Topotecan hydrochloride hydrate was purchased from Sigma-Aldrich (St. Louis, MO) and stock solutions were made in dimethyl sulfoxide (DMSO). All dilutions resulted in a final concentration of 0.05% DMSO within GBM growth media.
Antibodies and Reagents
Anti-HIF1α (H-206) rabbit polyclonal IgG was purchased from Santa Cruz Biotechnology (Dallas, TX). Anti-PD-L1 (E1L3N) rabbit monoclonal IgG and HRP-linked anti-rabbit and anti-mouse IgGs were purchased from Cell Signaling Technology (Danvers, MA). Anti-β-actin mouse monoclonal IgG was purchased from Sigma-Aldrich. TaqMan® probes for quantitative polymerase chain reaction (qPCR) of human CD274 (PD-L1), HIF1A (HIF1α), SLC2A1 (GLUT1), and ACTB (β-actin), as well as a TaqMan® Fast Universal PCR Master Mix, were purchased from Thermo Fisher Scientific (Waltham, MA).
Human GBM cell lines LN229 and Mz18 were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals Inc., Flowery Branch, GA), 100 U/mL penicillin, and 100 mg/mL streptomycin in 5% CO2 at 37°C.
3.5×105 GBM cells per well were plated into 6 well tissue culture treated plates and allowed to attach before being treated with topotecan in 0.05% DMSO for 12–14 h. Westerns were performed as has been previously described. Briefly, growth media was removed and the cells were gently washed with phosphate-buffered saline (PBS) before being lysed (100 mM Tris-Cl pH 7.4, 2% SDS, 50 mM EDTA, 20% glycerol). Samples were subsequently sonicated on ice and incubated at 95°C for 5 min. Protein concentrations were normalized using Pierce BCA Protein Assay (Thermo Fisher Scientific) prior to separation on 4–20% Tris-Glycine SDS-PAGE gels (Thermo Fisher Scientific). Images were taken with an Alpha Innotec FluorChem system (Alpha Innotech, San Leandro, CA). Protein expression levels were determined via densitometric analysis of the bands corresponding to the proteins of interest using ImageJ (NIH, Bethesda, MD). All densities were normalized to the corresponding β-actin levels and expressed as the ratio to the control (DMSO alone).
Quantitative Polymerase Chain Reaction (qPCR)
3.5×105 GBM cells per well were plated into 6 well tissue culture treated plates and allowed to attach before being exposed to topotecan in 0.05% DMSO for ~12–14 h. RNA was extracted using a RNeasy Mini Kit per the manufacturer’s instructions (Qiagen, Hilden, Germany). cDNAs were then generated using a high-capacity cDNA reverse transcription kit per the manufacturer’s instructions (Applied Biosystems, Foster City, CA). qPCR was conducted using a LightCycler® 480 II machine (F. Hoffmann-La Roche AG, Basel, Switzerland); ACTB was used as an endogenous control. All samples were tested in triplicate and the final expression fold changes relative to the DMSO control were calculated using the 2-ΔΔCT method.
All data are expressed as mean ± standard deviation (SD) of three independent experiments. To test for differences in topotecan-treated vs. DMSO control conditions, either one-way ANOVA with Dunnett’s post-hoc correction or a Student’s t-test was conducted. Values of p≤0.05 were deemed significant.