Abstract 5354: Single-cell sequencing resolves prevalence of PD-L1/2-expressing tumor-associated endothelial cells in human solid tumors

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Abstract BACKGROUND: The PD-1 ligands (PD-L1 and PD-L2) are immune checkpoint proteins that can be expressed by stromal cells including endothelial cells (ECs) within the tumor microenvironment (TME). Pe-clinical studies have implicated endothelial PD-L1 as a contributory therapeutic target of immune checkpoint inhibitor drugs especially when combined with anti-angiogenic drugs. However, the clinical prevalence and implications of endothelial PD-L1/2 expression in human solid tumors remain unexplored. HYPOTHESES: Endothelial PD-L1/2 expression occur at low frequencies in the TME overall, but may be upregulated in specialized EC phenotypes, e.g., tumor-associated high-endothelial venules (TU-HEVs) or tip-like ECs of angiogenic sprouting blood vessels. OBJECTIVES: (1) To explore the utility of single-cell profiling as a means to survey the levels of PD-L1 and PD-L2 transcription in ECs from clinical solid tumor specimens. (2) To identify potential EC subtypes in the TME that may be enriched for PD-L1/2 expression. METHODS: We performed single-cell RNA-seq (scRNA-seq) and single-cell ATAC-seq (scATAC-seq) on a pan-cancer cohort of 33 clinical core biopsies from 25 patients using the 10X Chromium Multiome kit. After performing quality control and normalization using Seurat (v4.3.0), there was a median of 4367 cells per sample (IQR: 2548-6025). RESULTS: Two orthogonal cell type classification methods - SingleR (v2.0.0) using the Human Primary Cell Atlas [PMID 24053356], and Seurat’s reference mapping function using the annotations from Bassez et al., 2021 [PMID 33958794] - identified 3, 829 ECs from 31 samples (median 102, IQR 43-141) with 99.7% concordance. The overall prevalence of ECs expressing PD-L1 or PD-L2 was low (1.4% and 2.6%, respectively). ECs were then classified into subtypes (arterial, venous, lymphatic, tip-like, TU-HEVs) by first clustering cells based on RNA expression using Seurat, and then assessing the expression of marker genes in each cluster against a published reference dataset Hua et al., 2022 [PMID: 36423635]. After subtyping, PD-L1 was enriched in lymphatic ECs (3.5%), and PD-L2 was enriched in lymphatic ECs (4.1%) and TU-HEVs (5.6%). CONCLUSIONS: This study suggests that endothelial PD-L1/2 expression can be found in clinical core biopsies of human solid tumors, and that the role of endothelial PD-L1/2 within the TME warrants further investigation especially in the context of combined anti-PD-1/PD-L1 plus anti-angiogenic therapy. Ongoing work aims to confirm these results at the protein level using spatial imaging in larger cohorts of specific cancer types. Citation Format: Cathy Yan, Florence T. Wu, Janessa Laskin, Cheryl Ho, Marco A. Marra. Single-cell sequencing resolves prevalence of PD-L1/2-expressing tumor-associated endothelial cells in human solid tumors [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 5354.

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