK/MAPK, and PI3K events. The qRT-PCR confirmed the downregulation of ERBB2 expression in the IKKα knockout cell line. A decrease in the cell proliferation rate was also observed when compared to the wildtype. There were no statistically significant results obtained from the hypomethylated regions and cell migration assay. Conclusion: Perturbations to IKKα expression result in epigenetic alterations in PDAC cell line via the NF-κB dependent and/or independent pathways. Subset genes such as NRG1, NRG3, ERBB4, and EGFR were also identified to be regulated by IKKα. Key words: IKKα, NF-κB, epigenetics, DNA methylation, PDAC

Background The non-immunogenic and immunosuppressive microenvironment of pancreatic ductal adenocarcinoma (PDAC) had been a major obstacle for immunotherapeutic interventions. Recent findings had shown that epigenetic changes play a critical role in altering immune contexture in PDAC. Therefore, this study aims to identify aberrant epigenetic changes that attribute in immunosuppressive state in PDAC, particularly focusing on tumour cell intrinsic factors that mediates PDAC cells resistant to cytotoxic T lymphocytes (CTL) mediated killing. Methods A total of 396065 CpG variables methylation data involving 102 patients derived from The Cancer Genome Atlas (TCGA) project were retrieved from International Cancer Genome Consortium (ICGC) portal. Student t-test was used to filter out the insignificant CpG variables. Differentially methylated CpG sites were subsequently identified through 3 independent supervised clustering (SVM, kNN, random tree) methods, mean-variance filtering and hierarchy clustering. The identified differentially methylated CpG sites were further annotated to their corresponding genes to obtain differentially methylated genes. Scatterplot was used to analyse correlation of methylation with gene expression to determine functional relevance of methylation at varying regulatory region. REACTOME over-representation analysis tool was used to identify enriched biological processes. Venn diagram was used to identify common differentially methylated genes between TCGA primary tumour and PDAC cell line datasets. Result Out of 9 differentially methylated genes that were significantly correlated to gene expression, 3 of the aberrant genes were deteced in both primary tumour and cell line datasets. Based on pathway enrichment analysis, TP53 regulated death receptor signalling pathways were enriched with hypermethylated genes while RUNX1, PPARα and RhoGTPases signalling were enriched with hypomethylated genes. Moreover, demethylating agent was observed to enhance cytolytic killing in PDAC cytotoxic T lymphocytes (CTL)-resistant cell lines in the presence of activated CD8+T cells Conclusion Hypomethylated Rab17 and PLCG; hypermethylated RGS12 were identified in both primary tumour and cell line model suggesting the potential of these intrinsic factors in contributing to the low cytolytic state in PDAC. Aberrant methylation event is evident to attribute in CTL resistance in pancreatic cancer cell lines. The deployment of DNMT inhibitor had shown to enhance the T cell mediated cytolytic killing in CTL resistant cell lines.

Pancreatic adenocarcinoma (PDAC) ranks high among the causes of cancer-related mortality. The prognosis of this disease has not improved significantly over the past 50 years despite advancements in imaging techniques, cancer genetics and treatment modalities. Likewise, emerging developments in immunotherapy have yet to bring significant clinical advantage among PDAC patients. Tumour-intrinsic immune evasion mechanisms of PDAC remained poorly understood. By leveraging on the large-scale genomic datasets reported by the Australia Pancreatic Cancer Project (PACA-AU) and The Cancer Genomic Atlas Project (TCGA-PAAD), we investigated the immunophenotype of PDAC and identified that tumour with low cytotoxic T cell (CTL) killing activity exhibited poorer clinical outcomes as compared to tumour with high CTL killing activity. Integrative transcriptomic analysis identified TWIST1, IL6R, MMP3, and HDAC5 as putative immune modulators, responsible for the tumour-cell intrinsic immune evasion in pancreatic cancers. Connectivity Map (CMap) analysis identified histone deacetylase (HDAC) inhibitors as potential candidate drugs that could target CTL resistant PDAC. Indeed, in vitro study demonstrated that entinostat and quisinostat enhanced CTL killing of CTL-resistant BxPC3 and SW1990 cells. Overall, these findings suggest the immunomodulatory effects of HDAC inhibitors in pancreatic cancer and warrant further investigation.

