GoKawiilCell lines
The syngeneic mouse GBM model SB28 was provided by H. Okada, and GL261 was obtained from the Developmental Therapeutic Program, NCI. CPA was provided by the Castro-Lowenstein Laboratory, and KR158 was provided by L. Deleyrolle. The mouse bladder cancer cell line MB49 was obtained from the Animal Tumour Core at the Cleveland Clinic. The mouse melanoma B16-F10 cells were a gift from T. Stappenbeck. The GBM patient-derived xenograft cell model L1 was obtained from B. Reynolds (originally from the laboratory of A. Vescovi) and GBM23 was obtained from E. Sulman.
After thawing, all cell lines were treated with 1:100 MycoRemoval agent (MP Biomedicals) and regularly tested for Mycoplasma spp. (Lonza). GBM cell lines were maintained in complete RPMI 1640 (Media Preparation Core, Cleveland Clinic) supplemented with 10% FBS (Thermo Fisher), 1% penicillin–streptomycin (Media Preparation Core) and GlutaMAX (Gibco). MB49 and B16-F10 cells were cultured in DMEM (Media Preparation Core, Cleveland Clinic) supplemented with 10% FBS, 1% penicillin–streptomycin, GlutaMAX and sodium pyruvate (Thermo Fisher Scientific). GBM patient-derived xenograft cells were cultured in DMEM/F12 medium (ThermoFisher) supplemented with 1% penicillin–streptomycin (ThermoFisher Scientific), 1% B27 without vitamin A (ThermoFisher), 20 ng ml–1 EGF (R&D Systems) and 20 ng ml–1 FGF (R&D Systems). Cells were cultured in humidified incubators at 37 °C and 5% CO 2 and were not allowed to exceed 15 passages.
Mice
All animals were kept in a specific pathogen-free facility of the Biological Resource Unit at the Lerner Research Institute, Cleveland Clinic, with a 12-h light–dark cycle. All animal procedures were performed in accordance with the guidelines and protocols approved by the Institutional Animal Care and Use Committee at the Cleveland Clinic. All mouse strains used in the study are listed in Supplementary Table 7.
Castration
Two weeks before tumour implantation, 5–6-week-old male mice underwent either castration or sham surgery. Mice were maintained under inhalation anaesthesia (2–2.5% isoflurane) through a nose cone and administered an ophthalmic lubricant to prevent corneal dryness. The scrotal area was disinfected using betadine and alcohol. A small horizontal incision was made in the skin of the scrotum and the inner skin membranes, and the testicles were exteriorized. Using resorbable vicryl sutures, testicular arteries were ligated, followed by the removal of testicles. The incision was closed using surgical clips (Fine Science Tools). For pain control, subcutaneous injections of buprenorphine (0.1 mg kg−1) and bupivacaine (5 mg kg–1) were administered. In sham-surgery mice, the same procedure was performed, excluding the ligation and removal of the testis.
Microglia depletion
Two weeks after castration, mice were fed either a control diet (AIN-76A) or a PLX3397-supplemented diet (660 mg kg–1; Research Diets) ad libitum. Mice then received intracranial tumour cell injections and remained on the assigned diet until the experimental end point.
Tumour implantation and treatments
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