Enhanced Efficacy of Simultaneous PD-1 and PD-L1 Immune Checkpoint Blockade in High-Grade Serous Ovarian Cancer.
Cancer research
Immune therapies have had limited efficacy in high-grade serous ovarian cancer (HGSC), as the cellular targets and mechanism(s) of action of these agents in HGSC are unknown. Here we performed immune functional and single-cell RNA sequencing transcriptional profiling on novel HGSC organoid/immune cell co-cultures treated with a unique bispecific anti-programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) antibody compared with monospecific anti-PD-1 or anti-PD-L1 controls. Comparing the functions of these agents across all immune cell types in real time identified key immune checkpoint blockade (ICB) targets that have eluded currently available monospecific therapies. The bispecific antibody induced superior cellular state changes in both T and natural killer (NK) cells. It uniquely induced NK cells to transition from inert to more active and cytotoxic phenotypes, implicating NK cells as a key missing component of the current ICB-induced immune response in HGSC. It also induced a subset of CD8 T cells to transition from naïve to more active and cytotoxic progenitor-exhausted phenotypes post-treatment, revealing the small, previously uncharacterized population of CD8 T cells responding to ICB in HGSC. These state changes were driven partially through bispecific antibody-induced downregulation of the bromodomain-containing protein BRD1. Small-molecule inhibition of BRD1 induced similar state changes and demonstrated efficacy , validating the co-culture results. Our results demonstrate that state changes in both NK and a subset of T cells may be critical in inducing an effective anti-tumor immune response and suggest that immune therapies able to induce such cellular state changes, such as BRD1 inhibitors, may have increased efficacy in HGSC. SIGNIFICANCE: This study indicates that increased efficacy of immune therapies in ovarian cancer is driven by state changes of NK and small subsets of CD8 T cells into active and cytotoxic states.
10.1158/0008-5472.CAN-20-1674
Delivery of a BET protein degrader via a CEACAM6-targeted antibody-drug conjugate inhibits tumour growth in pancreatic cancer models.
Nature communications
Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all cancers. To improve PDAC therapy, we establish screening systems based on organoid and co-culture technologies and find a payload of antibody-drug conjugate (ADC), a bromodomain and extra-terminal (BET) protein degrader named EBET. We select CEACAM6/CD66c as an ADC target and developed an antibody, #84.7, with minimal reactivity to CEACAM6-expressing normal cells. EBET-conjugated #84.7 (84-EBET) has lethal effects on various PDAC organoids and bystander efficacy on CEACAM6-negative PDAC cells and cancer-associated fibroblasts. In mouse studies, a single injection of 84-EBET induces marked tumor regression in various PDAC-patient-derived xenografts, with a decrease in the inflammatory phenotype of stromal cells and without significant body weight loss. Combination with standard chemotherapy or PD-1 antibody induces more profound and sustained regression without toxicity enhancement. Our preclinical evidence demonstrates potential efficacy by delivering BET protein degrader to PDAC and its microenvironment via CEACAM6-targeted ADC.
10.1038/s41467-024-46167-1
H. pylori-induced NF-κB-PIEZO1-YAP1-CTGF axis drives gastric cancer progression and cancer-associated fibroblast-mediated tumour microenvironment remodelling.
