The Application of Organoids in the Study of Antiviral Innate Immunity.

Antiviral innate immunity plays a critical role in the defense against viral infections, yet its complex interactions with viruses have been challenging to study using traditional models. Organoids, three-dimensional (3D) tissue-like structures derived from stem cells, have emerged as powerful tools for modeling human tissues and studying the complex interactions between viruses and the host innate immune system. This chapter summarizes relevant applications of organoids in antiviral innate immunity studies and provides detailed information and experimental procedures for using organoids to study antiviral innate immunity.

Generating ESC-Derived RGCs for Cell Replacement Therapy.

In several ocular diseases, degeneration of retinal neurons can lead to permanent blindness. Transplantation of stem cell (SC)-derived RGCs has been proposed as a potential therapy for RGC loss. Although there are reports of successful cases of SC-derived RGC transplantation, achieving long-distance regeneration and functional connectivity remains a challenge. To address these hurdles, retinal organoids are being used to study the regulatory mechanism of stem cell transplantation. Here we present a modified protocol for differentiating human embryonic stem cells (ESCs) into retinal organoids and transplanting organoid-derived RGCs into the murine eyes.

Generation of Induced-Primary Retinal Pigment Epithelium from Human Retinal Organoids.

Retinal pigment epithelium (RPE) cells derived from induced pluripotent stem cells (iPSCs) serve multiple roles, including among others, modeling RPE development in normal and pathological conditions, investigating mechanisms of RPE physiology, modeling retinal diseases involving the RPE, and developing strategies for regenerative therapies. We have developed a simple and efficient protocol to generate RPE tissue from human iPSCs-derived retinal organoids. The RPE tissue present in the retinal organoids is analogous to the native human RPE in differentiation timeline, histological organization, and key features of functional maturation. Building upon this system, we established a method to generate functionally mature, polarized RPE monolayers comparable to human primary RPE. This comprehensive protocol outlines the steps for isolating and culturing RPE tissue using retinal organoids. The outcome is a pure population of cells expressing mature RPE signatures and organized in a characteristic cobblestone monolayer featuring robust ultrastructural polarization. These RPE monolayers also exhibit the functional hallmarks of bona fide mature RPE cells, providing a suitable system to mimic the biology and function of the native human RPE.

Molecular pathology, developmental changes and synaptic dysfunction in (pre-) symptomatic human C9ORF72-ALS/FTD cerebral organoids.

A hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Human brain imaging and experimental studies indicate early changes in brain structure and connectivity in C9-ALS/FTD, even before symptom onset. Because these early disease phenotypes remain incompletely understood, we generated iPSC-derived cerebral organoid models from C9-ALS/FTD patients, presymptomatic C9ORF72-HRE (C9-HRE) carriers, and controls. Our work revealed the presence of all three C9-HRE-related molecular pathologies and developmental stage-dependent size phenotypes in cerebral organoids from C9-ALS/FTD patients. In addition, single-cell RNA sequencing identified changes in cell type abundance and distribution in C9-ALS/FTD organoids, including a reduction in the number of deep layer cortical neurons and the distribution of neural progenitors. Further, molecular and cellular analyses and patch-clamp electrophysiology detected various changes in synapse structure and function. Intriguingly, organoids from all presymptomatic C9-HRE carriers displayed C9-HRE molecular pathology, whereas the extent to which more downstream cellular defects, as found in C9-ALS/FTD models, were detected varied for the different presymptomatic C9-HRE cases. Together, these results unveil early changes in 3D human brain tissue organization and synaptic connectivity in C9-ALS/FTD that likely constitute initial pathologies crucial for understanding disease onset and the design of therapeutic strategies.

A reference for selecting an appropriate method for generating glioblastoma organoids from the application perspective.

Glioblastoma organoids (GBOs) serve as a powerful and reliable tool to study glioblastoma stem cells (GSCs) and glioblastoma (GBM). GBOs can be derived from different materials using different methods. To identify the predominant generation methods and the most applications of GBOs, we searched four databases (PubMed, Embase, Web of Science, and Wiley Online Laboratory) from August 2021 to August 2023. After screening, 42 out of 295 articles were included and analyzed. GBOs in these articles were generated using only one material, such as tumor tissues, tumor cells, and gene-edited multifunctional stem cells, or simultaneously using two materials, such as tumor cells and normal organoids. Methodologically, direct cultivation of GBM cells or tissues was the most commonly used method to generate GBOs. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) were the frequently used multifunctional stem cells to generate GBOs by simultaneously silencing P53, NF1, and PTEN using CRISPR/Cas9. In terms of applications, GBOs generated by direct cultivation of GBM tissue had the most applications, including molecular mechanisms, therapy, and culture technique. This review provides a theoretical reference for selecting an appropriate method to generate GBOs when studying GSCs and GBM.

