Single-cell sequencing and organoids: applications in organ development and disease

Recently, organoids have exhibited a multidimensional trend in innovative development [4, 35]. The introduction of gene editing technologies has significantly enhanced the precision modeling capabilities of organoid systems [36]. Technologies such as clustered regularly interspaced short palindromic repeats (CRISPR) and base editing enable the precise introduction of specific genetic mutations into organoids, simulating the onset and progression of monogenic hereditary diseases, as well as complex diseases [37, 38]. Additionally, innovations in engineered culture systems have significantly expanded the application scope of organoids [39]. The establishment of multi-organ co-culture systems has enabled the synergistic cultivation of organoids from different organs, simulating physiological interactions between organs [40]. Vascularized organoids partially address limitations in nutrient delivery and metabolic waste removal within traditional organoids by introducing endothelial cells (ECs), thereby enhancing the survival time and functional maturity of organoids [41].

Compared to traditional research models, organoids exhibit unique advantageous characteristics [42]. Unlike 2D cell cultures, the 3D structure of organoids can more authentically recapitulate cellular interactions, establishment of cell polarity, and formation of tissue structures within the in vivo microenvironment, providing an ideal platform for studying cell–cell and cell–matrix interactions. Compared to animal models, organoids offer numerous advantages, including lower costs, shorter research cycles, and fewer ethical restrictions [43, 44]. Notably, through the use of patient-derived iPSCs, they can circumvent species differences to achieve personalized modeling. Furthermore, organoids demonstrate exceptional scalability, enabling their use in high-throughput drug screening and toxicity testing, significantly enhancing the efficiency of drug development [2].

Organoids have become an ideal in vitro platform for studying organ development, disease mechanisms, and drug screening. With the continuous optimization and innovation of organoid culture systems, their application prospects within precision medicine, regenerative medicine, and toxicological evaluation will become even broader. In the future, greater breakthroughs are anticipated in simulating complex physiological and pathological processes, as well as enabling long-term dynamic monitoring through the deep integration of cutting-edge technologies, including single-cell sequencing, microfluidic chips, and organoids.

The introduction of single-cell sequencing has enabled the high-throughput sequence analysis of the genome, transcriptome, or epigenome of single cells. Its core advantage lies in its ability to uncover the heterogeneity masked by traditional bulk sequencing within cell populations [45, 46]. Next-generation sequencing has progressed from population-level to single-cell resolution, and subsequently to the integration of multi-omics and spatial localization. The origins of single-cell sequencing can be traced back to early exploratory studies on individual cells [47]. With the gradual advancement of molecular biology techniques and the increasing demand for research on cellular heterogeneity [48 –52], single-cell sequencing technologies began to emerge [53 –55]. The first single-cell transcriptome sequencing method was proposed in 2009. Since then, the technology has progressively evolved, from the first-generation, low-throughput, microwell plate-based methods to the current high-throughput, multi-omics integrated, and spatially resolved methods [56].

In the initial phase, bulk next-generation RNA sequencing (RNA-seq) laid the bioinformatics foundation for subsequent technologies by extracting and sequencing mixed RNA from a large number of cells, providing population-averaged gene expression profiles, but completely masking cellular heterogeneity [56 –59]. Single-cell RNA sequencing subsequently overcame this limitation, enabling the analysis of gene expression at the single-cell level. In 2011, single-cell tagged reverse transcription sequencing (STRT-seq) introduced cell barcoding, which supports the large-scale detection of various mixed cell samples, especially those with high heterogeneity [60, 61]. Switching mechanism at the 5' end of RNA template sequencing (SMART-seq) and the subsequent SMART-seq2 have improved the full-length transcriptome coverage and sensitivity of single-cell sequencing, addressing the challenges of whole-genome transcriptome analysis in rare cells [62 –64]. Furthermore, cell expression by linear amplification and sequencing (CEL-seq) enables multiplex analysis and study of different single cells [65].

Spatial transcriptomics technologies integrate gene expression with spatial location information, further compensating for the loss of spatial context inherent in single-cell technologies [83]. Single-cell sequencing has evolved from first-generation methods based on microplates to the current stage, which emphasizes high-throughput, multi-omics integration, and spatial resolution, becoming a core tool for deciphering cellular heterogeneity.

Single-cell sequencing enables the systematic and quantitative assessment of how well the cell type composition in an organoid matches that of the corresponding human tissue through transcriptomic profiling [6, 84, 85]. For instance, in brain organoids, single-cell sequencing can identify whether key cell subtypes, such as excitatory neurons, inhibitory neurons, and astrocytes, are present and reveal their correspondence to specific stages of fetal brain development. Additionally, pseudo-temporal analysis can reconstruct the cell differentiation trajectories within organoids, determining whether they accurately recapitulate the lineage development pathways observed in vivo [86]. Although single-cell sequencing does not directly provide spatial information, its integration with spatial transcriptomic data enables the indirect inference of spatial organization patterns of different cell populations within organoids, thereby comprehensively validating their structural and functional biomimicry [87].

The maturity of organoids is often limited by the imperfections of the culture systems [88]. Single-cell sequencing can precisely identify abnormal cell states caused by the absence of or imbalance in signaling factors. Comparing the cellular atlases of organoids under various culture conditions can screen for key factors that promote the differentiation or functional maturation of specific cell subtypes [89]. For example, in liver organoids, single-cell sequencing has revealed that the addition of specific morphogenetic factors induces the expression of functional hepatic enzyme systems, significantly enhancing their metabolic maturity [90]. This data-driven optimization strategy, combined with technologies such as CRISPR base editing, enables the construction of disease models harboring specific mutations within weeks, significantly shortening research cycles [91]. Utilizing patient-derived iPSCs to circumvent species differences demonstrates unique value in both ethical and economic aspects [92].

Significant heterogeneity exists among cells, even within the same batch of organoids [93]. The high resolution of single-cell sequencing enables the identification of rare cell populations that are obscured in bulk analyses, such as endocrine precursor cells or tissue-specific stem cells [94, 95]. Single-cell sequencing can also detect unintended differentiated cells resulting from suboptimal culture conditions, providing direct evidence for optimizing culture protocols and enhancing the fidelity of organoids [96]. Compared to traditional 2D cell models, which fail to recapitulate the complex interactions and structures in vivo, and animal models, which are associated with high costs, long research cycles, and stringent ethical constraints, organoids—with their 3D structure—can faithfully recapitulate the organ microenvironment. When combined with single-cell sequencing, organoids can support precise analysis of cellular heterogeneity, thereby significantly enhancing experimental reproducibility and data reliability [97, 98].

