Abstract P3-01-17: Transcriptomic and phenotypic profiling of circulating tumor cells from metastatic breast cancer patients

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Abstract Background: Metastatic breast cancer (MBC) is considered an incurable disease and understanding the molecular mechanisms at the basis of tumor progression has become mandatory. Circulating tumor cells (CTCs) constitute a population of rare cells that are shed in the bloodstream by primary and metastatic tumors and migrate towards distant organs. The molecular characterization of CTCs is a powerful tool to better investigate the mechanisms underlying metastasis. In this study, we combine phenotypic and transcriptional profiling of CTCs isolated from MBC patients to infer the mechanisms involved in their biology, also in relation to the metastatic site and their phenotypic features. Methods: Blood samples for CTC analysis were collected from luminal MBC patients recruited in the KENDO study (Protocol code: IRST174.19); CTCs were enriched using the RosetteSep CTC Enrichment Cocktail and frozen until downstream analyses as viable cells. To validate the antibody staining for phenotypic analysis, tests were conducted using the cancer cell line SKBR-3. In brief, SKBR-3 cells were spiked in peripheral blood of a healthy volunteer and enriched using RosetteSep CTC Enrichment Cocktail. Enriched SKBR-3 and patients’ CTCs were incubated with antibodies targeting CD45 (leukocyte marker) and epithelial markers (E-tag; EpCAM, E-cadherin) for phenotypic analysis using the DEPArray NxT platform. For transcriptomic profiling, single CTCs were isolated as single, viable cells. Libraries were prepared using the QIAseq UPX 3’ Transcriptome Kit and sequenced using MiSeq on V3-150 cycles cartridges. Bioinformatic and statistical analyses were carried out using the CLC Genomic Workbench. Results: Through feasibility analysis conducted on SKBR-3 cells, we demonstrate that our workflow involving phenotypic analysis on DEPArray NxT is an efficient tool to identify cancer cells (CD45-/E-tag+) and white blood cells (WBCs; CD45+/E-tag-). In MBC samples, we detected different cell populations: WBCs (CD45+/E-tag-), CTCs (CD45-/E-tag+), and dual-positive CTCs (CD45+/E-tag+; dpCTCs). Transcriptomic profiling was successfully performed on 37 single CTCs isolated from 7 patients. The transcriptional profile of CTCs is consistent with breast malignancy for different databases including ClinVar2019, DisGeNet, and Human Phenotype Ontology. Moreover, Gene Set Enrichment analysis highlighted pathways associated with synapse organization and calcium channel activity, suggesting the implication of the interplay between cancer cells and neurons in metastasis. Compared to a commercial RNA, CTCs express at high levels transcripts associated with cancer dissemination and genome architecture (i.e., STAG2 and H2AFZ). In CTCs from patients with bone metastasis we detected the expression of genes associated with metastatic osteotropism (i.e., S100A4, VAPA, HMGB1), while CTCs from a single patient with gastric metastasis expressed genes associated with gastric cancer metastasis and cell redox homeostasis. Transcriptional profiling of dpCTCs revealed the expression of transcripts encoding for members of the CD47/SIRPα axis, supporting their macrophage-cancer cell fusion origin, and increased glycogen biosynthetic and metabolic activity. Conclusions: Our data suggest the potential of CTC based transcriptomics in providing new insights on the clinical-biological evaluation of MBC and its mechanisms underlying metastatic cascade and organotropism. In addition, our data provide hints for the characterization of dpCTCs, a poorly studied population whose role in metastasis is still far to be fully elucidated. Citation Format: Michela Palleschi, Tania Rossi, Sara Bandini, Michele Zanoni, Michela Cortesi, Erika Bandini, Andrea Rocca, Giulia Gallerani, Ivan Vannini, Meropi Plousiou, Lorenzo Gerratana, Giovanni Tallini, Giovanni Martinelli, Ugo De Giorgi, Paola Ulivi. Transcriptomic and phenotypic profiling of circulating tumor cells from metastatic breast cancer patients [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-01-17.

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