Cell Culture and Cell Microarray
Human (A2058, A-375, G-361, RPMI-7951, SH-4, SK-MEL-1, SK-MEL-3, SK-MEL-24, and SK-MEL-28) cell lines were obtained directly from ATCC and were cultured following ATCC’s recommendations. The WM35, WM115, WM164, WM278, WM793, WM852, WM1341D, 451Lu, and 1205Lu cell lines were obtained from the Wistar Institute and cultured with tumor specialized media containing 2% FBS. The characteristics of these melanoma cell lines are highlighted in Supplemental Table 1.
To prepare cell pellet blocks, cells were grown in T-75 flasks to near confluence then washed 3 times with PBS and fixed with 10% neutral-buffered formalin (NBF) in flasks overnight. After fixation, cells were gently scraped and transferred to a conical tube and centrifuged to ~300 x g for 5 min at room temperature (RT). Formalin supernatant was then removed and pellets were washed with PBS. Finally, cell pellets were re-suspended in 80% ethanol and paraffin embedded in cell blocks. A tissue microarray (TMA), with 1.5 cm cores for each cell line, was then derived from the cell blocks. Two TMA slides were stained for MET and two were stained for PD-L1. The average percentage of positively stained cells and FastRed mean intensity were calculated.
Tissue Microarray and Patient Characteristics
TMA slides with 100 cores were purchased from Biomax (Cat. # ME1004g; US Biomax, Inc., Derwood, MD). Available clinicopathological characteristics are summarized in Supplemental Table 2. The mean age of the patients from this TMA was 50 years (range 0.5 - 84 years). The study included 58% (58) males and 42% (42) females. Staging (TNM and clinical staging) was only provided for 48 patients which include 42 cases of primary cutaneous melanoma and 6 cases of mucosal melanoma. Overall, the TMA included 42 cases with cutaneous malignant melanoma, 20 mucosal melanomas (including malignant melanoma from vulva, rectum, stomach and esophagus), 21 cases were obtained from metastatic sites including lymph nodes and 17 cases were benign melanocytic nevi.
Two TMA slides were stained for MET and two were stained for PD-L1. The average percentage of positively stained cells and FastRed mean intensity were calculated.
Unstained TMA sections (4 µm) were de-paraffinized and rehydrated using standard methods. For antigen retrieval, slides were incubated in 6.0 pH buffer (Reveal Decloaking reagent, Biocare Medical, Concord, CA) in a steamer for 30 min at 95-98°C, followed by a 20 min cooldown period. Subsequent steps were automated using an immunohistochemical staining platform (Intellipath, Biocare). Endogenous peroxidase activity was quenched by slide immersion in 3% hydrogen peroxide solution (Peroxidazed, Biocare) for 10 min followed by TBST rinse. A serum-free blocking solution (Background Sniper, Biocare Medical, Concord, CA) was placed on sections for 10 min. Blocking solution was removed and slides were incubated in primary antibody diluted in 10% blocking solution/90% TBST for 60 minutes at room temperature. Rabbit monoclonal anti-MET (clone D1C2 XP(R)(Cell Signaling, Denver, MA;1:50), followed with a TBST rinse and detection with Novocastra Novolink Polymer Kit (Leica Microsystems Inc., Buffalo Grove, IL) using the manufacturer’s specifications. Slides then proceeded with TBST rinse and detection with diaminobenzidine (DAB) (Covance, Dedham, MA). Slides were incubated for 5 min followed by TBS rinse then counterstained with CAT Hematoxylin (Biocare, Concord, CA) for 5 min. Finally, slides were dehydrated and coverslipped.
Rabbit monoclonal anti-PD-L1 (clone 28-8, Cell Marque, Rocklin, CA, 1:200) was followed by a TBST rinse and biotinylated anti-rabbit (Vector Labs, Burlingame, CA,1:200) was applied for 30 minutes. The slides were again rinsed with TBST and 4+ Streptavidin- Alkaline Phosphatase label (4+SA-AP) (Biocare Medical, Concord, CA, RTU) was applied for 30 minutes at room temperature. Slides proceeded to a TBST rinse and detection with WARP Red Chromagen (Biocare Medical, Concord, CA) according to manufacturers’ specifications. Following detection, slides were rinsed well in running tap water and counterstained for 1 minute in CAT Hematoxylin (1:2) (Biocare Medical, Concord, CA). Tap water rinse and 2 minutes in PureView PH Blue (Cancer Diagnostics, Durham, NC), 5 minute tap water rinse and then slides were air dried. When completely dry, slides were dipped in xylene and coverslipped with Permount mounting medium (Fisher, Fair Lawn, NJ).
TMA slides were scanned using an Aperio scanner, with a 40X objective. The high resolution images were analyzed using QuPath  version 0.1.2. The workflow consisted of (i) color deconvolution, (ii) identifying the TMA cores, (iii) segmenting the tissue region in each core, (iv) isolating nuclear and cytoplasmic regions of interest, (v) estimating the abundance of the deconvolved red component in each cell and finally (vi) calculating the percentage of positive cells in each core. 3-color deconvolution was performed using vectors calibrated visually on the image data, following the procedure outlined in the software documentation. All images were deconvolved using the same stain vectors. Steps (iii) to (v) were performed using the default algorithms in QuPath. For step (vi), each cell was considered positive if the red component intensity was above a threshold, calculated independently for each TMA slide as the average between the 5th and 95th percentile of red intensities across the slide. All algorithms and parameters for the analysis in QuPath were recorded in a script for repeatability (see Supplementary Material).
Statistical analysis was primarily descriptive. Correlation between percentage of MET- and PD-L1-positive cells was calculated using Pearson’s correlation coefficient (r) and its 95 % confidence interval calculated by the 2.5 and 97.5 percentiles of 500 bootstrap resamplings.