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KMT2D, a H3K4me1 methyltransferase primarily regulating enhancers, is a leading cause of KABUKI syndrome. This multisystem disorder leads to craniofacial and cognitive abnormalities, possibly through neural crest and neuronal lineages. However, the impacted cell-of-origin and molecular mechanism of KMT2D during the development of KABUKI disease remains unknown. Here we have optimized a brain organoid model to investigate neural crest and neuronal differentiation. To pinpoint KMT2D's enhancer target, we developed a genome-wide cis-regulatory element explorer (GREE) based on single-cell multiomic integration. Single cell RNA-seq revealed that KMT2D-knockout (KO) and patient-derived organoids exhibited neural crest deformities and GABAergic overproduction. Mechanistically, GREE identified that KMT2D targets a roof-plate-like niche cell and activates the niche cell-specific WNT3A enhancer, providing the microenvironment for neural crest and neuronal development. Interestingly, KMT2D-mutated mice displayed decreased WNT3A expression in the diencephalon roof plate, indicating impaired niche cell function. Deleting the WNT3A enhancer in the organoids presented phenotypic similarities to KMT2D-depletion, emphasizing the WNT3A enhancer as the predominant target of KMT2D. Conversely, reactivating WNT signaling in KMT2D-KO rescued the lineage defects by restoring the microenvironment. Overall, our discovery of KMT2D's primary target provides insights for reconciling complex phenotypes of KABUKI syndrome and establishes a new paradigm for dissecting the mechanisms of genetic disorders from genotype to phenotype., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Drug resistance is always a challenge in cancer treatment, whether for chemotherapy, targeting, or immunotherapy. Although tumor cell lines are derived from cancer patients, they gradually lost the original characteristics, including heterogeneity and tumor microenvironment (TME), during the long period of in vitro culturing. Therefore, it is urgent to use patient-derived tumor models instead of cancer cell lines to study tumor drug resistance. Herein, we developed a microarray device that serves as a platform for high-throughput and three-dimensional culture of breast cancer patient-derived organoids (BCOs) and investigated their resistance to adriamycin (ADM). Coupled with fluorescence microscopy, this system enabled on-chip drug response monitoring and cell viability assessment without the consumption of a large number of tumor cells. The organoids were divided into a resistant BCO group (RBCO) and a sensitive BCO group (SBCO) according to their half-inhibitory concentration (IC 50 ). Different from cancer cell lines, BCOs demonstrated obvious heterogeneity in drug treatment. Ivermectin (IVM), a broad-spectrum antiparasitic agent approved by the Food and Drug Administration (FDA), was observed to synergistically augment ADM-induced cytotoxicity in organoids. The BCO chip provides a promising platform for investigation of drug resistance and preclinical drug screening based on clinical samples.

Psoriasis is a prevalent, inflammatory skin disease without cure. Further research is required to unravel dysregulated processes and develop new therapeutic interventions. The lack of suitable in vivo and in vitro preclinical models is an impediment in the psoriasis research. Recently, the development of 3D skin models has progressed including replicas with disease-like features. To investigate the use of in vitro models as preclinical test tools, the study focused on treatment responses of 3D skin replicas. Cytokine-priming of skin organoids induced psoriatic features like inflammation, antimicrobial peptides (AMP), hyperproliferation and impaired differentiation. Topical application of dexamethasone (DEX) or celastrol (CEL), a natural anti-inflammatory compound reduced the secretion of pro-inflammatory cytokines. DEX and CEL decreased the gene expression of inflammatory mediators. DEX barely affected the psoriatic AMP transcription but CEL downregulated psoriasis-driven AMP genes. Subcutaneous application of adalimumab (ADM) or bimekizumab (BMM) showed anti-psoriatic effects via protein induction of the differentiation marker keratin-10. Dual blockage of TNF-α and IL-17A repressed the inflammatory psoriasis phenotype. BMM inhibited the psoriatic expression of AMP genes and induced KRT10 and cell-cell contact genes. The present in vitro model provides a 3D environment with in vivo-like cutaneous responses and represents a promising tool for preclinical investigations., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Heterozygosity for inverted formin-2 (INF2) mutations causes focal segmental glomerulosclerosis (FSGS) with or without Charcot-Marie-Tooth disease. A key question is whether the disease is caused by gain-of-function effects on INF2 or loss of function (haploinsufficiency). Despite established roles in multiple cellular processes, neither INF2 knockout mice nor mice with a disease-associated point mutation display an evident kidney or neurologic phenotype. Here, we compared responses to puromycin aminonucleoside (PAN)-induced kidney injury between INF2 R218Q and INF2 knockout mice. R218Q INF2 mice are susceptible to glomerular disease, in contrast to INF2 knockout mice. Colocalization, coimmunoprecipitation analyses, and cellular actin measurements showed that INF2 R218Q confers a gain-of-function effect on the actin cytoskeleton. RNA expression analysis showed that adhesion and mitochondria-related pathways were enriched in the PAN-treated R218Q mice. Both podocytes from INF2 R218Q mice and human kidney organoids with an INF2 mutation (S186P) recapitulate adhesion and mitochondrial phenotypes. Thus, gain-of-function mechanisms drive INF2-related FSGS and explain this disease's autosomal dominant inheritance.

The rational installation of pharmacophores targeting HSP90 and LSD1 axes has achieved significant anti-cancer capacity in prostate and colorectal cancer. Among the series of hybrids, inhibitor 6 exhibited remarkable anti-proliferative activity against prostate cancer cell lines PC-3 and DU145, with GI 50 values of 0.24 and 0.30 μM, respectively. It demonstrated notable efficacy in combinatorial attack and cell death initiation towards apoptosis. The cell death process was mediated by PARP induction and γH2AX signaling, and was also characterized as caspase-dependent and Bcl-xL/Bax-independent. Notably, no difference in eye size or morphology was observed in the zebrafish treated with compound 6 compared to the reference group (AUY922). The profound treatment response in docetaxel-resistant PC-3 cells highlighted the dual inhibitory ability in improving docetaxel sensitivity. Additionally, at a minimum concentration of 1.25 μM, compound 6 effectively inhibited the growth of patient-derived colorectal cancer (CRC) organoids for up to 10 days in vitro. Together, the designed HSP90/LSD1 inhibitors present a novel route and significant clinical value for anti-cancer drug therapy., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jing-Ping Liou reports financial support was provided by National Science and Technology, Taiwan. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

Exposure to micro- and nanoplastics (MNPs) has been implicated in potential cardiotoxicity. However, in vitro models based on cardiomyocyte cell lines lack crucial cardiac characteristics, while interspecies differences in animal models compromise the reliability of the conclusions. In addition, current research has predominantly focused on single-time point exposures to MNPs, neglecting comparative analyses of cardiac injury across early and late stages. Moreover, there remains a large gap in understanding the susceptibility to MNPs under pathological conditions. To address these limitations, this study integrated cardiac organoids (COs) and organ-on-a-chip (OoC) technology to develop the cardiac organoid-on-a-chip (COoC), which was validated for cardiotoxicity evaluation through multiple dimensions. Based on COoC, we conducted a dynamic observation of the cardiac damage caused by short- and long-term exposure to polystyrene nanoplastics (PS-NPs). Oxidative stress, inflammation, disruption of calcium ion homeostasis, and mitochondrial dysfunction were confirmed as the potential mechanisms of PS-NP-induced cardiotoxicity and the crucial events in the early stages, while cardiac fibrosis emerged as a prominent feature in late stages. Notably, low-dose exposure exacerbated myocardial infarction symptoms under pathological states, despite no significant cardiotoxicity shown in healthy models. In conclusion, these findings further deepened our understanding of PS-NP-induced cardiotoxic effects and introduced a promising in vitro platform for assessing cardiotoxicity.

