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Introduction

The intestinal tract contains a high concentration of microorganisms and neurons, which have a profound influence on both the physical and mental well-being of humans. The intestines are also known as the ‘second brain’ of the human body, and while they perform their normal functions, various intestinal diseases are inevitably present (1–3). These diseases can be broadly categorized into two groups: Functional diseases such as irritable bowel syndrome (IBS), functional dyspepsia and functional constipation, and organic diseases such as enterocolitis, ulcerative colitis (UC), Crohn's disease (CD) and various intestinal tumors (4–6). With the change in dietary habits and lifestyles (such as irregular eating, oversatiety or overhunger) of individuals, the incidence of IBS is on the rise, affecting ~20% of the general population. This not only diminishes the quality of life of individuals with the condition, but also causes a substantial healthcare burden (7,8). Furthermore, the prevalence of inflammatory bowel disease (IBD) has been increasing worldwide over the last century. Due to the impact of family history and weakened immune systems (9), there has been a notable rise in very early onset IBD, which is identified before the age of 6 years (10,11). This results in increased healthcare expenses for patients and can also hinder their career aspirations due to the enduring social stigma they face as they grow up. Chronic inflammation of the colon and accelerated renewal of epithelial cells can increase the risk of highly dysplastic growth, leading to the further development of colorectal cancer (CRC) (12). A previous study revealed that individuals with UC or CD are at a higher risk of developing both gastrointestinal and extraintestinal cancer (13,14). The etiology, severity and advancement of this intricate set of gastrointestinal disorders remain to be evaluated. For example, IBD might initially be misdiagnosed as IBS, leading to a delay in early treatment. The manifestations of IBD are varied and non-specific, with a wide range of differential diagnoses. The possibility of rapid growth and early metastasis of CRC is high, making early detection particularly crucial (15–19). Given the prevalence and challenges in diagnosing and treating various gastrointestinal diseases, contemporary medical treatment urgently needs more appropriate and optimized experimental methods to explore and uncover the developmental origins of diseases in order to effectively manage disease progression and provide improved patient care.

The human immune system is a protective system covering the whole body, which can maintain the immune homeostasis of the body, while excluding exogenous antigenic substances (20). The gastrointestinal tract, as the initial line of defense for the immune system, is closely associated with the occurrence of numerous diseases (21). The intestinal barrier has a heterogeneous structure, including mechanical, chemical, microbial and immune barriers (22,23). The intestinal mucosal barrier is the first line of defense against penetration of luminal contents and performs various biological functions (24), not only maintaining local intestinal function, but also facilitating communication with the nervous and endocrine systems via the bidirectional signaling pathways of the brain-gut axis (25). Additionally, the barrier is involved in immune, metabolic and emotional regulation, which is closely linked to the function of intestinal microorganisms (26). The human body, functioning as a superorganism, coexists in symbiosis with trillions of beneficial bacteria and eukaryotic cells. The advancement of the Human Microbiome Project and METAgenomics (27,28) of the human intestinal tract has offered novel technological resources for the research of these microorganisms. There are ~500-1,000 different types of bacteria in the human body, which can reproduce to about 100 trillion individual cells in an adult-roughly 10 times the total number of a person's body cells. These microorganisms benefit from the warm environment of the intestines and serve a crucial role in digestion, vitamin synthesis, immune system regulation, pathogen elimination, toxin removal and maintenance of the normal functioning of the intestines (29–32).

Since being awarded ‘Method of the Year 2020’ (33), spatial transcriptomics (ST) has gained popularity as a tool for studying disease mechanisms. When frozen tissue slices are attached to spatial transcriptomics slides, barcode primers bind and capture nearby mRNA from the tissue. The captured mRNA undergoes reverse transcription, resulting in cDNA containing spatial barcodes. By analyzing the sequence of spatial barcodes, the sequence transcribed by each mRNA can be mapped back to the starting position on the tissue slice, providing details of gene expression and its spatial distribution on the tissue slice (34,35). ST is often used in combination with other molecular research techniques such as proteomics, single-cell RNA sequencing (scRNA-seq), intestinal microbiomics and metabolomics. This approach has given researchers novel insights into the complexity of the mucosal immune system, and has provided increased opportunities for understanding intestinal disorders and identifying potential therapeutic targets. ST is the next wave in single-cell analysis, as research on diseases typically begins with single cells and then progresses to the spatial level (33). ST encompasses a range of techniques, such as in situ hybridization and targeted in situ sequencing (ISS). These methods, through multiple fluorescence in situ hybridization (FISH) or ISS, can preserve the three-dimensional spatial structure at the cellular resolution and reveal its functionality (36,37). In addition, there is the spatially-resolved transcript amplicon readout mapping (STARmap) technology, which combines hydrogel histochemistry, targeted signal amplification and ISS (38). These techniques allow exploration of the mechanisms of diseases, not only locally in the gut, but also in the nervous system to help understand brain-gut axis interactions. Fig. 1 illustrates the uses of ST in intestinal research.

Figure 1. Use of spatial transcriptomics in intestinal research.

In summary, ST can help understand the biological functions of the gut from multiple perspectives. First, generating high-resolution maps of neurons or the gut using ST indicates the development of the gut, the information exchange in the brain-gut axis and pain signaling. Second, with the assistance of ST, the physiopathological manifestations and healing of colonic tissues can be observed dynamically, and accurate judgments can be made about the development of intestinal diseases. Finally, spatially specifying changes in the gut microbiota helps understand the geographic features of the gut that have been altered by pathogens.

