Cholangiocarcinoma-on-a-chip: A human 3D platform for personalised medicine

Abstract

Background & aims: Cholangiocarcinoma (CCA) is a primary liver tumour characterised by a poor prognosis and limited therapeutic options. Available 3D human CCA models fail to faithfully recapitulate the tumour niche. We aimed to develop an innovative patient-specific CCA-on-chip platform.

Methods: A CCA tumour microenvironment was recapitulated on a microfluidic three-channel chip using primary CCA cells, cancer-associated fibroblasts (CAFs), endothelial cells, and T cells isolated from CCA specimens (n = 6). CAF and CCA cells were co-cultured in the central channel, flanked by endothelial cells in one lateral channel, recreating a tubular structure. An extensive characterisation of this platform was carried out to investigate its diffusion ability, hydrogel properties, and changes in matrix composition. Cell phenotype and functional properties were assessed.

Results: Primary cells seeded on the microfluidic device were shown to reproduce the architectural structure and maintain the original phenotype and functional properties. The tumour niche underwent a deep remodelling in the 3D device, with an increase in hydrogel stiffness and extracellular matrix deposition, mimicking in vivo CCA characteristics. T cells were incorporated into the device to assess its reliability for immune cell interaction studies. Higher T cell migration was observed using cells from patients with highly infiltrated tumours. Finally, the drug trial showed the ability of the device to recapitulate different drug responses based on patient characteristics.

Conclusions: We presented a 3D CCA platform that integrates the major non-immune components of the tumour microenvironment and the T cell infiltrate, reflecting the CCA niche. This CCA-on-chip represents a reliable patient-specific 3D platform that will be of help to further elucidate the biological mechanisms involved in CCA and provide an efficient tool for personalised drug testing.

Impact and implications: An innovative patient-specific cholangiocarcinoma (CCA)-on-chip platform was successfully developed, integrating the major components of the tumour microenvironment (tumour cells, cancer-associated fibroblasts, endothelial cells, and immune infiltrate) and faithfully mimicking the CCA niche. This CCA-on-chip represents a powerful tool for unravelling disease-associated cellular mechanisms in CCA and provides an efficient tool for personalised drug testing.

Keywords: Cell–cell crosstalk; Cholangiocarcinoma; Drug testing; Liver-on-chip; Microfluidics; Tumour microenvironment.

Graphical abstract

Fig. 1

CCA-on-CHIP. (A) AutoCAD chip design with pillar detail. At the bottom, the resin used for chip serial production with the schematic representation of the CCA-on-CHIP. (B) 3D reconstruction of the chip central channel. Left, the orthogonal view of CCA cells (green) and CAFs (red). Right, volume rendering of z-stack using IMARIS https://imaris.oxinst.com. Scale bar = 80 μm. (C) 3D reconstructions of the endothelial channel. Volume rendering of z-stack using IMARIS. Scale bar = 80 μm. CD31: magenta; nuclei: cyan; CCA cells: green; CAFs: red. (D) Gene expression analysis of CCA cells and CAFs in 2D monolayer and 3D platform. One-way ANOVA (mean ± SEM; n = 5 biological replicates). ∗p <0.05; ∗∗p <0.01; ∗∗∗∗p <0.0001. CAFs, cancer-associated fibroblasts; CCA, cholangiocarcinoma.

Fig. 2

Cell-laden hydrogel characterisation. (A) Pressure difference drop. Two-way ANOVA (mean ± SEM; n = 5 biological replicates). (B) Young’s modulus distribution. Two representative Young’s modulus force maps related to the local measurements. Mann-Whitney U test (median with IQR; n = five biological replicates). (C) Left, representative SEM images of the cell-laden hydrogel. Right, the hydrogel fibre images with the pore size distribution. Mann-Whitney U test (medians with interquartile range; n = 5 biological replicates). Scale bar = 1 μm ∗p <0.0001. CAFs, cancer-associated fibroblasts; CCA, cholangiocarcinoma.

Fig. 3

Collagen deposition analysis on-chip. (A) Gene expression of extracellular matrix (ECM) proteins in the co-culture of CCA primary cells and CAFs. Two-way ANOVA (mean ± SEM; n = 5 biological replicates). (B) Representative confocal images for collagen IV (green), phalloidin (red), and nuclei (cyan) for CCA primary cells and CAFs co-culture. Scale bar = 50 μm. (C) Left, representative confocal images for collagen IV (green), phalloidin (red) and nuclei (cyan) on Day 4 for CCA primary cells and CAFs-on-chip. Scale bar = 50 μm. MFI of collagen IV staining. One-way ANOVA (medians with IQR; n = 5 biological replicates). (D) Representative SEM images of the device on Day 4. Scale bar = 2 μm ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.0005; ∗∗∗∗p <0.0001. CAFs, cancer-associated fibroblasts; CCA, cholangiocarcinoma; MFI, mean fluorescence intensity.

