Spatial-omics: Novel approaches to probe cell heterogeneity and extracellular matrix biology

Abstract

Complex intercellular interactions as well as biomolecular and biomechanical cues from the extracellular matrix (ECM) profoundly affect cellular functions. Traditional transcriptomic and proteomic approaches have provided insight into disease progression by identifying discrete cellular subpopulations or microenvironmental signatures characteristic of normal or pathological tissues, however these techniques do not examine how a given cellular state relates to its interactions with neighboring cells or its surrounding ECM with multiparametric characterization (i.e. ECM alignment, mechanical forces, crosslinking, etc.). Emerging spatial-omic techniques can provide high-resolution mapping of expression profiles similar to scRNA-seq and mass spectroscopy directly within tissues. The ability to preserve the spatial context of cells within samples, their cellular geometry, as well as their surrounding ECM gives spatial-omics the opportunity to interrogate previously unexplored signaling modalities, which has the potential to revolutionize ECM research and our understanding of fibrotic diseases. In this review, we present current spatial transcriptomic and proteomic techniques and discuss how they may be applied to investigate cell-ECM interactions.

Keywords: Spatial transcriptomics; fibrosis; matrisome; multiplex imaging; spatial proteomics.

Figure 1:. Slide-seq multiplex imaging workflow

(A) Schematic of initial Slide-seq steps (A1) A rubber coated coverslip is covered in a monolayer of 10µm micro-particles (“beads”) to create a “puck”. Each bead is conjugated to its own unique barcode whose sequence is determined in situ via SOLiD (Sequencing by Oligonucleotide Ligation and Detection) for high resolution localization of each bead on the puck. (A2) Fresh frozen tissue can then be melted onto the puck, (A3) where the cells (approximately one cell/bead) overlap with the DNA barcoded beads. The tissue is digested and the mRNA of each cell is captured onto the DNA barcodes residing directly beneath the respective cell. (B) Schematic of library preparation for Slide-seq. (B1) mRNA are reverse transcribed to incorporate in a 3’-end, barcoded RNA-seq library preparation. Products are then amplified and undergo Illumina sequencing. (B2) Transcript profiles associated with a barcode sequence obtained from Illumina sequencing are mapped to their specific tissue location by matching the Illumina data to the barcode sequence from the initial in situ sequencing step in A1 [52].

Figure 2:. Co-Detection by indEXing (CODEX) multiplex imaging workflow.

(A) Top left moving right depicting a macro-view of CODEX workflow. (A1) Tissue section (either FFPE or fresh frozen) is simultaneously stained with the entire panel of antibodies tagged with their own unique oligonucleotide barcode by the user. Of note, the commercial system currently provides ~40 barcodes that can be conjugated to any antibody of interest. (A2) The stained section then undergoes several imaging cycles automated within the fluidics device where three fluorophores are imaged and subsequently removed via TCEP cleavage each cycle (14 cycles for 40 antigens). Images are then overlapped to create a ~40 protein multiplexed image from a single tissue section. (B) Bottom depicting cellular-level view of CODEX workflow with two fluorophores (for simplicity) and indexing technology. Each antibody is conjugated to a unique barcode with a 5’ overhang. The indexing nucleotide (denoted by underlining) and two nucleotides bound to fluorophores are added to the section. As shown in cycle 1, the indexing nucleotide is incorporating into all of the barcodes, while the fluorescent nucleotides are only able to bind to their respective pre-designated barcodes to image the antibodies denoted for that cycle. Fluorophores are removed to repeat the process. Barcodes for antibodies in earlier cycles will be shorter than later cycle oligo-tags. After the antigen of interest for that cycle is imaged, the oligonucleotide will no longer be indexed, as denoted by the lighter shading in cycle 2 [70]. Abbreviations: Antibody (Ab); nucleotide (nt); FFPE (Formalin Fixed Paraffin Embedded); Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)