Standardization of three-dimensional (3D) cell culture protocols by the liquid overlay technique for the evaluation of cytotoxicity in breast cancer cell lines

Name: Mileni Fátima RodriguesType: MSc dissertationPublication date: 31/03/2022Advisor:

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Examining board:

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Isabella dos Santos Guimarães External Examiner *
Leticia Batista Azevedo Rangel Internal Examiner *

Summary: reast cancer is the most common and the leading cause of cancer death among woman overall. This highly heterogeneous tumor has multiples subtypes with a wide range of clinical manifestations and therapies responses. The tumor behavior and their response to treatments are not exclusively determined by their intrinsic characteristics, but are influenced by the tumor microenvironment. The growing interest in developing in vitro cell culture models that mimics the tumor microenvironment led to the extensive use of three-dimensional cell culture. In this context, we proposed to standardize 3D culture protocols, verifying which one is best suited for the evaluation of cytotoxicity in breast cancer cell lines, in terms of applicability and effectiveness. The protocols used were based on the formation of spheroids by the liquid overlay technique, with agarose gel coated plates. We also evaluated the addition of reconstituted basement membrane (Geltrex®) in their formation. For MCF-7, spheroids were reproducibly obtained in 1.0% (w/v) agarose, both in the absence and in the presence of Geltrex® (concentration of 1.5% (v/v)). However, MDA-MB-231 was not able to form spheroids without the addition of the basement membrane, and the concentrations of 1.0% (w/v) agarose and 2.0% (v/v) Geltrex® were the best conditions for generating them. For both cell lines, the moment at which the basement membrane is incorporated into the medium directly interferes with the success in the structure formation: only the addition of Geltrex® 24 hours after plating allowed good results. After cells cultured in monolayer (2D) and 3D were treated with Paclitaxel for 72 hours, it was found that spheroids are less sensitive to treatments, with an estimated IC50 of 7.054x10-6 M (MCF-7 without Geltrex®), 1.751x10-5 M (MCF-7 with Geltrex®) and 5.053x10-6 M (MDA-MB-231 with Geltrex®), against IC50 of 3.962x10-8 M (MCF-7) and 9.502x10-8 M (MDA-MB-231) for 2D cells. These results demonstrate that the three-dimensionality of the model, which ensures greater cell-cell contact and hinders drug diffusion, and the presence of matrix components, are factors that influence the cancer cell response to taxane. Therefore, since MDA-MB-231 formed spheroids only in the presence of Geltrex®, and it mimics the basement membrane in vivo, this protocol was chosen as the most appropriate for cytotoxicity evaluation for MCF-7 and MDA-MB-231.Access to document

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