Metabolic dysfunction-associated steatohepatitis (MASH) is a form of chronic liver inflammation associated with metabolic syndrome, such as obesity and a major cause of hepatocellular carcinoma (HCC). Multi-biotics, a soymilk fermented with lactic acid bacteria, are known to alleviate obesity by lowering lipid profile. This study aimed to establish and characterize mouse organoids derived from MASH-related HCC models to evaluate drug responses, particularly focusing on Lenvatinib resistance. Organoids were developed using mouse liver tissues subjected to a choline-deficient L-amino acid-defined high-fat diet (CDAHFD) to mimic MASH-related HCC. The study evaluated the effect of multi-biotics, a fermented product, on tumor regression and drug sensitivity. While multi-biotics did not reduce tumor burden, they enhanced the response to Lenvatinib. Additionally, repeated treatment with Lenvatinib led to the development of drug-resistant organoids. Transcriptomic analysis of these resistant organoids identified key pathways related to KRAS signaling, inflammation, and epithelial-mesenchymal transition (EMT), revealing potential targets for overcoming Lenvatinib resistance. This study provides valuable insights into MASH-related HCC progression and drug resistance, offering a model for further therapeutic research., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Organoids are three-dimensional (3D) cultures, normally derived from stem cells, that replicate the complex structure and function of human tissues. They offer a physiologically relevant model to address important questions in cancer research. The generation of patient-derived organoids (PDOs) from various human cancers allows for deeper insights into tumor heterogeneity and spatial organization. Additionally, interrogating non-tumor stromal cells increases the relevance in studying the tumor microenvironment, thereby enhancing the relevance of PDOs in personalized medicine. PDOs mark a significant advancement in cancer research and patient care, signifying a shift toward more innovative and patient-centric approaches. This review covers aspects of PDO cultures to address the modeling of the tumor microenvironment, including extracellular matrices, air-liquid interface and microfluidic cultures, and organ-on-chip. Specifically, the role of PDOs as preclinical models in gene editing, molecular profiling, drug testing, and biomarker discovery and their potential for guiding personalized treatment in clinical practice are discussed., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

There is a paucity of human models to study immune-mediated host damage. Here, we utilized the GeoMx spatial multi-omics platform to analyze immune cell changes in COVID-19 pancreatic autopsy samples, revealing an accumulation of proinflammatory macrophages. Single-cell RNA sequencing (scRNA-seq) analysis of human islets exposed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or coxsackievirus B4 (CVB4) viruses identified activation of proinflammatory macrophages and β cell pyroptosis. To distinguish viral versus proinflammatory-macrophage-mediated β cell pyroptosis, we developed human pluripotent stem cell (hPSC)-derived vascularized macrophage-islet (VMI) organoids. VMI organoids exhibited enhanced marker expression and function in both β cells and endothelial cells compared with separately cultured cells. Notably, proinflammatory macrophages within VMI organoids induced β cell pyroptosis. Mechanistic investigations highlighted TNFSF12-TNFRSF12A involvement in proinflammatory-macrophage-mediated β cell pyroptosis. This study established hPSC-derived VMI organoids as a valuable tool for studying immune-cell-mediated host damage and uncovered the mechanism of β cell damage during viral exposure., Competing Interests: Declaration of interests R.E.S. is on the scientific advisory board of Miromatrix Inc. and Lime Therapeutics and is a consultant and speaker for Alnylam Inc. S.C. and T.E are the co-founders of OncoBeat, LLC. S.C. is a consultant of Vesalius Therapeutics and co-founder of iOrganBio., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Tumor deposits (TDs) represent a specific form tumor metastasis observed in colorectal cancer (CRC). The lack of successfully established cell lines for TDs, as well as the molecular mechanisms by which TDs occur remain largely unknown. Here, we established paired CRC organoids, including a human primary cancer organoid and its TD organoid, from a 46-year-old male patient with CRC. Further analysis revealed that, compared with primary tumor-derived cells, TD-derived cells exhibited enhanced proliferative, invasive and metastatic capabilities, and increased expression of stemness-related proteins. Furthermore, the present findings also demonstrated that TD-derived cells were more resistant to oxaliplatin or 5-FU. Transcriptomic profiling and qPCR revealed that TD-derived cells exhibited more alterations in fatty acid metabolism signaling and enhanced lipid synthesis ability compared to primary tumor-derived cells. Inhibition of lipid synthesis markedly decreased resistance to oxaliplatin in TD-derived cells. Taken together, the paired organoids established using CRC primary tumor and its TD specimens will provide valuable tools to study tumorigenicity, metastasis and chemoresistance in CRC. Notably, these models will provide novel insights to study tumor heterogeneity and lipid metabolism in CRC., (© 2024. The Author(s).)

Patient-derived organoids provide a unique model system to explore disease-causing mutations ex vivo. By using organoids from duodenal or colonic biopsies of pediatric patients with intestinal epithelial disorders, we can directly assay the patient cells to tailor treatment to their unique disease state. The advent of organoid technology from patients with severe intestinal disorders such as Congenital Diarrhea Enteropathies (CoDE) and Very-Early-Onset Inflammatory Bowel Disease (VEO-IBD) has allowed for rapid advances in the understanding of and the treatment of these monogenic disorders. Still, the expansion of these lines for scalable studies is not trivial, and success rates of expansion are variable between groups, and even lab members within the same group. These protocols have been validated on patients with CoDE or VEO-IBD and age-matched control patients. Here, we present our recommended protocols for the cultivation of organoids from pediatric patients with CoDE and VEO-IBD. These protocols have been validated on organoids generated from the duodenum (duodenoids), ileum (ileoids), colon (colonoids) and iPSC-derived intestinal colonoids from pediatric healthy donors or donors with CoDE or VEO-IBD (Gwilt et al., 2023). Using our modified culture media, extended culture times from biopsy preparation and thawing frozen lines, gentle passaging techniques with the incomplete removal of the organoids from the matrigel, and modified monolayer protocols (Maeda et al., 2023; Maeda et al., 2022), we have been able to successfully culture and expand several lines for more than 5 years. The conditions and protocols used here provide a basis for reproducible phenotypes, scaling for larger functional studies on patient lines, and for reproducibility of results between several investigators. We provide a useful starting point and troubleshooting guidelines for the optimization of culturing organoids from any patient with novel disease pathology., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Bugda Gwilt, Thiagarajah. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Most patients with prostate adenocarcinoma develop resistance to therapies targeting the androgen receptor (AR). Consequently, a portion of these patients develop AR-independent neuroendocrine (NE) prostate cancer (NEPC), a rapidly progressing cancer with limited therapies and poor survival outcomes. Current research to understand the progression to NEPC suggests a model of lineage plasticity whereby AR-dependent luminal-like tumors progress toward an AR-independent NEPC state. Genetic analysis of human NEPC identified frequent loss of RB1 and TP53, and the loss of both genes in experimental models mediates the transition to a NE lineage. Transcriptomics studies have shown that lineage transcription factors ASCL1 and NEUROD1 are present in NEPC. In this study, we modeled the progression of prostate adenocarcinoma to NEPC by establishing prostate organoids and subsequently generating subcutaneous allograft tumors from genetically engineered mouse models harboring Cre-induced loss of Rb1 and Trp53 with Myc overexpression (RPM). These tumors were heterogeneous and displayed adenocarcinoma, squamous, and NE features. ASCL1 and NEUROD1 were expressed within NE-defined regions, with ASCL1 being predominant. Genetic loss of Ascl1 in this model did not decrease tumor incidence, growth, or metastasis; however, there was a notable decrease in NE identity and an increase in basal-like identity. This study provides an in vivo model to study progression to NEPC and establishes the requirement for ASCL1 in driving NE differentiation in prostate cancer. Significance: Modeling lineage transitions in prostate cancer and testing dependencies of lineage transcription factors have therapeutic implications, given the emergence of treatment-resistant, aggressive forms of neuroendocrine prostate cancer. See related commentary by McQuillen and Brady, p. 3499., (©2024 American Association for Cancer Research.)

Brain size and cellular heterogeneity are tightly regulated by species-specific proliferation and differentiation of multipotent neural progenitor cells (NPCs). Errors in this process are among the mechanisms of primary hereditary microcephaly (MCPH), a group of disorders characterized by reduced brain size and intellectual disability. Biallelic citron rho-interacting serine/threonine kinase (CIT) missense variants that disrupt kinase function (CITKI/KI) and frameshift loss-of-function variants (CITFS/FS) are the genetic basis for MCPH17; however, the function of CIT catalytic activity in brain development and NPC cytokinesis is unknown. Therefore, we created the CitKI/KI mouse model and found that it did not phenocopy human microcephaly, unlike biallelic CitFS/FS animals. Nevertheless, both Cit models exhibited binucleation, DNA damage, and apoptosis. To investigate human-specific mechanisms of CIT microcephaly, we generated CITKI/KI and CITFS/FS human forebrain organoids. We found that CITKI/KI and CITFS/FS organoids lost cytoarchitectural complexity, transitioning from pseudostratified to simple neuroepithelium. This change was associated with defects that disrupted the polarity of NPC cytokinesis, in addition to elevating apoptosis. Together, our results indicate that both CIT catalytic and scaffolding functions in NPC cytokinesis are critical for human corticogenesis. Species differences in corticogenesis and the dynamic 3D features of NPC mitosis underscore the utility of human forebrain organoid models for understanding human microcephaly.

