Notably, the expression patterns of CD117 in PTC- and NTG-derived organoids were consistent with their corresponding tumor tissues. After cluster generation, the DNA libraries were sequenced on an Illumina HiSeq platform and 150bp paired-end reads were generated. A9165), nicotinamide (10 mM, Sigma-Aldrich, No. 2023 May;11(5):e006290. Groups sharing the same alphabets (a, b, c) above the bars are not significantly different from one another (P>.05), whereas those not sharing the same alphabets represent significant difference at P less than .05 by analysis of variance (ANOVA) plus Tukey multiple comparison test. All data generated or analyzed during this study are included in this published article or in the data repositories listed in References (20). The whole fluorescence intensity (FI) of EdU and Hoechst 33342 in each well were measured using the VICTOR Nivo Multimode Plate Reader (PerkinElmer Inc), and the proliferation levels were presented as a percentage of EdU-FI/Hoechst-FI. Broutier These organoid lines have been propagated by serial passaging and have been successfully cryopreserved and retried. Roerink 4A and 4B, Supplementary Table 1 and 2 [20]). 6 and Supplementary Fig. Organoid Culture. This site needs JavaScript to work properly. Passage numbers of the organoid lines were PTC-1_O, P4; PTC-2-I_O, P5; PTC-2-II_O, P5; PTC-3_O, P6; PTC-4-I_O, P4; PTC-4-II_O, P4; PTC-5_O, P6; PTC-6-A_O, P2 (culturing for 26 days); PTC-6-B_O, P8 (culturing for 121 days); PTC-7_O, P6; PTC-8_O, P6. Innovation: organoids-a better model for prostate cancer. Each individual specimen was split into several parts that were processed for organoid culture, histology diagnosis, and genomic analysis (Fig. A. Quantification of number of non-synonymous single nucleotide variations (SNV, pink) and insertion-deletions (Indel, orange) in each organoid sample compared to average total SNV and Indels of published whole-exome datasets (green). Our study demonstrates that the newly established PTC organoids may have potential to predict each individual patients response to treatment and support a potential strategy to attenuate tumor aggressiveness in women with ER-specific antagonists. Objective: To understand thyroid cancer, especially tumor heterogeneity, and to translate this knowledge to clinical applications, representative and robust preclinical thyroid cancer models are urgently needed. Gastric cancer samples were removed from the preservation solution and repeatedly rinsed with D-PBS (Solarbio, D1040) containing 100 U/mL penicillin-streptomycin-amphotericin B until the D-PBS was clear . , Holtzinger A, Jagan I, et al. National Library of Medicine As expected, after 4 months of culture, we found that the mutational landscape and CNVs of PTC-6_O (PTC-6-A_O: early passage, PTC-6-B_O: late passage) were stably maintained (see Fig. Similarly, the PTC-6-A/B_O line harboring the BRAF mutation represents the resistance to BRAFV600E-target drugs, highlighting the value of the combination of WES and organoid-based drug-screening assays in PTC organoids. 4A-4E). Epub 2014 Sep 23. These reagents were carefully diluted with dimethyl sulfoxide (DMSO) according to the respective manufacturers instructions to prepare stock solutions, and stored at 80 C. A, Heat map of logIC50 values for 24 anticancer drugs used to treat 11 PTC organoids derived from 8 patients by applying nonlinear regression. 3603), in duplicates. Organoid Cultures Derived From Patients With Papillary Thyroid Cancer Mutation effect predictions and annotations were performed by ANNOVAR and dbSNP. HY-12452), PHTPP (ER-selective antagonist, No. Careers. Prostate cancer patientderived organoids: detailed outcome from a , Chen GG, Vlantis AC, van Hasselt CA. Bottom: Copy number landscape of seven patient derived organoid lines and six publically available prostate cancer cell lines using array CGH . 2021 Feb 18;12(1):1117. doi: 10.1038/s41467-021-21300-6. Preclinical Models of Follicular Cell-Derived Thyroid Cancer: An Overview from Cancer Cell Lines to Mouse Models. The lack of in vitro prostate cancer models that recapitulate the diversity of human prostate cancer has hampered progress in understanding disease pathogenesis and therapy response. Breast Cancer Res Treat. After 2 days of culture, the organoids were incubated with E2 (10 nM or 100 nM), PPT (100 nM), DPN (100 nM), or G-1 (100 nM) at 37 C for another 2 days. Papillary thyroid cancer (PTC) has been one of the most frequent endocrine malignancies around the world. 