Abstract 3834: Establishment and characterization of 3D multicellular tumor spheroids as preclinical in vitro models of human colorectal cancer.

Abstract

Abstract Objective: Multicellular tumor spheroid is a well-known method for three-dimensional in-vitro culture that better represent in-vivo condition. We exploited this system to culture human established colorectal cell lines to investigate how the presence or absence of hypoxic and/or necrotic area, due to oxygen and nutrient gradients, can influence the gene expression profile and the responsiveness to drugs compared to 2D culture and in vivo xenografted cells. Methods: HT29 cell line was cultured as MCTS by hanging drop method. At size-defined time point spheroids were collected and analyzed for the presence of hypoxic and necrotic areas. The expression of a panel of genes related to tumor progression, metastatic behavior, and drug resistance was evaluated by real time PCR on MCTS at different growth stages in comparison to conventional 2D cultures and xenografts generated upon injection of HT29 cells in immunodeficient mice. Results: Based on the absence or presence of hypoxic and/or necrotic core, we identified three different MCTS growth stages: stage 1 (MCTS diameter size <200μm) defined by the absence of hypoxia/necrosis, stage 2 (300-350μm) characterized by the presence of hypoxic, but not apoptotic or necrotic areas, and stage 3 (>500μm) identified by the presence of both hypoxic and necrotic areas. The expression of a panel of genes, including KRAS, p53, and putative cancer stem cell markers CD133, CD44, CD24 was found to be upregulated in stage 1 MCTS, as compared to 2D cultures. Most interestingly a further upregulation of these genes was detected in stage 2 and especially in stage 3. Importantly expression levels in stage 3 were the most similar to those detected in xenografts. Conclusion: Our results showed that culture of established CRC cell lines offer the opportunity to investigate in standardized condition different MCTS stages possibly proving an improved model of in-vivo tumor growth. The drug-responsiveness of tumor cells cultured in 2D and 3D at different spheroid maturation stages is currently being assessed. Citation Format: Silvio Daester, Nunzia Amatruda, Diego Calabrese, Paul Zajac, Giulio Cesare Spagnoli, Giandomenica Iezzi, Valentina Mele, Manuele Giuseppe Muraro. Establishment and characterization of 3D multicellular tumor spheroids as preclinical in vitro models of human colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3834. doi:10.1158/1538-7445.AM2013-3834