The comprehensive integration of organoids and single-cell sequencing has driven fundamental research into the mechanisms underlying development and disease. It also has broad application prospects in drug development, as well as precision and regenerative medicine. With the continuous development and innovative integration of these two technologies, it is anticipated that they will bring further groundbreaking advancements to life science research.

The human brain is the most complex organ known among living organisms and represents the most advanced central nervous system (CNS) among life on earth. Previous studies on brain development and biology have often relied on rodent models, particularly mice and rats [107, 108]. While they have provided fundamental insights, the application of animal models in biomedical research is constrained by species differences. Additionally, 2D cell lines cannot recapitulate the hierarchical structure, spatial dimensions, cellular diversity, and intercellular interactions present in brain tissue. Moreover, the human brain exhibits more complex features, such as surface folding, forebrain expansion, layered organization, prolonged developmental processes, and richer cellular diversity, making it challenging to systematically elucidate human-specific gene regulatory mechanisms and pathways that control brain development [109 –111]. These issues limit research on the human CNS. Furthermore, while postmortem brain tissue is considered valuable for studying the human brain, it only reflects the terminal state of the brain and cannot be extended to the early stages of brain development or disease, restricting the investigation of dynamic changes at the cellular level [27].

Recently, brain organoids have progressively overcome limitations in model construction, gradually expanding into the mechanistic investigation of neural development and neurodegenerative diseases [1]. Single-cell sequencing has played a crucial role in this process, with research revealing the ability of organoids to recapitulate the diversity of cell types, lineage differentiation trajectories, and gene expression characteristics during human brain development at high resolution [112]. Furthermore, one study conducted single-cell sequencing at multiple levels, including transcriptomics, epigenomics, and spatial transcriptomics, using cortical organoids [105], revealing that the diversity of cell types during cortical organoid development approximates that during endogenous embryonic development, thereby reaffirming the significant research value of brain organoids as in vitro developmental models in studying human cortical development [105, 113].

The co-culture strategy and organ fusion technology have also successfully generated brain organoids with complex vascular networks, providing a new platform for studying vascular–neural interactions [117]. For example, a novel protocol has been introduced for producing vascularized brain organoids by co-culturing human multipotent progenitor cells with human umbilical vein ECs (HUVECs), demonstrating for the first time that the vascular system in vascularized brain organoids can connect with host blood vessels to construct a new functional vascularized system [118]. Other studies have independently induced blood vessels and brain organoids, then fused them to generate vascularized brain organoids [119]. Brain organoids with unique cortical regions and anteroposterior patterns have also been generated, enabling more detailed investigations into human-specific brain development and diseases [120]. These organoids possess a complex vascular network that integrates well with brain cells and exhibit more neural cell precursors than unfused brain organoids, thereby overcoming the limitations of existing models and providing a new platform for studying vascular–neural interactions in brain development and function.

Single-cell sequencing has expanded the applications of brain organoids in disease research. In neurodegenerative disease research, integrating organoids with single-cell multi-omics analysis can dynamically capture disease-related changes in cellular state, abnormal protein aggregation, and microenvironmental perturbations, providing a highly biomimetic platform for mechanistic research and drug screening in conditions such as Alzheimer's disease and Parkinson's disease [121, 122].

Given the above, the comprehensive integration of brain organoids and single-cell sequencing has advanced our understanding of fundamental biological questions in human brain development, including neurogenesis and cortical regionalization. It has also significantly expanded the application of brain organoids in modeling neurodevelopmental disorders and neurodegenerative diseases. This technological combination is driving neuroscience research from static descriptions toward dynamic mechanism analysis, establishing a novel platform for uncovering the cellular and molecular foundations of human brain development and disease.

As essential respiratory organs of the human body, the lungs have complex structures and functions that have long been the focus of biological and medical research. Traditional in vivo and in vitro models exhibit significant limitations in recapitulating human lung tissues and present notable biological discrepancies. The emergence of lung organoids has dramatically advanced our understanding of the lung tissue microenvironment and its role in repair processes following lung injury [123].

Studies have used organoid models of human fetal alveolar development to reveal cell fate patterns and the mechanisms underlying neonatal respiratory diseases [124]. In recent years, significant progress has been made in the field of lung organoid research, particularly in the long-term culture of alveolar cells. The alveolar epithelium is primarily composed of alveolar type 1 (AT1) and type 2 (AT2) cells, which are responsible for gas exchange and surfactant secretion, respectively [125]. One study developed a novel culture system that achieved the long-term expansion of human AT2 cells and their differentiation into AT1 cells [126]. This breakthrough provides a reliable in vitro model for studying alveolar-related diseases, including idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) [127].

The introduction of the air–liquid interface (ALI) culture approach has significantly enhanced the physiological relevance of lung organoids. By exposing organoids to an air environment, this approach more accurately simulates the in vivo airway microenvironment, thereby promoting key physiological processes, including the establishment of epithelial cell polarity, ciliogenesis, and mucus secretion [128]. ALI-cultured lung organoids have been successfully utilized in studying pathogen–host interactions, epithelial responses to air pollutants, and respiratory barrier functions, providing an essential tool for research on pulmonary epithelial biology [129, 130].

In mechanistic research on lung development, combining lung organoids with single-cell sequencing has revealed key regulatory networks [131]. Research indicates that Wnt signaling is a crucial upstream regulator of NK2 homeobox 1 (NKX2-1) levels [132, 133]. NKX2-1 binds directly to promoter regions of genes associated with alveolar differentiation-—surfactant protein C (SFTPC), lysosomal-associated membrane protein 3 (LAMP3), and solute carrier family 34 member 2 (SLC34A2)—promoting their transcription and thereby regulating the differentiation and functional maturation of alveolar cells [134]. Additionally, NKX2-1 interacts with other transcription factors, including forkhead box P1 (FOXP1), to coordinate the gene expression regulatory network during alveolar differentiation and maturation [135, 136]. Single-cell sequencing has been crucial in lung organoid research, providing a comprehensive analysis of the cellular composition and gene expression profiles of newly developed organoids (Fig. 3b). One study identified AT2 cells in differentiated alveolar organoids, further demonstrating that alveolar organoids can be used to study the differentiation and maturation processes of human alveolar cells [137].