Unveiling senescence-associated ocular pathogenesis via lacrimal gland organoid magnetic bioassembly platform and HMGB1-Box A gene therapy.

Dry eye disease (DED) is a multifactorial aging disorder leading to tear film insufficiency and instability. Yet, an important knowledge gap lingers in understanding senescence-associated ocular pathogenesis, due to limited in vitro translational lacrimal gland (LG) models. Consequently, this remains a major roadblock to discover effective therapies for the restoration of tear film secretion. Herein, the authors reported the magnetic bioassembly of two LG organoid platforms to recapitulate functional and aging states. Using a proof-of-concept approach, porcine primary LG cells were assembled into organoids via a magnetic 3D bioprinting (M3DB) platform. This platform could form reproducible LG organoids with epithelial hallmarks (AQP5+) and exhibit epithelial secretory functions (lysozyme activity). DNA damage-induced senescence and cell death was induced with etoposide, and LG organoid hypofunction and senescence-associated pathogenesis were observed. To confer DNA protection against aging, a novel gene therapy with Box A domain of high-mobility group box-1 (HMGB1-Box A) previously established by our group, was applied here to prevent LG cellular senescence for the first time. HMGB1-Box A transfection prevented LG organoids from senescence-associated pathogenesis at the transcriptomic, metabolomic and proteomic levels. Thus, M3DB platforms could generate functional and DNA damage-induced senescence LG organoids, and this latter damage could be prevented with HMGB1-Box A gene therapy.

Development of an automated 3D high content cell screening platform for organoid phenotyping.

The use of organoid models in biomedical research has grown substantially since their inception. As they gain popularity among scientists seeking more complex and biologically relevant systems, there is a direct need to expand and clarify potential uses of such systems in diverse experimental contexts. Herein we outline a high-content screening (HCS) platform that allows researchers to screen drugs or other compounds against three-dimensional (3D) cell culture systems in a multi-well format (384-well). Furthermore, we compare the quality of robotic liquid handling with manual pipetting and characterize and contrast the phenotypic effects detected by confocal imaging and biochemical assays in response to drug treatment. We show that robotic liquid handling is more consistent and amendable to high throughput experimental designs when compared to manual pipetting due to improved precision and automated randomization capabilities. We also show that image-based techniques are more sensitive to detecting phenotypic changes within organoid cultures than traditional biochemical assays that evaluate cell viability, supporting their integration into organoid screening workflows. Finally, we highlight the enhanced capabilities of confocal imaging in this organoid screening platform as they relate to discerning organoid drug responses in single-well co-cultures of organoids derived from primary human biopsies and patient-derived xenograft (PDX) models. Altogether, this platform enables automated, imaging-based HCS of 3D cellular models in a non-destructive manner, opening the path to complementary analysis through integrated downstream methods.

Developing patient-derived organoids to identify JX24120 inhibit SAMe synthesis in endometrial cancer by targeting MAT2B.

Endometrial cancer (EC) is one of the most common gynecologic malignancies, which lacking effective drugs for intractable conditions or patients unsuitable for surgeries. Recently, the patient-derived organoids (PDOs) are found feasible for cancer research and drug discoveries. Here, we established a series of PDOs from EC and performed drug repurposing screening and mechanism analysis for cancer treatment. We confirmed that the regulatory ß subunit of methionine adenosyltransferase (MAT2B) is highly correlated with malignant progression in endometrial cancer. Through drug screening on PDOs, we identify JX24120, chlorpromazine derivative, as a specific inhibitor for MAT2B, which directly binds to MAT2B (Kd = 4.724µM) and inhibits the viability of EC PDOs and canonical cell lines. Correspondingly, gene editing assessment demonstrates that JX24120 suppresses tumor growth depending on the presence of MAT2B in vivo and in vitro. Mechanistically, JX24120 induces inhibition of S-adenosylmethionine (SAMe) synthesis, leading to suppressed mTORC1 signaling, abnormal energy metabolism and protein synthesis, and eventually apoptosis. Taken together, our study offers a novel approach for drug discovery and efficacy assessment by using the PDOs model. These findings suggested that JX24120 may be a potent MAT2B inhibitor and will hopefully serve as a prospective compound for endometrial cancer therapy.

