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.

Objective: In cancer research, three dimensional (3D) cell culture models are rapidly gaining importance as they create a pragmatic microenvironment and mimic an in vivo system, which helps to understand cell-cell interactions. AlgiMatrix sponge (Invitrogen Corporation, Carlsbad, CA, USA) is a non toxic and biodegradable material made from alginate, which seems to construct a cell culture model that resembles normal cell characteristics and morphology. The main objective of the current study was to appraise the AlgiMatrix 3D cell culture system as a 3D invitro tumor model and to ascertain the anticancer activity of docetaxel. Methods: Cell titration was done to optimize seeding density (0.05, 0.10, 0.15, 0.25, 0.50, 0.75, 1.0, 1.25, 1.50, 3 & 6 million H460 cells/well using 6-well plate) and to determine the optimal incubation time for the formation of spheroids. On day 4, 9 & 13, the number and average size of the spheroids were determined using inverted phase contrast microscopy and imaging software. Further, optimized cell density (0.15 million cells/well) was used to determine the IC50 & IC90 values of docetaxel. The alamarBlue® assay was performed to determine the number of viable cells in control & treatment wells. Results: Optimized seeding density and the optimal incubation time was found to be 0.15 & 0.25 million H460 cells/well and 14 days respectively. Average size and the total number of the spheroids for 0.15 and 0.25 million H460 seeded cells was determined as shown in the table.     Time PointsAverage size of spheroids (µm)Total Number of spheroids 0.15 million0.25 million0.15 million0.25 million4th day177.88 ± 61.79220.69 ± 61.2748 ± 477 ± 89th day205.35 ± 78.03232.90 ± 82.8790 ± 8189 ± 1513th day232.27 ± 89.07263.94 ± 86.40153 ± 18228 ± 17 Total number of cells obtained by alamarBlue® assay on 14th day was 38.64 ± 4.94 & 42.92 ± 3.29 million cells/well for 0.15 & 0.25 million seeded cells respectively. The IC50 & IC90 concentration of the docetaxel was found to be 193.19 µM & 290.47 µM when ascertained with 0.15 million cells/well. Conclusion: These results strongly support that the AlgiMatrix 3D Cell Culture System may be used as an in-vitro tumor model and to evaluate cytotoxicity of anticancer drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3234.