A Patient-Derived Glioblastoma Organoid Model and Biobank Recapitulates Inter- and Intra-tumoral Heterogeneity

Jacob, Fadi; Salinas, Ryan; Zhang, Daniel Y.; Nguyen, Phuong; Schnoll, Jordan G.; Wong, Samuel Zheng Hao; Thokala, Radhika; Sheikh, Saad; Saxena, Deeksha; Prokop, Stefan; Liu, Di-ao; Qian, Xuyu; Petrov, Dmitriy; Lucas, Timothy; Chen, H. Isaac; Dorsey, Jay F.; Christian, Kimberly M.; Binder, Zev A.; Nasrallah, MacLean P.; Brem, Steven; O’Rourke, Donald M.; Ming, Guo‐li; Song, Hongjun

doi:10.1016/j.cell.2019.11.036

Cited by 700 publications

(862 citation statements)

References 93 publications

(96 reference statements)

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“…Human glioblastoma tissue obtained from surgical resection is micro-dissected into pieces and cultured in an optimized medium on an orbital shaker to generate glioblastoma organoids as described previously 11 and in Steps 1-19 of this protocol ( Fig. 2a).…”

Section: Generation Of Glioblastoma Organoids and Biobankingmentioning

confidence: 99%

“…Increased release of cytokines (e.g. IL-2, IFNγ, TNFα) from activated T cells should occur within 24-72 hours after co-culture in the presence of target antigen 11 .…”

Section: Evaluation Of Successful Gbo Co-culture With Car-t Cellsmentioning

confidence: 99%

“…(a) Diagram outlining the major steps to generate GBOs from resected tumor tissue with downstream applications and to prepare and co-culture CAR-T cells with GBOs. Procedures for genomic and transcriptomic analyses, drug treatments, and xenografts are not described (n.d.) in this protocol; refer to Jacob et al, 2020 for methods and sample data for these procedures 11 . Panel created with images from www.biorender.com.…”

Section: Data Availabilitymentioning

confidence: 99%

“…These organoid models and biobanks have furthered our understanding of tumor biology and shown promise for testing new therapeutic strategies 9,10 . We recently developed a protocol to generate glioblastoma organoids (GBOs) from surgically resected patient glioblastoma tissue with minimal perturbation 11 . We showed that GBOs recapitulate the heterogeneity of their parent tumors as evidenced by immunohistology, bulk and single-cell transcriptomics, and mutation analyses revealing the maintenance of diverse cell populations and their gene expression and mutational profiles.…”

Section: Introductionmentioning

confidence: 99%

“…GBOs exhibit rapid, aggressive infiltration of tumor cells when transplanted into adult rodent brains, a defining feature of human glioblastoma. We demonstrated the utility of GBOs to test personalized therapies by correlating GBO mutational profiles with responses to specific drugs and developed methods to co-culture GBOs with CAR-T cells to test killing efficacy and target specificity 11 . To better enable adoption of our methods in other laboratories, here we describe detailed procedures for generating, culturing, passaging, cryopreserving, and thawing GBOs (Figs.…”

Section: Introductionmentioning

confidence: 99%

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Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR-T cell testing