AB091. Establishment and application of brainstem glioma organoids.

BACKGROUND: Traditional preclinical experiments on brainstem gliomas mainly rely on patient-derived primary cell lines, but there are problems such as low success rate in establishment and inability to preserve tumor heterogeneity, which limit the clinical transformation. As a new type of in vitro tumor model, organoids have similar structure and function to the original tumor, requiring less tissue for cultivation, with short cycle and high success rate, which is particularly suitable for brainstem glioma biopsy. There is currently no precedent for the successful construction of brainstem glioma organoid models. This new established organoid provides us a more robust preclinical tool for comprehending the pathogenesis and conducting drug screening for this kind of disease. METHODS: Cultivate patient-derived brainstem glioma organoids in vitro, verify the genetic fidelity and consistency of the organoids through morphological experiments as well as sequencing technology. Then explore the evolutionary direction of multiple types of brainstem gliomas through pseudo-time series analysis. Complete drug screening, natural killer (NK) cell co-culture, oncolytic virus therapy, and other treatments based on organoids in vitro, and evaluate the efficacy. Complete co-culture of organoids and Institute of Cancer Research (ICR) mouse brain slices in vitro. Establish patient-derived organoid xenograft (PDOX) mouse models derived from organoids in vivo. RESULTS: The establishment of organoids of all types of brainstem gliomas was completed for the first time in the world, with a total of 41/48 organoid models derived from patients, with a success rate of 85.4%, covering all segments and pathological types. The results of morphological experiments and sequencing showed that the genetic characteristics of organoids were highly consistent with those of tumor tissues. Drug screening tests for temozolomide and panobinostat were completed in vitro, and NK cell co-culture and oncolytic virus therapy testing were achieved. Co-culture of brainstem glioma organoids and mouse brain slices was achieved in vitro. Furthermore, a PDOX model of brainstem glioma was established. CONCLUSIONS: Brainstem glioma organoids can be established maturely, stably, and reliably, and can be used for preclinical drug testing for patients. Animal models derived from brainstem glioma organoids have broad preclinical experimental value.

Harnessing the power of artificial intelligence for human living organoid research.

As a powerful paradigm, artificial intelligence (AI) is rapidly impacting every aspect of our day-to-day life and scientific research through interdisciplinary transformations. Living human organoids (LOs) have a great potential for in vitro reshaping many aspects of in vivo true human organs, including organ development, disease occurrence, and drug responses. To date, AI has driven the revolutionary advances of human organoids in life science, precision medicine and pharmaceutical science in an unprecedented way. Herein, we provide a forward-looking review, the frontiers of LOs, covering the engineered construction strategies and multidisciplinary technologies for developing LOs, highlighting the cutting-edge achievements and the prospective applications of AI in LOs, particularly in biological study, disease occurrence, disease diagnosis and prediction and drug screening in preclinical assay. Moreover, we shed light on the new research trends harnessing the power of AI for LO research in the context of multidisciplinary technologies. The aim of this paper is to motivate researchers to explore organ function throughout the human life cycle, narrow the gap between in vitro microphysiological models and the real human body, accurately predict human-related responses to external stimuli (cues and drugs), accelerate the preclinical-to-clinical transformation, and ultimately enhance the health and well-being of patients.

A comprehensive review of electrophysiological techniques in amyotrophic lateral sclerosis research.

Amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease, is characterized by progressive motor neuron degeneration, leading to widespread weakness and respiratory failure. While a variety of mechanisms have been proposed as causes of this disease, a full understanding remains elusive. Electrophysiological alterations, including increased motor axon excitability, likely play an important role in disease progression. There remains a critical need for non-animal disease models that can integrate electrophysiological tools to better understand underlying mechanisms, track disease progression, and evaluate potential therapeutic interventions. This review explores the integration of electrophysiological technologies with ALS disease models. It covers cellular and clinical electrophysiological tools and their applications in ALS research. Additionally, we examine conventional animal models and highlight advancements in humanized models and 3D organoid technologies. By bridging the gap between these models, we aim to enhance our understanding of ALS pathogenesis and facilitate the development of new therapeutic strategies.

Constructing condylar cartilage organoid to explore primary cilia functions.

