Cryopreservation of Circulating Tumor Cells for Enumeration and Characterization

Area of Science:

• Oncology
• Biotechnology
• Clinical Diagnostics

Background:

• Circulating tumor cells (CTCs) in blood samples can indicate cancer metastasis.
• Cryopreservation offers a method for long-term storage and future analysis of clinical samples.
• This technique enables the collection of samples over time for biomarker studies.

Purpose of the Study:

• To evaluate the impact of cryopreservation on the enumeration and biomarker integrity of circulating tumor cells (CTCs).
• To assess the feasibility of cryopreserving blood samples for future cancer biomarker analysis.
• To validate the cryopreservation protocol using both cell line-derived and clinical samples.

Main Methods:

• Blood samples were spiked with known numbers of tumor cells and cryopreserved using dimethyl sulfoxide (DMSO) in RPMI 1640 medium at -80°C.
• Flow cytometry was used to assess the effect of cryopreservation on key biomarkers: cytokeratin (CK), EpCAM, and CD45.
• Tumor cell recovery was quantified after various cryopreservation intervals (up to 6 months) and tested on clinical samples from breast cancer patients.

Main Results:

• Cryopreservation did not affect the expression of CK, CD45, and EpCAM biomarkers.
• High recovery rates (∼90%) were observed for samples with a high number of spiked tumor cells (∼500).
• Lower recovery rates (63%) were noted for samples with fewer tumor cells (median 43), with no significant impact at very low cell counts (median 3).
• No significant difference in CTC counts was observed between fresh and cryopreserved clinical samples, despite inherent variability.

Conclusions:

• Cryopreservation of circulating tumor cells (CTCs) is a viable method for clinical applications.
• A minor loss of CTCs may occur post-cryopreservation, particularly at lower cell concentrations.
• The integrity of key biomarkers is maintained, supporting the use of cryopreserved samples for future biomarker characterization.