Tumor-bearing mice were sacrificed approximately 35 days after orthotopic xenotransplantation. After the intracranial xenograft tumors were harvested, pieces of the tumors were dissociated and subcultured in vitro, and other pieces were used for pathological examination. Frozen sections were stained with H&E, and 4',6-diamidino-2-phenylindole (KeyGen, Nanjing, China) was used for nuclear labeling. The sections were then observed under both a fluorescence microscope and a confocal laser scanning microscope (ZEISS, Germany).
For cell subculture, minced tumor tissue was washed with cold phosphate-buffered saline, digested with 0.02% trypsin and cultured in DMEM containing 10% fetal bovine serum (FBS, Gibco). After the cells had been serially subcultivated in vitro for approximately two weeks, fluorescence-activated cell sorting was used to sort suspensions of RFP+ tumor cells and EGFP+ stromal cells, and EGFP-expressing tumor stromal cells were collected. Then, the limiting dilution method and single-cell cloning techniques were used to re-purify EGFP-expressing cells with high proliferation abilities from the different animal models [23 (link), 38 (link)]. The EGFP-expressing tumor stromal cells from Models I, II and III were named EGFP+ tumor microenvironment cells 1 (EGFP+TMEC1, EGFP+TMEC2 and EGFP+TMEC3, respectively).
For cell subculture, minced tumor tissue was washed with cold phosphate-buffered saline, digested with 0.02% trypsin and cultured in DMEM containing 10% fetal bovine serum (FBS, Gibco). After the cells had been serially subcultivated in vitro for approximately two weeks, fluorescence-activated cell sorting was used to sort suspensions of RFP+ tumor cells and EGFP+ stromal cells, and EGFP-expressing tumor stromal cells were collected. Then, the limiting dilution method and single-cell cloning techniques were used to re-purify EGFP-expressing cells with high proliferation abilities from the different animal models [23 (link), 38 (link)]. The EGFP-expressing tumor stromal cells from Models I, II and III were named EGFP+ tumor microenvironment cells 1 (EGFP+TMEC1, EGFP+TMEC2 and EGFP+TMEC3, respectively).
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