Abstract B081: Spatially-resolved, multiplexed (up to 800 plex) digital characterization of protein and mRNA abundance in FFPE tissue sections: Application to immuno-oncology
Authors: Dunaway Dwayne , Jung Jaemyeong , Merritt Chris , Sprague Isaac , Webster Philippa , Warren Sarah , Beechem Joseph 
Published: November 2016

Abstract
The Tumor Microenvironment (TME) has emerged as a key compartment that determines the overall effectiveness of cancer immunotherapy. Hence, it is very important to determine the abundance and location of key immune-regulators in the TME. Historically, standard immunohistochemistry (IHC) and immunofluorescence have been used to assess spatial heterogeneity of proteins and nucleic-acids in tissue slices. However, these techniques are inherently limited in utility because it has been difficult to quantify the abundance of multiple protein/nucleic-acids across a wide dynamic range. Here, we report the development and validation of a spatially-resolved protein and RNA detection platform with the potential to simultaneously quantify up to 800 targets with greater than 5 log10 of dynamic range from a single formalin-fixed paraffin-embedded (FFPE) slide. We demonstrate validation of this technology by characterization of a panel of immune proteins expressed in colorectal cancer samples, and we also demonstrate spatially resolved detection of RNA. In situ high-plex digital molecular profiling is enabled by the use of UV-photocleavable small indexing DNA-oligo tags that can be delivered to the target within the tissue via direct attachment to RNA binding probes or conjugation to primary antibodies and are quantified with the standard nCounter technology. A slide-mounted FFPE tissue section is bound with a multiplexed cocktail of oligo-labeled primary antibody or mRNA hybridization probes, and a microfluidic flow cell is attached to the slide. Low-plex (3 or 4 color fluorescence) visible wavelength probes are utilized to generate an overall view of the FFPE tissue slice morphology (e.g., nuclear staining probes, select antibody pairs such as anti-CD3 and anti-CD8). Using the visible wavelength morphology as a guide, regions of interest (ROI) in the tumor are identified (e.g., areas with tumor infiltrating leukocytes) and then sequentially illuminated with UV light to release the indexing-oligos off all the high-plex molecular profiling reagents. Using this approach and standard microscope instrumentation, the limits of detection enable near single cell resolution. Following each UV illumination cycle, the photocleaved indexing-oligos are released into the buffer-layer above the tissue slice, collected via microcapillary aspiration, and stored in an individual well of a microtiter-plate. The contents of each well can then be referenced back to the exact region of tumor that was illuminated by UV light. Oligos are then hybridized to the nCounter fluorescently labeled optical barcodes to permit ex-situ digital counting of as many as 800 different analytes localized within a single ROI in the tumor. As demonstration of the technology, simultaneous multiplexed detection of CD3, CD8, CD45R0, CD4, CD45, PD-1, PD-L1, Vista, TIM-3, B7-H3, Ki67 (plus additional key IO-targets) will be quantified from colorectal tumor biopsies using oligo-conjugated primary antibodies. Furthermore, we will demonstrate detection of key IO associated immune RNA targets using direct hybridization of oligo-labeled probes. The ability to measure DNA, RNA, and protein at up-to 800-plex from single slices of FFPE tissue may enable the discovery of key immune biomarkers in tumors and accelerate the development immunotherapy and their associated companion diagnostics.
Citation Format: Dwayne Dunaway, Jaemyeong Jung, Chris Merritt, Isaac Sprague, Philippa Webster, Sarah Warren, Joseph Beechem. Spatially-resolved, multiplexed (up to 800 plex) digital characterization of protein and mRNA abundance in FFPE tissue sections: Application to immuno-oncology [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B081.