Fig. 4

Overview of each ST seq technologies and Spatial transcriptome analysis of breast cancer using Visium. Overview of typical technologies based on microdissection (a). TIVA; this method introduces a photo-activated polyA oligo called a TIVA-tag into living cells in the tissue. This oligo has a cell penetrating peptide (CPP) that allows it to enter the cytoplasm. By irradiating the laser beam to cells of interest, the TIVA-tag is activated inside the cell and hybridizes with the cellular mRNA. The TIVA-tag-mRNA hybrid was purified from the selected cells with streptavidin, and the captured mRNA was analyzed by RNA-seq. ProximID; this method creates a cell structural unit containing two or three interacting cells by dissociating the tissue under mild conditions. Then, by performing scRNA-seq on cell structural units collected manually, it shows that we can find a new cell-cell interaction even if we do not know the constituent cell types in advance. Overview of typical technologies based on in situ hybridization (b). seqFISH; in this method, a primary probe incorporating four read probes (1–4) is hybridized per arbitrary mRNA. This expansion of the read sequence has dramatically increased the number of genes that can be analyzed. As in smFISH, fluorescently labeled read probes (indicated by a star) are bound to the read sequences and photographed for detection. After that, this reading probe is stripped, a new reading probe is hybridized, and imaging is performed. By repeating this operation 80 times per fluorescence, theoretically 24,000 genes can be detected. Overview of typical technologies based on in situ sequencing (c). STARmap; in this ISS method, it is possible to bypass the reverse transcription step by using a set of primers and PLPs that hybridize to a specific RNA, called SNAIL (Specific Amplification of Nucleic Acids via Intramolecular Ligation). The RCPs are amplified using the RCA method. After removing proteins and lipids to increase tissue permeability, sequencing was performed using the modified SBL method. Overview of typical technologies based on in situ capturing (d). Spatial Transcriptome; in this method, the oligo dT primer to trap poly A-RNA was pre-coated onto the glass slides on which the tissue sections were attached. Each primer was preloaded with a spatial barcode to identify the location of the coordinates on the glass slide. Upon attaching the tissue to a slide and disrupting the cell membrane, mRNA in the tissue was captured by nearby oligo dT primers. Adding reverse transcriptase to synthesize cDNA makes it possible to synthesize a gene library that reflects its localization. Because the determined sequence contains the coordinate information, the detailed expression information of the coordinates can be obtained. The 10x Genomics company acquired, developed, and improved this ST technology to 55 μm/diameter resolution and commercialized it under the name “10x Visium” at the end of 2018. Visium results for breast cancer. Hematoxylin and eosin staining (left). Three cancer cell populations, classified via non-hierarchical K-means clustering (k = 9), are shown in different colors (middle panel). Four non-cancerous areas are shown to represent gene expression reflecting the microenvironments of the corresponding cancer cells (right panel) [54] (e). PL photocleavable linker, CPP cell-penetrating peptide, PLP padlock probe, RCA rolling circle amplification, SBL sequencing-by-ligation, RT Reverse transcription