Abstract 2457: PICASSO: Ultra-multiplexed imaging technique for spatial protein analysis of tumor microenvironments

Abstract In recent cancer research in medicine, various combinatorial biomarkers are simultaneously visualized to study molecular heterogeneity of the tumor microenvironment in detail. These kinds of study aim to provide enhanced precise diagnostic system, optimal treatment decision, and more accurate prediction of survival or recurrence rates for each independent patient case. Conventional immunofluorescence-based approach, however, has been faced an intrinsic limitation in terms of spectral overlap among fluorophores, which allows only 3-4 fluorescent channels per single imaging cycle. Various alternatives, including mass cytometry or linear unmixing, have been proposed to overcome and enhance the multiplexing capability, while they could not totally replace the conventional immunofluorescence technique due to its requirement of specialized equipment and high barrier to entry. Here we report a state-of-the-art multiplexed imaging technique, termed PICASSO, which can perform an unprecedented high multiplexing capability even with a conventional microscopy platform. PICASSO is mainly composed of three parts; 1. antibody complex preformation technique for multiple target staining without host issue, 2. acquiring spectrally mixed images at different spectral ranges, in which each channel included one of those fluorophores’ emission peaks, indicating the number of image acquisitions equal to the number of fluorophores, and 3. fluorescent signals unmixing via mutual information (MI) minimization algorithm. Fifteen-color multiplexed imaging has been experimentally demonstrated within a single staining and imaging cycle via PICASSO. As a cyclic staining method, we demonstrated that PICASSO enables more than 40-color imaging within 3 staining and imaging rounds. Moreover, we demonstrated multiplexed imaging of mouse brains using commercialized bandpass filter-based microscopy. Other than mouse brain samples, various formalin-fixed paraffin-embedded (FFPE) specimens (e.g., breast, kidney, bladder, colon, prostate, thymus, etc.) could be utilized for PICASSO unmixing demonstration, which infers the feasibility of PICASSO technique in various pathological studies in medicine. In addition, PICASSO can be applied to 3D multiplexed imaging, mRNA multiplexed imaging, and large-area multiplexed imaging. We anticipate that PICASSO will provide a new perspective in multiplexed imaging field with high accessibility in cancer research and promote how we see and understand the molecular profiles of tumors and their microenvironments. Acknowledgments: This work was supported by Samsung Research Funding & Incubation Center for Future Technology (SRFC-IT1702-09). Citation Format: Junyoung Seo, Yeonbo Sim, Jeewon Kim, Hyunwoo Kim, In Cho, Hoyeon Nam, Young-Gyu Yoon, Jae-Byum Chang. PICASSO: Ultra-multiplexed imaging technique for spatial protein analysis of tumor microenvironments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2457.