Glioblastoma Multiforme Patient-derived Xenograft Research Articles

Reprogramming Glioblastoma Cells into Non-Cancerous Neuronal Cells as a Novel Anti-Cancer Strategy

Glioblastoma Multiforme (GBM) is an aggressive brain tumor with a high mortality rate. Direct reprogramming of glial cells to different cell lineages, such as induced neural stem cells (iNSCs) and induced neurons (iNeurons), provides genetic tools to manipulate a cell’s fate as a potential therapy for neurological diseases. NeuroD1 (ND1) is a master transcriptional factor for neurogenesis and it promotes neuronal differentiation. In the present study, we tested the hypothesis that the expression of ND1 in GBM cells can force them to differentiate toward post-mitotic neurons and halt GBM tumor progression. In cultured human GBM cell lines, including LN229, U87, and U373 as temozolomide (TMZ)-sensitive and T98G as TMZ-resistant cells, the neuronal lineage conversion was induced by an adeno-associated virus (AAV) package carrying ND1. Twenty-one days after AAV-ND1 transduction, ND1-expressing cells displayed neuronal markers MAP2, TUJ1, and NeuN. The ND1-induced transdifferentiation was regulated by Wnt signaling and markedly enhanced under a hypoxic condition (2% O2 vs. 21% O2). ND1-expressing GBM cultures had fewer BrdU-positive proliferating cells compared to vector control cultures. Increased cell death was visualized by TUNEL staining, and reduced migrative activity was demonstrated in the wound-healing test after ND1 reprogramming in both TMZ-sensitive and -resistant GBM cells. In a striking contrast to cancer cells, converted cells expressed the anti-tumor gene p53. In an orthotopical GBM mouse model, AAV-ND1-reprogrammed U373 cells were transplanted into the fornix of the cyclosporine-immunocompromised C57BL/6 mouse brain. Compared to control GBM cell-formed tumors, cells from ND1-reprogrammed cultures formed smaller tumors and expressed neuronal markers such as TUJ1 in the brain. Thus, reprogramming using a single-factor ND1 overcame drug resistance, converting malignant cells of heterogeneous GBM cells to normal neuron-like cells in vitro and in vivo. These novel observations warrant further research using patient-derived GBM cells and patient-derived xenograft (PDX) models as a potentially effective treatment for a deadly brain cancer and likely other astrocytoma tumors.

Abstract 6908: Integrating PDX GBM in vivo models with patient history and whole exome sequencing: Advancing relevance and precision in preclinical studies

Abstract Glioblastoma poses a significant challenge in oncology, necessitating improved translational models that faithfully replicate the intricate tumor microenvironment. Patient-derived xenograft (PDX) models, are key in glioblastoma multiforme (GBM) preclinical research. PDX models (orthotopic and subcutaneous) provide a more clinically relevant representation compared to CDX xenografts. Analyzing PDX tumors in their native environment allows for a nuanced understanding of GBM biology, tumor-stroma interactions, and treatment responses. This abstract explores the rationale and advantages of utilizing PDX GBM in vivo models coupled with patient diagnosis and treatment history, empowered by tumor whole exome sequencing (WES), to enhance the relevance of preclinical studies in evaluating GBM-specific treatments. TD2 has access to over 100 GBM PDX tumors models with fully annotated patient details and treatment history. Moreover, 68 of these PDXs also have WES data, that allows most appropriate model selection to ensure alignment with the therapeutic development strategy. Below is a detailed analysis of two GBM PDX models, GBM6 and GBM46, using patient history and WES data to define criteria for success in assessing novel preclinical therapeutic agents. GBM6, was obtained prior to treatment from a 65-year-old man with glioblastoma multiforme, that contains 642 mutations, including EGFR VIII amplification. GBM46, was obtained from a 55-year-old man with prior treatment, contained 646 mutations, including RAS pathway alterations and EGFR (MET and FLT3) amplification. When grown subcutaneously the anti-tumor activity of cetuximab against GBM6 and GBM46 resulted in 41% and 48% growth inhibition, respectively. Traditional treatments have also been evaluated in the orthotopic setting, with temozolomide (TMZ) showing a nominal improvement in lifespan (11%) as expected for the unmethylated MGMT found in the GBM6 model. By contrast, GBM46 from a patient with prior treatment with TMZ, OSI-774, radiation, and BCNU, exhibited a 58% increase in overall lifespan when treated with TMZ alone. Orthotopic PDX GBM models preserve many molecular characteristics, enabling the study of intrinsic GBM mutations for developing novel therapeutic strategies. While orthotopic models better capture GBM's dynamic nature, traditional xenograft systems remain valuable as rapid screening tools. Models with defined clinical treatment history and WES data allows for testing new agents in models that more closely resemble the clinical development path for a given new agent and aids in deciphering factors influencing therapeutic resistance. These models have the potential to reshape preclinical research paradigms, accelerating the translation of promising therapeutic agents increasing the likelihood of clinical benefit for patients with glioblastoma multiforme. Citation Format: Erin E. Trachet, Paul M. Gonzales, Stephen Gately. Integrating PDX GBM in vivo models with patient history and whole exome sequencing: Advancing relevance and precision in preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6908.

