Differential Angiogenic Potential of 3-Dimension Spheroid of HNSCC Cells in Mouse Xenograft

Int J Mol Sci. 2021 Jul 31;22(15):8245. doi: 10.3390/ijms22158245.


The experimental animal model is still essential in the development of new anticancer drugs. We characterized mouse tumors derived from two-dimensional (2D) monolayer cells or three-dimensional (3D) spheroids to establish an in vivo model with highly standardized conditions. Primary cancer-associated fibroblasts (CAFs) were cultured from head and neck squamous cell carcinoma (HNSCC) tumor tissues and co-injected with monolayer cancer cells or spheroids into the oral mucosa of mice. Mice tumor blood vessels were stained, followed by tissue clearing and 3D Lightsheet fluorescent imaging. We compared the effect of exosomes secreted from 2D or 3D culture conditions on the angiogenesis-related genes in HNSCC cells. Our results showed that both the cells and spheroids co-injected with primary CAFs formed tumors. Interestingly, vasculature was abundantly distributed inside the spheroid-derived but not the monolayer-derived mice tumors. In addition, cisplatin injection more significantly decreased spheroid-derived but not monolayer-derived tumor size in mice. Additionally, exosomes isolated from co-culture media of FaDu spheroid and CAF upregulated angiogenesis-related genes in HNSCC cells as compared to exosomes from FaDu cell and CAF co-culture media under in vitro conditions. The mouse tumor xenograft model derived from 3D spheroids of HNSCC cells with primary CAFs is expected to produce reliable chemotherapy drug screening results given the robust angiogenesis and lack of necrosis inside tumor tissues.

Keywords: angiogenesis; cancer-associated fibroblast; exosome; head and neck squamous cell carcinoma; oral squamous cell carcinoma; spheroid; tissue clearing.

MeSH terms

  • Animals
  • Cancer-Associated Fibroblasts / metabolism
  • Cancer-Associated Fibroblasts / pathology
  • Carcinoma, Squamous Cell / metabolism
  • Carcinoma, Squamous Cell / pathology*
  • Exosomes / metabolism
  • Female
  • Head and Neck Neoplasms / metabolism
  • Head and Neck Neoplasms / pathology*
  • Humans
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Mouth Neoplasms / metabolism
  • Mouth Neoplasms / pathology*
  • Neovascularization, Pathologic / metabolism
  • Neovascularization, Pathologic / pathology*
  • Primary Cell Culture / methods
  • Spheroids, Cellular / metabolism
  • Spheroids, Cellular / pathology*
  • Tumor Cells, Cultured
  • Xenograft Model Antitumor Assays / methods*
  • Xenograft Model Antitumor Assays / standards