Understanding the Tumor Microenvironment With a Precise Spatial Transcriptomics Platform

Visium HD 3’ enables unbiased whole transcriptome spatial profiling of Tumor microenvironment in fresh frozen cancer tissues at single cell scale resolution. Debashish Chitnis1, Marco Serra1, Josh Gu1, Anushka Gupta1, Nancy Conejo1, Aarushi Kalaimani1, Miriam Valencia1, Monica Nagendran1, Zixue Ma1, Govinda Kamath1, Joey Arthur1, Julia Cowen1, Anuj Patel1, David Sukovich1, Augusto M Tentori1 10x Genomics, Pleasanton, CA 3. Tissue microenvironment profile highlights tumor-stroma interaction in human breast cancer Please contact info@10xgenomics.com for inquiries or learn more at 10xgenomics.com References and Acknowledgments 1. Ali, A., Brown, V., Denley, S., Jamieson, N. B., Morton, J. P., Nixon, C., Oien, K. A. (2014). Expression of KOC, S100P, mesothelin and MUC1 in pancreatico-biliary adenocarcinomas: development and utility of a potential diagnostic immunohistochemistry panel. https://pmc.ncbi.nlm.nih.gov/articles/PMC4112611/?utm_source=chatgpt.com 2. 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Targeting macrophages in cancer immunotherapy. Retrieved from https://www.nature.com/articles/s41392-021-00506-6 Thanks to the Visium HD development team at 10x Genomics, Pleasanton, CA. (Histology team, Microscopy and Sequencing Core) #5301 1. Introduction Despite years of studies and effort, the best strategies for treating cancer and minimizing the complications of treatment remain unanswered questions. This gap in knowledge is partially due to the inability to dissect the complex heterogeneous tumor microenvironment (TME) and Stromal/immune compartment. Spatial transcriptomic technologies 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. 2. Methods 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’s array has a gapless design that enables integration of unsupervised gene expression clustering data with microscope 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 applications 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. Figure 1. Visium HD Spatial Gene Expression slide. The Visium HD Slide Capture Area consists of ~11 million 2 x 2 μm spatially-barcoded squares without gaps and the data was binned to 8 x 8 μm for visualization and analysis. Visium HD data is also available with the raw 2 μm data and other bin sizes. Figure 2. Visium HD 3’ assay workflow. Visium HD 3’ is compatible with H&E staining of fresh frozen (FF) tissues mounted on glass slides using routine histological workflows. This provides the flexibility to image and select target regions prior to whole transcriptome analysis. The Visium HD 3’ assay utilizes the CytAssist instrument for transcript capture. The captured transcript undergoes reverse transcription, second strand synthesis and finally cDNA amplification. This is followed by library construction, sequencing, data processing with Space Ranger, and visualization with Loupe Browser. Figure 3. (A) Unbiased gene expression based clustering (8μm bin) identifies key clusters differentiating the tumor and stromal environment in human breast cancer. Differential expression of the tumor and stromal cluster identifies spatial markers such as EPCAM, ERBB2, AR, MUC1 known to be overexpressed in breast cancers(3) and POSTN, ACTA2, LUM, MMP2, SFRP4 involved in modulating tumor-stroma interaction, extracellular matrix remodelling and immune evasion.(2) (D) Co-expression of these marker genes highlight the boundaries between tumor and stromal layers. (B) Annotation by pathologist of H&E stained image confirms the invasive carcinoma as identified with the unbiased clustering. (C) In addition, clustering based on nuclei based cell segmentation is further able to resolve the stromal compartment. Looking into the differentially expressed genes in these clusters manual cell type annotation was performed identifying tumor, endothelial, tumor associated macrophages (TAM), fibroblasts, mesenchymal and epithelial cell types. 