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A novel in vitro three-dimensional retinoblastoma model for evaluating chemotherapeutic drugs.

Mitra M, Mohanty C, Harilal A, Maheswari UK, Sahoo SK, Krishnakumar S - Mol. Vis. (2012)

Bottom Line: The antiproliferative effect of the drugs in the 3-D model was significantly lower than in the 2-D suspension, which was evident from the 4.5 to 21.8 fold differences in their IC(50) values.The collagen content of the cells grown in the 3-D model was 2.3 fold greater than that of the cells grown in the 2-D model, suggesting greater synthesis of the extracellular matrix in the 3-D model as the extracellular matrix acted as a barrier to drug diffusion.The microarray and miRNA analysis showed changes in several genes and miRNA expression in cells grown in the 3-D model, which could also influence the environment and drug effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu, India.

ABSTRACT

Purpose: Novel strategies are being applied for creating better in vitro models that simulate in vivo conditions for testing the efficacy of anticancer drugs. In the present study we developed surface-engineered, large and porous, biodegradable, polymeric microparticles as a scaffold for three dimensional (3-D) growth of a Y79 retinoblastoma (RB) cell line. We evaluated the effect of three anticancer drugs in naïve and nanoparticle-loaded forms on a 3-D versus a two-dimensional (2-D) model. We also studied the influence of microparticles on extracellular matrix (ECM) synthesis and whole genome miRNA-gene expression profiling to identify 3D-responsive genes that are implicated in oncogenesis in RB cells.

Methods: Poly(D,L)-lactide-co-glycolide (PLGA) microparticles were prepared by the solvent evaporation method. RB cell line Y79 was grown alone or with PLGA-gelatin microparticles. Antiproliferative activity, drug diffusion, and cellular uptake were studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole (MTT) assay, fluorescent microscope, and flow cytometry. Extra cellular matrix (ECM) synthesis was observed by collagenase assay and whole genome miRNA-microarray profiling by using an Agilent chip.

Results: With optimized composition of microparticles and cell culture conditions, an eightfold increase from the seeding density was achieved in 5 days of culture. The antiproliferative effect of the drugs in the 3-D model was significantly lower than in the 2-D suspension, which was evident from the 4.5 to 21.8 fold differences in their IC(50) values. Using doxorubicin, the flow cytometry data demonstrated a 4.4 fold lower drug accumulation in the cells grown in the 3-D model at 4 h. The collagen content of the cells grown in the 3-D model was 2.3 fold greater than that of the cells grown in the 2-D model, suggesting greater synthesis of the extracellular matrix in the 3-D model as the extracellular matrix acted as a barrier to drug diffusion. The microarray and miRNA analysis showed changes in several genes and miRNA expression in cells grown in the 3-D model, which could also influence the environment and drug effects.

Conclusions: Our 3-D retinoblastoma model could be used in developing effective drugs based on a better understanding of the role of chemical, biologic, and physical parameters in the process of drug diffusion through the tumor mass, drug retention, and therapeutic outcome.

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Related in: MedlinePlus

This figure shows the uptake of doxorubicin drug by Y79 cells either or not co-cultured with microparticles using flow cytometry. A: The flow cytometry histogram shows decreased uptake of drug by Y79 cells co-cultured with microparticles (3-D) when compared to Y79 cells cultured without microparticles at 4 h. B: Flow cytometry histogram shows decreased uptake of a drug by Y79 cells co-cultured with microparticles (3-D) when compared to Y79 cells cultured without microparticles at 24 h. The x-axis represent fluorescein isothiocyanate (FITC) intensity and the y-axis represents cell counts.
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f8: This figure shows the uptake of doxorubicin drug by Y79 cells either or not co-cultured with microparticles using flow cytometry. A: The flow cytometry histogram shows decreased uptake of drug by Y79 cells co-cultured with microparticles (3-D) when compared to Y79 cells cultured without microparticles at 4 h. B: Flow cytometry histogram shows decreased uptake of a drug by Y79 cells co-cultured with microparticles (3-D) when compared to Y79 cells cultured without microparticles at 24 h. The x-axis represent fluorescein isothiocyanate (FITC) intensity and the y-axis represents cell counts.

Mentions: After treatment with doxorubicin (2,500 ng/ml), significantly fewer (p<0.05) cells with PLGA–gelatin microparticles exhibited drug uptake compared to cells grown without microparticles. The difference in fluorescence intensity was 4.4 fold at 4 h (Figure 8A), which increased to 15.9 fold at 24 h (Figure 8B).


