Organoid Viability Research Articles

Characterization of Novel α-Mangostin and Paeonol Derivatives With Cancer-Selective Cytotoxicity.

α-Mangostin (aMan) and Paeonol (Pae) have shown anticancer and anti-inflammatory properties. However, these two natural compounds have no clinical value because of their low solubility and low membrane permeability. In this study, we screened chemically synthesized derivatives from these two natural compounds as potential novel chemicals that increase cancer cell cytotoxicity over nontransformed human cells. We found that two derivative compounds, named α-Mangostin-1 (aMan1) and Paeonol-1 (Pae1) more efficiently and more specifically induced cytotoxicity in HCT116, HT29, and SW48 colorectal cancer cell lines than the parental compounds. Both aMan1 and Pae1 arrested HCT116 cells in the G1 phase and HT29 and SW48 cells in the G2-M phase of the cell cycle. Both aMan1 and Pae1 induced apoptosis in human colorectal cancer cells, through a caspase-dependent mechanism. aMan1 and Pae1 induced selective transcriptional responses in colorectal cancer cells involving genes related to metabolic stress and DNA damage response signaling pathways. Finally, experiments on primary colon organoids showed that both derivatives were able to kill cancer-derived organoids without affecting the viability of organoids derived from healthy tissue, where the parental compounds and the currently used chemotherapeutic drug irinotecan failed. In conclusion, our findings expand the knowledge of natural compound derivatives as anticancer agents and open new avenues of research in the derivation of lead compounds aimed at developing novel chemotherapeutic drugs for colorectal cancer treatment that selectively target cancer, but not healthy cells.

Evaluation of growth, viability, and structural integrity of equine endometrial organoids following cryopreservation

Reproductive diseases in mares are a significant cause of subfertility and profound economic loss in the equine industry. Utilizing a 3D in vitro cell culture system that recapitulates the in vivo physiology will reduce time, cost, and welfare concerns associated with in vivo reproductive research in mares. If this 3D model is combined with effective cryopreservation, reproductive research on mares can occur year-round, which is not currently possible in this seasonal species. Endometrial organoids, 3D in vitro cell clusters that exhibit in vivo uterine physiology, have been established in mice, women, and mares. Here we report the first comprehensive assessment of cryopreservation of endometrial organoids in the domestic mare. Organoid growth rate was not affected by the type of freezing media. However, growth rate varied among non-cryopreserved controls, organoids cryopreserved at passage 0 (P0), and organoids cryopreserved at passage 3 (P3). Additionally, there was no difference in organoid viability among freezing media or freezing timepoint (passages). Furthermore, fresh and frozen-thawed organoids displayed positive immunohistochemical staining for ZO-1, which is a marker for intercellular tight junctions, and for periodic acid-Schiff staining as marker for organoid function through mucin production. Results demonstrate that equine endometrial organoids can be cryopreserved with 10% dimethyl sulfoxide with minimal detrimental effects while maintaining intercellular tight junctions (ZO-1) and secretory function. Availability of cryopreserved endometrial organoids may permit expanded research on uterine pathologies that negatively affect mare fertility and improve efficiency, reduce cost, and minimize animal welfare concerns associated with in vivo research in the domestic mare.

TMOD-06. CREATION OF PATIENT-DERIVED LOWER GRADE GLIOMA ORGANOID MODELS FOR PERSONALIZED TREATMENT RESPONSE ASSESSMENT

Abstract Creating in vitro models of lower grade glioma represents a major challenge in neuro-oncology research. There are few such models that are tractable and widely used, which has hindered understanding of the biology of these tumors. Recently, substantial progress has been made in generating patient-derived in vitro organoid models of high grade glioma, but modeling lower grade disease remains difficult. Based on our experience creating neurosphere cultures of lower grade astrocytomas from genetically engineered mice, we hypothesized that modifying patient-derived organoid generation protocols to incorporate physiological oxygen levels would allow establishment and propagation of lower grade glioma organoids. In this study, we show that this approach supports efficient organoid model generation from primary glioma specimens across all histological subtypes and tumor grades (WHO Grade I-IV, n = 20). These organoid models retain key characteristics of their respective parental tumors, including IDH mutations and other genetic alterations, metabolite profiles, intratumoral heterogeneity, cellular composition, and cytoarchitectural features. Importantly, lower grade glioma organoids can be cultured for months and reanimated after biobanking. Our high success rate ( >90%) in establishing organoid models from primary lower grade glioma tissue samples further highlighted opportunities for treatment response assessments. To perform longitudinal measurements of therapy-induced changes in glioma organoid viability, we designed a novel, non-invasive imaging assay (termed rapid apex imaging) to determine real-time treatment response in low and high grade gliomas. We evaluated longitudinal responses of glioblastoma and IDH1 R132H-positive Grade II astrocytoma organoids to temozolomide and olaparib with and without radiation treatment. We quantified topological changes in organoid structure by building a bioinformatics tool to translate imaging data into a cellularity metric as a biomarker of organoid response. Our work unveils an effective new method to create in vitro, personalized models of lower grade glioma that supports elucidation of treatment sensitivity profiles.

