Background Glioblastoma (GBM) is the most aggressive primary neuroepithelial tumor diagnosed in about 14,000 people in the United States each year. While immune-based cancer treatments have been successfully deployed in the clinic for hematological malignancies, treatment of solid tumors, like GBM, has proven more challenging. Targets for immunotherapy in GBM include PD-1, PD-L1, TIM-3 and LAG-3. A comprehensive understanding of the dynamic landscape of the tumor microenvironment (TME) will lead to identification of new biomarkers, development of new therapies and improved efficacy of current therapies.

The complex nature of the TME makes spatial multiplex immunohistochemistry (IHC) and immunofluorescence (IF) ideal for visualizing biomarker expression and cellular localization. The antibody’s specificity, sensitivity, and availability for multiplex IF is a critical success factor for TME characterization. Emerging automated staining and imaging platforms require qualified antibodies for accurate marker detection. Lunaphore COMET™ platform performs sequential immunofluorescence (seqIF™), which consists of sequential cycles of staining, imaging and elution.¹ COMET™ utilizes primary antibodies and fluorescently-conjugated secondary antibodies. We used seqIF™ to understand immune checkpoint marker expression in GBM.

Methods Formalin Fixed Paraffin Embedded (FFPE) tissue sections of human tonsil, kidney, and liver were stained with IHC validated antibodies from R&D Systems™, a Bio-Techne brand. Antigen retrieval and antibody incubation times were optimized on the COMET^TM platform.

Steps of Antibody Qualification on COMET^TM: 1) IHC validated antibodies are selected. 2) Specificity is confirmed by comparing staining on a positive and negative tissue sample, visual confirmation of subcellular localization, and overall expression pattern within the tissue. 3) Sensitivity is determined by staining tissues with different expression levels of the target protein. 4) Elution efficiency is determined by a secondary antibody only incubation using the integrated workflow on COMET™.

Results Using the antibody qualification process outlined above, we show tissue specific staining on liver endothelial cells and staining localized to sinusoids for Collagen IV. To further verify antibodies for use on Lunaphore COMET™, we show specific staining in additional tissues human tonsil and kidney. Importantly, we are able to simultaneously detect multiple immune checkpoint markers, like PD-L1, CTLA4, FASL, HLA-DRA, and IDO-1 in GBM.

Conclusions This study demonstrates a systematic qualification process for antibodies in multiplex immunofluorescence. We show successful staining of multiple immune checkpoint markers in GBM on Lunaphore COMET^TM. Rigorous antibody qualification for seqIF™ on COMET™ streamlines antibody selection and allows efficient TME profiling, facilitating a more comprehensive evaluation of solid tumors for biomarker discovery and immunotherapy development.

Acknowledgements We would like to thank the Lunaphore R&D team for their partnership on this project and the antibody qualification process.

Reference

1. Rivest François, et al. ‘Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics.’ Scientific Reports 2023 Oct 9;13(1):16994.