An organoid culture system better recapitulates the cellular structure, function, and interaction between cells and the extracellular matrix (ECM) than a two-dimensional (2D) culture system. We here constructed a condylar cartilage organoid to explore the regulatory role of primary cilia. Similar to the natural condylar cartilage, the condylar cartilage organoid exhibited abundant ECM and comprised superficial, proliferative, and hypertrophic zones. Primary cilia in the condylar cartilage organoid were shorter on average than those in the 2D culture chondrocytes, but their average length was equivalent to those in the natural condylar cartilage. Notably, primary cilia in each zone of the condylar cartilage organoid had an average length similar to that of primary cilia in the natural condylar cartilage. According to transcriptomic and biochemical analyses, the expression of cilia-related genes and cilia-related Hedgehog (HH) signaling differed between the condylar cartilage organoid and 2D culture systems. IFT88 knockdown promoted the protein levels of COL-Ⅹ, TRPV4, and HH signaling molecules in the condylar cartilage organoid, but decreased them in the 2D culture system. Notably, the protein levels of COL-Ⅹ, TRPV4, and HH signaling molecules increased in the superficial zone of the si IFT88 condylar cartilage organoid compared with the condylar cartilage organoid. However, the protein levels of aforementioned molecules were not significantly different in proliferative and hypertrophic zones. Collectively, we successfully constructed the condylar cartilage organoid with a better tissue structure and abundant ECM. Moreover, the condylar cartilage organoid is more suitable for exploring primary cilia functions.

3-D bioprinted human-derived skin organoids accelerate full-thickness skin defects repair.

The healing of large skin defects remains a significant challenge in clinical settings. The lack of epidermal sources, such as autologous skin grafting, limits full-thickness skin defect repair and leads to excessive scar formation. Skin organoids have the potential to generate a complete skin layer, supporting in-situ skin regeneration in the defect area. In this study, skin organoid spheres, created with human keratinocytes, fibroblasts, and endothelial cells, showed a specific structure with a stromal core surrounded by surface keratinocytes. We selected an appropriate bioink and innovatively combined an extrusion-based bioprinting technique with dual-photo source cross-linking technology to ensure the overall mechanical properties of the 3D bioprinted skin organoid. Moreover, the 3D bioprinted skin organoid was customized to match the size and shape of the wound site, facilitating convenient implantation. When applied to full-thickness skin defects in immunodeficient mice, the 3D bioprinted human-derived skin organoid significantly accelerated wound healing through in-situ regeneration, epithelialization, vascularization, and inhibition of excessive inflammation. The combination of skin organoid and 3D bioprinting technology can overcome the limitations of current skin substitutes, offering a novel treatment strategy to address large-area skin defects.

Viscoelasticity of Hyaluronic Acid Hydrogels Regulates Human Pluripotent Stem Cell-derived Spinal Cord Organoid Patterning and Vascularization.

Recently, it has been recognized that natural extracellular matrix (ECM) and tissues are viscoelastic, while only elastic properties have been investigated in the past. How the viscoelastic matrix regulates stem cell patterning is critical for cell-ECM mechano-transduction. Here, this study fabricated different methacrylated hyaluronic acid (HA) hydrogels using covalent cross-linking, consisting of two gels with similar elasticity (stiffness) but different viscoelasticity, and two gels with similar viscoelasticity but different elasticity (stiffness). Meanwhile, a second set of dual network hydrogels are fabricated containing both covalent and coordinated cross-links. Human spinal cord organoid (hSCO) patterning in HA hydrogels and co-culture with isogenic human blood vessel organoids (hBVOs) are investigated. The viscoelastic hydrogels promote regional hSCO patterning compared to the elastic hydrogels. More viscoelastic hydrogels can promote dorsal marker expression, while softer hydrogels result in higher interneuron marker expression. The effects of viscoelastic properties of the hydrogels become more dominant than the stiffness effects in the co-culture of hSCOs and hBVOs. In addition, more viscoelastic hydrogels can lead to more Yes-associated protein nuclear translocation, revealing the mechanism of cell-ECM mechano-transduction. This research provides insights into viscoelastic behaviors of the hydrogels during human organoid patterning with ECM-mimicking in vitro microenvironments for applications in regenerative medicine.

Protective Effect of Rosavin Against Intestinal Epithelial Injury in Colitis Mice and Intestinal Organoids.