Innovations in technical methods have also facilitated the development of lung organoids. A strategy has been established to direct the differentiation of human pluripotent stem cells (hPSCs) into distal lung organoids by sequentially differentiating them into definitive endoderm (DE), anterior foregut endoderm (AFE), ventral foregut endoderm, and lung bud organoids, ultimately forming lung organoids that recapitulate the process of lung development [138]. Another study established a stable culture system for human airway epithelial organoids, achieving the construction of a bipotent organoid model [123]. A further study established a stable human airway epithelial organoid culture system, achieving the construction of a dual-potent organoid system. Additionally, lung organoids constructed from hPSCs exhibited characteristics similar to those of embryonic developmental stages, including a multi-lineage cellular composition and complex tissue architecture. These organoids comprise multiple cell lineages, including mesenchymal and epithelial cells, and exhibit an ordered organization that resembles the structures of both proximal and distal airways [139].

Single-cell sequencing plays an indispensable role in lung organoid research. Applying single-cell sequencing to alveolar organoids enables the systematic elucidation of their cellular composition, lineage differentiation trajectories, and gene expression dynamics, effectively validating the model's similarity to actual tissue [132]. One study utilized single-cell sequencing to identify AT2 cells within alveolar organoids and elucidate their differentiation pathways, providing high-resolution data to support research into lung development and regeneration mechanisms [140]. As the technology continues to mature, lung organoids have expanded from fundamental developmental biology research into applied fields, including disease modeling and drug screening [141]. These advances provide a robust platform for deepening understanding of lung regeneration mechanisms, developing targeted therapeutic strategies, and advancing personalized medicine, heralding broad prospects in respiratory disease research and treatment development.

The human heart is a functional organ that develops during embryogenesis [142]. Reproducing the early stages of heart development in vitro presents particular challenges. Cardiac organoids, serving as crucial in vitro models for studying heart development and diseases, are currently primarily constructed from hiPSCs, offering new insights into heart development. Cardiac organoids began to emerge more frequently after 2017 [143, 144]. They were initially constructed using cardiomyocytes (CMs) derived from hiPSCs in conjunction with primary stromal cells, HUVECs, and human cardiac fibroblasts (CFs) [145, 146]. With advances in and the application of iPSCs, most contemporary cardiac organoids are formed either by directly differentiating embryoid bodies derived from iPSCs into CMs or by co-culturing multiple cardiac cell types derived from iPSCs [147 –150].

Physiologically, human cardiac organoids essentially mimic the in vivo distribution of cells, responses to external stimuli, and electrophysiological functions [151, 152]. However, primary stromal cells do not represent the genetic characteristics of specific patients, which limits the accuracy of disease modeling. To address this limitation, cardiac organoids have been constructed using CMs, CFs, and ECs derived from hiPSCs [153, 154]. This approach replicates patient-specific genetic variations and allows for the introduction of controlled genetic mutations or variations through gene-editing techniques, providing a means to model disease states and cardiac functions [155]. Cardiac organoids offer a foundation for studying cardiac development by simulating embryonic tissue and stage-specific 3D self-organizing structures in vitro.

One study established blood-generating heart-forming organoids by precisely supplementing cell cultures with multiple factors that promote hematopoiesis and vascular development: bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF). This approach ensures the orderly formation of organoids, successfully creating a three-layered structure comprising a core, a myocardial layer, and an outer layer. Each layer has distinct functions and collaborates to simulate the processes of heart and blood development, effectively recreating the embryonic anatomical layout [156]. In blood-generating heart-forming organoids, the angiogenesis process involves the VEGF signaling pathway and the classical TEK receptor tyrosine kinase (TEK/TIE2) signaling pathway. Specifically, VEGF binds to its receptor kinase insert domain receptor (KDR/VEGFR), which then activates the upstream Ras protein and further triggers the activation of the Raf-1 proto-oncogene, serine/threonine kinase (RAF1), initiating a cascade of signaling events. RAF1 activates downstream mitogen-activated protein kinase kinases 1 (MAP2K21/MEK1) and 2 (MAP2K2/MEK2), which then activate mitogen-activated protein kinases 1 (MAPK1/ERK2) and 3 (MAPK3/ERK1). This signaling pathway promotes EC migration and the formation of newly sprouting capillaries [157, 158]. ERK1 and ERK2 also activate cytosolic phospholipase A₂, leading to the release of arachidonic acid (Fig. 3c), which is then metabolized into prostaglandins and other molecules, thereby enhancing vascular permeability and supporting the dynamic regulation of vascular networks. This signaling pathway facilitates the establishment of a stable circulatory network within organoids, ensuring cellular metabolism and function, and thereby improving the physiological simulation of cardiac organoids [159]. Single-cell sequencing has been used to examine the gene expression profiles of blood-generating heart-forming organoids, accurately identifying myocardial cells, various EC types, hematopoietic progenitor cells, erythrocyte precursors, and leukocyte precursors [156].

Additionally, one study reported the successful cultivation of "miniature hearts" from stem cells that resembled early human embryonic hearts [160]. Measuring just 0.5 mm in diameter, these "miniature hearts" are organoids containing both myocardial and epicardial cells and can contract like human heart chambers when electrically stimulated. Another study developed multi-chamber heart organoids, which included the right ventricle (RV), left ventricle (LV), atrium, outflow tract (OFT), and atrioventricular canal (AVC), ultimately generating progenitor cell subpopulations with anterior and posterior heart chambers [161]. Single-cell sequencing and functional characterization revealed that the gene expression profiles of these heart organoids corresponded to different chambers during in vivo development, providing a platform for studying the coordinated development of organs.

The hPSC-derived heart-forming organoids encompass various aspects of the concurrent development of the heart, vascular system, and foregut, achieving the co-development of cardiac, endothelial, and multipotent hematopoietic tissues analogous to the in vivo embryonic hematopoietic region, thereby advancing in vitro hematopoiesis research [162]. Previously, the lack of humanized cardiac models that resemble myocardial injury and inflammatory responses has limited research into therapeutic drugs for myocardial ischemia/reperfusion (I/R) injury. One study also developed ventricular heart organoids from hiPSCs to simulate I/R injury induced by hypoxia/reoxygenation (H/R) [163]. The further development of these heart organoid models will facilitate a deeper exploration of the cooperative mechanisms of cardiac development and hematopoiesis, providing cellular and molecular targets for cardiovascular disease pathology.

