Abstract 1972: Utilization of flow cytometry for the characterization of mesenchymal and embryonic properties of circulating tumor cells

Abstract Background: Circulating tumor cells break off from the primary cancer tissue and enter into the bloodstream, where they reach the distant parts of the body where they can form metastases. The tumor cell could lose its adhesive properties and takes on a mobile, mesenchymal phenotype and they undergo epithelial-mesenchymal transition (EMT). The detection of these circulating tumor cells carries great diagnostic potential. Their identification is based on specific cellular markers such as cytokeratin or vimentin expression, which are key molecules in EMT, or SOX9 which plays a prominent role in the development of stem cell-like properties of tumor cells and carries therapy resistance potential. Aims: The aim of our study was to develop and optimize a technique suitable for the detection of circulating tumor cells and cells undergoing epithelial-mesenchymal or embryonic transition from blood. Materials and Methods: Magnetic cell separation technique (autoMACS Pro, Miltenyi) was performed in order to deplete CD45 immune cells from blood. Additionally, flow cytometry technique was used to detect fluorescent antibody labeled circulating tumor cells. CD45-AF700, CD15-PeVio770, CK-FITC, Vimentin-APC, SOX9-PE labeled antibodies were utilized. Peripheral whole blood of colorectal carcinoma patients (CRC) were analyzed, four of the patients were classified with grade II and grade III. Data was compared to healthy controls. Results: After the depletion of CD45 positive immune cells from the blood 5CTC/100000 counted cell were characterized as cytokeratin positive cell. Moreover, 50CTC/100000 counted cell was observed as vimentin positive cells. Additionally, SOX9 positive cells (6CTC/100000) were also detected from patients. These results indicates the presence of cancer cells with mesenchymal and stem cell characteristics in the circulation of cancer patients. Conclusion: Overall, magnetic separation is suitable technique for the enrichment of circulating tumor cells from whole blood. Moreover, flow cytometry is a key method for the detection of circulating tumor cells and cancer stem cells that have undergone embryonic transformation. With the sorting of these cells we hope that we can create the base of future diagnostic analysis of cancer patients. Citation Format: Tamás Ricárd Linkner, Zsófia Brigitta Nagy, Nikolett Szakállas, Eszter Farkas, Fruzsina Bányai, István Takács, Béla Molnár. Utilization of flow cytometry for the characterization of mesenchymal and embryonic properties of circulating tumor cells [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 1972.