Interfacing polymeric scaffolds with primary pancreatic ductal adenocarcinoma cells to develop 3D cancer models

Abstract

We analyzed the interactions between human primary cells from pancreatic ductal adenocarcinoma (PDAC) and polymeric scaffolds to develop 3D cancer models useful for mimicking the biology of this tumor. Three scaffold types based on two biocompatible polymeric formulations, such as poly(vinyl alcohol)/gelatin (PVA/G) mixture and poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) copolymer, were obtained via different techniques, namely, emulsion and freeze-drying, compression molding followed by salt leaching, and electrospinning. In this way, primary PDAC cells interfaced with different pore topographies, such as sponge-like pores of different shape and size or nanofiber interspaces. The aim of this study was to investigate the influence played by the scaffold architecture over cancerous cell growth and function. In all scaffolds, primary PDAC cells showed good viability and synthesized tumor-specific metalloproteinases (MMPs) such as MMP-2, and MMP-9. However, only sponge-like pores, obtained via emulsion-based and salt leaching-based techniques allowed for an organized cellular aggregation very similar to the native PDAC morphological structure. Differently, these cell clusters were not observed on PEOT/PBT electrospun scaffolds. MMP-2 and MMP-9, as active enzymes, resulted to be increased in PVA/G and PEOT/PBT sponges, respectively. These findings suggested that spongy scaffolds supported the generation of pancreatic tumor models with enhanced aggressiveness. In conclusion, primary PDAC cells showed diverse behaviors while interacting with different scaffold types that can be potentially exploited to create stage-specific pancreatic cancer models likely to provide new knowledge on the modulation and drug susceptibility of MMPs.

Keywords: 2D, Bi-dimensional; 3D, Three-dimensional; BCA, Bicinchoninic acid; BSA, Bovine serum albumin; Dd, double distilled; Ds, double stranded; ECM, Extracellular matrix; G, Gelatin; HRP, Horseradish peroxidase; K-ras, Kirsten rat sarcoma viral oncogene homolog; MMP, Matrix metalloproteinase; PBS, Phosphate buffer saline; PCR, Polymer-chain reaction; PDAC, Pancreatic ductal adenocarcinoma; PEOT/PBT, Poly(ethylene oxide terephthalate)/poly(butylene terephthalate); PVA, Poly(vinyl alcohol); PanIN, Pancreatic intraepithelial neoplasia; Pancreatic adenocarcinoma; Smad4, Mothers against decapentaplegic homolog 4; TME, Tumor microenvironment.; cancer; compression molding; electrospinning; emulsion and freeze-drying; metalloproteinase 2 (MMP-2); metalloproteinase 9 (MMP-9); polyethylene oxide terephthalate (PEOT); polyvinyl alcohol (PVA); scaffold.

Figure 1.

Isolation and characterization of the primary PDAC cell line NFCR01. (A) Micrograph of a pure population of epithelial PDAC cells, as obtained via contrast phase light microscopy (Original magnification 20×, scale bar = 50 μm). (B) Micrographs of laser microdissected epithelial cells from a PDAC fresh tissue to perform DNA extraction (Original magnification 10×). (C) K-ras gene mutation analysis of isolated cells showing G216T mutation in codon 12 of exon 2.

Figure 2.

SEM micrographs of the produced scaffolds: (A) PVA/G sponge, (B) PEOT/PBT sponge, and (C) PEOT/PBT fiber mesh. Zoomed-out micrographs highlight pore size and topography (A1–C1), while zoomed-in micrographs image pore surfaces (A2–C2).

Figure 3.

SEM micrographs of PDAC cell/scaffold constructs: (A) PVA/G sponge, (B) PEOT/PBT sponge, and (C) PEOT/PBT fiber mesh. Zoomed-out micrographs highlight interactions between cells and poral structures (A1–C1), while zoomed-in micrographs image single cells (A2–C2). Arrows indicate cells; arrowheads indicate scaffold surfaces.

Figure 4.

(A) Bar graph showing alamarBlue reduction percentage in PDAC cell/scaffold constructs along the culture time: PVA/G sponge, PEOT/PBT sponge, and PEOT/PBT fiber mesh. Data are reported as mean ± SD. Statistical analysis was performed at the endpoint (P > 0.05). (B) Bar graph showing total protein contents in PDAC cell/scaffold constructs at the culture endpoint. Data are reported as mean ± SD; asterisks indicate the following p values: * = 0.01; *** = 0.0001 and **** = 0.00005.

Figure 5.

Light micrographs of histologic sections of PDAC cell/scaffold constructs (A–C) and tumor tissue (D): (A) PVA/G sponge, (B) PEOT/PBT sponge, and (C) PEOT/PBT fiber mesh. (A1–D1) Hematoxylin and eosin (H&E) staining, showing cell morphology (original magnification 20×), IHC of (A2-D2) MMP-2 and (A3–D3) MMP-9 (original magnifications 40×). Arrows indicate organized clusters of cell with duct formation; “sc” indicates the scaffold material. (E–G) Some controls of IHC reactions: (E and F) negative controls, i.e., omitting the primary antibody MMP-2, (G) positive control of MMP-2 performed on a PDAC cell line. Scale bar = 50 μm.

Figure 6.

(A) Western blot results show the expression of MMP-2 and MMP-9 proteins, both in the active (cleaved) and inactive (full-length) forms in PVA/G sponge, PEOT/PBT sponge and PEOT/PBT fiber mesh. Molecular weight (Mw) scales are reported on the left. Arrows indicate the bands of interest. (B) Bar graph reporting the analysis of the band volume intensity for Western blot data (intensity × 10⁶) for the 3 cell/scaffold constructs.