Research Question: What influence does an intramural myoma have on the endometrium, and how is this mediated?, Design: Endometrium was collected from 13 patients with non-cavity-distorting intramural myomas (diameter ≤4 cm; International Federation of Gynecology and Obstetrics type 4) and 13 patients without myomas undergoing hysterectomy for benign cervical diseases with a similar clinical baseline. Endometrial organoids were established in vitro and induced to reach the secretory phase by oestrogen and progesterone. Transcriptome sequencing was conducted on endometrial organoids in both untreated and secretory stages from three individuals with myomas and three control participants. Immunofluorescence and real-time quantitative PCR (RT-qPCR) were performed on endometrial organoids from another 10 myoma patients and 10 control patients for validation., Results: The data revealed abnormally increased hormone receptor (PGR) levels in the untreated endometrial organoids with myomas, resulting in potentially abnormal glandular and vascular development. The aberrant responses to oestrogen and progestogen prompted further investigation into the secretory phase. The secretory endometrial organoids with myomas exhibited greater changes in acetyl-α-tubulin, ODF2 and TPPP, demonstrating likely decreased cilia, and COL6A1, used as a marker for increased extracellular matrix (ECM) modelling. Both untreated and secretory endometrial organoids with myoma showed an up-regulation of genes and pathways related to ECM mechanotransduction. The expression pattern of receptivity-related genes was disturbed in endometrial organoids with myoma., Conclusions: This study is the first to reveal that intramural myomas create an abnormal hormonal and mechanical environment in the untreated and secretory endometrial organoids. The intramural myomas negatively impacted gene expression relating to endometrial glands, blood vessels, cilia and ECM, indicating that intramural myomas impair endometrial decidualization and receptivity., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cancer cell clusters have a higher capacity for metastasis than single cells, suggesting cancer cell clusters have biological properties different from those of single cells. The nature of de novo cancer cell clusters that are newly formed from tumor masses is largely unknown. Herein, we generated small cell clusters from colorectal cancer organoids and tracked the growth patterns of the clusters up to four cells. Growth patterns were classified into actively growing and poorly growing spheroids (PG). Notch signaling was robustly activated in small clusters immediately after dissociation, and Notch signaling inhibition markedly increased the proportion of PG spheroids. Only a limited number of PG spheroids grew under growth-permissive conditions in vitro, but xenograft tumors derived from Notch inhibited clusters showed growth rates comparable to those of untreated spheroids. Thus, de novo clusters are composed of cells with interchangeable growth fates, which are regulated in a context-dependent manner by Notch signaling., (© 2024 The Author(s). Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

A variety of abnormal epithelial cells and immature and mature immune cells in thymic epithelial tumors (TETs) affect histopathological features, the degree of malignancy, and the response to treatment. Here, gene expression, trajectory inference, and T cell antigen receptor (TCR)-based lineage tracking are profiled in TETs at single-cell resolution. An original subpopulation of KRT14 + progenitor cells with a spindle cell phenotype is shown. An abnormal infiltration of immature T cells with a TCR hyper-rearrangement state is revealed, due to the lack of CCL21 + medullary epithelial cells. For thymic carcinoma, the novel biomarkers of MSLN, CCL20, and SLC1A5 are identified and observed an elevated expression of LAG3 and HAVCR2 in malignant tumorn-infiltrating mature T cells. These common features based on the single-cell populations may inform pathological reclassification of TETs. Meanwhile, it is found that macrophages (MACs) attract thymic tumor cells through the LGALS9-SLC1A5 axis, providing them with glutamine to elicit metabolic reprogramming. This MAC-based metabolic pattern can promote malignancy progression. Additionally, an interactive immune environment in TETs is identified that correlates with the infiltration of abnormal FOXI1 + CFTR - ionocytes. Collectively, the data broaden the knowledge of TET cellular ecosystems, providing a basis for tackling histopathological diagnosis and related treatment., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Human blood vessel organoids (hBVOs) offer a promising platform for investigating vascular diseases and identifying therapeutic targets. In this study, we focused on in vitro modeling and therapeutic target finding of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common form of hereditary stroke disorder caused by mutations in the NOTCH3 gene. Despite the identification of these mutations, the underlying pathological mechanism is elusive, and effective therapeutic approaches are lacking. CADASIL primarily affects the blood vessels in the brain, leading to ischemic strokes, migraines, and dementia. By employing CRISPR/Cas9 base-editing technology, we generated human induced pluripotent stem cells (hiPSCs) carrying Notch3 mutations. These mutant hiPSCs were differentiated into hBVOs. The NOTCH3 mutated hBVOs exhibited CADASIL-like pathology, characterized by a reduced vessel diameter and degeneration of mural cells. Furthermore, we observed an accumulation of Notch3 extracellular domain (Notch3ECD), increased apoptosis, and cytoskeletal alterations in the NOTCH3 mutant hBVOs. Notably, treatment with ROCK inhibitors partially restored the disconnection between endothelial cells and mural cells in the mutant hBVOs. These findings shed light on the pathogenesis of CADASIL and highlight the potential of hBVOs for studying and developing therapeutic interventions for this debilitating human vascular disorder., (© 2024. The Author(s).)

Serrated polyposis syndrome (SPS) presents with multiple sessile serrated lesions (SSL) in the large intestine and confers increased colorectal cancer (CRC) risk. However, the etiology of SPS is not known. SSL-derived organoids have not been previously studied but may help provide insights into SPS pathogenesis and identify novel biomarkers and chemopreventive strategies. This study examined effects of EGFR and COX pathway inhibition in organoid cultures derived from uninvolved colon and polyps of SPS patients. We also compared with organoids representing the hereditary gastrointestinal syndromes, Familial Adenomatous Polyposis (FAP) and Lynch syndrome (LS). Eighteen total organoid colon cultures were generated from uninvolved colon and polyps in SPS, FAP, LS, and non-syndromic screening colonoscopy patients. BRAF and KRAS mutation status was determined for each culture. Erlotinib (EGFR inhibitor) and sulindac (COX inhibitor) were applied individually and in combination. A 44-target gene custom mRNA panel (including WNT and COX pathway genes) and a 798-gene microRNA gene panel were used to quantitate organoid RNA expression by NanoString analysis. Erlotinib treatment significantly decreased levels of mRNAs associated with WNT and MAPK kinase signaling in organoids from uninvolved colon from all four patient categories and from all SSL and adenomatous polyps. Sulindac did not change the mRNA profile in any culture. Our findings suggest that EGFR inhibitors may contribute to the chemopreventive treatment of SSLs. These findings may also facilitate clinical trial design using these agents in SPS patients. Differentially expressed genes identified in our study (MYC, FOSL1, EGR1, IL33, LGR5 and FOXQ1) may be used to identify other new molecular targets for chemoprevention of SSLs., (© 2024. The Author(s).)

Perianal fistulizing Crohn's disease (CD) is a severe gastrointestinal disorder causing extensive mucosal damage with limited treatment options. Severe manifestations of the disease appear at higher rates in non-Europeans but the genetic and cellular mechanisms driving the disease phenotypes remain poorly understood. Herein, we tested whether pathologic determinants in the epithelial stem cell compartment could be detected at the transcript level in rectal organoids derived from a diverse patient population. Rectal organoid and mucosal cells from endoscopic biopsies of each patient having perianal fistulizing CD or no disease controls were prepared for and sequenced at the single cell level. After cell type annotations based on expressed marker genes, samples were analyzed by principal components, for differential transcript expression, cell type proportions, and pathway enrichment. After QC, we produced 77,044 rectal organoid cells (n = 13 patients; 8 CD, 5 controls) with high quality sequences that identified 10 distinct epithelial subtypes, that we compared to 141,367 mucosal epithelial cells (n = 29 patients; 18 CD, 11 controls). Consistent with mucosal epithelial cells, rectal organoids prominently displayed disease signatures represented by the stem and transit amplifying regions of the rectal crypt, including alterations in transcriptional signatures of metabolic, epigenetic, and proliferating pathways. Organoids also retained their gender- and ancestral-specific gene expression signatures. However, they lacked many of the inflammatory signatures observed in epithelial cells from diseased mucosa. Perianal CD patient derived rectal organoids reflect gene expression signatures related to disease, gender, and ancestry, suggesting they harbor inherent properties amenable to further patient-specific, disease-related experimentation., (© 2024. The Author(s).)

