Abstract 5658: From staining to analysis: End-to-end application of InSituPlexTM technology for multiplexed immuno-profiling in FFPE tumor samples
Authors: Manesse Mael , Bares Amanda , Boisvert Heike , Walter Stephanie 
Published: August 2018

Abstract
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.