Development and evolution of ST technology

Since the 1990s, with the rapid development of high-throughput sequencing technology, transcriptomics has gradually emerged as a discipline to study all transcribed RNAs in cells. Researchers are starting to fully uncover gene expression patterns and regulatory networks. However, conventional transcriptomics techniques are unable to determine the effects of different subcellular structures and environments on gene expression. To more fully understand the regulatory processes of gene expression, ST is rapidly emerging to reveal the spatial distribution, subcellular location and interactions of genes within the cell, providing novel perspectives for understanding the spatial regulatory mechanisms of gene expression (39).

The development of transcriptome technology has gone through three important stages: The first stage is transcriptome sequencing (bulk RNA) of a large number of mixed cells, which reveals the average expression level of a single gene in a large cell population, but fails to demonstrate the transcriptional expression level in a single cell (40). The second stage is scRNA-seq, which constructs the expression profile of each cell at the single-cell level, reflecting cell-to-cell heterogeneity (41). However, single-cell spatial location information is lost during the preparation of the suspension, resulting in changes in gene expression profiles of some single cells during enzymatic digestion or after leaving a specific microenvironment; this is one of the key features of organ function. Driven by this need, the Joakim Lundeberg research group proposed the concept of ST in 2016 and reported the first ST technique based on in situ capture of RNA (42). This provides important research tools in a number of fields, including tissue-cell function, microenvironment interaction, lineage tracing of developmental processes and disease pathology research. Subsequently, a series of techniques capable of high-throughput in situ RNA detection analysis have all been grouped into the category of ST techniques. Although the principles of these techniques vary, they have a common element with regard to recording information about the spatial position of the detected RNA molecules (43).

In the late 1990s, certain studies on ST started the development of ST using technologies such as laser capture microdissection (LCM), microarrays or RNA sequencing (RNA-seq) and single-molecule FISH (smFISH). Some techniques from the 1980s, although not called ‘spatial transcriptomics’, also acquired transcriptional information in space. Moses and Pachter (42) refer to this part of the development of ST technology as the ‘prequel era’ in their online ebook ‘Museum of Spacial Transcriptomics’.

The prequel era technology depicts the general technical characteristics of the spatial transcriptome: Imaging, localization and expression level. At the same time, one of the spatial transcriptomes that can see the ‘prequel era’ has been working on imaging and single cells (resolution), and the expression amount is often not obtained by high throughput. Although spatial transcriptomics at that time was limited by resolution, ease of operation, available software, rich databases and other factors, particularly the high-throughput expression data based on NGS not being available in the pre-transcriptomics era, causing numerous technologies of that era to decline. However, still, in the pre-transcriptomics era, numerous beneficial attempts were made, and it can even be said that it provided a reference for the spatial transcriptomics technology of the present era (39).

By contrast, Moses and Pachter (42) refer to the technology after the ‘prequel era’ as the ‘current era’ technology. The foundations of a number of the ‘current era’ technologies were established in the 1970s to the early 2000s. For example, microarray technology, first reported in 1995 (44), was initially used to quantify transcripts hybridizing to cDNA printed on slides, but was used to quantify the transcriptome of LCM samples in 1999 (45). Currently, popular technologies, including ST and 10× Genomics, rely on this microarray technology to capture transcripts from tissues mounted on microarray slides (46–48).

ST has undergone rapid development, with marked improvements in resolution and flux. The spatial transcriptome was declared to be the annual technical method by Nature Methods in 2020 and one of the seven noteworthy annual technologies by Nature magazine in 2022. In 2023, space omics became one of the 10 technologies selected by the World Economic Forum to have the highest potential and to have a positive impact on the world.

Advantages and disadvantages of the different ST techniques

Currently, the commonly used spatial transcriptome technologies are slide-DNA-seq, a method for capturing spatially resolved DNA sequences from intact tissue sections (49), ST, sequential FISH, multiple resistance error correction FISH (MERFISH), LCM-seq, geographical position sequencing (Geo-seq) and Topographic Single Cell Sequencing (Tomo-seq), which can be generally divided into four different types of strategies, each with their own advantages and disadvantages.

Computational strategies based on spatial reconstruction

Seurat Technology (50,51) is a computational spatial reconstruction strategy for cellular location inference by combining scRNA-seq and FISH technology. Specifically, the gene expression of individual cells is analyzed by Switching mechanism at 5′ end of the RNA transcript (Smart-seq) (52), then the cells are clustered into different types and signature genes are identified for each cell type. Subsequently, FISH is used to determine the spatial distribution of signature genes, and finally, the localization of cells is integrated with sequencing information and the spatial distribution information of signature cells. Smart-seq is a single-cell transcriptome sequencing technology for mRNA, which was developed by American and Swedish scientists, and can be used to perform large-scale sequencing of gene subtypes and alleles, among others, of single-cell RNA (52). The Smart-seq, which can generate full-length cDNA from single cells, is a landmark for in-depth studies of allele-specific expression, single nucleotide variant detection or splice variants. However, due to the technical limitations at the time of its creation, Smart-seq technology also has certain limitations, such as poor amplification efficiency for some low-abundance transcripts and the fact that the design of the primers may also affect the subsequent purification and require more cells for preliminary RNA extraction (53,54). This strategy reconstructs spatial information of tissues by combining scRNA-seq, in situ hybridization and prior knowledge, and presents a spatial trend or an overall layout of cells in a particular tissue, but does not fully match the actual cell coordinates (51).

Strategies based on laser microcutting

LCM (55) can cut individual cells or regions of interest in tissue sections at high resolution, and may be combined with transcriptome analysis by RNA-seq analysis, which can provide single-cell whole gene analysis and accurate location information. This technique for analyzing samples is not limited to use in mammals and can also be applied in plants (56,57). However, the spatial analysis technique based on LCM has certain limitations, including a large workload, high cost and the analysis of only a few cells (58).

