Organoid Model Systems Research Articles

Spheres of Influence: Insights into Salmonella Pathogenesis from Intestinal Organoids

The molecular complexity of host-pathogen interactions remains poorly understood in many infectious diseases, particularly in humans due to the limited availability of reliable and specific experimental models. To bridge the gap between classical two-dimensional culture systems, which often involve transformed cell lines that may not have all the physiologic properties of primary cells, and in vivo animal studies, researchers have developed the organoid model system. Organoids are complex three-dimensional structures that are generated in vitro from primary cells and can recapitulate key in vivo properties of an organ such as structural organization, multicellularity, and function. In this review, we discuss how organoids have been deployed in exploring Salmonella infection in mice and humans. In addition, we summarize the recent advancements that hold promise to elevate our understanding of the interactions and crosstalk between multiple cell types and the microbiota with Salmonella. These models have the potential for improving clinical outcomes and future prophylactic and therapeutic intervention strategies.

The power of one: advances in single-cell genomics in the kidney.

Single-cell genomics provide a powerful approach to investigate the intrinsic complexity of the kidney and understand the diverse cell types and states that exist during kidney development, homeostasis and disease. Several advances were made in 2019 that enhance our understanding of kidney immune cell states in health and disease and the quality of current kidney organoid model systems for studying human diseases.

BRCA1 mutation influences progesterone response in human benign mammary organoids

BackgroundWomen, who carry a germline BRCA1 gene mutation, have a markedly increased risk of developing breast cancer during their lifetime. While BRCA1 carriers frequently develop triple-negative, basal-like, aggressive breast tumors, hormone signaling is important in the genesis of BRCA1 mutant breast cancers. We investigated the hormone response in BRCA1-mutated benign breast tissue using an in vitro organoid system.MethodsScaffold-free, multicellular human breast organoids generated from benign breast tissues from non-carrier or BRCA1 mutation carriers were treated in vitro with a stepwise menstrual cycle hormone regimen of estradiol (E2) and progesterone (P4) over the course of 28 days.ResultsBreast organoids exhibited characteristics of the native breast tissue, including expression of hormone receptors, collagen production, and markers of luminal and basal epithelium, and stromal fibroblasts. RNA sequencing analysis revealed distinct gene expression in response to hormone treatment in the non-carrier and BRCA1-mutated organoids. The selective progesterone receptor modulator, telapristone acetate (TPA), was used to identify specifically PR regulated genes. Specifically, extracellular matrix organization genes were regulated by E2+P4+TPA in the BRCA1-mutated organoids but not in the non-carrier organoids. In contrast, in the non-carrier organoids, known PR target genes such as the cell cycle genes were inhibited by TPA.ConclusionsThese data show that BRCA1 mutation influences hormone response and in particular PR activity which differs from that of non-carrier organoids. Our organoid model system revealed important insights into the role of PR in BRCA1-mutated benign breast cells and the critical paracrine actions that modify hormone receptor (HR)-negative cells. Further analysis of the molecular mechanism of BRCA1 and PR crosstalk is warranted using this model system.

Development of Patient-Derived Preclinical Platform for Metastatic Pancreatic Cancer: PDOX and a Subsequent Organoid Model System Using Percutaneous Biopsy Samples

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal malignant tumor and more than 50% patients are diagnosed at metastatic stage. The preclinical model systems that reflect the genetic heterogeneity of metastatic tumors are urgently needed to guide optimal treatment. This study describes the development of patient-derived preclinical platform using very small sized-percutaneous liver gun biopsy (PLB) of metastatic pancreatic cancer, based on patient-derived xenograft (PDX)-mediated tissue amplification and subsequent organoid generation. To increase the success rate and shorten the tumor growth period, patient-derived orthotopic xenograft (PDOX) model was developed to directly implant threadlike PLB samples into the pancreas. The engraftment success rate of PDOX samples from 35 patients with metastatic PDAC was 47%, with these samples showing the potential to metastasize to distant organs, as in patients. The PDOX models retained the genetic alterations and histopathological features of the primary tumors. Tumor organoids were subsequently generated from first passage cancer cells isolated from F1 tumor tissue of PDOX that preserve the epithelial cancer characteristics and KRAS mutations of primary tumors. The response to gemcitabine of PDOX-derived organoids correlated with clinical outcomes in corresponding patients as well as PDOX models in vivo, suggesting that this PDOX-organoid system reflects clinical conditions. Collectively, these findings indicate that the proposed PDOX-organoid platform using PLB samples assessed both in vitro and in vivo could predict drug response under conditions closer to those found in actual patients, as well as enhancing understanding of the complexity of metastatic PDAC.

