Abstract 2825: High-plex, spatial RNA profiling of tumor infiltrating leukocytes and the tumor microenvironment of microsatellite instable colorectal cancer using GeoMx™ Digital Spatial Profiler
Authors: Norgaard Zachary , Zollinger Dan , Reeves Jason , Zhou Zoey , Kriner Michelle , McKay-Fleisch Jill , Bahrami Arya , Warren Sarah , Church Sarah , Merritt Chris , Hoang Margaret , Beechem Joseph 
Published: September 2020

Abstract
Immunotherapeutic intervention has revolutionized cancer treatment but improved understanding of immunomodulation in tumors is still necessary to expand the reach of these therapies and identify rational combination approaches. An important aspect of this process will be characterizing the molecular differences between tumor-infiltrating leukocytes (TILs) and stromal leukocytes (non-TILs) surrounding the same tissues. Most studies to date have focused on dissociated tissues, which means identifying the origin of the profiled leukocytes is only possible with post-hoc inference. High-plex profiling that retains spatial orientation has proven difficult in fixed tissues, preventing direct understanding of TIL localization beyond a handful of pre-selected targets. To explore the transcriptional profile of TILs in situ, we leveraged a high-plex (1,400+ gene) mRNA panel for the GeoMxTM Digital Spatial Profiler (DSP) to profile microsatellite instable (MSI) colorectal cancer (CRC) samples noted to have a high abundance of CD3+ TILs by 4-color immunofluorescence (IF).
In this study, more than 5,000 unique probes (3 to 10 probes per target mRNA) with coupled photocleavable oligonucleotide tags were hybridized to formalin-fixed paraffin-embedded (FFPE) tissue sections from these CRC samples. Regions of interest were selected inside (n = 6, per tumor) and outside (n = 6, per tumor) the tumor invasive margin focusing on tumor or stromal regions with high numbers of CD3+ cells. Within each region of interest, we created a custom segmentation strategy to specifically illuminate CD3+ cells, and then sequentially illuminate regions neighboring those cells. These additional custom masks (n = 47) were defined by extending multiple contours around the initially selected TILs and non-TILs to determine differences in the local environment of each population.
Collected photocleaved oligonucleotide tags were sequenced using the GeoMxTM NGS workflow. Targets included in the 1,400+ gene panel represent immune cell markers, checkpoint molecules, cytokines and chemokines, canonical cancer pathways, and biological signatures. We found that regions neighboring TILs express higher levels of known oncogenic pathways and stromal regions neighboring non-TILs were noted to have higher expression of ECM genes, confirming the specificity of the profiling approach. Furthermore, we found that TILs specifically up-regulate expression of cytolytic pathway genes, as well as several coinhibitory and costimulatory checkpoint genes. We also observe dysregulation of members of the adenosine metabolism pathway within the tumor regions profiled and TILs, but not in regions adjacent to the tumor itself. Together, our results demonstrate the feasibility of profiling specific cell populations with a high plex mRNA panel in situ in FFPE tissue, thus enabling pathway level differential expression analyses and exploration of key interactions between neighboring cell types while retaining their spatial context. For research use only. Not for use in diagnostic procedures.
Citation Format: Zachary Norgaard, Dan Zollinger, Jason Reeves, Zoey Zhou, Michelle Kriner, Jill McKay-Fleisch, Arya Bahrami, Sarah Warren, Sarah Church, Chris Merritt, Margaret Hoang, Joseph Beechem. High-plex, spatial RNA profiling of tumor infiltrating leukocytes and the tumor microenvironment of microsatellite instable colorectal cancer using GeoMx™ Digital Spatial Profiler [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2825.