Abstract 1149: Circulating tumor cells bind to leukocytes to promote metastasis formation in breast cancer

Abstract Circulating tumor cells (CTCs) in breast cancer have been established as the vehicles for metastatic spread and can be used to predict patient prognosis and response to treatment. Furthermore, the investigation of CTCs provides an opportunity to understand the metastatic process and relevant tumor biology that shapes that process. While many efforts have been undertaken to identify the molecular characteristics and biological features of CTCs, their relative rarity in circulation makes the study of CTCs challenging. Even rarer than single CTCs are CTC clusters including homotypic CTC-CTC clusters and heterotypic CTC-white blood cell (WBC) clusters. Heterotypic CTC clusters remain poorly understood, and the mechanisms by which these clusters form are incompletely known. Additionally, physical and signaling interactions between CTCs with immune cells in circulation may help promote metastasis in breast cancer. We employed multiparametric flow cytometry and FDA-approved CellSearch fluorescence imaging to analyze CTC-WBC clusters in liquid biopsies of advanced breast cancer patients to determine immune cell components in these clusters. We also sorted immune cells from breast cancer patient liquid biopsies to profile the overall circulating immune compartment using single-cell RNA sequencing. Furthermore, we employed in vitro and in vivo assays to confirm the role of adhesion molecules in CTC-immune cell clustering and metastatic lesion formation. Flow cytometry profiling revealed multiple immune cells such as neutrophils, monocytes, T cells, and others in the CTC-WBC clusters. A rare subset of T cells is significantly enriched in CTC-WBC clusters. Using single-cell RNA sequencing data, we identified a highly enriched receptor/ligand pair that drives CTC-WBC interactions. Using in vitro clustering assays and CRISPR/Cas9 mediated genetic knockouts, genetic depletion of the receptor/ligand in respective tumor cells and WBCs diminished the heterotypic CTC-WBC cluster formation and blocked spontaneous lung metastasis of breast cancer in mice. Finally, we demonstrated that blocking these interactions with a therapeutic neutralizing antibody reduces the amount of spontaneous CTC-WBC clusters in murine models of breast cancer and reduces metastatic lesion formation. This study identified novel interactions that promote CTC-WBC cluster formation and proved that these clusters play a key role in breast cancer metastasis. Additionally, we generated and analyzed a considerable amount of single-cell RNA sequencing data from breast cancer patient liquid biopsies that allowed us to correlate immune status with patient survival and CTC counts. Citation Format: David Scholten, Lamiaa El-Shennawy, Youbin Zhang, Yuzhi Jia, Huiping Liu. Circulating tumor cells bind to leukocytes to promote metastasis formation in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1149.