Pancreatic ductal adenocarcinoma (PDAC) presents challenges in detecting somatic mutations due to its complex cellular composition. This study investigated the utility of patient-derived organoids (PDOs) to overcome these obstacles and enhance somatic mutation identification. Surgically resected PDAC tumors and their paired PDOs from 21 patients were examined. Whole-exome sequencing (WES) of tumor tissue, organoids, and peripheral blood mononuclear cells was performed to identify somatic mutations. Our findings demonstrate that PDOs retained about 80% of the somatic mutations from the original tumors, showing high concordance in mutation types. PDOs exhibited increased tumor purity and uncovered key driver mutations, aiding in identifying clinically relevant genomic alterations. Moreover, eight cycles of FOLFIRINOX treatment did not significantly alter the mutational landscape at the DNA level, indicating the stability of the mutational profile after therapeutic pressure in patients. In conclusion, PDOs are potentially important tools for exploring the somatic mutational landscape of PDAC. While they can reveal mutations that may be challenging to detect through traditional biopsy sequencing due to the inherently low tumor purity of PDAC, it is important to note that PDOs may not always fully recapitulate all mutations found in primary tumors. Despite this limitation, PDOs can still offer critical insights into the genomic complexities of PDAC, which is crucial for the development of personalized vaccines and therapies for this disease., (© 2024. The Author(s).)

Despite advances in the treatment paradigm for patients with metastatic melanoma, melanoma brain metastasis (MBM) continues to represent a significant treatment challenge. The study of MBM is limited, in part, by shortcomings in existing preclinical models. Surgically eXplanted Organoids (SXOs) are ex vivo, three-dimensional cultures prepared from primary tissue samples with minimal processing that recapitulate genotypic and phenotypic features of parent tumors without an artificial extracellular scaffold. MBM SXOs were created by a novel protocol incorporating techniques for establishing glioma and cutaneous melanoma organoids. A BRAF V600K -mutant and BRAF-wildtype MBM sample were collected directly from the operating room. Organoids were cultured in an optimized culture medium without an artificial extracellular scaffold. Concurrently, matched patient-derived cell lines were created. Organoid growth was observed within 3-4 weeks, and MBM SXOs retained histological features of the parent tissue, including pleomorphic epithelioid cells with abundant cytoplasm, large nuclei, focal melanin accumulation, and strong SOX10 positivity. After sufficient growth, organoids could be manually parcellated to increase the number of replicates. Matched SXOs and cell lines demonstrated sensitivity to BRAF and MEK inhibitors. Further study using SXOs may improve the translational relevance of preclinical studies and enable the study of the metastatic melanoma tumor microenvironment., (© 2024. The Author(s).)

Hepatocellular carcinomas (HCCs) are highly heterogeneous malignancies. They are characterized by a peculiar tumor microenvironment and dense vascularization. The importance of signaling between immune cells, endothelial cells, and tumor cells leads to the difficult recapitulation of a reliable in vitro HCC model using the conventional two-dimensional cell cultures. The advent of three-dimensional organoid tumor technology has revolutionized our understanding of the pathogenesis and progression of several malignancies by faithfully replicating the original cancer genomic, epigenomic, and microenvironmental landscape. Organoids more closely mimic the in vivo environment and cell interactions, replicating factors such as the spatial organization of cell surface receptors and gene expression, and will probably become an important tool in the choice of therapies and the evaluation of tumor response to treatments. This review aimed to describe the ongoing and potential applications of organoids as an in vitro model for the study of HCC development, its interaction with the host's immunity, the analysis of drug sensitivity tests, and the current limits in this field.

Approximately 50% of patients with surgically resected early-stage lung cancer develop distant metastasis. At present, there is no in vivo metastasis model to investigate the biology of human lung cancer metastases. Using well-characterized lung adenocarcinoma (LUAD) patient-derived organoids (PDOs), we establish an in vivo metastasis model that preserves the biologic features of human metastases. Results of whole-genome and RNA sequencing establish that our in vivo PDO metastasis model can be used to study clonality and tumor evolution and to identify biomarkers related to organotropism. Investigation of the response of KRAS G12C PDOs to sotorasib demonstrates that the model can examine the efficacy of treatments to suppress metastasis and identify mechanisms of drug resistance. Finally, our PDO model cocultured with autologous peripheral blood mononuclear cells can potentially be used to determine the optimal immune-priming strategy for individual patients with LUAD., Competing Interests: Declaration of interests G.R. has financial relationships with Scanlan, AstraZeneca, and Medtronic. S.S. is a member of the AstraZeneca Advisory Board. J.M.I. has stock ownership in LumaCyte and is a consultant/advisory board member for Roche Genentech. M.J.B. is a consultant for AstraZeneca, Iovance Biotherapeutics, and Intuitive Surgical and receives research support from Obsidian Therapeutics. B.T.L. has served as an uncompensated advisor and consultant to Amgen, AstraZeneca, Boehringer Ingelheim, Bolt Biotherapeutics, Daiichi Sankyo, Genentech, and Lilly; has received research grants (institutional) from Amgen, AstraZeneca, Bolt Biotherapeutics, Daiichi Sankyo, Genentech, Hengrui USA, and Lilly; has received academic travel support from Amgen, Jiangsu Hengrui Medicine, and MORE Health; and has intellectual property rights as a book author at Karger Publishers and Shanghai Jiao Tong University Press. M.H.B. receives royalties from Globus Medical and DePuy Synthes. P.S.A. declares research funding from Atara Biotherapeutics; is a scientific advisory board member and consultant for ATARA Biotherapeutics, Bayer, Bio4T2, Carisma Therapeutics, Imugene, ImmPACT Bio, Johnson & Johnson, Orion, and Outpace Bio; has patents, royalties, and intellectual property on mesothelin-targeted chimeric antigen receptor and other T cell therapies, which have been licensed to Atara Biotherapeutics; and has an issued patent method for detection of cancer cells using virus and pending patent applications on PD-1 dominant negative receptor, a wireless pulse-oximetry device, and an ex vivo malignant pleural effusion culture system. MSK has licensed intellectual property related to mesothelin-targeted chimeric antigen receptors and T cell therapies to Atara Biotherapeutics and has associated financial interests. D.R.J. serves on a clinical trial steering committee for AstraZeneca and has research grant support from Merck., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