Abstract 6355: Urokinase plasminogen activator receptor-associated protein (uPARAP) is overexpressed in human glioblastomas and is a therapeutic target for antibody-drug conjugates (ADC)

Abstract Background: Glioblastoma Multiforme (GBM), primarily arising in the brain's frontal and temporal lobes, is a devastating malignancy originating from astrocytes. Despite treatment advancements, the prognosis remains bleak, with a median survival of 15-18 months and a 10% five-year survival rate.Our study explored a novel targeted approach to combat this challenge. uPARAP is an endocytic receptor expressed at low levels on selected mesenchymal cell types but highly overexpressed on the majority of Soft-Tissue- and Bone Sarcomas. It internalizes fragments of collagen and transfers them to the lysosome for degradation. Methods & Results: First, we analyzed formalin-fixed paraffin-embedded (FFPE) tumors from GBM patients through immunohistochemistry (IHC) staining. Interestingly, a majority of the samples showed strong but varying uPARAP expression. In parallel, we detected and quantified uPARAP receptor expression in GBM cell lines by flow cytometry, underscoring its potential as a therapeutic target.Next, we generated an antibody-drug conjugate (ADC) linked to monomethyl auristatin E (MMAE), a potent antimitotic agent. Confocal microscopy showed rapid internalization of the ADC in several GBM cell lines in vitro. Cellular cytotoxicity was demonstrated in Incucyte and Alamar Blue in vitro assays. Finally, anti-tumor activity of the uPARAP-ADC was studied in a GBM patient-derived xenograft (PDX) model in vivo, where it resulted in significant tumor regression with a tumor growth inhibition (TGI) of 92 and 97% at 3 and 6 mg/kg, respectively. Conclusions: Our findings show that uPARAP is overexpressed on GBM tumors and that a uPARAP-targeted ADC has anti-tumor activity in a PDX model of GBM. This research provides initial evidence that uPARAP-ADCs could become a novel treatment option for this highly aggressive disease. Citation Format: Ida Gregersen, Cecillie R. Løkke, Jette B. Lange, Pernille Barkholt, Christophé Côme, Tina Broberg, Marianne M. Petersen, Carmel M. Lynch, Dominik Mumberg, Niels Behrendt, Lars H. Engelholm. Urokinase plasminogen activator receptor-associated protein (uPARAP) is overexpressed in human glioblastomas and is a therapeutic target for antibody-drug conjugates (ADC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6355.

Abstract LB313: EGFRvIII-targeted alpha therapy shows significant therapeutic efficacy as both a single-agent and in combination with standard of care against preclinical GBM models

Abstract Targeted alpha therapy (TAT) is a rapidly advancing class of radiotherapeutics that can effectively deliver potent and local radiation to cancer cells while sparing the surrounding normal cells. TATs hold great promise for treatment-resistant tumors such as glioblastoma multiforme (GBM) due to the extensive DNA damage and cell death induced by alpha particles. GBM is an aggressive and lethal primary adult brain tumor that is highly resistant to external beam radiation and chemotherapy. Herein, we present the preclinical evaluation of a novel TAT for treatment of GBM targeting the most common tumour-specific mutant, epidermal growth factor receptor variant 3 (EGFRvIII). Our EGFRvIII TAT consists of a humanized EGFRvIII monoclonal antibody, a proprietary bifunctional chelate, and the alpha-emitting radionuclide, actinium-225 (225Ac). In vivo biodistribution and efficacy of our EGFRvIII TAT was evaluated in two aggressive orthotopic EGFRvIII-expressing GBM patient-derived xenograft models (PDXs; G06 and G39) with varying degrees of blood-brain-tumor barrier (BBTB) permeability. Imaging biodistribution studies were performed using an [111In]-anti-EGFRvIII agent analogous to our [225Ac] therapeutic candidate. SPECT/BLT imaging 96 h after intravenous administration of the imaging agent revealed high tumor-specific uptake of 50.2 %ID/cc in G39 tumors with a disrupted BBTB, and 10.4% ID/cc in G06 tumors with a relatively intact BBTB. Normal brain showed very low uptake (1.6-2.1% ID/cc), and all other normal organs were <10 % ID/cc except for highly perfused organs such as heart and liver, indicating no significant off-target uptake. [225Ac]-anti-EGFRvIII single-dose and fractionated multi-dose efficacy studies were conducted in G06 and G39 orthotopic PDX models, respectively. Survival analysis showed a dose-dependent increase in survival in response to treatment with [225Ac]-anti-EGFRvIII therapy, with a greater than 3-fold increase in survival compared to vehicle controls at doses of 200-400 nCi. Furthermore, the survival benefit of the 400 nCi single-dose or fractionated equivalent dose groups were comparable, demonstrating that efficacy was dependent upon the total administered radioactivity dose. Finally, we show that combined treatment of [225Ac]-anti-EGFRvIII with the standard of care (SoC), external beam radiation plus temozolomide, resulted in a significant survival advantage (>1.7-fold) compared to TAT or SoC therapy alone. Collectively, these results demonstrate that [225Ac]-anti-EGFRvIII is a highly selective and potent therapeutic for GBM which holds great potential as both a single-agent and in combination with SoC. Citation Format: Julie Metcalf, Alexander Nielsen, Suma Prabhu, Nicole Robinson, Sarah Marinacci, Natalie Grinshtein, Brigitte L. Theriault, Anne Marcil, Maria L. Jaramillo, Traian Sulea, Ivan A. Lessard, Maria Moreno, Christopher P. Leamon, Eric Burak, John Valliant. EGFRvIII-targeted alpha therapy shows significant therapeutic efficacy as both a single-agent and in combination with standard of care against preclinical GBM models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB313.

Secondary gliosarcoma: the clinicopathological features and the development of a patient-derived xenograft model of gliosarcoma

BackgroundGliosarcoma (GSM) is a distinct and aggressive variant of glioblastoma multiforme (GBM) with worse prognosis and few treatment options. It is often managed with the same treatment modalities with temozolomide (TMZ) as in GBM. However, the therapeutic benefits on GSM from such treatment regimen is largely unknown. Patient-derived xenograft (PDX) models have been used widely to model tumor progression, and subsequently to validate biomarkers and inform potential therapeutic regimens. Here, we report for the first time the successful development of a PDX model of secondary GSM.MethodsTissue obtained from a tumor resection revealed a secondary GSM arising from GBM. The clinical, radiological, and histopathological records of the patient were retrospectively reviewed. Samples obtained from surgery were cultured ex vivo and/or implanted subcutaneously in immunocompromised mice. Histopathological features between the primary GBM, secondary GSM, and GSM PDX are compared.ResultsIn explant culture, the cells displayed a spindle-shaped morphology under phase contrast microscopy, consistent with the sarcomatous component. GSM samples were subcutaneously engrafted into immunocompromised mice after single-cell suspension. Xenografts of serial passages showed enhanced growth rate with increased in vivo passage. We did not observe any histopathological differences between the secondary GSM and its serial in vivo passages of PDX tumors.ConclusionsOur PDX model for GSM retained the histopathological characteristics of the engrafted tumor from the patient. It may provide valuable information to facilitate molecular and histopathological modelling of GSM and be of significant implication in future research to establish precise cancer medicine for this highly malignant tumor.

