Breast tumors interfere with endothelial TRAIL at the pre-metastatic niche to promote cancer cell seeding

Purpose: To study the effects of endothelial cell (EC)-specific TRAIL depletion on lung endothelial cells. Here, we used RNA sequencing to explore the transcriptional changes of lung ECs upon deletion of TRAIL specifically in ECs. RNA from 2.000.000 sorted lung ECs (viability dye- CD45- CD31+) from EC-specific TRAIL WT and KO mice (Cdh5.iCreERT2xTnfsf10Lox/Lox) were isolated and the concentration as well as purity was determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies). Per sample, an amount of 3000ng of total RNA was used as input. Subsequently, preparation was performed with KAPA stranded mRNA seq according to the protocol and samples were pooled equimolar. Sequencing was performed as single end of 50bp on hiseq 4000 from Illumina. 35-40 Million reads were generated per sample. Raw FASTQ reads were mapped using STAR v2.7.1a and gene reads counts were estimated by RSEM v1.2.28 using GENCODE reference annotation of the mouse genome (GRCm38), version M24 (Ensembl 99). Gene reads counts were estimated by RSEM using GENCODE reference annotation of the mouse genome (GRCm38), version M24 (Ensembl 99). Differential expression of EC-specific TRAIL KO (EC-Tg/W) vs control (EC-W/W) was performed using the DESeq2 R package (v1.26.0). The significance was determined by padj < 0,05. The gene set enrichment analysis (GSEA) was performed using the pre-ranked gene list by log2 fold-change (EC-Tg/W vs EC-W/W control) and the MSigDB gene sets. Only gene sets containing between 5 and 500 genes were retained and the number of permutations was set at 1000. Overall design: 5x5 design in Cdh5.iCreERT2xTnfsf10Lox/Lox mice (5 W/W and 4 Tg/W) with sorted lung ECs RNA sequencing conducted