Prognostic and therapeutic implications of migrating tumor cells in glioblastomas

Glioblastoma is the most common and malignant type of primary brain tumor with a dismal prognosis and median survival of approximately 15 months. Inevitably, virtually all glioblastomas recur after standard-ofcare therapy consisting of maximal safe tumor resection and adjuvant radio- and chemotherapy with thealkylating agent temozolomide. The origin of tumor recurrence is found in the highly infiltrative growthpattern that glioblastomas notoriously exhibit, combined with high intrinsic resistance to therapy. As migrating tumor cells infiltrate the surrounding brain parenchyma, they escape both surgical resection and are betterguarded against adjuvant radio- and chemotherapy by a more intact blood-brain-barrier in the tumor periphery, and by localization at the outer margins of the radiation field. These migrating tumor cells that survivetherapeutic interventions therefore lay the foundation for eventual tumor recurrence, which ultimately leadsto patient death.

The overall aim of this thesis was to investigate migrating tumor cells in the context of prognostic biomarkers, considering specific expression in migrating tumor cells, and to investigate these cells in a therapeutic context. Further, this thesis aimed to investigate the anticancer effects of a novel drug compound,SB747651A, with affinity for protein kinases expressed in migrating glioblastoma cells, and to uncoverpotential phenotypic changes in migrating tumor cells following therapeutic intervention in the form of surgical tumor resection.

Study I in this thesis investigated the expression and prognostic value of two transcription factors, EGR1and EGR3, in a well-annotated glioma cohort including 207 patients. These two transcription factors haveshown prognostic value in multiple cancer types. A subset of 21 glioblastomas with P53 expression, whichwas utilized as a tumor cell marker, and inclusion of both central and peripheral tumor regions were investigated for intra-tumoral EGR1 and EGR3 staining patterns. Protein expression was assessed both as meanexpression in total tumor area, and specifically in migrating tumor cells. Relevant findings were elucidated byin silico analysis using publicly available glioma datasets. EGR1 and EGR3 protein expression was identifiedin all investigated tumors, but considerable inter-tumor variation was found. EGR1 protein expression positively correlated with WHO grade, but did not show prognostic value after adjusting for potential clinicalconfounders in Cox-regression analysis. EGR3 expression in peripheral migrating tumor cells was higherthan in central non-migrating cells, and high EGR3 protein expression was associated with poor overallpatient survival in a subgroup of glioblastoma patients with methylated MGMT-promoters. Future investigations should functionally assess a potential role of EGR3 in the process of tumor cell migration and validateits role as a prognostic biomarker in GBM.

Study II focused on the first basic and preclinical investigation of the small-molecule kinase inhibitorSB747651A in glioblastomas. With its multi-target affinity, this inhibitor also targets proteins associated withtumor cell migration, and may therefore be a mean to combat the migrating tumor cells responsible for tumorrecurrence. We investigated the anticancer efficacy of SB747651A in three well-characterized patient-derivedglioblastoma cell cultures and in a murine orthotopic xenograft model. SB747651A concentrations between 5-10 µM induced cell death in a concentration- and time dependent manner in all three investigated cellcultures, and addition of temozolomide yielded an additive effect on cell death. Treated tumor cells showedimpaired spheroid formation, reduced growth and cell migration, while phosphorylation of mTOR, CREB,GSK3 and GYS1 kinases as well as SOX2 protein expression was reduced after SB747651A treatment.Further, exposed cells accumulated intracellular glycogen and had increased generation of reactive oxygenspecies. Orthotopically xenografted mice lived significantly longer when treated with SB747651A for 5days/weekly with 25 mg/kg throughout 8 weeks compared to vehicle treated controls, and no adverse effectswere observed in vivo.

In study III, we aimed to investigate the isolated impact of surgical tumor resection on the phenotype ofresidual migrating tumor cells left behind in the resection cavity. To investigate this aspect, we developed apreclinical glioblastoma resection model using immunocompromised nude rats combined with small animalPET/CT imaging utilizing the radioisotope 18F-FET to monitor tumor growth and recurrence. Tumor cellsfrom primary and recurrent tumors were isolated and subject to total mRNA sequencing. Significantly deregulated genes and pathways were identified and further investigated in tissue specimens from the resectionmodel and from glioblastoma patients with paired primary/recurrent tumors. Functional investigations wereperformed in vitro with several patient-derived glioblastoma cell cultures. 18F-FET PET/CT scans couldsuccessfully detect growth of orthotopic glioblastoma xenografts 3-4 weeks after tumor cell implantation.Following detection of primary tumor growth, we successfully established preclinical glioblastoma resection,which closely mimics resection of patient glioblastomas, with few residual tumor cells left behind in thecavity. Comparisons of primary and recurrent tumors from our preclinical resection model revealed thatrecurrent tumors were more proliferative, showed increased infiltration of microglia/macrophages and upregulated multiple glioblastoma stem cell genes including SOX2, POU3F2, OLIG2 and Notch1 compared toprimary tumors. This upregulation was validated at the protein level both in recurrent xenografts and in earlyrecurrent patient glioblastomas indicating high translatability. Among the upregulated genes in recurrenttumors, pleiotrophin and its primary receptor PTPRZ1 were identified. Mechanistically, pleiotrophin couldinduce tumor cell proliferation and self-renewal in several patient-derived glioblastoma cell cultures, andinhibition of pleiotrophin with neutralizing antibodies abolished the pleiotrophin-mediated increase of tumorcell self-renewal. Further, pleiotrophin was able to induce expression of stem cell proteins SOX2, POU3F2and to a lesser extent OLIG2 in tumor cells, thereby making it a plausible mediator of the glioblastoma stemcell enrichment observed in recurrent tumors. Additionally, high pleiotrophin expression was associated withpoor overall patient survival.


In conclusion, this thesis shows that incorporating specific biomarker expression in migrating tumor cellscan be used to generate novel hypotheses regarding glioblastoma pathophysiology, however, unraveling thepotential prognostic value of biomarker expression in migrating tumor cells is complicated by lack of optimaltumor specific markers and sample size limitations. Migrating tumor cells pose a cell population with profound therapeutic implications, and should therefore be incorporated into future translational glioblastoma research to a greater extent. We provide novel insights into the implications of surgical tumor resection, andpropose that resection itself is an iatrogenic driver of glioblastoma stem cell enrichment among residual,formerly migrating, tumor cells in recurrent tumors. Application of more advanced preclinical models thatalso include tumor resection, as applied in this thesis, will most likely increase the translational success rateof novel findings to ultimately benefit glioblastoma patients through development of better and more efficienttherapeutic approaches.