Cell Culture and Human Tissues
Human neuroblastoma cell line LAN-1 was provided by Dr. Robert Seeger (Children's Hospital of Los Angeles, Los Angeles, CA), and NB1691 by Dr. Peter Houghton (St. Jude Children's Research Hospital, Memphis, TN). Human Burkitt's lymphoma cell line Daudi and adenocarcinoma cell line HeLa were purchased from American Type Culture Collection (Bethesda, MD). All cell lines were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2mM glutamine, 100 U/ml penicillin, and 100 ug/ml streptomycin at 37°C in a 5% CO2 incubator. Normal tissues as well as solid tumor samples of different histological types obtained at Memorial Sloan-Kettering Cancer Center (MSKCC) were snap frozen in liquid nitrogen. Written informed consent was obtained from the patients and/or their guardians in accordance to the guidelines of the institutional review board of MSKCC.
MoAbs 8H9 (murine IgG1) and 3E7 (murine IgG2b specific for L1-CAM) were produced against human neuroblastoma in our laboratory. They were purified by protein A (GE Healthcare, Piscataway, NJ) affinity chromatography before use. MAB1027 (anti-B7-H3 MoAb) was purchased from R&D System, Minneapolis, MN.
Whole Cell Lysates and Western blot
8H9-positive cell line LAN-1 and 8H9-negative cell line Daudi were grown to ~ 80% confluence. Cells were harvested using 2 mM EDTA and washed with ice-cold PBS. Cells were lysed on ice (20 min) in Triton Lysis Buffer (50 mM Tris-HCl, pH 7.2, 50 mM NaCl, 10% glycerol, 1% Triton X-100, and protease inhibitor cocktail tablets). The lysates were clarified by centrifugation at 14,000 rpm for 20 min at 4°C. ~25-50 ug whole cell lysates were analyzed by SDS-PAGE under nonreducing condition using Tris-Glycine Ready Gel System (Bio-Rad, Hercules, CA). After electrophoresis, samples were transferred onto Immun-Blot PVDF Membrane (Bio-Rad), blocked for one hour at room temperature (RT) with 10% dry milk in Tris-Buffered Saline Tween-20 (TBST), and incubated with primary antibodies (8H9 at 5-20 ug/ml, and MAB1027 at 5 ug/ml) for 3 hours at RT. The membrane was then washed with TBST, and incubated with secondary peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch, West Grove, PA). Bands were detected with SuperSignal West Pico Chemiluminescent Substrate (PIERCE, Rockford, IL).
For crude membrane preparation, LAN-1 cells were pipetted off the tissue culture dish, washed with ice-cold PBS, and lysed on ice in sucrose buffer (0.25 M sucrose, 5 mM Tris-HCl, pH 7.2, and protease inhibitor cocktail tablets) with a Dounce homogenizer (Kontes, Vineland, NJ). Upon centrifugation for 10 min at 1,000 g to pellet all nuclei, the supernatant was then ultracentrifuged at 100,000 g for 30 min in a Beckman L-70K (25,000 rpm, SW41Ti rotor) to separate the membrane particulate from the cytosolic fraction. The cytosolic fraction was adjusted to 1% Triton, while crude nuclear and membrane fractions were resuspended in Triton Lysis Buffer and clarified before use.
