Abstract 3693: Molecular and functional characterization of circulating CK+/CD45+ cells in breast cancer
Authors: Reduzzi Carolina , Nicolo Eleonora , Ozimski Lauren L. , Silvestri Marco , Gerratana Lorenzo , Serafini Mara S. , Molteni Elisabetta , Bayou Nadia , Strickland Amanda K. , Liu Huiping , Cappelletti Vera , Aceto Nicola , Cristofanilli Massimo 
Published: March 2024

Abstract
Background: Circulating tumor cells (CTCs) are considered a key element in metastatic progression and represent a validated prognostic biomarker in breast cancer (BC). CTCs in BC are normally defined as epithelial cells (positive for EPCAM or cytokeratin, CK) lacking the expression of the leukocyte marker CD45. Circulating cells expressing both EPCAM/CK and CD45 (dual positive, DPcells) have however been reported in BC but not investigated. It is believed that DPcells derive from the heterotypic cell fusion of tumor and immune cells, but this hypothesis has not been demonstrated yet. We recently reported, for the first time, the association of DPcells with worse survival in a cohort of 341 BC patients (pts) [Reduzzi et al. ASCO 2022] supporting their tumor origin. Here, we further investigated their malignancy and metastatic potential by molecular profiling and functional experiments.
Methods: The study consisted of 2 components: A) Single CTCs and DPcells were enriched from the blood of BC patients with the CellSearch platform, and isolated with the DEPArray system. Single cells underwent whole genome amplification and lowpass whole genome sequencing for single cell copy number alteration (CNA) profiling; B) DPcells and CTCs were investigated in the blood of immunodeficient (NSG) and immunocompetent (FVB) mouse models using various cell lines (LM2, 4T1, MVT1). To test their metastatic potential, DPcells from the primary tumor of FVB donor mice were transplanted into recipient NSG mice, and the metastatic burden in the lungs and liver quantified, in comparison with control CD45- tumor cells.
Results: We analyzed 73 DPcells and 18 CTCs collected from 16 BC pts: Aberrant genomes were observed in 28% and 93% of evaluable cells, respectively. When collected from the same pt, CTCs showed clonal CNA profiles, while DPcells were highly heterogeneous. In vivo, DPcells could only be detected in the blood of FVB mice but not in NSG mice. They were very rare, comprising of only about 3% of the total CTC count. When DPcells and control tumor cells were isolated from the primary tumor of 10 donor mice and transplanted into the tail vein of 10 recipient mice, we observed no significant differences in the metastatic colonization of the lungs and liver by the two population, indicating that DPcells do in fact possess metastatic ability, and to a similar extent as control cancer cells.
Conclusions: DPcells can have an aberrant genome, supporting their malignancy. Contrary to CTCs, DPcells’ CNA profiles are not clonal and, in vivo, they can only be observed in immunocompetent models; both observations support the tumor/immune fusion hypothesis. Finally, DPcells show metastatic potential in mice, in line with the association with worse survival we previously observed in BC pts. Overall DPcells seem a new CTC subpopulation that should not be neglected anymore. More studies are needed to better understand their origin and phenotypic/molecular features.
Citation Format: Carolina Reduzzi, Eleonora Nicolo, Lauren L. Ozimski, Marco Silvestri, Lorenzo Gerratana, Mara S. Serafini, Elisabetta Molteni, Nadia Bayou, Amanda K. Strickland, Huiping Liu, Vera Cappelletti, Nicola Aceto, Massimo Cristofanilli. Molecular and functional characterization of circulating CK+/CD45+ cells in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3693.