Capture and Filtration of Circulating Tumor Cells

Filtration of circulating tumor cells

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Why we need to capture tumor cells

How do we detect cancers without doing a biopsy? Can we continually monitor the progress of tumors without needing CT scans? What about monitoring a patient’s response to chemotherapy? It’s simple to take a patient’s blood but can these samples tell us if a patient has circulating tumor cells? All these questions relate to our topic for today.

As cancers grow, invade, and metastasize, some cells enter into the bloodstream. These circulating tumor cells (CTCs) give a picture of the patient’s health. A pathologist may be able to determine whether the patient’s latest chemotherapy regime had much effect on the cancer’s growth. One could even determine if a cancer has shifted from being a grade 1 tumor to a grade 3 or 4 by measuring the blood CTC count. Furthermore, using circulating tumor cells, we can design personalized medicine for the patient and even change the medicine once the patient’s disease morphs (as is the case with cancers).

Circulating tumor cells from cancer

Blood tests are the easiest and most accessible way to get samples with circulating tumor cells. Because of their ease of acquisition, they are repeatable and can be used to monitor patients more frequently than biopsies. However, blood tests also come with their own challenges.

Challenges of capturing tumor cells from blood draws

There are several challenges associated with capturing tumor cells in blood:

  • Circulating tumor cells are rare: The ratio of CTCs to normal cells is about 1: 1e9. Isolating such a rare cell type is incredibly hard!
  • Blood is complex: Blood from a cancer patient is a complex mixture of proteins, white cells, red cells, carbohydrates, platelets, single tumor cells, and tumor cell clusters.
  • Tumor cells morph: Because the cell surface markers of tumor cells change over time, a single biomarker cannot be used to identify circulating cells
  • Cells are fragile: Many downstream tests rely on intact tumor cells for analysis. So, fragile tumor cells and blood cells undergoing any kind of harsh purification strategies aren’t useful for downstream diagnostic analyses.
  • Tumor cells are complex: Not all tumor cells express the same surface markers. This is particularly challenging when using biochemical methods for isolation and purification of CTCs.

Complex blood mixture with circulating tumor cells

Biochemical capture of circulating tumor cells

By far the most common way to capture CTCs, is using antibodies or other biochemical tools. One of the most successful methods of capturing CTCs is CellSearch, a product made by Janssen Diagnostics (owned by Johnson and Johnson). Here, magnetic beads are coated with Anti-EpCam antibodies (epithelial cell adhesion molecule), which is enriched on tumor cells and not as common on blood cells (Red or White blood cells). After capturing the cells, magnetism is used to separate the beads from solution. SureCell is the only FDA approved capture device for assaying CTC concentrations in blood. However, even this approach has it’s limitations: tumor cells morph and so this assay may not always be accurate; additionally, whereas many epithelial cancers EpCAM, other cancers do not (such as melanomas).

Others have developed reverse binding approaches to isolate CTCs. Whereas most lymphocytes express CD45, CTCs do not. Hence one can remove lymphocytes from blood and leave behind CTCs. However, once again there are challenges (Tumor cells are complex and Blood is complex).

Biochemical capture of circulating tumor cells

Improving biochemical capture of CTCs

Using single biomarkers to identify CTCs is limited because of the inherent heterogeneity of tumor populations. However, using antibodies against several biomarkers may be a much more efficient and universal approach to capture all kinds of CTCs. Materials that bind multiple biomarkers may allow researchers to capture tumors using several hooks.

Mechanical capture methods for circulating tumor cells

Mechanical methods of capturing rely on using size to isolate circulating tumor cells from blood. Circulating tumor cells tend to be larger in size (10-20 um in diameter) than red blood cells (7-12 um in diameter). Using this to their advantage, researchers have developed microfluidic chips to separate CTCs from normal blood cells. As you can imagine, this is an interesting method since the surface biochemistry of cells doesn’t matter. However, tumor heterogeneity and the complexity of blood leads to several false positives. White blood cells tend to accumulate with CTCs when passed through these microfluidic channels.

Improving mechanical capture of CTCs

Mechanical capture methods can be augmented with biochemical capture methods by coating capture surfaces with multiple antibodies. With this combined approach, it is expected that the false-positive capture rate will decrease.

Mechanical filtration of circulating tumor cells

Software capture methods for CTCs

While not truly a method to capture CTCs, image analysis can be used to identify the number of circulating tumor cells within a blood smear. Researchers at labs like the Kuhn lab @ the Scripps institute in San Diego have successfully labeled CTCs with fluorescent markers and identified them. However, they are challenged with the speed of image analysis and the fragility of cells preventing downstream diagnostic analysis of intact CTCs.

Improving software analysis of CTCs

Image analysis is limited because of the speed of imaging and the sheer amount of cells that need to be scanned. By combining imaging with a cell filtration step, where mechanical or biochemical capture is utilized, it may be possible to further improve CTC capture and analysis via software-methods.

References
Review article on cancer management by CTC analysis

Summary of existing CTC capture methods by The Scientist

Label free physical capture of circulating tumor cells

Adhesion based tumor capture using nanotopography

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