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. 2013 Apr 23;8(4):e62630.
doi: 10.1371/journal.pone.0062630. Print 2013.

Multicellular tumor spheroids for evaluation of cytotoxicity and tumor growth inhibitory effects of nanomedicines in vitro: a comparison of docetaxel-loaded block copolymer micelles and Taxotere®

Andrew S Mikhail  1 , Sina Eetezadi, Christine Allen
Affiliations

Multicellular tumor spheroids for evaluation of cytotoxicity and tumor growth inhibitory effects of nanomedicines in vitro: a comparison of docetaxel-loaded block copolymer micelles and Taxotere®

Andrew S Mikhail et al. PLoS One. .

Abstract

While 3-D tissue models have received increasing attention over the past several decades in the development of traditional anti-cancer therapies, their potential application for the evaluation of advanced drug delivery systems such as nanomedicines has been largely overlooked. In particular, new insight into drug resistance associated with the 3-D tumor microenvironment has called into question the validity of 2-D models for prediction of in vivo anti-tumor activity. In this work, a series of complementary assays was established for evaluating the in vitro efficacy of docetaxel (DTX) -loaded block copolymer micelles (BCM+DTX) and Taxotere® in 3-D multicellular tumor spheroid (MCTS) cultures. Spheroids were found to be significantly more resistant to treatment than monolayer cultures in a cell line dependent manner. Limitations in treatment efficacy were attributed to mechanisms of resistance associated with properties of the spheroid microenvironment. DTX-loaded micelles demonstrated greater therapeutic effect in both monolayer and spheroid cultures in comparison to Taxotere®. Overall, this work demonstrates the use of spheroids as a viable platform for the evaluation of nanomedicines in conditions which more closely reflect the in vivo tumor microenvironment relative to traditional monolayer cultures. By adaptation of traditional cell-based assays, spheroids have the potential to serve as intermediaries between traditional in vitro and in vivo models for high-throughput assessment of therapeutic candidates.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. 3-D cultures as intermediary between 2-D cultures and animal models.
Intermediate in complexity, 3-D cultures permit the systematic, high-throughput assessment of formulation properties in a controlled environment that approximates important properties of in vivo tumors in the absence of complex parameters which may confound data interpretation.
Figure 3
Figure 3. Characterization of micelle morphology and size.
a) Transmission electron micrograph (Scale bar in represents 100 nm) and b) size distribution of BCM+DTX as determined by dynamic light scattering at 37°C.
Figure 2
Figure 2. In vitro assays used in this study for analysis of formulation efficacy in spheroids.
Figure 4
Figure 4. Drug release.
Release of docetaxel from dialysis bags containing BCM+DTX, Taxotere®, and DTX in DMSO, n = 3.
Figure 5
Figure 5. Spheroid packing density and growth.
a) Cells per HeLa and HT29 spheroid of given volume, n = 12. b) Growth of HeLa and HT29 spheroids, n = 6. Data was fit using the Gompertz equation for tumor growth. The dashed lines indicate spheroid properties used in the studies.
Figure 6
Figure 6. Cytotoxicity of Taxotere® and BCM+DTX in spheroid and monolayer cultures.
Viability of a) HeLa and b) HT29 cells cultured as monolayers and spheroids as measured using the APH assay. Data is expressed as the percent viability relative to untreated controls and fit to the Hill equation. c) Cytotoxicity of blank PEG-b-PCL micelles as a function of copolymer concentration. Each plot represents the mean of three independent experiments ± SD (n = 3).
Figure 7
Figure 7. Inhibition of spheroid growth.
a) Sequential images of the same HeLa and HT29 spheroids following treatment with BCM+DTX at a concentration of 20 ng/mL. Bars represent 100 µm. Growth inhibition of HeLa (b,c) and HT29 (d,e) MCTS by BCM+DTX and Taxotere® at concentrations of 2, 20 and 200 ng/mL. Cells were re-treated after two weeks (arrow). Box represents expanded region of plots b) and d). Data is expressed as the mean volume of six spheroids (n = 6) ± SD. “*” represents a significant difference between BCM 20 and TAX 20, p<0.05.
Figure 8
Figure 8. Histological assessment of spheroid microenvironment.
HeLa (a–c) and HT29 (d–f) MCTS cross-sections stained with H&E (a, d), Ki67 proliferation marker (b, e) and EF5 (c, f), a marker of hypoxia. Scale bars represent 100 µm. g) Properties of the spheroid microenvironment and their spatial distribution. “++”, “+”, and “–”, indicate high, intermediate and low levels of the corresponding feature, respectively.
Figure 9
Figure 9. Spatial distribution of proliferating cells in spheroids.
Ki67 positive signal distribution relative to radial position in a) HeLa and b) HT29 MCTS as a percent of total positive stain, n = 6.
Figure 10
Figure 10. Clonogenic potential of cells following treatment.
Clonogenic survival of HeLa and HT29 cells following 24 h treatment with 20 ng/mL of BCM+DTX or Taxotere® as a) monolayers, b) disaggregated spheroids and c) intact spheroids.
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Grants and funding

A. Mikhail is the recipient of post-graduate scholarships from NSERC and the Government of Ontario. S. Eetezadi is funded by the NSERC CREATE Biointerfaces training program and holds an Ontario Trillium scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.