Htb 85

Human Osteosarcoma cell lines MG63 (ATCC^® CRL1427™), SAOS-2 (ATCC^® HTB-85™), U2-OS (ATCC^® HTB-96™); Human Adenocarcinoma cell lines isolated from breast cancers MDA-MB-468 (ATCC^® HTB231™); and Human Glioblastoma cell lines U87 (ATCC^® HTB14™) were purchased from American Type Culture Collection (ATCC) and used for this study.
MG63 cell line was cultured in DMEM F12-GlutaMAX™ Modified Medium (Gibco) supplemented with 10% Foetal Bovine Serum (FBS) (Gibco) and 1% of penicillin/streptomycin mixture (pen/strep) (100 U/mL–100 μg/mL, Gibco). SAOS-2 cell line was cultured in McCoy’s 5A Modified Medium (Gibco) supplemented with 15% and 10% FBS, respectively, and 1% pen/strep. U2-OS cells were cultured using McCoy’s 5 A (modified) medium supplemented with 10% FBS and 1% Pen/Strep. MDA MB 468 cells were cultured in growth media using RPMI 1640 (Gibco), 10% FBS and 1% Pen/Strep; and U87 cells were grown in a complete medium composed of MEM-nucleosides no-ascorbic-acid medium (Gibco), 10% FBS and 1% Pen/Strep.
Cells were kept in an incubator at 37°C under controlled humidity and 5% CO₂ atmosphere conditions. Cells were detached from culture flasks by trypsinization and centrifuged. The cell number and viability were determined by Trypan Blue Dye Exclusion test and all cell handling procedures were performed under laminar flow hood in sterility conditions.

Tumor tissue samples with their matched normal tissue were obtained from the International DIPG/DMG Registry as described previously [34 (link)]. Briefly, patients with DIPG were informed and consented to the IRB-approved protocol (Study ID: 2016-3357) at Cincinnati Children’s Hospital Medical Center. Diagnosis of DIPG was based on clinical symptoms and imaging characteristics on pretreatment magnetic resonance imaging (MRI). Primary normal human foreskin fibroblasts (HFF) strains (ATCC CRL-2091), human cervical carcinoma cell lines (HeLa), and the human osteosarcoma cell lines (Saos-2) (ATCC HTB-85) were purchased from ATCC. HFF and HeLa cells were cultured in DMEM (Gibco) supplemented with 10% FBS, while Saos-2 cells were cultured in McCoy’s 5A medium supplemented with 15% FBS. Pediatric GBM cell lines, CCHMC-DIPG-1, R0315-GBM, SJ-HGGX42 and HSJD-GBM002, were cultured in neurosphere stem cell media as described elsewhere [35 (link),36 (link)]. KNS42 cells were cultured in DMEM (Gibco) supplemented with 10% FBS. KNS42 cells were obtained from the JCRB (Japan Cancer Research Resources) cell bank. HSJD-GBM002 cells were generous gifts from Dr. Angel Montero Carcaboso. SJ-HGGX42 cells were obtained from PBTP (https://pbtp.stjude.cloud). ATR inhibitor VE-822 (S7102) and CHK1 inhibitor (Chir-124) (S2683) were purchased from Selleckchem.

The human osteosarcoma cell lines SaOS-2 (HTB-85) and 143-B (CRL-8303) cells were obtained from ATCC (Gaithersburg, MD, USA). In order to enable visualization of tumor cells within mouse tissues, in vivo and ex vivo, 143-B cells were sequentially transduced with the LacZ gene, mCherry gene and Firefly luciferase gene as described recently (hereafter referred to as 143-B cells) [52 (link),53 (link),54 (link)]. Osteosarcoma cells were cultured in 1:1 DMEM (4.5 g/L glucose) and HamF12 medium (61965026 and 1765029, Thermo Fisher Scientific, Paisley, UK) and supplemented with 10% heat-inactivated fetal bovine serum (FBS, 10500, Thermo Fisher Scientific, Paisley, UK), hereafter referred to as complete medium. The cells were grown in 5% CO₂ and 95% air in humidified conditions at 37 °C. Cells were confirmed to be mycoplasma negative and authenticated using short tandem repeat DNA profiling (Microsynth, Balgach, Switzerland) and compared to the German Collection of Microorganisms and Cell Cultures database.

