Ln229

Human glioma cell lines LN229, LN308, U87, LN229 and human normal glial cell line HEB were purchased from American Type Culture Collection (ATCC, Manassas VA, USA). All cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Life Technologies, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, NY, USA). Cells were incubated at 37 °C in humidified air containing 5% CO₂. Cell passage was performed using trypsin until 80%-90% of confluence.

The human GBM cell lines U87(RRID: CVCL_0022), LN229 (RRID: CVCL_0393), U251 (RRID: CVCL_0021), and A172 (RRID: CVCL_0131) and human embryonic kidney cell line (293FT, RRID: CVCL_6911) were purchased from ATCC. The cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin, and streptomycin. All cells within passages 8 to 15 were used and passed the detection of mycoplasma contamination by Myco-Lumi^TMMycoplasma Kit (Beyotime, China).

LN18, LN229, T98G (T98), U87-MG (U87) and U251 cell lines were purchased from the ATCC, USA, and cultured in DMEM medium (Dulbecco’s modified Eagle medium, Thermo Fisher Scientific, USA). Primary glioblastoma cell lines (WG4 and WG9) were derived as described [43 (link)] and cultured in DMEM/F12 GlutaMAX media (Thermo Fisher Scientific, USA). Normal human astrocytes (NHA, Lonza) were cultured in medium containing ABM^TM Basal Medium (CC-3187) and AGM^TM SingleQuots^TM Supplements (CC-4123) from Lonza.. All culture media were supplemented with 10% FBS (Gibco, USA), antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin) and cultured in a humidified atmosphere of CO₂/air (5%/95%) at 37 °C. Cells were treated with Temozolomide (Sigma, USA) alone or with combination with: Olaparib, 3-aminobenzamide, Rucaparib, Tioguanine or Etoposide. Aforementioned compounds were dissolved in DMSO. Irradiation of UV-C light was used with 30 J/m² dose. For reagent specifications and catalogue numbers see the Supplementary Table S1.

Human glioma A172, LN18, LN229, and T98G cell lines were purchased from ATCC (USA). SNB19, U251MG, and U87MG cells were purchased from the Chinese Academy of Sciences Cell Bank (China). The human glioma TJ905 cell line was isolated from human GBM tissue and cultured in DMEM/F12 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA). All other glioma cell lines were cultured in DMEM (Gibco, USA) supplemented with 10% FBS and incubated in 5% CO₂ at 37 °C.

GBM6 (Mayo Clinic), GBM43 (Mayo Clinic), DBTRG-05MG (ATCC), U-251 (ATCC), T98-G (ATCC), and LN-229 (ATCC) GBM cells; HUVEC cells (ATCC); THP-1 human monocytes (ATCC); and human astrocytes (ScienCell) were verified using short tandem repeat (STR) profiling, passaged under 6 times, and confirmed mycoplasma free. Breast CAFs were provided by the Breast Cancer Now Tissue Bank (London, United Kingdom). GBM cells were cultured in DMEM/F-12 plus 10% FBS and 1% penicillin/streptomycin at 37°C. HUVECs were grown in EGM-2 media (Lonza, catalog CC-3162). THP-1 cells were grown in complete RPMI with HEPES. human astrocytes were grown in Gibco Astrocyte Medium (Thermo Fisher).
To generate GSC-containing neurospheres, GBM cells were grown in NM, consisting of DMEM/F12 (Gibco, Thermo Fisher Scientific) supplemented with 20 ng/ml EGF (Peprotech), 20 ng/mL bFGF (Peprotech), and 2% GEM21/neuroplex (Gemini Bio-Products). When comparing CAF_CM to NM, CAF_CM was generated by replacing the media of cultured CAFs with NM for 72 hours, after which media was collected and centrifuged at 300g for 5 minutes, followed by filtration through a 40 μm filter.