P08.09 A one-shot multiplex immunofluorescence platform for spatial analysis of heterogeneity in vascular immune niches in clear cell renal cell carcinoma

Introduction Clear cell renal cell carcinoma (ccRCC) is characterized by a highly heterogeneous tumor vasculature, which varies in density and architecture and is associated with prognosis and response to anti-angiogenic therapy and immune checkpoint inhibition. 1 2 Despite its clinical relevance, the molecular basis of vascular heterogeneity in ccRCC remains poorly understood. Conventional immunohistochemistry and vessel density measurements do not cover the full complexity of endothelial phenotypes and their spatial interactions with tumor and immune cells.3–5 This study aims to enable extended spatial characterization of endothelial subtypes in ccRCC using high-throughput multiplex immunofluorescence combined with computational image analysis.Material and methods An optimized one-shot multiplex immunofluorescence protocol for formalin-fixed paraffin-embedded (FFPE) tissues was established, enabling simultaneous detection of up to nine markers in a single staining round. The approach combines antibody-oligonucleotide conjugation and conventional secondary antibody staining to maximize sensitivity. Marker panels include endothelial, tumor, immune, stromal, and proliferation markers relevant to the vascular microenvironment in ccRCC. Whole-slide imaging was performed using widefield fluorescence microscopy for rapid acquisition of large tissue areas. A fully automated image analysis pipeline was developed, including denoising, spectral unmixing, background subtraction, tissue segmentation, and single-cell quantification. Vascular architectures were classified using pattern recognition algorithms.Results The staining protocol yielded reproducible, high signal-to-noise detection of all markers across full-slide FFPE sections with minimal autofluorescence and preserved tissue morphology. The one-shot workflow enabled staining and imaging of multiple whole-slide samples within 48 hours, substantially increasing throughput compared with sequential multiplexing approaches. The computational pipeline reliably separated spectrally overlapping signals, enabled robust tissue and cell segmentation, and supported quantitative spatial analysis at single-cell resolution, demonstrating the technical suitability of the platform for large-scale spatial profiling.Conclusion This work establishes a scalable, high-throughput multiplex immunofluorescence and analysis workflow optimized for FFPE ccRCC tissues. The platform provides a robust technical foundation for future systematic investigation of endothelial heterogeneity and tumor-microenvironment interactions and is readily extendable to larger cohorts and additional cancer types.References Toma MI, et al. Low-branching vessel architecture shapes immune cell niches and predicts immune responses in renal cancer. medRxiv. 2025;2025-06.Canete-Portillo S, et al. Vascular architectural patterns in clear cell renal cell carcinoma and clear cell papillary renal cell carcinoma. Virchows Archiv. 2021;479(6):1187–96.Cheng SH, et al. Prognostic role of microvessel density in patients with renal cell carcinoma: a meta-analysis. International Journal of Clinical and Experimental Pathology 2014;7(9):5855.Schraml P, et al. Specific immune cell and lymphatic vessel signatures identified by image analysis in renal cancer. Modern Pathology 2019;32(7):1042–52.Rautiola J, et al. Association of angiopoietin-2 and Ki-67 expression with vascular density and sunitinib response in metastatic renal cell carcinoma. PLoS One. 2016;11(4):e0153745.