LCM-seq is a specific technique involving cutting of frozen tissue or paraffin-embedded tissue, or single-cell cutting, combined with subsequent chip or NGS to obtain the transcriptomic data of single cells (59). In 2017, Chinese scientists established Geo-seq combined with LCM and scRNA-seq technology, which overcomes the problem of insufficient amounts of mRNA from a small number of cells and can achieve cell-level accuracy; this can be used not only for 3D reconstruction of a transcription map, but also to study the transcriptomic information of a small number of tissues or cells with special structures (60). In 2018, American scientists developed the TSCS technology. They used lasers to capture single cells, amplified the whole genome of the single cells, and attached independent label sequences. This increased the experimental throughput. After mixed sequencing, they matched the cell positions to tissues using labels and computational methods (61).

Tomo-seq technology was inspired by clinical multi-dimensional imaging technology. A research team sequenced all zebrafish embryos in three different directions and reconstructed different sections of the same axis to form complete 3D embryos by computational methods. However, this method has high requirements for computational methods, and necessitates three identical samples, which is not applicable in clinical practice. The accuracy also needs to be improved (62).

Strategies based on fluorescence imaging

With in situ transcriptomics based on fluorescence imaging, cell detection flux is no longer a problem. Early on, in 2015, Chen et al (43) proposed the MERFISH technique. MERFISH technology has been improved in both cell flux and mRNA species, and may be used for >1 million cells (63) and 10,000 mRNAs. This technique largely increases detectable regions, but also has novel or small sequence variants that cannot be detected, with higher error rates. The method also has limitations such as the requirement for preselected genes (64).

SmFISH uses a single probe sequence to bind the same transcript, using the fluorescent color molecules on the probe to display the expression level of the target molecule in the original image. Although smFISH has high sensitivity and subcellular spatial resolution, this method is susceptible to background influence and low flux due to its weak light signal (65). In 2014, researchers improved the invention of seqFISH, which uses multiple rounds of hybridization to assign unique barcodes to individual transcripts within a single cell. By performing consecutive rounds of hybridization, imaging and probe stripping, a distinct barcode is generated for each mRNA molecule, allowing for spatial transcriptomic mapping through color recognition (66). However, increasing the number of genes requires increasing the number of hybridization rounds, which is expensive and time-consuming and requires super-resolution microscopy (67).

Strategies based on the spatial barcode of the oligonucleotides

The spatial barcode-based strategies can also enable high-throughput detection of cells and are not limited to known sequences. These strategies mainly consist of ST technology (34) and Slide-seq technology (68).

In 2016, Spatial transcriptomics (ST) printed 1,007 pre-defined points on a 6.2×6.6 mm array, each with a spatial barcode, including paste part, sequencing fragments, position tags, random unique molecular identifier and mRNA capture poly (T), and each barcode was captured with mRNA molecules for library construction and sequencing to obtain a transcriptome spatial map (34). ST Technologies pioneered a newera of combining in situ RNA amplification with spatial barcode arrays. In 2018, 10× Genomics(https://www.10×genomics.com/) acquired ST Technologies, and in 2019, they released 10× Visium, reducing the distance from point to point from 200 to 100 microns for higher precision. ST indicates genome-wide spatial expression at the micrometer scale, but does not achieve single-cell resolution.

In 2019, Professors Alice Y. Ting and Howard Y. Chang from Stanford University introduced a new RNA localization detection technique called APEX-seq (69). The use of peroxidase APEX2 can produce covalent biotinylation of RNA molecules and has different sensitivities in different organelle regions, which allows the realization of transcriptome studies on different organelles. However, as cells are required to express APEX2, which is an engineered soybean ascorbate peroxidase, it cannot be used in clinical practice.

In 2020, NanoString Technologies released the GeoMx Digital Spatial Profiler, which is conjugated to DNA Oligo on antibodies or RNA (70). Each DNA Oligo corresponds to one target, with multiple reactions achieved through multiple targets. When the antibody or RNA is bound to the target on the tissue, ultraviolet light is used to cut off the linker between DNA Oligo and the antibody or RNA, thus releasing DNA Oligo for the next quantification of the transcriptome and proteome.

High-definition ST (HDST) and Slide-seq can implement spatial barcodes on microbeads (68,71). Slide-seq links spatial barcodes on a batch of 10-µm microbeads, and mRNA molecules in tissue sections are captured by microbead binding, which are then processed using ‘sequencing by oligo ligation detection’ sequencing to map tissue space by spatial barcode. HDST is a microwell arranged with 2 micron, each containing a microbead, which links a spatial barcode on the bead, bound to the mRNA in the tissue, and then allows Illumina, Inc. Sequencing.

In 2021, space omics technology was a research focus. The group of Professor Rong Fan from Yale University invented DBiT-seq spatial transcriptomics, which is a method of adding barcodes in a different way (72). Tissues are first attached to slides, with a minimum inter-hole distance of 10 micrometers achieved using microfluidic technology. Two sets of liquid containing barcodes are then flowed through the tissue sequentially from two axes, binding to mRNA and protein molecules in the tissue to form a 2D spatial barcode. Reverse transcription is performed inside the cells, and cDNA is collected for subsequent sequencing (73). Although the method can achieve simultaneous capture of the transcriptome and proteome, there may be barcode contamination, and the pore spacing can only be 10 µm, limiting the accuracy of the technique (74).

Chinese scientists established Stereo-seq space omics technology on a BGI sequencing platform. The technique is based on the DNA Nanoballs (DNB) sequencing technology. It works by depositing DNB containing random barcode sequences on a lithoetched modified chip. The nanosphere spacing can be 500 or 715 nm, using a rolling ring to amplify the barcode pool, and obtain the coordinate code of the DNB after the first round of sequencing. Then, by hybridization, connecting the molecular coding and polyT sequence, the tissue was loaded onto the chip and the barcode was captured with mRNA for a second round of sequencing to finally obtain the transcriptome map (75).

