Krishna M. Talasila

EGFR wild-type amplification and activation promote invasion and development of glioblastoma independent of angiogenesis

Angiogenesis is regarded as a hallmark of cancer progression and it has been postulated that solid tumor growth depends on angiogenesis. At present, however, it is clear that tumor cell invasion can occur without angiogenesis, a phenomenon that is particularly evident by the infiltrative growth of malignant brain tumors, such as glioblastomas (GBMs). In these tumors, amplification or overexpression of wild-type (wt) or truncated and constitutively activated epidermal growth factor receptor (EGFR) are regarded as important events in GBM development, where the complex downstream signaling events have been implicated in tumor cell invasion, angiogenesis and proliferation. Here, we show that amplification and in particular activation of wild-type EGFR represents an underlying mechanism for non-angiogenic, invasive tumor growth. Using a clinically relevant human GBM xenograft model, we show that tumor cells with EGFR gene amplification and activation diffusely infiltrate normal brain tissue independent of angiogenesis and that transient inhibition of EGFR activity by cetuximab inhibits the invasive tumor growth. Moreover, stable, long-term expression of a dominant-negative EGFR leads to a mesenchymal to epithelial-like transition and induction of angiogenic tumor growth. Analysis of human GBM biopsies confirmed that EGFR activation correlated with invasive/non-angiogenic tumor growth. In conclusion, our results indicate that activation of wild-type EGFR promotes invasion and glioblastoma development independent of angiogenesis, whereas loss of its activity results in angiogenic tumor growth.

Analysis of the Growth Dynamics of Angiogenesis-Dependent and -Independent Experimental Glioblastomas by Multimodal Small-Animal PET and MRI

The hypothesis of this study was that distinct experimental glioblastoma phenotypes resembling human disease can be noninvasively distinguished at various disease stages by imaging in vivo. Methods: Cultured spheroids from 2 human glioblastomas were implanted into the brains of nude rats. Glioblastoma growth dynamics were followed by PET using 18F-FDG, 11C-methyl-l-methionine (11C-MET), and 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) and by MRI at 3–6 wk after implantation. For image validation, parameters were coregistered with immunohistochemical analysis. Results: Two tumor phenotypes (angiogenic and infiltrative) were obtained. The angiogenic phenotype showed high uptake of 11C-MET and 18F-FLT and relatively low uptake of 18F-FDG. 11C-MET was an early indicator of vessel remodeling and tumor proliferation. 18F-FLT uptake correlated to positive Ki67 staining at 6 wk. T1- and T2-weighted MR images displayed clear tumor delineation with strong gadolinium enhancement at 6 wk. The infiltrative phenotype did not accumulate 11C-MET and 18F-FLT and impaired the 18F-FDG uptake. In contrast, the Ki67 index showed a high proliferation rate. The extent of the infiltrative tumors could be observed by MRI but with low contrast. Conclusion: For angiogenic glioblastomas, noninvasive assessment of tumor activity corresponds well to immunohistochemical markers, and 11C-MET was more sensitive than 18F-FLT at detecting early tumor development. In contrast, infiltrative glioblastoma growth in the absence of blood–brain barrier breakdown is difficult to noninvasively follow by existing imaging techniques, and a negative 18F-FLT PET result does not exclude the presence of proliferating glioma tissue. The angiogenic model may serve as an advanced system to study imaging-guided antiangiogenic and antiproliferative therapies.

The angiogenic switch leads to a metabolic shift in human glioblastoma.

Background Invasion and angiogenesis are major hallmarks of glioblastoma (GBM) growth. While invasive tumor cells grow adjacent to blood vessels in normal brain tissue, tumor cells within neovascularized regions exhibit hypoxic stress and promote angiogenesis. The distinct microenvironments likely differentially affect metabolic processes within the tumor cells. Methods In the present study, we analyzed gene expression and metabolic changes in a human GBM xenograft model that displayed invasive and angiogenic phenotypes. In addition, we used glioma patient biopsies to confirm the results from the xenograft model. Results We demonstrate that the angiogenic switch in our xenograft model is linked to a proneural-to-mesenchymal transition that is associated with upregulation of the transcription factors BHLHE40, CEBPB, and STAT3. Metabolic analyses revealed that angiogenic xenografts employed higher rates of glycolysis compared with invasive xenografts. Likewise, patient biopsies exhibited higher expression of the glycolytic enzyme lactate dehydrogenase A and glucose transporter 1 in hypoxic areas compared with the invasive edge and lower-grade tumors. Analysis of the mitochondrial respiratory chain showed reduction of complex I in angiogenic xenografts and hypoxic regions of GBM samples compared with invasive xenografts, nonhypoxic GBM regions, and lower-grade tumors. In vitro hypoxia experiments additionally revealed metabolic adaptation of invasive tumor cells, which increased lactate production under long-term hypoxia. Conclusions The use of glycolysis versus mitochondrial respiration for energy production within human GBM tumors is highly dependent on the specific microenvironment. The metabolic adaptability of GBM cells highlights the difficulty of targeting one specific metabolic pathway for effective therapeutic intervention.

