Abstract
Culture of cancerous cells in standard monolayer conditions poorly mirrors growth in three-dimensional architectures typically observed in a wide majority of cancers of different histological origin. Multicellular tumor spheroid (MCTS) culture models were developed to mimic these features. However, in vivo tumor growth is also characterized by the presence of ischemic and necrotic areas generated by oxygenation gradients and differential access to nutrients. Hypoxia and necrosis play key roles in tumor progression and resistance to treatment. To provide in vitro models recapitulating these events in highly controlled and standardized conditions, we have generated colorectal cancer (CRC) cell spheroids of different sizes and analyzed their gene expression profiles and sensitivity to treatment with 5FU, currently used in therapeutic protocols. Here we identify three MCTS stages, corresponding to defined spheroid sizes, characterized by normoxia, hypoxia, and hypoxia plus necrosis, respectively. Importantly, we show that MCTS including both hypoxic and necrotic areas most closely mimic gene expression profiles of in vivo-developing tumors and display the highest resistance to 5FU. Taken together, our data indicate that MCTS may mimic in vitro generation of ischemic and necrotic areas in highly standardized and controlled conditions, thereby qualifying as relevant models for drug screening purposes.
Highlights
Screening of novel anti-cancer agents is usually performed on cells from the US National Cancer Institute (NCI) 60 panel, a collection of established tumor cell lines representing nine distinct human tumor types [1], cultured in two-dimensional (2D) monolayers
Previous studies reported that a hypoxic core begins to be formed in spheroids larger than 200μm and that a necrotic core is detectable in Multicellular tumor spheroid (MCTS) larger than 500μm [26]
3D2-3, but not 3D1 stage MCTS or cells cultured in 2D, displayed expression of hypoxia-inducible factor 1α (HIF-1α) protein, typically detectable in hypoxic areas [32], similar to tumor xenografts generated in vivo upon s.c. injection of the same cells in immunodeficient animals (Figure 2E-2H and Supplementary Figure S2)
Summary
Screening of novel anti-cancer agents is usually performed on cells from the US National Cancer Institute (NCI) 60 panel, a collection of established tumor cell lines representing nine distinct human tumor types [1], cultured in two-dimensional (2D) monolayers. These cells are easy to propagate and amenable to high-throughput studies. Fluctuations of hypoxia and re-oxygenation differentially influence tumor cells and have broad ranging implications for tumor gene expression profiles, progression, stress response, and signal transduction [14,15,16]. Resistance to radiotherapy or www.impactjournals.com/oncotarget chemotherapeutic agents such as cisplatin or doxorubicin, has been suggested to be frequently associated with hypoxia [4, 17,18,19,20,21,22,23]
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