The collection and analysis of exosomes

 

Jeya Chelliah B.VSc Ph.D.

Collecting exosomes from cancer patients for diagnostic, prognostic, or therapeutic monitoring purposes involves a series of steps designed to isolate these nanoscale extracellular vesicles from various bodily fluids. The process is non-invasive or minimally invasive, making it an attractive option for patient monitoring over time. Here’s an overview of how exosomes can be collected and isolated from cancer patients:

1. Sample Collection

Exosomes can be isolated from a wide range of bodily fluids, including but not limited to:

• Blood (Serum/Plasma): The most common source for exosome isolation due to the ease of collection and the high volume of exosomes present. Blood samples are usually collected via venipuncture.
• Urine: Another valuable source for exosome collection, especially for urogenital cancers.
• Saliva: Increasingly recognized for head and neck cancers, among others.
• Cerebrospinal Fluid (CSF): For central nervous system cancers.
• Ascites: Fluid collected from the peritoneal cavity, often in advanced cancer stages.
• Pleural Effusions: Fluid collected from the pleural space around the lungs, in cases of lung cancer or metastases to the pleura.

2. Sample Processing

To prepare the samples for exosome isolation, initial processing steps are required:

• Centrifugation: The collected fluid is first subjected to low-speed centrifugation to remove cells and cell debris. This step might be repeated or followed by increased speed centrifugation to further clarify the sample.
• Filtration: The supernatant from centrifugation is often filtered through a 0.22 µm filter to remove any remaining larger particles.

3. Exosome Isolation

Several techniques are available for isolating exosomes from processed bodily fluids, each with its advantages and limitations:

• Differential Ultracentrifugation: The traditional method, involving a series of centrifugation steps at increasingly higher speeds to pellet exosomes.
• Size-Exclusion Chromatography (SEC): This method separates exosomes based on size, allowing for the collection of exosome-rich fractions.
• Polymeric Precipitation: Commercial kits are available that precipitate exosomes from the sample using a polymer. This method is straightforward but may co-isolate non-exosomal proteins.
• Immunoaffinity Capture: Targets specific markers on the exosome surface, allowing for the isolation of specific exosome populations. This method is highly selective but can be more costly and time-consuming.

4. Exosome Verification and Analysis

After isolation, it’s crucial to verify the presence of exosomes and assess their quantity and quality:

• Nanoparticle Tracking Analysis (NTA): Measures the size distribution and concentration of exosomes.
• Transmission Electron Microscopy (TEM): Provides visual confirmation of exosome morphology.
• Western Blot: Used to detect exosomal markers (e.g., CD9, CD63, and CD81) and ensure the isolated particles are indeed exosomes.

5. Downstream Applications

Once isolated and verified, exosomes can be analyzed for their content, including proteins, DNA, and RNA, to provide insights into the cancer’s molecular characteristics, monitor disease progression, or assess response to therapy.

The collection and analysis of exosomes from cancer patients hold significant promise for improving cancer diagnosis, prognosis, and treatment monitoring. As technologies advance, the efficiency and specificity of exosome isolation and analysis methods are expected to improve, further enhancing their utility in clinical oncology.