3D Glioblastoma Research Articles

Abstract 5231: Dianhydrogalactitol (VAL-083) exhibits strong efficacy in GBM tumors with different (epi)genetic background and treatment history

Abstract Background: Standard-of-care for glioblastoma (GBM) includes surgery, radiation and temozolomide (TMZ). Nearly all tumors recur and 5-year survival is less than 3%. Unmethylated promoter status for O6-methylguanine-DNA-methyltransferase (MGMT) is a validated biomarker for TMZ-resistance. Second-line treatment with bevacizumab has failed to improve survival and GBMs escape treatment by inducing intratumor hypoxia. VAL-083 is a bi-functional DNA-targeting agent that readily crosses the blood-brain barrier and accumulates in brain tumor tissue. VAL-083 induces DNA interstrand crosslinks at N7-guanine, leading to double-strand breaks and cancer cell-death in GBM cells, independent of MGMT. VAL-083 is currently in Phase II clinical trial for the treatment of MGMT promoter unmethylated GBM, both recurrent and treatment-naïve (NCT02717962, NCT03050736), and it remains to be seen if it shows enhanced anti-tumor effect compared to TMZ. Based on its unique monosaccharide backbone structure, VAL-083 may also benefit from bevacizumab-induced GLUT transporter upregulation leading to enhanced uptake and anti-tumor activity. Methods: The cytotoxic effect of VAL-083 and TMZ was verified in 3D GBM organoids derived from 18 patient-derived orthotopic xenograft (PDOX) GBM models of different (epi)genetic background. Cell responses to drugs were calculated as the area under the curve (AUC). We further evaluated VAL-083 ability to decrease tumor growth in vivo in a MGMT-unmethylated, temozolomide-resistant recurrent GBM PDOX model. Mice were grouped into control, bevacizumab, VAL-083, and VAL-083+bevacizmab. Tumor progression was measured by MRI and histopathological assessment. Results: GBM organoids showed only partial response to TMZ. As expected, MGMT- methylated GBMs were less resistant in comparison to MGMT-unmethylated GBMs. VAL-083 was generally more effective than TMZ and response to VAL-083 was not dependent on MGMT promoter methylation status. Responses to TMZ and VAL-083 were comparable between treatment-naïve PDOXs and PDOXs derived from patients previously treated with TMZ and radiation. VAL-083 led to dramatic reduction of tumor growth in vivo (-83% for VAL-083 group, -90% for VAL-083 + bevacizumab). The analysis of tumor growth in time showed further reduction of tumor progression upon combined treatment. Histological assessment showed increased DNA damage (H2AX-P) in tumor cells. H2AX-P was only slightly increased in certain zones of the normal brain, close to meninges and subventricular zone, to a much lower extend in comparison to tumor cell, which was in line with the low toxicity of VAL-083. Citation Format: Anna Golebiewska, Anaïs Oudin, Virginie Baus, Ann-Christin Hau, Eliane Klein, Anne Steino, Jeffrey A. Bacha, Simone P. Niclou, Dennis M. Brown. Dianhydrogalactitol (VAL-083) exhibits strong efficacy in GBM tumors with different (epi)genetic background and treatment history [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5231.

A 3D Spheroid Model for Glioblastoma.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

High-resolution tomographic analysis of in vitro 3D glioblastoma tumor model under long-term drug treatment.

Glioblastoma multiforme (GBM) is a lethal type of brain tumor that often develop therapeutic resistance over months of chemotherapy cycles. Recently, 3D GBM models were developed to facilitate evaluation of drug treatment before undergoing expensive animal studies. However, for long-term evaluation of therapeutic efficacy, novel approaches for GBM tissue construction are still needed. Moreover, there is still a need to develop fast and sensitive imaging methods for the noninvasive assessment of this 3D constructs and their response to drug treatment. Here, we report on the development of an integrated platform that enable generating (i) an in vitro 3D GBM model with perfused vascular channels that allows long-term culture and drug delivery and (ii) a 3D imaging modality that enables researchers to noninvasively assess longitudinal fluorescent signals over the whole in vitro model.