Clinical and translational medicine
BACKGROUND:Gastric cancer (GC) is one of the most common tumours in East Asia countries and is associated with Helicobacter pylori infection. H. pylori utilizes virulence factors, CagA and VacA, to up-regulate pro-inflammatory cytokines and activate NF-κB signaling. Meanwhile, the PIEZO1 upregulation and cancer-associated fibroblast (CAF) enrichment were found in GC progression. However, the mechanisms of PIEZO1 upregulation and its involvement in GC progression have not been fully elucidated. METHODS:The CAF enrichment and clinical significance were investigated in animal models and primary samples. The expression of NF-κB and PIEZO1 in GC was confirmed by immunohistochemistry staining, and expression correlation was analysed in multiple GC datasets. GSEA and Western blot analysis revealed the YAP1-CTGF axis regulation by PIEZO1. The stimulatory effects of CTGF on CAFs were validated by the co-culture system and animal studies. Patient-derived organoid and peritoneal dissemination models were employed to confirm the role of the PIEZO1-YAP1-CTGF cascade in GC. RESULTS:Both CAF signature and PIEZO1 were positively correlated with H. pylori infection. PIEZO1, a mechanosensor, was confirmed as a direct downstream of NF-κB to promote the transformation from intestinal metaplasia to GC. Mechanistic studies revealed that PIEZO1 transduced the oncogenic signal from NF-κB into YAP1 signaling, a well-documented oncogenic pathway in GC progression. PIEZO1 expression was positively correlated with the YAP1 signature (CTGF, CYR61, and c-Myc, etc.) in primary samples. The secreted CTGF by cancer cells stimulated the CAF infiltration to form a stiffened collagen-enrichment microenvironment, thus activating PIEZO1 to form a positive feedback loop. Both PIEZO1 depletion by shRNA and CTGF inhibition by Procyanidin C1 enhanced the efficacy of 5-FU in suppressing the GC cell peritoneal metastasis. CONCLUSION:This study elucidates a novel driving PIEZO1-YAP1-CTGF force, which opens a novel therapeutic avenue to block the transformation from precancerous lesions to GC. H. pylori-NF-κB activates the PIEZO1-YAP1-CTGF axis to remodel the GC microenvironment by promoting CAF infiltration. Targeting PIEZO1-YAP1-CTGF plus chemotherapy might serve as a potential therapeutic option to block GC progression and peritoneal metastasis.
10.1002/ctm2.1481
NUDT1 promotes the accumulation and longevity of CD103 T cells in primary biliary cholangitis.
Journal of hepatology
BACKGROUND & AIMS:Pyruvate dehydrogenase (PDC)-E2 specific CD8 T cells play a leading role in biliary destruction in PBC. However, there are limited data on the characterization of these autoantigen-specific CD8 T cells, particularly in the liver. Herein, we aimed to identify pathogenic intrahepatic CD8 T-cell subpopulations and investigate their immunobiology in PBC. METHODS:Phenotypic and functional analysis of intrahepatic T-cell subsets were performed by flow cytometry. CD103 T cell frequency was evaluated by histological staining. The transcriptome and metabolome were analyzed by RNA-seq and liquid chromatography-mass spectrometry, respectively. Cytotoxicity of T cells against cholangiocytes was assayed in a 3D organoid co-culture system. Moreover, the longevity (long-term survival) of T cells in vivo was studied by 2-octynoic acid-BSA (2OA-BSA) immunization, Nudt1 conditional knock-out and adoptive co-transfer in a murine model. RESULTS:Intrahepatic CD103 T (CD69CD103CD8) cells were significantly expanded, hyperactivated, and potentially specifically reactive to PDC-E2 in patients with PBC. CD103 T cell frequencies correlated with clinical and histological indices of PBC and predicted poor ursodeoxycholic acid response. NUDT1 blockade suppressed the cytotoxic effector functions of CD103 T cells upon PDC-E2 re-stimulation. NUDT1 overexpression in CD8 T cells promoted tissue-residence programming in vitro; inhibition or knockdown of NUDT1 had the opposite effect. Pharmacological blockade or genetic deletion of NUDT1 eliminated CD103 T cells and alleviated cholangitis in mice immunized with 2OA-BSA. Significantly, NUDT1-dependent DNA damage resistance potentiates CD8 T-cell tissue-residency via the PARP1-TGFβR axis in vitro. Consistently, PARP1 inhibition restored NUDT1-deficient CD103 T cell durable survival and TGFβ-Smad signaling. CONCLUSIONS:CD103 T cells are the dominant population of PDC-E2-specific CD8 T lymphocytes in the livers of patients with PBC. The role of NUDT1 in promoting pathogenic CD103 T cell accumulation and longevity represents a novel therapeutic target in PBC. LAY SUMMARY:Primary biliary cholangitis (PBC) is a rare inflammatory condition of the bile ducts. It can be treated with ursodeoxycholic acid, but a large percentage of patients respond poorly to this treatment. Liver-infiltrating memory CD8 T cells recognizing the PDC-E2 immunodominant epitope are critical in the pathogenesis of PBC. We identifed the key pathogenic CD8 T cell subset, and worked out the mechanisms of its hyperactivation and longevity, which could be exploited therapeutically.
