Modelling of pancreatic cancer biology: transcriptomic signature for 3D PDX-derived organoids and primary cell line organoid development

Abstract

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...

Three-Dimensional Cell Culture Systems in Radiopharmaceutical Cancer Research

“…Similar to other 3D culturing methods, organoids are grown encapsulated or on top of scaffolds such as collagen or Matrigel™ [ 34 , 76 ]. Together, scaffold material and media containing growth factors mimic a natural, growth promoting cell environment [ 77 ]. In this manner, human pancreas duct cells can be cultured in vitro up to five months [ 46 ] while remaining genetically stable [ 78 ].…”

Powerful quantifiers for cancer transcriptomics

“…The traditional alternative to the heterogeneous tissue is to create, profile separately and compare immortalized cell lines from normal and cancer samples[ 51 ]. However, regardless of such advantages as low-cost and repeatability, the value of the immortalized cell lines for clinical research is limited due to significant genetic modifications undergone during passages[ 52 ] and immortalization procedure. Cell lines are usually developed from advanced cancer stages, unsuitable to understand cancer progression.…”

“…The cellular heterogeneity of the organoids can be explored using single-cell RNA sequencing[ 63 ]. Instead of primary cultures, some investigators try to create 3D organoids from cancer patient derived xenografts[ 52 , 60 , 64 ].…”

Cell Culture Based in vitro Test Systems for Anticancer Drug Screening

“…Organoids from primary lung cancer tissues demonstrated the high reproduction levels of histological and genetic characteristics of in situ tissue and their high ability for using them in patient-specific drug trials (Kim et al, 2019). Organoid manner was used for modeling PDAC from patient derived xenografts (PDX) tumors (Nelson et al, 2020) and organoids derived from patient prostate cancer bone metastasis (Lee et al, 2020). Organoids derived from patients with bladder cancer were tested with epirubicin, mitomycin C, gemcitabine, vincristine, doxorubicin, and cisplatin, this model was presented as a prospective model of human bladder cancer (Mullenders et al, 2019; Figure 1C).…”