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. 2016 Jul 19;7(29):45745-45756.
doi: 10.18632/oncotarget.9935.

The relevance of using 3D cell cultures, in addition to 2D monolayer cultures, when evaluating breast cancer drug sensitivity and resistance

Susan Breslin  1 , Lorraine O'Driscoll  1
Affiliations

"VSports" The relevance of using 3D cell cultures, in addition to 2D monolayer cultures, when evaluating breast cancer drug sensitivity and resistance

Susan Breslin et al. Oncotarget. .

Abstract

Solid tumours naturally grow in 3D wherein the spatial arrangement of cells affects how they interact with each other. This suggests that 3D cell culture may mimic the natural in vivo setting better than traditional monolayer (2D) cell culture, where cells are grown attached to plastic. Here, using HER2-positive breast cancer cell lines as models (BT474, HCC1954, EFM192A), the effects of culturing cells in 3D using the poly-HEMA method compared to 2D cultures were assessed in terms of cellular viability, response/resistance to anti-cancer drugs, protein expression and enzyme activity. Scanning electron microscopy showed the morphology of cells in 3D to be substantially different to those cultured in 2D. Cell viability in 3D cells was substantially lower than that of cells in 2D cultures, while 3D cultures were more resistant to the effects of HER-targeted (neratinib) and classical chemotherapy (docetaxel) drugs. Expression of proteins involved in cell survival, transporters associated with drug resistance and drug targets were increased in 3D cultures. Finally, activity of drug metabolising enzyme CYP3A4 was substantially increased in 3D compared to 2D cultures. Together this data indicates that the biological information represented by 3D and 2D cell cultures is substantially different i VSports手机版. e. 3D cell cultures demonstrate higher innate resistance to anti-cancer drugs compared to 2D cultures, which may be facilitated by the altered receptor proteins, drug transporters and metabolising enzyme activity. This highlights the importance of considering 3D in addition to 2D culture methods in pre-clinical studies of both newer targeted and more traditional anti-cancer drugs. .

Keywords: 3D cell culture; breast cancer; drug resistance mechanisms; monolayer culture; targeted drugs. V体育安卓版.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Different morphology of each cell line in 2D and 3D culture
SEM images show how the morphology of cells differs substantially when grown in 2D compared to 3D cultured cells. For all lines, cells grew attached to the cover-slip when using standard culture methods. Both BT474 and HCC1954 cells form a tightly packed spheroid when grown in 3D, while EFM192A cells form a less organised 3D structure when cultured under the same conditions. Scale bars are shown on all images.
Figure 2
Figure 2. 2D compared to 3D cell viability
Starting with the same cell numbers and time of culture, cells cultured in 3D have significantly decreased viability compared to those cultured as 2D monolayers. Graphs represent triplicate biological repeats and are displayed as mean ± SEM, where ***p < 0.001.
Figure 3
Figure 3. 2D v 3D cell sensitivity to neratinib and docetaxel
3D cells are less sensitive to the effects of A. neratinib (Ner) and B. docetaxel compared to 2D cultured cells. Graphs represent triplicate biological repeats and are displayed as mean ± SEM, where *p < 0.05; **p < 0.01; ***p < 0.001. NT = not treated with drug.
Figure 4
Figure 4. Immunoblots of pAkt, Akt, pErk and Erk expression in 2D and 3D cultures
A. Akt, pAkt and Erk level were all significantly higher in 3D cultures of each of the cell lines compared to their 2D counterparts, while pErk levels were significantly reduced. B. Densitometry of respective immunoblots. Graphs represent triplicate biological repeats and are displayed as mean ± SEM, where *p < 0.05; **p < 0.01.
Figure 5
Figure 5. Immunoblots of EGFR family members in 2D and 3D cultures
A. EGFR, pEGFR, HER2, pHER2, HER3 and HER4 were all increased in 3D cultured cells compared to their 2D counterparts, with the exception that EGFR/pEGFR were undetected in EFM192A cells grown in either format. B. Densitometry of respective immunoblots. Graphs represent triplicate biological repeats and are displayed as mean ± SEM, where *p < 0.05; **p < 0.01; **p < 0.001.
Figure 6
Figure 6. Immunoblots of drug transporter expression in 3D compared to 2D cultures
A. P-glycoprotein (PGP) level were increased in 3D cultures of all three cell lines, but breast cancer resistance protein (BCRP) levels were not consistently changed between 3D and 2D. B. Densitometry of respective immunoblots. Graphs represent triplicate biological repeats and are displayed as mean ± SEM, where *p < 0.05; **p < 0.01.
Figure 7
Figure 7. CYP3A4 activity and protein in 3D compared to 2D cells
A. Cells grown in 3D have significantly higher CYP3A4 activity compared to 2D cultured cells. B. CYP3A4 protein quantities are increased in 3D cells compared to cells grown in 2D. Graphs represent triplicate biological repeats and are displayed as mean ± SEM, where *p<0.05, **p < 0.01***p < 0.001.
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