3D Co-culture of Cancer-Associated Fibroblast with Oral Cancer Organoids.

Limits of the available model have, in the long term, challenged the studies of cancer-associated fibroblasts (CAFs) and their interplay with cancer stem cells, one of the key driving forces of patients with oral squamous cell carcinoma (OSCC). In the present study, the 3-dimensional organoid model was employed to investigate whether co-culture with paralleled CAFs promotes stem-like properties of OSCC. Experimentally, tumor organoids and paralleled CAFs were generated from patients with OSCC. Then, the CD44 cells in organoids were sorted out by flow cytometry and co-cultured with CAFs in Matrigel. The results showed that co-culture with CAFs enhances the organoid-forming ability of CD44 cells and that this effect was blocked when production or uptake of lactate in CAFs or CD44 cells was inhibited. Furthermore, the expression levels of CD44 and OCT-4 were detected in organoids incubated with lactate, via immunofluorescence or Western blot assay. The results demonstrated that treatments with lactate promote the organoid-forming ability of CD44 cells, as well as the expression of CD44 and OCT-4 at the protein level in OSCC organoids.