Reversible chemoresistance of pancreatic cancer grown as spheroids

Abstract

Better in vitro models are needed to identify active drugs to treat pancreatic adenocarcinoma (PAC) patients. We used 3D hanging drop cultures to produce spheroids from five PAC cell lines and tested nine FDA-approved drugs in clinical use. All PAC cell lines in 2D culture were sensitive to three drugs (gemcitabine, docetaxel and nab-paclitaxel), however most PAC (4/5) 3D spheroids acquired profound chemoresistance even at 10 µM. In contrast, spheroids retained sensitivity to the investigational drug triptolide, which induced apoptosis. The acquired chemoresistance was also transiently retained when cells were placed back into 2D culture and six genes potentially associated with chemoresistance were identified by microarray and confirmed using quantitative RT-PCR. We demonstrate the additive effect of gemcitabine and erlotinib, from the 12 different combinations of nine drugs tested. This comprehensive study shows spheroids as a useful multicellular model of PAC for drug screening and elucidating the mechanism of chemoresistance.

Keywords: Abraxane; Organoids; cancer associated fibroblasts (CAFs); chemosensitivity; combination drug therapy; multidrug resistance (MDR); pancreatic cancer.

Figure 1

Spheroids cultured in hanging drop plates. PAC cells, A6L, Panc 215, and A10.7 formed compact and single spheroids (A-C). Human pancreatic duct epithelial (HPDE) cells formed multiple spheroids (D). Cancer associated fibroblasts (CAFs), CAF19 (E) and CAF35 (E), both formed compact and single spheroids. PAC cells, Panc 247 (G) and PK-9 (H), did not form spheroids. Spheroids were photographed by phase microscopy. Scale bar: 100 μm.

Figure 2

PAC cells chemoresponses in 2D and 3D Culture. Percent survival of PAC, HPDE, and CAF cells cultured in 2D or 3D are displayed after treatment with gemcitabine (gem), docetaxel (dtx), nab-paclitaxel (Nab-Ptx), or triptolide (tri) at the concentration of 100 nM or 10 μM (Mean ± SD). All growth assays were done in six replicates. *: p<0.05, **: p<0.01, ***: p<0.001

Figure 3

Chemosensitivity of PAC cells when cultured back to 2D from 3D. Chemosensitivity of control PAC cells grown in 2D culture (‘2D → 2D’) compared to those placed in 2D culture after spheroids growth (‘3D → 2D’) at Day 0, 1 or 4. Percent survival of Panc 10.05 cells (A, B) and TS0111 cells (C, D) with gemcitabine (A, C) or docetaxel (B, D) (Mean ± SD). All growth assays were done in six replicates. *: p<0.05, **: p<0.01, ***: p<0.001

Figure 4

Drug accumulation in 4 different PAC cells in 2D and 3D culture. Gemcitabine (A) and docetaxel (B) were added and incubated after eight hours incubation at the concentration of 10 μM (Mean ± SD). Each sample was tested as 3 biologic replicates. *: p<0.05, **: p<0.01, ***: p<0.001

Figure 5

Heatmap of genes differentially expressed in 2D vs. 3D. Cluster analysis based on 100 pairs of genes whose expression best separates cells grown in 2D culture from those grown in 3D culture. Blue and yellow cells in the heatmap indicate which of the two genes in the pair is more highly expressed. Only after identifying the 100 genes, the xenograft sample was analysed. PDX: Patient-derived xenograft. Each sample was tested in duplicate (r: biologic replicates). For gene names, see supplemental tables S2,S3.

Figure 6

Chemoresponses to chemotherapy combinations in 2D or 3D culture. Gemcitabine based chemotherapy (A-C) and 5-FC based chemotherapy (D-G) were evaluated in three PAC cell lines (Mean ± SD). Arrows indicate additive effects by gemcitabine+erlotinib. Arrowheads indicate additive effects of 5-fluorouracil with any of the three other drugs in the FOLFIRINOX (leucovorin, irinotecan, oxaliplatin, or FOLFIRINOX itself). All growth assays were done in six replicates. Gemcitabine (Gem), Nab-paclitaxel (Nab-Ptx), Erlotinib (Erl), F: 5-fluorouracil, L: leucovorin, I: irinotecan, O: oxaliplatin. *: p<0.05, **: p<0.01, ***: p<0.001

Figure 7

The structure of mixed spheroids. A6L+CAF35 mixed spheroids were fixed, sectioned, and stained for haematoxylin and eosin (A, HE), vimentin (B, Vim), and cytokeratin 19 (C, Ck19). Thin shell of PAC cells (300 cells) surrounds CAF spheroids (3,000 cells) (D). A6L percentage in the mixed spheroids as determined by quantification of microsatellite DNA after plating A6L:CAF35 cells at varying ratios (3 biologic replicates, Mean ± SD) (E). Individual CAF35 cells (mCherry) were added to preformed A6L spheroids (EGFP). Note that CAF35 spheroids are found in the centres of preformed A6L spheroids (F). Scale bars: 100 μm. A6L+CAF35 mixed spheroids cultured for 7 days (G) or 34 days (H). Arrowheads in G indicate early basolateral polarization. Arrows in H indicate cells with basolateral polarization. Scale bars: 50 μm. Human PAC tissue (I). Scale bars: 20 μm.