Abstract 2603: Single cell RNA sequencing-based identification of molecular drivers in circulating tumor cell cluster formation and lung metastasis

Abstract

Abstract Introduction: Circulating tumor cells (CTCs) mediate metastases, which account for 90% of solid tumor-related mortality. Compared to single cells, clustered CTCs mediate metastasis at a 20-100 times higher efficiency and are associated with lower overall survival in breast cancer (BC). We have recently identified a new mechanism of CTC cluster formation through cellular aggregation instead of cohesive shedding, and demonstrated that CTC clusters have enhanced stemness (Cancer Discovery, 2019). However, the cellular heterogeneity and molecular mechanisms underlying CTC cluster formation and polyclonal metastasis have yet to be fully elucidated. We hypothesize that molecular drivers of metastasis initiation enhance cancer stemness and CTC cluster formation, and serve as a novel therapeutic target for BC metastasis. Methods: Using single cell RNA sequencing, we compared tumor cells from the primary breast tumor site and lung metastases of BC patient-derived xenografts. We identified genes specifically expressed in the lung metastases and determined their functional importance in CTC clustering, cancer stemness, and lung colonization. We performed proteomic and transcriptomic analyses as well as machine learning to elucidate the downstream signaling pathways and protein structural basis involved in CTC cluster formation and lung metastasis. Finally, we explored therapeutic intervention options in blocking CTC cluster formation and lung metastasis. New results: Compared to the primary breast tumor cells, we identified a stemness gene signature enriched in a subpopulation of the CD44+ lung metastases, with 30-60 fold higher expression of CD34, CD36, ICAM1, VCAM1, ZEB1, ALDH1A1, TGFBR2 and TSPAN8. Analysis of patient blood samples (N=40) revealed that CD44 and many of these new candidate proteins were enriched in CTC clusters in comparison to single CTCs. We then examined the CSC-related properties of these tumor cells, such as tumorigenesis, sphere formation, and lung metastasis. Knockdown of selected surface molecules (e.g. ICAM1) significantly reduced the efficiency of lung metastasis of BC cells in vivo. A subset of ICAM1+/CD44+ tumor cells had increased stemness and tumor growth upon orthotopic implantation in vivo. Knockdown of ICAM1 dramatically reduced the self-renewal ability in mammosphere formation of breast tumor cells in vitro. In addition, our studies also revealed that these tumor cells cluster through CD44 and other surface protein-mediated homophilic binding between two neighboring tumor cells. Neutralizing antibodies significantly blocked tumor cluster formation and lung colonization. Conclusions: We identified new molecular mediators of CTC aggregation and lung metastasis in BC. We anticipate that specific blockade of tumor clustering could decrease cancer progression and improve survival of BC patients. Citation Format: Rokana Taftaf, Xia Liu, Salendra Singh, Yuzhi Jia, David Scholten, Youbin Zhang, Andrew Davis, Carolina Reduzzi, Yue Cao, Yang Shen, Massimo Cristofanilli, William A. Muller, Vinay Varadan, Huiping Liu. Single cell RNA sequencing-based identification of molecular drivers in circulating tumor cell cluster formation and lung metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2603.