4. Unbiased clustering and tissue-specific marker gene expression identifies small intestine remnant in pancreatic cancer tissue 5mm 2mm Tumor cells Endothelial cells TAM (Tumor Associated Macrophages) Fibroblasts Mesenchymal cells Epithelial cells 2mm 500μm Tumor EPCAM ERBB2 AR MUC1 Stroma POSTN KRT7 ACTA2 LUM MMP2 SFRP4 Spatial marker co-expression 2mm 500μm 5mm Tumor Organ: Ampulla of Vater Tumor Grade: Well Differentiated Immune Aggregate Small Intestine Invasive Carcinoma Figure 4. (A) Unbiased clustering (8μm bin) identifies two clusters pertaining to morphologically distinct spatial regions. (B) Co-expression of tissue specific markers such as Muc1, s100P(1) for pancreatic cancer and APOA4, APOA1, REG3A, ALDOB known to be expressed in the enterocytes in villi of small intestine and involved in lipid and energy metabolism.(4,5) (C) Annotation by pathologist of H&E stained image and anatomy of the tissues indicate that a portion of the small intestine was removed along with the pancreas while performing the tumor biopsy. This is fairly common when tumors form in the head of the pancreas, which connects to the duodenum region of the intestine. A. C. D. A. 5mm B. C. Small Intestine and Pancreas Anatomy Small Intestine Markers APOA4, APOA1, REG3A, ALDOB Pancreatic Cancer Markers Muc1, s100P 6. Deeper dive into the tumor microenvironment of human lung adenocarcinoma 2 mm 500μm A. 2mm Tumor cells EIF3L MAL2 RPSA OCLN MUC1 M2 TAMs SPP1 FOLR2 CD163 CCL13 Figure 6. (A) Unbiased nuclei based cell segmentation based clustering identifies key clusters. We annotate four cell types (tumor associated macrophages, tumor cells, plasma/B cells, fibroblasts) based on differential marker gene analysis. (B) M2 TAM’s or pro-tumorigenic macrophage identified based on CCLl13, SPP1, CD163, FOLR2.(6,7) Co-expression of tumor cell markers and M2 TAM’s is indicative of tumor progression, metastasis and immune invasion in lung adenocarcinoma. (C) IGFBP5, POSTN, and COL3A1 are integral to fibroblast activation in the tumor microenvironment of lung cancer.(8) The interaction between these genes and the tumor cells in the stromal component is in line with identification of M2 TAMs. These factors point to fibroblast-to-Cancer associated fibroblast conversion in the extracellular matrix (ECM).(9) (D) Plasma/B cell cluster are identified with expression of the IG genes. B-cells in the TME can activate TAMs via cytokine signaling promoting M2 polarization of macrophages.(10) 5. Tumor-stroma-immune interactions in pancreatic cancer tumor-microenvironment. A. Figure 5. (A) Unbiased gene expression (8μm bin) based clustering identifies key immune, stroma,tumor associated clusters. Spatial markers were identified by differential gene expression analysis. We use these key gene markers to show co-expression of invasive carcinoma with (B) immune aggregates, (C) B-cells/Plasma and (D) Stroma/Fibroblasts. This is also inline with the annotation by pathologist - Fig 4. (c) B. Cancer activated Fibroblasts (CAF) IGFBP5 POSTN COL3A1 2mm 500μm 2mm 500μm Plasma/B cells IGKC IGHG3/4 IGHA1/2 IGLC2 JCHAIN C. D. B. C. 5mm 1mm 1mm D. Stroma/Fibroblasts ISLR MMP11 COL3A1 1mm B-cell/Plasma IGLC3 IGHG3 IGHG4 B. 1mm Invasive carcinoma Muc1 s100P Immune aggregate CD48/52/79A MS4A1 CR2 CXCR4 Abstract Presentation Number 2mm 500μm 9. Conclusion 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 may assist with the development of innovative therapeutic strategies and precision medicine approaches, ultimately contributing to improve outcomes for cancer patients. Tumor cells Plasma/B cells Fibroblasts TAM (Tumor associated macrophages) 500μm © 2025 10x Genomics, Inc. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.