mRNA and Protein Expression Analysis

For mRNA expression, qRT-PCR analysis was performed using predefined TaqMan probes (Supplementary Data 1) chosen from the Thermo Fisher Scientific database (http://www.thermofisher.com). Using the High Capacity cDNA Reverse Transcription kit (Thermofisher), 1.5 µg of total RNA was reverse transcribed in a final volume of 50 µl. qPCR reactions were done using the high throughput BioMark HD system (Fluidigm) following manufacturer’s instructions. Pre-amplifications of 6 ng cDNA were performed using PreAmp Master Mix (Fluidigm) with a primer mix combining each primer used in the present study except the 18S probe due to it very high gene expression level. Expression data (Ct values) were acquired using the Fluidigm Real Time PCR Analysis software. The mean of 5 housekeeping genes (18S, ACTB, CLTC, GAPDH, and TBP) was used for the normalization of expression data. For protein expression, preparation of cells lysates from 30 frozen tumor samples and reverse phase protein array (RPPA) were performed as previously described^{42 (link)}. Array was revealed with the anti-PD-L1 monoclonal antibody (clone E1L3N; Cell Signaling Technology).