2 [20]). B-Raf proto-oncogene, serine/threonine kinase (BRAF), the most frequently mutated gene in PTC (22) and the well-known driver gene associated with the mitogen-activated protein kinase (MAPK) signaling pathway, was mutated in 5 of 8 patients (Fig. Renal tumouroids: challenges of manufacturing 3D cultures from patient E, Representative gene copy number variations (CNVs) of PTC tumors and the paired organoids (red, gain; blue, loss; green, diploid). FOIA To plate organoids for drug sensitivity screening, PTC organoid lines were harvested 7 days after passaging and passed through a 70-m cell strainer to eliminate large organoids. As first shown in colorectal cancer (6), tumor organoids faithfully maintained the characteristic of tumor tissues from which they were derived. Abbreviations: Akt, protein kinase B; BRAF, B-Raf proto-oncogene, serine/threonine kinase; Cat., catalog; MEK, mitogen-activated protein kinase kinase; mTOR, mammalian target of rapamycin; PDGFR, platelet-derived growth factor receptor ; VEGFR, vascular endothelial growth factor receptor. Left behind was a cancer organoid - a 3D mass . The complete medium for PTC organoids consisted of AdDMEM/F12+++ supplemented with B-27 (1, Gibco, No. Epub 2020 Feb 21. This could possibly be due to the intratumor diversification of the tumor samples. N0636), noggin (100 ng/mL, PeproTech, No. 4A and 4D [20]). In addition, PTC-6-A_O and PTC-6-B_O represent the early-passage (passage 8) organoid lines originating from the tumors of patient 6, respectively. Epub 2022 Dec 26. 2023 Jul;200(2):193-201. doi: 10.1007/s10549-023-06973-5. 3D organ-on-a-chip: The convergence of microphysiological systems and organoids. The corresponding patients normal blood was also sequenced and used as a reference. In agreement with the absence of 3 ERs in both tumor-organoid pairs of PTC-8 as stated earlier, E2 and 3 ER agonists all showed no stimulatory effects on cell proliferation of organoid lines (Fig. Kim doi: 10.1136/jitc-2022-006290. For each patient, the sex, age, tumor size, and clinical stage were recorded when available (Table 1). Organoid in colorectal cancer: progress and challenges Endocrinol Metab (Seoul). Drug sensitivity testing and organoid cell . Organoids in cancer research | Nature Reviews Cancer 2023 Jun 5;20(5):353-68. doi: 10.20892/j.issn.2095-3941.2023.0061. 01 February 2021 Article history PDF Split View Cite Permissions Share Abstract Context Papillary thyroid cancer (PTC) has been one of the most frequent endocrine malignancies around the world. ab39742) were applied to sections and incubated at 4 C overnight. Organoid cultures derived from patients with advanced prostate cancer Zhao Z, Chen X, Dowbaj AM, Sljukic A, Bratlie K, Lin L, Fong ELS, Balachander GM, Chen Z, Soragni A, Huch M, Zeng YA, Wang Q, Yu H. Nat Rev Methods Primers. 356231), and 6 drops of 45-L Matrigel-cell suspension were allowed to solidify in one well of a prewarmed 6-well plate at 37 C for 10 minutes. Two organoid cultures from different donors were obtained. The PDOs can be used to build a biobank and perform single-cell sequencing, drug screening and genetic analyses. Several studies have been conducted using two-dimensional cultured cell lines; however, these cells do not represent a study model of patient tumors very well. SZSM202011017), and the China Postdoctoral Science Foundation (grant No. Notably, PTC organoids preserve the histopathological profiles and genomic heterogeneity of the originating tumors. After 2 days of treatments, cell proliferation of PTC organoids was measured by 5-ethynyl-2deoxyuridine (EdU) uptake according to the manufacturers instructions. The culture of human PTC organoids requires the presence of the potent mitogenic factor EGF even though PTC-1_O and PTC-2-I_O lines both harbor the BRAFV600E mutation. The present study used established and modified culture conditions to report successful generation and characterization of patientderived organoids from fresh primary tissue specimens of patients with treatmentnave . 