Tissues are complex cellular environments, made up of a vast array of cell types and biomolecules all interacting with each other to carry out the functions of the organ. Traditionally, techniques for the analysis of biomolecules such as metabolites, glycans, and proteins involved the homogenization of tissue, destroying all spatial information. These traditional methods inhibited the complete understanding of complex intra- and extracellular molecular interactions. Mass Spectrometry Imaging (MSI), on the other hand, not only preserves the spatial information of biomolecules in tissue but also allows for multiple classes of analytes to be detected from the same tissue section through sequential analysis at a near-single-cell resolution. This enables us to derive a more complete picture of molecular interactions across different classes of biomolecules. The protocol presented here outlines the steps for performing mass spectrometry imaging of metabolites, N-linked glycans, and tryptic peptides sequentially from the same tissue section at a 20 µm resolution. Conscientious consideration of the order in which the classes of analytes are imaged, along with careful handling of the sections to ensure integrity, allows for multiple high-quality images to be collected from the same section. These data can subsequently be integrated with other spatial omics data (transcriptomics, immunohistochemistry, etc.) collected from serial sections, where the same cell can be analyzed in these adjacent sections.

The functionality of cells within a host does not depend on isolated signals. Instead, all components, from the smallest RNA molecules to the largest proteins, must operate in harmony and coordination within the human body. As such, the importance of approaches that integrate cellular-level spatial investigations across multiple omics is paramount to understanding cell-cell interactions and the progression of disease. Dissecting the proteome and transcriptome in the same spatial assay helps us understand how not only what a cell is being instructed to carry out (RNA), but also how it is executing those instructions in the context of the microenvironmental niche it finds itself in (protein). This manuscript is focused on integrating sequential immunofluorescence (SeqIF) with RNA in situ hybridization (ISH) with an on-tissue microfluidics driven system for high-throughput protein and RNA investigation in a spatial context (seqRNA-ISH+seqIF) that will allow up to 12 RNA and 24 protein targets in a single run with additional protein targets possible to be added via sequential runs. This method provides a sequential targeted multiomics platform that does not require consideration of fluorophore compatibility and extensive optimization for higher plexing. This allows one to understand what messages the cell is priming or is sending into its microenvironment. This targeted approach helps to validate whole transcriptome methods while examining the interactions between the RNA and proteins in a more precise manner.

When using traditional probe-based in situ hybridization (ISH) imaging detection methods, microRNAs (miRNAs) are difficult to capture due to their size, which ranges from 19 to 25 nucleotides. However, as miRNAs are key epigenetic regulators that contribute to normal physiology and numerous pathological conditions, understanding the spatiotemporal behavior of miRNAs is important to unravel their function at the cellular and subcellular levels. This creates a clear need for adequate detection and visualization techniques. Therefore, we created MicroRNA Amplification and Recognition through Locked-nucleic-acid in situ hybridizatioN (MARLIN), which was developed and optimized utilizing multiplex immunofluorescence staining, and locked nucleic acid (LNA) probes targeted to a miRNA. This method can quantify miRNA with multiplexed protein expression, which allows the quantification of intracellular and extracellular miRNA within the tumor microenvironment (TME) with high sensitivity and reproducibility. The combined use of miRNA detection and protein capture assays can also provide a comprehensive understanding of cellular functions and regulatory mechanisms within that TME.

The functionality of cells within a host does not depend on isolated signals. Instead, all components, from the smallest RNA molecules to the largest proteins, must operate in harmony and coordination within the human body. As such, the importance of approaches that integrate cellular-level spatial investigations across multiple omics is paramount to understanding cell-cell interactions and the progression of disease. Dissecting the proteome and transcriptome in the same spatial assay helps us understand how not only what a cell is being instructed to carry out (RNA), but also how it is executing those instructions in the context of the microenvironmental niche it finds itself in (protein). This manuscript is focused on integrating sequential immunofluorescence (SeqIF) with RNA in situ hybridization (ISH) with an on-tissue microfluidics driven system for high-throughput protein and RNA investigation in a spatial context (seqRNA-ISH+seqIF) that will allow up to 12 RNA and 24 protein targets in a single run with additional protein targets possible to be added via sequential runs. This method provides a sequential targeted multiomics platform that does not require consideration of fluorophore compatibility and extensive optimization for higher plexing. This allows one to understand what messages the cell is priming or is sending into its microenvironment. This targeted approach helps to validate whole transcriptome methods while examining the interactions between the RNA and proteins in a more precise manner.

When using traditional probe-based in situ hybridization (ISH) imaging detection methods, microRNAs (miRNAs) are difficult to capture due to their size, which ranges from 19 to 25 nucleotides. However, as miRNAs are key epigenetic regulators that contribute to normal physiology and numerous pathological conditions, understanding the spatiotemporal behavior of miRNAs is important to unravel their function at the cellular and subcellular levels. This creates a clear need for adequate detection and visualization techniques. Therefore, we created MicroRNA Amplification and Recognition through Locked-nucleic-acid in situ hybridizatioN (MARLIN), which was developed and optimized utilizing multiplex immunofluorescence staining, and locked nucleic acid (LNA) probes targeted to a miRNA. This method can quantify miRNA with multiplexed protein expression, which allows the quantification of intracellular and extracellular miRNA within the tumor microenvironment (TME) with high sensitivity and reproducibility. The combined use of miRNA detection and protein capture assays can also provide a comprehensive understanding of cellular functions and regulatory mechanisms within that TME.

The ability to detect protein and mRNA in the same assay helps us understand how the cell is priming for an arrival and what cues it receives as it travels and interacts in the environment it finds itself in. Higher-plexed imaging now permits characterization of spatially resolved mRNA (spatial transcriptomics) and protein (spatial proteomics) simultaneously. To avoid redundancy, these analytes can be analyzed in a single tissue section. There are many technologies that allow for many proteins and few mRNAs or the inverse, but as the central acting analytes are proteins, this manuscript is focused on integrating protein imaging mass cytometry (IMC) with mRNA in situ hybridization (ISH) utilizing metal probes. This allows us to understand what messages the cell is priming or is sending rapidly into the world as it communicates in its microenvironment. It also overcomes limitations related to the detection of secreted proteins or challenging markers that are usually hard to quantify and visualize utilizing antibodies only. This protocol also minimizes the need for compensations or post-processing seen in higher plex fluorescent-based methods, as there is no fluorescent spectral spillover in a metal probe-based detection system, nor are there tissue-specific autofluorescence considerations.