Exosome-based liquid biopsies in cancer: opportunities and challenges

Abstract

Liquid biopsy in cancer has gained momentum in clinical research and is experiencing a boom for a variety of applications. There are significant efforts to utilize liquid biopsies in cancer for early detection and treatment stratification, as well as residual disease and recurrence monitoring. Although most efforts have used circulating tumor cells and circulating tumor DNA for this purpose, exosomes and other extracellular vesicles have emerged as a platform with potentially broader and complementary applications. Exosomes/extracellular vesicles are small vesicles released by cells, including cancer cells, into the surrounding biofluids. These exosomes contain tumor-derived materials such as DNA, RNA, protein, lipid, sugar structures, and metabolites. In addition, exosomes carry molecules on their surface that provides clues regarding their origin, making it possible to sort vesicle types and enrich signatures from tissue-specific origins. Exosomes are part of the intercellular communication system and cancer cells frequently use them as biological messengers to benefit their growth. Since exosomes are part of the disease process, they have become of tremendous interest in biomarker research. Exosomes are remarkably stable in biofluids, such as plasma and urine, and can be isolated for clinical evaluation even in the early stages of the disease. Exosome-based biomarkers have quickly become adopted in the clinical arena and the first exosome RNA-based prostate cancer test has already helped >50 000 patients in their decision process and is now included in the National Comprehensive Cancer Network guidelines for early prostate cancer detection. This review will discuss the advantages and challenges of exosome-based liquid biopsies for tumor biomarkers and clinical implementation in the context of circulating tumor DNA and circulating tumor cells.

Keywords: cell-free DNA (cfDNA); circulating tumor DNA (ctDNA); circulating tumor cell (CTC); exosome; extracellular vesicle; liquid biopsy.

Figure 1.. ctDNA, exosomes, and CTCs in circulation.

The molecular constituents from the tumor are released into biofluids at various stages of the tumor development through very different mechanisms and represent different biological entities. ctDNA is released from the dying tumor cells through apoptosis and/or necrosis, exosomes are actively released from cells at each stage of tumor formation, whereas CTCs are intact cancer cells disseminated from the tumor at later stages. Exosomes are involved in promoting the growth, vascularization, and even the dissemination and metastatic process of cancer. Elements are not drawn to scale. CTCs, circulating tumor cells; ctDNA, circulating tumor DNA.

Figure 2.. Exosomes enable cancer specific enrichment.

Proteins overexpressed in a tumor are not necessarily cancer-specific when isolated from the blood or other biofluid. Non-cancerous tissues can produce the same marker and generate a variable amount of the marker in normal individuals. By enriching exosomes with a tissue-specific (or cancer-specific) marker, higher sensitivity and/or specificity can be achieved. There are also known neoantigens (for example EGFRv3) that can be used to isolate cancer-specific exosomes.^,

Figure 3.. Multi-analyte liquid biopsy approach utilizing exosomes for increased performance.

Combining features from cfDNA and exosomes have clear synergies to achieve increased liquid biopsy performance. The exosome fraction contains RNA and DNA (exoDNA) that give increased copy numbers of mutations and enable splice variant analysis of RNA,^– fusions,^, and transcriptome analysis.^, Combining the ctDNA mutations with cfDNA methylation and/or exosome analysis can enable a more complete picture of the tumor-derived biomarkers and potentially confer a better diagnostic performance. Exosomes from various sources (exemplified by the colors) can also be enriched to increase tissue specificity of the biomarker. cfDNA, cell-free DNA; ctDNA, circulating tumor DNA.

Figure 4.

(A) Exosome-based research publications. The interest in exosomes for diagnostics, therapeutics and as an intricate part of intercellular communication has significantly increased in recent years. (B) Overview of a total of 71 exosome-based clinical trials registered worldwide in ClinicalTrials.gov (as of 22 October 2020) in diagnostics and translational biomarkers for various cancers, manually curated through searches with keywords (exosomes, extracellular vesicle, EVs, and microvesicles) with the elimination of both duplicate hits and those for therapeutic applications.