RETRACTED ARTICLE: Inhibition of autophagic flux differently modulates cannabidiol-induced death in 2D and 3D glioblastoma cell cultures

Radiotherapy combined with chemotherapy is the major treatment modality for human glioblastoma multiforme (GBM). GBMs eventually relapse after treatment and the average survival of GBM patients is less than two years. There is some evidence that cannabidiol (CBD) can induce cell death and increases the radiosensitivity of GBM by enhancing apoptosis. Beside initiation of death, CBD has been demonstrated as an inducer of autophagy. In the present study, we address the question whether CBD simultaneously induces a protective effect in GBM by upregulating autophagy. Addition of chloroquine that suppressed autophagic flux to 2D GBM cultures increased CBD-induced cell death, presenting proof for the protective autophagy. Blockage of autophagy upregulated radiation-induced cytotoxicity but only modestly affected the levels of cell death in CBD- or CBD/γ-irradiated 3D GBM cultures. Furthermore, CBD enhanced the pro-apoptotic activities of JNK1/2 and MAPK p38 signaling cascades while partially downregulated the pro-survival PI3K-AKT cascade, thereby changing a balance between cell death and survival. Suppression of JNK activation partially reduced CBD-induced cell death in 3D GBM cultures. In contrast, co-treatment of CBD-targeted cells with inhibitors of PI3K-AKT-NF-κB, IKK-NF-κB or JAK2-STAT3 pathways killed surviving GBM cells in both 2D and 3D cultures, potentially improving the therapeutic ratio of GBM.

Radiation Responses of 2D and 3D Glioblastoma Cells: A Novel, 3D-specific Radioprotective Role of VEGF/Akt Signaling through Functional Activation of NHEJ.

Glioblastoma is resistant to conventional treatments and has dismal prognosis. Despite promising in vitro data, molecular targeted agents have failed to improve outcomes in patients, indicating that conventional two-dimensional (2D) in vitro models of GBM do not recapitulate the clinical scenario. Responses of primary glioblastoma stem-like cells (GSC) to radiation in combination with EGFR, VEGF, and Akt inhibition were investigated in conventional 2D cultures and a three-dimensional (3D) in vitro model of GBM that recapitulates key GBM clinical features. VEGF deprivation had no effect on radiation responses of 2D GSCs, but enhanced radiosensitivity of GSC cultures in 3D. The opposite effects were observed for EGFR inhibition. Detailed analysis of VEGF and EGF signaling demonstrated a radioprotective role of Akt that correlates with VEGF in 3D and with EGFR in 2D. In all cases, positive correlations were observed between increased radiosensitivity, markers of unrepaired DNA damage and persistent phospho-DNA-PK nuclear foci. Conversely, increased numbers of Rad51 foci were observed in radioresistant populations, indicating a novel role for VEGF/Akt signaling in influencing radiosensitivity by regulating the balance between nonhomologous end-joining and homologous recombination-mediated DNA repair. Differential activation of tyrosine kinase receptors in 2D and 3D models of GBM explains the well documented discrepancy between preclinical and clinical effects of EGFR inhibitors. Data obtained from our 3D model identify novel determinants and mechanisms of DNA repair and radiosensitivity in GBM, and confirm Akt as a promising therapeutic target in this cancer of unmet need.

Transcriptomic Profiling of 3D Glioblastoma Tumoroids for the Identification of Mechanisms Involved in Anticancer Drug Resistance

