Organotypic Culture of Acinar Cells for the Study of Pancreatic Cancer Initiation

Abstract

The carcinogenesis of pancreatic ductal adenocarcinoma (PDA) progresses according to multi-step evolution, whereby the disease acquires increasingly aggressive pathological features. On the other hand, disease inception is poorly investigated. Decoding the cascade of events that leads to oncogenic transformation is crucial to design strategies for early diagnosis as well as to tackle tumor onset. Lineage-tracing experiments demonstrated that pancreatic cancerous lesions originate from acinar cells, a highly specialized cell type in the pancreatic epithelium. Primary acinar cells can survive in vitro as organoid-like 3D spheroids, which can transdifferentiate into cells with a clear ductal morphology in response to different cell- and non-cell-autonomous stimuli. This event, termed acinar-to-ductal metaplasia, recapitulates the histological and molecular features of disease initiation. Here, we will discuss the isolation and culture of primary pancreatic acinar cells, providing a historical and technical perspective. The impact of pancreatic cancer research will also be debated. In particular, we will dissect the roles of transcriptional, epigenetic, and metabolic reprogramming for tumor initiation and we will show how that can be modeled using ex vivo acinar cell cultures. Finally, mechanisms of PDA initiation described using organotypical cultures will be reviewed.

Keywords: KRAS; acinar-to-ductal metaplasia; epigenetic reprogramming; metabolic reprogramming; organotypic culture; pancreas; pancreatic cancer.

Figure 1

Anatomy and histology of the pancreas. In (A) anatomic organization of the GI tract. The pancreas is magnified on the right, along few representative structures within the organ. (B) shows the cellular heterogeneity of the pancreas, which is a composite of exocrine (acini), endocrine (Langherans’ islets) and stromal elements. (C) shows the modular units of the exocrine pancreas (acini). Morphology of an acinar cell is magnified on the right.

Figure 2

PDA progression and in vitro modelling of disease initiation. In (A) step-wise model of PDA progression. Upon mutation of KRAS, pancreatic acinar cells undergo acinar-to-ductal metaplasia (ADM), a cell transforming event which initiates the carcinogenic cascade. Cells progressively acquire dysplastic features and accrue genetic mutations. Acinar cell-derived pre-cancerous lesions are termed Pancreatic Intraepithelial Neoplasms (PanIN) that are defined by different histological grades and eventually evolve to carcinomas. In (B) acinar cells isolated from mice (genotypes indicated), immediately embedded in Matrigel and observed over time. Duct-like cystic structures (arrows) are observed after few days. In (C) time course analysis reveals that KC-derived primary acinar cells undergoing ADM ex vivo (Matrigel-embedded) upregulate duct-specific genes (Krt19 and Sox9), and suppress acinar-specific genes (Cpa1 and Amy2). Data for each gene are normalized to the first time point (12 h post isolation).

Figure 3

Ex vivo acinar-to-ductal metaplasia (ADM) as a model for PDA initiation. In (A) schematic representation of ADM from acinar explants. Once isolated, acinar cells undergo transdifferentiation to acquire morphological and molecular features typical of ductal cells. Gene mutations (i.e., KRAS) and growth factors (i.e., TGFα, EGF) induce ADM ex vivo. The impact of additional genetic aberrations, signaling events or chemical compounds can be examined by several means (listed in orange): the ability to form ductal-like structure can be evaluated at end point, as shown in B and C. In this way, impact for tumor initiation can be inferred. In (B,C), two different metrics to score the tumor-initiating potential of KRAS mutation. In (B) percentage of acinar- and ductal-like structures is assessed by morphological evaluation of multiple samples. Acinar cells derived from Pdx1-Cre; LSL-KrasG12D (KC) mice form significantly more duct when cultured ex vivo (day 2 post isolation, in Matrigel). Percentage in white indicates the fraction of duct-like structures arising from cultures of acinar cells derived from either Pdx1-Cre or KC mice (on the total number of multi-cellular structures analyzed). In (C) quantitative PCR shows increased expression of duct-specific Cytokeratin-19 (Krt19) in cultures derived from either Pdx1-Cre or KC pancreata (at day 2 post isolation, in Matrigel, triplicates).

Figure 4

Cellular reprogramming during acinar-to-ductal metaplasia (ADM). In (A) ex vivo-isolated acinar cells transition to a tumor-initiating ductal phenotype. Cell identity is impacted: undergoing ADM, the cell loses its secretory function (apical granules disappear), changes morphology (acquire a more elongated, brick-like structure). Gene expression and markers are dynamically regulated, as shown in the lower panel. Transition in cell identity is a continuum, which progresses through de-differentiation into a progenitor-like cell type (in yellow) and culminates in a duct-like cell (in green). (B) Acinar-to-Ductal Metaplasia is critically supported by both epigenetic and metabolic reprogramming. Mechanisms for active cellular remodeling are necessary to switch cell identity across two terminally-differentiated states.