In idiopathic pulmonary fibrosis (IPF), epithelial abnormalities are present including bronchiolization and alveolar cell dysfunction. We hypothesized that the IPF microenvironment disrupts normal epithelial growth and differentiation. We mimicked the soluble factors within an IPF microenvironment using an IPF cocktail (IPFc), composed of nine factors which are increased in IPF lungs (CCL2, IL-1β, IL-4, IL-8, IL-13, IL-33, TGF-β, TNFα, and TSLP). Using IPFc, we asked whether the soluble factor milieu in IPF affects epithelial growth and differentiation and how IPFc compares to TGF-β alone. Epithelial growth and differentiation were studied using mouse lung organoids (primary Epcam+ epithelial cells co-cultured with CCL206 fibroblasts). Organoids exposed to IPFc and TGF-β were re-sorted into epithelial and fibroblast fractions and subjected to RNA sequencing. IPFc did not affect the number of organoids formed. However, pro-surfactant protein C expression was decreased. On these parameters, TGF-β alone had similar effects. However, RNA sequencing of re-sorted organoids revealed that IPFc and TGF-β had distinct effects on both epithelial cells and fibroblasts. IPFc upregulated goblet cell markers, whereas these were inhibited by TGF-β. Although both IPFc and TGF-β increased extracellular matrix gene expression, only TGF-β increased myofibroblast markers. VEGF-C and Wnt signaling were among the most differentially regulated signaling pathways by IPFc versus TGF-β. Interestingly, Wnt pathway activation rescued Sftpc downregulation induced by IPFc. In conclusion, IPFc alters epithelial differentiation in a way that is distinct from TGF-β. Alterations in Wnt signaling contribute to these effects. IPFc may be a more comprehensive representation of the soluble factor microenvironment in IPF., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Biallelic loss of cyclin-dependent kinase 12 (CDK12) defines a metastatic castration-resistant prostate cancer (mCRPC) subtype. It remains unclear, however, whether CDK12 loss drives prostate cancer (PCa) development or uncovers pharmacologic vulnerabilities. Here, we show Cdk12 ablation in murine prostate epithelium is sufficient to induce preneoplastic lesions with lymphocytic infiltration. In allograft-based CRISPR screening, Cdk12 loss associates positively with Trp53 inactivation but negatively with Pten inactivation. Moreover, concurrent Cdk12/Trp53 ablation promotes proliferation of prostate-derived organoids, while Cdk12 knockout in Pten-null mice abrogates prostate tumor growth. In syngeneic systems, Cdk12/Trp53-null allografts exhibit luminal morphology and immune checkpoint blockade sensitivity. Mechanistically, Cdk12 inactivation mediates genomic instability by inducing transcription-replication conflicts. Strikingly, CDK12-mutant organoids and patient-derived xenografts are sensitive to inhibition or degradation of the paralog kinase, CDK13. We therein establish CDK12 as a bona fide tumor suppressor, mechanistically define how CDK12 inactivation causes genomic instability, and advance a therapeutic strategy for CDK12-mutant mCRPC., Competing Interests: Declaration of interests A.M.C. co-founded and serves on scientific advisory boards (SABs) of Lynx Dx, Flamingo Therapeutics, Medsyn Pharma, Oncopia Therapeutics, and Esanik Therapeutics. A.M.C. is an advisor to Aurigene Oncology Limited, Proteovant, Tempus, Rappta, and Ascentage. C.J.L. received research funding from AstraZeneca, Merck KGaA, Artios, and NeoPhore and consultancy, SAB membership, or honoraria payments from FoRx, Syncona, Sun Pharma, Gerson Lehrman Group, Merck KGaA, Vertex, AstraZeneca, Tango, 3rd Rock, Ono Pharma, Artios, Abingworth, Tesselate, Dark Blue Therapeutics, Pontifax, Astex, NeoPhore, Glaxo Smith Kline, and Dawn Bioventures. C.J.L. has stock in Tango, Ovibio, Hysplex, and Tesselate. C.J.L. is named inventor on patents describing use of DNA repair inhibitors and stands to gain from their development and use. J.C. is an advisor for Exai Bio. F.Y.F. has served on SAB or received consulting fees from Astellas, Bayer, Celgene, Clovis Oncology, Janssen, Genentech Roche, Myovant, Roivant, Sanofi, and Blue Earth Diagnostics. F.Y.F. is also an SAB member for Artera, ClearNote Genomics, Serimmune, and BMS (Microenvironment Division). K.D. is an advisor for Kinoteck Therapeutics and has received financial support from Livzon Pharmaceutical Group. Patents for CDK12/13 degraders/inhibitors used here have been filed by the University of Michigan and Shanghai Institute of Organic Chemistry, with A.M.C., K.D., X.W., J.Y., Y. Chang, and J.C.T. as co-inventors., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Crohn's disease (CD) is a complex and heterogeneous condition with no perfect preclinical model or cure. To address this, we explore adult stem cell-derived organoids that retain their tissue identity and disease-driving traits. We prospectively create a biobank of CD patient-derived organoid cultures (PDOs) from colonic biopsies of 53 subjects across all clinical subtypes and healthy subjects. Gene expression analyses enabled benchmarking of PDOs as tools for modeling the colonic epithelium in active disease and identified two major molecular subtypes: immune-deficient infectious CD (IDICD) and stress and senescence-induced fibrostenotic CD (S2FCD). Each subtype shows internal consistency in the transcriptome, genome, and phenome. The spectrum of morphometric, phenotypic, and functional changes within the "living biobank" reveals distinct differences between the molecular subtypes. Drug screens reverse subtype-specific phenotypes, suggesting phenotyped-genotyped CD PDOs can bridge basic biology and patient trials by enabling preclinical phase "0" human trials for personalized therapeutics., Competing Interests: Declaration of interests S.D. and P.G. have a patent on the methodology., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Epilepsies are disorders of the brain characterised by an imbalance in electrical activity, linked to a disruption in the excitation and inhibition of neurons. Progress in the epilepsy research field has been hindered by the lack of an appropriate model, with traditionally used 2D primary cell culture assays and animal models having a number of limitations which inhibit their ability to recapitulate the developing brain and the mechanisms behind epileptogenesis. As a result, the mechanisms behind the pathogenesis of epilepsy are largely unknown. Brain organoids are 3D aggregates of neural tissue formed in vitro and have been shown to recapitulate the gene expression patterns of the brain during development, and can successfully model a range of epilepsies and drug responses. They thus present themselves as a novel tool to advance studies into epileptogenesis. In this review, we discuss the formation of brain organoids, their recent application in studying genetic epilepsies, hyperexcitability dynamics and oxygen glucose deprivation as a hyperexcitability agent, their use as an epilepsy drug testing and development platform, as well as the limitations of their use in epilepsy research and how these can be mitigated., (© 2024. The Author(s).)

Precedential evidence ascertaining the overexpression of LSD1 and HDACs in colorectal cancer spurred us to design a series of dual LSD1-HDAC inhibitors. Capitalizing on the modular nature of the three-component HDAC inhibitory model, tranylcypromine as a surface recognition motif was appended to zinc-binding motifs via diverse linkers. A compendium of hydroxamic acids was generated and evaluated for in vitro cytotoxicity against HCT-116 cells (human colorectal cancer cell lines). The most potent cell growth inhibitor 2 (GI 50 = 0.495 μMm HCT-116 cells) shows promising anticancer effects by reducing colony formation and inducing cell cycle arrest in HCT-116 cells. It exhibits preferential inhibition of HDAC6, along with potent inhibition of LSD1 compared to standard inhibitors. Moreover, Compound 2 upregulates acetyl-tubulin, acetyl-histone H3, and H3K4me2, indicative of LSD1 and HDAC inhibition. In vivo , it demonstrates significant antitumor activity against colorectal cancer, better than irinotecan, and effectively inhibits growth in patient-derived CRC organoids.

Genomic-oriented oncology has improved tumor classification, treatment options, and patient outcomes. However, genetic heterogeneity, tumor cell plasticity, and the ability of cancer cells to hijack the tumor microenvironment (TME) represent a major roadblock for cancer eradication. Recent biotechnological advances in organotypic cell cultures have revolutionized biomedical research, opening new avenues to explore the use of cancer organoids in functional precision oncology, especially when genomics alone is not a determinant. Here, we outline the potential and the limitations of tumor organoids in preclinical and translational studies with a particular focus on lung cancer pathogenesis, highlighting their relevance in predicting therapy response, evaluating treatment toxicity, and designing novel anticancer strategies. Furthermore, we describe innovative organotypic coculture systems to dissect the crosstalk with the TME and to test the efficacy of different immunotherapy approaches, including adoptive cell therapy. Finally, we discuss the potential clinical relevance of microfluidic mini-organ technology, capable of reproducing tumor vasculature and the dynamics of tumor initiation and progression, as well as immunomodulatory interactions among tumor organoids, cancer-associated fibroblasts (CAFs) and immune cells, paving the way for next-generation immune precision oncology.

Sarcomas are rare malignancies with over 100 distinct histological subtypes. Their rarity and heterogeneity pose significant challenges to identifying effective therapies, and approved regimens show varied responses. Novel, personalized approaches to therapy are needed to improve patient outcomes. Patient-derived tumor organoids (PDTOs) model tumor behavior across an array of malignancies. We leverage PDTOs to characterize the landscape of drug resistance and sensitivity in sarcoma, collecting 194 specimens from 126 patients spanning 24 distinct sarcoma subtypes. Our high-throughput organoid screening pipeline tested single agents and combinations, with results available within a week from surgery. Drug sensitivity correlated with clinical features such as tumor subtype, treatment history, and disease trajectory. PDTO screening can facilitate optimal drug selection and mirror patient outcomes in sarcoma. We could identify at least one FDA-approved or NCCN-recommended effective regimen for 59% of the specimens, demonstrating the potential of our pipeline to provide actionable treatment information., Competing Interests: Declaration of interests A.S., J.Y., P.C.B., and N.C.F. are founders and owners of Icona BioDx. A.S. is a founder and owner of MiRiO., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Ovarian cancer is the most lethal gynecological cancer. As the primary treatment, chemotherapy has a response rate of only 60-70% in advanced stages, and even lower as a second-line treatment. Despite guideline recommendations, which drugs will be most effective remains unclear. Thus, a strategy to prioritize chemotherapy options is urgently needed. Cancer organoids have recently emerged as a method for in vitro drug testing. However, limited clinical correlations have been assessed with test results from cancer organoids, particularly in gynecological cancers. We therefore aimed to generate patient-derived organoids (PDOs) of ovarian cancer, to assess their drug sensitivities and correlations with patient clinical outcomes., Methods: PDOs were generated from fresh tumors obtained during surgical resection, which was then cultured under matrix gel and appropriate growth factors. Morphological and molecular characterization of PDOs were assessed by phase contrast microscopy and paraffin-embedded histopathology. Expressions of PAX8, TP53, WT1, CK7, and CK20 were tested by immunohistochemical staining and compared with parental tumor tissues and the human protein atlas database. PDOs were subjected to in vitro drug testing to determine drug sensitivity using Titer-Glo ® 3D Cell Viability Assay. PDO viability was measured, and area under the curve calculated, to compare responses to various compounds. Correlations were calculated between selected patients' clinical outcomes and in vitro drug testing results., Results: We established 31 PDOs. Among them, 28 PDOs can be expanded, including 15, 11, and 2 from ovarian, endometrial, and cervical cancers, respectively. The PDOs preserved the histopathological profiles of their originating tumors. In vitro drug testing of 10 ovarian cancer PDOs revealed individual differential responses to recommended drugs, and interpersonal heterogeneity in drug sensitivity, even with the same histology type. Among four patients who were platinum sensitive, resistant, or refractory, PDO drug responses correlated well with their clinical courses., Conclusion: In vitro drug testing using ovarian cancer organoids is feasible and correlates well with patient clinical responses. These results may facilitate development of precision chemotherapy and personalized screening for repurposed or new drugs., (© 2024. The Author(s).)

