Abstract 4220: Combined light sheet and multispectral imaging facilitates the detection of tumor-associated tertiary lymphoid structures in the KP NSCLC model

Abstract Tertiary lymphoid structures (TLS) have recently emerged as a predictive biomarker in cancer immunotherapy. Their consideration in preclinical drug development is impeded by rare and transient appearance and random distribution, making them difficult to analyze with conventional sampling methods. We applied whole-organ tissue clearing and 3D staining of B and T cell clusters to comprehensively identify TLS in a KP GEMM of NSCLC using 3D Light Sheet Fluorescence Microscopy (3D LSFM). To further investigate cellular composition of TLS we performed 3D LSFM-educated sectioning of relevant ROIs and cyclic immunofluorescence of the same samples. 3D LSFM facilitated correlation of tumor features and location with T cell infiltration as well as the analysis of number, size and location of TLS. The majority of TLS were located in proximity to bronchi instead of intratumorally. LSFM-guided multiplex immunofluorescence revealed further details on cellular composition and maturation status of TLS. This combination of 3D and 2D imaging modalities offers a holistic approach for the identification of TLS in whole murine tumors or organs. The technology could help studying effects of immunotherapy on de novo formation or maturation of TLS. Citation Format: Nils O'Brien, Birte Appelt, Marie-Lena Moerbitz, Joerg P. Mueller, Frank Herting, Pablo Umaña, Claudio Murgia, Sara Colombetti, Thomas Pöschinger. Combined light sheet and multispectral imaging facilitates the detection of tumor-associated tertiary lymphoid structures in the KP NSCLC model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4220.