A novel in vitro three-dimensional retinoblastoma model for evaluating chemotherapeutic drugs.
OBJECTIVE
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.
Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu, India.
Moutushy
M
Mitra
Chandana
C
Mohanty
Anju
A
Harilal
Uma K
UK
Maheswari
Sanjeeb Kumar
SK
Sahoo
Subramanian
S
Krishnakumar
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001672:Biocompatible Materials / Q000737:chemistry; D016190:Carboplatin / Q000494:pharmacology*; D018929:Cell Culture Techniques / Q000379:methods; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004058:Diffusion; D004317:Doxorubicin / Q000494:pharmacology*; D005047:Etoposide / Q000494:pharmacology*; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D020869:Gene Expression Profiling; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D019344:Lactic Acid / Q000737:chemistry; D035683:MicroRNAs / Q000096:biosynthesis*; D053758:Nanoparticles / Q000737:chemistry; D020411:Oligonucleotide Array Sequence Analysis; D010316:Particle Size; D011100:Polyglycolic Acid / Q000737:chemistry; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D016062:Porosity; D012175:Retinoblastoma / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D023822:Tissue Engineering / Q000379:methods; D054457:Tissue Scaffolds
D000970:Antineoplastic Agents; D001672:Biocompatible Materials; D035683:MicroRNAs; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D005047:Etoposide; D004317:Doxorubicin; D016190:Carboplatin