Inclusion of cancer-associated fibroblasts in drug screening assays to evaluate pancreatic cancer resistance to therapeutic drugs

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is characterised by a pro-inflammatory stroma and multi-faceted microenvironment that promotes and maintains tumorigenesis. However, the models used to test new and emerging therapies for PDAC have not increased in complexity to keep pace with our understanding of the human disease. Promising therapies that pass pre-clinical testing often fail in pancreatic cancer clinical trials. The objective of this study was to investigate whether changes in the drug-dosing regimen or the addition of cancer-associated fibroblasts (CAFs) to current existing models can impact the efficacy of chemotherapy drugs used in the clinic. Here, we reveal that gemcitabine and paclitaxel markedly reduce the viability of pancreatic cell lines, but not CAFs, when cultured in 2D. Following the use of an in vitro drug pulsing experiment, PDAC cell lines showed sensitivity to gemcitabine and paclitaxel. However, CAFs were less sensitive to pulsing with gemcitabine compared to their response to paclitaxel. We also identify that a 3D co-culture model of MIA PaCa-2 or PANC-1 with CAFs showed an increased chemoresistance to gemcitabine when compared to standard 2D mono-cultures a difference to paclitaxel which showed no measurable difference between the 2D and 3D models, suggesting a complex interaction between the drug in study and the cell type used. Changes to standard 2D mono-culture-based assays and implementation of 3D co-culture assays lend complexity to established models and could provide tools for identifying therapies that will match clinically the success observed with in vitro models, thereby aiding in the discovery of novel therapies.

Keywords: CAFs; Drug screening; PDAC chemotherapy; PDAC resistance.

Fig. 1

Isolated CAFs secrete collagen and are insensitive to gemcitabine and paclitaxel treatments compared to pancreatic cancer cell lines in standard 2D culture conditions. (A) Col1a1 secretion from three established pancreatic cancer cell lines and CAFs isolated from three different patients (R3072, R3088 and R3134) was determined by ELISA. The level of Col1a1 secreted by the cancer cell lines was below the acceptable limit of detection for the assay, precluding statistical analysis. (B–E) Dose–response curves following gemcitabine treatment of pancreatic cancer cell lines (B) and CAFs (R3072) (C) or paclitaxel treatment of pancreatic cancer cell lines (D) and CAFs (R3088) (E). Cells were exposed to increasing concentrations of gemcitabine or paclitaxel and cell viability measured at 72 h using CellTiter-Glo. Data were fitted to a sigmoidal dose–response curve, and IC50 was determined using GraphPad prism. For PDAC cell lines, the data are representative of three independent experiments ± SD performed in triplicate and normalised to a DMSO control set to 100%. For CAFs, the data are representative of three independent experiments using three biological replicates (R3088, R3072 and R3134) ± SD performed each in triplicate and normalised to a DMSO control set to 100%. (F–G) Graphs depicting the mean pIC₅₀ in molar (M) ± SEM for multiple assays of gemcitabine and paclitaxel in pancreatic cancer cell lines

Fig. 2

Chemotherapeutic pulsing with gemcitabine and paclitaxel to mimic clinical dosing in vitro. (A and C) The graphs show the cell viability of pancreatic cancer cell lines after a pulse with gemcitabine (A) or paclitaxel (C) representing 0.5 cMAX or 0.1 cMAX, data was analysed using DMSO as a control set to 100%. (B and D) The graphs show the cell viability of CAFs after a pulse with gemcitabine (B) or paclitaxel (D) representing 0.5 cMAX or 0.1 cMAX. At 24, 48, 72 and 96 h cell viability was determined using CellTiter-Glo. The data are shown as ± SEM of at least 2 independent experiments performed in triplicate and normalised to a DMSO control set to 100%. P value determined by one-way ANOVA with post hoc Dunnett’s test. The * symbol refers to the 0.5 cMAX condition with *P ≤ 0.05 and **0.01. The Φ symbol refers to the 0.1 cMAX condition with Φ P ≤ 0.05

Fig. 3

The addition of CAFs to 2D screening models reduces the anti-proliferative effect of gemcitabine on PANC-1 cells. (A) Schematic representation of a transwell co-culture model in which the two cell populations are separated by a physical barrier. In this model, CAFs were placed in the bottom chamber and PANC-1 cells were on the transwell insert (1:1 ratio). (B) Dose–response curve showing the efficacy of gemcitabine in killing PANC-1 cells in a transwell co-culture model of CAFs and PANC-1 cells. Cells were cultured for 72 h in the presence of gemcitabine. The cell viability of PANC-1 cells was measured using CellTiter-Glo. The data are shown as mean ± SD of one assay for two CAFs (R3088 and R3072) and normalised to a DMSO control set to 100%. (C) Schematic representation of the direct 2D co-culture model with an image depicting anti-αSMA-488 labelled CAFs (Green), anti-CTK-594-labelled PANC-1 cells (Yellow) and nuclei (Blue). The average nuclei count was measured using an Operetta (PerkinElmer), counting 4 randomly assigned areas of interest/well. (D) Dose–response curve showing the direct 2D cell viability assay using three different ratios of CAF to PANC-1 cells compared to PANC-1 cells alone. Cells were exposed to gemcitabine and DMSO as control. At 72 h, cell viability was determined by counting nuclei which were associated with positive pCTK staining (considered PANC1 cells) and which were not associated with areas of positive αSMA staining (considered CAFs). The data are shown as mean ± SD of one assay performed in triplicate

Fig. 4

The addition of CAFs to a 3D co-culture model of pancreatic cancer cell lines confers resistance to gemcitabine. (A) Schematic representation of the assay formats utilised: a 2D standard mono-culture cell viability assay, a 3D mono-culture assay of pancreatic cancer cell lines and a 3D co-culture assay of pancreatic cancer cell lines combined with CAFs (R3008). (B) Dose–response curves of the different assay formats described above which were treated with various concentrations of gemcitabine. At 72 h cell viability was determined using CellTiter-Glo. The data are shown as ± SD of one assay performed in triplicate and normalised to a DMSO control set to 100%