# Viable Cryopreservation Strategy for Extending the Timeframe of Circulating Tumor Cell Detection in Breast Cancer Clinical Trials

## TL;DR

This study shows that freezing blood samples after a specific step allows for reliable detection of cancer cells in the blood, even after a year, which could help in large cancer research studies.

## Contribution

The study identifies a viable cryopreservation strategy for preserving CTCs in breast cancer research for extended periods.

## Key findings

- Cryopreservation after Ficoll gradient separation allows over 81% recovery of CTCs after one year.
- Cryopreservation at later stages leads to undetectable CTCs or cellular debris.
- The method supports multicenter clinical trials by extending the processing timeframe for CTC detection.

## Abstract

Circulating tumor cells (CTCs) hold recognized prognostic value in various cancers, including breast cancer, where their presence correlates with survival outcomes. However, the typical 24 h window for blood processing and CTC isolation poses a logistical challenge, particularly for multicenter studies. This study aimed to evaluate cryopreservation at different stages of CTC isolation and immunocytological detection to extend the blood sample processing period. Using spiked peripheral blood samples with MDA-MB-231, SKBR3, and MCF7 breast cancer cell lines, four distinct cryopreservation points were assessed: following Ficoll gradient separation, immunomagnetic separation, cytocentrifugation, and cytokeratin labeling. Our findings demonstrated that cryopreservation of the mononuclear and granulocytic cell fraction after double-density Ficoll gradient separation was the only viable method for subsequent CTC detection. This approach allowed for consistent recovery of CK+ CTCs, with an average recovery rate of over 81% after one year of cryopreservation. In contrast, cryopreservation at later stages resulted in undetectable CTCs or only cellular debris. In conclusion, cryopreservation following density gradient centrifugation is a feasible strategy for delaying CTC isolation and immunocytological analysis in breast cancer research, facilitating its application in multicenter clinical trials.