Cecilia Krona

Case-specific potentiation of glioblastoma drugs by pterostilbene

Glioblastoma multiforme (GBM, astrocytoma grade IV) is the most common malignant primary brain tumor in adults. Addressing the shortage of effective treatment options for this cancer, we explored repurposing of existing drugs into combinations with potent activity against GBM cells. We report that the phytoalexin pterostilbene is a potentiator of two drugs with previously reported anti-GBM activity, the EGFR inhibitor gefitinib and the antidepressant sertraline. Combinations of either of these two compounds with pterostilbene suppress cell growth, viability, sphere formation and inhibit migration in tumor GBM cell (GC) cultures. The potentiating effect of pterostilbene was observed to a varying degree across a panel of 41 patient-derived GCs, and correlated in a case specific manner with the presence of missense mutation of EGFR and PIK3CA and a focal deletion of the chromosomal region 1p32. We identify pterostilbene-induced cell cycle arrest, synergistic inhibition of MAPK activity and induction of Thioredoxin interacting protein (TXNIP) as possible mechanisms behind pterostilbenes effect. Our results highlight a nontoxic stilbenoid compound as a modulator of anticancer drug response, and indicate that pterostilbene might be used to modulate two anticancer compounds in well-defined sets of GBM patients.

Establishment and characterization of an orthotopic patient-derived Group 3 medulloblastoma model for preclinical drug evaluation

Medulloblastomas comprise a heterogeneous group of tumours and can be subdivided into four molecular subgroups (WNT, SHH, Group 3 and Group 4) with distinct prognosis, biological behaviour and implications for targeted therapies. Few experimental models exist of the aggressive and poorly characterized Group 3 tumours. In order to establish a reproducible transplantable Group 3 medulloblastoma model for preclinical therapeutic studies, we acquired a patient-derived tumour sphere culture and inoculated low-passage spheres into the cerebellums of NOD-scid mice. Mice developed symptoms of brain tumours with a latency of 17–18 weeks. Neurosphere cultures were re-established and serially transplanted for 3 generations, with a negative correlation between tumour latency and numbers of injected cells. Xenografts replicated the phenotype of the primary tumour, including high degree of clustering in DNA methylation analysis, high proliferation, expression of tumour markers, MYC amplification and elevated MYC expression, and sensitivity to the MYC inhibitor JQ1. Xenografts maintained maintained expression of tumour-derived VEGFA and stromal-derived COX-2. VEGFA, COX-2 and c-Myc are highly expressed in Group 3 compared to other medulloblastoma subgroups, suggesting that these molecules are relevant therapeutic targets in Group 3 medulloblastoma.

Image-Based Detection of Patient-Specific Drug-Induced Cell-Cycle Effects in Glioblastoma:

Image-based analysis is an increasingly important tool to characterize the effect of drugs in large-scale chemical screens. Herein, we present image and data analysis methods to investigate population cell-cycle dynamics in patient-derived brain tumor cells. Images of glioblastoma cells grown in multiwell plates were used to extract per-cell descriptors, including nuclear DNA content. We reduced the DNA content data from per-cell descriptors to per-well frequency distributions, which were used to identify compounds affecting cell-cycle phase distribution. We analyzed cells from 15 patient cases representing multiple subtypes of glioblastoma and searched for clusters of cell-cycle phase distributions characterizing similarities in response to 249 compounds at 11 doses. We show that this approach applied in a blind analysis with unlabeled substances identified drugs that are commonly used for treating solid tumors as well as other compounds that are well known for inducing cell-cycle arrest. Redistribution of nuclear DNA content signals is thus a robust metric of cell-cycle arrest in patient-derived glioblastoma cells.

Abstract 4813: In vivo modeling of high grade glioma for oncology drug development

In a multi-disciplinary project, patient derived glioblastoma stem-cell cultures (GSCs) have been established and characterized extensively with the goal of applying computational efforts to integrate results from high-throughput screens of drugs and RNAi with genomic and transcriptional profiling to predict the most successful therapy for individual glioma patients. The main goal of this study was to develop a platform for testing the tumor-initiating capacity of GSCs in mouse brain and to label the cells with GFP-luciferase to enable non-invasive quantification of tumor growth by in vivo bioluminescence imaging. Adherently grown GFP-luciferase labeled glioblastoma stem-cell cultures were dissociated and injected stereotactically into immunodeficient mice. Tumor growth was monitored by IVIS imaging for up to 40 weeks and brains were collected for histopathological and immunohistochemical stainings. Automatic quantification and growth pattern analysis of tumor cells in brain sections was set up based on human cell specific staining and a CellProfiler Analyst’s machine learning classifier with a manual observer correlation of 0.86. GSC xenograft tumors with a wide range of histopathological features and biological behaviors recapitulating high grade astrocytoma was confirmed in mice injected with 14 of the 29 glioblastoma cell cultures (48%). Glial lineage markers, such as Sox2, GFAP, and Olig2, were expressed both in patient tumors and patient derived xenografts. Individual glioblastoma cell cultures were either characterized by formation of condensed tumors (8/14, 57%) or diffuse infiltrative growth through the brain parenchyma (6/14, 43%). In summary, we present a valuable mouse model for preclinical studies of glioma. Integrative analysis of molecular profiles with growth pattern data has the potential to unravel genes that distinguish tumors with more infiltrative growth. Our biobank of luciferase labeled GSCs may be useful for longitudinal monitoring of tumor growth dynamics in the setting of therapies and provides a foundation for testing the effect of predicted drug vulnerabilites in orthotopic xenografts mouse models. Citation Format: Cecilia Krona, Soumi Kundu, Karl Holmberg-Olausson, Riasat Islam, Rashmi Ramachandra, Ludmila Elfineh, Sven Nelander. In vivo modeling of high grade glioma for oncology drug development [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 4813. doi:10.1158/1538-7445.AM2017-4813