Combining single-cell sequencing and cardiac organoids has deepened our understanding of cardiac development and provided new tools and perspectives for studying developmental disorders. As these techniques continue to advance, cardiac organoids will have an increasingly critical role in cardiovascular disease mechanism research, drug screening, and personalized medicine.

The liver is a vital organ responsible for producing and storing amino acids and vitamins, as well as performing various functions, including metabolism and detoxification. It comprises two types of epithelial cells: hepatocytes and bile duct cells. Monocultured hepatocytes do not remain stable in the long term. Multicellular liver tissue cultures are better suited to drug screening, disease modeling, and transplantation therapy than hepatocyte monocultures. However, these model systems are non-proliferative and have limitations in scalability [164]. In 2013, one study successfully established a mouse-derived liver organoid system by adding components such as hepatocyte growth factor (HGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) to the culture system [165]. However, significant differences exist between the effects of various drugs in animal models and in clinical use [166]. Human liver organoids derived from adult or fetal hepatocytes, hepatocyte-like cells (HLCs) derived from hiPSCs, and reprogrammed human hepatocytes can reproduce the key morphological, functional, and gene expression characteristics of mature hepatocytes in vivo [167].

Notably, some studies have established a stable culture system for adult hepatocyte organoids by optimizing culture conditions, thereby addressing the limitation in maintaining the functions of primary hepatocytes over the long term. In 2019, one study reported a newly developed method that utilizes a fully defined serum-free and feeder-free medium to produce hESC-derived expandable hepatic organoids [168]. These organoids stably maintained the bipotent phenotype of hepatic stem/progenitor cells, allowing them to differentiate into functional hepatocytes or cholangiocytes. Another study combined microengineering techniques with stem cell biology to generate well-functioning liver organoids from hiPSCs [169]. In 2022, a study thoroughly explored and optimized the culture medium for hiPSC-derived liver organoids, identifying four essential growth factors needed for the various stages of liver organoid differentiation: activin A, BMP4, oncostatin M (OSM), and HGF [170].

Liver organoids successfully recapitulated the zonation phenomenon of liver lobules, a critical feature for hepatic metabolic functions [171]. Single-cell sequencing has revealed the central regulatory role of the Wnt/catenin beta 1 (CTNNB1/β-catenin) signaling gradient in establishing zonation [172]. Analyzing the spatial distribution of gene expression in hepatocytes within organoids revealed that subpopulations of hepatocytes with distinct metabolic functions exhibit distribution patterns similar to those in vivo, providing new insights into the molecular basis of liver zonation [173]. The liver organoids demonstrate significant potential in high-throughput functional genomics studies. One study utilized liver organoids to conduct large-scale CRISPR screens, systematically identifying key gene networks involved in liver development and disease [174]. These studies have deepened our understanding of liver biology and also provided valuable resources for drug target discovery.

HGF primarily initiates signal transduction by binding to its transmembrane receptor, MET proto-oncogene, receptor tyrosine kinase (MET/c-Met) [175]. Upon binding to HGF, c-Met dimerizes, activating its intracellular tyrosine kinase activity. In a standard hepatic organoid culture system, the addition of exogenous HGF results in a rapid increase in the phosphorylation of c-Met, indicating the activation of its signaling pathway. This phosphorylation recruits a series of downstream signaling molecules, including GRB2-associated-binding protein 1 (GAB1). Additionally, the activated c-Met/GAB1 complex can activate the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway [176]. PI3K phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) on the cell membrane to produce phosphatidylinositol-3,4,5-trisphosphate (PIP3), a secondary messenger that recruits AKT to the cell membrane, where it is phosphorylated (activated). AKT activation promotes the expression of proteins, such as cyclin D1 (CCND1), facilitating the progression of hepatic organoid cells from the G1 to the S phase of the cell cycle, thereby promoting their proliferation [177]. Cell proliferation is crucial during the development of hepatic organoids, allowing them to increase in size and form structures resembling liver tissue (Fig. 3d).

One study also employed co-culture techniques to establish a culture model of adult mouse hepatobiliary organoids, significantly enhancing their long-term expansion capability [178]. Another study reported tracking temporal transcriptomic and phenotypic changes in cultures of human-derived adult hepatocytes, comparing them to fetal-derived hepatocytes, which led to the first observation of the lack of proliferation initiation and inhibition of lipid metabolism in adult hepatocyte-based organoid cultures [90]. A further study observed that supplementation with growth-related factors, such as interleukin 6 (IL6) and nuclear receptor subfamily 1 group H member 4 (NR1H4/FXR), enabled the in vitro culturing of organoids with adult hepatocyte characteristics. Additionally, single-cell sequencing of the fetal and adult liver highlighted their high complexity and cellular heterogeneity, identifying new cell subpopulations. An in-depth analysis of individual cells precisely identified regulatory genes crucial for this developmental maturation process, providing critical evidence for further research into the standard development mechanisms of the liver and the pathogenesis and therapeutic strategies for related liver diseases.

Intestinal development is a highly complex and fascinating biological process, with intestinal organoids playing a crucial role in this field. Advanced single-cell sequencing technologies have been used to investigate the differentiation trajectories of intestinal epithelial cells in detail. In 2009, one study reported the successful in vitro cultivation of mouse leucine-rich-repeat-containing G protein-coupled receptor 5 (LGR5)⁺ intestinal stem cells into mouse small intestinal organoids, recapitulating the 3D structures of the crypt- and villus-like epithelial regions [23]. The subsequent optimization of the culture conditions enabled the accurate simulation of the physiological state of mouse small intestinal epithelium, allowing for long-term in vitro culture [179]. Additionally, one study demonstrated the in vitro simulation of embryonic intestinal development using hPSCs, where hPSC-derived DE differentiated into midgut and hindgut endoderm, thereby facilitating gut tube-like morphogenesis [180].

The in vitro cultivation of isolated intestinal stem cells into intestinal organoids depends on recreating the microenvironment needed for the proliferation, differentiation, and migration of intestinal stem cells. In the native intestine, the niche for intestinal stem cells is a complex structure composed of various intestinal cells, primarily Paneth cells, intestinal stromal cells, fibroblasts, lymphocytes, and nerve cells [181]. One study utilized single-cell transcriptomic mapping of respiratory and gastrointestinal (GI) endodermal organogenesis to elucidate cellular states and benchmark human intestinal organoids, demonstrating the influence of mesenchymal stem cells (MSCs) on intestinal development [182]. Another study demonstrated that activation of the bone morphogenetic protein (BMP) signaling pathway generates colonic organoids with early hematopoietic features [183]. With their capacity for self-renewal and multidirectional differentiation, hPSCs can be used to generate a diverse cell types both in vitro and in vivo. However, existing differentiation protocols often suffer from low efficiency and functional inadequacy. To overcome these challenges, the roles of different signaling pathways, particularly the BMP signaling pathway, in stem cell differentiation have been explored. The BMP signaling pathway was found to be rapidly activatable, effectively guiding hPSCs toward differentiation into intestinal organoids.