American scientists have released the Seq-Scope spatial group on the Illumina platform. This method involves two rounds of sequencing. In the first round, sequencing is done on the Illumina sequencing platform to amplify clusters on a chip with fixed labels. The distance between clusters can be achieved at 0.6–0.8 micrometers. After amplification, a restriction endonuclease is used to expose the Oligo-dT sequence. Following the capture of mRNA molecules in the tissue, a second round of sequencing is conducted to obtain the transcriptome map (76).

The evolution of the development of spatial transcriptomics is summarized in Fig. 2. In 1996, Emmert-Buck et al (55) proposed the LCM technique. In 1998, Femino et al (65) combined digital imaging microscopy and FISH technology. In 2002, Levsky et al (77) used a combined marker approach in order to increase the number of identifiable transcripts. In 2012, Lubeck and Cai (78) reported on the use of super-resolution microscopy and combinatorial markers combined. Subsequently, Ke et al (79) proposed the ISS method. In 2015, Lee et al (80) proposed and developed the fluorescence ISS technology. In 2014, Lubeck et al (67) developed the sequential hybridization technology. Subsequently, Chen et al (43) developed a method called MERFISH. In 2018, Wang et al (38) developed the STARmap method. Subsequently, Vickovic et al (70) proposed the HDST sequencing method and at the same time, Rodriques et al (68) invented a similar Slide-Seq method. In 2019, 10 Genomics introduced the Visium spatial transcriptome technology.

Figure 2. Evolution of the development of spatial transcriptomics. In 1996, Emmert-Buck et al (55) proposed the LCM technique. In 1998, Femino et al (65) combined digital imaging microscopy and FISH technology. In 2002, Levsky et al (77) used a combined marker approach in order to increase the number of identifiable transcripts. In 2012, Lubeck and Cai (78) combined super-resolution microscopy and combinatorial markers. In 2013, Ke et al (79) proposed an ISS method. In 2014, Lubeck et al (67) developed the sequential hybridization technology. In 2015, Lee et al (80) proposed and developed fluorescence ISS technology. In 2015, Chen et al (43) developed a method known as MERFISH. In 2018, Wang et al (38) developed the STAR map method. In 2019, Vickovic et al (70) proposed the HDST sequencing method, while contemporaneously, Rodriques et al (68) invented a similar Slide-Seq method. In 2019, 10× Genomics introduced the Visium spatial transcriptome technology. APEX-seq, ascorbate peroxidase sequencing; BaristaSeq, barcode in situ targeting sequencing; BOLORAMIS, barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel in situ analyses; DBiT-seq, deterministic Barcoding in Tissue for spatial omics sequencing; DSP, digital Signal Processing; FISH, fluorescence in situ hybridization; FISSEQ, fluorescent in situ sequencing; Geo-seq, geographical position sequencing; HDST, high-definition ST; ISS, in situ sequencing; LCM, laser capture microdissection; MERFISH, multiple resistance error correction FISH; NGS, next-generation sequencing; NICHE-seq, combines photoactivatable fluorescent reporters, two-photon microscopy and single-cell RNA sequencing (scRNA-seq) to infer the cellular and molecular composition of niches.; osmFISH, ouroboros smFISH; ProximID, cell structures that interact with each other are manually dissociated into single cells and analyzed individually; RCA, rolling circle amplification; RNAscope, in situ hybridization technique for detecting target RNA within intact cells; ROI, region of interest; S1000, BMKMANU S1000; sci-Space, single-cell gene expression differences with spatial context; Seq-SCOPE, sub-micron resolution sequencing; seqFISH, sequential fluorescence in situ hybridization; seqFISH+, upgraded seqFISH; Slide-seq, slide sequencing; Slide-seq V2; smFISH, single-molecule FISH; ST, spatial transcriptomics; STAR map, spatially resolved transcript amplicon readout mapping; Stereo-seq, spatial enhanced resolution omics-sequencing; TIVA, transcriptome in vivo analysis; Tomo-seq, topographic single cell sequencing.

A spatial view of human intestinal development

The gut is the site of immune cell initiation at birth (81) and is closely associated with individual developmental abnormalities and illnesses. The gut is subsequently symbiotic with the gut microbiota, and these work together to supply nutrients to the body and maintain immune function (82,83). Marked advancements have been made in scRNA-seq, which have helped enhance the knowledge of the various types of cells in the gut and their biological characteristics (84). However, to the best of our knowledge, the molecular mechanisms underlying the formation of the unique intestinal morphology through the interplay of multiple cell types remain unclear. During the formation of the proto-gut embryo, three primary germ layers, the ectoderm, mesoderm and endoderm, are generated (85–88). Once the primary germ layers of cells are formed, the endoderm undergoes intricate changes and extensive folding to create the embryonic gut tube (89). The intestinal loop is shaped around Carnegie stage (CS) 14 and then moves into the space outside the body of the embryo at CS 16, returning to the peritoneal cavity at post-conceptual week (PCW) 11. Between PCWs 8 and 12, the pseudostratified columnar epithelial cells give rise to chorionic villi and crypt structures, establishing a self-renewal circuit maintained by ESCs (90,91). Whereas various animal models have identified distinct intestinal cells and regional tissues driving the formation of the aforementioned structures, suggesting differential features across species (92,93), the mechanisms driving this process in humans are not fully understood. To gain a more precise understanding of human tissues, Fawkner-Corbett et al (94) used high-throughput scRNA-seq combined with ST technology to map high-resolution organ development by acquiring whole-layer intestinal tissues, which revealed previously unknown disease-related phenotypes in the adult intestinal tract and localized transcriptional signatures spatially to different feature regions. This offers a valuable tool for further exploration of the molecular origins and heritability of intestinal diseases (94).