CBMT-33. VISUALIZING THE METABOLISM OF GLIOBLASTOMA PATIENT-DERIVED ORTHOTOPIC XENOGRAFTS BY MASS SPECTROMETRY IMAGING

Abstract INTRODUCTION. Glioblastoma multiforme (GBM) has a dismal prognosis, which has been attributed to extensive inter- and intra-tumoural heterogeneity. To investigate the metabolic heterogeneity of GBM, we employed desorption electrospray ionization (DESI) imaging on snap frozen samples of GBM patient-derived orthotopic xenografts (PDOX) in rat brains, following infusion of [U-13C]glucose. METHODS. Athymic nude rats (n=3) were infused with [U-13C]glucose (0.4 mg/g body weight bolus, 0.012 mg/g/min at 300 µL/h infusion [1]) for 40, 80 and 120 min, followed by cardiac puncture to measure blood [U-13C]glucose levels using 13C NMR spectroscopy. A second set of rats (n=2) were implanted intracranially with patient-derived GBM cells. The presence of tumour was confirmed by T2-weighted MRI. One animal was infused with [U-13C]glucose for 2 h, while another with [12C]glucose. The brains were snap frozen in liquid nitrogen followed by analysis with DESI imaging in negative ion mode (35 µm resolution). RESULTS. After a 2 h infusion, 43% of the circulating glucose was 13C labelled. Unsupervised spatial clustering of the MSI data resulted in segmentation of normal brain from tumour. Levels of [12C and 13C]glucose were high in the peri-tumoural region and low within the tumour. [12C and 13C]lactate showed the opposite distribution, indicating that the rate of glucose utilization within the tumour exceeded the rate of delivery. The peri-necrotic and peri-tumoural areas had higher levels of glutamate compared to the rest of the tumour. CONCLUSIONS. The results demonstrate that the PDOX is metabolically heterogeneous and differs from surrounding brain. While the tumour was predominately glycolytic, the presence of glutamate in peri-necrotic and peri-tumoral regions indicates the presence of increased TCA cycle activity. Comparison of MSI data with histological staining of brain sections should yield additional information about the relationship between the tumour and its microenvironment.

Abstract 89: Development and characterization of a panel of orthotopic glioblastoma multiforme (GBM) patient-derived xenograft (PDX) mouse models for drug efficacy evaluation

Abstract Background Glioblastoma multiforme (GBM) is an aggressive cancer type with poor prognosis and survival. The lack of effective treatment may be due to the complex molecular composition and the heterogeneity of the tumors. Subcutaneous patient derived xenograft (PDX) mouse models are widely used in drug development. However, the models fail at modeling the complex microenvironment in the brain and the impact of the blood brain barrier on drug bioavailability. Here we report the development and characterization a panel of orthotopic PDX mouse models. Methods Low passage subcutaneous tumors from ten different PDX GBM models (ST108, ST112, ST146, ST545, ST610, ST1388, ST2473, ST3537, ST3713, and ST3720) were implanted orthotopically. Tumor development and growth was monitored by T2-weighted magnetic resonance imaging (MRI). Mice were treated with either temozolomide or vehicle when tumor take was confirmed on an individual basis by MRI. Tumor treatment response was evaluated by MRI, and the final end-point was survival by humane endpoints. Whole brains and tumors were formalin fixed or snap frozen for histological evaluation of markers of invasiveness and cancer stem cells (Nestin, CD44, SOX2, and CXCR4). In addition, the radiosensitivity of the models was characterized by fractionated external radiation therapy (XRT) delivered as 2 Gy QD x5 or sham in the subcutaneous setting. Expression of EGFR, EGFRvIII, MGMT and mGluR3 were evaluated by qPCR. Results The models displayed a wide range in expression levels of EGFR, EGFRvIII, MGMT and mGluR3. Mice treated with XRT showed a variable treatment outcome on both tumor volume and survival. The impact of XRT on prolongation of median survival ranged from 45% to 141%. In the orthotopic setting a variable range of sensitivities was observed in the models ranging from sensitive (ST610 and ST2473) to highly resistant (ST112). Histologic evaluation showed typical characteristics of GBM tumors such as pseudopalisading cells. Furthermore, cell populations positive for cancer stem cell and invasiveness markers were detected by immunofluorescence imaging. Conclusion A panel of GBM PDX models was characterized for several molecular markers and the panel reflected the heterogeneity of human GBM tumors. The models displayed varying sensitivity towards XRT and TMZ. The established panel of orthotopic GBM PDX models can be used as a platform for testing of new drugs in a setting that more closely mimics the GBM tumor microenvironment and impact of the blood brain barrier. Citation Format: Carsten H. Nielsen, Maria Z. Alfsen, Michael J. Wick, Melissa Rundle, Johann M. Gudbergsson, Mette M. Jensen, Lotte K. Kristensen, Mark U. Juul, Kyriakos P. Papadopoulos, Andreas Kjaer. Development and characterization of a panel of orthotopic glioblastoma multiforme (GBM) patient-derived xenograft (PDX) mouse models for drug efficacy evaluation [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 89.

