Periodic Reporting for period 1 - NeuroblastORG (Developing neural crest organoids with inducible neuroblastoma to model chemoresistance in MYCN amplification related high-risk cases)
Periodo di rendicontazione: 2023-03-01 al 2025-02-28
Neuroblastoma (NB) is the most common cancer in infants and a leading cause of cancer-related mortality in children, with 90% of cases occurring in patients under five years old. A subset of NB, classified as high-risk, is characterized by the amplification of the MYCN oncogene, which drives tumor aggressiveness and significantly worsens prognosis. Despite advancements in treatment, recurring high-risk NB remains challenging to manage, often resulting in poor patient outcomes.
One of the key obstacles in developing effective therapies is chemotherapy resistance. Investigating the mechanisms behind acquired resistance is complicated by the limited availability of high-risk NB patient samples and suitable model systems. While mouse models are frequently used, species-specific differences in sympathoblast development—the embryonic precursors to NB tumor cells—can lead to misleading results.
The NeuroblastORG project aimed to address this gap by establishing a human model using miniature organ-like structures, called organoids that can accurately represent the neural crest (NC) lineage, from which sympathoblasts originate. By recapitulating human-specific developmental pathways, this model enables the generation of sympathoblast-like cells, providing a more physiologically relevant system for studying MYCN-driven NB. This approach allows researchers to systematically explore drug efficacy and uncover mechanisms underlying chemoresistance in high-risk NB cases, ultimately paving the way for improved therapeutic strategies.
One of the key obstacles in developing effective therapies is chemotherapy resistance. Investigating the mechanisms behind acquired resistance is complicated by the limited availability of high-risk NB patient samples and suitable model systems. While mouse models are frequently used, species-specific differences in sympathoblast development—the embryonic precursors to NB tumor cells—can lead to misleading results.
The NeuroblastORG project aimed to address this gap by establishing a human model using miniature organ-like structures, called organoids that can accurately represent the neural crest (NC) lineage, from which sympathoblasts originate. By recapitulating human-specific developmental pathways, this model enables the generation of sympathoblast-like cells, providing a more physiologically relevant system for studying MYCN-driven NB. This approach allows researchers to systematically explore drug efficacy and uncover mechanisms underlying chemoresistance in high-risk NB cases, ultimately paving the way for improved therapeutic strategies.
As part of the NeuroblastORG project, researchers developed a method to generate neural crest organoids—three-dimensional (3D) cell clusters grown from human induced pluripotent stem cells (hiPSCs). These organoids mimic early human development and contain sympathoblast-like cells, the embryonic precursors to neuroblastoma.
After receiving training in CRISPR/Cas9 gene-editing technology, the ERA fellow created genetically modified hiPSC lines. She inserted either the MYCN oncogene, known to drive aggressive neuroblastoma, or a GFP fluorescent reporter gene as a control into the genome of two different hiPSC lines. To allow the investigation of gender-specific differences in NB formation and acquisition of chemoresistance both male and female hiPSC lines were used. This resulted in the successful generation of eight transgenic hiPSC lines, which maintained their ability to form neural crest organoids.
MYCN-overexpressing cells within these organoids were extensively characterized using immunochemistry and RNA sequencing, offering valuable insights into how this oncogene drives tumor progression. Additionally, the ERA fellow received training on patient-derived neuroblastoma sample collection and processing. RNA sequencing was performed on samples collected from both low- and high-risk neuroblastoma cases to reveal similarities between the primary tumor and the NB organoid model. The neuroblastoma organoid system proved to be a promising tool for testing potential therapeutic compounds, allowing researchers to evaluate their effectiveness against MYCN-overexpressing neuroblastoma-like cells within a tissue-like structure.
Beyond the scientific achievements, the project fostered academic growth. The ERA fellow was able to strengthen her leadership skills by attending the ‘Laboratory Leadership for group leaders’ course organized by EMBO. Another objective of the fellowship was to strengthen the mentoring skills of the ERA fellow. This was achieved through the supervision of two PhD students and a senior molecular biology scientist.
After receiving training in CRISPR/Cas9 gene-editing technology, the ERA fellow created genetically modified hiPSC lines. She inserted either the MYCN oncogene, known to drive aggressive neuroblastoma, or a GFP fluorescent reporter gene as a control into the genome of two different hiPSC lines. To allow the investigation of gender-specific differences in NB formation and acquisition of chemoresistance both male and female hiPSC lines were used. This resulted in the successful generation of eight transgenic hiPSC lines, which maintained their ability to form neural crest organoids.