Numerous research studies have used scRNA-seq techniques to identify the diversity of intestinal epithelial, mesenchymal and immune cell types in the adult intestines (95–97). This has led to the exploration of several important questions using this information. Initially, the process of crypt-villus axis formation was elucidated. By scoring the activity of all modules (in the field of transcriptomics, modules can be understood as collections of genes with similar expression patterns), it was found that modules derived from endothelial cells, fibroblasts and pericytes are located deep in the tissue; epithelial-specific modules expressing Hedgehog pathway genes (such as the convolution donor receptor LDL receptor related protein 5) (94), are located in the vicinity of the canaliculus, and genes expressed by modules containing myofibroblasts (such as WNT2B) emerge later and are abundant at PCW 12, indicating that the ISC-myofibroblast signaling circuit is established only after crypt formation. These findings suggest that ST may partially restore the disrupted morphological gradients and increased physical distances during intestinal development (93). Furthermore, it can determine the hierarchical structure and differentiation functions of fibroblasts and myofibroblast subtypes. After classifying fibroblasts (98), intestinal fibroblasts were divided into different functional zones according to the characteristics of different phenotypes along the crypt structure to the villus axis (99). According to such anatomical zoning, as the main determinant of intestinal fibroblast heterogeneity, one of the original studies identified human colonic fibroblasts with numerous fibroblast subcharacteristics, Wnt differential expression and bone morphogenic protein signaling genes, which reflect the specific location along the anatomical axis of crypt villi (100). Antibodies in colon mesenchymal cells of UC patients were further investigated using scRNA-Seq and flow sorting to enrich CD90 cells. This study identified four populations of colonic stromal fibroblasts (S1-S4). After classifying fibroblasts, the analysis of various morphogens with different cells and genes expression revealed that the submucosal structural cells were mainly composed of S1 cells. S2 fibroblasts mainly contribute to maintaining the epithelial crypt ecological niche and promoting epithelial formation (101,102), whereas S3 fibroblasts are more commonly found in large vessels, emphasizing their role in forming the intestinal vascular support ecotone (93). S4 fibroblasts are closely associated with immune follicular cells in the adult colon and exhibit characteristics of follicular reticulocytes, which are crucial for the formation of lymphoid structures and are closely linked to the pathogenesis of UC (98). The development of blood vessels and nerves in the intestines, the formation of Peyer's patches (PP), gut-associated lymphoid tissue (GALT), and the immune system processes have been established (94). PPs are secondary lymphoid organs that interact with the external environment via the intestinal lumen. Spatial transcriptome analysis showed that fetal type 3 lymphocytes (innate lymphoid cells) express IL7RA and inhibitor of DNA binding 2, crucial genes for PP formation in the mouse uterus (103). Additionally, deficiencies in GDNF family receptor α3 also result in developmental disorders of PP. A study confirmed that GALT formation occurs in the small intestine prenatally and in the colon after birth (104), and lymphoid structures can be visualized by mapping factors enriched for B-cell-related genes onto colonic tissue. Enrichment of the NF-κB and TNF-α pathways has been detected in regions of immune and inflammatory activity, closely matching the spatial distribution of lymphoid clusters (105).

Landscape of molecular regionalization of the colon under physiological and pathological conditions

The intestinal epithelium maintains the normal physiology of the intestinal tract through continuous regeneration, and disruption of the regeneration process of intestinal epithelium can lead to pathogen translocation, which in turn mediates various chronic intestinal diseases (106). scRNA-seq technology has broadened the understanding of the cell types, cell subpopulations and cell states present in physiological and pathological states (107). scRNA-seq has also enabled the detection of subpopulations of cells abnormally driven (activation of KRAS in lung cancer is a poor prognostic indicator) by disease conditions, which is of considerable importance in the study of disease mechanisms (108,109). One study revealed that even though mice given dextran sodium sulfate (DSS) showed improvement in symptoms and body weight after a period of DSS discontinuation, the inflammatory changes in the colon did not fully return to normal levels (110). Therefore, it is crucial to analyze the transcriptomic landscape of the mucosal healing process in detail. Parigi et al (111) used ST to reveal previously unrecognized molecular regionalization in the colon of healthy mice. The study revealed a spatially organized transcriptional program for regionalized mucosal healing, demonstrating that factors in the proximal and distal colon have distinct functions and are involved in different regulatory processes, which was determined through dataset integration and associated pathway analyses. At the same time, the integration of longitudinal and ST data to observe the expression status of different modules at different stages can reveal the dynamic timing of colon tissue healing (110,111). Studies have revealed that clusterin accumulated during the repair phase following intestinal injury. Clusterin is seldom present in a healthy state, and further verification is needed to determine its association with infection. Multimodal spatial analysis will help identify important events related to tissue damage repair (111,112).

Macrophages are resident immune cells that exist in two primary states following activation: M1 macrophages and M2 macrophages. The functions and appearances of these macrophages vary depending on the microenvironment and location, and there is ongoing debate with regard to their roles and characteristics (113). What has been demonstrated is a higher expression of the M1 population in IBD samples, and that the phenotype and function of intestinal macrophages are related to their spatial distribution in the gut (114). ST has revealed the transcriptional status of macrophages in healthy and inflammatory states, defined specific markers for M2 macrophages, and clarified cell types and differences in the spatial distribution of different cells during intestinal inflammation (99). For example, ST showed that M1 macrophages and neutrophils were located near the mucosal surface in areas with intestinal ulcers. Spatial analysis has verified the diversity of macrophage populations and highlighted their interaction with inflammatory fibroblasts (115). This interaction enhances colony stimulating factor 2 expression and stimulates macrophage activation, leading to the release of IL-6 and TNF (115), which serve a role in the inflammatory process, and this spatial analysis technique is essential for comprehending the interactions between several specific cells.

