Medulloblastoma (MB) is the most common malignant brain tumor in children and among the subtypes, Group 3 MB has the worst outcome. Here, we perform an in vivo, patient-specific screen leading to the ...identification of Otx2 and c-MYC as strong Group 3 MB inducers. We validated our findings in human cerebellar organoids where Otx2/c-MYC give rise to MB-like organoids harboring a DNA methylation signature that clusters with human Group 3 tumors. Furthermore, we show that SMARCA4 is able to reduce Otx2/c-MYC tumorigenic activity in vivo and in human cerebellar organoids while SMARCA4 T910M, a mutant form found in human MB patients, inhibits the wild-type protein function. Finally, treatment with Tazemetostat, a EZH2-specific inhibitor, reduces Otx2/c-MYC tumorigenesis in ex vivo culture and human cerebellar organoids. In conclusion, human cerebellar organoids can be efficiently used to understand the role of genes found altered in cancer patients and represent a reliable tool for developing personalized therapies.
Brain tumors are the leading cause of cancer‐related death in children. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of pediatric brain cancers are ...limited and hard to establish. We present a protocol that enables efficient generation, expansion, and biobanking of pediatric brain cancer organoids. Utilizing our protocol, we have established patient‐derived organoids (PDOs) from ependymomas, medulloblastomas, low‐grade glial tumors, and patient‐derived xenograft organoids (PDXOs) from medulloblastoma xenografts. PDOs and PDXOs recapitulate histological features, DNA methylation profiles, and intratumor heterogeneity of the tumors from which they were derived. We also showed that PDOs can be xenografted. Most interestingly, when subjected to the same routinely applied therapeutic regimens, PDOs respond similarly to the patients. Taken together, our study highlights the potential of PDOs and PDXOs for research and translational applications for personalized medicine.
Synopsis
Patient‐derived organoids (PDOs) and patient‐derived xenograft organoids (PDXOs) have been established through direct in vitro culture of primary tumors and patient‐derived xenograft (PDX)‐derived tumors with the goal to better model pediatric tumors of the central nervous system.
PDOs recapitulate cellular heterogeneity, histological features, mutational profiles (number of variants, microsatellite instability, tumor mutational burden) of the corresponding parental tumor even after 1 year of in vitro culture
ZFTA‐RELA EPN‐ and G3 MB‐PDOs recapitulate patients' response to routinely applied therapeutic regimens
Several PDOs and PDXOs were derived from human tumors for which there are currently very limited in vivo and in vitro models available
PDOs and PDXOs are available to the research community, and can be subjected to genetic manipulations, to drug screening, and translational applications for personalized medicine
Patient‐derived organoids (PDOs) and patient‐derived xenograft organoids (PDXOs) have been established through direct in vitro culture of primary tumors and patient‐derived xenograft (PDX)‐derived tumors with the goal to better model pediatric tumors of the central nervous system.
In the second half of the last century, the American dent hybrids began to be widely grown, leading to the disappearance or marginalization of the less productive traditional varieties. Nowadays the ...characterization of traditional landraces can help breeders to discover precious alleles that could be useful for modern genetic improvement and allow a correct conservation of these open pollinated varieties (opvs). In this work we characterized the ancient coloured cultivar "Millo Corvo" typical of the Spanish region of Galicia. We showed that this cultivar accumulates high amounts of anthocyanins (83.4 mg/100g flour), and by TLC (Thin Layer Chromatography) and HPLC (High Pressure Liquid Chromatography) analysis, we demonstrated that they mainly consisted of cyanidin. Mapping and sequencing data demonstrate that anthocyanin pigmentation is due to the presence of the red color1 gene(r1), a transcription factor driving the accumulation of this pigment in the aleurone layer. Further chemical analysis showed that the kernels are lacking in carotenoids, as confirmed by genetic study. Finally a DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging ability test showed that Millo Corvo, even though lacking carotenoids, has a high antioxidant ability, and could be considered as a functional food due to the presence of anthocyanins.
