Streptomycin

U87-MG, U251-MG, and U373-MG (Uppsala) GBM cell lines were obtained by Sigma-Aldrich (St. Louis, MO, USA) (HPA Culture Collections). The A172 GBM cell line was obtained by American Type Culture Collection (ATCC, Manassas, VA, USA). Cell lines were not used beyond passage 20. U251 Cells were cultivated in Roswell Park Memorial Institute (RPMI) 1640 medium (Biochrom, Berlin, Germany) containing 10% fetal calf serum (FCS; Biochrom, Berlin, Germany), 100 IU/ml penicillin, and 100 µg/ml streptomycin (Biowest, Nuaillé, France). All other cells and cell lines were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) low glucose (Biowest, Nuaillé, France) containing 10% FCS (Biochrom, Berlin, Germany), 100 IU/ml penicillin, and 100 µg/ml streptomycin (Biowest, Nuaillé, France). Primary GBM cells derived from a primary GBM tumor biopsy were obtained from the University Hospital Cologne, genetically characterized and cultured as previously described (Haas et al. 2018 (link)).

Six human BC cell lines: MCF-7 and T47-D (luminal A-like), BT474 (luminal B/HER2-negative), SKBR3 (HER2-enriched), MDA-MB-231, and MDA-MD-468 (TNBC), and the normal breast epithelium cell line (184A1) were purchased from ATCC (Manassas, VA, USA). All tumor cell lines were maintained in 75 cm² flask (SPL Life Science, Gyeonggi, Korea) fed with Dulbecco Modified Eagle Medium/Ham F12 (1:1) with L-glutamine and 15 mM HEPES media (Biowest, Nauaillé, France) supplemented with 10% Fetal Bovine Serum (Biowest, Nauaillé, France), 50 U/mL penicillin and 50 mg/mL streptomycin (Biowest, Nauaillé, France). The cell line 184A1 was maintained in Mammary Epithelial Cell Growth Medium (MEGM Bullekit^TM Lonza, Basel, Switzerland) supplemented with growth factors (MEGMTM SingleQuots^TM Supplements, Lonza, Basel, Switzerland), and 1% penicillin and streptomycin (Biowest, Nauaillé, France). Cells were incubated in humidified incubators at 37 °C and 5% CO₂.
Once a year, or when cells were thawed out, a mycoplasma test was carried out following the instructions of the Venor^®GeM One Step kit (Minerva BioLabs, Berlin, Germany). Cells were maintained in culture for a few weeks, enough time for a couple of passages, and needed experiments.

The established human cervical cancer cell lines SiHa and HeLa were obtained from American Type Culture Collection (ATCC). All cells were maintained in Dulbecco's modified Eagle's medium (DMEM, HyClone, Thermo Scientific, USA) supplemented with 10% fetal bovine serum (Gibco, Life technologies, USA), 100 U/ml penicillin (Biowest, Nuaillé, France), and 100 U/ml streptomycin (Biowest, Nuaillé, France) and incubated at 37°C in a humidified atmosphere with 5% CO₂.
The primary human cervical cells were obtained from the cervical cancer patients at Fudan University Shanghai Cancer Center (FUSCC). The primary cells were maintained in Gibco^® Medium 199 (Gibco, Life technologies, USA), supplemented with 1μg/ml epidermal growth factor (EGF), 15% fetal bovine serum (Gibco, Life technologies, USA), 100 U/ml penicillin (Biowest, Nuaillé, France), and 100 U/ml streptomycin (Biowest, Nuaillé, France) and incubated at 37°C in a humidified atmosphere with 5% CO₂.