Introduction: Rhodiola species have been utilized as functional foods in Asia and Europe for promoting health. Research has demonstrated that Rhodiola has the potential to alleviate inflammatory bowel disease (IBD) in animal models. However, the specific active components and the underlying mechanism for ameliorating intestinal damage remain unclear. This study aims to explore the relieving effect of Rosavin (Rov), a known active constituent of Rhodiola, in IBD and the regulatory mechanisms. Methods: The therapeutic effect of Rov was evaluated using a murine model of acute colitis induced by dextran sulfate sodium salt (DSS). Inflammatory cytokines and neutrophil activation markers were measured by corresponding kits. Immunohistochemistry, immunofluorescence, TUNEL, and EdU assays were applied to investigate the tight conjunction proteins expression, epithelial marker expression, number of apoptotic cells, and epithelial proliferation, respectively. The protection effect of Rov on gut epithelial injury was assessed using TNF-α-induced intestinal organoids. Additinally, RNA sequencing was applied to observe the genetic alteration profile in these intestinal organoids. Results: Oral administration of Rov significantly attenuated weight loss and restored colon length in mice. Notably, Rov treatment led to decreased levels of pro-inflammatory cytokines and neutrophil activation markers while increasing anti-inflammatory factors. Importantly, Rov restored intestinal despair by increasing the number of Lgr5+ stem cells, Lyz1+ Paneth cells and Muc2+ goblet cells in intestines of colitis mice, displaying reduced epithelial apoptosis and recovered barrier function. In TNF-α-induced intestinal organoids, Rov facilitated epithelial cell differentiation and protected against TNF-α-induced damage. RNA sequencing revealed upregulation in the gene expression associated with epithelial cells (including Lgr5+, Lyz1+ and Muc2+ cells) proliferation and defensin secretion, unveiling the protective mechanisms of Rov on the intestinal epithelial barrier. Discussion: Rov holds potential as a natural prophylactic agent against IBD, with its protective action on the intestinal epithelium being crucial for its therapeutic efficacy.

Limosilactobacillus fermentum SLAM 216- Derived Extracellular Vesicles Promote Intestinal Maturation in Mouse Organoid Models.

Probiotics, when consumed in adequate amounts, can promote the health of the host and beneficially modulate the host's immunity. Particularly during the host's early life, the gut intestine undergoes a period of epithelial maturation in which epithelial cells organize into specific crypt and villus structures. This process can be mediated by the gut microbiota. Recent studies have reported that the administration of probiotics can further promote intestinal maturation in the neonatal intestine. Therefore, in this study, we investigated the effects of extracellular vesicles derived from the Limosilactobacillus fermentum SLAM 216 strain, which is an established probiotic with known immune and anti-aging effects on intestinal epithelial maturation and homeostasis, using mouse small intestinal organoids. As per our findings, treatment with L. fermentum SLAM 216-derived LF216EV (LF216EV) has significantly increased the bud number and size of organoid buds. Furthermore, extracellular vesicle (EV) treatment upregulated the expression of maturation-related genes, including Ascl2, Ephb2, Lgr5, and Sox9. Tight junctions are known to have an important role in the intestinal immune barrier, and EV treatment has significantly increased the expression of genes associated with tight junctions, such as Claudin, Muc2, Occludin, and Zo-1, indicating that it can promote intestinal development. This was supported by RNA sequencing, which revealed the upregulation of genes associated with cAMP-mediated signaling, which is known to regulate cellular processes including cell differentiation. Additionally, organoids exposed to LF216EV exhibited upregulation of genes associated with maintaining brain memory and neurotransmission, suggesting possible future functional implications.

Prostatic lineage differentiation from human embryonic stem cells through inducible expression of NKX3-1.

BACKGROUND: Understanding the lineage differentiation of human prostate not only is crucial for basic research on human developmental biology but also significantly contributes to the management of prostate-related disorders. Current knowledge mainly relies on studies on rodent models, lacking human-derived alternatives despite clinical samples may provide a snapshot at certain stage. Human embryonic stem cells can generate all the embryonic lineages including the prostate, and indeed a few studies demonstrate such possibility based on co-culture or co-transplantation with urogenital mesenchyme into mouse renal capsule. METHODS: To establish a stepwise protocol to obtain prostatic organoids in vitro from human embryonic stem cells, we apply chemicals and growth factors by mimicking the regulation network of transcription factors and signal transduction pathways, and construct cell lines carrying an inducible NKX3-1 expressing cassette, together with three-dimensional culture system. Unpaired t test was applied for statistical analyses. RESULTS: We first successfully generate the definitive endoderm, hindgut, and urogenital sinus cells. The embryonic stem cell-derived urogenital sinus cells express prostatic key transcription factors AR and FOXA1, but fail to express NKX3-1. Therefore, we construct NKX3-1-inducible cell line by homologous recombination, which is eventually able to yield AR, FOXA1, and NKX3-1 triple-positive urogenital prostatic lineage cells through stepwise differentiation. Finally, combined with 3D culture we successfully derive prostate-like organoids with certain structures and prostatic cell populations. CONCLUSIONS: This study reveals the crucial role of NKX3-1 in prostatic differentiation and offers the inducible NKX3-1 cell line, as well as provides a stepwise differentiation protocol to generate human prostate-like organoids, which should facilitate the studies on prostate development and disease pathogenesis.

