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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE199461 | GEO | 2023/04/13

REPOSITORIES: GEO

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Project description:Purpose: Transport processes in the renal collecting duct are responsible for precise regulation of blood pressure and body fluid composition. The collecting duct is composed of at least three cell types, type A intercalated cells (A-IC), type B intercalated cells (B-IC) and principal cells (PC). To identify the genes that are selectively expressed in each cell type, including cell-surface receptors, transcription factors, transporters and secreted proteins, we used cell surface markers necessary for isolation of each of the three cell types using fluorescence-activated cell sorting and carried out single-cell RNA-Seq to measure the mRNA species in each of these cell types. Methods: We enriched each of the three cell types using fluorescence-activated cell sorting. Subsequently, we carried out single-cell RNA-Seq of those cells using a microfluidic chip (Fluidigm C1 system). Single-cell cDNA libraries were constructed for paired-end sequencing and sequenced on Illumina HiSeq3000 platform. In addition, we also microdissected mouse CCDs and cTALs and carried out single-tubule RNA-Seq. Reads were mapped to mouse Ensembl Genome by STAR and transcript abundances were calculated in the units of transcripts per million (TPM) using RSEM (https://github.com/deweylab/RSEM). Single-cell RNA-Seq data anaysis were carried out using Seurat package in R. Results and conclusion: Single-cell cDNA libraries were constructed for paired-end sequencing and at least 10-million sequence reads per cell were mapped to the mouse genome (Ensembl, GRCm38.p5). On average, cells were sequenced to a depth of 3000 genes (TPM>1). Unsupervised clustering analysis revealed five different cell types, namely type A intercalated cells (n=87), type B intercalated cells (n=23), principal cells (74), proximal tubule cells (n=19), and non-epithelial cells (n=32). The identified patterns of gene expression among A-ICs, B-ICs and PCs provide a foundation for understanding physiological regulation and pathophysiology in the renal collecting duct.

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

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