Among various types of brain tumors, glioblastoma is the most malignant and highly aggressive brain tumor that possesses a high resistance against anticancer drugs. To understand the underlined mechanisms of tumor drug resistance, a new and more effective research approach is required. The three dimensional (3D) in vitro cell culture models could be a potential approach to study cancer features and biology, as well as screen for anti-cancer agents due to the close mimicry of the 3D tumor microenvironments. With our developed 3D alginate scaffolds, Ilumina RNA-sequencing was used to transcriptomically analyze and compare the gene expression profiles between glioblastoma cells in traditional 2-dimensional (2D) monolayer and in 3D Ca-alginate scaffolds at day 14. To verify the reliability and accuracy of Illumina RNA-Sequencing data, ATP-binding cassette transporter genes were chosen for quantitative real-time polymerase chain reaction) verification. The results showed that 7,411 and 3,915 genes of the 3D glioblastoma were up-regulated and down-regulated, respectively, compared with the 2D-cultured glioblastoma. Furthermore, the Kyoto Encyclopaedia of Genes and Genomes pathway analysis revealed that genes related to the cell cycle and DNA replication were enriched in the group of down-regulated gene. On the other hand, the genes involved in mitogen-activated protein kinase signaling, autophagy, drug metabolism through cytochrome P450, and ATP-binding cassette transporter were found in the up-regulated gene collection. 3D glioblastoma tumoroids might potentially serve as a powerful platform for exploring glioblastoma biology. They can also be valuable in anti-glioblastoma drug screening, as well as the identification of novel molecular targets in clinical treatment of human glioblastoma.

ATIM-48 (LTBK-05). MULTIDIMENSIONAL PERSONALIZED RESPONSE ASSESSMENT TO MICROGLIA MODULATORS IN GLIOBLASTOMA BIOREACTORS

Abstract BACKGROUND Recently, strategies harnessing the non-neoplastic immune tumor microenvironment (iTME), consisting of macrophages and microglia (TAMs), as well as adaptive immune cells have been employed to treat glioblastoma (GBM). To evaluate the effect of local TAM-modulating therapies in combination with T-cell checkpoint inhibitors, we generated 3D GBM perfusion bioreactor cultures from patient-derived samples. We report patient- and tumor region specific responses to microglia modulators and checkpoint inhibitors using multidimensional fluorescent microscopy techniques, and multiplexed cytokine measurements, Subsequently, we aime at identifying responders versus non-responders as well as predictive markers of treatment response. METHODS Fresh, neuronavigated GBM biopsies from tumor center and periphery were placed into perfusion bioreactors and cultured for 7 days. Explants were treated with combinations of TAM and T-cell modulating drugs including anti-PD1 and anti-CD47 antibodies and their combination. Tissue was harvested for histology, and supernatants were processed for multiplexed cytokine analysis. Multidimensional CODEX technology analysis using a customized TAM centric 54 marker panel was implemented, and a map of individualized response criteria to specific immunotherapies developed. RESULTS Multiplex cytokine analysis showed a dominance of proinflammatory cytokines (CCL2, CCL3, CCL4 and PAI-1) in the periphery of the tumor at baseline. We further observed that the tumor periphery was more responsive to treatments confirming the efficacy of the treatment after tumor resection. Using CODEX, we identified specific cell types responding to the treatment and undergoing phenotypic changes. Moreover, dynamic shifting of T-cell checkpoint expression levels under treatment pointed to potential resistance mechanisms in a subset of tumors. Further, we identified region-specific cytokine release as a response to the treatment in a series of 8 patient-specific explant cultures. In summary, we present an in-depth profiling of the GBM-region specific iTME at baseline and document its dynamic response under innate/adaptive immune modulators using CODEX. CONCLUSION The proposed approach serves as a patient-tailored ex vivo “Clinical Trial” by stratifying the individual patient’s iTME response.

P12.09 Multidimensional Personalized Response Assessment to Microglia Modulators in Gioblastoma Bioreactors