Abstract 2161: Characterization of a panel of orthotopic glioblastoma multiforme (GBM) patient-derived xenograft (PDX) mouse models and efficacy evaluation of temozolomide and external radiation therapy

Abstract Background Glioblastoma multiforme (GBM) is an aggressive cancer type with poor prognosis. The lack of effective treatment may be due to the complex molecular composition and the heterogeneity of the tumors. Standard of care treatment is surgery, external radiation therapy (XRT) and chemotherapy with temozolomide (TMZ); however, the response rate varies widely. Subcutaneous patient-derived xenograft (PDX) models are increasingly used during drug development. Implantation of GBM PDX tumors orthotopically in the brain displays an advantage as the tumors develop in a microenvironment that resembles that of the original patient tumor. In addition, the impact of the blood brain barrier (BBB) in terms of reduced drug bioavailability is also represented in the orthotopic GBM models. The aim of this study was to develop and characterize a panel of orthotopic GBM PDX models. The models were then used to study the efficacy of TMZ and XRT. Methods A panel of 14 PDX GBM models was characterized for EGFR, EGFRvIII, MGMT and mGluR3 expression. Selected PDX GBM models were furthermore characterized for GBM stem cell markers by flow cytometry. Low passage subcutaneous tumors from seven PDX GBM models (ST108, ST112, ST146, ST545, ST610, ST1388 and ST2473) were used for assessment of XRT sensitivity. Mice were randomized to receive XRT (2 Gy/day for 5 days) or sham. Furthermore, subcutaneous tumors were enzymatically digested and used for intracranial stereotactic injection in nude mice. Tumor take and growth rate was determined by T2-weighted magnetic resonance imaging (MRI) and 18F-fluoro-ethyl-L-tyrosine (18F-FET) PET imaging. At confirmed tumor take, mice were treated with TMZ (20 mg/mg 5 days on/2 days off x3 or 100mg/kg/day for 5 days) or whole brain XRT (2 Gy/day for 5 days). Control groups received vehicle or sham XRT. Final endpoint was survival by humane endpoints and tumors were fixed in formalin for histologic evaluation. Results A wide variation in expression levels of EGFR, EGFRvIII, MGMT and mGluR3 was observed in the PDX GBM panel. Three models had EGFRvIII mutation and all models had variable expression levels of EGFR. XRT showed a variable efficacy outcome on both tumor volume and survival in the PDX GBM models in the subcutaneous and orthotopic setting. TMZ showed efficacy in ST610 and ST2473 whereas ST146 and ST112 were resistant towards TMZ. Histologic evaluation showed typical characteristics of GBM tumors. Conclusion A panel of GBM PDX models was characterized for several molecular markers and the panel reflected the heterogeneity of human GBM tumors. The orthotopic GBM PDX models showed a wide range of sensitivity towards both TMZ and XRT. The established panel of orthotopic GBM PDX models can be used as a platform for testing of new drugs in a setting that more closely mimic the GBM tumor microenvironment and impact of the BBB. Citation Format: Mette Munk Jensen, Michael J. Wick, Lotte K. Kristensen, Maria Z. Alfsen, Laura A. Paludan, Mark Juul, Melissa Rundle, Kyriakos P. Papadopoulos, Andreas Kjær, Carsten H. Nielsen. Characterization of a panel of orthotopic glioblastoma multiforme (GBM) patient-derived xenograft (PDX) mouse models and efficacy evaluation of temozolomide and external radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2161.

Combinatorial Drug Testing in 3D Microtumors Derived from GBM Patient-Derived Xenografts Reveals Cytotoxic Synergy in Pharmacokinomics-informed Pathway Interactions

Glioblastoma multiforme (GBM), the most common form of primary malignant brain cancer in adults, is a devastating disease for which effective treatment has remained elusive for over 75 years. One reason for the minimal progress during this time is the lack of accurate preclinical models to represent the patient’s tumor’s in vivo environment, causing a disconnect in drug therapy effectiveness between the laboratory and clinic. While patient-derived xenografts (PDX’s or xenolines) are excellent human tumor representations, they are not amenable to high throughput testing. Therefore, we developed a miniaturized xenoline system (microtumors) for drug testing. Nineteen GBM xenolines were profiled for global kinase (kinomic) activity revealing actionable kinase targets associated with intracranial tumor growth rate. Kinase inhibitors for these targets (WP1066, selumetinib, crizotinib, and cediranib) were selected for single and combination therapy using a fully human-derived three-dimensional (3D) microtumor model of GBM xenoline cells embedded in HuBiogel for subsequent molecular and phenotype assays. GBM microtumors closely resembled orthotopically-implanted tumors based on immunohistochemical analysis and displayed kinomic and morphological diversity. Drug response testing could be reproducibly performed in a 96-well format identifying several synergistic combinations. Our findings indicate that 3D microtumors can provide a suitable high-throughput model for combination drug testing.

Abstract 2805: A panel of orthotopic glioblastoma multiforme (GBM) patient derived xenograft (PDX) mouse models for efficacy evaluation of drugs

