CTC Separation: Circulating Tumor Cells Isolation Methods and CTC Isolation from Whole Blood
Updated on Aug 21, 2023 Share
CTC Separation
When a cancerous tumor is present in the human body, some of the tumor cells can be shed into blood or lymph vessels, where they travel through the bloodstream alone or in clusters. These cells are called circulating tumor cells (CTCs). Scientists can collect these cells for research purposes through a process called CTC separation.
CTC separation is the process of CTC isolation from whole blood samples. Residual blood cells such as red blood cells (RBCs), white blood cells (WBCs), and other substances can all have a negative effect on the clarity of downstream experimental results. Therefore, it is important to remove them from a CTC sample when preparing for research.
CTC Structure
CTCs are both identified and differentiated by their composition. These cells have a nucleus and test positive for the cytoplasmic expression of cytokeratin, a type of protein that facilitates cell growth. They also test negative for the CD45 protein, which helps to restrict cell growth. Beyond the typical indications of a cancer cell, size plays a vital role in CTC detection within a sample.
The circulating tumor cell size can provide hints as to which form of cancer may be present in a patient’s body. Typically measured in microns (µm), which are equal to one millionth of a standard meter, CTCs vary in size depending on where the host tumor is located. For example, one study on prostate cancer found CTCs to be between 8-16 µm, while a study on non-small-cell-lung cancer found the average CTC size to be 30 µm. The ability to identify the origin of CTCs by their size and composition is one of the many reasons they’re beneficial to the medical community.
CTC Significance
One impactful research field for CTC separation is the detection of tumor cells in their stage of circulation through the body. CTC separation plays an important role assisting oncologists in early cancer diagnoses because the cells can often be detected in the blood before imaging is able to detect a tumor. CTC separation can also help doctors determine what specific type of treatment a patient should receive for the cancer they have, allowing for more individualized and effective treatments.
Additional benefits include the ability to study how cancer spreads and metastasizes, how different cancers affect the body, and how to identify the threat level of a tumor from the bloodstream. This means CTC separation has the great opportunity to positively influence patient treatment and outcome.
To properly study CTCs, it is critical that they can be isolated and purified. This process of isolation and purification can be performed through cell separation techniques or with CTC Enrichment.
CTC Isolation Methods
As far as cell separation techniques are concerned, CTCs are typically isolated using the following methods:
- Magnetic activated cell sorting (MACS)
- Density-gradient centrifugation
- Fluorescence activated cell sorting (FACS)
- Buoyancy activated cell sorting (BACS)
Each of these methods have advantages and disadvantages associated with them.
MACS
Magnetic activated cell sorting, or MACS, is one approach to CTC separation. This technique uses antibody-antigen receptors to link target cells with magnetic beads. Once the beads have been attached to their target, the sample is flown through a magnetic field that holds onto the magnetic bead-bound targets, isolating them as the remainder of the sample passes through.
While MACS protocols are commonly used, the harsh magnetic field can cause fragile cells to rupture or lyse. Once the cell membrane is destroyed, extracellular debris may cause clumping in the sample. Clumping and cell death can both decrease the overall throughput of an experiment. In addition, MACS requires the use of specialized equipment and consumables like magnetic beads, rare earth magnets, and columns, as well as the appropriately trained personnel to properly use, store, and maintain it.
Density-Gradient Centrifugation
An alternative to MACS is density-gradient centrifugation. This is a process that uses a mechanical device called a centrifuge to spin samples at high speeds. As the solution rotates, similar particles group together based on relative density and form isolated populations. The addition of a substance that only certain cells can filter through–a density gradient–allows for smooth separation of particles based on their physical properties.
While centrifugation is known for its ability to process large samples at once, it is not always the most practical choice when dealing with smaller samples or delicate cells that can be damaged by the forces involved.
FACS
Another CTC separation method is fluorescence activated cell sorting, or FACS. FACS uses a flow cytometer machine to sort cells based on their physical characteristics after assessing them with a series of lasers and sensors.
FACS is useful when sorting complex samples that require multiple isolated cell populations, but isn’t always practical. FACS requires expensive machinery, large amounts of time, and can cause cells to rupture when passing through the flow cytometer.
MACS, centrifugation, and FACS all have hidden costs and the potential to damage cell populations. Luckily, there is one more CTC separation technique that addresses many of the issues with these traditional methods.
BACS
Akadeum Life Sciences has developed an innovative technology called BACS. BACS, or buoyancy activated cell sorting, uses microscopic bubbles to float unwanted substances to the top of a solution for removal, leaving behind the enriched sample. By binding target cells to the bubbles with antigen-antibody receptors, the removal of unwanted cells like red blood cells (RBCs) becomes easy and efficient.
Using a BACS depletion workflow, Akadeum’s functionalized microbubbles are gently mixed into the sample where they bind to the unwanted cells, floating them to the top of the sample container for removal. The remainder of the sample remains untouched and ready for downstream use. Akadeum’s Human Red Blood Cell Depletion microbubbles use a fast and easy 3-step workflow that takes only 10 minutes to complete while maintaining the health and physiology of cells.
BACS and CTC Separation
Enriching for rare cells from blood or tissue preparations presents significant sample preparation challenges. Current methods for CTC preparation frequently result in significant residual RBC contamination, negatively impacting downstream analysis, or are harsh and damaging to cells. Microbubble technology can remove up to 99% of contaminating RBCs in under 10 minutes start-to-finish while maintaining the health and physiology of cells of interest. By binding to the surface markers identified by specific antigens in the bubbles, the removal of RBCs and other unwanted cells is fast and easy. Akadeum’s Red Blood Cell Depletion Microbubbles allow for enrichment of rare target populations in 10 minutes, enabling higher efficiency of downstream applications like single-cell analysis.
To learn more about how Akadeum’s microbubbles can be used to enrich for rare circulating cells like CTCs, you can download our recent poster presentation, Enrichment of Rare Circulating Cells Using Microbubble-Based Preparation, that Akadeum recently presented at the 12th Scientific Workshop of the Early Detection Research Network, 20th Year of EDRN: Making Cancer Detection Possible.