Improvements on the Liquid Biopsy: Exosomes

Liquid biopsies have revolutionized cancer diagnostics by alleviating the traditional requirement of tissue biopsies. Beyond the cost benefits of liquid biopsies, in comparison to tissue, liquid biopsies are both easy to collect and less invasive, thus enabling more frequent and real-time assessment of tumor mutational status.  Further, liquid biopsies are thought to provide more holistic information of tumor mutational status by overcoming challenges of tumoral heterogeneity within a single tumor location as well as in the scenario of multiple metastatic lesions—a limitation of tissue biopsy.

Liquid biopsy approaches have matured over time. Early methods included circulating tumor cells (CTCs) and more recently, assessing circulating tumor DNA (ctDNA). Certainly, each approach has unique advantages and disadvantages. For example, CTCs provide the complete package of information, specifically DNA, RNA, and proteins. Further, as CTCs are intact cells, all that information could be assessed in a spatial context as well (ie, localization to various cellular compartments). That said, CTCs have been plagued by their low abundance in circulation, and doubts have been raised if CTCs have the colonization and metastatic potential once thought.¹ ctDNA is the most common approach to liquid biopsies today, with several FDA-approved tests on the market. ctDNA is released from dying cells through either apoptosis or necrosis and is found in greater abundance than CTCs. In theory, ctDNA may represent the mutational profile of all tumor loci in the body, thus avoiding the problem of tumor heterogeneity associated with tissue biopsy. Disadvantages of ctDNA include low prevalence in the blood, especially in early-stage cancers, largely limiting its utility to more advanced malignancies.

Enter the exosome.

Exosomes are small (30 - 200 nm) snippets of cells that are released into all biofluids in the body, such as blood, urine, cerebrospinal fluid, etc. They are part of a broad class of extracellular vesicles released into biofluids through either fusion with the plasma membrane and then exocytosis of multivesicular bodies, or direct budding of small cytoplasmic protrusion from the cell surface.² Their biological function is thought to be one of the main intercellular communication pathways, joining endocrine, paracrine, neuronal, and contact-mediated signaling. As exosomes are essentially small packets of cellular membrane and cytoplasm, they contain multiple analytes amenable to diagnostic testing, including DNA, RNA, proteins, lipids, oligosaccharides, and other metabolites.² In contrast to ctDNA, where only two copies of DNA are released per cancer cell following apoptosis or necrosis, exosomes are released at a rate of more than 20,000 exosomes per 48 hours from living cancer cells.³ This represents a significant advantage as not only are exosomes in much greater quantity than ctDNA, but the biological information they carry comes from the cells we are most concerned about…the surviving cancer cells.

While exosomes contain multiple biological analytes, most of the diagnostic progress related to exosomes has leveraged exosomal RNA, particularly messenger RNA (mRNA). Unlike free mRNA, exosomal RNA are protected from degradation and do not require special preservatives for collection. In fact, plasma can be stored for multiple days at room temperature and/or processed through multiple freeze-thaw cycles without significant degradation of exosomal DNA or RNA.⁴ Upon isolation, exosomal mRNA can be reverse transcribed and processed using routine downstream methods from reverse transcription polymerase chain reaction to next-generation sequencing. Beyond being a more abundant source to detect point mutations and indels, like other RNA sources, exosomal RNA have been used to detect alternatively spliced isoforms of RNA (ie, Arv7 and MET exon 14 skipping),^5,6 and fusion transcripts such as EML4-ALK.⁷

The true power of an exosome powered liquid biopsy comes from the improved sensitivity when combined with ctDNA. Recent studies have shown that combining exosomal RNA with ctDNA increases the total number of mutant copies detected within a sample. One clinically relevant study demonstrated this enhancement for assessing epidermal growth factor receptor (EGFR) activating mutations in lung cancer. Specifically, there was a near 10-fold increase in mutant copies detected when combining exosomal RNA and ctDNA (24 copies/mL versus 234 copies/mL).⁸ The potential implications of this improved sensitivity are not limited to better detection of clinically relevant variants in late-stage cancer but could extend to early-stage cancers as exosomes are released in such abundance from living cancer cells. This enhanced performance is not the future, as there are existing commercially available exosome isolation kits as well as diagnostic tests leveraging exosomes as a source of biological analytes.^9,10,11

As researchers and pathologists continue to utilize exosomes, there are obvious enhancements that can be applied. First, as exosomes are essentially cell membrane bound packets of cytoplasm, they contain cell surface markers that can be leveraged for further enrichment of tissue-specific exosomes. The significant advantage this provides is the enhancement of “signal to noise” from the off-target cells. While currently challenging and requiring optimization of antibodies and technique, studies have shown that further enrichment is possible from patient plasma samples.² One can imagine enrichment approaches can be generalized, such as pancytokeratins for the isolation of carcinomas, or more specific applications such as targeting CA 125 or CA 19-9 enriching for exosomal mRNA from ovarian or pancreatic carcinomas respectively.¹²