Abstract BACKGROUND Recently, strategies harnessing the non-neoplastic, immune tumor microenvironment (iTME) consisting of myeloid-derived macrophages and yolk sac derived microglia (termed TAMs) as well as adaptive immune components have been employed to treat glioblastoma (GBM). To evaluate the effect of TAM-modulating therapies in combination with T-cell checkpoint inhibitor approaches, we generated 3D GBM bioreactor cultures from patient-derived samples. Here, we report patient-tailored, tumor region specific response assessment to microglia modulators and T-cell checkpoint inhibitors using multidimensional fluorescent microscopy techniques MATERIAL AND METHODS GBM tissue fragments from the tumor center and periphery were placed into perfusion bioreactors shortly after resection and cultured for up to 3 weeks. Control conditions included non-perfused cultures of the same tissue. Cultures were treated with combinations of TAM and T-cell modulating, FDA approved drugs including anti-PD1, anti-CTLA4 and anti-CD47 antibodies. Tissue was harvested for histology, RNA extraction, and supernatants were processed for multiplexed cytokine analysis. Multidimensional CODEX technology analysis using a customized TAM/microglia-centric 50 marker panel was implemented, and a map of individualized response criteria to specific immunotherapies developed. RESULTS We were able to cultivate viable GBM tissue with intact iTME. Tumor cell proliferation and invasion capacity were preserved for up to 3 weeks. Conventional immunohistochemistry confirmed the presence of TAMs and T cells. Treatment with immunomodulators resulted in a profound polarization shift of TAMs. Furthermore, cytokine analysis confirmed proinflammatory immune responses in most assessed samples. We present preliminary data of the CODEX analysis of our combinatorial immunotherapies in a series of 8 patient-specific explant samples. CONCLUSION GBM tissue could be incubated in the perfused 3D bioreactor model and kept viable for up to 21 days. The proposed model allows patient-tailored testing of immunomodulatory drugs by taking into account the patients individual iTME response. GBM tissue could be incubated in the perfused 3D bioreactor model and kept viable for up to 21 days. The proposed model allows patient-tailored testing of immunomodulatory drugs by taking into account the patients individual iTME response.

Abstract 53: Ex vivo models of glioblastoma: a comparison of 3D tissues and patient-derived xenografts to clinical response

Abstract Standard first-line treatment of newly diagnosed Glioblastoma (GBM) is primarily radiotherapy and Temozolomide (TMZ) with a strong NCCN recommendation of enrollment in clinical trials. The only biomarker that can currently provide a stratification of strong and poor responders to standard of care is methylation of MGMT which indicates a median PFS of 10.3 months for methylated patients and 5.3 months for unmethylated patients. Development of patient-specific in vitro models of GBM for rapid testing of therapeutic options may yield more efficacious therapies and faster, more accurate assignation of therapies to each patient. To that end, we have developed a multi-faceted, patient-based 3D GBM model with modularity that facilitates increasing levels of model complexity such as the inclusion of immune cell components. Stable populations of glioma stem cells (GSC) from 24 of 41 patient samples have been successfully established, verified for stemness through limited dilution in in vitro and in vivo studies, and cultured long-term with minimal molecular changes, as determined from genetic analyses including RT-PCR arrays and MGMT methylation status, flow cytometry, and IHC. These cell populations have been used to establish and validate our 3D model system as well as generate comparable patient-derived xenografts (PDX). KIYATEC’s 3D microtumor in our 3DKUBE™ perfusion system provides a moderate throughput, dynamic system that is easily controlled to establish complex microtumors. GSC cell populations were cultured in monoculture only, co-cultured with human brain endothelial cells (HBEC), and tri-cultured with HBEC and CD14+ peripheral blood mononuclear cells (PBMC). Three microtumors were characterized by drug response to TMZ and axitinib, IHC, and molecular profiling including RNA expression and MGMT methylation status. Interestingly, increasing the complexity of the microtumor was capable of reestablishing the primary tumor MGMT methylation status if it were lost during culturing. We also generated PDX models from the same 3 patient tumor tissues as the above 3D models. PDX are a low throughput, time consuming, and expensive model that are still utilized for many systemic drug response studies and therefore a good comparator to the 3D tissue models we established. In vitro drug response in the 3D tissue models has been compared to both the matched PDX in vivo drug response and the patient’s clinical response to TMZ and MGMT methylation. Our data supports that KIYATEC’s complex patient-derived GBM model can be successfully used to identify, screen, and characterize novel treatments of GBM. Citation Format: Ashley M. Smith, Lillia Holmes, Lacey E. Dobrolecki, Charles Kanos, Stephen Gardner, Philip Hodge, Michael Lynn, Jeff Edenfield, Michael T. Lewis, Howland E. Crosswell, Teresa M. DesRochers. Ex vivo models of glioblastoma: a comparison of 3D tissues and patient-derived xenografts to clinical response [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 53.