Human induced pluripotent stem cell derived kidney organoids as a model system for studying cryopreservation

The ability to cryopreserve organs would have an enormous impact in transplantation medicine. To investigate organ cryopreservation strategies, experiments are typically done on whole organs, or on cells in 2D culture. Whole organs are not amenable to high throughput investigation, while conventional 2D culture is limited to a single cell type and lacks the complexity of the whole organ. In this study, we examine kidney organoids as a model system for studying cryopreservation. Consistent with previous studies, we show that kidney organoids comprised of multiple cell types can be generated in 96-well plates, with an average of about 8 organoids per well. We present a live/dead staining and image analysis method for quantifying organoid viability and show that this method can be used for assessing cryoprotectant toxicity. Our results highlight the potential for using organoids for high throughput investigation of cryopreservation approaches.

Real-Time Non-Invasive and Direct Determination of Lactate Dehydrogenase Activity in Cerebral Organoids-A New Method to Characterize the Metabolism of Brain Organoids?

Organoids are a powerful tool in the quest to understand human diseases. As the developing brain is extremely inaccessible in mammals, cerebral organoids (COs) provide a unique way to investigate neural development and related disorders. The aim of this study was to utilize hyperpolarized 13C NMR to investigate the metabolism of COs in real-time, in a non-destructive manner. The enzymatic activity of lactate dehydrogenase (LDH) was determined by quantifying the rate of [1-13C]lactate production from hyperpolarized [1-13C]pyruvate. Organoid development was assessed by immunofluorescence imaging. Organoid viability was confirmed using 31P NMR spectroscopy. A total of 15 organoids collated into 3 groups with a group total weight of 20–77 mg were used in this study. Two groups were at the age of 10 weeks and one was at the age of 33 weeks. The feasibility of this approach was demonstrated in both age groups, and the LDH activity rate was found to be 1.32 ± 0.75 nmol/s (n = 3 organoid batches). These results suggest that hyperpolarized NMR can be used to characterize the metabolism of brain organoids with a total tissue wet weight of as low as 20 mg (<3 mm3) and a diameter ranging from 3 to 6 mm.

O47: IKK-ALPHA AS A POTENTIAL NOVEL TARGET FOR TREATMENT OF COLORECTAL CANCER

Abstract Introduction There is substantial evidence linking NF-κB and cancer-related processes. The aim of this study was to investigate the role of IKKα, the main catalytic component of non-canonical NF-κB pathway in colorectal cancer (CRC). Method Immunohistochemistry and immunofluorescence was used to examine expression of IKKα in 1030 patients undergoing resection for stage I-III CRC. To assess IKKα inhibition, organoids were prepared from wild type (WT) mouse colon, mouse models of CRC (Apc and Apc.KRAS.pT53.TGFbR2 (AKPT)) and patient derived human organoids. These were treated with an IKKα inhibitor, SU1433 and organoid size and cell viability assessed. Result High cytoplasmic expression of IKKα was associated with increasing T stage (p=0.012), poor tumour differentiation (p=0.010), tumour necrosis (p=0.013) and low proliferation status (p=0.013). High punctate IKKα expression was associated with reduced CSS (HR1.20 95%CI 1.02-1.42, p&amp;lt;0.001). Immunofluorescence showed IKKα was co-located with a Golgi-related structure. SU1433 did not significantly impact on WT (C57/B16) organoid viability up to a concentration of 1uM, however organoid size and cell viability was significantly reduced in a dose-dependent manner in organoids from both Apc and AKPT mouse models. A similar reduction was observed in patient derived human organoids. Conclusion Punctate IKKα expression was associated with poor cancer specific survival in CRC patients. The relationship between IKKα and the Golgi requires further investigation. Inhibition with SU1433, impacted on CRC mouse and patient derived human organoid size and cell viability. These results suggest that IKKα following further investigation could be employed as a novel therapeutic target in CRC. Take-home message IKKα is up-regulated in patients undergoing surgery for CRC, this is associated with poor survival outcome and inhibition of IKKα and has presented itself as a novel therapeutic target. The relationship between IKKα and the Golgi has raised interesting questions regarding the molecular behaviour of IKKα and this warrants further investigation.

Organoid microphysiological system preserves pancreatic islet function within 3D matrix.

Three-dimensional (3D) multicellular organoids recapitulate the native complexities of human tissue better than traditional cellular monolayers. As organoids are insufficiently supported using standard static culture, microphysiological systems (MPSs) provide a key enabling technology to maintain organoid physiology in vitro. Here, a polydimethylsiloxane-free MPS that enables continuous dynamic culture and serial in situ multiparametric assessments was leveraged to culture organoids, specifically human and rodent pancreatic islets, within a 3D alginate hydrogel. Computational modeling predicted reduced hypoxic stress and improved insulin secretion compared to static culture. Experimental validation via serial, high-content, and noninvasive assessments quantitatively confirmed that the MPS platform retained organoid viability and functionality for at least 10 days, in stark contrast to the acute decline observed overnight under static conditions. Our findings demonstrate the importance of a dynamic in vitro microenvironment for the preservation of primary organoid function and the utility of this MPS for in situ multiparametric assessment.

