Abstract 5301: Visium HD 3’ enables unbiased whole transcriptome spatial profiling of tumor microenvironment in fresh frozen cancer tissues at single cell-scale resolution

Abstract Spatial transcriptomic technologies demonstrate the ability to comprehensively investigate the complexity of the tumor microenvironment, providing crucial insights into the interplay of different cellular and acellular components that orchestrate the tumor progression, angiogenesis, immune evasion, and metastasis. Here, we introduce Visium HD 3’, a novel assay for unbiased spatial gene expression profiling of fresh frozen tissue sections mounted on a standard glass slide. The Visium HD slide’s array has a gapless design that enables integration of unsupervised gene expression clustering data with high-resolution H&E images from the same tissue section, allowing precise profiling of finer anatomical features with high tissue coverage at single cell-scale resolution. Furthermore, this novel reverse transcription-based assay generates cDNA products compatible with both short reads and long reads sequencing, extending its utility beyond gene expression analysis to enable immune profiling and the discovery of isoforms and novel transcripts on a spatial level unlocking deeper insights into cancer diversity. Our research focused on deciphering the spatial heterogeneity of gene expression patterns within the tumor microenvironment, shedding light on the interactions between cancer cells, stromal cells, and vasculature. Visium HD 3’ was performed on fresh frozen human breast, pancreatic, liver, and lung cancer tissues. Spatial profiles of clinically-relevant cancer biomarkers such as AR, MUC1, CDH1, and ERBB2 were able to highlight tumor-prone regions in the tissues. Expression of known prognostic angiogenic markers such as VEGF, FGFR, PDGF was inline with the tumor region, indicating tumor growth and metastasis. Additionally, targeting stromal markers such as POSTN, LUM, MMP1, ACTA2, and SFRP4 revealed a distinct boundary between the tumor and stromal layer. The high resolution of this technology further enabled profiling of macrophage and T cell infiltration in cells surrounding the tumor by identification of immune markers like CD4, CD68, CD3E, PTPRC and CCL5. Our study highlights the importance of exploring the spatial organization of the entire transcriptome in cancer tissues and demonstrates that Visium HD 3’ platform is a powerful tool for unraveling the complexities of the tumor microenvironment. These findings lay the groundwork for the development of innovative therapeutic strategies and precision medicine approaches, ultimately contributing to improve outcomes for cancer patients. Citation Format: Debashish Chitnis, Marco Serra, Josh Gu, Anushka Gupta, Nancy Conejo, Aarushi Kalaimani, Govinda Kamath, Zixue Ma, Monica Nagendran, Joey Arthur, Julia Cowen, Anuj Patel, David Sukovich, Augusto M. Tentori. Visium HD 3’ enables unbiased whole transcriptome spatial profiling of tumor microenvironment in fresh frozen cancer tissues at single cell-scale resolution [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 5301.