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1503 Optimization of an Integrated MultiOmyx-RNAscope hyperplex assay for co-detection and characterization of multiple protein and RNA biomarkers within the tumor microenvironment (TME)
  1. Courtney Todorov,
  2. Sandra Lam,
  3. Harry Nunns,
  4. Kevin Gallagher,
  5. Juliana Wortman,
  6. Elaine Yeung,
  7. Eric Leones,
  8. Flora Shafi,
  9. Erinn Parnell and
  10. Qingyan Au
  1. NeGenomics Laboratories, Aliso Viejo, CA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Spatial analysis of protein or gene expression is vital to understand the distribution, phenotypes, and interactions between cells within TME. Traditionally, multiplexed spatial analysis has been performed using methods to detect either protein or RNA separately. Combining spatial analysis of protein-RNA on a single specimen is a powerful method to identify the cellular source of secreted proteins, characterize the cytokine signature, study gene expression in specific cell types as defined by protein biomarkers, or map the distribution of CAR-T+ cells within the TME. We have previously demonstrated validation of an integrated workflow to co-detect RNA and protein in a single FFPE slide using the MultiOmyxTM and RNAscope platforms. MultiOmyx is a proprietary immunofluorescence (IF) platform for the visualization and characterization of up to 60 protein biomarkers in a single FFPE section. RNAscope Multiplex is a highly sensitive fluorescent in-situ hybridization (ISH) assay that can detect up to 3 RNA markers in a single FFPE section. A unique feature of the Integrated MultiOmyx-RNAscope assay is the presence of a protease pretreatment step, which is required for RNAscope ISH staining but can damage proteins and consequently interfere with downstream antibody-antigen binding. We previously demonstrated robustness of an Integrated assay to characterize infiltrating lymphocytes within TME. However, subsequent testing of additional protein biomarkers with the Integrated assay has shown some incompatibility or suboptimal signal to noise (S/N). Individual optimization steps to improve protein biomarker S/N and compatibility are time consuming and can be limited by antibody clone availability. Therefore, we decided to globally optimize the Integrated MultiOmyx-RNAscope assay to improve overall protein biomarker performance.

Methods Optimization of both antigen retrieval and protease pretreatment steps was performed to improve protein biomarker performance. A validation of the optimized Integrated MultiOmyx-RNAscope assay was then completed using a 2-plex ISH 18-plex IF biomarker panel on FFPE human CRC samples. Expression of each ISH and protein biomarker was quantified using the proprietary MultiOmyx Analytics pipeline and inter/intra run coefficient of variation (CV) were calculated for the precision assessment.

Results The optimized Integrated assay demonstrated improved protein biomarker staining/compatibility without compromising RNA ISH signal. Additionally, all markers evaluated showed highly reproducible results and passed successful criteria for precision evaluation. These results therefore demonstrate a highly robust assay with even improved performance observed for some markers previously evaluated.

Conclusions Therefore, we successfully optimized the Integrated MultiOmyx-RNAscope assay for co-detection of protein/RNA in single specimen thereby improving assay development turnaround and protein biomarkers performance/compatibility.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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