Genotypic and phenotypic diversity, which generates heterogeneity during disease evolution, is common in cancer. The identification of features specific to each patient and tumor is central to the development of precision medicine and preclinical studies for cancer treatment. However, the complexity of the disease due to inter- and intratumor heterogeneity increases the difficulty of effective analysis. Here, we introduce a sequential deep learning model, preprocessing to organize the complexity due to heterogeneity, which contrasts with general approaches that apply a single model directly. We characterized morphological heterogeneity using microscopy images of patient-derived organoids (PDOs) and identified gene subsets relevant to distinguishing differences among original tumors. PDOs, which reflect the features of their origins, can be reproduced in large quantities and varieties, contributing to increasing the variation by enhancing their common characteristics, in contrast to those from different origins. This resulted in increased efficiency in the extraction of organoid morphological features sharing the same origin. Linking these tumor-specific morphological features to PDO gene expression data enables the extraction of genes strongly correlated with intertumor differences. The relevance of the selected genes was assessed, and the results suggest potential applications in preclinical studies and personalized clinical care., (© 2024. The Author(s).)

Human pancreatic cancer is characterized by the molecular diversity encompassing native duct-like and squamous cell-like identities, but mechanisms underlying squamous transdifferentiation have remained elusive. To comprehensively capture the molecular diversity of human pancreatic cancer, we here profiled 65 patient-derived pancreatic cancer organoid lines, including six adenosquamous carcinoma lines. H3K27me3-mediated erasure of the ductal lineage specifiers and hijacking of the TP63-driven squamous-cell programme drove squamous-cell commitment, providing survival benefit in a Wnt-deficient environment and hypoxic conditions. Gene engineering of normal pancreatic duct organoids revealed that GATA6 loss and a Wnt-deficient environment, in concert with genetic or hypoxia-mediated inactivation of KDM6A, facilitate squamous reprogramming, which in turn enhances environmental fitness. EZH2 inhibition counterbalanced the epigenetic bias and curbed the growth of adenosquamous cancer organoids. Our results demonstrate how an adversarial microenvironment dictates the molecular and histological evolution of human pancreatic cancer and provide insights into the principles and significance of lineage conversion in human cancer., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Background and Aims: Patient-derived tumor organoids (PDTOs) are a promising new disease model in pancreatic cancer for use in personalized medicine. However, the overall success rate (SR) of establishing these cultures from EUS-guided biopsies is unknown., Methods: We searched relevant database publications reporting SRs of PDTO establishment from pancreatic cancer. The primary outcome was SR stratified on tissue acquisition method (EUS-guided biopsies, percutaneous biopsies, and surgical specimens)., Results: Twenty-four studies were identified that included 1053 attempts at establishing PDTOs. Overall SR was 63% (95% confidence interval [CI], 54%-72%). Pooled SRs of PDTO establishment from EUS-guided biopsies, percutaneous biopsies, and surgical specimens were 60% (95% CI, 43%-76%), 36% (95% CI, 14%-61%), and 62% (95% CI, 48%-75%), respectively, and did not differ significantly (P = .1975)., Conclusion: The SR of PDTO establishment from EUS-guided biopsies is comparable to that from surgical specimens. Both techniques are suitable for tissue acquisition for PDTOs in clinical and research settings. (PROSPERO registration number: CRD42023425121.)., Competing Interests: Disclosure The following authors disclosed financial relationships: J. G. Karstensen: consultant for SNIPR BIOME, Boston Scientific, and Ambu. P. Vilmann: consultant for Medi-Globe GmbH. All other authors disclosed no financial relationships. This research was funded by the Danish Cancer Society (grant no. 2021 R322-A17350); the European Research Executive Agency under the project Training in Translational Protocols for Minimal Invasive Diagnosis and Therapy in Pancreatico-Biliary Cancers—TRIP (grant agreement no. 101079210); and Arvid Nilssons Fond, Tømrermester Jørgen Holm og hustru, Elisa f. Hansens Mindelegat, Dagmar Marshalls Fond, and Lægeforeningens Forskningsfond., (Copyright © 2024 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.)

Cancer stem cells (CSC) are thought to be responsible for cancer phenotypes and cellular heterogeneity. Here we demonstrate that the human colon cancer cell line DLD1 contains two types of CSC-like cells that undergo distinct morphogenesis in the reconstituted basement membrane gel Matrigel. In our method with cancer cell spheroids, the parent cell line (DLD1-P) developed grape-like budding structures, whereas the other (DLD1-Wm) and its single-cell clones dynamically developed worm-like ones. Gene expression analysis suggested that the former mimicked intestinal crypt-villus morphogenesis, while the latter mimicked embryonic hindgut development. The organoids of DLD1-Wm cells rapidly extended in two opposite directions by expressing dipolar proteolytic activity. The invasive morphogenesis required the expression of MMP-2 and CD133 genes and ROCK activity. These cells also exhibited gastrula-like morphogenesis even in two-dimensional cultures without Matrigel. Moreover, the two DLD1 cell lines showed clear differences in cellular growth, tumor growth and susceptibility to paclitaxel. This study also provides a simple organoid culture method for human cancer cell lines. HT-29 and other cancer cell lines underwent characteristic morphogenesis in direct contact with normal fibroblasts. Such organoid cultures would be useful for investigating the nature of CSCs and for screening anti-cancer drugs. Our results lead to the hypothesis that CSC-like cells with both invasive activity and a fetal phenotype, i. e. oncofetal CSCs, are generated in some types of colon cancers., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: K. M. acted an advisor for Kishikasei Co. Ltd (Yokohama Japan) for 2 years (2021–2022). All other authors declare no competing interests. Patent application: Japanese Patent application in pending: 2021-049395; Date: May 23, 2021; Patent Title: Methods for producing organoid structures of cancer cell lines, medium additives for producing the organoids, and a method for screening anticancer drug candidates using the organoids; Applicants: Kanagawa Prefectural Hospital Organization, Kishikasei and K. M.; Inventors: K. M., Y. M. and Daisuke Maezawa., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Early dissemination of primary pancreatic ductal adenocarcinoma (PDAC) is the main cause of dismal prognosis as it highly limits possible treatment options. A number of PDAC patients experience distant metastasis even after treatment due to the metastatic clones. We aimed to demonstrate the molecular architecture of borderline resectable PDAC manifests cancer dissemination of PDAC., Methods: Here, 36 organoids isolated from primary tumor masses of PDAC patients with diverse metastatic statues are presented. Whole-exome sequencing and RNA sequencing were performed and drug responses to clinically relevant 18 compounds were assessed., Results: Our results revealed that borderline resectable PDAC organoids exhibited distinct patterns according to their metastatic potency highlighted by multiple genetic and transcriptional factors and strong variances in drug responses., Conclusions: These data suggest that the presence of metastatic PDAC can be identified by integrating molecular compositions and drug responses of borderline resectable PDAC., (© 2024. The Author(s).)

