Improved Characterization of Circulating Tumor Cells and Cancer-Associated Fibroblasts in One-Tube Assay in Breast Cancer Patients Using Imaging Flow Cytometry

SIMPLE SUMMARY: Liquid biopsy is a promising but challenging tool for potentially upgrading cancer patient diagnostics and providing new insights into tumor biology. Here, we applied a unique approach to detect CTCs and cCAFs in a one-tube assay using imaging flow cytometry, enabling improved enumeration, multimarker-based phenotyping, and detailed morphological characterization of those rare cells. We identified new, putatively EMT-related phenotypes of negative CTCs for both epithelial and mesenchymal markers and showed that EMT-related CTCs might contribute to breast cancer progression, whereas a coincidence of CTCs and cCAFs might be a signature of metastasis. ABSTRACT: Circulating tumor cells (CTCs) and circulating cancer-associated fibroblasts (cCAFs) have been individually considered strong indicators of cancer progression. However, technical limitations have prevented their simultaneous analysis in the context of CTC phenotypes different from epithelial. This study aimed to analyze CTCs and cCAFs simultaneously in the peripheral blood of 210 breast cancer patients using DAPI/pan-keratin (K)/vimentin (V)/alpha-SMA/CD29/CD45/CD31 immunofluorescent staining and novel technology—imaging flow cytometry (imFC). Single and clustered CTCs of different sizes and phenotypes (i.e., epithelial phenotype K+/V− and epithelial–mesenchymal transition (EMT)-related CTCs, such as K+/V+, K−/V+, and K−/V−) were detected in 27.6% of the samples and correlated with metastases. EMT-related CTCs interacted more frequently with normal cells and tended to occur in patients with tumors progressing during therapy, while cCAFs coincided with CTCs (mainly K+/V− and K−/V−) in seven (3.3%) patients and seemed to correlate with the presence of metastases, particularly visceral ones. This study emphasizes the advantages of imFC in the field of liquid biopsy and highlights the importance of multimarker-based analysis of different subpopulations and phenotypes of cancer progression-related cells, i.e., CTCs and cCAFs. The co-detection of CTCs and cCAFs might improve the identification of patients at higher risk of progression and their monitoring during therapy.