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).
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.
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.