APOE4 ACCELERATES NEURODEGENERATION AND EXACERBATES ALZHEIMER’S DISEASE RELATED PATHOLOGY IN CEREBRAL ORGANOIDS

APOE4 is the strongest genetic risk factor for late-onset Alzheimer's disease (AD) compared to the common APOE3 allele. The cerebral organoids derived from human induced pluripotent stem cells (iPSCs) have been shown to be a novel model system closely resembling the physiological condition of human brains. To understand how human apoE isoforms differentially impact neuron viability and AD-related pathologies, we developed iPSC lines with different APOE genotypes from individuals with either normal cognition or sporadic AD and further into cerebral organoids. We found that cerebral organoids from AD patients with APOE ε4/4 genotype showed accelerated differentiation pattern and had enhanced neuronal apoptosis in the ventricular zone-like area at day 90 when compared to those from normal controls or AD with the APOE ε3/3 genotype. Cerebral organoids from AD patients also had increased AD-related pathologies including the levels of Aβ40, Aβ42, and phosphorylation tau. More interestingly, we found that apoE4 exacerbates tau pathology in a disease status-independent manner. Transcriptomic analysis by RNA-sequencing revealed an enrichment of apoptosis pathway in cerebral organoids from AD patients with the APOE ε4/4 genotype. Together, our results using the cerebral organoids suggest that apoE4 accelerates neurodegeneration in addition to impacting AD-related pathology in a human-relevant model system. Both the cerebral organoid model system and the information on apoE4-relevant disease pathways should help the development of new therapeutics for AD.

Abstract 3745: Mechanisms of HIF2-mediated small intestine radioprotection

Abstract Pancreatic cancer is the fourth-leading cause of cancer-related deaths in the United States, and it is projected to become the second by 2030. The 5-year overall survival rate for locally advanced pancreatic cancer (LAPC) remains < 5%. Chemotherapy regimens often fail due to poor drug delivery and penetration into the tumor. Because surgery is not feasible by definition in LAPC, radiation therapy (RT) is the only option to achieve local control. However, irradiating the pancreas is challenging because potentially curative doses are limited by the neighboring small intestine, which can only tolerate a maximum of 50Gy. A selective radioprotector of the intestinal tract could allow pancreatic cancer patients to receive a higher, and potentially definitive, dose of radiation by increasing the duodenum’s tolerance to survive radiation injury. The EGLN prolyl hydroxylases are cellular oxygen sensors that regulate cell survival and metabolism through the degradation of hypoxia-inducible factors (HIFs) in the presence of oxygen. Our group has shown that HIF2 stabilization through genetic or pharmacologic inhibition of the EGLN family prevents death from radiation-induced gastrointestinal toxicity in mice without protecting the primary tumor. To understand the mechanism by which HIF2 mediates this protection, we generated intestinal organoids from mice with a Cre-inducible allele that expresses a non-degradable form of human HIF1 or HIF2. Whole transcriptomic analysis revealed HIF1 and HIF2 have distinct gene responses in the small intestinal crypt. HIF2 induced the expression of known radiation modulators and genes involved in oxidative damage response, tissue healing, and intestinal homeostasis such as the non-canonical Wnt5a. We validated HIF2 drives Wnt5a expression in multiple organoid model systems. We then generated knockouts (KO) of Wnt5a or Ror2, its cognate receptor, using the Cre/lox system to test if Wnt5a is necessary and sufficient for radioprotection. By using the spheroid formation assay, a modified clonogenic assay, we found Wnt5a KO organoids had decreased crypt regeneration following RT. Our preliminary data suggest Wnt5a protects the duodenum from RT by increasing intestinal stem cell survival. Citation Format: Carolina J. Garcia Garcia, Suman Govindaraju, Marimar de la Cruz Bonilla, Cullen M. Taniguchi. Mechanisms of HIF2-mediated small intestine radioprotection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3745.

