Recellularized colorectal patient-derived scaffold as in vitro pre-clinical 3D model for drug screening

Purpose: Colorectal cancer (CRC), the third most common cancer diagnosed in both men and women shows a highly ineffective therapeutic management. In contrast, CRC is a rare pediatric tumor, representing only 1% of all pediatric malignancies, with an incidence of approximately 1 per million. In this context, an urgent needing not yet addressed is the random assignment to adjuvant chemotherapy of high-risk stage II and stage III colon cancer patients, both young and adults, without any predictive factor of efficacy. Secondly, in the field of drug discovery the critical step is the preclinical evaluation of drug cytotoxicity, efficacy, and efficiency. We purpose to develop a patient-derived 3D preclinical model useful for drug evaluation that can mimic in vitro the patient's disease. Methods: Surgically resected healthy colon mucosa and matched CRC were decellularized by a detergent-enzymatic treatment (DET). DET scaffolds were recellularized with HT29 cells. Qualitative and quantitative characterization of matched recellularized samples were evaluated through histology, immunofluorescences and DNA amount quantification. Chemosensitivity test was performed using increasing concentration of 5-Fluorouracil (5-FU), range 0.1 µM to 100 µM. In vivo studies were carried out using the zebrafish (Danio rerio) animal model. For cancer xenograft assays, HT29 cells were injected into the duct of Couvier and subsequently incubated in 96-well plates with different concentrations of 5-FU. Drug absorption and perfusion along fresh and DET tumor scaffolds were evaluated qualitative using autofluorescence of doxorubicin (doxo, 594nm) and quantitative calculated by Darcy’s law. Buffy coat-derived monocytes were cultured with DET scaffolds and macrophages lineage markers were evaluated with flow cytometry. Results: Decellularization protocol allowed the preservation of original structure and ultrastructure (SEM analysis). Five days after recellularization with HT29 cell line, the 3D CRC model exhibited reduced sensitivity to 5-FU treatments compared with conventional 2D culture. Calculated IC50 resulted in 11.5 µM and 1.3 µM of 5-FU, respectively. In the zebrafish transplantation model, HT29 extravasation was detected after 4 days post injection. Moreover, we obtained a 5-FU IC50 comparable with that observed in the 3D CRC model. Using confocal microscopy, we demonstrated that doxorubicin diffuses through the volume of 3D CRC model and co-localize with the cell nuclei which repopulate the 3D CRC scaffold. Finally, we observed that monocytes exposed to tumor decellularized ECM differentiated towards a pro-tumoral anti-inflammatory macrophage-like profile. Conclusion: 3D CRC model could be preclinical reliable tool to bridge the gap between in vitro, in vivo and ex vivo drug testing assays. The 3D CRC model, translated in the pediatric setting, could help clinicians and oncologists to identify the most suitable treatment for the patient.