Abstract 2780: Development of automated multiplex immunofluorescence protocols for tumor microenvironment evaluation
Authors: Masabanda Julio S. , Wang Sherry , Vargas Joseph , Ramos Jason 
Published: July 2021

Abstract
Introduction: In recent years, the ability to detect several proteins on a single slide in Multiplex Immunofluorescence (mIF) experiments has become common place. This is possible thanks to factors such as development of antibodies of high quality, improvements in fluorescent technology, and automation of IHC staining. One of the advantages of multiplexing protein detection is to gain maximal data per tissue section, which is critical when samples are limited. Understanding co-expression and spatial organization of multiple targets within preserved tissue architecture is also important, especially in tumor microenvironment (TME) analysis. mIF plays a key role in research fields like tumor immunology where it is needed to catalog subsets of immune and cancer cells within the TME. Ultimately, this will enable the development of personalized, combinatorial therapeutic interventions.
Materials and Methods: Selected antibody panels: Tonsil Panel 1: p40, CD3, CD8, CD20 & CD68 Tonsil Panel 2: LAG3 [BC40], LAG3 [CAL26], CD57, CD19, FOXP3, T-bet, PD-L1, PD-1 & GATA-3 Colon Cancer Panel: Granzyme B, CD8, CDX2, CD3 & Pan Cytokeratin Plus Melanoma Panel: Melan A, CD3, SOX10, CD8 & Granzyme B Detection was performed with Biocare MACH 2 Universal HRP Polymer. Fluorochromes: Opal 480, Opal 520, Opal 570, Opal 620, Opal 650 VALENT (Biocare Medical) fully automated staining platform was utilized to stain slides. Image acquisition and analysis was performed on an Olympus BX61 microscope coupled with the ASI FISH imaging system. In the workflow, a proprietary room temperature (RT) stripping reagent was utilized instead of the heat stripping step commonly used between antibody detection incubations.
Results and Discussion: The antibodies used have been previously optimized for standard IHC protocols on VALENT using DAB. However, the sensitivity of tyramide detection is very high and for most of the antibodies, the incubation times had to be drastically reduced to minimize non-specific staining and the potential bleed-through to other filters for neighboring fluors. Additionally, Opal fluorochromes were diluted between 1:200 and 1:300. The RT stripping reagent helps in the preservation of tissue integrity even after the sequential detection of 5 antibodies. Additionally, by using this reagent, a rigorous order of primary antibody application is no longer needed. Finally, this reagent dimmed some autofluorescence coming from red blood cells. Following some brief optimization steps, we obtained strong and specific staining patterns similar to ones obtained with individual IHC experiments visualized with DAB.
Conclusion: We have demonstrated the use on an automated platform of a RT stripping solution. Additionally, this reagent can be used with different antibodies in order to effectively characterize the TME in many cancers.
Citation Format: Julio S. Masabanda, Sherry Wang, Joseph Vargas, Jason Ramos. Development of automated multiplex immunofluorescence protocols for tumor microenvironment evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2780.