10.1016/j.jhep.2022.06.014
Natural Hydrogels Support Kidney Organoid Generation and Promote In Vitro Angiogenesis.
Advanced materials (Deerfield Beach, Fla.)
To date, strategies aiming to modulate cell to extracellular matrix (ECM) interactions during organoid derivation remain largely unexplored. Here renal decellularized ECM (dECM) hydrogels are fabricated from porcine and human renal cortex as biomaterials to enrich cell-to-ECM crosstalk during the onset of kidney organoid differentiation from human pluripotent stem cells (hPSCs). Renal dECM-derived hydrogels are used in combination with hPSC-derived renal progenitor cells to define new approaches for 2D and 3D kidney organoid differentiation, demonstrating that in the presence of these biomaterials the resulting kidney organoids exhibit renal differentiation features and the formation of an endogenous vascular component. Based on these observations, a new method to produce kidney organoids with vascular-like structures is achieved through the assembly of hPSC-derived endothelial-like organoids with kidney organoids in 3D. Major readouts of kidney differentiation and renal cell morphology are assessed exploiting these culture platforms as new models of nephrogenesis. Overall, this work shows that exploiting cell-to-ECM interactions during the onset of kidney differentiation from hPSCs facilitates and optimizes current approaches for kidney organoid derivation thereby increasing the utility of these unique cell culture platforms for personalized medicine.
10.1002/adma.202400306
Emerging enterococcus pore-forming toxins with MHC/HLA-I as receptors.
Cell
Enterococci are a part of human microbiota and a leading cause of multidrug resistant infections. Here, we identify a family of Enterococcus pore-forming toxins (Epxs) in E. faecalis, E. faecium, and E. hirae strains isolated across the globe. Structural studies reveal that Epxs form a branch of β-barrel pore-forming toxins with a β-barrel protrusion (designated the top domain) sitting atop the cap domain. Through a genome-wide CRISPR-Cas9 screen, we identify human leukocyte antigen class I (HLA-I) complex as a receptor for two members (Epx2 and Epx3), which preferentially recognize human HLA-I and homologous MHC-I of equine, bovine, and porcine, but not murine, origin. Interferon exposure, which stimulates MHC-I expression, sensitizes human cells and intestinal organoids to Epx2 and Epx3 toxicity. Co-culture with Epx2-harboring E. faecium damages human peripheral blood mononuclear cells and intestinal organoids, and this toxicity is neutralized by an Epx2 antibody, demonstrating the toxin-mediated virulence of Epx-carrying Enterococcus.
10.1016/j.cell.2022.02.002
Establishing mouse and human oral esophageal organoids to investigate the tumor immune response.
Disease models & mechanisms
Organoid culture systems are very powerful models that recapitulate in vivo organ development and disease pathogenesis, offering great promise in basic research, drug screening and precision medicine. However, the application of organoids derived from patients with cancer to immunotherapeutic research is a relatively untapped area. Esophageal cancer is one of the most lethal malignancies worldwide, including two major pathological subtypes: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma. ESCC shares many biological and genomic features with oral squamous cell cancers. Herein, we provide a versatile protocol for the establishment and maintenance of oral and esophageal organoid cultures derived from both murine and human samples. We describe culture conditions for organoids derived from normal tongue, esophagus and gastroesophageal junction, esophageal cancer and Barrett's esophagus. In addition, we establish an ex vivo model by co-culturing patient tumor-derived organoids and autologous CD8+ T lymphocytes to assess CD8+ T cell-mediated tumor killing. Our protocol can also be modified for organoid establishment from other squamous epithelia and carcinomas. The co-culture model can serve as a template for studies of other tumor-immune cell interactions and the efficacy of immune checkpoint blockade therapy.
10.1242/dmm.050319
Metastatic-niche labelling reveals parenchymal cells with stem features.