An Intestine/Liver Microphysiological System for Drug Pharmacokinetic and Toxicological Assessment.

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests phase of drug developing process because they are genetically human and recapitulate the interplay among tissues. In this study, the human intestinal barrier (emulated by a co-culture of Caco-2 and HT-29 cells) and the liver equivalent (emulated by spheroids made of differentiated HepaRG cells and human hepatic stellate cells) were integrated into a two-organ chip (2-OC) microfluidic device to assess some acetaminophen (APAP) pharmacokinetic (PK) and toxicological properties. The MPS had three assemblies: Intestine only 2-OC, Liver only 2-OC, and Intestine/Liver 2-OC with the same media perfusing both organoids. For PK assessments, we dosed the APAP in the media at preset timepoints after administering it either over the intestinal barrier (emulating the oral route) or in the media (emulating the intravenous route), at 12 µM and 2 µM respectively. The media samples were analyzed by reversed-phase high-pressure liquid chromatography (HPLC). Organoids were analyzed for gene expression, for TEER values, for protein expression and activity, and then collected, fixed, and submitted to a set of morphological evaluations. The MTT technique performed well in assessing the organoid viability, but the high content analyses (HCA) were able to detect very early toxic events in response to APAP treatment. We verified that the media flow does not significantly affect the APAP absorption whereas it significantly improves the liver equivalent functionality. The APAP human intestinal absorption and hepatic metabolism could be emulated in the MPS. The association between MPS data and in silico modeling has great potential to improve the predictability of the in vitro methods and provide better accuracy than animal models in pharmacokinetic and toxicological studies.

Butyrate enhances the efficacy of radiotherapy via FOXO3A in colorectal cancer patient‑derived organoids.

Enhancing the radioresponsiveness of colorectal cancer (CRC) is essential for local control and prognosis. However, consequent damage to surrounding healthy cells can lead to treatment failure. We hypothesized that short-chain fatty acids (SCFAs) could act as radiosensitizers for cancer cells, allowing the administration of a lower and safer dose of radiation. To test this hypothesis, the responses of three-dimensional-cultured organoids, derived from CRC patients, to radiotherapy, as well as the effects of combined radiotherapy with the SCFAs butyrate, propionate and acetate as candidate radiosensitizers, were evaluated via reverse transcription-quantitative polymerase chain reaction, immunohistochemistry and organoid viability assay. Of the three SCFAs tested, only butyrate suppressed the proliferation of the organoids. Moreover, butyrate significantly enhanced radiation-induced cell death and enhanced treatment effects compared with administration of radiation alone. The radiation-butyrate combination reduced the proportion of Ki-67 (proliferation marker)-positive cells and decreased the number of S phase cells via FOXO3A. Meanwhile, 3/8 CRC organoids were found to be non-responsive to butyrate with lower expression levels of FOXO3A compared with the responsive cases. Notably, butyrate did not increase radiation-induced cell death and improved regeneration capacity after irradiation in normal organoids. These results suggest that butyrate could enhance the efficacy of radiotherapy while protecting the normal mucosa, thus highlighting a potential strategy for minimizing the associated toxicity of radiotherapy.

Abstract 1146: Development of CRISPR human Skp2 knock-in in the prostate of mouse and the associated prostate organoids for testing Skp2 targeting agents

Abstract S-phase kinase associated protein 2 (Skp2) is a promising drug target as studies have demonstrated that Skp2 is required for spontaneous tumor development that occurs in the retinoblastoma protein (pRb), Pten or p53 deficient mice. We therefore have aimed to establish CRISPR human Skp2 (hSkp2) knock-in in the prostate of mouse to examine its role in prostate carcinogenesis. In addition, Skp2 overexpressing organoids are being developed for convenient and efficient screening of specific Skp2 inhibitors. cDNA of hSkp2 and porcine teschovirus-1 (P2A) was introduced into immediate upstream of mouse probasin coding sequencing using CRISPR knock-in targeting approach to establish transgenic mice specifically overexpressing hSkp2 in the prostate glands. Quantitative PCR, western blot and immunohistochemistry (IHC) were used to identify the expression levels of Skp2 and histopathological analyses were performed after H&amp;E staining. Prostate glands were dissected and digested for organoid culture, then morphological features and viability of organoids were evaluated following treatments of potential Skp2 inhibitors. Prostate glands and other tissues from mice of three transgenic lines which were genotyped positive were dissected and hSkp2 was detected in ventral, dorsal and lateral prostate lobes, but not in testis, kidney, spleen, and liver. Line 10092 was found to have the highest expression of hSkp2 among these three lines. Prostate weight slightly increased in transgenic mice compared to that of wild-type control mice. Hyperplasia, prostatic intraepithelial neoplasia (PIN), and carcinoma were noted, which suggested that overexpression of hSkp2 in the prostate of mice promoted proliferation and prostate tumorigenesis. Organoids that were derived from the prostate of hSkp2 transgenic mice recapitulated prostatic phenotypic features and maintained a high level of hSkp2. A novel Flavokawain A derivative was found to selectively alter the morphology of hSkp2 organoids and decrease the viability. Prostatic lesions found in the hSkp2 knock-in mice demonstrated a potential role of hSkp2 in early prostate carcinogenesis and organoids overexpressing hSkp2 have the utility for screening out compounds targeting Skp2 in prostate cancer. Citation Format: Liankun Song, Shan Xu, Kia Arabzadehkaffash, Ali Fazelpour, Dongjun Fu, Matthew Tippin, Xiaolin Zi. Development of CRISPR human Skp2 knock-in in the prostate of mouse and the associated prostate organoids for testing Skp2 targeting agents [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1146.

