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RNASeq analysis of endothelial cells isolated from B16F0 tumours treated with IgG/ 15C4.

ABSTRACT: LRG1 is a novel proangiogenic and profibrotic molecule that is upregulated in pathologies characterised by abnormal angiogenesis such as AMD and cancer. Blocking LRG1 by genetic models or by our in-house developed function blocking antibody leads to vascular normalsation. In this study, we have treated B16F0 melanoma tumours with 15C4, our function blocking antibody, or with IgG as a control. The tumours were collected at day 17 and the endothelial cell population was isolated by FACS sorting the CD31 positive cells. RNA was isolated and used for the preparation of cDNA libraries. Expression profile of the endothelial cell population was assessed by Illumina sequencing. Differenital expression analysis was conducted using the DESeq2 package in R.

ORGANISM(S): Mus musculus

PROVIDER: GSE184816 | GEO | 2021/09/27

REPOSITORIES: GEO

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Project description:As a type of secreted membrane vesicle, exosomes are emerging as an important mode of cell-to-cell communication. The objective of this study was to compare the abundance of transcripts present in the parental B16F0 cell to transcripts present in exosomes isolated from B16F0 conditioned media. Identifying local mechanisms of immunosuppression is a key barrier for expanding the clinical benefit of cancer immunotherapy. While exosomes are emerging as a new mode of intercellular communication, their role in establishing a malignant tissue niche remains unclear. Similar to the sculpting of tumor antigens during oncogenesis, a related hypothesis is that proteins secreted by malignant cells are shaped by somatic evolution. To test this hypothesis, we characterized the biological influence of tumor-derived exosomes on immune cell function. In particular, we analyzed exosomes from three melanoma models: B16F0, a non-immunogenic model of malignant melanoma; Cloudman S91, a model of immunogenic melanoma; and Melan-A, an immortalized melanocyte cell line. Using electron microscopy, exosomes derived from all three cell lines were morphologically similar. The exosomes contained receptors derived from the parent cell as demonstrated by IL12RB2 expression on B16F0 exosomes and intact mRNAs. Furthermore, transcript profiling of B16F0 exosomes and cells suggested that exosomal mRNA is enriched for mRNAs that target immune-related pathways, including Ptpn11 that inhibited T cell proliferation and Dnmt3a that inhibited T cell production of IFN-gamma. Functionally, B16F0 exosomes dose-dependently suppressed cell proliferation and the expression of IL12RB2 in primary CD8+ T cells. In contrast, Cloudman S91 exosomes promoted T cell proliferation and Melan-A exosomes had a negligible effect on primary CD8+ T cells. Collectively, the results are consistent with somatic editing of exosomal payloads and suggest that exosomes establish a density-dependent field effect by altering the activity of immune cells that enter the tumor microenvironment.

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RNASeq analysis of endothelial cells isolated from B16F0 tumours treated with IgG/ 15C4.

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