A novel in vitro three-dimensional retinoblastoma model for evaluating chemotherapeutic drugs.

Mitra M, Mohanty C, Harilal A, Maheswari UK, Sahoo SK, Krishnakumar S - Mol. Vis. (2012)

This figure shows the uptake of doxorubicin drug by Y79 cells either or not co-cultured with microparticles using flow cytometry. A: The flow cytometry histogram shows decreased uptake of drug by Y79 cells co-cultured with microparticles (3-D) when compared to Y79 cells cultured without microparticles at 4 h. B: Flow cytometry histogram shows decreased uptake of a drug by Y79 cells co-cultured with microparticles (3-D) when compared to Y79 cells cultured without microparticles at 24 h. The x-axis represent fluorescein isothiocyanate (FITC) intensity and the y-axis represents cell counts.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369889&req=5

f8: This figure shows the uptake of doxorubicin drug by Y79 cells either or not co-cultured with microparticles using flow cytometry. A: The flow cytometry histogram shows decreased uptake of drug by Y79 cells co-cultured with microparticles (3-D) when compared to Y79 cells cultured without microparticles at 4 h. B: Flow cytometry histogram shows decreased uptake of a drug by Y79 cells co-cultured with microparticles (3-D) when compared to Y79 cells cultured without microparticles at 24 h. The x-axis represent fluorescein isothiocyanate (FITC) intensity and the y-axis represents cell counts.
Mentions: After treatment with doxorubicin (2,500 ng/ml), significantly fewer (p<0.05) cells with PLGA–gelatin microparticles exhibited drug uptake compared to cells grown without microparticles. The difference in fluorescence intensity was 4.4 fold at 4 h (Figure 8A), which increased to 15.9 fold at 24 h (Figure 8B).

Bottom Line: The antiproliferative effect of the drugs in the 3-D model was significantly lower than in the 2-D suspension, which was evident from the 4.5 to 21.8 fold differences in their IC(50) values.The collagen content of the cells grown in the 3-D model was 2.3 fold greater than that of the cells grown in the 2-D model, suggesting greater synthesis of the extracellular matrix in the 3-D model as the extracellular matrix acted as a barrier to drug diffusion.The microarray and miRNA analysis showed changes in several genes and miRNA expression in cells grown in the 3-D model, which could also influence the environment and drug effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu, India.

ABSTRACT

Purpose: Novel strategies are being applied for creating better in vitro models that simulate in vivo conditions for testing the efficacy of anticancer drugs. In the present study we developed surface-engineered, large and porous, biodegradable, polymeric microparticles as a scaffold for three dimensional (3-D) growth of a Y79 retinoblastoma (RB) cell line. We evaluated the effect of three anticancer drugs in naïve and nanoparticle-loaded forms on a 3-D versus a two-dimensional (2-D) model. We also studied the influence of microparticles on extracellular matrix (ECM) synthesis and whole genome miRNA-gene expression profiling to identify 3D-responsive genes that are implicated in oncogenesis in RB cells.

Methods: Poly(D,L)-lactide-co-glycolide (PLGA) microparticles were prepared by the solvent evaporation method. RB cell line Y79 was grown alone or with PLGA-gelatin microparticles. Antiproliferative activity, drug diffusion, and cellular uptake were studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole (MTT) assay, fluorescent microscope, and flow cytometry. Extra cellular matrix (ECM) synthesis was observed by collagenase assay and whole genome miRNA-microarray profiling by using an Agilent chip.

Results: With optimized composition of microparticles and cell culture conditions, an eightfold increase from the seeding density was achieved in 5 days of culture. The antiproliferative effect of the drugs in the 3-D model was significantly lower than in the 2-D suspension, which was evident from the 4.5 to 21.8 fold differences in their IC(50) values. Using doxorubicin, the flow cytometry data demonstrated a 4.4 fold lower drug accumulation in the cells grown in the 3-D model at 4 h. The collagen content of the cells grown in the 3-D model was 2.3 fold greater than that of the cells grown in the 2-D model, suggesting greater synthesis of the extracellular matrix in the 3-D model as the extracellular matrix acted as a barrier to drug diffusion. The microarray and miRNA analysis showed changes in several genes and miRNA expression in cells grown in the 3-D model, which could also influence the environment and drug effects.

Conclusions: Our 3-D retinoblastoma model could be used in developing effective drugs based on a better understanding of the role of chemical, biologic, and physical parameters in the process of drug diffusion through the tumor mass, drug retention, and therapeutic outcome.

Show MeSH
Related in: MedlinePlus