Nature
Direct investigation of the early cellular changes induced by metastatic cells within the surrounding tissue remains a challenge. Here we present a system in which metastatic cancer cells release a cell-penetrating fluorescent protein, which is taken up by neighbouring cells and enables spatial identification of the local metastatic cellular environment. Using this system, tissue cells with low representation in the metastatic niche can be identified and characterized within the bulk tissue. To highlight its potential, we applied this strategy to study the cellular environment of metastatic breast cancer cells in the lung. We report the presence of cancer-associated parenchymal cells, which exhibit stem-cell-like features, expression of lung progenitor markers, multi-lineage differentiation potential and self-renewal activity. In ex vivo assays, lung epithelial cells acquire a cancer-associated parenchymal-cell-like phenotype when co-cultured with cancer cells and support their growth. These results highlight the potential of this method as a platform for new discoveries.
10.1038/s41586-019-1487-6
Co-Culture System of Human Enteroids/Colonoids with Innate Immune Cells.
Staab Janet F,Lemme-Dumit Jose M,Latanich Rachel,Pasetti Marcella F,Zachos Nicholas C
Current protocols in immunology
Human intestinal enteroids derived from adult stem cells offer a relevant ex vivo system to study biological processes of the human gut. They recreate cellular and functional features of the intestinal epithelium of the small intestine (enteroids) or colon (colonoids) albeit limited by the lack of associated cell types that help maintain tissue homeostasis and respond to external challenges. In the gut, innate immune cells interact with the epithelium, support barrier function, and deploy effector functions. We have established a co-culture system of enteroid/colonoid monolayers and underlying macrophages and polymorphonuclear neutrophils to recapitulate the cellular framework of the human intestinal epithelial niche. Enteroids are generated from biopsies or resected tissue from any segment of the human gut and maintained in long-term cultures as three-dimensional structures through supplementation of stem cell growth factors. Immune cells are isolated from fresh human whole blood or frozen peripheral blood mononuclear cells (PBMC). Monocytes from PBMC are differentiated into macrophages by cytokine stimulation prior to co-culture. The methods are divided into the two main components of the model: (1) generating enteroid/colonoid monolayers and isolating immune cells and (2) assembly of enteroid/colonoid-immune cell co-cultures with separate apical and basolateral compartments. Co-cultures containing macrophages can be maintained for 48 hr while those involving neutrophils, due to their shorter life span, remain viable for 4 hr. Enteroid-immune co-cultures enable multiple outcome measures, including transepithelial resistance, production of cytokines/chemokines, phenotypic analysis of immune cells, tissue immunofluorescence imaging, protein or mRNA expression, antigen or microbe uptake, and other cellular functions. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Seeding enteroid fragments onto Transwells for monolayer formation Alternate Protocol: Seeding enteroid fragments for monolayer formation using trituration Basic Protocol 2: Isolation of monocytes and derivation of immune cells from human peripheral blood Basic Protocol 3: Isolation of neutrophils from human peripheral blood Basic Protocol 4: Assembly of enteroid/macrophage or enteroid/neutrophil co-culture.
10.1002/cpim.113
Gut microbiota promotes stem cell differentiation through macrophage and mesenchymal niches in early postnatal development.
Immunity
Intestinal stem cell maturation and development coincide with gut microbiota exposure after birth. Here, we investigated how early life microbial exposure, and disruption of this process, impacts the intestinal stem cell niche and development. Single-cell transcriptional analysis revealed impaired stem cell differentiation into Paneth cells and macrophage specification upon antibiotic treatment in early life. Mouse genetic and organoid co-culture experiments demonstrated that a CD206 subset of intestinal macrophages secreted Wnt ligands, which maintained the mesenchymal niche cells important for Paneth cell differentiation. Antibiotics and reduced numbers of Paneth cells are associated with the deadly infant disease, necrotizing enterocolitis (NEC). We showed that colonization with Lactobacillus or transfer of CD206 macrophages promoted Paneth cell differentiation and reduced NEC severity. Together, our work defines the gut microbiota-mediated regulation of stem cell niches during early postnatal development.
10.1016/j.immuni.2022.11.003
Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.