5). , Gardner L, Hughes J, et al. In our study, drug screening of human PTC organoid lines demonstrated patient-specific sensitivities to drugs. , Bonofiglio D, Albanito L, et al. MAB-0572), thyroglobulin (Tg, 1:200, Abcam, No. Surgically resected PTC primary tissues were dissociated and processed for organoid derivation. There is a pressing need for clinically relevant preclinical thyroid cancer models for personalized therapy because of the lack of in vitro models that faithfully represent the biology of the parental tumors. B and F, Representative confocal images of cell proliferation in PTC organoids induced by 10 nM and 100 nM E2, 100 nM propylpyrazole triol (PPT), 100 nM DPN (2,3-bis(4-hydroxyphenyl)-propionitrile), and 100 nM GPER-selective agonist (G-1). Mutation features were clustered with 30 known mutation features in COSMIC, in which the sample mutation pattern was described by known features. The site is secure. Dong Chen and others, Organoid Cultures Derived From Patients With Papillary Thyroid Cancer, The Journal of Clinical Endocrinology & Metabolism, Volume 106, Issue 5, May 2021, Pages 14101426, https://doi.org/10.1210/clinem/dgab020. Each data point represents the meanSEM of 3 technical duplicates. Relationship between genetic alterations and clinicopathological characteristics of papillary thyroid carcinoma. Patient-derived tumor organoids (PDOs) currently represent important modeling tools in pre-clinical investigation of malignancies. Cell viability was detected after 6 days of incubation by using the CellTiter-Glo 3D Reagent (Promega, No. Surgically resected PTC primary tissues were dissociated and processed for organoid derivation. These data suggest that mutations in the RAS/BRAF/MAPK pathway alone were insufficient to compensate for the absence of EGF, and additional molecular variations in EGF signal pathways may be required. 5) (20). FOIA For instance, we observed that PTC-1_O and PTC-2_O appeared as dense spheres consisting of multiple layers of cells, whereas PTC-3_O exhibited a hollow morphology with cells that appear to be wreathed (Fig. Bethesda, MD 20894, Web Policies Y and transmitted securely. 4E). Cell nuclei were counterstained with Hoechst 33342 solution. Mutational landscape of patient-derived prostate, Figure 2. OS often spreads to lungs, an optimized model for studying. In our study, we described a 3D culture system for successful and efficient generation of patient-derived PTC organoid lines in vitro. The components of culture medium for human PTC organoids were based on the previously reported culture conditions for human breast cancer organoids (12) and mouse normal thyroid organoids (19). Furthermore, PTC organoids developed in our study showed both cohesive and discohesive structures in the same culture, with the cohesive shapes accounting for the vast majority in all cultures. 2022 Dec;37(6):830-838. doi: 10.3803/EnM.2022.1636. However, ER has been shown to exert inhibitory actions (40). Figure 1. 2 [20]). However, study research on colorectal cancer organoids found that 6 of 14 lines with the KRAS mutation remained dependent on EGF (27). F, Time-lapse imaging sequence of PTC organoids. Patient-Derived Organoids: The Beginning of a New Era in Ovarian Cancer 4A) were sensitive to the BRAFV600E inhibitor vemurafenib and dabrafenib, whereas other lines with wild type for BRAF were resistant (Fig. government site. 55 Two effective anti-tumor compounds that suppress CBC organoids growth were . The minced tissues were digested with collagenase type II (5 mg/mL, Gibco, No. Front Cell Dev Biol. Furthermore, we screened different culture media to find the minimum requirement for the establishment of PTC organoid lines. Although most PTC patients have a favorable prognosis, a subgroup of patients die, especially when disease recurrence occurs. Magr L, Verstegen MMA, Buschow S, van der Laan LJW, Peppelenbosch M, Desai J. J Immunother Cancer. 1E). Recently, Sondorp et al (32) reported the establishment of normal thyroid organoids with a Wnt3a condition medium. The mutational profiles of PTC-2-I_O were highly concordant with those of PTC-2_T, but distant from PTC-2-II_O. 6E). Search for other works by this author on: Department of Breast and Thyroid Surgery, Shenzhen Second Peoples Hospital, Department of Nuclear Medicine, Peking University Shenzhen Hospital. 4) (20). Organoid models derived from patients with malignant - Springer , de Witte CJ, Lhmussaar K, et al. PTC, papillary thyroid cancer; NTG, nodular thyroid goiter; Normal, nontumor normal tissue; T, tumor; O, organoid. HY-107216), and G15 (GPER-selective antagonist, No. Patient-derived xenografts and organoids model therapy response in prostate cancer. Development of glioblastoma organoids and their applications in personalized therapy. The light blue line represents the . We observed both cohesive (dense and solid structures) and discohesive (irregularly shaped structures) organoids within the same culture derived from the same specimen (Fig. The lack of in vitro prostate cancer models that recapitulate the diversity of human prostate cancer has hampered progress in understanding disease pathogenesis and therapy response. In addition to histological conservation, subsequent marker expression analysis revealed that organoids and their matched tumors exhibited similar staining patterns for CK19, galectin-3, Tg, Ki-67, CD117, and TTF-1 (see Fig. The process, from receiving the sample in the research lab, to having a culture in the incubator, in preparation for the organoids to form, takes around 4 hours. 