In the current study, we examined a panel of serially passaged glioblastoma xenografts, in the context of an intracranial tumor therapy response model, to identify associations between glioblastoma molecular characteristics and tumor sensitivity to the epidermal growth factor receptor (EGFR) kinase inhibitor erlotinib. From an initial evaluation of 11 distinct glioblastoma xenografts, two erlotinib-sensitive tumors were identified, each having amplified EGFR and expressing wild-type PTEN. One of these tumors expressed truncated EGFRvIII, whereas the other expressed full-length EGFR. Subsequent cDNA sequence analysis revealed the latter tumor as expressing an EGFR sequence variant with arginine, rather than leucine, at amino acid position 62; this was the only EGFR sequence variant identified among the 11 xenografts, other than the aforementioned vIII sequence variant. EGFR cDNAs were then examined from 12 more xenografts to determine whether additional missense sequence alterations were evident, and this analysis revealed one such case, expressing threonine, rather than alanine, at amino acid position 289 of the extracellular domain. This glioblastoma was also amplified for EGFR, but did not display significant erlotinib sensitivity, presumably due to its lacking PTEN expression. In total, our study identified two erlotinib-sensitive glioblastoma xenografts, with the common molecular characteristics shared by each being the expression of wild-type PTEN in combination with the expression of amplified and aberrant EGFR. [Mol Cancer Ther 2007;6(3):1167 -74]

Activating epidermal growth factor receptor (EGFR) mutations are common in many cancers including glioblastoma. However, clinical responses to EGFR inhibitors are infrequent and short-lived. We show that the Src family kinases (SFK) Fyn and Src are effectors of oncogenic EGFR signaling, enhancing invasion and tumor cell survival in vivo. Expression of a constitutively active EGFR mutant, EGFRvIII, resulted in activating phosphorylation and physical association with Src and Fyn, promoting tumor growth and motility. Gene silencing of Fyn and Src limited EGFR-and EGFRvIIIdependent tumor cell motility. The SFK inhibitor dasatinib inhibited invasion, promoted tumor regression, and induced apoptosis in vivo, significantly prolonging survival of an orthotopic glioblastoma model expressing endogenous EGFRvIII. Dasatinib enhanced the efficacy of an anti-EGFR monoclonal antibody (mAb 806) in vivo, further limiting tumor growth and extending survival. Examination of a large cohort of clinical samples showed frequent coactivation of EGFR and SFKs in glioblastoma patients. These results establish a mechanism linking EGFR signaling with Fyn and Src activation to promote tumor progression and invasion in vivo and provide rationale for combined anti-EGFR and anti-SFK targeted therapies. [Cancer Res 2009;69(17):6889-98]

The results of these experiments indicate that BLI monitoring can be used as a surrogate for predicting survival benefit from TMZ treatment, permits early determination of relative survival benefit associated with distinct TMZ therapeutic regimens, and offers a means of investigating secondary/salvage therapy efficacy following tumor regrowth from initial therapy.

Despite the use of radiation and chemotherapy, the prognosis for children with diffuse brainstem gliomas is extremely poor. There is a need for relevant brainstem tumor models that can be used to test new therapeutic agents and delivery systems in pre-clinical studies. We report the development of a brainstem-tumor model in rats and the application of bioluminescence imaging (BLI) for monitoring tumor growth and response to therapy as part of this model. Luciferase-modified human glioblastoma cells from five different tumor cell sources (either cell lines or serially-passaged xenografts) were implanted into the pontine tegmentum of athymic rats using an implantable guide-screw system. Tumor growth was monitored by BLI and tumor volume was calculated by three-dimensional measurements from serial histopathologic sections. To evaluate if this model would allow detection of therapeutic response, rats bearing brainstem U-87 MG or GS2 glioblastoma xenografts were treated with the DNA methylating agent temozolomide (TMZ). For each of the tumor cell sources tested, BLI monitoring revealed progressive tumor growth in all animals, and symptoms caused by tumor burden were evident 26–29 days after implantation of U-87 MG, U-251 MG, GBM6, and GBM14 cells, and 37–47 days after implantation of GS2 cells. Histopathologic analysis revealed tumor growth within the pons in all rats and BLI correlated quantitatively with tumor volume. Variable infiltration was evident among the different tumors, with GS2 tumor cells exhibiting the greatest degree of infiltration. TMZ treatment groups were included for experiments involving U-87 MG and GS2 cells, and in each case TMZ delayed tumor growth, as indicated by BLI monitoring, and significantly extended survival of animal subjects. Our results demonstrate the development of a brainstem tumor model in athymic rats, in which tumor growth and response to therapy can be accurately monitored by BLI. This model is well suited for pre-clinical testing of therapeutics that are being considered for treatment of patients with brainstem tumors.