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. 2016 Nov:108:129-42.
doi: 10.1016/j.biomaterials.2016.08.041. Epub 2016 Sep 2.

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

Matthew J Ware  1 , Vazrik Keshishian  1 , Justin J Law  1 , Jason C Ho  1 , Carlos A Favela  2 , Paul Rees  3 , Billie Smith  4 , Sayeeduddin Mohammad  4 , Rosa F Hwang  5 , Kimal Rajapakshe  6 , Cristian Coarfa  6 , Shixia Huang  6 , Dean P Edwards  6 , Stuart J Corr  7 , Biana Godin  8 , Steven A Curley  1
Affiliations

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

Matthew J Ware et al. Biomaterials. 2016 Nov.

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 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 PSC-PDAC cell interactions so as to inhibit cancer progression.

Keywords: 3D tumor microenvironment; Human pancreatic stellate cells; Pancreatic cancer; Stroma.

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Figures

Figure 1
Figure 1. Co-culture of PANC-1 and Capan-1 cells with PSCs in 2-D monolayer settings
PDAC cells were labeled with CellTracker CMFDA Green™ before being co-cultured with unlabeled PSCs. Brightfield, FITC fluorescence and color picro-sirius red images of Panc-1 cells alone, Capan-1 cells alone, PSCs alone, PANC-1 and PSCs (co-cultured in 2:1 ratio) and Capan-1 and PSCs (co-cultured in 2:1 ratio) (Scale bars = 1000μm)
Figure 2
Figure 2. Proteomic analysis of PDAC/PSCs interaction
A) Hierarchical clustering of RPPA data of (left panel) PSC in PANC-1 media versus untreated PCSs and (right panel) PANC-1 in PSC media versus untreated PANC-1 (differentially expressed proteins, t test P < 0.05, fold change exceeding 1.25 times). B) Activated stroma related pathways enriched by the core-culture signatures. Data are displayed as the –log of the P value for each pathway.
Figure 3
Figure 3. Brightfield microscopy characterization of micro-tumors co-cultured with and without PSCs
A) Brightfield images of PANC-1, AsPc-1, MIA PaCa-2, Capan-1 and BxPc-3 tumors at 7-day time-point. (White scale bar = 1000μm and red scale bar = 500μm m) B) Diameter of PDAC spheroids with and without PSCs at 10-day time-point. C) Optical density of PDAC spheroids with and without PSCs at 10-day time-point (p<0.01). D) Schematic representing the increase in density of tumor due to collagen fibers and PSCs packing regions between PDAC cells.
Figure 4
Figure 4. Scanning electron microscopy of tumor surface
Scanning electron micrograph of A) PANC-1 spheroid B) Capan-1 spheroid, C) PANC-1 PDAC-SS, D) Capan-1 PDAC-SS and E) Orthotopic PDAC tumor from mouse model.
Figure 5
Figure 5. Collagen content of PDAC tumors
A) Picro Sirius staining of various PDAC spheroids with and without PSCs, B) Quantification of collagen positive regions per unit area of tumor. (white and red scale bars= 500μm and yellow scale bars = 200μm *p<0.05).
Figure 6
Figure 6. Staining of the stroma fibrosis
Immunohistochemistry of PDAC stroma collagen components in PDAC-SS, as compared to orthotopic PDAC mouse tumors and human grade 2 stage 1 PDAC. Tissues sections stained for collagen 1, collagen 3, and fibronectin and smooth muscle actin (SMA) (Black scale bars = 500μm, red scale bars = 200μm).
Figure 7
Figure 7. Diffusion of molecules through PANC-1 spheroids with and without PSCs
FITC fluorescent molecule (MW=389.382 g/mol) was used as a model for gemcitabine (MW=263.198 g/mol) to visualize the diffusion through PANC-1 spheroids with and without PSCs. (A) FITC fluorescent images of PANC-1 spheroid regions with and without PSCs/collagen component B) quantification of the fluorescent intensity per unit area of spheroid after 24h of FIT exposure (p<0.05) and C) Cross-sectional FITC fluorescence from spheroid border through to spheroid centre (lower inlaid images display typical position of intensity measurement across centre of spheroid) D) LC-MS quantification of gemcitabine content normalized to spheroid surface area (spheroid assumed to be perfectly spherical during normalization) in PANC-1 spheroids with and without PSCs (Scale bars = 300μm, * indicates p<0.05).
Figure 8
Figure 8
Histological analysis after 24h exposure of gemcitabine. Structural integrity and of PDAC spheroids with and without PSCs after 24h gemcitabine exposure. A) Color images of H&E stained histological slides show (left) zoomed representative image of a PANC1 spheroid without PSCs and (right) a PANC1 spheroid with PSCs after 24h gemcitabine exposure. B) Quantification of circularity and solidity of PANC-1 spheroids with and without PSCs. (scale bars = 1000μm, p<0.01) (Robustness of segmentation algorithm is shown in Figure S7). *p<0.05.
Figure 9
Figure 9
Histological analysis after 120h exposure of gemcitabine. Proliferative index, measured by Ki67 staining, of PDAC spheroids with and without PSCs after 120h gemcitabine exposure. Color images of Ki67 stained histological slides show (left) PANC1 spheroids without PSCs and (right) PANC1 spheroids with PSCs after 0h (top row) and 120h (bottom row) gemcitabine exposure.
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