Abstract Background: Patients with glioblastoma multiforme (GBM) have a poor prognosis and few treatment options; hence new treatments are needed. Subcutaneous patient derived xenograft (PDX) models are increasingly used for efficacy studies in drug development. However, orthotopic implantation confers a translational advantage as the cancer develops in a microenvironment more closely mimicking that of the original patient tumor. Also the major impact of the blood brain barrier that must be taking into account when targeting brain tumors as GBM in terms of drug bioavailability is better represented in the orthotopic models. The aim of this study was therefore to develop a panel of orthotopic GBM PDX models for pre-clinical efficacy studies of new drugs. The models were then used to study the efficacy of standard of care such as temozolomide (TMZ) and external radiation therapy (XRT). Methods: Low passage subcutaneous tumors from six different PDX GBM models designated ST108, ST112, ST146, ST545, ST610 and ST2473 were digested and used for intracranial stereotactic injection in nude mice. Tumor take and growth was determined by T2-weighted magnetic resonance imaging (MRI). At confirmed tumor take mice were either treated with TMZ (100mg/kg/day for 5 days) or whole brain XRT (2 Gy/day for 5 days). Control groups receiving vehicle or sham XRT were included depending on treatment regiment. Final endpoint was survival by humane endpoints and tumors were fixed in formalin for histological evaluation. Results: MRI confirmed tumor take in all models within 5 weeks of implantation. The take rate was > 80% across all models. TMZ showed efficacy in the orthotopic ST610 GBM PDX model evaluated by MRI on day 14 (16.2±2.9 mm3 vs. 76.8±13.1 mm3, p=0.016), whereas the ST146 model displayed resistance to TMZ on day 14 (12.7±5.6 mm3 vs. 26.5±11.9 mm3, p=0.26). The median survival was 60 days vs. 14 days in the ST610 model (TMZ vs. vehicle, p=0.0005) and 27 days vs. 13 days in the ST146 model (TMZ vs. vehicle, p=0.007). XRT showed efficacy in the orthotopic ST2473 model. Tumor volume was significantly smaller in treated vs. sham animals 11 days after inclusion (6.9±1.4 mm3 vs. 28.9±3.3 mm3, p=0.001). Also, a survival benefit was observed in XRT treated animals compared to sham. Histology confirmed the presence of orthotopic tumors and typical GBM pathology characteristics such as pseudopalisading tumor cells surrounding necrosis and micro vascular proliferation were identified. Conclusion: Six different orthotopic GBM PDX models were established from low passage subcutaneous PDX models. Models sensitive and resistant to TMZ were identified and histological GBM characteristics were identified. Together, the established panel of orthotopic PDX models can be used as a relevant translational platform for testing of new drugs in a setting that more closely mimics the GBM tumor microenvironment and the impact of the blood brain barrier in patients. Citation Format: Mette M. Jensen, Camilla S. Knudsen, Lotte K. Kristensen, Mette K. Nedergaard, Michael J. Wick, Kyriakos P. Papadopoulos, Anthony W. Tolcher, Andreas Kjaer, Carsten H. Nielsen. A panel of orthotopic glioblastoma multiforme (GBM) patient derived xenograft (PDX) mouse models for efficacy evaluation of drugs [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 2805. doi:10.1158/1538-7445.AM2017-2805

Abstract A16: Combinatorial drug testing using 3D microtumors derived from GBM PDX reveals cytotoxic synergisms in pharmacokinomics-informed pathway interactions

Abstract Introduction: Drug testing of Glioblastoma multiforme (GBM) has revealed that many promising preclinical therapies fail when translated to the clinic. Serious limitations in preclinical models (often monolayer, serum-treated immortalized cell models) likely contribute to this translational failure. Although patient-derived xenografts (PDX), in which patient tumors are serially passaged in immunocompromised mice, are potentially superior model systems of human disease, high throughput drug studies in GBM PDX tumors are not feasible due to time, cost and throughput. To overcome these challenges, we have developed a GBM PDX MicroTumor system in which GBM PDX cells are grown in a novel 3D extracellular matrix material (HuBiogel™, Vivo Biosciences Inc., Birmingham, AL). We hypothesized that this 3D MicroTumor system would better emulate in vivo PDX tumor growth while providing a high throughput assay system for drug combination studies that can be performed in a more rapid timeframe. Methods: Six GBM PDX tumor “xenolines” were selected from the UAB Brain Tumor Animal Model Core to represent the 4 known molecular subtypes of GBM: Classical (JX10, X1016, X1046); Proneural (XD456); Neural (JX10) and Mesenchymal (JX22P). Tumor cells were prepared as single cell suspensions from subcutaneously passaged tumors and embedded in HuBiogel™ beads to form MicroTumors using Neuro-basal media devoid of serum. Single and combinatorial drug cytotoxicity studies were performed using MTT mitochondrial viability assays and Calcein AM imaging. Small molecule inhibitors selected for drug testing were: 1) selumetinib-targeting MEK1/2; 2) crizotinib-targeting c-MET and ALK; 3) cediranib-targeting VEGFR, FLT-1, FLT-4, c-KIT, and PDGFR; and 4) WP1066-targeting JAK2/STAT3. Combinatorial interactions were determined by Chou-Talalay combination index (CI) calculation using Calcusyn software. Global kinase signaling (kinomic profiling) was assessed using paired total cell lysates of GBM-PDX from orthotopic tumors grown in athymic nu/nu mice and as 3D MicroTumors. Testing was performed on the PamStation12 high content peptide microarray platform within the UAB Kinome Core. Results: Following optimization of model system conditions, 4 drugs were tested at multiple concentrations to identify effective dose ranges. While crizotinib, cediranib and WP1066 demonstrated significant cytotoxicity in at least 1 of the GBM-PDX MicroTumors tested, selumetinib was ineffective in producing cytotoxicity as a monotherapy. However, in combinatorial drug testing, selumetinib was effective in promoting synergistic activity (CI<1.0) when combined with each of the other drugs in several of the GBM-PDX tested. Conversely, WP1066 demonstrated some antagonistic interactions with the other drugs in certain tumors. Overall, cediranib and crizotinib demonstrated the most consistent level of efficacy both as monotherapies and combinatorial therapies across the GBM-PDX MicroTumors. Kinomic profiles were compared between GBM-PDX MicroTumors and matched GBM-PDX tumor xenolines grown as orthotopic intracranial tumors to confirm kinase signaling fidelity between the model systems. Paired t-test of kinomic phosphopeptide probes revealed only 20 of 246 peptides significantly differed (FDR<0.05) between the model systems suggesting a high level of signaling preservation within the MicroTumors. Conclusions: The GBM-PDX 3D MicroTumor provides a relatively high throughput drug screening model system that displays similar kinase signaling pathway activation as compared to their in vivo intracranial counterpart. Specific synergistic drug combinations were identified through MicroTumor testing. Ongoing drug combination testing of intracranially implanted GBM-PDX will determine how robustly the MicroTumor system predicts in vivo efficacy. Citation Format: Christopher D. Willey, Ashley N. Gilbert, Rachael Shevin, Catherine P. Langford, Christine W. Duarte, Raj Singh, Joshua C. Anderson, G. Yancey Gillespie. Combinatorial drug testing using 3D microtumors derived from GBM PDX reveals cytotoxic synergisms in pharmacokinomics-informed pathway interactions. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A16.