BMPs belonging to the transforming growth factor-beta (TGF-β) superfamily play a crucial role in human intestinal development [184]. Research on intestinal organoids has demonstrated that bone morphogenetic protein 2 (BMP2) significantly increases the levels of phosphorylated SMAD family members 1 (p-SMAD1), 5 (p-SMAD5), and 8 (p-SMAD8), a key step in the BMP signaling pathway. BMP2 induces the expression of SATB homeobox 2 (SATB2), a chromatin remodeling protein that influences gene expression patterns. BMP2 also promotes the expression of posterior homeobox (HOX) genes, which are responsible for establishing cell fate along the anterior–posterior axis and play significant roles in organ morphogenesis during embryonic development. Therefore, BMP2 affects intestinal organ morphogenesis by regulating the expression of these factors, thereby playing a crucial role in the patterning of the intestinal tube. Organoids treated with BMP2 exhibit marker expression patterns consistent with the distal intestine after long-term culture in vitro [185]. When transplanted in vivo, human intestinal organoids and colon organoids retain their regional characteristics, expressing region-specific markers and hormones, with gene expression patterns that are highly consistent with those of the corresponding human intestinal regions. Furthermore, colonic organoids treated with BMP2 can produce colon-specific endocrine cells, further elucidating the critical role of the BMP signaling pathway (Fig. 3e).

The intestinal epithelium harbors various rare cell types that, despite their extremely low proportions, play crucial functional roles, which are difficult to effectively replicate with traditional models [186]. The integration of intestinal organoids and single-cell sequencing technologies has provided a breakthrough tool for the study of these cells. Among them, the research on enteroendocrine cells (EECs) is most representative [187]. These cells constitute approximately 1% of the intestinal epithelial cells but are key components of the gut-brain axis, regulating various physiological functions including appetite and insulin release [188]. Single-cell sequencing technology, through systematic analysis of human intestinal development, has revealed the complexity of signaling pathways like EGF/ERBB and identified key factors involved in the differentiation of rare cell types [188, 189]. The construction of the single-cell transcriptomic atlas of human EEC tissues has significantly advanced the in-depth study of rare intestinal cells, providing a crucial resource for understanding the functions of these cells under both physiological and pathological conditions [190].

The development of genome editing technologies, such as CRISPR/Cas9, has transformed intestinal organoids from developmental models into functional research platforms [191, 192]. Studies have successfully simulated hereditary intestinal diseases, such as cystic fibrosis and familial adenomatous polyposis, by introducing specific gene knockouts or pathogenic mutations in organoids [191, 193]. These disease-specific organoids exhibit highly humanized characteristics, providing reliable tools for disease mechanism research and drug screening, effectively compensating for the significant physiological differences between traditional animal models and humans. Particularly noteworthy is the use of CRISPR technology to genetically label specific cell types, LGR5 + intestinal stem cells and EECs, enabling researchers to track the fate determination and differentiation pathways of these cells in real-time during organoid development [23, 194]. This technology revealed the dynamic changes in the lineage of intestinal cells. It enabled the visualization and sorting of rare cells through the construction of a gene reporter system, providing high-purity cell samples for subsequent functional studies [195]. The combination of intestinal organoids and single-cell technologies has significantly deepened our understanding of intestinal development and the mechanisms that maintain homeostasis. It has also led to breakthroughs in precision medicine and the development of novel drugs for intestinal diseases.

The mammalian kidney comprises thousands to millions of nephrons and a branching network of collecting ducts connected to them [196, 197]. During embryonic kidney development, nephron and ureteric bud (UB) progenitors mutually induce further differentiation of progenitor cells and branching of the collecting duct, ultimately forming a complex nephron-collecting duct network structure [198, 199]. Kidney organoids are 3D cell aggregates derived from in vitro stem cell culture, replicating key aspects of kidney development and forming glomerular structures [200]. Recently, studies on kidney organoids have evolved from primarily developing nephron organoids to modeling UB or collecting duct organoids, thereby simulating human kidneys [201 –204]. Advances in kidney organoid technology have led to the formation of organoids with more specialized cell types, reduced heterogeneity, and increased structural complexity [205]. One critical factor in this process is the interaction between the UB and the metanephric mesenchyme (MM). The UB originates from the Wolffian duct, while the MM forms as the UB invades the embryonic body [185]. The interaction between the UB and the MM promotes kidney morphogenesis and determines the final structure of the nephron.

Kidney organoids have evolved from early models primarily establishing nephron organoids to more complex organoids capable of simulating ureteric buds or collecting ducts [206]. These advances have enabled kidney organoids to incorporate more specialized cell types, exhibit increased structural complexity, and significantly reduce heterogeneity. Among these developments, simulating interactions between the UB and MM has become critical, as this process plays a central role in kidney morphogenesis and the formation of nephron structure in vivo [207]. However, early kidney organoids exhibited notable limitations, including the absence of a vascular system and ineffective connections between nephrons and collecting ducts. To overcome these challenges, researchers have developed multiple innovative strategies. Flow culture on microfluidic chips significantly enhances the maturity of kidney organoids, as evidenced by the development of more mature capsules and tubular compartments, along with improved cell polarity and the expression of adult genes [208, 209]. Furthermore, kidney organoids cultured using kidney extracellular matrix hydrogels exhibit extensive vascular networks and self-derived ECs [210]. Single-cell sequencing analysis confirms their more mature glomerular development patterns and higher similarity to human kidneys [211].