Abstract 3914: High-throughput drug screening of personalized chordoma organoids: A case series of six patients

Abstract Chordoma is a rare malignant tumor that arises from embryonic remnants of the notochord (Sahyouni et al, J Neurosurg Spine, 2018). This tumor is highly chemotherapy resistant, and there are no known compounds which have demonstrated clinically significant remissions (Gelderblom et al, Oncologist, 2008). With local therapies (surgery and/or radiation), local recurrence rates are high (∼40%). Repeated surgeries with or without radiation have dismal results (Fuch et al, J Bone Joint Surg, 2005). Systemic therapies are largely ineffective and often highly toxic. Given its rarity, there is limited randomized clinical data regarding effective treatments. Moreover, less than ten validated chordoma cell lines and fewer PDX models are currently available, further hindering drug development and discovery studies (Bosotti et al, Sci Reports, 2017). Here, we take advantage of tumor organoids to establish chordoma models that can be rapidly and effectively screened ex vivo to identify effective therapies. Tumor organoids are capable of reproducing many crucial features of the cancer they are generated from, including heterogeneity, cell organization as well as drug response. We have developed an automated high-throughput screening platform to test the response of patient-derived tumor organoids to hundreds of therapeutic agents, with results available within a week from surgery, a timeline compatible with therapeutic decision-making (Phan et al, Communications Biol, 2019). Here we show how our platform can be effectively used to investigate drug susceptibilities and heterogeneity in chordoma. Fresh tumour tissue from n=6 patients obtained from needle biopsies or surgical resections was processed to single cell suspension prior to establishing organoids according to our unique geometry. We performed a drug screening of up to 230 compounds on seven samples established from n=6 patients and determined organoid viability after a 48h exposure to the drugs. Our data suggest the potential efficacy of particular compounds that target the mTOR and PI3K pathways. We will show validation as well as histopathology of the organoids including staining for S100, Ki-67 and brachyury that was observed also in the pathology of specimens derived from the same patient. Overall, we have successfully established organoids from chordoma patients with a 100% success rate and 7/7 specimens growing and yielding biologically and therapeutically relevant models. Our methodology allows us to perform large drug screenings including for low grade chordomas, which are typically refractory to growth in immunocompromised mice as patient-derived xenografts. Determining the clinical efficacy of any new therapeutic approach is challenging in chordoma due to its natural history and slow growth rates. Our organoid-based functional sensitivity profile may be used to tailor therapy to each patient and to potentially rule out ineffective drugs, thus sparing ineffective therapies and associated toxicities. Citation Format: Ardy Davarifar, Huyen T. Nguyen, Nasrin Tavanaie, Jane Yanagawa, Robert Damoiseaux, Noah Federman, Nicholas Bernthal, Scott Nelson, Francis Hornicek, Alice Soragni. High-throughput drug screening of personalized chordoma organoids: A case series of six patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3914.

Abstract B18: Patient-derived xenograft and organoids models of prostate cancer

Abstract Therapy resistance and metastatic processes in prostate cancer (PCa) remain undefined, mainly due to the lack of experimental models representing different disease stages. We aim to provide functional experimental and preclinical drug tools applicable for direct use with patient-derived material. Biopsies from metastatic PCa were used for the establishment of patient-derived xenografts (PDXs) by subcutaneous implantation. In vivo tumor growth kinetic in response to androgens was assessed by surgical castration and testosterone supplementation. RNA and whole-exome sequencing (WES) and organoid drug screens were used to characterize the genomic, transcriptomic, and functional properties. Organoid culture methodology was adapted in order to set up an automated medium-throughput organoid drug screen (Nexus Theragnostics automated platform) for screening standard-of-care compounds and candidates for drug repurposing. A novel case of PCa xenograft model derived from soft tissue metastasis (PNPCa) was established. RNAseq and WES confirmed transcriptomic and genomic similarity to the primary tumor. PNPCa harbors BRAC2 and CHD1 mutations and shows SPOP-like and FOXA1-like transcriptomic signature with microsatellite instability. PNPCa tumor growth is inhibited after androgen deprivation by castration, while androgen replacement leads to tumor reformation. No spontaneous tumor growth occurred after prolonged period of castration; however, scarce micrometastases were found in the bone marrow and lymph nodes. The treatment-naive and androgen-dependent properties of the PNPCa made it a candidate model for identification of potent drug compounds. PNPCa-derived organoid screens on standard-of-care and 74 FDA-approved compounds showed that mTOR pathway and multi-tyrosine kinase inhibitors, used for clinical treatment of other cancer types, have high impact on PCa organoid viability. Application of the organoid drug screen panel on two additional metastatic PDXs of advanced disease has allowed shortlisting of compounds for repurposing use in patient-derived organoid screens that can be routinely performed in a timeframe of few weeks, and thus, provide information on treatment decision. Treatment response of a treatment-naive, early metastatic PCa case (PNPCa PDX) highlights the potential of organoid screens to assess therapy response with extended applications for personalized screens. Citation Format: Sofia Karkampouna, Federico la Manna, Maria R. De Filippo, Mirjam Kiener, Marta De Menna, Eugenio Zoni, Joel Grosjean, Irena Klima, Andrea Garofoli, Marco Bolis, Jean-Philippe Theurillat, Vera Genitsch, David Keller, Booij Tijmen, Christian U. Stirnimann, Andrea Sboner, Charlotte K.Y. Ng, Salvatore Piscuoglio, Martin Spahn, Mark A. Rubin, George N. Thalmann, Marianna Kruithof-de Julio. Patient-derived xenograft and organoids models of prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B18.