Vincristine Impairs Microtubules and Causes Neurotoxicity in Cerebral Organoids

The advance of nanotechnology in drug delivery systems has allowed central nervous system (CNS) accumulation of several anti-tumor agents with poor brain penetration but also lead to concerns about central neurotoxicity. Vincristine is commonly administered as an effective anti-brain tumor drug. It is known to act by interfering with microtubule dynamics, but models for detailed elucidation of its mechanism of neurotoxicity are limited. Here we generated cerebral organoids using human-induced pluripotent stem cells (iPSCs) for evaluation of neurotoxic mechanisms. Cerebral organoids were treated with different concentrations of vincristine for 48 h and their expansion was measured. We also assayed various cell markers, microtubule associated proteins, and matrix metalloproteinases (MMP) in cerebral organoids. After treatment for 48 h, we observed dose-dependent neurotoxicity, including reduced neuron and astrocyte numbers at high concentration. Vincristine treatment also impaired the microtubule-associated protein tubulin, and fibronectin, and downregulated MMP10 activity. Further analysis using the STRING database found that, both MMP10 and fibronectin bind with MMP9 experimentally, and text-mining indicated an interaction between MMP10 and fibronectin. Our organoid model system allowed quantitative investigation of the effects of vincristine treatment. Our findings indicated vincristine exhibited dose-dependent neurotoxicity, inhibited fibronectin, tubulin, and MMP10 expression in cerebral organoids.

Abstract 2854: Reconstruction of BRAFV600E driven colorectal carcinogenesis and identification of novel drug combinations involving BRAF and RTK inhibitors

Abstract BRAF mutations are found in approximately 10% of colorectal cancers (CRC) and are associated with an aggressive, less-differentiated and therapy-resistant phenotype. Inhibitors targeting BRAFV600E elicit only limited survival benefits when used as single agents. This unresponsiveness was mechanistically attributed to the relief of negative feedbacks on the epidermal growth factor receptor (EGFR) and initiated dual and triple combinatorial trials. These trials often involve the combination of BRAF inhibitors (BRAFi) and/or MEK inhibitors with anti-EGFR antibodies such as cetuximab or panitumumab. Although first results of these dual or triple therapies demonstrated improved efficacy, the response rates still were heterogeneous. Here, we show that BRAFi upregulate a variety of receptor tyrosine kinases (RTK) in CRC cell lines, including not only the EGFR, but also human epidermal growth factor receptor (HER) 2 and HER3. Importantly, combination of BRAFi (vemurafenib, dabrafenib or encorafenib) with inhibitors dually targeting the EGFR and HER2 (lapatinib, canertinib or afatinib) significantly reduced the metabolic activity and proliferation of CRC cells. Similarly, genetic depletion of HER2 and HER3 re-sensitized CRC cells to BRAF inhibition. Interestingly, BRAF inhibition also led to increased levels of the GRB2-associated binders (Gab) 1 and Gab2, two important mediators of RTK signaling. The Gab2 upregulation was directly dependent on the loss of BRAFV600E signaling and not caused by “off-target” effects, as demonstrated by allele-specific shRNA mediated BRAFV600E knockdown. These findings suggest new escape mechanisms for current treatment regimens and indicate that targeted therapy in BRAF mutant CRC could benefit from broad RTK pathway blockade. Importantly, the BRAF/HER family inhibitor combination was also more effective in murine intestinal MouseT1 cells (originating from a Vil-Cre;BrafLSL-V637E/+;Tp53LSL-R172H/+ mouse model), which indicates a species-independent phenomenon. These novel and our previous findings that BRAFV600E suppresses features of epithelial differentiation and effector function prompted us to establish crypt organoid cultures from mice carrying conditional BraffloxV600E/+ and/or Tp53LSL-R172H/+ knock-in alleles, either singly or in combination. Using a Vil-CreERT2 transgene, we were able to induce expression of oncogenic BRAF and dominant-negative p53R172H in organoids by 4-hydroxy-tamoxifen mediated Cre activation. We demonstrate that the sudden expression of BRAFV600E and TP53R172H induce marked morphological and molecular changes in small and large intestinal crypts. This organoid model system represents an excellent tool to better understand key characteristics of BRAF mutant CRC such as intrinsic aggressiveness, poor differentiation and resistance to BRAFi. Citation Format: Nadine Reischmann, Ricarda Herr, Sebastian Halbach, Miriam Heizmann, Hauke Busch, Melanie Boerries, Tilman Brummer. Reconstruction of BRAFV600E driven colorectal carcinogenesis and identification of novel drug combinations involving BRAF and RTK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2854.

Establishment of an in vitro organoid model of dermal papilla of human hair follicle.

