Introduction. Cell-based assays using three-dimensional (3D) cell cultures may reflect the antitumor activity of compounds more accurately, since these models reproduce the tumor microenvironment better. Methods. Here, we report a comparative analysis of cell behavior in the two most widely employed methods for 3D spheroid culture, forced floating (Ultra-low Attachment, ULA, plates) and hanging drop (HD) methods, using the RT4 human bladder cancer cell line as a model. The morphology parameters and growth/metabolism of the spheroids generated were first characterized, using four different cell-seeding concentrations (0.5, 1.25, 2.5 and 3.75 x 104 cells/mL), and then, subjected to drug resistance evaluation. Results. Both methods generated spheroids with a smooth surface and round shape in a spheroidization time of about 48 hours, regardless of the cell-seeding concentration used. Reduced cell growth and metabolism was observed in 3D cultures compared to two-dimensional (2D) cultures. The optimal range of spheroid diameter (300-500 μm) was obtained using cultures initiated with 0.5 and 1.25 x 104 cells/mL for the ULA method and 2.5 and 3.75 x 104 cells/mL for the HD method. RT4 cells cultured under 3D conditions also exhibited a higher resistance to doxorubicin (IC50 of 1.00 μg/mL and 0.83 μg/mL for the ULA and HD methods, respectively) compared to 2D cultures (IC50 ranging from to 0.39-0.43). Conclusions. Comparing the results, we concluded that the forced floating method using ULA plates was considered more suitable and straightforward to generate RT4 spheroids for drug screening/cytotoxicity assays. The results presented here also contribute to the improvement in the standardization of the 3D cultures required for widespread application.

Patient-derived xenograft (PDX) models allow for personalized drug selection and the identification of drug resistance mechanisms in cancer cells. However, PDX models present technical disadvantages, such as long engraftment time, low success rate, and high maintenance cost. On the other hand, tumor spheroids are emerging as an in vitro alternative model that can maintain the phenotype of cancer cells long enough to perform all assays and predict a patient’s outcome. The present work aimed to describe a simple, reproducible, and low-cost 3D in vitro culture method to generate bladder tumor spheroids using human cells from PDX mice. Cancer cells from PDX BL0293 and BL0808 models, previously established from advanced bladder cancer, were cultured in 96-well round-bottom ultra-low attachment (ULA) plates with 5% Matrigel and generated regular and round-shaped spheroids (roundness > 0.8) with a diameter larger than 400 μm and a hypoxic core (a feature related to drug resistance in solid tumors). The responses of the tumor spheroids to the antineoplastic drugs cisplatin, gemcitabine, and their combination were similar to tumor responses in in vivo studies with PDX BL0293 and BL0808 mice. Therefore, the in vitro 3D model using PDX tumor spheroids appears as a valuable tool that may predict the outcome of in vivo drug-screening assays and represents a low-cost strategy for such purpose.