Enhanced comprehension of the spatial dynamics of the gut microbiota

The gut microbiota is an integral part of the human body, and although a portion is highly conserved, the dynamic microbiota changes in response to age, and physiological and pathological states, and participates in processes such as neuromodulation, immunity and metabolism (116,117). The human gut consists mainly of Bacteroidota and Bacillota bacterial phyla, and an imbalance between these two phyla is considered to be a sign of microbial dysbiosis, which is closely linked to the pathogenesis of IBD (118). Dysfunction of the microbiota, such as an increase in facultative anaerobes and a decrease in obligate anaerobes, occurs during periods of active intestinal inflammation (119). Currently, the study of gut microbes is dominated by the use of genomics, which provides a comprehensive understanding of microbial categories and certain functions (120,121), but it does not offer spatial information about the microbes. The spatial organization of gut microbes can affect various properties such as colonization, metabolism and stability of the internal environment (122,123). It has been found that the distribution and abundance of different phyla is heterogeneous in different regions of the gut and that the microbial composition differs considerably between species (124).

The stomach, being the most acidic part of the digestive system, has fewer bacterial species. In the transition to the duodenum and ileum, the pH markedly increases and the oxygen pressure gradient decreases, creating a more favorable environment for bacterial growth. Although study results have indicated that the bacterial diversity in this area is either lower or equal to that of the stomach, there is a notable increase in the abundance of specific microbiota (125,126). This low diversity environment protects the intestinal tract to a certain extent, while bacterial overgrowth can potentially contribute to the onset of conditions such as IBS or functional dyspepsia (127). By the time it reaches the cecum and proximal colon, the intestinal lumen becomes intensely anaerobic and transit time is slowed due to the decreased pH, as a result of fermentation of fibers and complex polysaccharides. Dietary fibers provide ample nutrients for the growth of microbes, leading to a marked increase in both the quantity and variety of microbial species (128,129). In the human colon, there are higher concentrations of Bacteroidota, Pseudomonadota and Bacillota, and processes that shape the gut microbiota are thought to be mostly niche-driven (122). The intestinal epithelium has folds, villi and invaginations on its surface, known as crypts, which offer specialized protection to the intestine, but the crypts are heterogeneous in their response to injury. Paneth cells colonize the crypts of the small intestine and secrete antimicrobial peptides to achieve multi-level management of the intestinal flora and to maintain a stable intestinal flora structure. Since the large intestine lacks Paneth cells, it is guarded at the entrance of the colonic crypts by cup cells, which secrete mucin and form a mucus barrier to protect the epithelial cells (130). In addition, it has been previously reported that chemokines with antimicrobial activity against Escherichia coli and Salmonella enterica are found in colonic crypt compartments, which serve an important role in coordinating the normal influx of immune cells and inflammation in the intestine (131). Various microorganisms inhabit crypts, and some also extend their colonization beyond these areas, including into the PP, a specific region where bacteria directly interact with host tissues. This site serves as a sampling location for the microbiota of the immune system. Here, specialized cells access microbial and environmental antigens, passing them to antigen-presenting dendritic cells, participating in the immune process (132).

The degree of bacterial spatial organization in the gut microbiota varies, and the peristaltic contractions of the intestines can promote the fusion of bacteria in the gut. Therefore, even in the colon where the content flow rate is low, there is a high degree of mixing in the gut microbiota (133,134). A sample block sampling technique used to characterize the spatial organization of microbial communities at the 10–30-µm scale found well-mixed sites scattered with micron-scale clusters of specific taxa (126), which may be related to the heterogeneity of food and plant particles within the lumen (131,135). Bifidobacterium pseudolongum has been proved to colonize starch granules and plant granules, and Bacteroides colonizes undigested plant granules and is spatially segregated into multiple small colonies, suggesting that mucosal communities based on different intestinal regions could be useful in determining the role of mucosal geography in driving colonization of epithelial surfaces (136,137). Alterations in the microbiota are strongly associated with various human illnesses, such as IBD and CRC, as well as skin and mental health conditions (138,139). Clarifying the geographical distribution of gut microbiota affected by pathogens will provide novel insights for the understanding of host health and disease (140).

Identifying molecular features of human injury receptors and enhancing the understanding of brain-gut-related hypothalamic functions

IBD and IBS are commonly viewed as gastrointestinal conditions resulting from heightened sensitivity in the colon, along with dysfunction in other visceral and somatic organ (141,142). The interaction of sensory signals between multiple organs, known as ‘cross-organ sensitization’, is caused by the dorsal root ganglia transmitting sensory data for integrated processing (143). Injury receptors are located in the dorsal root ganglion and trigeminal ganglion, where they receive sensory signals related to bodily injury. These receptors are the first neurons in the pain pathway and express a range of receptors, allowing them to respond to various stimuli (144). In cases of cross-organ sensitization linked to intestinal dysfunction, the sensitization of colonic afferent neurons can lead to the cross-activation of reflex pathways in other uninjured organs, with the release of substance P, calcitonin gene-related peptide and excitatory amino acids to the peripheral organs, which activate the injury receptors and lower their thresholds (143). The excitability of these neurons increases in both acute and chronic pain states, and their excited phenotype changes are directly associated with the chronic pain state, so these neurons are key targets for the treatment of pain (144). ST enables researchers to draw high-resolution maps of human sensory neurons in the dorsal root ganglion, which can help identify more effective drug targets, therefore opening up novel avenues for pain management (145).

