Abstract 2029: Insight of 3D multicellular tumor spheroids for innovative culture models of potential relevance for the screening of anti-CRC compounds

Abstract

Abstract OBJECTIVE - Multicellular tumor spheroid is a well-known method for 3D 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 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 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 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 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 next generation sequencing and drug-responsiveness of tumor cells cultured in 2D and 3D at different spheroid maturation stages is currently being evaluated. Citation Format: Silvio Däster, Nunzia Amatruda, Diego Calabrese, Paul Zajac, Giulio C. Spagnoli, Giandomenica Iezzi, Valentina Mele, Manuele Giuseppe Muraro. Insight of 3D multicellular tumor spheroids for innovative culture models of potential relevance for the screening of anti-CRC compounds. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2029. doi:10.1158/1538-7445.AM2014-2029