Abstract 1144: Development of imaging mass cytometry (IMC) as a tool to characterize circulating tumor cells (CTCs) in pre-clinical mouse models

Abstract Metastasis is in part due to circulating tumor cells (CTCs), especially CTC clusters, which are associated with poor prognosis and treatment resistance. CTCs increase in numbers after biopsy, surgery, and during neo-adjuvant treatment. This may represent not only a window into cancer progression but also novel targets for intervention. The development of methods for the functional characterization of CTCs is, therefore, imperative toward assessing and achieving these goals. This project addresses three specific challenges. First, while many advancements in techniques to isolate CTCs have been made, each is associated with physical manipulations that may alter CTC characteristics. Second, while CTCs from patients may provide valuable insights, only preclinical mouse models permit experimental manipulations that will lead to functional understanding of CTCs. Lastly, because CTCs are rare, most studies have for technical reasons focused on analysis of nucleic acids rather than proteins. Here we present multiplex imaging mass cytometry (IMC) of metal-labeled antibodies, which allows for the simultaneous detection of more than 40 proteins in situ, as a tool to analyze single versus cluster CTCs with small volumes (<100 µl) of blood from mice in an exclusive single-step process. Through three stages of validation, we developed a panel of antibodies that targets human proteins, including cytokeratins, epithelial-mesenchymal (E/M) markers, signaling molecules, transcription factors, and indicators of cell death. First, human cancer cell lines that represent a spectrum of E/M phenotypes were analyzed by IMC in parallel to Western analysis. Next, antibodies were validated on cell lines that were spiked into naive mouse blood, followed by CTCs in the blood of mice bearing SUM149 or MDA-MB-231 xenograft tumors. Expression of a GFP-luciferase reporter provided independent information on CTC quantities. Lastly, the panel was used to characterize CTCs generated by a patient-derived xenograft (PDX) mouse model. By first manually locating CTCs as DNA+/PanCK+/CD45- cells, we also developed an automated a custom AI Halo cell segmentation classifier trained to identify CTCs. Downstream analysis was accomplished through adaptation of an open-sourced Bodenmiller lab pipeline for single-cell marker quantification. Our data show that both single CTCs and CTC clusters can be identified and distinguished with the IMC platform. We also observed that blood collected by cardiac puncture yields a higher number of CTCs and CTC clusters compared to the blood collected by tail vein puncture. Taken together, IMC can be used as a single-step approach to detect and characterize human CTCs in preclinical models. Future investigations will apply this technology to investigate the effect of genetic manipulations and pharmacological interventions on CTC cluster formation and metastasis. Citation Format: Milind Pore, Balamurugan Kuppusamy, Devynn Breen, Paul Mallory, Abigail Atkinson, Ashley Cardamone, Christine Newkirk, Lois McKennett, Shikha Sharan, William Bocik, Esta Sterneck. Development of imaging mass cytometry (IMC) as a tool to characterize circulating tumor cells (CTCs) in pre-clinical mouse models [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 1144.