Fabrication of 3D calcium-alginate scaffolds for human glioblastoma modeling and anticancer drug response evaluation.

The three-dimensional (3D) cell culture model has been increasingly used to study cancer biology and screen for anticancer agents due to its close mimicry to in vivo tumor biopsies. In this study, 3D calcium(Ca)-alginate scaffolds were developed for human glioblastoma cell culture and an investigation of the responses to two anticancer agents, doxorubicin and cordycepin. Compared to the 2D monolayer culture, glioblastoma cells cultured on these 3D Ca-alginate scaffolds showed reduced cell proliferation, increased tumor spheroid formation, enhanced expression of cancer stem cell genes (CD133, SOX2, Nestin, and Musashi-1), and improved expression of differentiation potential-associated genes (GFAP and β-tubulin III). Additionally, the vascularization potential of the 3D glioblastoma cells was increased, as indicated by a higher expression of tumor angiogenesis biomarker (VEGF) than in the cells in 2D culture. To highlight the application of Ca-alginate scaffolds, the 3D glioblastomas were treated with anticancer agents, including doxorubicin and cordycepin. The results demonstrated that the 3D glioblastomas presented a greater resistance to the tested anticancer agents than that of the cells in 2D culture. In summary, the 3D Ca-alginate scaffolds for glioblastoma cells that were developed in this study offer a promising platform for anticancer agent screening and the discovery of drug-resistant mechanisms of cancer.

Gene signatures of quiescent glioblastoma cells reveal mesenchymal shift and interactions with niche microenvironment.

BackgroundGlioblastoma (GBM), a highly malignant brain tumor, invariably recurs after therapy. Quiescent GBM cells represent a potential source of tumor recurrence, but little is known about their molecular underpinnings.MethodsPatient-derived GBM cells were engineered by CRISPR/Cas9-assisted knock-in of an inducible histone2B-GFP (iH2B-GFP) reporter to track cell division history. We utilized an in vitro 3D GBM organoid approach to isolate live quiescent GBM (qGBM) cells and their proliferative counterparts (pGBM) to compare stem cell properties and therapy resistance. Gene expression programs of qGBM and pGBM cells were analyzed by RNA-Seq and NanoString platforms.FindingsH2B-GFP-retaining qGBM cells exhibited comparable self-renewal capacity but higher therapy resistance relative to pGBM. Quiescent GBM cells expressed distinct gene programs that affect cell cycle control, metabolic adaptation, and extracellular matrix (ECM) interactions. Transcriptome analysis also revealed a mesenchymal shift in qGBM cells of both proneural and mesenchymal GBM subtypes. Bioinformatic analyses and functional assays in GBM organoids established hypoxia and TGFβ signaling as potential niche factors that promote quiescence in GBM. Finally, network co-expression analysis of TCGA glioma patient data identified gene modules that are enriched for qGBM signatures and also associated with survival rate.InterpretationOur in vitro study in 3D GBM organoids supports the presence of a quiescent cell population that displays self-renewal capacity, high therapy resistance, and mesenchymal gene signatures. It also sheds light on how GBM cells may acquire and maintain quiescence through ECM organization and interaction with niche factors such as TGFβ and hypoxia. Our findings provide a starting point for developing strategies to tackle the quiescent population of GBM.FundNational Institutes of Health (NIH) and Deutsche Forschungsgemeinschaft (DFG).

Refining Radiation for the Next Century.

![][1] Wilhelm Roentgen's report more than a century ago describing X-ray imaging fostered in the application of ionizing radiation (IR) for cancer therapy. Today, about half of all the patients with cancer are treated with radiotherapy and it remains one of our most effective

Gelatin Methacryloyl Hydrogels in the Absence of a Crosslinker as 3D Glioblastoma Multiforme (GBM)-Mimetic Microenvironment.