Fig. 4

GEM/CDPP exposure analysis. (A) At the top, representative confocal images of CCA cells in 3D mono-culture and in 3D co-culture on chip after drug treatment (48 h). Scale bar = 100 μm. At the bottom, dose–response curves for CCA primary cells (n = 5 patients) in 2D culture, 3D mono-culture and 3D co-culture. Two-way ANOVA (mean ± SEM; n = 5 biological replicates). Gene expression analysis for YAP and TAZ in 3D mono-culture and 3D co-culture. Mann-Whitney U test (mean ± SEM; n = 5 biological replicates). (B) Dose–response curves for 3D mono-culture and 3D co-culture on chip in early (ER; n = 3 patients) and late (LR; n = 2 patients) recurrence patients. Two-way ANOVA (mean ± SEM; n = 5 biological replicates). (C) Percentage of cell viability for the 3D mono-culture and the 3D co-culture in ER and LR patients. Two-way ANOVA (mean ± SEM; n = 5 biological replicates). (D) Percentage of cell viability in the 3D co-culture for CAFs and CCA cells singularly in ER and LR patients. Two-way ANOVA (mean ± SEM; n = 3 biological replicates). ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.0005; ∗∗∗∗p <0.0001. CAFs, cancer-associated fibroblasts; CCA, cholangiocarcinoma; CDDP, cisplatin; ER, early recurrence; GEM, gemcitabine; LR, late recurrence; TAZ, transcriptional coactivator with PDZ-binding motif; YAP, yes-associated protein.

Fig. 5

Immune cells-on-chip. (A) Workflow of T cell migration assay on-chip. (B) MFI for stimulated and unstimulated T cells migrated in the hydrogel. Two-way ANOVA (mean ± SEM; n = 3 biological replicates). (C) Left, 3D surface rendering of z-stack using IMARIS. Scale bar = 15 μm. Right, representative confocal images of the migrating T cells in the tumour compartment. Scale bar = 20 μm. (D) 3D surface rendering of the device using IMARIS. Scale bar = 25 μm. (E) Left, representative confocal images of T cell migration with the medium supplemented with cytokines (CXCL9; CXCL10; CXCL11), compared with the chip-conditioned medium. Scale bar = 50 μm. Right, MFI of the green channel within the tumour compartment. Mann-Whitney U test (mean ± SEM; n = 5 biological replicates). T cells: green; CCA cells and CAFs: red. ∗∗∗∗p <0.0001. CXCL9, C-X-C motif chemokine ligand 9; CXCL10, C-X-C motif chemokine ligand 10; CXCL11, C-X-C motif chemokine ligand 11; MFI, mean fluorescence intensity.

Fig. 6

T cell migration assay on-chip. (A) T cells migration through the tumour compartment (red) in 3D mono-culture of CCA cells alone and 3D co-culture of CCA cells and CAFs (n = 6 patients). Scale bar = 50 μm. Right, MFI of the green channel within the tumour compartment. Mann-Whitney U test (mean ± SEM; n = 5 biological replicates). (B) 3D surface rendering of the z-stack using IMARIS in the 3D mono-culture (i) and 3D co-culture (ii). T cells: green; CCA cells and CAFs: red. White arrows: interactions between T cells and CCA cells. Scale bar = 10 μm. (C) Gene expression analyses of T cell attractive and immunosuppressive molecules. Mann–Whitney U test (mean ± SEM; n = 5 biological replicates). (D) Left, representative confocal images of low- (COLD; n = 3 patients) and high-infiltrating (HOT; n = 3 patients) CCA patients. Scale bar = 50 μm. Right, representative IHC images (CD3+ cells) of HOT and COLD patients. Scale bar = 100 μm. (E) Gene expression analyses of T cell attractive and immunosuppressive molecules in patients designated HOT and COLD. Two-way ANOVA (mean ± SEM; n = 5 biological replicates). ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.0005; ∗∗∗∗p <0.0001. CAFs, cancer-associated fibroblasts; CCA, cholangiocarcinoma; IHC, immunohistochemistry; MFI, mean fluorescence intensity; TME, tumour microenvironment.