Abstract A84: Stereotactic injections of primary medulloblastomas as a representative model of the different subgroups for preclinical drug screening

Established tumor cell lines are extensively used for screening of novel therapeutic compounds. However, during long-term culturing cells adjust to the conditions and acquire additional genetic aberrations, which may cause them to differ significantly from the original tumor. In vitro conditions may also fail to recapitulate aspects of the tumor microenvironment that may have an impact on cell fate, for example, drug clearance by the circulation, which would lead to renewed cell cycling and the heterogeneity of the tumor cell population versus the homogenous population of cells in culture. Predisposed genetic mouse models more closely recapitulate most aspects of human disease but they usually require complex breeding schemes and may suffer from incomplete tumor penetrance and a variable age of tumor onset. In order to establish a new medulloblastoma model for therapeutic studies, we have acquired primary cultures of medulloblastoma tumors representing the four different subtypes of medulloblastomas (Wnt, Shh, group 3 and group 4). Cells were grown as neurospheres and characterized regarding stem cell markers and immunological status. Early passages were collected and for each primary line, two NOD scid mice were orthotopically injected with 20.000 and 200.000 cells, respectively. Mice were closely monitored for symptoms of brain tumor burden (weight loss, hunched posture, uncoordinated movement, lethargy, poor balance and/or paralysis) during six months. Mice injected with cells from the SHH subgroup and group 3 medulloblastomas developed symptoms of brain tumors with a latency of 13-25 weeks. A positive correlation was observed between tumor latency and number of injected cells. Brains were collected and processed for histopathological confirmation of tumor prevalence, whole genome sequencing and gene expression analysis. Neurosphere cultures were also re-established to create a reproducible model for pre-clinical therapeutic trials on human medulloblastoma by serial orthotopic transplantation. Citation Format: Cecilia Krona, Cecilia Dyberg, Emma Sanden, Anna Darabi, Malin Wickstrom, Paul Northcott, John Inge Johnsen, Peter Siesjo. Stereotactic injections of primary medulloblastomas as a representative model of the different subgroups for preclinical drug screening. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A84.

Abstract 3302: Antisense oligonucleotide depletion of the mitotic kinesin Eg5 by direct delivery to the brain could be a useful strategy for treating glioma tumors.

To date, all approved chemotherapeutic agents which target the mitotic cell division interfere with spindle microtubule dynamics, leading to mitotic arrest and apoptosis. While effective, these drugs are subject to resistance mechanisms and they are also associated with a variety of side effects, including neurotoxicity. Their use in treating nervous system tumors is therefore not warranted. One strategy to target mitosis, without damaging microtubules in non-dividing neurons, would be to inhibit key mitotic components, such as the mitotic kinesin Eg5, which is required for establishing a normal bipolar mitotic spindle. We have shown that glioblastoma cells depleted for Eg5 arrest in the next mitosis. After a prolonged arrest, they may slip out and become multinucleated, which will likely prevent further successful divisions or they may go into apoptosis. Further, mitotic arrest and induction of apoptosis in Eg5 depleted glioblastoma cells occur independent of p53, Rb-signalling and the PI3K-pathway suggesting that Eg5 is a potential therapeutic target for glioblastoma patients with different underlying genetic abnormalities We have also tested the clinical feasibility of using a cell cycle targeting antisense oligonucleotide based therapy delivered directly to the central nervous system (CNS) as a novel treatment for glioblastoma tumors. This work has demonstrated that intraventricular administration of ASOs can efficiently target cells in the CNS and be delivered to glioma-initiating neural stem cells transplanted into the cortex of naive mice as well as to glioblastoma tumors in a genetically predisposed mouse model. This strategy is therefore a potential route of administration for treating glioblastoma tumors which originate in the CNS. Direct targeting of mitotic components in the brain should have a limited toxicity to non-cycling neurons and as a benefit, as long as the blood-brain barrier is intact direct CNS delivery should have minimal dose-limiting toxicity outside of the CNS. Ongoing studies will determine the effect of Eg5 inhibition on glioblastoma growth in vivo. Citation Format: Cecilia C. Krona, Jihane Boubaker, Dinorah Friedmann-Morvinski, Alex Wong, Melissa McAlonis-Downes, Erich Koller, Aneeza S. Kim, Gene Hung, Frank Rigo, Seung Chun, Benjamin Vitre, Frank Bennett, Inder Verma, Don W. Cleveland. Antisense oligonucleotide depletion of the mitotic kinesin Eg5 by direct delivery to the brain could be a useful strategy for treating glioma tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3302. doi:10.1158/1538-7445.AM2013-3302