Comparisons between Animal Models and Intestinal Crypt Cultures when Investigating the Impact of Fatty Acids on Gene Expression in Intestinal Epithelial Cells

Developmental delay of UDP‐glucuronosyltransferase 1A1 (UGT1A1) expression is central toward the development of neonatal hyperbilirubinemia, observed clinically as increased total serum bilirubin (TSB) in newborns. UGT1A1 is the sole enzyme responsible for the glucuronidation and detoxification of bilirubin. We have developed a murine model that resembles neonatal hyperbilirubinemia. In humanized UGT1 (hUGT1) mice, the expression of intestinal but not hepatic UGT1A1 is inversely associated with TSB levels. In addition, the expression of intestinal UGT1A1 is closely associated with proliferation and maturation of intestinal epithelial cells (IECs). It is known that fatty acids (FAs), the major component of breast milk, plays an important role in intestinal development. We have treated hUGT1 neonatal mice with oleic acid (OA, 16:1), a monounsaturated fatty acid. Following OA exposure, TSB levels in hUGT1 neonatal mice were reduced significantly, and RT‐qPCR analysis demonstrated that intestinal human UGT1A1 was induced, along with increased expression of Cyp4a10, a target gene of peroxisome proliferator‐activated receptor alpha (PPARα). In addition, IEC maturation markers including sucrase‐isomaltase (Sis) and alkaline phosphatase 3 (Akp3) are also induced. To examine the mechanism of OA induction, intestinal crypt cells were isolated from hUGT1 mice. The organoids were exposed to different fatty acids, including OA, palmitoleic acid (PA), arachidonic acid (AA), linoleic acid (LA), and docosahexaenoic acid (DHA). Gene expression parameters were monitored 24 hours after exposure. We found that OA‐ and PA‐treated organoids have similar survival rates in comparison to controls, but at similar concentrations of LA, DHA or AA cell death and decreased organoid viability was observed. At lower FA concentrations, RT‐qPCR analysis demonstrated that the UGT1A1 gene was not induced in any of the samples, including the IEC maturation markers Sis and Akp3. However, increased expression of the Cyp4a10 gene resulted in DHA, AA, and LA‐treated organoids. By comparing the in vivo and in vitro experiments, it suggests that unlike Cyp4a10, the regulation of UGT1A1 by fatty acids is associated with the development of the intestinal tract. Under current culture conditions, the application of intestinal organoid cultures in the study of developmental‐associated events is limited.Support or Funding InformationSupported in part by NIH grants GM126074 and ES010337