The lpa1 mutations in maize are caused by lesions in the ZmMRP4 (multidrug resistance-associated proteins 4) gene. In previous studies (Raboy et al. in Plant Physiol 124:355—368, 2000; Pilu et al. in ...Theor Appl Genet 107:980—987, 2003a; Shi et al. Nat Biotechnol 25:930—937, 2007), several mutations have been isolated in this locus causing a reduction of phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate, or InsP6) content and an equivalent increasing of free phosphate. In particular, the lpa1-241 mutation causes a reduction of up to 90% of phytic acid, associated with strong pleiotropic effects on the whole plant. In this work, we show, for the first time to our knowledge, an interaction between the accumulation of anthocyanin pigments in the kernel and the lpa mutations. In fact the lpa1-241 mutant accumulates a higher level of anthocyanins as compared to wild type either in the embryo (about 3.8-fold) or in the aleurone layer (about 0.3-fold) in a genotype able to accumulate anthocyanin. Furthermore, we demonstrate that these pigments are mislocalised in the cytoplasm, conferring a blue pigmentation of the scutellum, because of the neutral/basic pH of this cellular compartment. As a matter of fact, the propionate treatment, causing a specific acidification of the cytoplasm, restored the red pigmentation of the scutellum in the mutant and expression analysis showed a reduction of ZmMRP3 anthocyanins' transporter gene expression. On the whole, these data strongly suggest a possible interaction between the lpa mutation and anthocyanin accumulation and compartmentalisation in the kernel.
Medulloblastoma and high-grade glioma represent the most aggressive and frequent lethal solid tumors affecting individuals during pediatric age. During the past years, several models have been ...established for studying these types of cancers. Human organoids have recently been shown to be a valid alternative model to study several aspects of brain cancer biology, genetics and test therapies. Notably, brain cancer organoids can be generated using genetically modified cerebral organoids differentiated from human induced pluripotent stem cells (hiPSCs). However, the protocols to generate them and their downstream applications are very rare. Here, we describe the protocols to generate cerebellum and forebrain organoids from hiPSCs, and the workflow to genetically modify them by overexpressing genes found altered in patients to finally produce cancer organoids. We also show detailed protocols to use medulloblastoma and high-grade glioma organoids for orthotopic transplantation and co-culture experiments aimed to study cell biology in vivo and in vitro, for lineage tracing to investigate the cell of origin and for drug screening. The protocol takes 60-65 d for cancer organoids generation and from 1-4 weeks for downstream applications. The protocol requires at least 3-6 months to become proficient in culturing hiPSCs, generating organoids and performing procedures on immunodeficient mice.
Abstract
Central nervous system (CNS) tumors are the most common solid tumors in the pediatric population and the main cause of death among childhood cancers. They present common features related to ...the development and produce symptoms based on the age of the child, their location and rate of tumor growth. Tumors of embryonal origin, such as medulloblastoma or primitive neuroectodermal tumor, have a higher incidence in younger patients, whereas older ones tend to present tumors of glial origin. Most of CNS-embryonal tumors are highly malignant and consequently leading to poor prognoses. Despite the understanding of the underlying molecular landscape of these tumors that has helped in clinical advances to improve life expectancies of the patients, there is still an emerging need to minimize morbidity and manage the long-term therapy effects. Developing new models to better understand the heterogeneity of these tumors and to test personalized treatment strategies remains a clinical challenge. We are willing to answer to this medical need with Patient-Derived Organoids (PDOs) established through the direct in vitro culture of primary tumors and patient derived xenograft (PDX)-derived tumors. We developed a method for generating PDOs from a variety of pediatric brain cancers that recapitulates the cellular heterogeneity, histological features and mutational profiles of the corresponding parental tumors, also confirmed by the DNA methylation profile analysis, nowadays used for an accurate routine CNS tumors diagnosis. Through this new model, we confirmed the action of some clinically used drugs, obtaining a proof of concept of their possible application as a reliable tool for drug screening. Further applications derive from the enrichment of PDOs culture media for possible tumor-specific antigens that could be useful to define tumor markers identifiable with diagnostic tests or as candidates for new therapies.