Background and Aims: Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disorder without effective medical treatment which is characterized by inflammation and fibrotic structures around the bile ducts. Biliary epithelial cells (cholangiocytes) are the target and potential disease drivers in PSC, yet little is known if cholangiocytes from PSC patients differ from non-PSC controls. To characterize cholangiocytes at early rather than end-stage disease, cholangiocyte organoids (COs) were derived from diseased bile ducts of PSC patients and compared to organoids generated from disease controls., Methods: Cholangiocytes were obtained during endoscopic retrograde cholangiopancreatography (ERCP) brushing of diseased bile duct areas and expanded as organoids using previously established culture methods. Stable CO lines were analyzed for cell type identity, basic cholangiocyte function, and transcriptomic signature., Results: We demonstrate that cholangiocytes, derived from the damaged area within the bile ducts of PSC patients, can be expanded in culture without displaying functional or genetic disease-related features. We further show that COs from patients who later were diagnosed with dysplasia exhibit higher expression of the cancer-associated genes PGC, FXYD2, MIR4435-2HG, and HES1., Conclusions: Our results demonstrate that PSC organoids are largely similar to control organoids after culture and highlight the significance of COs as a tool for regenerative medicine approaches as well as their potential for discovering new potential biomarkers for diagnosing cholangiocarcinoma., (© 2024. The Author(s).)

Around 13% of women will likely develop breast cancer during their lifetime. Advances in cancer metabolism research have identified a range of metabolic reprogramming events, such as altered glucose and amino acid uptake, increased reliance on glycolysis, and interactions with the tumor microenvironment (TME), all of which present new opportunities for targeted therapies. However, studying these metabolic networks is challenging in traditional 2D cell cultures, which often fail to replicate the three-dimensional architecture and dynamic interactions of real tumors. To address this, organoid models have emerged as powerful tools. Tumor organoids are 3D cultures, often derived from patient tissue, that more accurately mimic the structural and functional properties of actual tumor tissues in vivo, offering a more realistic model for investigating cancer metabolism. This review explores the unique metabolic adaptations of breast cancer and discusses how organoid models can provide deeper insights into these processes. We evaluate the most advanced tools for studying cancer metabolism in three-dimensional culture models, including optical metabolic imaging (OMI), matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), and recent advances in conventional techniques applied to 3D cultures. Finally, we explore the progress made in identifying and targeting potential therapeutic targets in breast cancer metabolism.

Ex vivo assessment of drug response with conventional cell viability assays remains the standard practice for guiding initial therapeutic choices. However, such ensemble approaches fail to capture heterogeneities in treatment response and cannot identify early markers of response. Here, we leverage Raman spectroscopy (RS) as an accurate, low-cost, extraction-free, and label-free approach to track metabolic changes in cancer cells, spheroids, and organoids in response to cisplatin treatment. We identified 12 statistically significant metabolites in cells and 19 metabolites in spheroids and organoids as a function of depth. We show that the cisplatin treatment of 4T1 cells and spheroids results in a shift in metabolite levels; metabolites including nucleic acids such as DNA, 783 cm -1 with p = 0.00021 for cells; p = 0.02173 for spheroids, major amino acids such as threonine, 1338 cm -1 with p = 0.00045 for cells; p = 0.01022 for spheroids, proteins such as amide III, 1248 cm -1 with p = 0.00606 for cells; p = 0.00511 for spheroids serve as early predictors of response. Our RS findings were also applicable to canine-derived organoids, showing spatial variations in metabolic changes as a function of organoid depth in response to cisplatin. Further, the metabolic pathways such as tricarboxylic acid (TCA)/citric acid cycle and glyoxylate and dicarboxylate metabolism that drive drug response showed significant differences based on organoid depth, replicating the heterogeneous treatment response seen in solid tumors where there is a difference from the periphery to the tumor core. Our study showcases the versatility of RS as a predictive tool for treatment response applicable from cells to organotypic cultures, that has the potential to decrease animal burden and readout time for preclinical drug efficacy.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by de novo mutations in the MECP2 gene. Although miRNAs in extracellular vesicles (EVs) have been suggested to play an essential role in several neurological conditions, no prior study has utilized brain organoids to profile EV-derived miRNAs during normal and RTT-affected neuronal development. Here we report the spatiotemporal expression pattern of EV-derived miRNAs in region-specific forebrain organoids generated from female hiPSCs with a MeCP2:R255X mutation and the corresponding isogenic control. EV miRNA and protein expression profiles were characterized at day 0, day 13, day 40, and day 75. Several members of the hsa-miR-302/367 cluster were identified as having a time-dependent expression profile with RTT-specific alterations at the latest developmental stage. Moreover, the miRNA species of the chromosome 14 miRNA cluster (C14MC) exhibited strong upregulation in RTT forebrain organoids irrespective of their spatiotemporal location. Together, our results suggest essential roles of the C14MC and hsa-miR-302/367 clusters in EVs during normal and RTT-associated neurodevelopment, displaying promising prospects as biomarkers for monitoring RTT progression., (© 2024. The Author(s).)

Single-nuclei RNA sequencing (snRNA-seq) allows for obtaining gene expression profiles from frozen or hard-to-dissociate tissues at the single-nuclei level. Here, we describe a protocol to obtain snRNA-seq data of pancreatic tumors from orthotopically grafted organoid-derived mouse models. We provide details on the establishment of these mouse models, the isolation of single nuclei from pancreatic tumors, and the analysis of the snRNA-seq datasets. For complete details on the use and execution of this protocol, please refer to Mucciolo et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2024. The Author(s).)

Perturbation of cell polarity is a hallmark of pancreatic ductal adenocarcinoma (PDAC) progression. Scribble (SCRIB) is a well-characterized polarity regulator that has diverse roles in the pathogenesis of human neoplasms. To investigate the impact of SCRIB deficiency in PDAC development and progression, Scrib expression was genetically ablated in well-established mouse models of PDAC. Scrib loss in combination with KrasG12D did not influence development of pancreatic intraepithelial neoplasms in mice. However, Scrib deletion cooperated with KrasG12D and concomitant Trp53 heterozygous deletion to promote invasive PDAC and metastatic dissemination, leading to reduced overall survival. Immunohistochemical and transcriptome analyses revealed that Scrib-null tumors display a pronounced reduction of collagen content and an abundance of cancer-associated fibroblasts (CAF). Mechanistically, IL1α levels were reduced in Scrib-deficient tumors, and Scrib knockdown downregulated IL1α in mouse PDAC organoids (mPDO), which impaired CAF activation. Furthermore, Scrib loss increased YAP activation in mPDOs and established PDAC cell lines, enhancing cell survival. Clinically, SCRIB expression was decreased in human PDAC, and SCRIB mislocalization was associated with poorer patient outcome. These results indicate that SCRIB deficiency enhances cancer cell survival and remodels the tumor microenvironment to accelerate PDAC development and progression, establishing the tumor suppressor function of SCRIB in advanced pancreatic cancer.  Significance: SCRIB loss promotes invasive pancreatic cancer development via both cell-autonomous and non-cell-autonomous processes and is associated with poorer outcomes, denoting SCRIB as a tumor suppressor and potential biomarker for the prediction of recurrence., (©2024 American Association for Cancer Research.)

Aggregated α-synuclein (α-SYN) proteins, encoded by the SNCA gene, are hallmarks of Lewy body disease (LBD), affecting multiple brain regions. However, the specific mechanisms underlying α-SYN pathology in cortical neurons, crucial for LBD-associated dementia, remain unclear. Here, we recapitulated α-SYN pathologies in human induced pluripotent stem cells (iPSCs)-derived cortical organoids generated from patients with LBD with SNCA gene triplication. Single-cell RNA sequencing, combined with functional and molecular validation, identified synaptic and mitochondrial dysfunction in excitatory neurons exhibiting high expression of the SNCA gene, aligning with observations in the cortex of autopsy-confirmed LBD human brains. Furthermore, we screened 1280 Food and Drug Administration-approved drugs and identified four candidates (entacapone, tolcapone, phenazopyridine hydrochloride, and zalcitabine) that inhibited α-SYN seeding activity in real-time quaking-induced conversion assays with human brains, reduced α-SYN aggregation, and alleviated mitochondrial dysfunction in SNCA triplication organoids and excitatory neurons. Our findings establish human cortical LBD models and suggest potential therapeutic drugs targeting α-SYN aggregation for LBD.

