In this report, we study and compare three murine glioma cell lines and tumors following LDH-A shRNA knockdown (KD). The objective was to explore and compare the effect of LDH-A knockdown (KD) on the expression levels of LDH-A and LDH-B mRNA, protein, LDH enzymatic activity, the effect on the LDH isoenzyme profiles and the impact of these changes on the growth of cancer cells in vitro and in vivo.
Cells and culture conditions
The GL261 murine glioblastoma cell line was obtained from NCI depository (23, 24). The ALTS1C1 (ALT) murine glioblastoma cell line derived from SV40 large T antigen-transfected astrocytes was kindly provided by Dr. Chiang (Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Taiwan) (25) and the CT2A high-grade murine astrocytoma cell line was kindly provided by Dr. Seyfried (Biology Department, Boston College, Boston) (26). These cell lines were cultured in DMEM media supplemented with 25 mM glucose, 10% FCS, 4 mM glutamine, and penicillin/streptomycin. LDH-A KD (knock-down) and NC (negative control) cells, derived from each cell line, were grown in the same media and 2.5 mg/L of puromycin.
Generation of LDH-A knockdown and control cell lines
GL261, CT2A and ALTS1C1 cells were transfected with Sure Silencing shRNA plasmids (QIAGEN, Frederick, MD, USA) to specifically knock-down expression of the mouse LDH-A gene as described previously (27). Stably transduced clones (KD cell lines) were developed, along with a control (NC) cell line bearing a scrambled shRNA. Based on the previous experience (12, 28), we decided to use the most effective shRNAs (shRNA-2) to develop LDH-A KD cells in murine glioma cells. The transfection of GL261 cancer cells with shRNA-2 resulted in a significant knock-down effect for LDH-A (approximately 10% of that in wild type cells), while bulk CT2A and ALTS1C1 cells transfected with shRNA-2 had a significantly less level of LDH-A knock-down (40-60%). In order to enrich the level of LDH-A knock-down we used a sub-cloning strategy for CT2A and ALTS1C1 cell lines (27).
All immunoblotting experiments were performed as described previously (11, 29). Cell lines underwent protein extraction using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) with protease & phosphatase inhibitors cocktail (1:100, Thermo Scientific Halt Protease & Phosphatase Inhibitor Single-Use Cocktail). Protein concentrations were determined by Pierce BCA protein assay (Thermo Fisher Scientific, Waltham, MA, USA). The proteins in equivalent amounts (10-40 µg/well) were separated by electrophoresis in a NuPAGE gradient 4-12% Bis-Tris Gel (Invitrogen, Carlsbad, CA, USA) and were immuno-blotted with anti-LDH-A antibody (#2012S, Cell Signaling Technology, Danvers, MA, USA) at a 1:1,000 dilution and anti-ß-actin antibody (Sigma life science, #A2103, USA) at a 1:5,000 dilution antibodies. Bound primary antibodies were visualized with either appropriate horseradish peroxidase–conjugated secondary antibodies (1:2,000) using enhanced chemiluminescence reagent (Western Lightning-ECL) or with Eu-labelled antibody using ScanLater Western Blot Assay kit and SpectraMax ID5 (Molecular Devices, US).
LDH enzyme activity
Total LDH enzyme activity was assessed using the Cytotoxicity Detection Kit PLUS (LDH) (Roche Diagnostics) as described before (27).
Proliferation assay in vitro
2×105 cells were seeded in 3 mL culture media in 6-well plates, followed by counting cells at 3 different time points, 48, 72, and 96 hours after seeding cells using Countess automated cell counter (Thermo Fisher Scientific, Waltham, MA, USA). Each sample was triplicated, and media were changed every other day.
The animal protocol was approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. Two strains of mice were used in animal experiments. First, 1×106 cells in 100 µL PBS were injected into the right flank of immunocompromised Hsd: Athymic Nude Foxn/nu (Envigo) or athymic nu/nu male mice (Charles River Laboratories). Second, 1×106 cells in 100 µL PBS mixed with 100 µL matrigel were injected subcutaneously into the right flank of immunocompetent C57BL/6 male mice (Charles River Laboratories). The volume (V) of subcutaneous tumors was calculated from caliper measurements, where V = (π/6) × x × y × z where x, y, and z are 3 orthogonal diameters. Doubling times were calculated by the equation of trend lines using GraphPad Prism.
Zymography, a common method to detect isoenzymes, was used to detect tissue-specific differences in LDH isoenzymes. This approach can directly observe 5 isozyme bands in the active state (30). Based on their different electrophoretic motility, all LDH isoenzymes can be identified as LDH1 (B4 or H4), LDH2 (B3A1 or H3M1), LDH3 (B2A2 or H2M2), LDH4 (B1A3 or H1M3), and LDH5 (A4 or M4). The buffer system at pH 8.6 was chosen for the best separation of the five LDH isoenzymes (30-33). Because the B polypeptide has more acidic amino acid residues than the A polypeptide, LDH1/B has the highest migration rateand LDH5/A has the lowest migration rate. The electrophoretic mobilities of the LDH isoenzymes are: LDH 1/B > LDH 2 > LDH 3 > LDH 4 > LDH 5/A.
Immunohistochemical staining and image analyses
Dissected tumors were placed into 4% paraformaldehyde for further immunohistochemistry (IHC). The immunofluorescent (IF) staining was performed at Molecular Cytology Core Facility of MSKCC using Discovery XT processor (Ventana Medical Systems). 5 µm thick, paraffin-embedded sections were stained for H&E and LDH-A and LDH-B staining. The sections of tumors from nude or immunocompetent mice were stained by anti-LDH-A, anti-LDH-B. The small 5-day tumors from GL261 NC and KD were stained with immune markers: anti-CD68 antibody (Catalog No. TA1518, Boster), anti-CD4 (Catalog No. AF554, R & D Systems) and anti-CD3 antibody (Catalog No. A0452, Dako). Quantification of morphological characteristics was performed using trainable Weka Segmentation (Image J segmentation plugin) to assess the fraction of viable tumor cells, stroma, hemorrhage and necrosis in the H&E sections. The same approach was used to quantify LDH-A and LDH-B staining (28, 34).
Results are presented as mean ± standard error unless otherwise specified. Statistical significance was determined by a two-tailed Student t-test. A p-value of <0.05 was considered significant. All data presented for T cells assessment using IF staining were analyzed using GraphPad Prism (version 7.0; GraphPad Software) and are presented as mean +/- SD. Results were analyzed using the unpaired Student’s t-test, and statistical significance was defined as p<0.05.