With a five-year survival rate of 9%, pancreatic ductal adenocarcinoma (PDAC) is the deadliest of all cancers. The rapid mortality makes PDAC difficult to research, and inspires a resolve to create reliable, tractable cellular models for preclinical cancer research. Organoids are increasingly used to model PDAC as they maintain the differentiation status, molecular and genomic signatures of the original tumour. In this paper, we present novel methodologies and experimental approaches to develop PDAC organoids from PDX tumours, and the simultaneous development of matched primary cell lines. Moreover, we also present a method of recapitulating primary cell line cultures to organoids (CLOs). We highlight the usefulness of CLOs as PDAC organoid models, as they maintain similar transcriptomic signatures as their matched patient-derived organoids and patient derived xenografts (PDX)s. These models provide a manageable, expandable in vitro resource for downstream applications such as high throughput screening, functional genomics, and tumour microenvironment studies. With a rapid progression and fatal outcome, pancreatic cancer is one of the deadliest of all cancers. Long-term survivors are limited to those with resected early stage tumours; however, overall survival rate is a dismal 9%, with a median survival time of 7-11 months 1,2. As pancreatic cancer is notoriously asymptomatic at an early stage, 80% of patients are diagnosed after the cancer has metastasised, making them ineligible for resection, which is the only curative treatment. The number of cases of pancreatic cancer has been steadily rising since 2004 3. It is currently the fourth most common cause of cancer death in the US, and by 2030 it is estimated that it will surpass breast and colorectal cancer to become the second most common cause of death by cancer 4. The majority of pancreatic cancers are in the exocrine pancreas (95%) known as pancreatic ductal adenocarcinoma (PDAC), and 5% are in the endocrine pancreas 5. The leading epidemiological factors include smoking, obesity, type II diabetes mellitus and acute pancreatitis, which account for approximately 25% of PDAC cases 6-9. A limitation in the understanding of the disease progression and development of effective treatments in PDAC may be due the lack of in vitro patient tumour representative models. Established 2D cell lines are the most widely used model for the development and testing of chemotherapeutics for over 50 years 10. The ability of cell lines to grow indefinitely makes them a low-cost, repeatable model, easy to manipulate and are an important base for discovery and proof-of-concept studies. Their importance in cancer research is indisputable, however, their use as a robust clinical model is questionable 11. During passaging, cell lines undergo genetic modifications, such as copy number variation and point mutations 12. Cell lines also have a high level of homogeneity, which does not represent the heterogenetic nature of PDAC tumours, and not all cancer subtypes are well represented as t...

Background. Pancreatic cancer is a devastating disease; its lethality is related to rapid growth and tendency to invade adjacent organs and metastasize at an early stage. Objective. The aim of this study was to identify miRNAs and their gene targets involved in the invasive phenotype in pancreatic cancer to better understand the biological behaviour and the rapid progression of this disease. Methods. miRNA profiling was performed in isogenic matched high invasive and low-invasive subclones derived from the MiaPaCa-2 cell line and validated in a panel of pancreatic cancer cell lines, tumour, and normal pancreas. Online miRNA target prediction algorithms and gene expression arrays were used to predict the target genes of the differentially expressed miRNAs. miRNAs and potential target genes were subjected to overexpression and knockdown approaches and downstream functional assays to determine their pathological role in pancreatic cancer. Results. Differential expression analysis revealed 10 significantly dysregulated miRNAs associated with invasive capacity (Student’s t-tests; P value <0.05; fold change = ±2). The expression of top upregulated miR-135b and downregulated let-7c miRNAs correlated with the invasive abilities of eight pancreatic cancer cell lines and displayed differential expression in pancreatic cancer and adjacent normal tissue specimens. Ectopic overexpression of let-7c decreased proliferation, invasion, and colony formation. Integrated analysis of miRNA-mRNA using in silico algorithms and experimental validation databases identified four putative gene targets of let-7c. One of these targets, SOX13, was found to be upregulated in PDAC tumour compared with normal tissue in TCGA and an independent data set by qPCR and immunohistochemistry. RNAi knockdown of SOX13 reduced the invasion and colony formation ability of pancreatic cancer cells. Conclusion. The identification of key miRNA-mRNA gene interactions and networks provide potential diagnostic and therapeutic strategies for better treatment options for pancreatic cancer patients.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death worldwide. This high mortality rate is due to the disease’s lack of symptoms, resulting in a late diagnosis. Biomarkers and treatment options for pancreatic cancer are also limited. In order to overcome this, new research models and novel approaches to discovering PDAC biomarkers are required. In this review, we outline the hereditary and somatic causes of PDAC and provide an overview of the recent genome wide association studies (GWAS) and pathway analysis studies. We also provide a summary of some of the systems used to study PDAC, including established and primary cell lines, patient-derived xenografts (PDX), and newer models such as organoids and organ-on-chip. These ex vitro laboratory systems allow for critical research into the development and progression of PDAC.