PD47-11 RAPID ORGANOID DEVELOPMENT, DRUG SCREENING, AND NEOADJUVANT CHEMOTHERAPY RESPONSE PREDICTION FOR PATIENTS WITH LOCALLY-ADVANCED BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology IV (PD47)1 Apr 2020PD47-11 RAPID ORGANOID DEVELOPMENT, DRUG SCREENING, AND NEOADJUVANT CHEMOTHERAPY RESPONSE PREDICTION FOR PATIENTS WITH LOCALLY-ADVANCED BLADDER CANCER Kathryn A Marchetti*, Nathan Merrill, Nathalie Vandecan, Liwei Bao, Xu Cheng, Aaron Udager, Lindsey A Herrel, Jeffery Montgomery, Khaled Hafez, Todd Morgan, Alon Weizer, Ajjai Alva, Matthew Soellner, Sofia Merajver, and Samuel Kaffenberger Kathryn A Marchetti*Kathryn A Marchetti* More articles by this author , Nathan MerrillNathan Merrill More articles by this author , Nathalie VandecanNathalie Vandecan More articles by this author , Liwei BaoLiwei Bao More articles by this author , Xu ChengXu Cheng More articles by this author , Aaron UdagerAaron Udager More articles by this author , Lindsey A HerrelLindsey A Herrel More articles by this author , Jeffery MontgomeryJeffery Montgomery More articles by this author , Khaled HafezKhaled Hafez More articles by this author , Todd MorganTodd Morgan More articles by this author , Alon WeizerAlon Weizer More articles by this author , Ajjai AlvaAjjai Alva More articles by this author , Matthew SoellnerMatthew Soellner More articles by this author , Sofia MerajverSofia Merajver More articles by this author , and Samuel KaffenbergerSamuel Kaffenberger More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000934.011AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Neoadjuvant chemotherapy prior to radical cystectomy (RC) for muscle-invasive urothelial carcinoma is the standard of care despite a small absolute survival benefit and the potential for disease progression during chemotherapy. While progress has been made in the prediction of sensitivity to platinum-based chemotherapies, providing more accurate, personalized, and clinically-relevant chemotherapy response prediction is an unmet need. We present our early and ongoing experience with rapid, organoid-based drug-screening. METHODS: From patients undergoing a transurethral resection of bladder tumor or RC, 1 gram of tumor was procured and divided between DNA/RNA sequencing, organoid drug-screening, and patient-derived xenografts. Tissue was dissociated, filtered, and resuspended in organoid media for serial passage and drug screening. Drugs were tested at the maximum plasma concentration in human trials, so to provide physiologic relevance. Results were normalized to control such that a value of “100” indicated no difference in organoid viability compared to control, and a value of 0 indicated complete response. The number of drugs screened was dependent upon tissue available, but was often 20-30. RESULTS: Thus far, 25 patients have undergone rapid organoid development and drug testing. Drug response analyses were available on average 5-10 days following the procedure. Secondary to limited space, select drug response data from the 20 most recent patient organoid (org) samples are presented (Figure 1). All patients have DNA and RNA sequencing and PDX models in progress. CONCLUSIONS: Rapid organoid development and drug screening allows for the determination of neoadjuvant chemotherapy response within 5-10days. Use of this technique on tissue provided during disease work-up may further guide selection of effective therapeutic agents in patients with bladder cancer. This would overall minimize the morbidity of standard of care therapies. Source of Funding: none © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e929-e929 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kathryn A Marchetti* More articles by this author Nathan Merrill More articles by this author Nathalie Vandecan More articles by this author Liwei Bao More articles by this author Xu Cheng More articles by this author Aaron Udager More articles by this author Lindsey A Herrel More articles by this author Jeffery Montgomery More articles by this author Khaled Hafez More articles by this author Todd Morgan More articles by this author Alon Weizer More articles by this author Ajjai Alva More articles by this author Matthew Soellner More articles by this author Sofia Merajver More articles by this author Samuel Kaffenberger More articles by this author Expand All Advertisement PDF downloadLoading ...

Evaluation of cisplatin-induced injury in human kidney organoids.

Acute kidney injury (AKI) remains a major global healthcare problem, and there is a need to develop human-based models to study AKI in vitro. Toward this goal, we have characterized induced pluripotent stem cell-derived human kidney organoids and their response to cisplatin, a chemotherapeutic drug that induces AKI and preferentially damages the proximal tubule. We found that a single treatment with 50 µM cisplatin induces hepatitis A virus cellular receptor 1 (HAVCR1) and C-X-C motif chemokine ligand 8 (CXCL8) expression, DNA damage (γH2AX), and cell death in the organoids but greatly impairs organoid viability. DNA damage was not specific to the proximal tubule but also affected the distal tubule and interstitial cell populations. This lack of specificity correlated with low expression of proximal tubule-specific SLC22A2/organic cation transporter 2 (OCT2) for cisplatin. To improve viability, we developed a repeated low-dose regimen of 4 × 5 µM cisplatin over 7 days and found this caused less toxicity while still inducing a robust injury response that included secretion of known AKI biomarkers and inflammatory cytokines. This work validates the use of human kidney organoids to model aspects of cisplatin-induced injury, with the potential to identify new AKI biomarkers and develop better therapies.

IKKα as a potential novel target for treatment of colorectal cancer.

174 Background: Colorectal cancer (CRC) is a heterogeneous disease leading to different survival outcomes for patients with the same stage of disease. The non-canonical NF-κB pathway has been shown to have a key role in tumorigenesis, and the aim of this study was to investigate the role of IKKα, the main catalytic component of this pathway in CRC. Methods: A tissue microarray was retrospectively constructed from a patient cohort (1033) with stage I-III CRC who underwent surgery. IHC was utilised to examine cytoplasmic and punctate IKKα expression and determine any association with clincopathological features and cancer specific survival (CSS). To assess IKKα inhibition, organoids were prepared from wild type (WT) mouse colon, mouse models of CRC (Apc and Apc.KRAS.pT53.TGFbR2 (AKPT)) and patient derived human organoids. These were treated with an IKKα inhibitor, SU1433 and organoid size and cell viability assessed. Results: High cytoplasmic expression of IKKα was associated with increasing T stage (p = 0.012), poor tumour differentiation (p = 0.010), tumour necrosis (p = 0.013) and low proliferation status (p = 0.013) but was not associated with CSS. High punctate IKKα expression associated with tumour differentiation (p = 0.001), necrosis (p = 0.004), proliferation (p = 0.044) and MMR competence (p &lt; 0.001) and was also significantly associated with reduced CSS (HR1.20 95%CI 1.02-1.42, p &lt; 0.001). SU1433 did not significantly impact on WT (C57/B16) organoid viability up to a concentration of 1 uM, however organoid size and cell viability was significantly reduced in a dose dependent manner in organoids from both Apc and AKPT mouse models. A similar reduction was observed in patient derived human organoids. Conclusions: Punctate IKKα expression was associated with poor cancer specific survival in CRC patients, and inhibition with SU1433, impacted on CRC mouse and patient derived human organoid size and cell viability. These results suggest that, following further investigation and confirmation, IKKα may be employed as a novel therapeutic target in CRC.