The WFS1-ZnT3-Zn2+ Axis Regulates the Vicious Cycle of Obesity and Depression.

Obesity, a growing global health concern, is closely linked to depression. However, the neural mechanism of association between obesity and depression remains poorly understood. In this study, neural-specific WFS1 deficiency exacerbates the vicious cycle of obesity and depression in mice fed a high-fat diet (HFD), positioning WFS1 as a crucial factor in this cycle. Through human pluripotent stem cells (hESCs) neural differentiation, it is demonstrated that WFS1 regulates Zn2+ homeostasis and the apoptosis of neural progenitor cells (NPCs) and cerebral organoids by inhibiting the zinc transporter ZnT3 under the situation of dysregulated lipid metabolism. Notably, riluzole regulates ZnT3 expression to maintain zinc homeostasis and protect NPCs from lipotoxicity-induced cell death. Importantly, riluzole, a therapeutic molecule targeting the nervous system, in vivo administration prevents HFD-induced obesity and associated depression. Thus, a WFS1-ZnT3-Zn2+ axis critical is demonstrated for the vicious cycle of obesity and depression and that riluzole may have the potential to reverse this process against obesity and depression.

Establishment and long-term expansion of adult hepatobiliary organoids co-cultured with liver endothelial cells.

The liver has a unique ability to regenerate in response to injury or disease with hepatocytes and biliary epithelial cells (BECs) driving the regenerative response. Liver progenitor cells (LPCs) also play role in regeneration with the ability to differentiate into either hepatocytes or BECs. However, during chronic liver disease, the regenerative capacity of the liver is impaired. The use of LPCs is a promising therapeutic strategy for patients with chronic liver diseases. LPCs can be expanded in vitro as self-renewing organoids, however, most approaches to LPC organoids do not include critical cells from the LPC niche in 3D organoid cultures. In this study, we highlight the role of liver endothelial cells (LiECs), as a part of LPC niche, in supporting the hepatobiliary organoids in long-term culture even in the absence of defined growth supplements, such as Wnt agonists. Furthermore, LiECs alter the gene expression profile of hepatobiliary organoids involved in inflammation, migration, extracellular matrix organization, and receptor signaling pathway through paracrine manner. Our findings expand the role of LiECs for regulating stemness of LPCs and elucidate a role for niche cells in a LPC organoid co-culture model with a reduction in growth supplements.

Identification of TPI1 As a potential therapeutic target in pancreatic cancer with dependency of TP53 mutation using multi-omics analysis.

Mutations of KRAS, CDKN2A, TP53, and SMAD4 are the four major driver genes for pancreatic ductal adenocarcinoma (PDAC), of which mutations of KRAS and TP53 are the most frequently recognized. However, molecular-targeted therapies for mutations of KRAS and TP53 have not yet been developed. To identify novel molecular targets, we newly established organoids with the Kras mutation (KrasmuOR) and Trp53 loss of function using Cre transduction and CRISPR/Cas9 (Krasmu/p53muOR) from murine epithelia of the pancreatic duct in KrasLSL-G12D mice, and then analyzed the proteomic and metabolomic profiles in both organoids by mass spectrometry. Hyperfunction of the glycolysis pathway was recognized in Krasmu/p53muOR compared with KrasmuOR. Loss of function of triosephosphate isomerase (TPI1), which is involved in glycolysis, induced a reduction of cell proliferation in human PDAC cell lines with the TP53 mutation, but not in PDAC or in human fibroblasts without TP53 mutation. The TP53 mutation is clinically recognized in 70% of patients with PDAC. In the present study, protein expression of TPI1 and nuclear accumulation of p53 were recognized in the same patients with PDAC. TPI1 is a potential candidate therapeutic target for PDAC with the TP53 mutation.