Recellularized Colorectal Cancer Patient-derived Scaffolds as in vitro Pre-clinical 3D Model for Drug Screening

Abstract

Colorectal cancer (CRC) shows highly ineffective therapeutic management. An urgent unmet need is the random assignment to adjuvant chemotherapy of high-risk stage II and stage III CRC patients without any predictive factor of efficacy. In the field of drug discovery, a critical step is the preclinical evaluation of drug cytotoxicity, efficacy, and efficiency. We proposed a patient-derived 3D preclinical model for drug evaluation that could mimic in vitro the patient's disease. Surgically resected CRC tissue and adjacent healthy colon mucosa were decellularized by a detergent-enzymatic treatment. Scaffolds were recellularized with HT29 and HCT116 cells. Qualitative and quantitative characterization of matched recellularized samples were evaluated through histology, immunofluorescences, scanning electron microscopy, and DNA amount quantification. A chemosensitivity test was performed using an increasing concentration of 5-fluorouracil (5FU). In vivo studies were carried out using zebrafish (Danio rerio) animal model. Permeability test and drug absorption were also determined. The decellularization protocol allowed the preservation of the original structure and ultrastructure. Five days after recellularization with HT29 and HCT116 cell lines, the 3D CRC model exhibited reduced sensitivity to 5FU treatments compared with conventional 2D cultures. Calculated the half maximal inhibitory concentration (IC₅₀) for HT29 treated with 5FU resulted in 11.5 µM in 3D and 1.3 µM in 2D, and for HCT116, 9.87 µM in 3D and 1.7 µM in 2D. In xenograft experiments, HT29 extravasation was detected after 4 days post-injection, and we obtained a 5FU IC₅₀ fully comparable to that observed in the 3D CRC model. Using confocal microscopy, we demonstrated that the drug diffused through the repopulated 3D CRC scaffolds and co-localized with the cell nuclei. The bioengineered CRC 3D model could be a reliable preclinical patient-specific platform to bridge the gap between in vitro and in vivo drug testing assays and provide effective cancer treatment.

Keywords: 3D culture model; colorectal cancer; decellularization; drug test; extracellular matrix; response to treatment..

Figure 1

Characterization of matched 3DN and 3DT HT29-recellularized samples. (A) histological characterization of sections stained with hematoxylin and eosin (H&E), collagen IV staining (Col IV), and periodic acid-Shiff (PAS) (scale bar = 200 µm). Scanning electron microscopy (SEM) analysis performed on 3DN and 3DT (Scale bar = 10 µm). (B) DNA amount quantification in fresh samples, after decellularization and 5 days of culture after seeding of HT29 cells, in both 3DN and 3DT. (C) IF staining in 3DT and 3DN and relative quantifications: Ki67, as proliferation marker; E-cadherin, as epithelial marker; vimentin, as mesenchymal marker; laminin to highlight basement membrane; DAPI to counterstain the nuclei (scale bar = 100 µm) (* p-value < 0.05; ** p-value < 0.01).

Figure 2

Effect of 5-fluorouracil (5FU) and FOLFIRI ((leucovorin + 5-fluorouracil (5FU) + irinotecan) treatments on HT29-cells cultured in a 3D model. (A) Comparison between percentages of viable cells (by absorbance fold-change detection) after administration of 5FU at 1-10-100 μM in 2D cultures and in both 3DN and 3DT models. (B) Calculation of 5FU and FOLFIRI 3D IC₅₀ by nonlinear regression. (C) MIB1 immunohistochemistry before and after administration of 3D-calculated IC₅₀ in both 3DN and 3DT; comparison of percentages of MIB1⁺ cells before and after treatment (scale bar = 100 µm). (D) EdU staining as a marker of proliferation and TUNEL staining as a marker of apoptosis, before and after 5FU treatment in 3DN and relative quantification (scale bar = 100 µm). (E) EdU and TUNEL before and after 5FU treatment in 3DT and relative quantification (scale bar = 100 µm) (* p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001).

Figure 3

Effect of 5FU treatment on in vivo zebrafish model. (A) Tg(fli1:EGFP) zebrafish embryo (with green fluorescent vessels) xenotransplantated with Dil marked HT29 cells (red), injected into the duct of Cuvier (white asterisk). (B) The monitoring of zebrafish embryos showed viable cells after 24-48-72 h post-injection. (C) Analysis of Dil⁺ HT29 injected cells in zebrafish embryos, pre-treatment (time 0), after 24 h of treatment with DMSO (control group), IC₅₀ 2D, IC₅₀ 3D, and relative quantifications (scale bar = 100 µm). (D) Analysis of Dil⁺ HT29 injected cells in zebrafish embryos, pre-treatment (time 0), after 48 h of treatment with DMSO (control group), IC₅₀ 2D, IC₅₀ 3D, and relative quantifications (scale bar = 100 µm) (* p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001).

Figure 4

Drug diffusion evaluation. (A) Thickness (µm) of a 3DT. (B) Doxorubicin (doxo; red) diffusion assay in 3DT, repopulated with HT29 ZSgreen positive cells (green). In the enlargement, the same fixed samples were evaluated with immunofluorescence to underline the co-localization between drug (doxo) and nuclei (DAPI). Absence of non-specific binding between doxo and extracellular matrix (ECM) (scale bar = 100 µm). (C) Quantification of co-localized signals. (D) In-house developed permeability device. (E) Experimental results of the filtration process carried out on the tumor colon fresh, 3DT, and decellularized (decell). Open circles refer to the experimental values, and solid black lines to the Equation (2) fitted to experimental data, and the charts report the estimated values of permeability K (mm4/Ns). (F) Quantification of permeability measurement obtained from fresh, 3DT, and decellularized (decell) tumor samples. (* p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001).