Abstract 5658: From staining to analysis: End-to-end application of InSituPlexTM technology for multiplexed immuno-profiling in FFPE tumor samples

Introduction: The development of new and improved cancer diagnostic and therapeutic methods relies on a deep understanding of the protein expression profile in the tumor microenvironment. Conventional immunohistochemistry (IHC) methods only provide insights into a limited number of markers of interest, prompting the need for the development of multiplexed methods to assess and characterize multiple cell phenotypes and their spatial context in a single sample. We have developed an approach to enable high-multiplexing detection of protein markers in FFPE tumor samples using an integrated workflow with the Leica Bond RX autostainer and Leica Versa whole slide scanner. Methods: A cocktail of primary antibodies of interest, modified with unique, addressable DNA barcodes, is applied to tissue samples in a single staining step. The barcodes are then amplified simultaneously to improve sensitivity. Finally, a cocktail of fluorescently labeled oligonucleotides, complementary to the barcodes on the antibodies, is used to tag the different targets of interest for multiplexed imaging. Formalin-fixed, paraffin-embedded samples from both human tonsil and primary tumor biopsies were stained manually and automatically on the BondRX, using a panel of antibodies specific to cancer immuno-profiling markers. Images were acquired using the Leica Versa whole slide scanner, and analyzed with Leica Aperio Image Analysis and Indica Labs Halo software. Results: Compared to typical multiplex IHC methods, the InSituPlex single staining step offers multiple advantages. First, the workflow preserves the integrity and antigenicity of the samples by avoiding any antibody stripping steps and streamlining the assay development process by eliminating the need to optimize staining order. In addition, the total assay time stays constant regardless of the multiplexing level. Adding targets to the panel does not increase staining time, facilitating the optimization of higher multiplex assays, as well as the development of streamlined automated assays. A fully automated staining protocol was developed on the BondRX by modifying assay parameters such as reagent concentrations and volumes, optimizing the timing of each step, and evaluating the impact on the resulting images in terms of specificity, sensitivity and overall signal to background ratio. After optimization, the automated staining protocol delivered similar or better results compared to the manual assay protocol, while decreasing the total assay time by approximately 40%. The optimized protocol was subsequently used to carry out a multiplexed analysis of immune cell phenotypes in tumor samples. Conclusions: the InSituPlex technology enables multiplexed immune-profiling in tumor FFPE sections, and provides a streamlined, high-throughput workflow with existing automated staining and imaging instrumentation. Citation Format: Mael Manesse, Amanda Bares, Heike Boisvert, Stephanie Walter. From staining to analysis: End-to-end application of InSituPlexTM technology for multiplexed immuno-profiling in FFPE tumor samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5658.