Introduction: Circulating Tumor Cells (CTCs) have attracted significant attention as a new class of "liquid biopsy", enabling longitudinal and non-invasive disease monitoring to capture an overall snapshot of individual disease. Primary breast tumors are highly heterogeneous due to their genetic instability. Thus, the presence of heterogeneous populations of CTCs is expected. A number of attempts have been made to address CTC's phenotypic heterogeneity using different methods and approaches. For comprehensive understanding of distinct CTC phenotypes, transcriptomic and cell surface marker studies using single cell analysis separated CTCs into different subgroups with epithelial-, mesenchymal-, and stem-like signatures. These studies concluded that the prevalence of stem-like CTCs is associated with poor prognosis. Immunofluorescence is an ideal approach to understand the heterogeneity of 3 - 4 markers in a large number of distinct CTCs; however, such approach does not address the presence of overlapping phenotypic signatures. The collection method of CTCs adds another layer of complexity since the most commonly adopted CTC enumeration strategy relies on affinity-based selection using antibody against EpCAM, which, although abundantly expressed on epithelial cancer cells, automatically eliminates a large fraction of non-epithelial CTCs. To better understand the whole landscape of this heterogeneous disease, the use of marker-independent unbiased methods to collect CTCs is critical to obtain the overall landscape of phenotypic heterogeneity.

Aim: We aimed to investigate the heterogeneity of breast CTCs on a single cell level using size exclusion- based enrichment principal.

Methods: Whole blood was collected from metastatic breast cancer patients under IRB approved protocol. CTCs were isolated on porous membrane (8 µm) under negative pressure (ISET®) from 10 mL of blood. CTCs were fixed on the porous membrane and used for multiplexed immunofluorescence to investigate the expression of epithelial (CK and EPCAM), mesenchymal (Vimentin, Snail and TWIST), and stem-like markers (CD44, ALDH1, and CD133) on CTCs.

Results: CTCs were isolated on ISET filter from all subtypes, Stage IV patients. CTCs were identified based on their morphological and phenotypical characteristics with our CTC criteria of size of nucleus ≥16 µm, triple negative for α-smooth muscle actin, fibroblast associated protein, and CD45. The CTCs were further characterized based on their mesenchymal and stem-like signatures to understand their heterogeneity.

Conclusions: Multiplex immunofluorescence staining of CTCs collected using the size-based enrichment approach enabled us to identify the heterogeneity of CTCs. The phenotypic heterogeneity represents a corner stone for future studies to identify a subset of CTCs that may be associated with breast cancer patient prognosis.

Citation Format: Kamal M, Zhang R, Walker M, Squires R, Talbert B, Dooley W, Razaq W, Tanaka T. A multiplexed immunofluorescence identifies phenotypic heterogeneity of circulating tumor cells in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-18.