8600 Rockville Pike Organoids | Nature Reviews Methods Primers Financial Support: This work was supported by the National Key R&D Program of China (grant No. Abbreviations: F, female; M, male; NA, not available; NTG, nodular thyroid goiter; PTC, papillary thyroid cancer. Human primary liver cancer-derived organoid cultures for disease modeling and drug screening. Overall, our studies using ER-selective agonists and antagonists demonstrate that ER is the predominant mediator for E2-induced cell proliferation in PTC organoid lines. TTF-1, a marker of thyroid differentiation, was present in all tumor tissues, their corresponding organoids, and nontumor tissues. For permissions, please e-mail: journals.permissions@oup.com. This is probably due to the intratumor heterogeneity. Effects of estradiol (E2) and estrogen receptor (ER) modulators on cell proliferation of papillary thyroid cancer (PTC) organoids derived from female patients. Floating in culture medium, the tumour samples survived. Hernndez-Llodr S, Segals L, Safont A, Juanpere N, Lorenzo M, Fumad L, Rodrguez-Vida A, Cecchini L, Bellmunt J, Lloreta-Trull J. Prostate. Disclaimer. Cancer immunotherapies immune checkpoint inhibitors as patient-derived organoids are under investigation as a personalized platform for drug discovery and for predicting clinical efficacy of chemotherapies and targeted agents. SOP40104: Cryopreservation of Patient-Derived Organoid (PDOrg) Cultures; SOP30108: Implantation of Patient-Derived in vitro Material (2D and 3D cell cultures) for Generation of Cell Line Xenografts (CLXs) PDMR Genomics. To determine whether cell proliferation of organoids will respond to estrogen stimulation and to study estrogen receptors that are involved in this process, we treated PTC organoid lines derived from female patients with E2, either in the presence or absence of the selective ER antagonists, or with the selective ER agonists. Le Pennec Chen D, Su X, Zhu L, Jia H, Han B, Chen H, Liang Q, Hu C, Yang H, Liu L, Li P, Wei W, Zhao Y. J Transl Med. For immunostaining, paraffin slides were deparaffinized and subjected to 3% hydrogen-peroxide buffer for 15 minutes to remove the endogenous peroxidase activity. Although most PTC patients have excellent overall survival, a subgroup of patients die, particularly when disease recurrence occurs. To test the critical composition of medium required for organoid formation, freshly resected PTC tissues were dissociated and digested into single cells, and cultured in the absence of the indicated compound from the complete medium for 10 days. Nat Methods. All rights reserved. 2). Epub 2018 Jan 31. See also Supplementary Table 1 (20). Organoid Cultures Derived from Patients with Advanced Prostate Cancer - ScienceDirect Volume 159, Issue 1, 25 September 2014, Pages 176-187 Resource Organoid Cultures Derived from Patients with Advanced Prostate Cancer Dong Gao 1 13 , Ian Vela 1 2 13 , Andrea Sboner 3 4 8 13 , Phillip J. Iaquinta 1 13 , Wouter R. Karthaus 5 , Anuradha Gopalan 6 , Whole-exome sequencing (WES) libraries were generated using Agilent SureSelect Human All Exon V6 kit (Agilent Technologies) following the manufacturers recommendations and index codes were added to each sample. The lack of in vitro models to reflect the primary disease has become a bottleneck for further study on thyroid cancer. Patient-derived organoids can be established with a high success rate from minimal tissue core specimens acquired with endoscopic-ultrasound-guided techniques and assembled into platforms, representing tens to hundreds of cancers each conserving specific features, expanding the types of patient samples that can be propagated and analyzed in the . However, we noticed that PTC-5_T harbored the BRAF missense mutation, which was absent in PTC-5_O. For passaging, organoids were resuspended in 5-mL TrypLE Express with incubation at 37 C for approximately 5 minutes. The primary histological types of thyroid cancer include papillary thyroid cancer (PTC), follicular thyroid cancer, Hrthle cell thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer. 36, 51-54 Yuan et al. Organoids were passaged at a 1:2 to 1:4 dilution every 2 weeks.