Improved characterization of circulating tumor cells and cancer-associated fibroblasts in breast cancer patients using imaging flow cytometry

Circulating tumor cells (CTCs) and circulating cancer-associated fibroblasts (cCAFs) have been individually considered as 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 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 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 7 (3.3%) patients and seemed to correlate with the presence of metastases, particularly visceral ones. This study emphasizes advantages of imFC in the field of liquid biopsy and highlights the importance of multimarker detailed analysis of different subpopulations and phenotypes of cancer progression-related cells i.e. CTCs and cCAFs. Co-detection of CTCs and cCAFs might improve the identification of patients at higher risk of progression and their monitoring during therapy. Simple SummaryLiquid biopsy is promising but challenging tool potentially upgrading cancer patients diagnostics and bringing new insights into tumor biology. Here, we applied a unique approach to detect CTCs and cCAFs in one-tube assay using imaging flow cytometry enabling improved enumeration, multimarker-based phenotyping and detailed morhopological characterization of those rare cells. We showed that EMT-related CTCs might contribute to breast cancer progression, whereas coincidence of CTCs and cCAFs might be signature of metastasis.