Human hair dermal papilla (DP) cells are specialized mesenchymal cells that play a pivotal role in hair regeneration and hair cycle activation. The current study aimed to first develop three-dimensional (3D) DP spheroids (DPS)with or without a silk-gelatin (SG) microenvironment, which showed enhanced DP-specific gene expression, resulting in enhanced extracellular matrix (ECM) production compared with a monolayer culture. We tested the feasibility of using this DPS model for drug screening by using minoxidil, which is a standard drug for androgenic alopecia. Minoxidil-treated DPS showed enhanced expression of growth factors and ECM proteins. Further, an attempt has been made to establish an in vitro 3D organoid model consisting of DPS encapsulated by SG hydrogel and hair follicle (HF) keratinocytes and stem cells. This HF organoid model showed the importance of structural features, cell-cell interaction, and hypoxia akin to in vivo HF. The study helped to elucidate the molecular mechanisms to stimulate cell proliferation, cell viability, and elevated expression of HF markers as well as epithelial-mesenchymal crosstalks, demonstrating high relevance to human HF biology. This simple in vitro DP organoid model system has the potential to provide significant insights into the underlying mechanisms of HF morphogenesis, distinct molecular signals relevant to different stages of the hair cycle, and hence can be used for controlled evaluation of the efficacy of new drug molecules.

Verteporfin exhibits YAP-independent anti-proliferative and cytotoxic effects in endometrial cancer cells.

Endometrial Carcinoma (EMCA) is the most common gynecologic malignancy and the fourth most common malignancy in women in the United States. Yes-associated protein (YAP) is a potent transcription coactivator acting via binding to the TEAD transcription factor, and plays a critical role in organ size regulation. Verteporfin (VP), a benzoporphyrin derivative, was identified as an inhibitor of YAP-TEAD interaction. We investigated the therapeutic efficacy and mechanism of VP in EMCA. The efficacy of VP on cell viability, cytotoxicity and invasion was assayed in EMCA cell lines. An organoid model system was also developed to test the effect of VP on apoptotic markers in an in vitro model system. Treatment with VP resulted in a decrease in cell viability, invasion and an increase in cytotoxicity of EMCA cells. These effects occurred as early as 15 minutes following treatment. Similarly, VP treatment versus vehicle control increased apoptosis in human organoid model systems. Quantitative RT-PCR, cDNA based RTPCR array analysis and western blotting were performed to investigate the mechanism of VP action. The cytotoxic and anti-proliferative effects appeared to be independent of its effect on YAP. Our results suggest that VP is a promising chemotherapeutic agent for the treatment of endometrial cancer.

Gastric Organoids: An Emerging Model System to Study Helicobacter pylori Pathogenesis.

Helicobacter research classically uses fixed human tissue, animal models or cancer cell lines. Each of these study objects has its advantages and has brought central insights into the infection process. Nevertheless, in model systems for basic and medical research, there is a gap between two-dimensional and most often transformed cell cultures and three-dimensional, highly organized tissues. In recent years, stem cell research has provided the means to fill this gap. The identification of the niche factors that support growth, expansion and differentiation of stem cells in vitro has allowed the development of three-dimensional culture systems called organoids. Gastric organoids are grown from gastric stem cells and are organized epithelial structures that comprise all the differentiated cell types of the stomach. They can be expanded without apparent limitation and are amenable to a wide range of standard laboratory techniques. Here, we review different stem cell-derived organoid model systems useful for Helicobacter pylori research and outline their advantages for infection studies.

Abstract A04: Identification of novel pancreatic cancer-specific biomarkers with organoid models

Abstract Pancreatic ductal adenocarcinoma (PDA) is almost uniformly lethal and surgical intervention is the only cure. Unfortunately, the majority of patients are ineligible for resection because of the advanced stage of disease by the time of diagnosis. This is due in part to that lack of diagnostic tools, especially for families with elevated risk. Although considered an intractable and aggressive malignancy, progression from the initiating events to overt cancer requires approximately 12 years, and advancement to metastatic disease takes another 5 – 7 years. This latency indicates that there is a window for early detection and intervention. The PDA biomarker, CA19-9, is measured in the blood to follow tumor burden longitudinally, but is neither sensitive nor specific enough to be used for diagnosis. In particular, the use of CA19-9 in PDA diagnosis is problematic given the elevation of CA19-9 in benign conditions, such as pancreatitis, that have a higher incidence than PDA. To improve the diagnostic performance of CA19-9, we identified tissue- and cancer-specific protein carriers of the CA19-9 carbohydrate modification using murine and human organoid model systems of pancreatic cancer disease progression. To induce CA19-9 expression in murine organoids, two human gylcosyl transferases that were deficient in mice were ectopically expressed. CA19-9 protein carriers specific to malignancy were identified relative to normal, proliferating ductal cells. Some CA19-9 carriers that were shared between normal and malignant pancreatic ductal organoids were also detected in the sera of patients with benign pancreatic disease, highlighting their inability to distinguish malignancy from pancreatitis. Importantly, several of the tumor-specific novel CA19-9 carriers identified in organoids also discriminated between benign and malignant disease in human patient sera, and are nominated for clinical validation. Overall, organoid model systems allow identification of biomarkers that discriminate between normal proliferation and malignant conditions of multiple gastrointestinal tissues, providing a robust platform for biomarker discovery. Citation Format: Dannielle Engle, Herve Tiriac, Rivera Keith, Michael Ludwig, Chang-il Hwang, Kenneth Yu, Darryl Pappin, David Tuveson.{Authors}. Identification of novel pancreatic cancer-specific biomarkers with organoid models. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A04.

