Abstract B42: Identification of molecular drivers in circulating tumor cell cluster formation and lung metastasis

Abstract

Abstract Introduction: Breast cancer (BC) is the leading type of cancer and the second leading cause of cancer-related deaths among women in the US. Metastasis accounts for 90% of solid tumor-related mortality and is mainly mediated by hematogenous spread of circulating tumor cells (CTCs). Compared to single cells, clustered CTCs mediate metastasis at a 20-100 times higher efficiency and are associated with lower overall survival. 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 breast cancer patient-derived xenograft. We identified genes with differential expression levels in the lung mets and determined their functional importance in CTC clustering, cancer stemness, and lung colonization. Upon the candidate gene modulation, we performed proteomic and transcriptome analyses to elucidate the downstream signaling pathways 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- to 60-fold higher expression of CD34, CD36, VCAM1, ZEB1, ALDH1A1, TGFBR2, TSPAN8, and ICAM1. Patient blood analyses (N=40) revealed that CD44 and many of these new candidate proteins are 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 from the signature genes significantly reduced the efficiency of lung metastasis of BC cells in vivo. A subset of these tumor cells had increased stemness and highest tumor growth upon orthotopic implantation in vivo. Knockdown of the signature genes 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. Conclusion: We identified new molecular mediators of CTC aggregation and lung metastasis in breast cancer. We anticipate that a specific blocking of tumor clustering could decrease cancer progression and improve survival of breast cancer patients. Citation Format: Rokana Taftaf, Xia Liu, Salendra Singh, Yuzhi Jia, David Scholten, Massimo Cristofanilli, William A. Muller, Vinay Varadan, Huiping Liu. Identification of molecular drivers in circulating tumor cell cluster formation and lung metastasis [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B42.