With less than 10% of the 5-year survival rate, pancreatic ductal adenocarcinoma (PDAC) is known to be one of the most lethal types of cancer. Current literature supports that gemcitabine is the first-line treatment of PDAC. However, its poor cellular penetration, acquired and intrinsic chemoresistance is common despite the broad usage of gemcitabine. Photodynamic therapy (PDT), a more selective and minimally invasive treatment, may be used synergistically with gemcitabine to reduce the doses utilised and dose-related side effects. The anti-pancreatic cancer evaluation of gemcitabine and rhenium(I), Re(I) pyta complex as a single agent does not show much difference between with/without PDT treatment. In contrast, Re(I) bisquinolinyl complex was to the PDAC (SW1990 and BxPC3) at light irradiation regimen of 365 nm for 2.5 minutes and 1.3 J/cm2 (IC50 without PDT: > 100 μM; IC50 with PDT for BxPC3: 42.06 μM; IC50 with PDT for SW1990: 61.75 μM). Combinatorial therapy with gemcitabine and either Re(I) pyta complex or Re(I) bisquinolinyl complex was then performed. All the tested combinations indicated synergism at most ratios and no significant difference in synergism was observed between with/without PDT. To improve the cytotoxicity of Re(I) pyta complex, it was conjugated with gemcitabine and successfully killed PDAC. With the aim to enhance cellular uptake and therapeutic efficiency, potent Re(I) bisquinolinyl complex and gemcitabine were encapsulated into liquid crystalline nanoparticles (LCNPs) system. All LCNPs formulations were found to be homogeneous, with both drugs encapsulated efficiently, in nano-sized (159 to 173 nm) and stable within the acceptable range of zeta potential (+1.06 to -10 mV). Around 70% of gemcitabine and 90% of Re(I) bisquinolinyl complex were found to be entrapped efficiently in the synthesised LCNPs. The release rate of gemcitabine or/and Re(I) bisquinolinyl complex loaded into LCNPs were evaluated in vitro and the much hydrophilic gemcitabine was released at a faster rate than the much lipophilic Re(I) complex. LCNPs loaded with gemcitabine and Re(I) bisquinolinyl complex in 1:1 ratio illustrated the best anti-pancreatic cancer activity among the synthesised LCNPs formulations (IC50 of BxPC3: 0.15 μM; IC50 of SW1990: 0.76 μM) through apoptosis. This work highlights the relevance of drug combinations, drug conjugation and formulations with LCNPs strategies to improve therapeutic potency. Future in vivo validations will be helpful to further bridge this work from bench to bedside.

Background Immunosuppressive microenvironment of pancreatic cancer pancreatic ductal adenocarcinoma cell (PDAC) and pancreatic stellate cell (PSC) comprise myeloid derived suppressor cells (MDSC) which greatly contribute in impaired antitumour response, as well as progression of tumour. It was previously (unpublished) reported that physical contact of PDAC and PSC with peripheral blood mononuclear cells (PBMC) can convert PBMC into MDSC, causing suppression of T cells proliferation and activity. Little is known on the role of PDAC or combined with PSC in MDSC differentiation. In order to better mimic the tumour microenvironment, the effects of PDAC and PSC on PBMC was investigated via incubation with conditioned media prepared from supernatant of PDAC and PSC co-culture. Methods PBMCs were incubated with conditioned media prepared from supernatant of PDAC and PSC co-culture. The differentiation of PBMC to MDSC and their subtypes, polymorphonuclear-MDSC (p-MDSC) and mononuclear-MDSC (m-MDSC) were analysed using flow cytometer. Results This study had shown that secreted molecules from PDAC and PSC alone can induce the differentiation of MDSC and thus increase the percentage of MDSC in the total cell population. Secreted molecules from PDAC and PSC combination can also increase the ratio of p-MDSC to m-MDSC. Conclusion The secreted molecules in conditioned media alone is able to increase the ratio of p-MDSC : m-MDSC, even though there is no physical contact between PDAC, PSC and PBMC.