gender: M age at diagnosis: 37 cohort: Wistar cell type: primary cutaneous melanoma tissue
Treatment protocol
N/A None
Growth protocol
This cohort includes human cell lines (n=5, in triplicate). WM35, WM983, and WM1552c were grown in 2% tumor media containing a 4:1 mixture of MCDB 153 medium with 1.5 g/L sodium bicarbonate and Leibovitz's L-15 medium with 2 mM L-glutamine supplemented with 0.005 mg/ml bovine insulin, 1.68 mM CaCl2, and 2% fetal bovine serum. . YUPEET and YUCHIME were grown in bFGF media (OPTIMEM, 5% FBS, 10ng/ml bFGF, 1ng/ml heparin, 0.1mM dbcAMP, 0.1mM IBMX.) This cohort includes human tissue obtained from FFPE (n=205), fresh frozen human tissue (n=24), and human cell lines (n=12).
Extracted molecule
protein
Extraction protocol
Cell were lysed using lysis Buffer (1% Triton X‐100, 50mM HEPES, pH 7.4, 150mM NaCl, 1.5mM MgCl2, 1mM EGTA, 100mM NaF, 10mM Na pyrophosphate, 1mM Na3VO4, 10% glycerol, containing freshly added protease and phosphatase inhibitors) for 30 minutes while shaking. The cell lysates were centrifuged at 14,000 rpm for 10 minutes at 4C. Supernatant was collected, pellet discarded. Protein concentration was quantified by Qubit and adjusted to 1.5 ug/ul. (using lysis buffer to dilute). The cell lysate was mixed with (4XSDS + B‐Me) sample buffer without bromophenol blue (3 parts of cell lysate plus one part of 4XSDS sample buffer). The samples were boiled for 5 minutes, and stored in –80C until sample submission. RNA was obtained from the FFPE human tissue samples using the RecoverAll Total Nucleic Acid Isolation kit. RNA was obtained from the fresh frozen human tissue samples using the Qiagen Rneasy kit. Cell lines were pelleted and RNA was harvested using the Qiagen Rneasy kit.
Label
NA
Label protocol
NA Specimens were labeled with decode assignment which was linked to tumor origin.
Hybridization protocol
Serially diluted cellular proteins are arrayed on nitrocellulose‐coated slides and probed with validated antibodies that recognize signaling molecules in their functional state. Signals are captured by tyramide dye deposition and a DAB colorimetric reaction. 200 ng RNA was hybridized to a custom NanoString panel code set according to the recommendations of the manufacturer (NanoString Technologies, Inc., Seattle, Washington, USA).
Scan protocol
Data is collected and quantitative analysis is performed using custom “Supercurve”software developed for this purpose. The values derived from the slope and intercept are expressed relative to standard control cell lysates or control peptides on the array. Thesevalues indicate the levels of protein expression and modification (phosphorylation or cleavage based on antibody specificity) Following hybridization, the target-probe complexes were purified and immobilized on the nCounter prep station. Digital counts for each gene-specific target RNA were then acquired on the nCounter detection analyzer and normalized to account for differences in tissue mRNA quantity as well as slight differences in assay efficiency.
Data processing
Each dilution curve was fitted with a logistic model (“Supercurve Fitting” developed by the Department of Bioinformatics and Computational Biology in MD Anderson Cancer Center, “http://bioinformatics.mdanderson.org/OOMPA”). This fits a single curve using all the samples (i.e., dilution series) on a slide with the signal intensity as the response variable and the dilution steps are independent variable. The fitted curve is plotted with the signal intensities – both observed and fitted ‐ on the y‐axis and the log2‐concentration of proteins on the x‐axis for diagnostic purposes. The protein concentrations of each set of slides were then normalized for protein loading. Correction factor was calculated by: 1) median‐centering across samples of all antibody experiments; and 2) median‐centering across antibodies for each sample. The nSolver analysis software version 4.0 (NanoString Technologies) was used for extraction of raw digital counts of expression, checking the quality of the data, normalizing expression values using housekeeping genes, and generating heatmaps.
