Multiplex quantitative analysis of stroma-mediated cancer cell invasion, matrix remodeling, and drug response in a 3D co-culture model of pancreatic tumor spheroids and stellate cells

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is a stroma-rich carcinoma, and pancreatic stellate cells (PSCs) are a major component of this dense stroma. PSCs play significant roles in metastatic progression and chemoresistance through cross-talk with cancer cells. Preclinical in vitro tumor model of invasive phenotype should incorporate three-dimensional (3D) culture of cancer cells and PSCs in extracellular matrix (ECM) for clinical relevance and predictability.

Methods: PANC-1 cells were cultured as tumor spheroids (TSs) using our previously developed minipillar chips, and co-cultured with PSCs, both embedded in collagen gels. Effects of PSC co-culture on ECM fiber network, invasive migration of cancer cells, and expression of epithelial-mesenchymal transition (EMT)-related proteins were examined. Conditioned media was also analyzed for secreted factors involved in cancer cell-PSC interactions. Inhibitory effect on cancer cell invasion was compared between gemcitabine and paclitaxel at an equitoxic concentration in PANC-1 TSs co-cultured with PSCs.

Results: Co-culture condition was optimized for the growth of TSs, activation of PSCs, and their interaction. Increase in cancer cell invasion via ECM remodeling, invadopodia formation and EMT, as well as drug resistance was recapitulated in the TS-PSC co-culture, and appeared to be mediated by cancer cell-PSC interaction via multiple secreted factors, including IL-6, IL-8, IGF-1, EGF, TIMP-1, uPA, PAI-1, and TSP-1. Compared to gemcitabine, paclitaxel showed a greater anti-invasive activity, which was attributed to suppresion of invadopodia formation in cancer cells as well as to PSC-specific cytotoxicity abrogating its paracrine signaling.

Conclusions: Here, we established 3D co-culture of TSs of PANC-1 cells and PSCs using minipillar histochips as a novel tumoroid model of PDAC. Our results indicate usefulness of the present co-culture model and multiplex quantitative analysis method not only in studying the role of PSCs and their interactions with tumor cell towards metastatic progression, but also in the drug evaluation of stroma-targeting drugs.

Keywords: 3D co-culture; Cancer invasion; Matrix remodeling; Paclitaxel; Pancreatic stellate cell; Tumor microenvironment; Tumor spheroids.

Fig. 1

Optimization of co-culture conditions for TSs and PSCs. a A schematic illustration of cancer cells-PSCs co-culture using minipillar chips and 96-well plates and subsequent confocal microscopy following immunofluorescence staining. b The relationship between EMT status (levels of E-cadherin and vimentin expression; green) and spheroid surface roughness among five pancreatic cancer cell lines. c Comparison of the number and size of spheroids among five pancreatic cancer cell lines. d Morphology and viability (calcein AM) of TSs and PSCs when co-cultured. Cells were grown in the absence (b and c) and presence (d) of PSCs for 6 days. Staining of whole TSs and PSCs was carried out during cultivation in the well plates, and optical sections were acquired at 2 μm (b), 10 μm (c) and 5 μm (d) intervals and stacked into a z-projection. Data represent the mean ± SD of three independent experiments. Scale bars: 50 μm

Fig. 2

Activation of PSCs when co-cultured with PANC-1 spheroids. a Changes in the cell shape and length when co-cultured with PANC-1 spheroids. b The expression of α-SMA and TGF-β1 increased in PSCs co-cultured with TSs. Immunostaining was done after 7 day of culture in 96-well plates. Optical sections were acquired at 5 μm (a) and 1.5 μm (b) intervals and stacked into a z-projection. Data represent the mean ± SD of three independent experiments. Minimum of 50 cells were analyzed per field. Scale bars: 200 μm. * p < 0.05, **p < 0.01

Fig. 3

Changes in distribution and morphology of matrix protein fibers. The effects of PSC co-culture on the expression level and thickness of type I collagen (a) and fibronectin (b). Yellow arrowheads indicate the ECM bundle found in the periphery of TSs. c Difference angle distribution of collagen fibers between normal (non-invaded) matrix and matrix proximal to membrane protrusion (Invadopodia). After 7 days of culture, immunostaining was performed on cryo-sections of TSs (a and b) or on the whole TSs (c). Optical sections were acquired at 1 μm intervals and stacked into a z-projection. Data represent the mean ± SD of three independent experiments. Scale bars: 50 μm. * p < 0.05, ***p < 0.001