Combined EZH2 and BCL2 Inhibitors As Precision Therapy for Genetically Defined DLBCL Subtypes

DLCBL has recently been classified into genetically defined subtypes based on groupings of particular genetic lesions (Chapuy et al, Nat Med 2018; Schmitz et al, NEJM 2018). One predominant cluster, C3 or EZB, is defined by mutations in the chromatin modifying genes EZH2, KMT2D, and CREBBP as well as alterations in BCL2 including mutations and/or translocation of BCL2 to the IgH enhancer. Since tumors in this cluster are likely dependent on both EZH2 and BCL2, and these oncogenes carry out their effects through distinct mechanisms and pathways, targeting both of these oncogenes is a rational therapeutic approach. We hypothesized that EZH2 inhibition and BCL2 inhibition would be synergistic in DLBCL with characteristics of the C3/EZB cluster. To test this, we evaluated the EZH2 inhibitor tazemetostat and the BCL2 inhibitor venetoclax in DLBCL cells, 3D lymphoma organoids, and patient derived xenografts. To assess the effect of combination therapy with tazemetostat and venetoclax, we administered each drug alone and the combination in a panel of DLBCL cell lines, including cells with and without EZH2 mutation and BCL2 translocation. In DLBCL cells with both a BCL2 translocation and EZH2 mutation, the combination resulted in increased killing compared to either drug alone (Figure 1, SUDHL6 (p&amp;lt;0.005), WSU-DLCL2 (p&amp;lt;0.005), and OCI-LY1 (p&amp;lt;0.005)). In contrast, in cells with WT EZH2 and no BLC2 translocation, the effect of the combination was not different than either drug alone. To evaluate for synergy, cells were exposed to increasing doses of each drug alone and the combination. The combined response was evaluated using the Chou-Talalay method. Synergy between tazemetostat and venetoclax was observed in SUDLH-6 (CI value 0.03), WSU-DLCL2 (CI 0.26) and OCI-LY1 (CI 0.06) but not in Farage and LY7 both of which have WT EZH2 and no BCL2 translocation. Since cell lines in suspension do not reflect lymph node architecture, we developed a 3D lymphoma "organoid" culture system that consists of extracellular matrix, lymphoma cells, and stromal cells (Tian et al, Biomaterials 2015; Beguelin et al, Nat Commun 2017). GCB-DLBCLs express integrin αvβ3 that may bind to RGD peptides or vitronectin in tumor extracellular matrix. Based on this, we developed matrix metalloproteinase (MMP)-degradable 3D lymphoma hydrogels functionalized with RGD peptides. To generate organoids, we homogeneously encapsulated DLBCL cells (40,000/gel) in 10 µL hydrogel droplets fabricated in individual wells of a 96-well plate. We established two organoid systems to evaluate response to tazemetostat/venetoclax combination therapy: 1) OCI-LY1 organoids; 2) patient derived xenograft (PDX) organoids. The PDX organoids were generated from a patient tumor after propagation in NSG mice. The PDX tumor harbors both a BCL2 translocation and EZH2 mutation. Organoid viability was evaluated using immunofluorescence for calcein AM (live cells) and ethidium homodimer (dead cells) as well as flow cytometry. In both OCI-Ly1 organoids and PDX organoids, tazemetostat and venetoclax had minimal activity as single agents, however the combination resulted in significant cell killing (Figure 2). To investigate potential mechanisms of synergy, we evaluated RNA-seq profiles of a panel of DLBCL cell lines (n=26) treated with vehicle vs. EZH2 inhibitor (Brach et al, Mol Cancer Ther 2017). Preliminary data suggest that EZH2 inhibition induces expression of pro-apoptotic proteins genes including BCL2L11 (Wilcoxon p=0.01), BAD (p=0.02), BMF (p&amp;lt;0.01), BCL2L13 (p=0.02), and BCL2L14 (p&amp;lt;0.01). BCL2 inhibition with venetoclax may be further enhancing pro-apoptotic signals and lymphoma cell death, especially in C3/EZB DLBCL tumors with dependence on BCL2. In summary, using novel model systems, we have demonstrated that BCL2 inhibition combined with EZH2 inhibition results in synergistic anti-tumor effect that is anticipated to be especially effective as precision therapy for the newly identified cluster 3/EZB DLBCL subtype. A clinical trial of this combination is currently in development. Disclosures Melnick: Janssen: Research Funding; Constellation: Consultancy; Epizyme: Consultancy. Roth:Janssen: Consultancy; ADC Therapuetics: Consultancy; Merck: Membership on an entity's Board of Directors or advisory committees.