Cancer cell
Microsatellite stable (MSS) colorectal cancers (CRCs) are often resistant to anti-programmed death-1 (PD-1) therapy. Here, we show that a CRC pathogen, Fusobacterium nucleatum (Fn), paradoxically sensitizes MSS CRC to anti-PD-1. Fecal microbiota transplantation (FMT) from patients with Fn-high MSS CRC to germ-free mice bearing MSS CRC confers sensitivity to anti-PD-1 compared to FMT from Fn-low counterparts. Single Fn administration also potentiates anti-PD-1 efficacy in murine allografts and CD34-humanized mice bearing MSS CRC. Mechanistically, we demonstrate that intratumoral Fn generates abundant butyric acid, which inhibits histone deacetylase (HDAC) 3/8 in CD8 T cells, inducing Tbx21 promoter H3K27 acetylation and expression. TBX21 transcriptionally represses PD-1, alleviating CD8 T cell exhaustion and promoting effector function. Supporting this notion, knockout of a butyric acid-producing gene in Fn abolishes its anti-PD-1 boosting effect. In patients with MSS CRC, high intratumoral Fn predicts favorable response to anti-PD-1 therapy, indicating Fn as a potential biomarker of immunotherapy response in MSS CRC.
10.1016/j.ccell.2024.08.019
Engineered bacteria detect tumor DNA.
Science (New York, N.Y.)
Synthetic biology has developed sophisticated cellular biosensors to detect and respond to human disease. However, biosensors have not yet been engineered to detect specific extracellular DNA sequences and mutations. Here, we engineered naturally competent to detect donor DNA from the genomes of colorectal cancer (CRC) cells, organoids, and tumors. We characterized the functionality of the biosensors in vitro with coculture assays and then validated them in vivo with sensor bacteria delivered to mice harboring colorectal tumors. We observed horizontal gene transfer from the tumor to the sensor bacteria in our mouse model of CRC. This cellular assay for targeted, CRISPR-discriminated horizontal gene transfer (CATCH) enables the biodetection of specific cell-free DNA.
10.1126/science.adf3974
Neuromedin U programs eosinophils to promote mucosal immunity of the small intestine.
Science (New York, N.Y.)
Eosinophils are granulocytes that play an essential role in type 2 immunity and regulate multiple homeostatic processes in the small intestine (SI). However, the signals that regulate eosinophil activity in the SI at steady state remain poorly understood. Through transcriptome profiling of eosinophils from various mouse tissues, we found that a subset of SI eosinophils expressed neuromedin U (NMU) receptor 1 (NMUR1). Fate-mapping analyses showed that NMUR1 expression in SI eosinophils was programmed by the local microenvironment and further enhanced by inflammation. Genetic perturbation and eosinophil-organoid coculture experiments revealed that NMU-mediated eosinophil activation promotes goblet cell differentiation. Thus, NMU regulates epithelial cell differentiation and barrier immunity by stimulating NMUR1-expressing eosinophils in the SI, which highlights the importance of neuroimmune-epithelial cross-talk in maintaining tissue homeostasis.
10.1126/science.ade4177
Organoids and organs-on-chips: Insights into human gut-microbe interactions.
Puschhof Jens,Pleguezuelos-Manzano Cayetano,Clevers Hans
Cell host & microbe
The important and diverse roles of the gut microbiota in human health and disease are increasingly recognized. The difficulty of inferring causation from metagenomic microbiome sequencing studies and from mouse-human interspecies differences has prompted the development of sophisticated in vitro models of human gut-microbe interactions. Here, we review recent advances in the co-culture of microbes with intestinal and colonic epithelia, comparing the rapidly developing fields of organoids and organs-on-chips with other standard models. We describe how specific individual processes by which microbes and epithelia interact can be recapitulated in vitro. Using examples of bacterial, viral, and parasitic infections, we highlight the advantages of each culture model and discuss current trends and future possibilities to build more complex co-cultures.
10.1016/j.chom.2021.04.002
Long-term expanding human airway organoids for disease modeling.
The EMBO journal
Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway organoids represent versatile models for the study of hereditary, malignant, and infectious pulmonary disease.
10.15252/embj.2018100300
A Microwell-Based Intestinal Organoid-Macrophage Co-Culture System to Study Intestinal Inflammation.