Single-cell sequencing technology has had a pivotal impact on kidney organoid research, particularly in deciphering key transcription factor networks. Taking WT1 as an example, this transcription factor is crucial for podocyte development and functional maintenance [212]. WT1 expression increases with the development of mesenchyme and podocyte progenitors, eventually becoming restricted to mature podocytes [213, 214]. One study utilized single-cell sequencing of fetal kidneys and kidney organoids to identify a range of WT1 target genes essential for podocyte development and structural maintenance, including BMP-binding endothelial regulator (BMPER), paired box 2 (PAX2), and membrane-associated guanylate kinase, WW and PDZ domain containing 2 (MAGI2), which regulate the Wnt signaling pathway, myosin heavy chain 9 (MYH9), which maintains actin filament organization; and NPHS1 adhesion molecule, nephrin (NPHS1), which regulates cell junction assembly [212]. Heterozygous missense mutations in WT1 prevented it from activating target genes like MAGI2, MYH9, and NPHS1, leading to delayed podocyte development and cellular structural damage. This observation has been confirmed through single-cell sequencing and functional analysis of kidney organoids derived from iPSCs from patients with WT1 mutations. Moreover, correcting the WT1 mutation in patients using the CRISPR/Cas9 system could rescue the podocyte phenotype, underscoring the pathogenic role of WT1 mutations in abnormal podocyte development [207]. Another study found that WT1 mutations disrupting the podocyte phenotype were often limited to the role of single genes. A further study has elucidated the epigenomic landscape of WT1 in human podocyte development at a single-cell resolution from a systemic perspective, revealing the key mechanistic role of WT1 in regulating podocyte development and maintaining podocyte structure and function during kidney development and kidney organoid differentiation [212]. These findings highlight the advantages and value of kidney organoids as models of the human kidney (Fig. 3f).

With the deep integration of single-cell multi-omics analysis and gene editing technologies, kidney organoids have broad application prospects in simulating kidney development, studying disease mechanisms, and drug screening [215]. Future research directions include improving the vascularization of organoids, enhancing structural complexity, and better simulating the physiological functions of the kidney [215]. The combination of kidney organoids and single-cell technology will continue to drive the transformation of kidney developmental biology from descriptive research to mechanistic understanding, providing breakthroughs for precision medicine in kidney diseases.

In summary, the deep integration of single-cell sequencing and organoid technology has opened up new avenues for understanding the cellular and molecular basis of organ development. Through systematic application in key organ models such as the brain, lung, heart, liver, intestine, and kidney, this technological combination has revealed cellular heterogeneity, lineage differentiation trajectories, and gene regulatory networks at high resolution, demonstrating its advantages in simulating human-specific developmental processes and complex disease mechanisms. Current models still face challenges in vascularization, functional maturity, and standardization, but engineered culture strategies and multi-omics integrated analysis are continuously driving them toward higher physiological relevance. In the future, with the further integration of cutting-edge technologies, for instance, spatial transcriptomics, gene editing, and live imaging, organoids will transition from static simulation to dynamic analysis, continuously providing a robust platform for developmental biology, disease mechanism research, and precision medicine, ultimately driving a paradigm shift in life science research.

Lung cancer (LC) exhibits significant complexity and heterogeneity, encompassing non-small cell LC (NSCLC) and small cell LC, with diverse mutational profiles. Tumor heterogeneity poses a considerable challenge in LC treatment, as substantial genetic, epigenetic, and phenotypic differences exist among tumors, complicating the development of precise and effective treatment methods [174]. Organoid models retain the genetic, transcriptional, morphological, and drug sensitivity characteristics of the parental tumors [19]. The tumor microenvironment in LC organoids includes various cell types, including immune, vascular endothelial, and stromal cells. Single-cell sequencing can elucidate the roles of these cells in the development of tumors. For example, tumor-associated macrophages (TAMs) exhibit different polarization states within LC organoids, where M2-type TAMs promote tumor cell proliferation and metastasis by secreting cytokines, name a interleukin 10 (IL-10) [220].

The Wnt/β-catenin pathway plays a pivotal role in LC progression [221], with Wnt family member 3 A (Wnt3A) acting as a key activator. The binding of Wnt3A to frizzled class receptors and co-receptors LDL receptor-related protein 5/6 (LRP5/6) at the cell membrane initiates a cascade of complex and ordered signaling events [222]. This cascade involves the activation of disheveled segment polarity proteins, which disrupt the stability of the β-catenin destruction complex composed of AXIN, APC regulator of Wnt signaling pathway (APC), GSK3β, and casein kinase 1 (CK1) [223, 224]. Normally, GSK3β activity phosphorylates β-catenin, marking it for ubiquitin–proteasome degradation. However, Wnt3A signaling prevents its degradation, allowing β-catenin to accumulate in the cytoplasm. Then, β-catenin translocates to the nucleus, interacting with the T cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors [225], forming a transcriptionally active complex that targets promoters of genes crucial for stemness and proliferation, cellular myelocytomatosis (c-Myc), CCND1, and survivin [221]. For example, as an important oncogene, c-Myc activation can drive cell cycle progression, promote metabolic reprogramming, and enhance cell proliferation capacity [226]. CCND1 helps to regulate the transition from the G1 phase to the S phase of the cell cycle, accelerating cellular proliferation [227]. By activating the expression of these genes, Wnt3A successfully enhances the activity of the Wnt/β-catenin pathway [228], establishing a robust molecular foundation for maintaining stem cell pluripotency, which endows tumor cells with a strong potential for self-renewal and proliferation advantage, facilitating the progressive evolution of LC cells from normal to malignant states. By enhancing tumor cell proliferation, invasion, and metastatic potential, this process thereby exerts a profound influence on LC pathogenesis, clinical outcomes, and ultimately, the efficacy of therapeutic interventions.

Establishing a biobank of LC organoids facilitates the screening of anticancer drugs and uncovers potential differences in resistance [229], as well as varied responses to erlotinib and crizotinib [220, 228]. In immunotherapy, co-culture models of organoids and immune cells enable the identification of immune activation strategies and the study of tumor immune microenvironment responses. Moreover, LC organoids can facilitate personalizing cancer treatment by assessing drug toxicity through comparisons with normal lung organoids, providing patients with precise therapeutic methods.

Breast cancer (BC) is a highly heterogeneous disease with multiple molecular subtypes, including luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)-positive, and triple-negative (TNBC). Traditional 2D cell cultures and animal models often fail to replicate BC complexity [230]. BC organoids effectively preserve the heterogeneity of tumor tissues, with distinct morphological, cellular, and gene expression profiles across different subtypes, reflecting intratumoral diversity [231]. They faithfully retain the genetic characteristics of tumors, including mutations, gene amplifications, and chromosomal abnormalities, which are crucial for studying BC pathogenesis, predicting therapeutic responses, and developing personalized treatments. Single-cell sequencing has revealed that BC organoids retain the characteristics of primary BCs (PBCs) to varying degrees across different molecular subtypes. While BC organoids can closely retain the characteristics of PBCs in hormone receptor (HR)-positive and HER2-positive BC, they differ significantly from PBCs in stemness, hypoxia-related pathways, and drug sensitivity in TNBC [232]. This discrepancy may be related to oxygen levels in the culture conditions. Single-cell sequencing can further delineate the characteristics of various cell subpopulations within the tumor microenvironment, among others immune cells and stromal cells.

