gms | German Medical Science

Objective: Glioblastoma is the most common primary malignant brain tumor in adults. It is highly resistant to treatment as the invasiveness limits complete resection, the dynamic tumor genome, multiple pathways driving the malignant phenotype, and the blood brain barrier, which limits the availability of drugs to the tumor. Evidence suggests a significant role for NF-kB in the gliomagenesis and the mechanism of treatment resistance making NF-kB a potentially potent target for treatment. However, increasing evidence suggests a great cellular heterogeneity in glioma, making it important to understand which glioma cell types are dynamically driven by certain molecular pathways, and how that relates to their function in progression and resistance, Therefore, we established an in vivo reporter system based on NF-κB responsive promoter (RE) elements driving green fluorescent protein (gfp) to monitor the expression of this transcription factor.

Methods: Ten tandem repeats of the NF-κB RE (GGGACTTTC) were designed, annealed and ligated into the HIV-1 based lentiviral vector pLVX Puro (Clontech Laboratories, Inc. A Takara Bio Company, CA USA) by replacing the CMV promoter with NFkB elements. GFP was subcloned into the multiple cloning site of pLVX Puro. The integrity of the plasmid was verified by sequencing analysis. Its reliability as a NF-κB reporter was verified in vivo and in vitro. Using a chronic cranial window mouse model two-photon microscopy allows for consecutive in vivo imaging to observe different biological processes such as tumor growth, angiogenesis, and tumor repair after laser ablation and radiotherapy.

Results: While cells grown under stem like conditions in vitro were highly NF-κB reporter positive, only few cells were positive in the established tumor in vivo, and that this changed dynamically depending on distinct states of tumor progression, and in response to lesions in the tumor area that were repopulated - "repaired" - by tumor cells. Furthermore, we were able to show that TNFa induced NF-κB reporter activity significantly in vitro. Radiotherapy led to a significant increase in reporter GFP expression in vivo as well as in vitro, to the extent that most cells became reporter positive. Pre-existing reporter-positive cells differed from reporter negative cells with respect to resistance against cytotoxic stress. NFκB reporter positivity did not colocalize with established “stem cell” markers Mushashi, Nestin and Sox2 using immunohistochemistry on tumor slices grown in our mouse model.

Conclusion: Our results suggest a role for NF-κB expression during GBM resistance to treatment and progression after radiation, in a heterogeneous manner. We will further analyze cell fate and cell morphology during irradiation and tumor “repair” with regard to their NF-κB positivity, and will include this data in the presentation, including inhibitory strategies of the NF-κB pathway.