Abstract
BackgroundThree-dimensional (3D) in-vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in-vivo conditions. Taking the advantages of 3D culture, we have developed the in-vitro tumor model for anticancer drug screening.MethodsCancer cells grown in 6 and 96 well AlgiMatrix™ scaffolds resulted in the formation of multicellular spheroids in the size range of 100–300 µm. Spheroids were grown in two weeks in cultures without compromising the growth characteristics. Different marketed anticancer drugs were screened by incubating them for 24 h at 7, 9 and 11 days in 3D cultures and cytotoxicity was measured by AlamarBlue® assay. Effectiveness of anticancer drug treatments were measured based on spheroid number and size distribution. Evaluation of apoptotic and anti-apoptotic markers was done by immunohistochemistry and RT-PCR. The 3D results were compared with the conventional 2D monolayer cultures. Cellular uptake studies for drug (Doxorubicin) and nanoparticle (NLC) were done using spheroids.ResultsIC50 values for anticancer drugs were significantly higher in AlgiMatrix™ systems compared to 2D culture models. The cleaved caspase-3 expression was significantly decreased (2.09 and 2.47 folds respectively for 5-Fluorouracil and Camptothecin) in H460 spheroid cultures compared to 2D culture system. The cytotoxicity, spheroid size distribution, immunohistochemistry, RT-PCR and nanoparticle penetration data suggested that in vitro tumor models show higher resistance to anticancer drugs and supporting the fact that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro.ConclusionThe results from our studies are useful to develop a high throughput in vitro tumor model to study the effect of various anticancer agents and various molecular pathways affected by the anticancer drugs and formulations.
Highlights
Most in-vitro cell cultures are grown in two dimensional (2D) environments
The porous nature of this system provides the required space for the cells to grow them as in vitro lung tumor models and other organ types based on the types of cells incorporated
Cell titrations for H460, A549, H1650 and H1650 human non small cell lung cancer (NSCLC) cell lines were done by using different cell concentrations (0.05, 0.10, 0.15, 0.25, 0.50, 0.75, 1.0, 1.25, 1.50, 3 & 6 million cells/well in a 6-well plate) to determine the optimal seeding density and time required for the formation of spheroids
Summary
Most in-vitro cell cultures are grown in two dimensional (2D) environments. Most cell cultures are grown in 2D environments, they do not accurately recapitulate the structure, function, or physiology of living tissues [1,5]. Cancer researchers typically rely on 2D in-vitro studies and small animal models to study the complex mechanisms of tumor angiogenesis, invasion, and metastasis [6]. Animal models are accurate representative of tumor environment, they are considerably less amenable to large-scale screening. Three-dimensional (3D) in-vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in-vivo conditions. Taking the advantages of 3D culture, we have developed the invitro tumor model for anticancer drug screening
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