Role of exosomal micrornas in regulating the immune response to lung cancer

Abstract

Lung cancer is one of the most frequent malignant neoplasms and the main cause of cancer death. Despite medical advances, survival remains low in non-small cell lung cancer (NSCLC), the commonest type. Macrophages play a substantial role in tumour progression because of their plasticity during activation and tumour infiltration. M1 polarised macrophages are associated with increase survival in lung cancer but tumour-associated macrophages (TAM) are distinct in their potential to promote or hinder tumour development. There has been increasing recognition of molecular drivers of cancer and amongst them, exosomes are thought to modulate the wider tumour micro-environment. Exosomes are extracellular vesicles secreted by all cell types and facilitate remodelling, immune escape promotion and tumour development in the tumour microenvironment. Exosomes can transport a diverse cargo of RNA, DNA, and protein. MicroRNAs form a potentially important exosomal cargo which may affect entire cellular pathways of recipient cells, such as macrophages. Identifying, which miRNAs are associated with these processes may be essential in management of lung cancer patients.
I propose that TAMs are affected by exosomes generated by tumours, and this affects their ability to respond to the tumour. The hypothesis is that differentially expressed specific exosomal miRNAs derived from NSCLC tumour alters the phenotype of macrophages resulting in immune regulation of macrophages in the tumour microenvironment. In this project, I characterised tumour derived exosomes (TDE) and their paired normal lung tissue derived exosomes (NDE) and shown that I can sequence their microRNA cargo. The differential expression testing with DESeq2 of TDE and NDE identified 465 differentially expressed miRNAs. Eight miRNAs (miR-21-5p, miR-100-5p, miR-101-3p, miR-126a-5p, miR-133a-3p, miR-149-5p, miR193a-3p, and miR-205-5p) were statistically significantly differentially expressed and involved in signaling pathways related to polarisation of macrophages towards the anti-inflammatory phenotype M2. Those pathways were PI3K/Akt/mTOR, TLRs/NF-κB, JAK/STAT and JNK/MAPK.
I subsequently compared and correlated the targeted mRNA of the TDE versus NDE differentially expressed miRNAs with the differentially expressed mRNA from tumour associated macrophages (TAM) and non-tumour associated macrophages (NTAM). Eleven miRNAs (miR-1-3p, miR-105-5p, miR-126a-3p, miR-16-5p, miR-193a-3p, miR-21-5p, miR2682-5p, miR-30a-3p, miR-34a-5p, miR-503-5p, miR-9-5p) were identified. The dysregulated mRNA and their signaling pathways were like the affected pathways from differentially expressed miRNAs in the TDE and NDE comparison group. These were PI3K/Akt/mTOR, TLRs/NF-κB and JAKSTAT signaling pathway. I subsequently exposed in vitro derived macrophages to TDE and their paired NDE. My results indicated that macrophages exposed to TDE had a milder inflammatory profile (dysregulation of IL6, SOCS-1, Serpin-B1 and CCL18) than those exposed to paired NDE. These macrophages were subsequently subjected to Next Generation Sequencing (NGS)/RNA to evaluate their transcriptome (mRNA and microRNA) expression. The aim was to establish if miRNA cargo can affect macrophage’s phenotype. The miRNA signature of those macrophages showed one significant differentially expressed miRNA (miR-451a) which is known to be dysregulated in cancer and has anti-inflammatory effect. My work has potential impact in identifying the effects of tumour derived exosomes and particularly their miRNAs in macrophage’s polarisation in the tumour microenvironment.

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Identifiers

ORCID for Tilman Sanchez-Elsner: orcid.org/0000-0003-1915-2410

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