Higher percentages of CD221 (IGF-1 receptor)-positive EVs detected by bead-based and singleEV flow cytometry in blood from breast cancer survivors: preliminary results during lifestyle intervention

Introduction: Among techniques for single EV analysis, flow cytometry (FC) has a high potential for clinical application due to its high throughput and multiplex fluorescence capability. Indeed, bead-based FC has also been used to interrogate EV surface proteins. Particularly, IGF-1R (CD221), a key member of the IGF axis, known for its oncogenic role in multiple cancer lineages, was investigated on both cells and EVs in blood from breast cancer survivors since IGF-1/IGF-1R pathway is highly breast cancer subtype context-dependent. Methods: An FC method for analyzing the expression and activation of the IGF-1 receptor is currently set up in our laboratory, giving good results on myeloid cells. The plasma of donors and BC survivors was investigated at different time points for EV detection and CD221 positivity. BC patients are enrolled in the MoviS Trial (ClinicalTrials.gov: NCT04818359) and follow a 3-month aerobic exercise and nutritional education program. All reagents were spun at 21000 g, following general guidelines. We stained blood plasma (centrifuged at 2500 g) with a lipophilic cationic dye (LCD, BD Biosciences) recently identified as a generic EV tracer, highly useful when samples containing heterogeneous EV population need to be analyzed or when EVs may not express the classical tetraspanin markers (e.g., CD9, CD81, CD63). Rosetta Calibration beads and the application of both an SSC or fluorescent trigger threshold were employed. The capture bead technique was applied using Exostep (Immunostep) for bead-based evaluation. Results: Preliminary results show higher percentages of CD221+ EVS in blood from BC survivors concerning age-matched donors. Furthermore, these C 221+ EVS mainly belong to the pool of CD81 negative EVs. Although further tests are needed (also to increase adherence to the MISEV guidelines), this finding is confirmed by Exostep data. Other experiments are ongoing to confirm data and to add more MISEV technical approaches. Summary/Conclusion: The IGF-1 receptor is an important immunomodulator, emerging as an attractive target for cancer therapy. These preliminary findings will be integrated with the characterization of the isolated EVs and nanoparticle tracking analysis, aiming at consolidating current results and coupling them with data on the expression and activation of the IGF-1 receptor on immune cells.