This article focused on the application of exosomes to liquid biopsies via exosomal mRNA, but as previously mentioned, exosomes contain a plethora of biomolecules that can be analyzed. Studies have shown successful isolation and detection of cancer-specific protein exosomal biomarkers such as EGFRvIII in brain cancer¹³ and HER2 in breast cancer,¹⁴ which through further studies may prove to have clinical utility. In addition, another biomolecule, exosomal DNA has the potential of enabling the detection of genomic rearrangements and copy number changes¹⁵ starting to round out the complete complement of biological information commonly utilized in cancer diagnosis and management. Beyond cancer, as exosomes are released by all living cells, there are numerous nononcology applications being explored today in areas such as transplant rejection, cardiovascular disease, neurodegenerative disease, fetal or prenatal diagnostics, and immunological disease.¹ This all sets the stage for the ultimate utility of exosomes as an easily collected, single source of various biomolecules, enabling true multiomic approaches to liquid biopsy in cancer and beyond. One can imagine multiomic approaches that achieve enhanced specificity and sensitivity while also providing more biological and clinically actionable information than any individual biomolecule can alone.

1. Rodrigues P, Vanharanta S. Circulating tumor cells: come together, right now, over metastasis. Cancer Discov. 2019;9:22-24.
2. Yu W, Hurley J, Roberts D, et al. Exosome-based liquid biopsies in cancer: opportunities and challenges. Ann Oncol. 2021;32(4):466-477
3. Balaj L, Lessard R, Dai L, et al. Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences. Nat Commun. 2011;2:180.
4. Enderle D, Spiel A, Coticchia CM, et al. Characterization of RNA from exosomes and other extracellular vesicles isolated by a novel spin column-based method. PLoS One. 2015;10(8):e0136133.
5. Del Re M, Biasco E, Crucitta S, et al. The detection of androgen receptor splice variant 7 in plasma-derived exosomal RNA strongly predicts resistance hormonal therapy in metastatic prostate cancer patients. Eur Urol. 2017;71(4):680-687.
6. Cortot AB, Kherrouche Z, Descarpentries C, et al. Exon 14 deleted MET receptor as a new biomarker and target in cancers. J Natl Cancer Inst. 2017;109(5):djw262.
7. Reclusa P, Laes J-F, Malapelle U, et al. EML4-ALK translocation identification in RNA exosomal cargo (ExoALK) in NSCLC patients: a novel role for liquid biopsy. Transl Cancer Res. 2019;8:S76-S78.
8. Krug AK, Enderle D, Karlovich C, et al. Improved EGFR mutation detection using combined exosomal RNA and circulating tumor DNA in NSCLC patient plasma. Ann Oncol. 2018;29(3):700-706.
9. EXO-NET Pan-Exosome Capture and Nucleic Acid Extraction Solutions. Promega. Accessed July 3, 2025. https://www.promega.com/products/nucleic-acid-extraction/rna/exo-net-pan-exosome-capture-and-maxwell-ht-mirna-extraction-kits/?catNum=CS3419A01&cs=y&gad_source=1&gad_campaignid=21871599277&gbraid=0AAAAAD_rg19ZqKvOU7V2TX6m9mbHwi8SD&gclid=EAIaIQobChMIvMDzp4bTjQMVcSdECB1h2SKBEAAYASAAEgLC1PD_BwE
10. Exosome Isolation. Qiagen. Accessed July 3, 2025. https://www.qiagen.com/us/product-categories/discovery-and-translational-research/exosomes-ctcs/exosomes?cmpid=PC_QF_SP_rna-purification-sales_0321_SEA_GA&gad_source=1&gad_campaignid=12501942132&gbraid=0AAAAAD-RrCyOkUY6qzRTi9qrS20rY_FH4&gclid=EAIaIQobChMIvMDzp4bTjQMVcSdECB1h2SKBEAAYAiAAEgKWAvD_BwE
11. QuantideX qPCR ESR1 exoMutation Kit. Bio-Techne Asuragen. Accessed July 3, 2025.  https://asuragen.com/portfolio/oncology/quantidex-esr1-exomutation-kit/
12. Im H, Shao H, Park YI, et al. Label-free detection and molecular profiling of exosomes with a nano-plasmonic sensor. Nat Biotechnol. 2014;32:490-495.
13. Al-Nedawi K, Meehan B, Micallef J, et al. Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Nat Cell Biol. 2008;10(5):619-624.
14. Grasso L, Wyss R, Weidenauer L, et al. Molecular screening of cancer-derived exosomes by surface plasmon resonance spectroscopy. Anal Bioanal Chem. 2015;407(18):5425-5432.
15. Lee DH, Yoon H, Park S, et al. Urinary exosomal and cell-free DNA detects somatic mutation and copy number alteration in urothelial carcinoma of bladder. Sci Rep. 2018;8:14707.

Jordan Laser, MD, is a board certified anatomic, clinical, and molecular genetic pathologist. Jordan is the Senior Director of Clinical and Medical Affairs at Bio-Techne as well as the CEO and Founder of Laser Laboratory Consulting. Previously, he served as Chief Laboratory Officer at Everly Health. The laboratory services at Everly Health support a wide range of consumer-initiated at-home tests. Having roots in New York, Jordan spent his first 11 years of practice as a pathology leader at Northwell Health. His expertise includes molecular and genomic medicine, laboratory management, healthcare finance, and standards and regulations.