10x Visium: A breakthrough enabling Omics analysis at spatial level

Contributors: Aditya Mahadevan Iyer, Philge Philip, Debamitra Chakravorty, Tahseen Abbas, Shashi Rekha Thummala, Indu Gangwar, Jeffin Rockey, Prathik KV, Akshay Subramanian, Uzma Saeed, Jitesh Pillai, Puneet Saxena, Chandra Sekhar Pedamallu

Cell-cell interactions have played a significant role in the development of multicellular organisms. Understanding the spatial location of genes along with their expression can provide valuable insights for dissecting cellular functions and phenotypic state. This has been enabled by spatial transcriptomics that unravel the tissue architecture and provide a clear picture of the biological landscape. Interestingly, recent technological advancements have made spatial transcriptomics more accessible. The need for Spatial Transcriptomics (ST) technology can be understood in the context of its application to crucial areas such as the tumor microenvironment (TME), tumor heterogeneity, developmental biology, neuroscience, and regenerative medicine.

Out of the three broad Spatial transcriptomics technologies (namely, Laser capture microdissection (LCM) based approaches, Image-based in situ transcriptomics and Spatial barcoding-based transcriptomics), the barcoding-based ST methods that employ next-generation sequencing are at the forefront.

While our earlier whitepaper provides a generic sense of the Spatial omics, its application benefits and how Excelra takes it further, providing custom solutions, this collateral focusses sharply on the 10x Visium technology, detailing on technical aspects as well as certain use-cases where the Visium is comes handy.

Spatial barcoding-based transcriptomics

The spatial barcode-based transcriptomic approaches involve the sequencing of the RNA species at the whole transcriptome level. They offer unbiased and high-throughput analytical solutions. In this technology, the tissue sections are immobilized on the glass slides along with the reverse transcription primers with poly-T, which bind to the poly-A of the mRNA from the tissue sections. The primers also contain the spatial barcodes and unique molecular identifiers (UMIs) that represent the coordinates of each array. When the tissue is permeabilized, the mRNA molecules in the tissue cells get diffused into microwells (100 μm in size) on the slides and get hybridized with primers. The reverse transcription reagents will be added to the tissue to synthesize the cDNA molecules, which are then visualized using the Cy3-labeled nucleotides. The tissue section is removed by enzymes and the cDNA molecules remain hybridized on the glass side (Stahl et al. 2016).

Visium is the most widely applied spatial omics technologies that can sequence tissues of 6mm × 6mm in size. Each spot on the chip can be measured at the resolution of ~100 µm containing about 2–10 cells. In 2019, this method got further developed by 10× Genomics and commercialized as “10× Genomics Visium”. Its application benefits lead to exploring the new functionality of the organelles and may enhance our understanding in the field of spatiotemporal molecular medicine (Figure 1).

The latest Visium HD technology offers high sensitivity and single-cell scale resolution by improvement in spot resolution to 2 x 2 um squares, providing even more granular insights into tissue composition and cellular interactions. This high-definition mapping is crucial for identifying rare cell types and understanding complex biological processes.

Spatial barcoding-based transcriptomics

The spatial barcode-based transcriptomic approaches involve the sequencing of the RNA species at the whole transcriptome level. They offer unbiased and high-throughput analytical solutions. In this technology, the tissue sections are immobilized on the glass slides along with the reverse transcription primers with poly-T, which bind to the poly-A of the mRNA from the tissue sections. The primers also contain the spatial barcodes and unique molecular identifiers (UMIs) that represent the coordinates of each array. When the tissue is permeabilized, the mRNA molecules in the tissue cells get diffused into microwells (100 μm in size) on the slides and get hybridized with primers. The reverse transcription reagents will be added to the tissue to synthesize the cDNA molecules, which are then visualized using the Cy3-labeled nucleotides. The tissue section is removed by enzymes and the cDNA molecules remain hybridized on the glass side (Stahl et al. 2016).

Visium is the most widely applied spatial omics technologies that can sequence tissues of 6mm × 6mm in size. Each spot on the chip can be measured at the resolution of ~100 µm containing about 2–10 cells. In 2019, this method got further developed by 10× Genomics and commercialized as “10× Genomics Visium”. Its application benefits lead to exploring the new functionality of the organelles and may enhance our understanding in the field of spatiotemporal molecular medicine (Figure 1).

The latest Visium HD technology offers high sensitivity and single-cell scale resolution by improvement in spot resolution to 2 x 2 um squares, providing even more granular insights into tissue composition and cellular interactions. This high-definition mapping is crucial for identifying rare cell types and understanding complex biological processes.

Figure 1: Flowchart of 10X Visium technology and its related analyses [Adapted from Plant biotechnology research with single-cell transcriptome: recent advancements and prospects (Ali et al., Plant Cell Reports, 2024)]

Potential Solutions & Applications

Thanks to the growing interest in the ST domain, wherein in the last 5 years tremendous amount of work has been done while exploring application benefits of Visium technology. The following section highlights some of the use cases where Visium has opened new frontiers in the research field.