In approximately 15%–20% of BC cases, overexpression or amplification of the HER2 gene plays a crucial role in tumorigenesis. In BC organoids, the HER2 signaling pathway is typically aberrantly activated through heterodimerization with other HER family members, activating downstream pathways such as Ras/Raf/MEK/ERK and PI3K/AKT/mTOR, which promotes tumor cell proliferation, migration, and invasion [233, 234]. Despite the significant therapeutic efficacy of HER2-targeted drugs like trastuzumab and pertuzumab in HER2-positive BC, resistance remains a clinical challenge. Therefore, the regulation and resistance of the HER2 signaling pathway in BC organoids are vital for developing new anti-HER2 therapeutic strategies. The PI3K/AKT/mTOR pathway is frequently hyperactivated in BC [235, 236], which is closely associated with tumor cell proliferation, metabolism, and resistance [237]. In BC organoids, various factors can activate this pathway, overexpression of HER2 or loss of phosphatase and tensin homolog (PTEN). Modulating the PI3K/AKT/mTOR pathway significantly impacts the growth and survival of BC organoids [238]. Multiple inhibitors targeting this pathway are currently under development and evaluation in clinical trials.

In BC, patient-derived organoids (PDOs) are considered promising models to overcome the limitations of cell lines and patient-derived xenografts (PDXs) [239]. One study aimed to validate the efficacy of PDOs in predicting patient drug responses by establishing a biobank of BC PDOs [240, 241]. Another study utilized PDOs to explore personalized treatments for refractory BC. It successfully demonstrated that the sensitivity of PDOs to microtubule-targeting drugs can predict distant recurrence-free survival in invasive breast cancer treated with adjuvant chemotherapy [242]. Its findings suggest that PDOs can inform personalized treatment decisions for patients with advanced BC.

Liver cancer, as a severe malignant tumor, holds a significant position in both incidence and mortality rates globally. The most common type of primary liver cancer is Hepatocellular Carcinoma (HCC), while cancer originating from the bile ducts is termed Cholangiocarcinoma [243]. HCC is a malignant disease that poses a serious threat to human health, with high incidence and mortality. A liver cancer organoid biobank comprised of 399 tumor organoids derived from 144 patient samples revealed a degree of mutation concordance between tumor tissues and organoids [244]. However, some exhibited tumor evolution during passaging, highlighting cellular heterogeneity. Organoids from different patients and regions of the same tumor showed differences in gene expression and mutation profiles, further underscoring this heterogeneity [245]. The complex tumor microenvironment in primary liver cancer can be further explored using organoid models to study intercellular interactions [246].

Multiple signaling pathways and associated proteins play critical roles during HCC development and progression. The overexpression of cellular jun-transforming protein (c-Jun) can mediate resistance to lenvatinib via the JUN N-terminal kinase (JNK) and β-catenin signaling pathways. The synthesized compound PKUF-01, composed of lenvatinib and veratramine, exhibited synergistic inhibitory effects on resistant cells. The histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) upregulates the expression of PTEN and inhibits the AKT signaling pathway, thereby reversing lenvatinib resistance in HCC cells [244]. In minimal residual disease (MRD) of HCC, programmed cell death 1 ligand 1 (PD-L1)-positive M2 macrophages interact with cancer stem cells through the TGF-β signaling pathway, sustaining tumor cell persistence. Combined blockade of PD-L1 and TGF-β signaling pathways effectively prevents HCC recurrence. The aberrant activation or dysregulation of these pathways and proteins collectively contributes to HHC development, progression, drug resistance, and recurrence.

Utilizing HCC organoid models, research has proposed that collaboration among different cellular subpopulations within tumors leads to drug resistance, offering new insights and strategies for treating HCC. Combining patient-derived tumor organoids with single-cell sequencing and drug screening assays has revealed drug resistance mechanisms in HCC [247]. Pharmacogenomic analysis of organoids has enabled the identification of biomarkers predictive of drug response, to name a few lenvatinib resistance. In-depth investigations of resistance can facilitate the development of novel combination therapies [248]. For example, combining lenvatinib with HDAC or AKT inhibitors synergistically inhibited tumor cell proliferation and induced apoptosis, enhancing therapeutic efficacy [249]. This approach supports personalized medicine in treating HCC by tailoring strategies to the unique characteristics of a patient's tumor, including genetic mutations, gene expression profiles, and drug sensitivities, thereby improving treatment precision and effectiveness.

Gastric Cancer (GC) ranks among the most prevalent and deadly malignant tumors worldwide. GC organoids serve as tumor surrogates, effectively mimicking the tumor microenvironment and facilitating research into tumor biology. They overcome the limitations of traditional GC cell lines, which often fail to represent the characteristics of the original tumor tissue accurately [250]. The application of high-throughput single-cell sequencing to tumor organoids has expedited the identification of tumor driver mutations, dysregulated programs, and molecular subtypes [251]. One study used a diffuse-type GC PDO-monocyte/macrophage co-culture system, discovering that aurora kinase inhibitors (AURKi) induce senescence in diffuse GC. Another study demonstrated that senescent cells secrete C–C motif chemokine ligand 2 (CCL2/MCP-1), which triggers the polarization of locally accumulated monocyte/macrophages towards the M2 phenotype, creating an immunosuppressive microenvironment that weakens the innate immune capacity of macrophages to eliminate tumor cells [252].

GC involves several critical signaling pathways. The MEK/ERK and signal transducer and activator of transcription 3 (STAT3) pathways play crucial roles in the growth and survival of gastric precancerous lesions. Dual inhibition of these pathways can suppress the stem cell and dysplastic features of precancerous lesions, offering new strategies for treating GC [253]. The claudin 18 (CLDN18)-Rho GTPase activating protein 26 (ARHGAP26) fusion gene is frequently recurrent in diffuse GC, inducing the formation of characteristic "signet-ring cells" and promoting the activation of ras homolog family member A (RHOA) and its downstream signaling, notably the focal adhesion kinase (FAK) and YAP signaling pathways. Co-inhibition of the FAK and YAP/TEAD pathways significantly impedes tumor growth, providing a basis for evaluating the therapeutic potential of related inhibitors [254]. Additionally, N-acetyltransferase 10 (NAT10) is overexpressed in GC and is associated with poor prognosis. NAT10 regulates RNA dynamics through condensate formation and interacts with splicing factor serine- and arginine-rich splicing factor 2 (SRSF2), affecting the splicing pattern of the m⁶A reader YTH N⁶-methyladenosine RNA binding protein F1 (YTHDF1) [255].

