Abstract

To power immuno-oncology discovery, it is highly beneficial
to explore healthy and tumor tissues with immuno-oncology markers using
multiplexed analysis. The Hyperion™ Imaging System uses novel technology for
tissue imaging that enables multiplexed analysis of protein expression in a
single tissue sample. This methodology uses tissue sections stained with a
cocktail of antigen-specific antibodies conjugated to different metal isotopes.
In this study, we demonstrate how to generate high-parameter images with highly
relevant immuno-oncology markers on the Hyperion Imaging System.

To detect multiple markers in one panel, we optimized the
tissue staining protocol for signal detection and tissue preservation. For
formalin-fixed, paraffin-embedded (FFPE) tissue staining, antigen retrieval
conditions (temperature and incubation time) were optimized. We determined that
antigen retrieval conditions of 96 °C for 30 minutes in basic retrieval
solution enabled detection of nuclear markers such as FoxP3, along with other
surface and cytoplasmic markers.  To
verify these methods, we also generated equivalent data to compare these
results with immunofluorescence on FFPE tissue sections and examined co-localization
and anti-localization of the antibodies with previously verified counter stains.

Using these optimized staining protocols, we generated
images from various normal and tumor tissues (including diffuse large B cell
lymphoma, colon adenocarcinoma, and bladder urothelial carcinoma) to show a
combination of 5 structural, 1 cancer, 3 nuclear, and 18 immuno-oncology
markers simultaneously. Together with other tissue architectural details,
different immune cell types were identified in both normal and tumor tissues.
This image resolution allowed for the visualization of proteins in the
membranous, cytoplasmic, and nuclear cell compartments. Therefore, our data
demonstrate that the Hyperion Imaging System provides a high-parameter imaging
solution at sub-cellular resolution to characterize the immune repertoire in the
tumor microenvironment.