U87-MG, U251-MG, and U373-MG (Uppsala) GBM cell lines were obtained by Sigma-Aldrich (HPA Culture Collections) in 2015 and not used beyond passage 20. U251 Cells were cultivated in RPMI 1640 (Biochrom) containing 10% FCS (Biochrome), 100 IU/ml penicillin, and 100 µg/ml streptomycin (Biowest). All other cells and cell lines were cultivated in DMEM low Glucose (Biowest) containing 10% FCS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Primary GBM cells derived from a primary GBM tumor biopsy were obtained from the University Hospital Cologne. IDH1 mutation and MGMT promoter methylation status [52 (link)] of all primary cells and cell lines used is displayed in Table 1.Table 1

IDH1 mutation and MGMT promoter methylation status of GBM cell lines and primary cells

Cells	IDH1	MGMT promoter
U251-MG	Wild-type	Methylated
U373-MG	Wild-type	Methylated
U87-MG	Wild-type	Methylated
Primary culture	Wild-type	Methylated

Immortalized Human Gingival Keratinocytes (ihGK, Applied Biological Materials Inc., Richmond, BC, Canada) and Immortalized Human Gingival Fibroblasts-hTERT (ihGF, Applied Biological Materials Inc) were grown at 37 °C and 5% CO₂ atmosphere. The culture medium was renewed twice per week. Keratinocytes were cultured on tissue culture flasks for sensitive adherent cells (Sarstedt, Germany) using Dulbecco’s modified Eagle’s medium (DMEM) without magnesium and calcium (Gibco, Grand Island, NY, USA) and Ham’s F12 (Biowest, Nuaille, France) in a 2:3 proportion, supplemented with 0.01 mg/mL insulin (Sigma-Aldrich), 0.4 ng/mL hydrocortisone (Sigma-Aldrich), 6.7 ng/mL selenium (Sigma-Aldrich), 0.01 μg/mL human epidermal growth factor (ThermoFisher Scientific, Waltham MA, USA), 1M HEPES buffer (Biowest), 5.5 μg/mL transferrin (Sigma-Aldrich), 0.1 nM cholera toxin (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), 5% (v/v) fetal bovine serum embryonic stem cells tested (FBS, Biowest), and 100 µg/mL penicillin and 100 µg/mL streptomycin (Biowest). Fibroblasts were cultured in DMEM low glucose (Biowest) and Ham’s F12 (Biowest) in a 2:1 proportion, supplemented with 10% (v/v) FBS (Biowest) and 100 µg/mL penicillin, as well as 100 µg/mL streptomycin (Biowest).

Commercial Pluronic^® F68 (Mw 8400 Da), N,N′-dicarbonyldiimidazole (CDI), ethanol, sodium carbonate, resazurin, hydrocortisone and McCoy’s 5A cell medium were acquired from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile was purchased from Carlo Erba (Val de Reuil, France). Commercial branched PEI 99% (Mw 1800 Da) and acryloyl chloride were acquired from Alfa Aesar (Haverhill, MA, USA). Regenerated cellulose dialysis tubing (MWCO 3500 Da) was acquired from Orange Scientific (Braine l’Alleud, Belgium). Cellulose acetate syringe filters were purchased from Filter-Lab^® (Barcelona, Spain). A DNA plasmid kit was obtained from Qiagen (Hilden, Germany), and U2OS and SCC-9 cell lines were purchased from ATCC. DMEM:F12, fetal bovine serum (FBS) (10%), penicillin and streptomycin were obtained from Biowest (Nuaille, France). A DC protein assay was acquired from Biorad (Hercules, CA, USA). resazurin sodium salt was obtained from Sigma-Aldrich (St. Louis, MO, USA).

PBMCs were obtained from fasting blood samples provided anonymously from 39 healthy adult volunteers who signed an informed consent form. The study followed the Helsinki Declaration for medical research involving human subjects and was approved by the Virgen Macarena-Virgen del Rocío University Hospital ethical committee (reference number 2012PI/200). Blood samples were collected in BD Vacutainer^® CPT™ Mononuclear Cell Preparation Tubes (BD Biosciences, San Jose, CA, USA) containing sodium heparin, and PBMCs were isolated immediately by centrifugation. Cells were then cultured at 1 × 10⁶ cells/mL in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 U/mL streptomycin (all from Biowest) in the presence or absence of two different concentrations of WGPHs (0.25 and 0.5 mg/mL) and incubated at 37 °C in a 5% CO₂ humidified atmosphere. In addition, PBMCs were stimulated with 8 µg/mL of phytohaemagglutinin-P (PHA) (Sigma-Aldrich, St. Louis, MO, USA), a T cell proliferative stimulus, a T cell proliferative stimulus, with or without WGPHs to analyze proliferation and cytokine production.