Published primary research articles that examined NIS protein levels in human breast tumor samples were included in the survey. Six articles examined NIS protein in breast cancer by immunohistochemistry [10
] of which one performed Western blot analysis [12
]. Publications based solely on cell lines, animal models or review articles were excluded from the study.
Nine tissue microarrays composed of 210 breast cancers were examined. Three cores of 1 mm in diameter corresponding to different regions of each tumor were included in the array. Of the 210 cases, 192 were evaluable for this study.
Affinity purified #331 and #442 as well as non-purified #836 polyclonal human NIS antibodies were custom-generated against the myelin basic protein (MBP)-human NIS fusion protein (amino acids 468-643 of human NIS) (Sigma Genesys, The Woodlands, TX). FP-13 monoclonal human NIS antibody was raised against the same antigen [16
]. VJ1 NIS monoclonal mouse antibody recognizing the extracellular loop of human NIS was kindly provided by Dr. Sabine Costagliola, Institute of Interdisciplinary Research, Free University of Brussels, Brussels, Belgium [17
]. The PA716 polyclonal rat NIS antibody raised against the rat NIS peptide (amino acids 603-618) was kindly provided by Dr. Bernard Rousset, Institut National de la Santé et de la Recherche Mèdicale, Lyon, France [18
Paraffin-embedded tissue sections or tissue microarrays of 4 μm thickness were heated in a 60°C oven for 1 hour, cooled to room temperature, deparaffinized with xylene and rehydrated with graded ethanol solutions. Endogenous peroxidase was blocked by incubating tissues with 3% hydrogen peroxide in water for 5 minutes. Antigen retrieval was then performed using steaming Target Retrieval Solution (Dako Cytomation, Denmark) with a pH of 6.1 for 30 minutes. The following immunostaining procedures were performed by the Dako Autostainer (Dako Cytomation, Denmark). Briefly, tissues were incubated with either #442 (1:25), #836 (1:300) or VJ1 (1:10) primary human NIS antibodies for one hour. Tissues were then rinsed in Tris-Buffered Saline Tween-20 (TBST) buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl and 0.05% Tween-20) and blocked for endogenous biotin using the Biotin Blocking System (Dako Cytomation, Denmark). A Labeled Streptavidin-Biotin (LSAB) Complex Detection System (Dako Cytomation, Denmark) was used for signal amplification per the manufacturer’s protocol. Tissues were washed and incubated with diaminobenzidene (DAB) chromagen (Dako Cytomation, Denmark) for 5 minutes for visualization. Tissues were rinsed in distilled water, counterstained with hematoxylin, rinsed, immersed in 1% NH3OH solution, rinsed, dehydrated through graded ethanol solutions and dipped in xylene. Slides were mounted with Micromount solution (Surgipath Medical Industries, Richmond, IL).
Interpretation of immunohistochemical staining
The level of cell surface NIS in each case was scored on a scale of 0, 1+, 2+, 3+, using criteria analogous to the evaluation of Her-2/neu staining. Absent staining was scored as 0; weak cell surface staining was scored as 1+; weak to moderate cell surface staining was scored as 2+; and strong cell surface NIS staining was scored as 3+. Cytoplasmic staining was also scored on a scale of 0, 1+, 2+ and 3+. Cases with scores of 2+ and 3+ were considered positive for intracellular staining.
MCF-10A breast epithelial cells were cultured in a 1:1 solution of DMEM and Ham’s F-12 media (Gibco), 10% horse serum, 1% penicillin/streptomycin and 1% hormone mixture (20 ng/mL EGF, 10ug/mL bovine insulin, 0.2 mM sodium bicarbonate and 0.5 ug/mL hydrocortisone). MCF-7 breast cancer cells were cultured in a 1:1 solution of DMEM and Ham’s F12 medium, 10% FBS and 1% penicillin/streptomycin. MB-231 and SK-Br-3 breast cancer cell lines were each cultured in RPMI media with 10% FBS and 1% penicillin/streptomycin. FRTL-5 immortalized rat thyroid cells were cultured in Coon’s modified F-12 media with 5% calf serum, 2 mM glutamine, 1% penicillin/streptomycin, 10 mM NaHCO3, and 6H hormone mixture (1 mU/mL bovine TSH, 10 ug/mL bovine insulin, 10 nM hydrocortisone, 10 ng/mL somatostatin, 5 μg/mL transferrin and 2 ng/mL L-glycyl-histidyl-lysine). All cells were maintained in a 37°C incubator with 5% CO2.
Western blot analysis and deglycosylation
Western blot analysis was performed as described [20
] with the following modifications. Protein extracts of 100 μg were subjected to 10% SDS-PAGE and transferred to a nitrocellulose membrane. The primary NIS antibodies used were: #331 human NIS (1:1000), FP-13 human NIS (1:5000) or PA716 rat NIS antibody (1:1500). HRP-conjugated anti-rabbit IgG secondary antibody (1:4000) was used for #331 and PA716 polyclonal NIS antibodies and anti-mouse IgG secondary antibody (1:4000) was used for FP-13 monoclonal NIS antibody.
For deglycosylation, 200 μg of protein were denatured with buffer (0.5% SDS and 1% B-mercaptoethanol) and incubated with 500 units of Peptide N-Glycosidase F (PNGase-F) (New England Biolabs, Ipswitch, MA). The deglycosylation reaction was enhanced by the addition of reducing sample buffer.
Radioactive iodide uptake assay (RAIU)
RAIU assay was performed as described in Knostman et al. [19
] with modifications. Counts per minute (CPM) were normalized to cell number. NIS-mediated RAIU was confirmed by comparing with the addition of NaClO4
NIS inhibitor in parallel experiments. Fisher Rat thyroid (FRTL-5) cells, an immortalized rat thyroid follicular cell line, served as a positive control.