Program

PO2-10-31

Comparative analysis of a two-dimensional and three dimensional model of BT-20 triple-negative breast carcinoma cells in response to an antiproliferative agent

[Speaker] Jie Wang:1
[Co-author] Duncan Allan Duncan:1, Iman Van Den Bout:2, Werner Cordier:1
1:Pharmacology, University of Pretoria, South Africa, 2:Physiology, University of Pretoria, South Africa

Background

Triple-negative breast cancer lacks oestrogen, progesterone and human epidermal growth factor-receptor-2 receptors, thus no specific target is available for antineoplastic treatment. Within solid tumors, blood supply creates an oxygen and nutrient gradient.Solid tumors use a three-dimensional (3D) structure to fit for this gradient. Traditional two-dimensional (2D) culturing techniques fail to replicate this environment. The aim of the study is to comparatively assess the effect of doxorubicin on BT-20 triple-negative breast carcinoma cells in a 2D and 3D model of cell growth.

Methods

BT-20 spheroids (40 000 cells/well) were grown using liquid overlay. Volume was assessed using microscopy, while viability was observed by live-dead staining. Metabolism was determined using resazurin. Cytotoxicity of doxorubicin was determined in monolayers by sulforhodamine B staining after 72 h. Monolayer cultures and spheroids (day 4) were exposed to the IC25, IC50 and IC75 for 72 h, and cellular kinetics and proteomic markers (p53) compared through flow cytometry and western blot.

Results

Spheroid decreased in size over 10 days. Central necrosis appaeared at day 4 and didn't be detected at day 7 and 10. Spheroid metabolism was not detected after day 4, however, resorufin was observed in spheroid. Doxorubicin displayed an IC25, IC50 and IC75 of 1.4, 3.6 and 11.75 μM. Sub G1 phase raised in treated monolayer cells and spheroid at IC75 and G1 phase in the 2D and 3D groups showed reduction during analysis excluded sub G1. Volume of p53 protein increased after treatment compared with negative control, except monolayer cell at IC50, IC75 and spheroid at IC75, in which no loading control were detected.

Conclusion

Spheroids displayed structural integrity and viability during the culturing period. Based on the reduced volume, compaction is taking place. Reszaurin is not applicable to see viability in spheroids, as resorufin couldn't be released. After 10 days spheroid may begin to disintegrate. Cell cycle would be blocked after treatment for DNA repair protein to rectify damage, which can be explain by increase of p53. Protein may appear denaturation at high concentration of drug. Spheroids presented with a higher resistance towards doxorubicin, where reductions were only evident at the IC75.

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