CSIG-11OLIG2-MEDIATED REGULATION OF GLIOMA INVASION

Glioblastoma multiforme (GBM) is a highly aggressive, infiltrative, and lethal brain tumor. GBM patients have an overall survival of <1 year, despite major research efforts in diagnosis and treatment. Almost all GBM patients experience tumor recurrence, for which there is currently no standard of care. A major challenge with GBM is that invasive glioma cells extend beyond the surgically resected regions typically defined based on magnetic resonance imaging. These invasive cells are resistant to adjuvant therapies, lead to tumor recurrence and contribute to poor prognosis in GBM patients. It has been proposed that migrating tumor cells undergo a switch from proliferation to invasion. However, the molecular and cellular mechanism involved in the regulation of this switch from proliferation to invasion/migration in GBM is poorly understood. We have previously shown that the lineage-specific transcription factor, Olig2 plays a critical role in glioma formation. Here we examined the differential role of Olig2 in promoting the switch from proliferation to invasion. We utilized genetically relevant mouse model of as well as GBM patient-derived xenograft (PDX) models to investigate the role of Olig2 in promoting glioma invasion. We show that Olig2 expressing glioma cells are highly invasive and migratory irrespective of their TCGA molecular subtype. Knockdown of Olig2 significantly decreases the ability of GBM PDX-derived cells to invade matrigel matrix. We find that Olig2 expressing cells are present both at the proliferating tumor core as well as at the invasive rim in GBM patient tissues. Mechanistically, we find that Olig2 regulates the expression of epithelial-to-mesenchymal transition (EMT) genes such as Twist1, Snai1, Zeb1, Stat3, CD44 and Vimentin and genes involved in invasion regulation like TGFβ2 and Epha2. Our data provides evidence for a role of Olig2 in both proliferation and invasion.

Abstract 312: Profiling drug sensitivity and kinomic pathways utilizing a novel human tumor derived MicroTumor assay

Abstract Introduction: At present, drug screening studies are commonly performed using monolayer or spheroid culture and xenograft models of tumor cell lines. However these do not fully replicate the primary tumor's microenvironment and fail to accurately predict clinical endpoints. Vivo Biosciences has developed a novel MicroTumor 3D matrix based assay system that emulates primary tumor multicellular growth and biology ex vivo, providing an advanced drug screening platform. We postulated that MicroTumors established from patient-derived xenograft (PDX) tumors will allow for accurate analysis of drug response and preserve molecular signaling of parent tumors. Glioblastoma multiforme (GBM), the most common primary brain malignancy, was used to test our hypothesis. MicroTumors were evaluated by comparing kinome activation profiles of GBM-MicroTumors with corresponding parental orthotopically implanted PDX; and determining single and combination treatment effects of small molecule kinase inhibitors (SMI) on GBM MicroTumors. Methods: We investigated 6 GBM PDX tumor lines representing the 4 known molecular subtypes: Classical (JX10, X1016, X1046); Proneural (XD456); Mesenchymal (JX22P); and Neural (JX10). Four SMIs (primary kinase target indicated) were studied: WP1066 (JAK2), selumetinib (MEK1/2), crizotinib (c-MET, ALK), and cediranib (VEGFR, FLT-1, FLT-4, c-KIT, PDGFR). MTT assays and Calcein AM imaging were used for cytotoxicity assessment and PamStation 12 Kinomic analyses were performed (UAB Kinome Core). Results: Kinomic analyses of GBM orthotopic PDX and GBM-MicroTumors revealed similar kinase signaling profiles based on comparison of commonly shared, most-variant phosphopeptides. Upstream kinase analyses identified these peptides as substrates of EGFR, AXL, ZAP70 and MERTK kinases. Initial drug response studies demonstrated dose dependency and PDX-specific responses for each drug used independently informing doses for ongoing combination studies. Interestingly, the least cytotoxic drug across all 6 MicroTumors, selumetinib, did impact MicroTumor morphology observed with Calcein AM imaging. Conclusions: We identified kinomic alterations that may correlate MicroTumor and patient tumor biology and guide the use of molecularly targeted SMIs. SMI activities towards these targets highlighted that this novel 3D translational model for GBM can provide relevant drug sensitivity information. Future studies with in vivo PDX tumors will examine the most promising SMI combinations based on MicroTumor data. We believe this two-stage approach (Microtumor screening to predict PDX sensitivities) will improve preclinical drug screening in GBM and other cancers. Citation Format: Christopher D. Willey, Ashley N. Gilbert, Rachael Shevin, Catherine P. Langford, Raj Singh, Joshua C. Anderson, G. Yancey Gillespie. Profiling drug sensitivity and kinomic pathways utilizing a novel human tumor derived MicroTumor assay. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 312. doi:10.1158/1538-7445.AM2015-312

Abstract 1468: Characterization of patient-derived xenograft (PDX) models to evaluate clinical and therapeutic responses of glioblastoma multiforme