The mental, nervous, endocrine and immune regulation via the brain-gut axis serves an important role in the onset of certain intestinal diseases (146). The hypothalamus serves a leading role in regulating the basic social behaviors of the body and homeostatic functions, and is involved in the regulation of anxiety, depression, immune inflammation and gut microbiota (147,148). Studies have found that the volume of the hypothalamus in patients with IBD was larger than that of patients in the control group, and that it is also more strongly connected to other brain functional areas (149,150). This excessive connectivity can lead to the activation of abnormal anxiety circuits, mediating emotional disorders (151). However, the molecular characteristics of the development of the human hypothalamus are still unclear, and there is limited knowledge about the structure, spatial organization and function of the hypothalamic nucleus. The imaging-based single-cell transcriptomics method combines gene expression profiles with activity markers to directly image and analyze multiple RNAs in their natural cellular environment, thereby mapping out their spatial structure (152). By uniformly slicing the preoptic area, researchers have created a cellular map of the mouse hypothalamic preoptic area, identifying rare cell types and describing the spatial organization of specific neuronal cell types. This revealed their functions in different behaviors, providing potential insight into how different cell types communicate in physiological and pathological states (63). At the same time, ST can reveal the spatiotemporal transcriptional profile and cell type characteristics of human hypothalamic development. Mapping the spatiotemporal transcriptome of human hypothalamic development has demonstrated the asynchrony of spatial development of different neurons and neuroglial cells, identified key regulatory genes that neural progenitor cells and neural epithelial cells, and provided a deeper understanding of cellular network organization, circuit formation and the mechanisms of hypothalamic dysfunction (63,153). Analyzing the spatial transcription of key areas of the brain and gaining a comprehensive understanding of the connectivity patterns of the nervous system of the brain can provide valuable insights for studying the brain-gut axis, potentially offering novel therapeutic targets for the treatment of brain-gut-related diseases (153,154).

Applications in the study of CRC

During the progression of tumors, the interactions between cancer cells and other cell populations promote the heterogeneity of tumors. Experimental findings have demonstrated that the tumor microenvironment (TME) serves an important role in the development of cancer, and its spatial distribution and specific host-microbe cell interactions to some extent influence the progression of tumors (155,156). Single-cell sequencing of the genome, transcriptome and epigenome has contributed to the understanding of the internal structure of tumors, and more advanced ST is expected to decipher the complex principles and mechanisms of gene activity in three dimensions, which will have profound implications for life science research (157).

CRC is the third most common malignant tumor globally, and due to its high mortality rate due to metastasis, nearly 900,000 individuals die from CRC each year (158). Since CRC only presents with noticeable symptoms in the late stages, it is particularly important to identify relevant oncogenes and improve the early detection rate and early treatment of CRC (159,160). The combined application of multi-omics is important in studying the biological characteristics of the early local spread and distant metastasis of CRC (161). Cancer-associated fibroblasts (CAFs) are a major component of the stromal cells in numerous malignant tumors (109,162). In order to determine the interaction between the TME and CAFs in the pathogenesis of CRC, researchers have combined ST and bulk RNA-seq. Two types of CAFs were identified: Myo-CAFs and inflammatory CAFs. The latter not only promoted the progression and metastasis of tumors, but were also associated with the poor prognosis of the tumor (163). Certain studies have also found that spatial analysis can reveal the molecular and immunological characteristics of early-stage CRC, identify biomarkers associated with disease progression and prognosis, and reveal genes related to CRC invasion, providing a deeper understanding of the biological processes associated with tumor malignancy (164,165). The ST and GeoMx digital spatial profiling technologies of 10× Visium can identify the identity and location of the internal microbial community within tumors. The technologies can also detect areas with low vascularization, high levels of immune suppression and association with malignant tumors. Research has shown that the degree of vascularization in bacteria-colonized regions is lower than in bacteria-negative regions, with decreased expression of smooth muscle actin, decreased proliferation levels and downregulation of Ki-67 and p90 RSK. The bacteria-colonized microniches significantly increases the phosphorylation levels of JNK, ERK1, ERK2 and P38 in CRC tumors, revealing signal pathways that are activated in response to bacteria. The results indicated that the infected areas of CRC tumor tissue have lower proliferative potential compared to uninfected areas (166–168).

The liver metastasis of CRC poses a significant challenge for clinicians, with 80% of CRC cases experiencing tumor metastasis to the liver before the primary tumor can be detected clinically, which is closely associated with low survival rates (169,170). There is controversy regarding whether or not aging cancer cells serve a positive role in the progression and metastasis of tumors. Researchers have used ST and multi-database integration to comprehensively map the whole transcriptome cell atlas of CRC and related liver metastasis. They analyzed the molecular specificity within the TME and the transcriptional heterogeneity of senescent cancer cells, which could identify the aging-dependent cancer ecosystem in CRC liver metastasis and potentially provide therapeutic targets for CRC-related cancer types (171,172).

Applications in intestinal diseases

As one of the eight regions of the digestive system, the gastrointestinal tract serves an important role in absorption, digestion, excretion, endocrine, nutritional metabolism, immune balance and other functions. Due to the diversity of intestinal cells, dissecting the characteristics, functions and internal operation of the cells in the intestinal mucosa has been a technical challenge (173,174). The advent of ST provides a higher-resolution research tool for developmental and cancer-related studies of the gut, adding new dimensions to the understanding of multiple gastrointestinal diseases (94). Table I shows that different ST technologies are applied to different intestinal diseases, and the corresponding conclusions are drawn from different materials, indicating that they are widely used in intestinal diseases (111,175–181).

Table I. Applications of spatial transcriptomics in intestinal diseases.