Abstract IA11: Pancreatic cancer biology and medicine

Abstract Pancreatic cancer remains the most deadly of the common malignancies. To increase our basic understanding of this disease and advance medical approaches, we have recently co-developed with Hans Clevers mouse and human organoid model systems. Our organoid cultures representative of either normal or neoplastic ductal cells facilitate the systematic analysis of neoplastic biochemical cascades, provide a platform to explore therapeutic and diagnostic strategies, and also enable the reconstitution of tumor microenvironment interactions. We have found that Nrf2, a principal regulator of redox metabolism, promotes proliferation in KRAS mutant pancreatic ductal cells in part by regulating the reduced state of the cellular proteome. As a consequence of proteome oxidation, central pathways of homeostasis are impaired, including protein translation. The genes with markedly impaired translation include a number of critical proto-oncogenes. Additionally, diagnostic and therapeutic approaches have revealed new avenues to explore for early detection and disease treatment. Finally, the co-culture of organoids with pancreatic stellate cells promotes the activation of each cell type and results in the production of extracellular matrix. Co-cultures reveal distinct subtypes of stellate cells and provide an opportunity to identify and target novel mediators of the tumor microenvironment. Citation Format: David Tuveson.{Authors}. Pancreatic cancer biology and medicine. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr IA11.

Abstract 3165: Metastatic tumor evolution delineates clinically important oncogenic drivers in diffuse gastric cancer.

Abstract We conducted a detailed genetic and functional analysis of personalized tumor evolution in a case of diffuse gastric cancer. Leveraging the unique genetics of an individual with a confirmed germline mutation in the causative CDH1 tumor suppressor gene, we identified candidate drivers for a primary tumor in the stomach and a metastatic recurrence in the left ovary, referred to as a Krukenberg tumor. Using genome sequencing, we demonstrate that the primary and metastatic tumors share the majority of cancer driver events including biallelic CDH1 and TP53 mutations consistent with a common genetic origin. The primary gastric cancer displayed amplification of FGFR2, a gene that encodes the fibroblast growth factor (FGF) receptor 2 with multiple small molecule inhibitors in clinical therapy development. Tumor evolution and genetic divergence was clearly evident with the ovarian metastasis lacking the FGFR2 amplification and displaying its own subset of mutations and genomic aberrations. Namely, the metastatic tumor had unique biallelic events affecting TGFBR2, a critical gene in the TGF-β (tumor growth factor) signaling pathway that has not previously implicated in diffuse gastric cancer. To determine the independent oncogenic attributes of these separate pathways, we used a diffuse gastric cancer cell line and organoid model system to replicate and study the context of the known and candidate drivers found in the index patient's primary versus metastatic sites. We demonstrated that in the context of biallelic CDH1 and TP53 functional loss, the addition of either FGFR2 or TGFBR2 drivers independently lead to gastric cancer. This result supports the independent cancer driver roles of the FGF and TGF-β signaling pathways in metastatic tumor evolution in diffuse gastric cancer. Our study has implications regarding personalized cancer medicine approaches in patients with metastatic gastric cancer. Citation Format: Lincoln D. Nadauld, Sarah Garcia, Georges Natsoulis, John M. Bell, Erik Hopmans, Hua Xu, Reetesh K. Pai, John F. Regan, James M. Ford, Calvin J. Kuo, Hanlee Ji. Metastatic tumor evolution delineates clinically important oncogenic drivers in diffuse gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3165. doi:10.1158/1538-7445.AM2013-3165