Abstract BACKGROUND/PURPOSE The Mayo Clinic has developed a large panel of patient-derived xenografts (PDX) from patients with glioblastoma multiforme (GBM). Here we report initial molecular profiling of the Mayo PDX panel and a comparison of patient and PDX molecular profile and response to therapy. METHODS &amp; MATERIALS Clinical data was retrieved by retrospective chart review. Animal data from all PDX investigations conducted between 2004-2014 were retrieved from experimental logs and subsequently consolidated into a database for analysis. RESULTS From 1999 to 2014, 182 patient tumor samples of varying histological grades were attempted for xenograft with 73 resulting viable PDX lines. Viable xenografts were only produced from WHO grade IV tumor specimens, yielding an overall success rate of 49% for these tumors. GBM patients that produced viable xenografts compared to those that did not exhibited a trend for decreased overall survival (P = 0.18). There was no significant association between successful xenografting and whether tissue was from newly diagnosed (45/93%) or recurrent (20/40; 50%) tumors. Patient age &amp;gt; 45 at diagnosis was correlated with increased PDX viability in GBMs (p = 0.05). Of the viable PDX models analyzed, EGFR mutation was identified in 17 lines, TERT mutation was found in 13 lines, IDH mutation in 1 line, and MGMT hypermethylation in 25 lines. RNAseq was performed on orthotopic tumor samples from 53 PDX models. After excluding contaminating murine sequence reads, expression analysis demonstrated 32 models with a mesenchymal phenotype. Array comparative genomic hybridization was performed on 9 patient samples and derivative early, mid and late passage PDX tumors. Using unbiased hierarchical clustering, there was a high concordance between patient and xenograft models. Within the PDX panel, fractionated radiation (RT) alone and RT combined with temozolomide (TMZ) was tested in orthotopic tumors in 38 lines. The overall median survival benefit (ratio of median survival for treated vs. placebo) in PDX lines treated with RT only was 1.6 (range: 0.9-2.5) and with RT/TMZ was 2.5 (range: 1.1 - 8.9). There was a positive association between observed patient survival and the corresponding survival benefit in the PDX for subjects treated with RT/TMZ (r = 0.2; n = 17). Response to adjuvant TMZ was evaluated in 42 tumor lines, and response to bevacizumab was tested in 33 tumor lines, and correlations with clinical treatment response are being evaluated. CONCLUSIONS: The Mayo GBM PDX panel is widely used in the neuro-oncology community. The initial molecular analysis suggests a good correlation between patient and PDX at the level of genotypic characteristics and therapeutic sensitivity. Citation Format: Dioval A. Remonde, Brett L. Carlson, Mark A. Schroeder, Brock Armstrong, Sen Peng, Lisa Evers, Paul A. Decker, Jeanette Eckel Passow, Michael E. Berens, Nhan L. Tran, Robert B. Jenkins, Jann N. Sarkaria. Characterization of patient-derived xenograft (PDX) models to evaluate clinical and therapeutic responses of glioblastoma multiforme. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1468. doi:10.1158/1538-7445.AM2015-1468

Abstract 3227: Advanced imaging with PET and MRI in Temozolomide sensitive and resistant subcutaneous and orthotopic patient-derived xenograft (PDX) glioblastoma models

Abstract Background: Patients with glioblastoma multiforme (GBM) have a poor prognosis and few treatment options; hence new treatment strategies are needed. Recently, we adopted standard clinical protocols using magnetic resonance imaging (MRI) and positron emission tomography (PET) with the amino acid PET-tracer (18F-FET) for monitoring of tumor development in orthotopic cell-line GBM models. However, patient-derived xenograft (PDX) models are increasingly used, as they are more predictive of the therapeutic response in patients. Orthotopic PDX models confer a translational advantage as the cancer cells develop in a microenvironment that is similar to the original patient tumor. The aim of this study was to establish Temozolomide sensitive and resistant subcutaneous and orthotopic PDX GBM models for pre-clinical efficacy studies of new drugs. Furthermore, the performance of 18F-FET PET as an imaging biomarker of therapeutic response was evaluated in the orthotopic PDX models. Methods: Patient biopsy material from a Temozolomide sensitive (ST610) and a Temozolomide resistant (ST146) patient were implanted subcutaneously in nude mice and tumor growth was measured by caliper after Temozolomide (100 mg/kg/day) or control treatment. Orthotopic PDX GMB models were established from ST610 and ST146 subcutaneous tumors. Tumors were enzymatically digested and used for intracranial stereotactic injection in nude mice (n = 16 / model). MRI and 18F-FET PET imaging were used to determine tumor take (TT), tumor growth, and treatment response. At confirmed TT, mice were treated with Temozolomide (100mg/kg/day) or control. The primary endpoint was survival. Secondary endpoints were treatment response using MRI and 18F-FET PET. Results: Temozolomide sensitive and a Temozolomide resistant subcutaneous PDX models were established. Temozolomide inhibited tumor growth in the sensitive model whereas no effect was seen in the resistant model. In addition, contrast enhanced MRI confirmed orthotopic tumor development after implantation. PET imaging as early as two weeks after intracranial implantation detected increased 18F-FET uptake in tumor areas. Finally, response to Temozolomide was followed by 18F-FET PET in the orthotopic models. Conclusion: The current study confirms the predictive value of the GBM PDX models. By using the orthotopic GBM PDX models, translational imaging techniques can be evaluated and the potential of tracers like 18F-FET as imaging biomarkers of therapeutic response can be assessed. Together, the established subcutaneous and orthotopic PDX models can be used as a relevant translational platform for testing of new drugs. Citation Format: Mette K. Nedergaard, Carsten H. Nielsen, Kyriakos P. Papadopoulos, Anthony W. Tolcher, Michael J. Wick, Andreas Kjaer. Advanced imaging with PET and MRI in Temozolomide sensitive and resistant subcutaneous and orthotopic patient-derived xenograft (PDX) glioblastoma models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3227. doi:10.1158/1538-7445.AM2015-3227

Delineation of MGMT Hypermethylation as a Biomarker for Veliparib-Mediated Temozolomide-Sensitizing Therapy of Glioblastoma.

