Robust Automated Separation of Circulating Tumor Cells and Cancer-Associated Fibroblasts for Enhanced Liquid Biopsy in Breast Cancer

Abstract

Circulating tumor cells (CTCs) are a key biomarker in cancer diagnostics, offering critical insights into metastasis and treatment responses. Although several automated CTC isolation systems have been developed, a thorough comparison of their performance with diverse cell types remains lacking. In addition to CTCs, simultaneous tumor microenvironment (TME) analysis can be valuable for formulating cancer treatment strategies. This includes investigating circulating cancer-associated fibroblasts (cCAFs), which offer a minimally invasive, real-time status of the TME, enabling frequent monitoring of cancer metastasis and treatment response. However, the automated and simultaneous isolation of CTCs and cCAFs has been unexplored. This research systematically evaluated the performance of FDA-registered automated CTC isolation systems with cancer cells of heterogeneous phenotypes, a breast cancer CTC cell line, as well as clinical samples from 27 breast cancer patients. The continuous centrifugal microfluidic system (CTCeptor) demonstrated superior recovery rates and enriched CTCs with broader size and surface marker heterogeneity compared to other positive selection-based technologies, isolating significantly more CTCs from the blood of cancer patients and achieving high detection rates. Notably, since the system relies on an unbiased isolation method, it also isolated cCAFs from patient blood, which were detected at frequencies 10 times higher than CTCs in early-stage breast cancer patients. For the first time, this study identified key CAF markers, highlighting the potential of cCAFs as a biomarker for early diagnosis and prognosis. The ability of this automated system to efficiently isolate both CTCs and cCAFs represents a significant advancement in liquid biopsy and precision oncology.