Abstract 5254: Multi-plexed spatial interaction analysis of the tumor microenvironment in head and neck cancers

Abstract Head and neck cancers are the 7th most common cancer type globally accounting for over 900, 000 cases annually. With increasing numbers of systemic and targeted therapies, including immune checkpoint inhibitors (ICI), it has become increasingly important to understand the complex tumour microenvironment (TME) milieu dictating clinical responses to therapy. In this study, we profiled 40 whole slide, head and neck cancer tissues from patients prior to ICI therapy and profiled the TME for spatial interactions using the NaveniPlex in situ proximity ligation assay (isPLA, Navinci ). Using the Leica BOND RX, formalin-fixed, paraffin-embedded (FFPE) tissue sections were prepared by baking, deparaffinization, and epitope unmasking. Staining was then carried out using the NaveniPlex isPLA technology on the Leica BOND RX instrument. To assess interactions between PD1/PD-L1, CD8/MHCI, and CD3+ T cells, three pairs of Navenibodies were employed simultaneously. Briefly, the NaveniPlex isPLA protocol involves tissue blocking, followed by incubation with primary Navenibodies, ligation, and rolling circle amplification (RCA). Each Navenibody pair generates a distinct RCA product. The RCA products for PD1/PD-L1, CD8/MHCI, and CD3+ T cells were visualized using oligonucleotides conjugated to Atto647N, Atto550, and AlexaFluor488, respectively.Our spatial interaction analysis for PD1/PDL1, CD8/MHCI and CD3+ T-cells showed distinct TME interactions associated with responders and non-responders to ICI therapy. Interactions mapped with cellular phenotypes revealed the cellular neighbourhoods and communities at which these interactions may be useful prognostically. Taken together, this study provides a methodological framework to profile pre-ICI treatment tissues to identify spatial interactions associated with clinical endpoints. Citation Format: Vahid Yaghoubi Naei, Sara Bodbin, Habib Sadeghirad, Rafael Tubelleza, James Monkman, Agata Wicher, Subham Basu, Arutha Kulasinghe. Multi-plexed spatial interaction analysis of the tumor microenvironment in head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5254.