Isolating and Characterizing Circulating Tumor Cells: A Detailed Protocol
Understanding cancer progression and treatment response hinges on a deep dive into the characteristics of circulating tumor cells (CTCs). You can gain valuable insights by isolating these rare cells from a patient’s blood sample and analyzing their molecular profile. This detailed protocol outlines a robust method for CTC isolation and characterization, offering a pathway to personalized cancer management.
Preparing Your Cells for Analysis
first, enriching the CTC fraction is crucial. Following enrichment, cells are carefully washed with phosphate-buffered saline (PBS) and then gently centrifuged at 558g for 5 minutes. This ensures a clean cell pellet ready for further processing.
Next, resuspend the cell pellet in 100 μl of fixative solution (medium A from the Fix&Perm kit). Another PBS wash (5 minutes at 368g, followed by centrifugation) prepares the cells for permeabilization.
Antibody Staining and Incubation
Now, it’s time to introduce the antibodies that will allow you to identify and isolate specific CTC populations. Resuspend the cell pellet in 100 μl of permeabilization solution (medium B from the Fix&Perm kit) along with an anti-HER3 monoclonal antibody (2 µg/ml, clone RTJ2). Incubate this mixture for 20 minutes at room temperature.
Following incubation, add a cocktail of reagents:
A secondary goat anti-mouse AF488 antibody (1:800 dilution).
Staining reagents containing cytokeratin (CK)-phycoerythrin (targeting CK 8, 18, and 19).
CD45-allophycocyanin antibodies.
Incubate this final mixture for 20 minutes in the dark at room temperature. A final PBS wash removes unbound antibodies.
Cell Sorting and Collection
resuspend the cell pellet in 300 μl of PBS and store it at +4°C until ready for sorting. Before sorting, add Hoechst 33342 (16.7 µg/ml) to stain the cell nuclei.
Utilize a BD FACS ARIA III cell sorter, equipped with 405 nm, 488 nm, 561 nm, and 640 nm lasers. Optimize the system with a pressure of 138 kPa, a 100-μm nozzle, and yield precision mode.
Defining Your CTC Populations
Cell sorting begins by identifying Hoechst-positive (Hoechst+) elements. Then, select for CD45-APC-negative (CD45−) events, effectively excluding white blood cells.sort and collect three key CTC populations into a 96-well plate:
- Hoechst+CD45−CK+HER3− cells
- Hoechst+CD45−CK+HER3+ cells
- Hoechst+CD45−CK−HER3+ cells
As a control, include 20 Hoechst+CD45+CK−HER3− cells. centrifuge the plates at 280g for 10 minutes and freeze them at -20°C for at least 30 minutes.
Data Considerations
For extensive analysis, CTC count data should be available for a sufficient number of patients.In one study, data was available for 34 patients, with 27 of those patients having assessments at multiple time points. This longitudinal data is invaluable for tracking treatment response and disease progression.
This protocol provides a solid foundation for isolating and characterizing CTCs. Remember, meticulous technique and careful optimization are key to obtaining reliable and meaningful results. By leveraging this approach, you can unlock critical insights into your patient’s cancer and