Fig. 4

The effect of PSC co-culture on cell invasion from PANC-1 TSs. a Changes in size and shape of the spheroids under PSC co-culture conditions. b Three distinct stages of progressive invasion of cancer cells out of TSs. The cells with nuclear elongation are indicated by white arrowheads and nuclear aspect ratio was compared between cells remaining within spheroids and cells invading matrix. c Increased fraction of TSs with invasive morphology under PSC co-culture conditions. Invadopodia (yellow arrowheads) and dispersed single cells (yellow asterisks) can be seen around some spheroids. Sectional areas containing minimum of 30 spheroids were analyzed for their invasion stage of non-invasive, invadopodia, or invasive as defined in (b). The cell invasion was compared calculated the number of dispersed single cells found in the matrix outside spheroids and compared between mono-culture TSs and TSs co-cultured with PSCs on day 9 of culture. Nuclear DAPI (blue) or F-actin (red) staining was performed on cryo-sections of TS (a) or on whole TSs (b and c). Confocal optical sections were acquired at 1 μm (a and b) or 7 μm (c) intervals and stacked into a z-projection. TS: tumor spheroid. Data represent the mean ± SD of three independent experiments. Scale bars: 50 μm. ***p < 0.001

Fig. 5

Increased expression of the EMT-related proteins in PANC-1 TSs under PSC co-culture conditions. a Changes in the expression levels of E-cadherin, vimentin, α-SMA, TGF-β1 and β-catenin as determined by immunofluorescence and western blot analysis. b Increased expression of Ki-67 under PSC co-culture conditions. Immunofluorescence staining was performed on cryo-sections of TSs following 7 days of culture. Optical sections were acquired at 1 μm intervals and stacked into a z-projection. Fluorescence intensity was measured from whole section of spheroid cultures and normalized by DAPI intensity. Individual TSs were shown in the insert. Data represent the mean ± SD of three independent experiments. Scale bars: 50 μm. * p < 0.05, **p < 0.01***p < 0.001

Fig. 6

Increased secretion of chemokines and cytokines in CM of PANC-1 TS co-cultured with PSCs. a Representative images of human cytokine array analysis in CM of PANC-1 TS under mono-culture and PSC co-culture conditions. Two factors were detected only in co-culture CMs (yellow boxes); five showed over 5-fold increase under co-culture conditions (red boxes). In the graph are the factors showing increase by more than 2-folds. b Upregulation of the TGF-β1 and EMT-related cytokines in CM under TS-PSC co-culture conditions. CM: conditioned media; POS: positive control; NEG: negative control

Fig. 7

Paclitaxel inhibits the invasion of cancer cells and suppresses the viability of PSCs. a Changes in cell invasion and EMT factor expression in TSs when exposed to GEM or PTX. Staining was performed on whole TSs (calcein AM and F-actin) and cryo-sections (vimentin and TGF-β1). Yellow arrowhead and asterisk indicate invadopodia and dispersed single cells, respectively. b Changes in cell morphology and fibroblast activation factors of PSCs exposed to GEM or PTX. c Changes in the expression of four EMT-related cytokines in the CM following drug treatment. d Schematic illustration of the proposed mechanism of PTX-induced inhibition of reciprocal activation and cytokine cross-talk between TSs and PSCs. Optical sections were acquired at 1.5 μm intervals and stacked into a z-projection. Drug effect was compared at the concentrations of both drugs producing 30% decrease in viability (IC₃₀) after 72 h exposure, i.e., 180 μM of GEM and 3 μM of PTX (Additional file 3: Figure S3-a). TS: tumor spheroid; GEM: gemcitabine; PTX: paclitaxel; CM: conditioned media. Data represent the mean ± SD of three independent experiments. Scale bars: 100 μm; *p < 0.05, **p < 0.01, ***p < 0.001 as compared to the control group