Abstract 1921: Stem-like clusters of CD44+ circulating tumor cells seed metastases in breast cancer

Abstract

Abstract Background: Circulating tumor cells (CTCs) are cells shed from primary tumor and circulate in the peripheral blood. They are considered the seeds of metastasis. Compared to single CTCs, clusters of multiple CTCs possess 50 times higher metastatic capacity in mouse breast cancer models. However, the mechanisms underlying the metastatic promotion effect of CTC clusters are unclear. In addition, whether clustered CTCs have cancer stem cell (CSC) properties and what stem cell markers they express have not been determined. Methods: Immunohistochemistry (IHC) was used to detect CTCs in vascular structures, and the stem cell markers in single and clustered CTCs. Lung metastases were either monitored by bioluminescence imaging (BLI) or visualized by fluorescence microscopy. Stem cell properties were examined by mammosphere assays in vitro and tumorigenic assays in vivo. Clustering assay was performed by culturing cells in Poly-HEMA coated plates (for cell lines) or collagen-coated plates (for primary cells derived from patient-derived breast tumor xenografts, PDXs), and then monitored by IncuCyte live cell dynamic imaging. Results: CD44+ enriched circulating tumor cell (CTC) clusters were found in the lung/liver vascular structures in vivo in both metastatic breast cancer patients and PDXs that develop spontaneous lung metastases. Comparing to single CD44+ cells, clustered CD44+ cells formed more mammospheres, increased tumorigenic potential, and promoted lung colonization. Combining siRNA-mediated knockdown and CRISPR/Cas-based knockdout, we found that CD44 is required for breast tumor cell cluster formation and lung colonization upon tail vein seeding. During cluster formation, EGFR was activated, which improved both stemness and survival of clustered CTCs. Anti-EGFR antibody mimicked CD44 knockdown to inhibit cluster formation of PDX-derived tumor cells. Administration of EGFR inhibitor Erlotinib efficiently inhibited CD44+ cells-mediated spontaneous metastases to the lungs without affecting primary tumor growth. Conclusions: Our data provide new insights into the cellular and molecular mechanisms of stem-like clustered CD44+ cells-seeded metastasis, and implicate that targeting CD44+ CTC clusters by inhibition of EGFR activity could be a new therapeutic strategy to treat metastatic breast cancer. Citation Format: Xia Liu, Wenjing Chen, Rokana Taftaf, Huiping Liu. Stem-like clusters of CD44+ circulating tumor cells seed metastases in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1921. doi:10.1158/1538-7445.AM2017-1921