HER2 positive breast adenocarcinoma cells (SKBR-3), ERα positive breast adenocarcinoma cells (MCF-7) used as cancer negative control, murine fibroblasts cells (NIH-3T3) used as negative control. All cell lines were obtained from the American Type Culture Collection (HTB85, ATCC, Manassas, VA, United States). After thawing, SKBR-3 cell line was cultured in McCoy’s 5A Medium Modified (Sigma-Aldrich), 10% of Fetal Bovine Serum (FBS, EuroClone), 1% of L-Glutamine (Lonza), 2% sodium pyruvate (Lonza), 0.4% antibiotics (Lonza) and 0.1% fungizone. MCF-7 cell line was cultured in Minimum Essential Medium Eagle (Sigma-Aldrich), 10% of Fetal Bovine Serum (EuroClone), 1% sodium pyruvate (Lonza), 1% Non-Essential Aminoacids (EuroClone), 1% Bovine Insulin (Sigma-Aldrich), 1% L-Glutamine (Lonza) and 0.4% antibiotics (Lonza). NIH-3T3 cell line was cultured in Dulbecco’s Modified Eagle Medium High Glucose 4.5 mg/mL (Sigma-Aldrich), 10% of Bovine Calf Serum (BCS, EuroClone) and 1% of L-Glutamine (Lonza). All cell lines were incubated at 37°C in 5% CO₂, routinely trypsinized (Trypsin EDTA solution 1X, Lonza) after confluence, counted and seeded into wells.

Human embryonic kidney 293 (HEK293), cervical cancer (HeLa 1211) and sarcoma osteogenic (SAOS 2) cell lines were purchased from the ATCC (#CRL‐1573.3, #CCL‐2 and #HTB‐85, respectively). All cell lines were maintained in Dulbecco's Modified Eagle Medium (Sigma, Madrid, Spain, #D‐5796) supplemented with 10% FBS (Biowest, Nuaillé, France, #S1810‐500), and were cultured at 37 °C with 5% CO₂.
pBABE‐SAOS 2 and hTERT‐SAOS 2 stable cell lines were obtained after transfection of the SAOS 2 cell line with the plasmids pBABE‐puro or pBABE‐puro‐hTERT from Addgene (Teddington, UK; #1764 and #1771, respectively) and Lipofectamine 2000 (Invitrogen, Madrid, Spain, #11668‐027) following the manufacturer's instructions. Then, several stable clones were selected with puromycin.

Cancer‐associated fibroblasts (CAFs) and the osteosarcoma cell line Saos‐2 (ATCC® HTB‐85) were used to produce cell‐derived extracellular matrices. Matrices were prepared, as described previously (Oliver‐Kozup et al., 2013). Briefly, CAFs and Saos‐2 cells were cultured in high‐glucose Dulbecco's Modified Eagle Medium with 10% of fetal bovine serum and 1% of penicillin and streptomycin at 37°C in an atmosphere of 5% of CO₂ throughout the experiment. To produce the extracellular matrix for specific tests, cells were grown as follows: (i) for matrix characterization via immunofluorescence or for GFP expressing GAS (GFP‐GAS) attachment assays, cells were grown on 15‐mm glass coverslips inserted into wells of (24‐well) tissue culture plates and (ii) for crystal violet biofilm assay, matrix characterization by ELISA or by Ponceau S staining, cells were growth in plastic wells without glass coverslips. Cells were seeded at 50,000 cells per well, grown until confluent and then detached through treatment with 5 mM of ethylene glycol tetraacetic acid (EGTA) and removed from wells. Samples were washed gently with PBS and wells or coverslips were subsequently used for assessment.