International journal of molecular sciences
The mammalian intestinal epithelium contains more immune cells than any other tissue, and this is largely because of its constant exposure to pathogens. Macrophages are crucial for maintaining intestinal homeostasis, but they also play a central role in chronic pathologies of the digestive system. We developed a versatile microwell-based intestinal organoid-macrophage co-culture system that enables us to recapitulate features of intestinal inflammation. This microwell-based platform facilitates the controlled positioning of cells in different configurations, continuous in situ monitoring of cell interactions, and high-throughput downstream applications. Using this novel system, we compared the inflammatory response when intestinal organoids were co-cultured with macrophages versus when intestinal organoids were treated with the pro-inflammatory cytokine TNF-α. Furthermore, we demonstrated that the tissue-specific response differs according to the physical distance between the organoids and the macrophages and that the intestinal organoids show an immunomodulatory competence. Our novel microwell-based intestinal organoid model incorporating acellular and cellular components of the immune system can pave the way to unravel unknown mechanisms related to intestinal homeostasis and disorders.
10.3390/ijms232315364
Human iPSC-derived glia models for the study of neuroinflammation.
Journal of neuroinflammation
Neuroinflammation is a complex biological process that plays a significant role in various brain disorders. Microglia and astrocytes are the key cell types involved in inflammatory responses in the central nervous system. Neuroinflammation results in increased levels of secreted inflammatory factors, such as cytokines, chemokines, and reactive oxygen species. To model neuroinflammation in vitro, various human induced pluripotent stem cell (iPSC)-based models have been utilized, including monocultures, transfer of conditioned media between cell types, co-culturing multiple cell types, neural organoids, and xenotransplantation of cells into the mouse brain. To induce neuroinflammatory responses in vitro, several stimuli have been established that can induce responses in either microglia, astrocytes, or both. Here, we describe and critically evaluate the different types of iPSC models that can be used to study neuroinflammation and highlight how neuroinflammation has been induced and measured in these cultures.
10.1186/s12974-023-02919-2
Macrophage-organoid co-culture model for identifying treatment strategies against macrophage-related gemcitabine resistance.
Journal of experimental & clinical cancer research : CR
BACKGROUND:Gemcitabine resistance (GR) is a significant clinical challenge in pancreatic adenocarcinoma (PAAD) treatment. Macrophages in the tumor immune-microenvironment are closely related to GR. Uncovering the macrophage-induced GR mechanism could help devise a novel strategy to improve gemcitabine treatment outcomes in PAAD. Therefore, preclinical models accurately replicating patient tumor properties are essential for cancer research and drug development. Patient-derived organoids (PDOs) represent a promising in vitro model for investigating tumor targets, accelerating drug development, and enabling personalized treatment strategies to improve patient outcomes. METHODS:To investigate the effects of macrophage stimulation on GR, co-cultures were set up using PDOs from three PAAD patients with macrophages. To identify signaling factors between macrophages and pancreatic cancer cells (PCCs), a 97-target cytokine array and the TCGA-GTEx database were utilized. The analysis revealed CCL5 and AREG as potential candidates. The role of CCL5 in inducing GR was further investigated using clinical data and tumor sections obtained from 48 PAAD patients over three years, inhibitors, and short hairpin RNA (shRNA). Furthermore, single-cell sequencing data from the GEO database were analyzed to explore the crosstalk between PCCs and macrophages. To overcome GR, inhibitors targeting the macrophage-CCL5-Sp1-AREG feedback loop were evaluated in cell lines, PDOs, and orthotopic mouse models of pancreatic carcinoma. RESULTS:The macrophage-CCL5-Sp1-AREG feedback loop between macrophages and PCCs is responsible for GR. Macrophage-derived CCL5 activates the CCR5/AKT/Sp1/CD44 axis to confer stemness and chemoresistance to PCCs. PCC-derived AREG promotes CCL5 secretion in macrophages through the Hippo-YAP pathway. By targeting the feedback loop, mithramycin improves the outcome of gemcitabine treatment in PAAD. The results from the PDO model were corroborated with cell lines, mouse models, and clinical data. CONCLUSIONS:Our study highlights that the PDO model is a superior choice for preclinical research and precision medicine. The macrophage-CCL5-Sp1-AREG feedback loop confers stemness to PCCs to facilitate gemcitabine resistance by activating the CCR5/AKT/SP1/CD44 pathway. The combination of gemcitabine and mithramycin shows potential as a therapeutic strategy for treating PAAD in cell lines, PDOs, and mouse models.
10.1186/s13046-023-02756-4