3D platforms are important for monitoring tumor progression and screening drug candidates to eradicate tumors such as glioblastoma multiforme (GBM), a malignant type of human brain tumor. Here, a new strategy is reported that exploits visible-light-induced crosslinking of gelatin where the reaction is carried out in the absence of an additional crosslinker. Visible light-induced crosslinking promotes the design of cancer microenvironment-mimetic system without compromising the cell viability during the process and absence of crosslinker facilitates the synthesis of the unique construct. Suspension and spheroid-based models of GBM are used to investigate cellular behavior, expression profiles of malignancy, and apoptosis-related genes within this unique network. Furthermore, sensitivity to an anticancer drug, Digitoxigenin, treatment is investigated in detail. The data suggest that U373 cells, in sparse or spheroid form, have significantly decreased expressions of apoptosis-activating genes, Bad, Puma, and Caspase-3, and a high expression of prosurvival Bcl-2 gene within GelMA hydrogels. Matrix-metalloproteinase genes are also upregulated within GelMA, suggesting positive contribution of gels on extracellular remodeling of cancer cells. This unique photocurable gelatin holds great potential for clinical translation of cancer research through the analysis of 3D malignant cancer cell behavior, and hence for more efficient treatment methods for GBM.

Abstract 1129: Sigma receptors: Novel targets for the treatment of highly malignant tumors

Abstract Introduction. More than 7 million patients are diagnosed with cancer every year, and approximately 24 million new cases of cancer will probably be registered by 2035. Over the past decades, numerous cancer targets have been identified and, despite promising results in this field, the discovery of novel targeted therapies is still a largely unmet need. Sigma receptors (SRs), more highly expressed in tumor cells than in normal cells, have aroused the interest of the scientific community because of increasing evidence of their involvement in proliferation and survival signaling pathways of cancer cells. Glioblastoma (GBM) and pancreatic cancer (PCa) are two of the most aggressive and lethal solid tumors. GBM patients usually relapse within 7-10 months of the end of first-line treatment with median survival of about 14.6 months while PCa is the fourth most fatal cancer in both men and women, with median survival of 6-12 months. Little or no improvement in prognosis has been obtained over the past 20 years. Thus, new approaches to the treatment of both tumors are urgently needed. In the present work, 3D GBM primary cultures endowed with stemness features and PCa cell lines were used to evaluate the antitumor activity of a dual S1R and S2R ligand RC-106, a novel sigma receptor modulator. Methods. RC-106 tissue distribution studies were performed in mice. PCa cell lines were assessed by cytotoxic assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay). Molecular analyses were performed by qRT-PCR and Western blot. Apoptosis and cell cycle were analyzed by flow cytometry. GBM primary cultures were isolated from surgical tumor samples. A rotatory cell culture system (RCCS) was used to obtain 3D cell cultures. Growth and morphology of the colonies were monitored by open-source AnaSP and ReViSP software tools. Molecular analyses were performed by flow cytometry and qRT-PCR. 3D cell viability was measured using CellTiter-Glo® 3D Cell Viability Assay. Apoptosis and cell cycle were analyzed by flow cytometry. Results. RC-106 preferentially accumulate in mice pancreas and brain after intravenous administration, supporting the use of sigma receptor modulators as a novel therapy for these tumors. Treatment with RC-106 strongly inhibited cell proliferation and survival regardless of the type of cancer investigated. 25 μM of the drug impaired the in vitro clonogenic ability of tumor cells, an effect lasting for up to 42 days, the longest time tested. RC-106 also led to the induction of substantial apoptosis mediated by caspase 3 and 9 activation, and to a modulation of the Akt pathway. Furthermore, treatment with RC-106 significantly reduced the growth of human GBM and PCa spheroids. Conclusions. (RC-106) represents the hit compound of a new class of dual-action ligands targeting S1R and S2R potentially useful for the treatment of cancer disease. Citation Format: Michela Cortesi, Sara Pignatta, Simona Collina, Andrea Rocca, Alice Zamagni, Chiara Arienti, Michele Zanoni, Luigino Tosatto, Daniela Bartolini, Evandro Nigrisoli, Annamaria Marra, Marta Rui, Anna Tesei. Sigma receptors: Novel targets for the treatment of highly malignant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1129. doi:10.1158/1538-7445.AM2017-1129