Pancreatic cancer is a highly lethal disease. Therapeutic resistance to chemotherapy is a major cause of treatment failure and recurrence in pancreatic cancer. Organoids derived from cancer stem cells (CSC) are promising models for the advancement of personalised therapeutic responses to inform clinical decisions. However, scaling-up of 3D organoids for high-throughput screening is time-consuming and costly. Here, we successfully developed organoid-derived cell lines (2.5D) from 3D organoids; the cells were then expanded and recapitulated back into organoids known as cell line organoids (CLOs). The 2.5D lines were cultured long term into 2D established cell lines for downstream comparison analysis. Experimental characterisation of the models revealed that the proliferation of CLOs was slightly faster than that of parental organoids. The therapeutic response to chemotherapeutic agents in 3D CLOs and organoids showed a similar responsive profile. Compared to 3D CLOs and organoids, 2D cell lines tended to be less responsive to all the drugs tested. Stem cell marker expression was higher in either 3D CLOs or organoids compared to 2D cell lines. An in vivo tumorigenicity study found CLOs form tumours at a similar rate to organoids and retain enhanced CSC marker expression, indicating the plasticity of CSCs within the in vivo microenvironment.

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Pancreatic ductal adenocarcinoma (PDAC) is the deadliest of all cancers, with a five-year survival rate of 8%. Decades of research has done little to improve patient outcomes. To identify key therapeutic strategies, appropriate research preclinical models are critical for testing the efficacy of drugs or downstream functional genomic analysis. Current methods of growing PDAC established cell lines in a 2D monolayer do not fully represent how the tumor cells grow in vivo as they fail to recapitulate the physiology of tumors in vivo due to different architecture, adherence structures and biochemical gradients. Patient-derived xenografts can retain the histological and genetic characteristics of their donor tumor and can remain stable across passages. Three-dimensional (3D) organoids grown ex vivo are tractable also retain the architecture, genotype and phenotype of a patients’ tumor. However, a detailed comparative assessment of the utility of organoids, matched 2D cell lines and 3D cell organotypic models to assess the complex biology, interactions, response to therapeutic regimens has not been performed. Using our unique protocol, we have established PDAC organoids and isogenetically matched 2D cell lines derived from PDAC PDX tumors. To establish if these matched 2D cell lines grown in 3D can recapitulate the organoid model, we cultured the matched cell lines in 3D organoid media in non-adherent, Matrigel containing cultures. The expression of PDAC histological markers between the original tumor, the PDX tumor, organoid and matched cell line in 3D and 2D has identified differential expression of tumor initiating cell (TIC) markers. RNA-seq transcriptomics of the 2D cell line models, matched recapitulated 3D organotypic models, organoids, PDX-tumor and patient tumor will reveal the application of these models for ex vivo downstream functional analysis. Citation Format: Shannon R. Nelson, Sandra Roche, Fiona O'Neill, AnneMarie Larkin, John Crown, Naomi Walsh. Assessment of pancreatic cancer 3D organotypic cultures as models for functional preclinical analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 44.