In drug screening, establishing GC organoid biobanks enables in vitro personalized drug testing [256]. This approach can help identify gene expression signatures associated with chemosensitivity or resistance, facilitating the development of predictive models. One study using GC organoids showed that those sensitive to 5-fluorouracil (5-FU) or oxaliplatin exhibited upregulation of tumor suppressor genes or pathways, whereas organoids resistant to 5-FU exhibited increased proliferation and invasion [256]. In immunotherapy, it has been observed that drug-induced senescent cells can affect macrophage function. For example, a study using diffuse GC PDO-monocyte/macrophage co-cultures demonstrated that AURKi-induced senescent cells secreted CCL2/MCP-1, promoting the M2 polarization of monocytes/macrophages and weakening their innate immune capacity, suggesting a new therapeutic strategy [252]. Another study examined 5-FU-resistant GC organoids and identified KH RNA binding domain-containing signal transduction associated 3 (KHDRBS3) as a promising biomarker for predicting treatment response and prognosis [257]. In personalized medicine, GC organoids reflect patient-specific tumor characteristics, aiding in the tailored selection of chemotherapeutic agents. Different GC subtypes respond variably to drugs. For example, patients with poorly differentiated intestinal-type GC may benefit from combined 5-FU and veliparib treatment [254]. Organoid models hold the potential for advancing precision treatment in GC.

Traditional research models face limitations in simulating the complexity of CRC, whereas the advent of organoid models has brought significant breakthroughs to CRC. CRC organoids encompass tumor types in various states [252]. Single-cell sequencing of CRC organoids has revealed the heterogeneity of tumor cells, demonstrating differences in gene expression and cellular functions among different PDOs. For example, single-cell sequencing of CRC liver metastasis (CRLM) organoids identified significant differences in spontaneous differentiation between those derived from primary CRC and those derived from liver metastases (LMs). Tumor stem cells within LM organoids possessed higher self-renewal potential than those in CRC organoids. Cell trajectory analyses indicated that tumor stem cells in CRC organoids tend to undergo differentiation, while mature cells in LM organoids exhibit dedifferentiation tendencies, underscoring the self-renewal capacity of tumor stem cells in LMs [258]. Additionally, one study has revealed distinct state transitions that CRC cells undergo during metastasis and how these changes influence adaptability and treatment response. Using single-cell sequencing to dissect the intrinsic plasticity and environmental adaptability of cells, it identified transcription factors that play critical roles in regulating gene transcription to name a fewprospero homeobox 1 (PROX1) [259].

Several key signaling pathways and proteins are involved in CRC pathogenesis. NLR family pyrin domain containing 12 (NLRP12) inhibits the Wnt/β-catenin pathway by interacting with serine/threonine kinase 38 (STK38), thereby suppressing CRC development and progression [260]. Research has demonstrated that Nlrp12 deficiency increased CRC incidence and significantly elevated activation of the Wnt/β-catenin pathway, independent of the gut microbiota composition. This finding indicates that the NLRP12/STK38/GSK3β signaling axis may be a promising therapeutic target for CRC [260]. Further investigation revealed that NLRP12 inhibits the Wnt/β-catenin pathway by interacting with STK38, which acts as a crucial regulator in controlling the phosphorylation of GSK3β. Under normal conditions, GSK3β phosphorylates β-catenin, marking it for proteasomal degradation and maintaining low Wnt/β-catenin pathway activity. The interaction between NLRP12 and STK38 stabilizes the activity of GSK3β, allowing it to continuously phosphorylate β-catenin effectively, thereby preventing abnormal activation of the Wnt/β-catenin pathway. However, this inhibitory effect is lost in the absence of NLRP12, leading to β-catenin accumulating within the cell, excessive activation of the Wnt/β-catenin pathway, and promotion of CRC development [261].

Cetuximab is an anti-EGFR antibody commonly used to treat CRC without KRAS proto-oncogene GTPase (KRAS), NRAS proto-oncogene GTPase (NRAS), or B-Raf proto-oncogene serine/threonine kinase (BRAF) mutations [262]. However, despite the presence of these biomarkers, the response to cetuximab varies significantly, and predictive biomarkers remain limited. Previous preclinical studies have mainly relied on cell lines, which lack tumor complexity and often produce data that are difficult to translate due to the absence of the patient's tumor microenvironment [263]. In order to address these limitations, studies have employed PDX models, which more closely mimic human tumor biology, to identify reliable predictive markers for cetuximab sensitivity in CRC. One study has made significant progress, demonstrating the value of using a large, well-characterized PDX collection and integrative multi-omics approaches in predicting cetuximab sensitivity [263]. Furthermore, research on personalized treatment for metastatic CRC has explored the feasibility and clinical efficacy of using tumor-derived organoids and in vitro drug sensitivity testing to provide customized treatment plans for patients [264]. These advances suggest that integrating advanced models and testing methods can enhance the precision of targeted cancer therapies.

In summary, single-cell sequencing technology has provided a notable high-resolution perspective for tumor organoid research, systematically revealing the high heterogeneity characteristics of major cancer types such as lung cancer, breast cancer, liver cancer, gastric cancer, and colorectal cancer. This technology precisely deciphers the gene expression profiles and signaling pathway networks within tumor cells and deeply describes the functional states and interactions of immune and stromal cells in the tumor microenvironment. By constructing disease-specific organoid biobanks and integrating multi-omics analysis, studies have successfully identified key signaling axes, including Wnt/β-catenin, HER2 and NLRP12/STK38/GSK3β, providing novel targets for the development of targeted therapies and combination drug strategies. Despite ongoing challenges in model standardization and clinical translation, the deep integration of tumor organoids with single-cell technologies is driving cancer research to leap from traditional population-level studies to single-cell precision, laying a solid technical foundation for drug screening, efficacy prediction, and the development of personalized treatment plans in the era of precision medicine.