Generation of an in vitro 3D PDAC stroma rich spheroid model

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent desmoplastic/stromal reaction, which contributes to the poor clinical outcome of this disease. Therefore, greater understanding of the stroma development and tumor-stroma interactions is highly required. Pancreatic stellate cells (PSC) are myofibroblast-like cells that located in exocrine areas of the pancreas, which as a result of inflammation produced by PDAC migrate and accumulate in the tumor mass, secreting extracellular matrix components and producing the dense PDAC stroma. Currently, only a few orthotopic or ectopic animal tumor models, where PDAC cells are injected into the pancreas or subcutaneous tissue layer, or genetically engineered animals offer tumors that encompass some stromal component. Herein, we report generation of a simple 3D PDAC in vitro micro-tumor model without an addition of external extracellular matrix, which encompasses a rich, dense and active stromal compartment. We have achieved this in vitro model by incorporating PSCs into 3D PDAC cell culture using a modified hanging drop method. It is now known that PSCs are the principal source of fibrosis in the stroma and interact closely with cancer cells to create a tumor facilitatory environment that stimulates local and distant tumor growth. The 3D micro-stroma models are highly reproducible with excellent uniformity, which can be used for PDAC-stroma interaction analysis and high throughput automated drug-screening assays. Additionally, the increased expression of collagenous regions means that molecular based perfusion and cytostaticity of gemcitabine is decreased in our Pancreatic adenocarcinoma stroma spheroids (PDAC-SS) model when compared to spheroids grown without PSCs. We believe this model will allow an improved knowledge of PDAC biology and has the potential to provide an insight into pathways that may be therapeutically targeted to inhibit PSC activation, thereby inhibiting the development of fibrosis in PDAC and interrupting PSCPDAC cell interactions so as to inhibit cancer progression.

Antifibrotic effects of pentoxifylline improve the efficacy of gemcitabine in human pancreatic tumor xenografts

“…Evaluation of antifibrotic drugs for their synergistic interaction should be further validated in genetically engineered mouse models before clinical evaluation . Tumor models in vitro , such as 3D culture of cancer cells with myofibroblast creating desmoplastic tumor tissue, may serve as an efficient tool for screening of antifibrotic agents, which warrants further studies …”

“…(48) Tumor models in vitro, such as 3D culture of cancer cells with myofibroblast creating desmoplastic tumor tissue, may serve as an efficient tool for screening of antifibrotic agents, which warrants further studies. (49) 2 Burris HA III, Moore MJ, Andersen J et al Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial. J Clin Oncol 1997; 15: 2403-13.…”

Methods and Model Systems Used to Study Pregnant Human Uterine Smooth Muscle

“…Additionally, the 3D platform can provide cells with an environment comprising the growth factors, cytokines and some extracellular matrix proteins produced by the cells during their growth. Over the past several years, immense effort has been put into the creation of various 3D cell culture platforms, for instance protein gels such as Matrigel and collagen, which reconstruct extracellular matrix composition [42]; polymer scaffolds, which mimic tissue structure and material properties [43]; hanging drop spheroids, which use water tension in liquid droplets to aggregate cells into spheroids [44][45][46]; round bottom plates, which use plate geometry to aggregate cells in spherical bottom wells [46]; as well as nano-patterned plates. Although these 3D cell culture platforms recreate tissue environments to varying degrees, they still have technical and cost limitations; for instance, long production times, the need for specialized equipment, as well as the produced 3D structures primarily being limited to spheroids, as opposed to more complex shapes that would better enable investigations into uterine smooth muscle contractility, such as rings or strips [47].…”

Establishment and Characterization of Paired Primary Cultures of Human Pancreatic Cancer Cells and Stellate Cells Derived from the Same Tumor

“…Similar to cancer cell lines, research on PSCs has mainly been conducted using a few commercially available PSC lines, which differ phenotypically and in their interactions with cancer cells compared to primary established PSCs, as shown in our recent study [25]. Several recent reports on various 2D and 3D models attempting to mimic the microenvironment of PDACs have invariably used PCC and PSC combinations of primary cells and immortalized cells obtained from different tumors [26][27][28][29][30]. This prompted us to establish an experimental model of selective pairs of primary PCCs and primary PSCs obtained from the same surgically resected human PDAC tumor.…”