Purpose: Pseudomyxoma peritonei (PMP) is a rare and poorly understood malignant condition characterized by the accumulation of intra-abdominal mucin produced from peritoneal metastases. Currently, cytoreductive surgery remains the mainstay of treatment but disease recurrence and death after relapse frequently occur in patients with PMP. New therapeutic strategies are therefore urgently needed for these patients., Experimental Design: A total of 120 PMP samples from 50 patients were processed to generate a collection of 50 patient-derived organoid (PDO) and xenograft (PDX) models. Whole exome sequencing, immunohistochemistry analyses, and in vitro and in vivo drug efficacy studies were performed., Results: In this study, we have generated a collection of PMP preclinical models and identified druggable targets, including BRAFV600E, KRASG12C, and KRASG12D, that could also be detected in intra-abdominal mucin biopsies of patients with PMP using droplet digital PCR. Preclinical models preserved the histopathological markers from the original patient sample. The BRAFV600E inhibitor encorafenib reduced cell viability of BRAFV600E PMP-PDO models. Proof-of-concept in vivo experiments showed that a systemic treatment with encorafenib significantly reduced tumor growth and prolonged survival in subcutaneous and orthotopic BRAFV600E-PMP-PDX mouse models., Conclusions: Our study demonstrates for the first time that systemic targeted therapies can effectively control PMP tumors. BRAF signaling pathway inhibition represents a new therapeutic opportunity for patients with BRAFV600E PMP who have a poor prognosis. Importantly, our present data and collection of preclinical models pave the way for evaluating the efficacy of other systemic targeted therapies toward extending the promise of precision oncology to patients with PMP., (©2024 The Authors; Published by the American Association for Cancer Research.)

Vascular organoids (VOs), derived from induced pluripotent stem cells (iPSCs), hold promise as in vitro disease models and drug screening platforms. However, their ability to faithfully recapitulate human vascular disease and cellular composition remains unclear. In this study, we demonstrate that VOs derived from iPSCs of donors with diabetes (DB-VOs) exhibit impaired vascular function compared to non-diabetic VOs (ND-VOs). DB-VOs display elevated levels of reactive oxygen species (ROS), heightened mitochondrial content and activity, increased proinflammatory cytokines, and reduced blood perfusion recovery in vivo. Through comprehensive single-cell RNA sequencing, we uncover molecular and functional differences, as well as signaling networks, between vascular cell types and clusters within DB-VOs. Our analysis identifies major vascular cell types (endothelial cells [ECs], pericytes, and vascular smooth muscle cells) within VOs, highlighting the dichotomy between ECs and mural cells. We also demonstrate the potential need for additional inductions using organ-specific differentiation factors to promote organ-specific identity in VOs. Furthermore, we observe basal heterogeneity within VOs and significant differences between DB-VOs and ND-VOs. Notably, we identify a subpopulation of ECs specific to DB-VOs, showing overrepresentation in the ROS pathway and underrepresentation in the angiogenesis hallmark, indicating signs of aberrant angiogenesis in diabetes. Our findings underscore the potential of VOs for modeling diabetic vasculopathy, emphasize the importance of investigating cellular heterogeneity within VOs for disease modeling and drug discovery, and provide evidence of GAP43 (neuromodulin) expression in ECs, particularly in DB-VOs, with implications for vascular development and disease., (© The Author(s) 2024. Published by Oxford University Press.)

Experimental models play a pivotal role in biomedical research, facilitating the understanding of disease mechanisms and the development of novel therapeutics. This is particularly true for neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and motor neuron disease, which present complex challenges for research and therapy development. In this work, we review the recent literature about experimental models and motor neuron disease. We identified three main categories of models that are highly studied by scientists. In fact, experimental models for investigating these diseases encompass a variety of approaches, including modeling the patient's cell culture, patient-derived induced pluripotent stem cells, and organoids. Each model offers unique advantages and limitations, providing researchers with a range of tools to address complex biological questions. Here, we discuss the characteristics, applications, and recent advancements in terms of each model system, highlighting their contributions to advancing biomedical knowledge and translational research.

KRAS mutations in pancreatic ductal adenocarcinoma (PDAC) are suggested to vary in oncogenicity but the implications for human patients have not been explored in depth. We examined 1,360 consecutive PDAC patients undergoing surgical resection and find that KRAS G12R mutations are enriched in early-stage (stage I) disease, owing not to smaller tumor size but increased node-negativity. KRAS G12R tumors are associated with decreased distant recurrence and improved survival as compared to KRAS G12D . To understand the biological underpinnings, we performed spatial profiling of 20 patients and bulk RNA-sequencing of 100 tumors, finding enhanced oncogenic signaling and epithelial-mesenchymal transition (EMT) in KRAS G12D and increased nuclear factor κB (NF-κB) signaling in KRAS G12R tumors. Orthogonal studies of mouse Kras G12R PDAC organoids show decreased migration and improved survival in orthotopic models. KRAS alterations in PDAC are thus associated with distinct presentation, clinical outcomes, and biological behavior, highlighting the prognostic value of mutational analysis and the importance of articulating mutation-specific PDAC biology., Competing Interests: Declaration of interests E.M.O., research funding: Genentech/Roche, BioNTech, AstraZeneca, Arcus, Elicio, Parker Institute, NIH/NCI, Pertzye; consulting/DSMB: Boehringer Ingelheim, BioNTech, Ipsen, Merck, Novartis, AstraZeneca, BioSapien, Astellas, Thetis, Autem, Novocure, Neogene, BMS, Tempus, Fibrogen, Merus, Agios (spouse), Genentech-Roche (spouse), Eisai (spouse). G.M.C.:A full listing of G.M.C.’s interests can be found at http://arep.med.harvard.edu/gmc/tech/html. C.E.M., founder: Onegevity, Twin Orbit, and Cosmica Biosciences; consulting: Nanostring. L.E.D., research funding/consulting: Revolution Medicines; scientific advisory board: Mirimus. R.C., research funding: Sanofi; consulting/DSMB: Boston Scientific., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Intratumour heterogeneity has been shown to play a role in the malignant progression of cancer. The clonal evolution in primary cancer has been well studied, however, that in metastatic tumorigenesis is not fully understood. In this study, we established human colon cancer-derived organoids and investigated clonal dynamics during liver metastasis development by tracking barcode-labelled subclones. Long-term subclone co-cultures showed clonal drift, with a single subclone becoming dominant in the cell population. Interestingly, the selected subclones were not always the same, suggesting that clonal selection was not based on cell intrinsic properties. Furthermore, liver tumours developed by co-transplantation of organoid subclones into the immunodeficient mouse spleen showed a progressive drastic reduction in clonal diversity, and only one or two subclones predominated in the majority of large metastatic tumours. Importantly, selections were not limited to particular subclones but appeared to be random. A trend towards a reduction in clonal diversity was also found in liver metastases of multiple colour-labelled organoids of mouse intestinal tumours. Based on these results, we propose a novel mechanism of metastasis development, i.e. a subclone population of the disseminated tumour cells in the liver is selected by neutral selection during colonization and constitutes large metastatic tumours., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Parkinson's disease (PD) is a neurodegenerative condition characterized by the loss of dopaminergic neurons and the accumulation of Lewy-body protein aggregates containing misfolded α-synuclein (α-syn) in a phosphorylated form. The lack of effective models for drug screens has hindered drug development studies for PD. However, the recent development of in vitro brain-like organoids provides a new opportunity for evaluating therapeutic agents to slow the progression of this chronic disease., Methods: In this study, we used a 3D brain-like organoid model to investigate the potential of repurposing Tilorone, an anti-viral drug, for impeding the propagation of α-synucleinopathy. We assessed the effect of Tilorone on the uptake of fluorescently labeled α-syn preformed fibrils (sPFF) and sPFF-induced apoptosis using confocal microscopy. We also examined Tilorone's impact on the phosphorylation of endogenous α-syn induced by pathogenic sPFF by immunoblotting midbrain-like organoid extracts. Additionally, quantitative RT-PCR and proteomic profiling of sPFF-treated organoids were conducted to evaluate the global impact of Tilorone treatment on tissue homeostasis in the 3D organoid model., Results: Tilorone inhibits the uptake of sPFF in both mouse primary neurons and human midbrain-like organoids. Tilorone also reduces the phosphorylation of endogenous α-syn induced by pathogenic α-syn fibrils and mitigates α-syn fibril-induced apoptosis in midbrain-like organoids. Proteomic profiling of fibril-treated organoids reveals substantial alterations in lipid homeostasis by α-syn fibrils, which are reversed by Tilorone treatment. Given its safety profile in clinics, Tilorone may be further developed as a therapeutic intervention to alleviate the propagation of synucleinopathy in PD patients., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)