8H9 Antigen Affinity Purification
8H9 antigen was purified from LAN-1 cell extracts by immuno-affinity chromatography using MoAb 8H9. The 8H9 affinity column was prepared by covalently conjugating Fc portion of 8H9 to protein G on the gel matrix using Protein G IgG Plus Orientation Kit (PIERCE) according to the manufacturer's instructions. 4 mg of LAN-1 whole cell lysates or equivalent membrane fraction prepared as described above were incubated overnight at 4°C with 20 ul 8H9-protein G Sepharose (3 mg bound 8H9/ml beads). After extensive washing with Triton Lysis Buffer, the column was eluted sequentially with 50 mM Tris-HCl, pH 7.2 containing 1M NaCl (E-NaCl), 0.1 M Glycine-HCl, pH 2.8 (E-2.8) and pH 2.0 (E-2.0), SDS Sample Buffer (E-SDS: 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.005% Bromophenol blue), and SDS Sample Buffer plus boiling in water for 5 min (E-SDS-B). A small aliquot of eluates was monitored for the presence of 8H9 antigen by Western blot using 8H9 antibody. 25% of the eluate was also analyzed by silver staining (SilverQuest Silver Staining Kit, Invitrogen). Finally, 50% of the 8H9 antigen-positive eluate (E-2.0 fraction) was analyzed by colloidal Coomassie blue staining (GelCode Blue Stain Reagent, PIERCE), and the 8H9 antigen-positive band was collected and used for mass spectrometric identification by the Microchemistry and Proteomics Core Facility at MSKCC.
Detection of B7-H3 mRNA by quantitative (q)RT-PCR
Total RNA from normal tissues, as well as solid tumors detailed in were isolated using Trizol Reagent (Invitrogen) according to the manufacturer's instructions. One ul of cDNA synthesized from 1 ug of total RNA was used for real-time qPCR using Applied Biosystems (ABI) Sequence Detection System 7300 (Foster City, CA). B7-H3 (CD276) gene expression assay reagent (Hs00228846_m1) as well as 2 endogenous controls: hypoxanthine phosphoribosyltransferase 1 (HPRT1, 4326321E) and succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (SDHA, Hs00188166_ml) were purchased from ABI. Each sample was quantified using the comparative CT method (ABI) as a relative fold-difference compared to peripheral blood mononuclear cells (PBMC).
B7-H3 transcript was ubiquitously expressed in solid tumors and normal human tissues
Quantitation of miR-29
miR-29a, miR-29b, miR-29c, as well as the endogenous control RNU48 were purchased from ABI. A two-step RT-PCR with reverse transcription using a miRNA-specific primer, followed by qPCR with TaqMan probes was carried out according to the manufacturer's instructions.
Protein expression by immunofluorescence and flow cytometry
These procedures were previously described (18
). The blocking experiment was carried out by incubating 0 ~ 10 ug of recombinant human 4Ig-B7-H3 (R&D System) with 1 ug 8H9 or 0.1 ug 3E7 (as negative control) for 30 min at room temperature, before mixing with 106
M14 melanoma cells for immunofluorescence staining and flow cytometry analyses.
Luciferase Reporter Assay
Oligonucleotides corresponding to the miR-29a binding site in the B7-H3 3′UTR or a single-base mutant (illustrated in ) were synthesized (Integrated DNA Technologies, Coralville, IA) and inserted into the XbaI site immediately downstream from the stop codon of firefly luciferase of the pGL3-control vector (Promega, Madison, WI). HeLa cells were cotransfected in 24-well plates using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol, with 50 ng of the firefly luciferase reporter, 1 ng of the renilla luciferase reporter (pRL-CMV vector, Promega) as transfection control, and 100 nM (final) miRNA mimics (ThermoFisher Scientific, Lafayette, CO). Firefly and renilla luciferase activities were measured sequentially using dual-luciferase assays (Promega) 24 hrs after the transfection. The experiments were performed in triplicate.
miR-29a directly targets B7-H3 3′UTR and downregulates B7-H3 protein expression
NB1691 Transient Transfection
NB1691 cells were transfected in 6-well plates using DharmaFECT reagent (ThermoFisher Scientific) according to the manufacturer's instructions, with 100 nM (final) miRNA mimics, inhibitors, or mimics plus inhibitors (ThermoFisher Scientific). 24 hrs after transfection, cells were treated with 1 mg/ml Pronase E (E. Merck, Darmstadt, Germany) for 30 min at 37°C to strip off B7-H3 protein already on the cell surface, and another 48 hrs later newly expressed B7-H3 protein level were measured by 8H9 immunofluorescence staining followed by flow cytometry analyses as previously described (18