Summary Glioblastoma is the most common and deadly primary brain malignancy. Despite advances in precision medicine oncology (PMO) allowing the identification of molecular vulnerabilities in glioblastoma, treatment options remain limited, and molecular assays guided by genomic and expression profiling to inform patient enrollment in life-saving trials are lacking. Here, we generate four-dimensional (4D) cell-culture arrays for rapid assessment of drug responses in glioblastoma patient-derived models. The arrays are 3D printed with thermo-responsive shape memory polymer (SMP). Upon heating, the SMP arrays self-transform in time from 3D cell-culture inserts into histological cassettes. We assess the utility of these arrays with glioblastoma cells, gliospheres, and patient derived organoid-like (PDO) models and demonstrate their use with glioblastoma PDOs for assessing drug sensitivity, on-target activity, and synergy in drug combinations. When including genomic and drug testing assays, this platform is poised to offer rapid functional drug assessments for future selection of therapies in PMO.
Summary Robust patient-derived platforms that recapitulate the cellular and molecular fingerprints of glioblastoma are crucial for developing effective therapies. Here, we describe a chemically defined protocol for 3D culture and propagation of glioblastoma in 3D gliospheres, patient-derived organoids (PDOs), mouse brain orthotopic xenografts (PDOXs), and downstream drug and immunofluorescence assays. This simple-to-follow protocol allows assessing drug sensitivity, on-target activity, and combined drug synergy. Promising therapies can then be validated in PDOXs for translation in precision medicine oncology trials. For complete details on the use and execution of this protocol, please refer to Chadwick et al. (2020) and Patrizii et al. (2018) .
Glioblastoma multiforme (GBM) are the most common and deadly primary brain tumors. GBMs are thought to derive from neuroglial stem or progenitor cells. Although surgery, radiotherapy and concomitant or alternating chemotherapy followed by anti-angiogenic therapy are still the mainstay of treatment, individually tailored strategies based on tumor-intrinsic dominant genomic, transcriptomic, epigenetic and antigenic tumor profiles may ultimately improve outcome. GBM recurrence is thought to be caused by a dynamically changing population of slow-cycling stem-like cells that are capable of evading current therapeutics which spare the radio/chemoresistant cells. Due to the extreme heterogeneity of these tumors, the likelihood of success of new therapeutic strategies for GBM relies on utilizing tailored combinations of targeted genetic-, epigenetic- and/or immune-modulating therapies. Targeting epigenetic polycomb repressor complex 1/2 (PRC1/2) and their key drivers BMI1 and EZH2 is highly attractive to target GBM stem-like and progenitor cells. Since PRC2 activity is dependent on PRC1 chromatin association, targeting BMI1 is a priority for GBM. BMI1 is strongly upregulated in most GBM subtypes, and we and others have demonstrated that reducing BMI1 levels results in a decrease in tumor cell self-renewal and eliminate the stem-cell like phenotypes in other tumor types. We have therefore developed novel small molecule BMI1 inhibitors, named RU-A compounds, for GBM therapy. First, we demonstrate that these RU-A compounds could eliminate BMI1 at low nanomolar conc. We further determined using modeling and luciferase assays that RU-A compounds exert effects on the BMI1 RNA, suggesting that RU-A compounds might bind to a pocket within the BMI1 untranslated region. Strikingly, RU-A compounds demonstrate complete ablation of self-renewal of GBM stem-like cells in serial clonogenic, sphere assays, and GBM organoids, and sensitize GBM cells to chemotherapy. We demonstrate the anti-GBM and reduction of tumor initiation activities of these inhibitors in a series of patient-derived xenografts and patient derived orthotopic mouse models of GBM. Due to the high potency of these compounds, when injected in mice, there were significant reductions in intratumor BMI1 associated with elimination of stem cell-like phenotypes, and no hematopoietic toxicity or neurotoxicity detected. These novel RU-A compounds by altering the stem-like populations of GBM cells offer effective targeted agents that could be utilized alone or combined with other targeted and immune modulatory agents in clinical trials for GBM patients. Citation Format: Michelle Chadwick, Eric Huselid, Monica Bartucci, Michele Patrizii, Kelly Jara, Monal Mehta, John Gilleran, David Augeri, Hatem Sabaawy. Development of novel BMI1 inhibitors targeting glioblastoma stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3073.
In recent years, three-dimensional (3D) models of human brain derived from pluripotent embryonic and iPS cells have emerged as brain organoids, providing established models for brain development and genetic engineering. However, tumor organoids derived from adult glioblastoma multiforme (GBM) patients were seldomly formed. WHO grade IV GBM is a devastating locally invasive brain cancer with a median survival of 14.6 months. Standard of care for GBM includes surgical resection followed by radiotherapy plus concomitant and maintenance temozolomide (TMZ). Resistance to TMZ develops rapidly and neither dose-intensified TMZ nor anti-angiogenic approaches could improve survival. Genomic, transcriptomic and epigenetic profiling allowed classifying adult GBM into three groups: isocitrate dehydrogenase (IDH)-mutant (mut), 1p/19q co-deleted oligodendroglial GBM with best prognosis; IDH-mut, 1p/19q non-co-deleted astrocytic GBM with intermediate outcome; and IDH wild-type (WT) GBM with poor prognosis. Preclinical models that reflect these GBM profiles and heterogeneity are urgently needed to examine new therapies. Patient derived orthotopic xenografts (PDOXs) are thought to better mimic the GBM environment. Genetically engineered mouse models (GEMMs) make gliomas in mice with competent immune systems, but are laborious and expensive requiring compound genetics to mimic human GBM. Here, we describe a 3D serum-free organoid system in low-adherence plates derived from primary and sphere cultures that supports the long-term growth and expansion of GBM organoids for several months. We generated organoids from GBM with IDH-WT or IDH-Mut, EGFR amplification, +7q/-10q genotype, PTEN mutation, and EGFRvIII expression. GBM organoids are comprised of a heterogenous cell populations, thus mimicking the original tumor. GBM organoids could be generated from a single cell, therefore allowing to track intratumor heterogeneity. GBM organoids could be utilized for investigating aspects of GBM biology such as 3D cellular self-renewal showing expression of stem cell and differentiation targets, 3D cell cycle regulation including synchronized growth and phase cycling, 3D cell metabolism including intracellular adenosine triphosphate (ATP) level and protein synthesis rate, 3D cell invasiveness validated with IHC assays, and for evaluation of drug effects in the context of IDH, PI3K and EGFR aberrations. We have demonstrated that GBM sphere cells exhibit a solid growth pattern when implanted orthotopically into the mouse PDOXs. Since GBM organoids form tighter cell-cell contact, oxygen and nutrient gradients, we implanted GBM organoids in mouse orthotopic xenografts to demonstrate their tumor growth and invasive phenotypes. This human GBM organoid platform allows for novel preclinical therapeutic approaches to be assessed and provides personalized therapeutic options for individual GBM patients. Citation Format: Michelle Chadwick, Rachael Mfon, Kelly Jara, Shabbar Danish, Robert Aiken, Hatem E. Sabaawy. Single cell derived 3D organoids recapitulate the tumorigenic features of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4705.
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