Organoid culture containing cancer cells and stromal cells reveals that podoplanin-positive cancer-associated fibroblasts enhance proliferation of lung cancer cells

•We generated `hybrid cancer organoids` containing cancer cells and tumor-promoting CAFs.•Podoplanin (+) CAFs promoted cancer cell growth.•This technique is potentially an innovative cancer research tool. Podoplanin-positive cancer-associated fibroblasts (CAFs) play an important role in tumor progression. The aim of this study was to evaluate the effect of podoplanin (+) CAFs on the proliferation of cancer cells using a three-dimensional (3D) organoid model. We examined the success rate of organoid culture containing PC-9 cancer cells and CAFs. Thereafter, we compared the proliferating index (MIB-1 index) of PC-9 cells co-cultured with podoplanin-overexpressing CAFs and control CAFs using organoid specimens. Furthermore, we compared the MIB-1 labeling index of cancer cells in podoplanin (+) CAFs cases (n = 13) and podoplanin (-) CAFs cases (n = 14) using surgically resected adenocarcinoma specimens. Without CAFs, PC-9 cells did not form any organoid (success rate: 0%). When PC-9 cells were mixed with CAFs (1:10), the mixed cells generated round and steric aggregates (hybrid cancer organoids, success rate: 100%). In three independent experiments, the MIB-1 index of PC-9 cells in hybrid cancer organoids containing podoplanin-overexpressing CAFs was significantly higher than that of PC-9 cells in organoids containing control CAFs (Exp. 1: 40.4% vs. 24.4%; Exp. 2: 40.0% vs. 24.5%; Exp. 3: 40.3% vs. 25.2%; p