Cryo conservation preserves viability of circulating tumor cells from leukapheresis product for in vitro culture

Background:

Solid tumors are constantly releasing circulating tumor cells (CTCs) into the circulatory system. Their extremely low frequency is one of the main limiting factors to obtain CTCs for functional studies. To overcome this challenge CTCs are enriched prior to culturing from larger blood volumes or diagnostic leukapheresis (DLA) products.

Nonetheless, only few patients with growing CTCs are obtained. However, it is critical to collect larger cohorts for further understanding of the biology of CTCs. Therefore, we tested to grow CTCs from banked cryo-conserved DLA products.

Methods:

DLA samples of metastasized breast cancer patients were collected and CTC numbers were determined by CellSearch® analysis. Of eight CTC positive samples DLA product was analyzed directly after leukapheresis and after up to 18 months of cryo-conservation. CTC recovery was determined and morphology of detected CTCs was compared by evaluating DAPI and cytokeratin signals. Viable CTCs were enriched from fresh and thawed DLA products with the Parsortix™ system and cultured.

Results:

Cryo-conservation does not impact CTC morphology and quality. A total CTC loss of 15.2%± 9.7% was observed. However, the number of CTCs with intact nucleus and intact cytoplasm was reduced by only 6.0%± 3.8%. CTC cultures could be grown from both fresh and frozen samples in two cases with high CTC numbers.

Conclusions:

Cryo-conservation of DLA products enables storage of primary CTCs with low CTC loss while maintaining their quality and viability and will help to establish larger cohorts of cultivable primary CTCs for further functional studies.