MYCN-overexpressing cells within these organoids were extensively characterized using immunochemistry and RNA sequencing, offering valuable insights into how this oncogene drives tumor progression. Additionally, the ERA fellow received training on patient-derived neuroblastoma sample collection and processing. RNA sequencing was performed on samples collected from both low- and high-risk neuroblastoma cases to reveal similarities between the primary tumor and the NB organoid model. The neuroblastoma organoid system proved to be a promising tool for testing potential therapeutic compounds, allowing researchers to evaluate their effectiveness against MYCN-overexpressing neuroblastoma-like cells within a tissue-like structure.
Beyond the scientific achievements, the project fostered academic growth. The ERA fellow was able to strengthen her leadership skills by attending the ‘Laboratory Leadership for group leaders’ course organized by EMBO. Another objective of the fellowship was to strengthen the mentoring skills of the ERA fellow. This was achieved through the supervision of two PhD students and a senior molecular biology scientist.
To study neuroblastoma and develop effective treatments, researchers rely on different model systems. The most widely used models of neuroblastoma include transgenic mouse models, cancer cell lines, patient-derived neuroblastoma spheroid cultures and human induced pluripotent stem cell-derived 2D neural crest models. Developing human-relevant model systems is of high importance due to the differences in early sympathoblast development between humans and rodents, which can lead to inaccurate findings. Among the human models patient-derived tumor spheroids stand out due to their three-dimensional structure which resembles real tumors more closely than 2D cultures. However, patient-derived tumor spheroids show high variability between samples and cannot produce standardized cultures for large-scale drug testing.
While all state-of-the-art models offer critical insights, the establishment of a reproducible and scalable, human-relevant, complex (3D) model system for drug screening applications could lead to the discovery of novel, more-effective therapies for high-risk neuroblastoma patients. The NeuroblastORG project addressed this gap by developing a neuroblastoma organoid model, combining both MYCN-overexpressing tumor cells and healthy neural crest cells within a 3D tissue-like structure. These organoids can be generated in large quantities with high reproducibility, making them a powerful tool for testing new treatments and studying the earliest stages of high-risk neuroblastoma formation.
The NeuroblastORG project delivered a novel, patentable technology. This allowed the ERA fellow to get trained in the patenting process. As a result, the patent application has been submitted, and a Hungarian company expressed interest in licensing the patent and offering the platform as a screening service for new compounds developed by pharmaceutical companies.
Following the patenting process, the organoid model will be published to share its full characterization, detailing how well it replicates the molecular features of MYCN-amplified neuroblastoma and paving the way for future drug development efforts. Another publication describing a novel way for delaying therapy resistance was already published as part of the NeuroblastORG project. Additionally, the ERA fellow presented her work at two international conferences as invited speaker and at public events such as the Researcher’s Night and Neuroblastoma Awareness Day. These opportunities greatly strengthened the science communication skills of the ERA fellow.
During the NeuroblastORG project the ERA fellow was able to establish the Human Organoid Laboratory at the HUN-REN Research Centre for Natural Sciences and together with her team they will continue to investigate chemoresistance mechanisms developing within the neuroblastoma organoids upon chemotherapy.
While all state-of-the-art models offer critical insights, the establishment of a reproducible and scalable, human-relevant, complex (3D) model system for drug screening applications could lead to the discovery of novel, more-effective therapies for high-risk neuroblastoma patients. The NeuroblastORG project addressed this gap by developing a neuroblastoma organoid model, combining both MYCN-overexpressing tumor cells and healthy neural crest cells within a 3D tissue-like structure. These organoids can be generated in large quantities with high reproducibility, making them a powerful tool for testing new treatments and studying the earliest stages of high-risk neuroblastoma formation.
The NeuroblastORG project delivered a novel, patentable technology. This allowed the ERA fellow to get trained in the patenting process. As a result, the patent application has been submitted, and a Hungarian company expressed interest in licensing the patent and offering the platform as a screening service for new compounds developed by pharmaceutical companies.
Following the patenting process, the organoid model will be published to share its full characterization, detailing how well it replicates the molecular features of MYCN-amplified neuroblastoma and paving the way for future drug development efforts. Another publication describing a novel way for delaying therapy resistance was already published as part of the NeuroblastORG project. Additionally, the ERA fellow presented her work at two international conferences as invited speaker and at public events such as the Researcher’s Night and Neuroblastoma Awareness Day. These opportunities greatly strengthened the science communication skills of the ERA fellow.
During the NeuroblastORG project the ERA fellow was able to establish the Human Organoid Laboratory at the HUN-REN Research Centre for Natural Sciences and together with her team they will continue to investigate chemoresistance mechanisms developing within the neuroblastoma organoids upon chemotherapy.