Jacob

Ming

Song

2020

Preprint

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Short Summary: Detailed procedures for generating and biobanking glioblastoma organoids from resected patient tumor tissue and testing CAR-T cell efficacy by co-culture. Additional procedures for tissue processing, immunohistology, and detecting hypoxia gradients and actively proliferating cells. AbstractGlioblastoma tumors exhibit extensive inter-and intra-tumoral heterogeneity, which has contributed to the poor outcomes of numerous clinical trials and continues to complicate the development of effective therapeutic strategies. Current in vitro models do not preserve the cellular and mutational diversity of parent tumors and often require a lengthy generation time with variable efficiency. Here, we describe detailed procedures for generating glioblastoma organoids (GBOs) from surgically resected patient tumor tissue using a chemically defined medium without cell dissociation. By preserving cell-cell interactions and minimizing clonal selection, GBOs maintain the cellular heterogeneity of parent tumors. We include methods for passaging and cryopreserving GBOs for continued use, biobanking, and long-term recovery. We further describe procedures for investigating patient-specific responses to immunotherapies by co-culturing GBOs with chimeric antigen receptor (CAR) T cells. This protocol takes approximately 2-4 weeks to generate GBOs and 5-7 days to perform CAR-T cell co-culture. Competence with human cell culture, tissue processing, and immunohistology is required for optimal results.An ideal in vitro model for glioblastoma would maintain cellular diversity, preserve native cell-cell interactions, recapitulate defining histological characteristics, and maintain the transcriptomes and mutation profiles of parent tumors. To be useful for personalized medicine approaches, the model would need a quick generation time with high fidelity for success and be scalable to test potential therapeutics. This would require maintaining patient-derived glioblastoma tissue in culture with minimal perturbation. Towards this goal, we opted to directly culture micro-dissected tumor pieces without cell dissociation to preserve cell-cell interactions and minimize clonal selection. We start from resected patient tumor tissue that can be sampled from different tumor foci or subdivided into regional samples to analyze intra-tumoral heterogeneity. Through testing of many medium formulations, we learned that tumor pieces did not require basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), serum, or added extracellular matrix such as Matrigel to survive, form organoids, and proliferate at rates similar to parent tumors 11 . We optimized a chemically defined medium that contains basic nutrients and components needed to support glial cell health with few exogenous factors to minimize clonal selection induced by growth factors and decrease potential treatment confounders. This resulted in an optimized version of our medium used to maintain human brain organoid cultures 12,13 . We culture tumor pieces on an orbital shaker to inc...

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Section: Generation Of Glioblastoma Organoids and Biobankingmentioning

confidence: 99%

“…Increased release of cytokines (e.g. IL-2, IFNγ, TNFα) from activated T cells should occur within 24-72 hours after co-culture in the presence of target antigen 11 .…”

Section: Evaluation Of Successful Gbo Co-culture With Car-t Cellsmentioning

confidence: 99%

Section: Data Availabilitymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR-T cell testing

Jacob

Ming

Song

2020

Preprint

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Unravelling the mosaic: Epigenetic diversity in glioblastoma

Lucchini,

Constantinou,

Marino

2024

Molecular Oncology

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Glioblastoma is the most common primary malignant brain tumour. Despite decades of intensive research in the disease, its prognosis remains poor, with an average survival of only 14 months after diagnosis. The remarkable level of intra‐ and interpatient heterogeneity is certainly contributing to the lack of progress in tackling this tumour. Epigenetic dysregulation plays an important role in glioblastoma biology and significantly contributes to intratumour heterogeneity. However, it is becoming increasingly clear that it also contributes to intertumour heterogeneity, which historically had mainly been linked to diverse genetic events occurring in different patients. In this review, we explore how DNA methylation, chromatin remodelling, microRNA (miRNA) dysregulation, and long noncoding RNA (lncRNA) alterations contribute to intertumour heterogeneity in glioblastoma, including its implications for advanced tumour stratification, which is the essential first step for developing more effective patient‐specific therapeutic approaches.

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Assembly of glioblastoma tumoroids and cerebral organoids: a 3Din vitro model for tumor cell invasion

Kim,

Lee

et al. 2024

Molecular Oncology

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Glioblastoma (GBM) has a fatal prognosis because of its aggressive and invasive characteristics. Understanding the mechanism of invasion necessitates an elucidation of the relationship between tumor cells and the tumor microenvironment. However, there has been a scarcity of suitable models to investigate this. In this study, we established a glioblastoma‐cerebral organoid assembloid (GCOA) model by co‐culturing patient‐derived GBM tumoroids and human cerebral organoids. Tumor cells from the tumoroids infiltrated the cerebral organoids, mimicking the invasive nature of the parental tumors. Using time‐lapse imaging, various invasion patterns of cancer cells within cerebral organoids resembling a normal tissue milieu were monitored. Both single‐ and collective‐cell invasion was captured in real‐time. We also confirmed the formation of an intercellular tumor network and tumor–normal‐cell interactions. Furthermore, the transcriptomic characterization of GCOAs revealed distinct features of invasive tumor cells. Overall, this study established the GCOA as a three‐dimensional (3D) in vitro assembloid model to investigate invasion mechanisms and interactions between tumor cells and their microenvironment.

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A Patient-Derived Glioblastoma Organoid Model and Biobank Recapitulates Inter- and Intra-tumoral Heterogeneity

Cited by 700 publications

References 93 publications

Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR-T cell testing

Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR-T cell testing

Unravelling the mosaic: Epigenetic diversity in glioblastoma

Assembly of glioblastoma tumoroids and cerebral organoids: a 3Din vitro model for tumor cell invasion

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