https://openworks.mdanderson.org/sumexp21/1205/thumbnail.jp

Cuentas, Edwin Parra

Ibarguen, Heladio P

John, Davis Panachethu

McAllen, Salome A

Rodriguez, Saxon A

Tamegnon, Auriole

English

OpenWorks @ MD Anderson

Optimization of an Exploratory Oncology Panel for Immunoprofiling Tissues in the Tumor Microenvironment using CO-Detection by indEXing (CODEX) methodology Davis P. John1, Saxon A. Rodriguez2, Salome A. McAllen2, Edwin R. Parra21. The University of Texas at Dallas, Richardson, TX, 2. Department of Translational Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX• Phillips D, Schürch CM, Khodadoust MS, Kim YH, Nolan GP, Jiang S. Highly Multiplexed Phenotyping of Immunoregulatory Proteins in the Tumor Microenvironment by CODEX Tissue Imaging. Front Immunol. 2021 May 19;12:687673. doi: 10.3389/fimmu.2021.687673. PMID: 34093591; PMCID: PMC8170307.Acknowledgements: Research for this poster was supported by the MD Anderson Cancer Center’s Department of Translational Pathology. This department is responsible for the summer ITERT program. For further information, please contact Davis John at davisjohnpm@gmail.com or Dr. Edwin Parra Cuentas at ERParra@mdanderson.org  Methods • We hypothesize that the various markers will stain in similar patternswith both chromogenic IHC and CODEX, and that the antibodies willcorrectly recognize various lymphoid, myeloid, structural and immunecells in order to effectively depict the immunoprofile.Figure 1. Workflow of the application of the CODEX panel. ResultsFigure 5. Example of cycles staining in human reactive tonsil and non-small cell lung cancer case . BackgroundReferencesDiscussion and ConclusionHypothesis Multiplex Immunofluorescence and Digital Pathology LaboratorySecond cycleDAPICD3DAPICKDAPICD8DAPICD107DAPIKI67DAPICD20DAPICKKI67CD3CD20CD8CD107DAPICKDAPICKKI67CD3CD20CD8CD107Reactive tonsil Non-small cell lung cancerFirst cycleFirst cycleSecond cycle• Antibody selection• Panel design• Individual antibody optimization by Immunohistochemistry• Antibody conjugation with their oligonucleotide• Gel electrophoresis testing to validate proper conjugation• Individual antibody staining in CODEX • Group the antibodies in cycles and staining Figure 2. Antibodies included in the CODEX panel. • We successfully optimized the various markers to obtain similar staining patterns with both chromogenic IHC and CODEX. PD-L1 was predominantly expressed on the cell membranes of epithelial cells on tonsil tissue. Other immune checkpoint proteins were observed in various immune cells. In control tonsil, CK was expressed by epithelial cells, and among cells surrounding the germinal centers, the T cell marker CD3 was most abundant, followed by CD8, Granzyme B, CD45RO, FOXP3. PD-1 was predominantly distributed within the germinal center of the tonsil tissue. We are continuing to test the other markers in the panel.• Multiplexed immunophenotyping approaches reveal key insight into identifying therapeutic mechanisms. A comprehensive immunoprofile will advance our understanding of how various factors can determine disease progression, resistance and response to immunotherapies and facilitate the development of new treatment approaches.• Cancer immunotherapy is revolutionizing cancer treatment by aiding the natural ability of the immune system to identify and defend against tumors.• In order to better design cancer therapeutics and advance clinical care, the immunoregulatory proteins involved in immune checkpoints and immunosuppression must be immunoprofiled.  • We used highly multiplexed tissue imaging technologies, such as CODEX (CO-Detection by indEXing), which allows more than 40 antibody markers to be placed and analyzed on tissues.• Herein, we describe the optimization, validation and application of a 27-antibody panel used for multiplexed immunophenotyping. Figure 3. Heat map showing the presence of various markers in different cell-types. Figure 4. Percent of immune and tumor cells that express various immunoregulatory proteins. 

Optimization of an Exploratory Oncology Panel for Immunoprofiling Tissues in the Tumor Microenvironment using CO-Detection by indEXing (CODEX) Methodology

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1205&context=sumexp21

Optimization of an Exploratory Oncology Panel for Immunoprofiling Tissues in the Tumor Microenvironment using CO-Detection by indEXing (CODEX) Methodology

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