Illuminating Biomarker Profiles in Cellular Landscape with Genotyping & Phenotyping Spatial Multiomics by Omicsveu

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Introduction

Pathologists have developed the incredible ability to visually annotate the complex biology captured on a tissue slide. Leveraging the traditional H&E stain and a microscope, they can distinguish and classify areas affected with disease within tissue and reach crucial diagnostic conclusions. Due to the complexity of tissue biology, and the constant need for greater resolution to study that complexity, scientists sought to develop molecular tools for spatial tissue analysis of genotype and phenotype. They found that fluorescently labeled antibodies could localize a selection of known protein targets, providing a picture of cellular localization and post-translational gene expression in the tissue context. Moreover, they looked to merge transcriptomics or genomics and proteomics, providing tools that offer spatially resolved molecular annotation of gene expression and pathological gene signatures. This blog aims to explore the understanding of spatial transcriptomics, spatial proteomics, and its importance and advantage in identifying tumor grade and precise differentiation.

 

Before delving into spatial transcriptomics and spatial proteomics (Genotyping & Phenotyping), let us briefly review two individual techniques that form its foundation:

Immunofluorescence (IF): Immunofluorescence is a widely used microscopy technique that enables researchers to visualize specific proteins or molecules within cells. The process involves the use of antibodies conjugated to fluorescent dyes that bind to target molecules of interest. The resulting fluorescence antibody staining signals are detected and captured using a fluorescence microscope.

Fluorescence in situ Hybridization (FISH): FISH is a molecular cytogenetic technique used to detect and localize specific DNA or mRNA sequences within cells or tissues. Fluorescently labeled probes complementary to the target nucleic acids hybridize with them, allowing their precise localization and visualization under a fluorescence microscope.

The Power of Transcriptional and Translational Regulation Co-detection and Co-localization

While IF and FISH are potent techniques individually, they do have limitations. IF can provide specific protein localization, but often lacks the genomic context, making it challenging to understand how proteins are positioned relative to specific DNA or mRNA sequences. On the other hand, FISH allows researchers to observe nucleic acid sequences, but does not reveal their association with proteins or other cellular components.

Simultaneous detection of highly multiplexed proteins and mRNA in situ is important for understanding healthy and diseased states. The spatially resolved relationship of different cell populations, proteins, and mRNAs in their native tissue structure bears crucial information for disease diagnosis, pathogenesis, and treatment. Multiplex Immunofluorescence (mIF, Immunoplex) represents a powerful new platform for highly multiplexed proteins (up to 60) in situ detection. FISH (RNAveu) represents another multiplexing mRNA detection system at a single-copy sensitivity and high specificity where the unique double barcode probes and corresponding fluorophore conjugated oligonucleotide barcode enables concurrent signal amplification and background noise reduction. The combination of miRveu, RNAveu and ImmunoPlex to detect transcriptional and translational expression within the same tissue section can be implemented using NanoVIP®. NanoVIP®, a fully automated all-in-one slide-based staining instrument used in conjunction with our ImmunoPlex, miRveu, and RNAveu kits allow for the visualization of protein, miRNA and mRNA in the same spatial context (Figure 1).

Multiplex

Figure 1: Genotyping and Phenotyping for Co-localization of EGFR (AB); PD1 (mRNA) & miR-17 in Colon Carcinoma

Importance of tumor microenvironment

To better understand diseases like cancer, its growth and progression, spatial patterns which include breaking of normal tissue, invasion and metastasis are needed. For their evaluation, Genotyping and Phenotyping assays provide characterization of tumor microenvironment and cell-cell interaction as it provides genomic, transcriptomic and proteomic data in the spatial context. These studies will help to understand the evolution of tumor microenvironment which will clarify the heterogenic pattern of tumors for new therapeutic options.

Advantages of Genotyping & Phenotyping Assays

There are numerous advantages to understanding the transcriptional and translational relationship in a spatial context, some of these include the following:

  • Spatially map different cell types based on requirement in heterogeneous tissue samples while accessing their full gene expression profiles.
  • Refine anatomical tissue and cell atlas spatial maps through a more comprehensive characterization of cell types at both the protein and transcript level.
  • Determine the activation or exhaustion states of infiltrating immune cells in the tumor microenvironment by tracking the cellular sources of secreted proteins.

Identify new tumor biomarkers and drug targets with cell type.

Conclusion

In conclusion, Genotyping and Phenotyping assays represent a comprehensive approach that enhances our understanding of the intricate world within cells. By seamlessly integrating mIF and FISH, this hybrid technique illuminates the cellular landscape like never before. Its application in diverse research areas promises to unlock new discoveries and deepens our understanding of complex biological processes, ultimately advancing our knowledge of health, disease, and fundamental cellular biology. As technology continues to evolve, Genotyping and Phenotyping is set to remain at the forefront of cell biology, empowering scientists to unravel the mysteries of life at the cellular level.

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In Situ Sequencing: Benefits, Applications, and Protocol

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Sequencing technologies have expanded the possibilities to a single-cell – and even subcellular- resolution. The ability to read the sequence from intact tissues is critical to understand complex biological and pathological mechanisms. In situ sequencing offers an in-depth understanding of gene expression and its location.

What is In Situ Sequencing?

In situ sequencing (ISS) is a targeted and barcoded method where RNA is sequenced directly in a morphologically preserved tissue or cell sample. Unlike traditional sequencing methods, where samples are analyzed after extraction -where histological context is lost, ISS simultaneously analyses hundreds of mRNA transcripts and their spatial context at cellular and subcellular resolution. Developed by Mats Nilsson’s group at the Science for Life Laboratory at Stockholm University, ISS allows scientists to identify cell types by analyzing their gene expression profiles.