Abstract 4837: Development of an in vitro 3D glioblastoma model system for patient-specific drug response profiling

Abstract Glioblastoma (GBM) has a median survival of less than 2 years due to intra-tumoral and inter-patient heterogeneity, diffuse infiltration of adjacent brain tissue, and absence of effective therapies. Development of more efficacious therapies will require better GBM models for the testing and identification of novel agents; traditional 2D cell culture lacks biologic and clinical fidelity and orthotopic xenograft models are costly, low throughput, and time consuming. We have developed a complex, patient-specific 3D cultured GBM model to assay drug response that combines high-throughput drug response determination with neurosphere formation and next-generation sequencing (NGS). Neurosphere formation and the presence/quality of glioblastoma stem cells (GSCs) has been validate through limited dilution growth in vivo. Our 3D model system has been validated against the primary patient GBM tissue and/or patient-derived xenografts (PDX) and consists of histology, epigenetic and mRNA expression analysis, and comparison of in vivo drug response. This strategy enabled examination of MGMT methylation in relation to temozolomide response, and possible actionable genetic mutations with candidate targeted therapies. Using our 3D model, we have observed EGFR-amplified GBM sensitivity to the EGFR inhibitor afatanib, and PTEN mutant GBM sensitivity to dual PI3K/mTOR inhibitor dactolisib. These data suggest validation of clinical and molecular correlation with this new in vitro, patient-derived 3D GBM model for drug response profiling. Our data supports the further development and use of complex 3D models, neurosphere formation, and NGS profiling for patient-specific GBM analysis. This model system is currently being considered for preclinical assessment of novel therapies and may be a useful adjunct in future precision medicine applications to improve patient outcomes. Citation Format: Teresa DesRochers, Ashley Clark, Lauren O'Donnell, Qi Guo, Lillia Holmes, Lacey Dobrolecki, Michael Lewis, David Schammel, Jeff Edenfield, Charles Kanos, Fred Nelson, Steve Gardner, Michael Lynn, Philip Hodge, Christopher Corless, Paul Clark, Hal E. Crosswell, John Kuo. Development of an in vitro 3D glioblastoma model system for patient-specific drug response profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4837. doi:10.1158/1538-7445.AM2017-4837

Discovery and Characterization of R/S-N-3-Cyanophenyl-N'-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea, a New Histone Deacetylase Class III Inhibitor Exerting Antiproliferative Activity against Cancer Cell Lines.

A new series of N-aryl-N'-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)ureas bearing an alkoxycarbonylamino group at the 6-position were synthesized and examined as putative anticancer agents targeting sirtuins in glioma cells. On the basis of computational docking combined to in vitro sirtuin 1/2 inhibition assays, we selected compound 18 [R/S-N-3-cyanophenyl-N'-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea] which displays a potent antiproliferative activity on various glioma cell types, assessed by quantitative videomicroscopy, eventually triggering senescence. The impact on normal glial cells was lower with a selectivity index of >10. Furthermore, human U373 and Hs683 glioblastoma cell lines served to demonstrate the inhibitory activity of 18 against histone deacetylase (HDAC) class III sirtuins 1 and 2 (SIRT1/2) by quantifying acetylation levels of histone and non-histone proteins. The translational potential of 18 was validated by an NCI-60 cell line screen and validation of growth inhibition of drug resistant cancer cell models. Eventually, the anticancer potential of 18 was validated in 3D glioblastoma spheroids and in vivo by zebrafish xenografts. In summary, compound 18 is the first representative of a new class of SIRT inhibitors opening new perspectives in the medicinal chemistry of HDAC inhibitors.

EH1.2 Radiation responses of 2D and 3D glioblastoma cells: a novel, 3D-specific radioprotective role of VEGF/Akt signaling through functional activation of NHEJ

EH1.2 Radiation responses of 2D and 3D glioblastoma cells: a novel, 3D-specific radioprotective role of VEGF/Akt signaling through functional activation of NHEJ

A novel 3D human glioblastoma cell culture system for modeling drug and radiation responses.