>> Exploring the spatial and temporal dimensions of tissue formation and growth

10X Visium technology has garnered extensive usage in studying the development of embryos, tissues, and organs across various species, supported by a substantial body of literature [Rao et al, Garcia-Alonso et al]. Notably, it has advanced the understanding of embryogenesis. By analyzing tissues at different developmental stages, Visium can provide insights into the temporal dynamics of gene expression crucial for studying molecular processes like embryonic development, wound healing, and tissue regeneration. Visium supports both fresh-frozen and formalin-fixed, paraffin-embedded (FFPE) tissue sections, making it suitable for a wide range of studies. This versatility allows for the examination of archived clinical samples and the study of various tissue types.

>> Creating detailed maps of tissues or anatomical areas

Visium offers unbiased means to create spatial maps of tissues, leading to the development of comprehensive reference tissue atlases. Examples include human kidney tissues in various health and disease states, spatial multi-omics maps detailing cardiac remodeling, and detailed atlases of human lung structures. Moreover, Visium has also contributed to identifying spatial distribution of gene expression signatures in human dorsolateral prefrontal cortex [Maynard et al.].

>> Investigating the underlying genetic and cellular disruptions that lead to various diseases.

The 10X Visium platform has significantly advanced our understanding of genetic and cellular disruptions in various diseases by preserving the spatial context of gene expression within tissues. This helps in identifying how different cells interact within their native environments, which is crucial for understanding complex diseases like cancer. The platform’s high-density arrays of spatially barcoded capture probes enable precise tracking of gene expression. In diseases like Alzheimer’s, Visium has been used to identify specific gene expression changes near amyloid plaques, providing insights into inflammation and myelination processes. 10X Visium is thus assisting in more targeted and effective treatments.

>>Examining the diverse cell populations and their surrounding environments within tumors

The interplay between extrinsic immune cells and intrinsic tumor cells plays a pivotal role in shaping tumor progression and metastasis. Spatially, different tumors vary amongst themselves in tumor microenvironment organization and hierarchy [Satilmis et al] and external stimuli such as Chemotherapy initiate spatial reprogramming, thus emphasizing the need for understanding spatial architecture of TME.

In primary liver cancer (PLC), 10X Visium has been used to characterize stromal and immune cells distribution in TME from normal to leading-edge to tumor regions. This analysis led to the identification of different patterns of PLC spatial heterogeneities in different patients. Spatial analysis of stemness markers revealed PROM+ and CD47+ to be related to TME remodeling and tumor metastasis [Wu et al]. 10X Visium has also been used to understand tumor associated microbiota in oral squamous cell carcinoma and colorectal cancer.

>> Mapping disease and treatment-related biomarkers with spatial precision

Biomarkers can be identified through spatial omics that are prognostic or predictive of therapeutic response, highlighting the translational application of spatial transcriptomics. 10X Visium has revolutionized the identification and application of spatial microstructural biomarkers, significantly enhancing the accuracy and sensitivity of molecular, cellular, and microstructural biomarkers for improved diagnosis, prognosis, and treatment of diseases. Numerous examples demonstrate its impact across various conditions:

In hepatocellular carcinoma, a 6-gene signature has been identified using ST for predicting patient survival [Zhao et al]. CD56+ immune cells are predictive of outcomes following PD-1 checkpoint blockade in non-small cell lung cancer (NSCLC) [Zugazagoitia et al]. CDH12-enriched epithelial subpopulations predict poor-outcomes post-surgery and superior responses to immune checkpoint therapy in bladder cancer [Gouin et al].

In addition to gene signatures predicting response to immunotherapy, spatial features such as spatial organization of immune cells and identification of tumor invasive front can be biomarkers of prognosis and treatment response.

Future Perspectives and Conclusions

Advancements in 10X Visium underscore the transformative potential of the technology in not only unraveling spatially defined biomarkers but also in translating findings into clinical practice, enhancing personalized medicine approaches across diverse disease contexts. With the currently fast-moving progress and promising results from early adopted research projects, we can foresee the bright future of such new tools in understanding life at the most profound analytical level. 10X Visium has significantly amplified the capabilities of single cell sequencing technologies in identifying and characterizing cell types and states. Understanding the functional roles of different cell types necessitates their physical localization and interactions. The spatial dimension provided by 10X Visium enables precise analysis of cell populations, neighboring cell interactions, and overall cellular structural organization. Other than transcriptomics, the proteome, neuronal connectome, and 3D chromatin conformation are crucial to cell function, and methods like DBiT-seq and MERFISH, have been developed to profile these along with the transcriptome in the same cells.

The future holds lot of promises in this direction along with scope to integrate data into comprehensive databases. The introduction of modern querying using AI models and visualization techniques is the way forward. Despite progress, achieving this vision will likely require continued development and integration of diverse techniques and technologies. Integration of such high-dimensional technologies into routine clinical use is not an easy task as multiple steps remain before preclinical findings are integrated into clinical practice. Excelra where “data means more” is aiming to contribute towards this growth where our SMEs and customized Nextflow pipelines is assisting clients navigate through complex datasets and building actionable insights.

References

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