In Situ Sequencing Benefits

In situ sequencing is becoming the method of choice for scientists studying gene expression, pathological mechanisms, and new drug targets.

 

    • High efficiency in detecting multiple RNA molecules in a tissue section in a high-throughput manner, covering a comprehensive dynamic range
    • Provides spatial information of gene expression profiles for gene panels at subcellular resolution
    • Capability to include analysis of multiple genes across various tissue types
    • Ability to identify point mutations in preserved cells and tissue
    • High specificity for targeting single nucleotide variations within tissue, which makes it a helpful tool in pinpointing somatic mutations

In Situ Sequencing Applications

In Situ Sequencing as a Diagnostic Tool in Cancer

With ISS capabilities of multiplexing gene expression and the ability to identify point mutations in fixed cells and tissue, it is a powerful diagnostic tool for cancer tumors. The first such application was presented in the early 2013 study by Mats Nelisson when ISS was used across human breast cancer tissue. So far, ISS has been used to analyze many more genes across various tissues.

In Situ Sequencing Integration with Other Tools

ISS can be incorporated with other methods like single-cell RNA sequencing and Spatial Transcriptomics, which can be valuable for cell mapping and analysis of the cellular network. For example, a recent study used spatial transcriptomics and in situ sequencing to demonstrate the role of amyloid-beta plaques linked to Alzheimer’s disease.

In Situ Sequencing Protocol

In situ sequencing is a targeted method using padlock probes (PLPs), rolling circle amplification (RCA) and sequence by ligation. These are the primary steps of the current protocol:

 

    • mRNA generates cDNA by reverse transcription
    • PLP hybridization: A padlock probe hybridization to the targeted cDNA fragment
    • DNA ligation closes the two probe ends to form a circularized DNA
    • PLPs are amplified by targeted RCA, which generates Rolling Circle Products (RCPs)
    • RCPs are sequenced by ligation
    • The sample is read using imaging technologies, and each RCP reveals a color to the corresponding base
    • The process is repeated to achieve the number of preferred reads of bases.

Design Your In Situ Sequencing Experiment

At OmicsVeu, we developed NanoVIP®, an automated In Situ Sequencing solution. See the video to learn more about NanoVIP®.

Conclusion

In situ sequencing is a targeted image-based technology that can detect gene expression and provide location information within a cell sample or a tissue section. This tool is valuable for understanding the pathology and molecular mechanisms of diseases. So far, it has been applied in various areas, such as cancer diagnosis, gene expression profiling, and mutation detection. Providing gene expression images and spatial context using ISS results in an atlas-like map which has been used to develop The Human Developmental Cell Atlas. The project is a Swedish initiative developed by the Science for Life Laboratory, which aims to create a detailed molecular atlas of human cell types and show their spatial distributions in three dimensions.

References:

    • Chen, W.-T. et al. (2020) “Spatial transcriptomics and in situ sequencing to study alzheimer’s disease,” Cell, 182(4). Available at: https://doi.org/10.1016/j.cell.2020.06.038.
    • Hilscher, M.M. et al. (2021) “Correction to: In situ sequencing: A high-throughput, multi-targeted gene expression profiling technique for cell typing in tissue sections,” Methods in Molecular Biology [Preprint]. Available at: https://doi.org/10.1007/978-1-0716-0623-0_25.
    • In situ sequencing (2022) SciLifeLab. Available at: https://www.scilifelab.se/units/in-situ-sequencing/ (Accessed: December 4, 2022).
    • Ke, R., Mignardi, M., Pacureanu, A. et al.In situ sequencing for RNA analysis in preserved tissue and cells. Nat Methods10, 857–860 (2013). https://doi.org/10.1038/nmeth.2563
    • The Human Developmental Cell Atlas (no date) HDCA Sweden. Available at: https://hdca-sweden.scilifelab.se/ (Accessed: December 4, 2022).

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Accelerate The Pace Of Spatial Multi-omics Profiling With Genotyping & Phenotyping

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Infographic Transcript

Accelerate The Pace Of Spatial Multi-omics Profiling

For precise, accurate & clear insight to connect Genotype to Phenotype

OmicsVeu offers Genotyping & Phenotyping system consisting of optimized protocols, ready-to-use reagents & fully automated NanoVIP® instrument for the co-localization of protein-miRNA-DNA-mRNA.

NanoVIP® is an open system for the automation of any slide-based manual assay without any limitations. OmicsVeu NanoVIP®, an all-in-one plug-n-play automation, is the game changer in the world of fully automated spatial multi-omics systems for speeding up the research & development of new diagnostic modalities.

Proteomics & Genomics profiling elucidates precisely tumor microenvironment. An automated spatial multi-omics system for co-localization of Genotyping & Phenotyping provides cutting-edge solutions to understand the microenvironment with minimum hands-on time.

 

    • OmicsVeu, a pioneer in Genotyping & Phenotyping technology enables researchers to enhance & upgrade their understanding of tumor biology.
    • OmicsVeu provides a simple, reliable & easy-to-use method for the co-detection of protein-RNA-DNA-miRNA by the sequential use of immunofluorescence (IF) & fluorescent in situ hybridization (FISH) methodologies.
  • OmicsVeu helps generate enormous high-end multiplexing data sets.

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