What does GLIOscreen offer?
Our academic-based preclinical research service was established to evaluate (combinations of) drugs and other experimental therapies in patient-derived brain tumor cells, including astrocytomas, oligodendrogliomas, oligoastrocytomas, glioblastomas and ependymomas (together called gliomas). The basis of the services is formed by the Erasmus Brain Tumor Cell Culture Repository, a mounting tumor database currently containing over 350 patient-derived brain tumor samples, of the department of Neurosurgery & BrainTumorCenter, Erasmus Medical Center, Rotterdam, the Netherlands. The cell cultures are well-characterized in terms of molecular profiles and have been shown to closely resemble the genotype of the parental tumor.
GLIOscreen offers the unique opportunity to test agents in a clinically-relevant setting. The tumor cells are cultured under serum-free conditions and our culture database covers the spectrum of expression-based glioma subtypes as found in patients. GLIOscreen provides functional medium-throughput cell-based drug testing including IC50 determination, Chou-Talalay drug combination analysis, as well as growth kinetics and migration/invasion assessment by live-cell imaging. In vitro results can be further validated by GLIOscreen in vivo testing services including orthotopic xenograft tumor models with bioluminescence analysis. Moreover, the accessibility of molecular data of the parental tumor samples allows correlation of drug efficacy to molecular profiles. This can serve to identify subgroups of glioma patients that will benefit from specific treatments.
Serum free cell cultures
From every glioma resection performed at Erasmus MC department of Neurosurgery, which provides sufficient tumor material, tissue is transported to the lab under an IRB-approved protocol. Part of the tissue is stored directly at -80 C for molecular analysis of the original patient material. Remaining tumor is dissociated (mechanically and enzymatically) using an optimized protocol* and is placed in defined serum-free culture medium*. The genotypic profile of the original tumor is preserved for at least 15 passages in derived cell cultures under these culture conditions.
*based on stringent analysis and comparison of multiple dissociation protocols and culture medium formulas.
Genetic stability
Our culture of patient-derived glioma cells in serum-free medium was found to preserve the genetic profile of the tumor cells. As depicted in this figure of parallel established serum-free and serum-supplemented cultures, at passage 5 only the serum-free cultured cells had retained copy number alterations analogous to the parental tumor.
In vitro drug testing
Efficacy of chemical and biological compounds is assessed on panels of patient-derived glioma cells. Tumor cells are plated onto coated 96-wells plates in optimal culture medium* and are exposed to different concentrations and combinations of drugs. At multiple time points after treatment, viability is determined using a luminescent or fluorescent chemical viability assay* as well as by assessment of confluence by a kinetic live-cell imaging system. Together, these assays provide a reliable measure of cell viability, allowing IC50 calculation. Agents can be tested alone or in combination with each other. For combination studies, Chou-Talalay synergy quantification is applied. Valuable information can be obtained on potentially synergistic interactions between drugs.
*based on stringent analysis and comparison of multiple dissociation protocols, matrix coatings, culture medium formulas, and commercially available viability assays
For most agents large inter-tumoral variation is seen, which generally translates to responders, non-responders and intermediate responders. For example, the graph depicts variation in response to Temozolomide treatment. Whereas GS102 shows a dose-dependent decrease in viability upon TMZ treatment, GS265 remains completely refractory to this agent
GLIOscreen also offers the opportunity to identify compounds that increase the response rate to standard therapy. The graph below, for example, depicts an example of effective combination treatment. The percentage of glioma cell cultures (of 34 tested samples) that respond* to standard treatment of Temozolomide and radiotherapy increases by combining these therapies with each other or with an HDAC inhibitor and reaches almost 90% when applying the triple treatment of TMZ+RT+HDACi.
*Response is defined as more than 75% decrease in viability over 5 days.
Live-cell imaging system
Cell behavior (growth kinetics, morphology, monolayer confluence, migration) can also be closely monitored using a live-cell imaging system. In this example you'll find that treatment of GS102 cells with an HDAC inhibitor sensitizes these cells to radiation therapy leading to growth arrest and apoptosis in the combination treated cells.
Molecular profiles
In the era of personalized medicine, GLIOscreen drug screening service can serve to identify (subgroups of) glioma patients that will benefit from specific treatments. Stratification of patients in phase II/III clinical studies according to predicted response may increase the probability of demonstrating therapeutic activity of novel agents or treatment regimens. Molecular data (RNA/DNA) of parental tumors can be analyzed in the context of response to a specific treatment, allowing the identification of (a set of) genes that predict response (“predictor profiles”).
In vivo drug testing
Our GLIOscreen in vivo testing services offer the possibility to test agents in subcutaneous and orthotopic xenograft tumor models. Diversity in molecular subtype, survival and growth patterns of intracranial tumors allows validation of predicted response in a more clinically-relevant setting. Moreover, delivery and micro-environmental aspects are incorporated in this model. Luciferase-expressing xenograft models are available allowing close monitoring of intracranial growth and response to treatment by in vivo bioluminescence analysis.
GLIOscreen R&D
The systematic analysis of culture success rate of our optimized culture protocol, reveals that 37% of all gliomas cannot be cultured under serum-free conditions. These are predominantly grade II,III and secondary grade IV gliomas.
Alternative in vitro models are being developed for growth or sustainment of low-grade glioma, including various 3D model systems. In addition, alternative culture conditions are being evaluated to support growth of a specific subpopulation of high grade glioma for which current protocols are not suitable.
Serum-free culture success of glial tumors is related to specific molecular profiles and expression of extracellular matrix-associated gene modules.
Neuro-Oncology 2013 (in press)
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