Limits...
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.

Show MeSH

Related in: MedlinePlus

This figure shows the anti-proliferative effect of drug loaded nanoparticles on Y79 cells cultures in Two-dimensional (2-D) or three dimensional (3-D) patterns. A: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to native carboplatin when compared to Y79 cells cultured without microparticles (2-D). B: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.0001) to Carboplatin loaded nanoparticles when compared to Y79 cells cultured without microparticles (2-D). C: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to native etoposide when compared to Y79 cells cultured without microparticles (2-D). D: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to etoposide-loaded Np when compared to Y79 cells cultured without microparticles (2-D). E: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to native doxorubicin when compared to Y79 cells cultured without microparticles (2-D). F: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to doxorubicin loaded nanoparticles when compared to Y79 cells cultured without microparticles (2-D). G: Fresh retinoblastoma tumor cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to native carboplatin when compared to Y79 cells cultured without microparticles (2-D). H: Fresh retinoblastoma tumor cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.0001) to native etoposide when compared to Y79 cells cultured without microparticles (2-D). I: Fresh retinoblastoma tumor cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to native doxorubicin when compared to Y79 cells cultured without microparticles (2-D). Error bars represent standard deviation obtained from triplicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3369889&req=5

f7: This figure shows the anti-proliferative effect of drug loaded nanoparticles on Y79 cells cultures in Two-dimensional (2-D) or three dimensional (3-D) patterns. A: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to native carboplatin when compared to Y79 cells cultured without microparticles (2-D). B: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.0001) to Carboplatin loaded nanoparticles when compared to Y79 cells cultured without microparticles (2-D). C: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to native etoposide when compared to Y79 cells cultured without microparticles (2-D). D: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to etoposide-loaded Np when compared to Y79 cells cultured without microparticles (2-D). E: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to native doxorubicin when compared to Y79 cells cultured without microparticles (2-D). F: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to doxorubicin loaded nanoparticles when compared to Y79 cells cultured without microparticles (2-D). G: Fresh retinoblastoma tumor cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to native carboplatin when compared to Y79 cells cultured without microparticles (2-D). H: Fresh retinoblastoma tumor cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.0001) to native etoposide when compared to Y79 cells cultured without microparticles (2-D). I: Fresh retinoblastoma tumor cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to native doxorubicin when compared to Y79 cells cultured without microparticles (2-D). Error bars represent standard deviation obtained from triplicates.

Mentions: Cytotoxicity of model anticancer drugs was determined in Y79/RB cells grown with or without PLGA–gelatin microparticles following 48 h of drug treatment. Cell numbers were similar in both models at the time of treatment, and the inhibition in cell growth was calculated according to the respective untreated controls. IC50 values of drugs and drug-loaded nanoparticles were significantly higher in Y79 cells with PLGA–gelatin microparticles than in cells grown without PLGA–gelatin microparticles (Figure 7). The differences in the IC50 values observed were 4.5 to 21.8 fold depending upon the drug and drug-loaded nanoparticles (Table 2). Similarly, we observed significantly higher IC50 values of native drugs in RB tumor cells with PLGA–gelatin microparticles compared to RB cells grown without PLGA–gelatin microparticles (Figure 7G-I). The differences in the IC50 values of native drugs in RB tumor cells observed were 2.4 to 13.4 fold (Table 2).


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 anti-proliferative effect of drug loaded nanoparticles on Y79 cells cultures in Two-dimensional (2-D) or three dimensional (3-D) patterns. A: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to native carboplatin when compared to Y79 cells cultured without microparticles (2-D). B: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.0001) to Carboplatin loaded nanoparticles when compared to Y79 cells cultured without microparticles (2-D). C: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to native etoposide when compared to Y79 cells cultured without microparticles (2-D). D: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to etoposide-loaded Np when compared to Y79 cells cultured without microparticles (2-D). E: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to native doxorubicin when compared to Y79 cells cultured without microparticles (2-D). F: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to doxorubicin loaded nanoparticles when compared to Y79 cells cultured without microparticles (2-D). G: Fresh retinoblastoma tumor cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to native carboplatin when compared to Y79 cells cultured without microparticles (2-D). H: Fresh retinoblastoma tumor cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.0001) to native etoposide when compared to Y79 cells cultured without microparticles (2-D). I: Fresh retinoblastoma tumor cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to native doxorubicin when compared to Y79 cells cultured without microparticles (2-D). Error bars represent standard deviation obtained from triplicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: This figure shows the anti-proliferative effect of drug loaded nanoparticles on Y79 cells cultures in Two-dimensional (2-D) or three dimensional (3-D) patterns. A: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to native carboplatin when compared to Y79 cells cultured without microparticles (2-D). B: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.0001) to Carboplatin loaded nanoparticles when compared to Y79 cells cultured without microparticles (2-D). C: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to native etoposide when compared to Y79 cells cultured without microparticles (2-D). D: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to etoposide-loaded Np when compared to Y79 cells cultured without microparticles (2-D). E: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to native doxorubicin when compared to Y79 cells cultured without microparticles (2-D). F: Y79 cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to doxorubicin loaded nanoparticles when compared to Y79 cells cultured without microparticles (2-D). G: Fresh retinoblastoma tumor cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to native carboplatin when compared to Y79 cells cultured without microparticles (2-D). H: Fresh retinoblastoma tumor cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.0001) to native etoposide when compared to Y79 cells cultured without microparticles (2-D). I: Fresh retinoblastoma tumor cells co-cultured with microparticles (3-D) shows decreased sensitivity (p<0.001) to native doxorubicin when compared to Y79 cells cultured without microparticles (2-D). Error bars represent standard deviation obtained from triplicates.
Mentions: Cytotoxicity of model anticancer drugs was determined in Y79/RB cells grown with or without PLGA–gelatin microparticles following 48 h of drug treatment. Cell numbers were similar in both models at the time of treatment, and the inhibition in cell growth was calculated according to the respective untreated controls. IC50 values of drugs and drug-loaded nanoparticles were significantly higher in Y79 cells with PLGA–gelatin microparticles than in cells grown without PLGA–gelatin microparticles (Figure 7). The differences in the IC50 values observed were 4.5 to 21.8 fold depending upon the drug and drug-loaded nanoparticles (Table 2). Similarly, we observed significantly higher IC50 values of native drugs in RB tumor cells with PLGA–gelatin microparticles compared to RB cells grown without PLGA–gelatin microparticles (Figure 7G-I). The differences in the IC50 values of native drugs in RB tumor cells observed were 2.4 to 13.4 fold (Table 2).

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