Comparative analysis of different three-dimensional culture systems for the establishment of an in vitro bladder cancer model for therapeutic-efficacy assays

Author(s)

Robson Luis Ferraz do Amaral

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2020-07-03

Examining board members

Kamilla Swiech Antonietto; Márcia Regina Cominetti; Silvya Stuchi Maria Engler

• Advisor: Kamilla Swiech Antonietto

Abstract

Different three-dimensional (3D) cell cultures techniques were evaluated in order to establish a bladder cancer model with the ability to recapitulate in vitro the tumor features observed in vivo. Using the bladder cancer cell line RT4, the first part of the project focused on comparing the scaffold-free 3D cell culture systems, employing forced floating (Ultra-low attachment plates, ULA) and hanging drop (HD plates) techniques, and scaffold-dependent systems, employing microcarriers and hydrogels (Matrigel, Alginate and HydroMatrix), to generate RT4 spheroids. These were characterized regarding morphological parameters, cell growth and metabolism. Only 3D cell culture using the forced floating and hanging drop techniques, and Matrigel (50% v/v) and HydroMatrix (0.25% v/v) scaffolds, were able to generate spheroids with suitable parameters (solidity>0.95; sphericity~0.90; rounding between 0.70- 0.90). In the ULA and HD cell cultures, a single spheroid was generated per well, with diameters ranging between 200 and 600 μm. The cell growth and, consequently, the metabolism observed in these spheroids were lower than in 2D cultures. The Matrigel and HydroMatrix cultures, on the other hand, generated several spheroids per well, with diameters that did not exceed 200 μm. The spheroids in Matrigel showed cell growth and metabolism higher than those observed in 2D cultures. RT4 spheroids generated in ULA plates and Matrigel were selected to assess the Doxorubicin (DOX) sensitivity and the expression of commonly overexpressed genes in solid bladder tumors. ULA plates\' spheroids were less sensitive to DOX treatment (IC50 8.62 ± 1.53 μM) than 2D (IC50 2.35 ± 0.39 μM) and Matrigel (IC50 3.31 ± 1.28 μM) cultures. Moreover, it was observed a higher expression of ALDH1A1 and HRAS genes in RT4 spheroids generated in ULA, two of the main genes involved in the mechanisms of resistance and recurrence of bladder tumors. Almost all the analyzed genes (HIF-1α, IFIT5, MDR-1, IL3, ALDH1A1 and HRAS) were more expressed in Matrigel cultures. In the second part of the project, the strategy based on the use of ULA cultures with 5% of Matrigel (v/v) was successfully applied to generate spheroids using primary human bladder tumor cells (BL0293 and BL0808) derived from PDX models (patient-derived xenografts). The sensitivity profiles of the BL0293 and BL0808 spheroids to Cisplatin and Gemcitabine were similar to those obtained previously in the in vivo tests using PDX models BL0293 and BL0808. In general, it was possible to conclude that both 3D cultures generated using the forced floating technique (ULA plates) and Matrigel scaffold were able to recapitulate in vitro some characteristics of the solid tumor in vivo and can be considered as promising 3D models of bladder cancer. The present work shows an unprecedented comparative analysis between the bladder tumor spheroids generated with different 3D cell culture techniques regarding the morphology and the ability to recap some features of the tumor in vivo. (AU)

• FAPESP's process: 15/05102-1 - Comparative analysis of different three-dimensional culture systems to establish an in vitro bladder cancer model for toxicological assays
• Grantee: Robson Luis Ferraz do Amaral
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)