Background.Glioblastoma (GBM) is the most common primary brain tumor, with dismal prognosis. The failure of drug–radiation combinations with promising preclinical data to translate into effective clinical treatments may relate to the use of simplified 2-dimensional in vitro GBM cultures.Methods.We developed a customized 3D GBM culture system based on a polystyrene scaffold (Alvetex) that recapitulates key histological features of GBM and compared it with conventional 2D cultures with respect to their response to radiation and to molecular targeted agents for which clinical data are available.Results.In 3 patient-derived GBM lines, no difference in radiation sensitivity was observed between 2D and 3D cultures, as measured by clonogenic survival. Three different molecular targeted agents, for which robust clinical data are available were evaluated in 2D and 3D conditions: (i) temozolomide, which improves overall survival and is standard of care for GBM, exhibited statistically significant effects on clonogenic survival in both patient-derived cell lines when evaluated in the 3D model compared with only one cell line in 2D cells; (ii) bevacizumab, which has been shown to increase progression-free survival when added to standard chemoradiation in phase III clinical trials, exhibited marked radiosensitizing activity in our 3D model but had no effect on 2D cells; and (iii) erlotinib, which had no efficacy in clinical trials, displayed no activity in our 3D GBM model, but radiosensitized 2D cells.Conclusions.Our 3D model reliably predicted clinical efficacy, strongly supporting its clinical relevance and potential value in preclinical evaluation of drug–radiation combinations for GBM.

Pharmacological inhibition of lipid droplet formation enhances the effectiveness of curcumin in glioblastoma

Increased lipid droplet number and fatty acid synthesis allow glioblastoma multiforme, the most common and aggressive type of brain cancer, to withstand accelerated metabolic rates and resist therapeutic treatments. Lipid droplets are postulated to sequester hydrophobic therapeutic agents, thereby reducing drug effectiveness. We hypothesized that the inhibition of lipid droplet accumulation in glioblastoma cells using pyrrolidine-2, a cytoplasmic phospholipase A2 alpha inhibitor, can sensitize cancer cells to the killing effect of curcumin, a promising anticancer agent isolated from the turmeric spice. We observed that curcumin localized in the lipid droplets of human U251N glioblastoma cells. Reduction of lipid droplet number using pyrrolidine-2 drastically enhanced the therapeutic effect of curcumin in both 2D and 3D glioblastoma cell models. The mode of cell death involved was found to be mediated by caspase-3. Comparatively, the current clinical chemotherapeutic standard, temozolomide, was significantly less effective in inducing glioblastoma cell death. Together, our results suggest that the inhibition of lipid droplet accumulation is an effective way to enhance the chemotherapeutic effect of curcumin against glioblastoma multiforme.

Delta-24-RGD Induces Cytotoxicity of Glioblastoma Spheroids in Three Dimensional PEG Microwells.

Glioblastoma (GBM) is the most aggressive brain tumor, with 12-15 months median survival time despite current treatment efforts. Among the alternative treatment approaches that have gained acceptance over the last decade is the use of replication-competent oncolytic adenoviruses, which are promising due to their relatively low toxicity and tumor-specific targeting. Three-dimensional (3D) tumor models can mimic the physiological microenvironment of GBM tumors and provide valuable information about the interaction between tumor cells and adenoviruses. Therefore, robust in vitro 3D tumor models are critical to investigate the mechanisms underlying tumor progression and explore the cytotoxicity effect of the adenovirus on tumor cells. In this study, we used a hydrogel microwell platform to generate in vitro 3D GBM spheroids and studied their interactions with the Delta-24-RGD adenovirus. The results showed that the cultured 3D spheroids were successfully infected by the Delta-24-RGD. A significant cell lysis was observed. Cell viability was decreased approximately 37%, 54% and 65% with 10, 50, and 100 MOIs, respectively. The infection of the Delta-24-RGD was found more effective on 3D spheroids when compared to 2D monolayer cell culture. These results implicate that our hydrogel microwell platform could provide a promising 3D model to investigate the oncolytic potential of the viruses in vitro.