PB2099 PATIENT‐SPECIFIC MULTIPLE MYELOMA MODEL USING 3D PRINTED ORGANOIDS FOR PREDICTING AND UNDERSTANDING TREATMENT RESPONSE

Background:Results of studies using current pre‐clinical multiple myeloma (MM) models are limited in their ability to incorporate the effects bone marrow microenvironment has on cell proliferation, cell death, and drug resistance. 3D constructs, or organoids, have recently been tested to better recapitulate MM tumors and their microenvironment using a combination of commercially available and primary cell cultures. Here we present the first reported viable patient‐derived organoids composed entirely from fresh tumor cells co‐cultured with the patient's own stromal stem cells. This patient‐specific model serves as a platform to study tumor behavior and treatment screening.Aims:We aim to construct a durable 3D in vitro model derived exclusively from patient's own myeloma and stromal cells to serve as a true patient‐specific model. We examined for adequate representation of the bone marrow microenvironment throughout the life of the organoid and feasibility of drug screening.MethodsPrimary cells Bone marrow (BM) aspirates were collected from MM patients undergoing treatment. In the first 24 hours, samples were treated with Ficoll‐paque solution for cell sorting. CD138+ cells were positively selected and isolated using coated magnetic beads. The CD138+ cells were then fluorescently tagged with nanocrystals into the cytoplasm. Total BM pellet and assorted cell count and viability was quantified by the NucleoCounter NC‐200 automated cell counter. 3D Organoids The isolated stromal and multiple myeloma cells were incorporated into an ECM‐based hydrogel system and high throughput bioprinted into 3D patient‐specific disk‐shaped organoids. The bioengineered set up consisted of a 96 well plate, were 10 μl of the ECM/cell mixture was dispensed. Each organoid consisted of 200,000 cells (cell density of 20 × 106 cell/ml). Each well was then exposed to UV for hydrogel crosslinking and organoids covered in tissue culture media that was changed on day 2 and 4. Drug treatment studies Organoids were screened in parallel with established treatment regimens (Figure 1). Their viability was assessed by live/dead and ATP metabolism assays, and corroborated by H&amp;E staining. The results described were performed in triplicate. Immunohistochemistry Parafin blocks of organoids were obtained at each evaluation time point using CD138+ for plasma cells, CD11b+ for stromal cells, and additional CD45+ for lymphocyte infiltrates.Results:We were able to build a microenvironment to sustain patient stromal cells and fresh tumor cells that allowed 3D models to live long enough for tumor studies and chemotherapy screening. A minimum of 6cc of aspirate is sufficient to bioprint a 96 well plate experiment per patient. Average time from sample aspiration to organoid development to initiation of drug protocols was 24 hours, with a metabolic viability of 7 days. Each group of patient‐specific organoids exhibited a mixed response to the three stablished therapies, but a low variability inside each regiment.Summary/Conclusion:Our organoid constructs provide the first reported in vitro patient‐specific 3D MM model that mimics in vivo environmental and cellular composition of a patient tumor niche. By maintaining patient‐derived organoids viable for 5‐7 days, chemotherapy screening to guide patient care could be a possibility in the near future. The ability to sort cells prior to performing organoid constructs had no impact on organoid viability and could allow for studies such as gene editing, CAR‐T therapy, or niche studies combining different cell types. Further testing is needed to optimize cell viability of different cell types.image

Prostate Stroma Increases the Viability and Maintains the Branching Phenotype of Human Prostate Organoids

SummaryThe fibromuscular stroma of the prostate regulates normal epithelial differentiation and contributes to carcinogenesis in vivo. We developed and characterized a human 3D prostate organoid co-culture model that incorporates prostate stroma. Primary prostate stromal cells increased organoid formation and directed organoid morphology into a branched acini structure similar to what is observed in vivo. Organoid branching occurred distal to physical contact with stromal cells, demonstrating non-random branching. Stroma-induced phenotypes were similar in all patients examined, yet they maintained inter-patient heterogeneity in the degree of response. Stromal cells expressed growth factors involved in epithelial differentiation, which was not observed in non-prostatic fibroblasts. Organoids derived from areas of prostate cancer maintained differential expression of alpha-methylacyl-CoA racemase and showed increased viability and passaging when co-cultured with stroma. The addition of stroma to epithelial cells in vitro improves the ability of organoids to recapitulate features of the tissue and enhances the viability of organoids.

A Simple Bioreactor-Based Method to Generate Kidney Organoids fromPluripotent Stem Cells.

SummaryKidney organoids made from pluripotent stem cells have the potential to revolutionize how kidney development, disease, and injury are studied. Current protocols are technically complex, suffer from poor reproducibility, and have high reagent costs that restrict scalability. To overcome some of these issues, we have established a simple, inexpensive, and robust method to grow kidney organoids in bulk from human induced pluripotent stem cells. Our organoids develop tubular structures by day 8 and show optimal tissue morphology at day 14. A comparison with fetal human kidneys suggests that day-14 organoid tissue most closely resembles late capillary loop stage nephrons. We show that deletion of HNF1B, a transcription factor linked to congenital kidney defects, interferes with tubulogenesis, validating our experimental system for studying renal developmental biology. Taken together, our protocol provides a fast, efficient, and cost-effective method for generating large quantities of human fetal kidney tissue, enabling the study of normal and aberrant kidney development.