Table I.

Applications of spatial transcriptomics in intestinal diseases.

First author, year	Technology	Disease or tissue	Conclusion	(Refs.)
Pelka et al, 2021	Slide-based sequencing and scRNA-seq	CRC	Revealed spatially organized multicellular immune centers in human CRC.	(175)
Parigi et al, 2022	LCM-seq and scRNA-seq	IBD	Visualized the gene expression landscape of the gut throughout injury and post-injury recovery, and described the spatial expression profile of IBD risk genes in the colon.	(111)
Frede et al, 2022	LCM-seq and scRNA-seq	UC	The expansion of B cells hindered the healing of IBD. Assessment of the influence of B-cell subsets on epithelial-stromal cell interactions.	(176)
Zhang et al, 2022	Immuno-LCM-RNA-seq	Small intestine lacteal cells	Showed partitioning of mouse small intestine lacteal cells.	(177)
Ghaddar and De, 2022	Neighbor-seq	Small intestinal epithelium, terminal respiratory tract and splenic white pulp	Analysis of cellular duplexes/multiplexes captured during standard scRNA-seq to infer cell-to-cell interactions. Proof of concept data in the small intestine.	(178)
Zhu et al, 2023	Slide-based sequencing and scRNA-seq	Gut microbiota	Intestinal-derived Akkermansia muciniphila may contribute to suppressing tumorigenesis by affecting the tumor commensal microbiome and reprogramming tumor metabolism.	(179)
Sun et al, 2023	LCM-seq and scRNA-seq	Gastric cancer	Identification of a ‘tumor-normal tissue interface’ region dominated by immune cells.	(180)
Tun et al, 2024	Seq-scope	IBD	Enterorome dysregulation driven by Caudovirales expansion affected intestinal immunity and dysfunction.	(181)

[i] LCM-seq, laser capture microdissection sequencing; scRNA-seq, single-cell RNA sequencing; Immuno-LCM-RNA-seq, laser capture microdissection sequencing combining with immunohistochemistry; Neighbor-seq, a method to identify and annotate the architecture of direct cell-cell interactions and relevant ligand-receptor signaling from the undissociated cell fractions in massively parallel single cell sequencing data; Seq-scope, a spatial barcoding technology with a resolution comparable to an optical microscope; CRC, colorectal cancer; UC, ulcerative colitis; IBD, inflammatory bowel disease.

Conclusions and prospects

The present review discusses the applications of ST technologies related to the gut. The rapid development of ST and its combined application with multi-omics has provided powerful technical support for studying the mechanisms involved in gut development and physiological pathology.

Due to the complex structure of the intestines, accurately mapping their spatial organization and molecular characteristics is crucial for understanding the progression of diseases. Various sequencing technologies have been continuously evolving, from scRNA-Seq to ST based on scRNA-Seq, enabling the visualization of the spatial positioning of tissues and cells. Given that low-resolution ST cannot meet higher imaging requirements, researchers have developed high-definition spatial genomics. HDST has been combined with powerful imaging modularization techniques through the modification of microbead arrays and the development of polynomial Bayesian classifiers, resulting in a marked increase in the resolution of the technique to 1,400 times higher than that of normal ST. Together with the highly specific nature of HDST signals, which can be interpreted through computational integration with morphological features and single-cell profiles, this has opened up novel avenues for high-resolution spatial analyses of cells and tissues (70,182). With the continuous development of sequencing technology, phenotypic information at the protein level on the cell surface has been gradually and well presented by novel technologies, and the use of DNA-barcoded antibodies allows for simultaneous analysis of the single-cell transcriptome and proteome, whereas NGS-based ST allows for the analysis of spatial multi-omics data. The single-cell multi-omics approach permits a more comprehensive understanding of intestinal biology and disease mechanisms, which facilitates the prediction of drug targets and clinical responses (183,184). More in-depth studies are already underway, including the use of ST to study epigenomic, proteomic or chromosomal structures (185), which can define neuroscience- and cancer-related cell types, provide spatial analyses that can present epigenetic traits at the single-cell level, and provide insight into the spatial state of protein molecules in tissues (186–188).

Numerous discoveries in the life sciences are closely related to the biological function of tissue and cell interactions, where spatial relationships between cells determine cell fate and diseases emerge as abnormalities in spatial structures within tissues (189). ST reconstructs spatial organization based on single-cell sequencing and combines it with other methods, such as different sequencing techniques, computer programming and visualization technology to present biological processes more intuitively. These technologies have facilitated new discoveries in various fields ranging from intestinal development to various diseases of the intestines.

Efficient research methods serving research should also serve the clinic, and the clinical application of ST is one of the important issues in translational medicine (190). Complete and efficient sampling and testing protocols, as well as the analysis and interpretation of data, are key to applying ST in clinical settings. A number of transcriptome-based commercial platforms are progressively being developed for applications that utilize artificial intelligence (AI) to provide open datasets and accurate data processing methods (echnologies such as SOMDE, SEDR and STAGATE), leveraging the potentials of AI, which will contribute to the clinical application of ST (68,191–193).

Acknowledgements

Not applicable.

Funding

The present study was supported by Chongqing Natural Science Foundation (grant no. cstc2021jcyj-msxmX0858).

Availability of data and materials

Not applicable.

Authors' contributions

YH and YG discussed the premise of the study and conceived the format. YG drafted the initial manuscript based on this discussion. CR completed the revisions of the manuscript. YH and YW generated the figures. YW revised the content of the manuscript. XY proposed the main idea of the article and coordinated the work to ensure the accuracy or completeness of the manuscript and address any issues that arise during the writing process. All authors have read and approved the final version of the manuscript. Data authentication is not applicable.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References