Sensitizing effects of poly-ADP-ribose polymerase inhibitors have been studied in several preclinical models, but a clear understanding of predictive biomarkers is lacking. In this study, in vivo efficacy of veliparib combined with temozolomide (TMZ) was evaluated in a large panel of glioblastoma multiforme (GBM) patient-derived xenografts (PDX) and potential biomarkers were analyzed. The efficacy of TMZ alone vs TMZ/veliparib was compared in a panel of 28 GBM PDX lines grown as orthotopic xenografts (8-10 mice per group); all tests of statistical significance were two-sided. DNA damage was analyzed by γH2AX immunostaining and promoter methylation of DNA repair gene O6-methylguanine-DNA-methyltransferase (MGMT) by Clinical Laboratory Improvement Amendments-approved methylation-specific polymerase chain reaction. The combination of TMZ/veliparib statistically significantly extended survival of GBM models (P < .05 by log-rank) compared with TMZ alone in five of 20 MGMT-hypermethylated lines (average extension in median survival = 87 days, range = 20-150 days), while the combination was ineffective in six MGMT-unmethylated lines. In the MGMT promoter-hypermethylated GBM12 line (median survival with TMZ+veliparib = 189 days, 95% confidence interval [CI] = 59 to 289 days, vs TMZ alone = 98 days, 95% CI = 49 to 210 days, P = .04), the profound TMZ-sensitizing effect of veliparib was lost when MGMT was overexpressed (median survival with TMZ+veliparib = 36 days, 95% CI = 28 to 38 days, vs TMZ alone = 35 days, 95% CI = 32 to 37 days, P = .87), and a similar association was observed in two nearly isogenic GBM28 sublines with an intact vs deleted MGMT locus. In comparing DNA damage signaling after dosing with veliparib/TMZ or TMZ alone, increased phosphorylation of damage-responsive proteins (KAP1, Chk1, Chk2, and H2AX) was observed only in MGMT promoter-hypermethylated lines. Veliparib statistically significantly enhances (P < .001) the efficacy of TMZ in tumors with MGMT promoter hypermethylation. Based on these data, MGMT promoter hypermethylation is being used as an eligibility criterion for A071102 (NCT02152982), the phase II/III clinical trial evaluating TMZ/veliparib combination in patients with GBM.

Genome-wide RNAi screens in human brain tumor isolates reveal a novel viability requirement for PHF5A

To identify key regulators of human brain tumor maintenance and initiation, we performed multiple genome-wide RNAi screens in patient-derived glioblastoma multiforme (GBM) stem cells (GSCs). These screens identified the plant homeodomain (PHD)-finger domain protein PHF5A as differentially required for GSC expansion, as compared with untransformed neural stem cells (NSCs) and fibroblasts. Given PHF5A's known involvement in facilitating interactions between the U2 snRNP complex and ATP-dependent helicases, we examined cancer-specific roles in RNA splicing. We found that in GSCs, but not untransformed controls, PHF5A facilitates recognition of exons with unusual C-rich 3' splice sites in thousands of essential genes. PHF5A knockdown in GSCs, but not untransformed NSCs, astrocytes, or fibroblasts, inhibited splicing of these genes, leading to cell cycle arrest and loss of viability. Notably, pharmacologic inhibition of U2 snRNP activity phenocopied PHF5A knockdown in GSCs and also in NSCs or fibroblasts overexpressing MYC. Furthermore, PHF5A inhibition compromised GSC tumor formation in vivo and inhibited growth of established GBM patient-derived xenograft tumors. Our results demonstrate a novel viability requirement for PHF5A to maintain proper exon recognition in brain tumor-initiating cells and may provide new inroads for novel anti-GBM therapeutic strategies.

Abstract A20: Genome-wide RNAi screens in human brain tumor isolates reveal a novel viability requirement for PHF5A

Abstract To identify key regulators of human brain tumor maintenance and initiation, we performed multiple genome-wide RNAi screens in patient-derived glioblastoma multiforme (GBM) stem cells (GSCs). These screens identified the PHD-finger domain protein PHF5A as differentially required for GSC expansion gene, as compared to untransformed neural stem cells (NSCs) and fibroblasts. Given PHF5A's known involvement in facilitating interactions between the U2 snRNP complex and ATP-dependent helicases, we examined cancer-specific roles in RNA splicing. We find that in GSCs, but not untransformed controls, PHF5A facilitates recognition of exons with unusual C-rich 3' splice sites in thousands of essential genes. PHF5A knockdown in GSCs, but not untransformed NSCs, astrocytes, or fibroblasts, inhibited splicing of these genes, leading to cell cycle arrest and loss of viability. Notably, pharmacologic inhibition of U2 snRNP activity phenocopied PHF5A knockdown both in GSCs and in multiple genetically transformed cell types, suggesting that a cancer-specific requirement for PHF5A activity may be generalizable to a wide range of cancers. Furthermore, PHF5A inhibition compromises GSC tumor formation in vivo and dramatically inhibits the growth of established GBM patient-derived xenograft tumors. Together, this work demonstrates an additional mechanism for maintaining splicing fidelity in cancer cells and also suggests that PHF5A activity may provide new in-roads for novel anti-GBM therapeutic strategies. Citation Format: Christopher G. Hubert, Robert K. Bradley, Yu Ding, Toledo M. Chad, Kyobi Skutt-Kakaria, Emily J. Girard, Jerry Davison, Jason Berndt, Philip Corrin, Ryan Basom, Jeffery J. Delrow, Thomas Webb, Steven M. Pollard, Jeongwu Lee, James M. Olson, Patrick J. Paddison. Genome-wide RNAi screens in human brain tumor isolates reveal a novel viability requirement for PHF5A. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A20.