Induction of hypoxia and necrosis in multicellular tumor spheroids is associated with resistance to chemotherapy treatment

Oncotarget. 2017 Jan 3;8(1):1725-1736. doi: 10.18632/oncotarget.13857.


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.

Keywords: hypoxia; multicellular tumor spheroids; necrosis; three-dimensional culture; tumor model.

MeSH terms

  • Animals
  • Antimetabolites, Antineoplastic / pharmacology*
  • Cell Hypoxia / physiology*
  • Colorectal Neoplasms / drug therapy
  • Colorectal Neoplasms / pathology*
  • Drug Resistance, Neoplasm / physiology*
  • Fluorouracil / pharmacology*
  • Gene Expression Profiling
  • HCT116 Cells
  • HT29 Cells
  • Humans
  • Mice
  • Mice, Inbred NOD
  • Mice, SCID
  • Necrosis / pathology*
  • Oxygen / metabolism
  • Spheroids, Cellular / physiology*
  • Tumor Cells, Cultured
  • Xenograft Model Antitumor Assays


  • Antimetabolites, Antineoplastic
  • Oxygen
  • Fluorouracil