Abstract
Central nervous system (CNS) tumors are the most common solid tumors in the pediatric population and the main cause of death among childhood cancers. They present common features related to ...the development and produce symptoms based on their location, rate of tumor growth and age of the child. Tumors of embryonal origin, such as medulloblastoma or primitive neuroectodermal tumor, have a higher incidence in younger patients, whereas older ones tend to present tumors of glial origin. Most of CNS-embryonal tumors are highly malignant and consequently leading to poor prognoses. Despite the understanding of the underlying molecular landscape of these tumors that has helped in clinical advances to improve life expectancies of the patients, there is still an emerging need to minimize morbidity and manage the long-term therapy effects. Developing new models to better understand the heterogeneity of these tumors and to test personalized treatment strategies remains a clinical challenge. We are trying to answer to this medical need with Patient-Derived Organoids (PDOs) established through the direct in vitro culture of primary tumors and patient derived xenograft (PDX)-derived tumors. We have developed a method for generating PDOs from a variety of pediatric brain cancers that recapitulates the cellular heterogeneity, histological features and mutational profiles of the corresponding parental tumors. We have obtained promising results from the DNA methylation profile analysis, nowadays used for an accurate routine CNS tumors diagnosis. Through this new model, we confirmed the action of some clinically used drugs, obtaining a proof of concept of their possible application as a reliable tool for drug screening. Another interesting application derives from the enrichment of PDOs culture media for possible tumor-specific antigens: their identification could be of pivotal importance for the definition of tumor markers identifiable with diagnostic tests or as candidates for new therapies.
Abstract
Three-dimensional (3D) cell culture systems have gained increasing interest in drug discovery and tissue engineering due to their evident advantages in providing more relevant information ...and more predictive data for in vivo tests. Willing to increase the knowledge about Medulloblastoma Brain tumor we are developing Human cerebellar organoids derived from human iPSC: three-dimensional cultures that include multiple stem cell types, cell organization and features of human cerebellum. Notably, Medulloblastoma is the most common brain tumor affecting infants and stands as a cause for a high percentage of morbidity and mortality among cancer patients. To identify new Medulloblastoma driver genes we set up an innovative way to modify human cerebellar organoid, developing the first in-vitro 3D model of Medulloblastoma. We are mimicking human pathological conditions modifying this organoids with different putative oncogenes. Notably thanks to this type of model, we are able to study the functions of new Medulloblastoma driver genes in modulating stem cells differentiation and commitment in a three-dimensional structure, during the development of the organoid and the tumor. We speculate that our new Medulloblastoma 3D culture systems hold great promise for applications in infant tumor research, cancer cell biology and drug discovery, being the first human 3D in-vitro model that resembles human pathological conditions.
Abstract BACKGROUND Medulloblastoma (MB), the most frequent childhood brain tumor, originates in the cerebellum. Despite treatment advances, recurrence affects 40%, with a mortality rate of 30%. ...Cancer stem cells drive tumor initiation, maintenance, and therapy resistance, in several tumors including MB (MBSC). In response to stress, stem cells activate mechanisms like the unfolded protein response (UPR) to maintain homeostasis and survive adverse conditions. The UPR restores endoplasmic reticulum balance, supporting cell function and survival. Recent studies have shown that the UPR plays an important role in tumorigenesis, however, its function in MBSC remains unknown. METHODS To understand the involvement of the UPR in stem cell maintenance, we have investigated the biological effects of its pharmacological inhibition on Group 3 (G3) MBSC. We selected three G3 MB cell lines (D283-Med, D341-Med and Med411), for in vitro studies. Cell line selection was based on their fidelity to the expected methylation class, corresponding to the subgroup of the parental tumors, and were grown in “stem-like” conditions as neurospheres. We evaluated drugs targeting the UPR stress program, identifying ONC201 as the most effective against MBSC. ONC201 is an imipridone compound that activates p53-independent apoptosis causing changes in gene expression similar to those caused by UPR. RESULTS Our findings revealed that ONC201 modulates protein synthesis via ATF4, a pivotal UPR molecule, with stronger induction observed in MB cells cultured under “stem-like” conditions. This ATF4 upregulation triggers the activation of pro-apoptotic programs. Additionaly, ONC201 was effective on reducing the stem-like features of MBSC, including self-renewal, stem cell markers expression, migration, and invasion potential. Notably, ONC201 demonstrates synergy with vincristine and methotrexate chemotherapy. CONCLUSIONS These results propose ONC201 as a promising agent for targeting G3 medulloblastoma by compromising the stem cell compartment. Further investigation is warranted to confirm its efficacy and potential clinical application.
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