Cathepsin L silencing enhances arsenic trioxide mediated in vitro cytotoxicity and apoptosis in glioblastoma U87MG spheroids

Despite improved treatment options, glioblastoma multiforme (GBM) remains the most aggressive brain tumour with the shortest post-diagnostic survival. Arsenite (As2O3) is already being used in the treatment of acute promyelocytic leukaemia (APL), yet its effects on GBM have not been evaluated in detail. In U87MG cell monolayers, we have previously shown that arsenite cytotoxicity significantly increases upon transient inhibition of lysosomal protease Cathepsin L (CatL). As multicellular spheroids more closely represent in vivo tumours, we aimed to evaluate the impact of permanent CatL silencing on arsenite treatment in U87MG spheroids. CatL was stably silenced using shRNA expression plasmid packed lentiviruses. By using metabolic- and cell viability assays, we demonstrated that long-term CatL silencing significantly increased arsenite cytotoxicity in U87MG spheroids. Silenced CatL also increased arsenite-mediated apoptosis in spheroids via elevated p53 expression, Bax/Bcl2 ratio and caspase 3/7 activity, though with lower efficacy than in monolayers. Arsenite cytotoxicity was enhanced by lower CatL activity, since similar cytotoxicity increase was also observed using the novel CatL inhibitor AT094. The results have significant translational impact, since stable CatL silencing would enable the application of lower systemic doses of arsenite to achieve the desired cytotoxic effects on GBMs in vivo.

Expansive growth of two glioblastoma stem-like cell lines is mediated by bFGF and not by EGF.

Abstract Background. Patient-derived glioblastoma (GBM) stem-like cells (GSCs) represent a valuable model for basic and therapeutic research. GSCs are usually propagated in serum-free Neural Basal medium supplemented with bFGF and EGF. Yet, the exact influence of these growth factors on GSCs is still unclear. Recently it was suggested that GBM stemlike cells with amplified EGFR should be cultured in stem cell medium without EGF, as the presence of EGF induced rapid loss of EGFR amplification. However, patient biopsies are usually taken into culture before their genomic profiles are defined. Thus, an important question remains whether GBM cells without EGFR amplification also can be cultured in stem cell medium without EGF. Meterials and methods. To address this question, we used two heterogeneous glioblastoma GSC lines (NCH421k and NCH644) that lack EGFR amplification. Results. Although both cell lines showed very low EGFR expression under standard growth conditions, bFGF stimulation induced higher expression of EGFR in NCH644. In both cell lines, expression of the stem cell markers nestin and CD133 was higher upon stimulation with bFGF compared to EGF. Importantly, bFGF stimulated the growth of both cell lines, whereas EGF had no effect. We verified that the growth stimulation by bFGF was either mediated by proliferation (NCH421k) or resistance to apoptosis (NCH644). Conclusions. We demonstrate that GSC cultures without EGFR amplification can be maintained and expanded with bFGF, while the addition of EGF has no significant effect and therefore can be omitted.

Tumor versus stromal cells in culture--survival of the fittest?

Two of the signature genetic events that occur in human gliomas, EGFR amplification and IDH mutation, are poorly represented in experimental models in vitro. EGFR amplification, for example, occurs in 40 to 50% of GBM, and yet, EGFR amplification is rarely preserved in cell cultures derived from human tumors. To analyze the fate of EGFR amplified and IDH mutated cells in culture, we followed the development over time of cultures derived from human xenografts in nude rats enriched for tumor cells with EGFR amplification and of cultures derived from patient samples with IDH mutations, in serum monolayer and spheroid suspension culture, under serum and serum free conditions. We observed under serum monolayer conditions, that nestin positive or nestin and SMA double positive rat stromal cells outgrew EGFR amplified tumor cells, while serum spheroid cultures preserved tumor cells with EGFR amplification. Serum free suspension culture exhibited a more variable cell composition in that the resultant cell populations were either predominantly nestin/SOX2 co-expressing rat stromal cells or human tumor cells, or a mixture of both. The selection for nestin/SMA positive stromal cells under serum monolayer conditions was also consistently observed in human oligodendrogliomas and oligoastrocytomas with IDH mutations. Our results highlight for the first time that serum monolayer conditions can select for stromal cells instead of tumor cells in certain brain tumor subtypes. This result has an important impact on the establishment of new tumor cell cultures from brain tumors and raises the question of the proper conditions for the growth of the tumor cell populations of interest.

Cathepsin L silencing increases As2O3 toxicity in malignantly transformed pilocytic astrocytoma MPA58 cells by activating caspases 3/7

&NA; Low‐grade, pilocytic astrocytomas are treated by resection, but additional therapy is necessary for those tumors with anaplastic features. Arsenic trioxide (As2O3) is emerging as an effective chemotherapeutic agent for treatment of malignant glioblastoma multiforme, where Cathepsin L silencing enables lower, less harmful As2O3 concentrations to achieve the desired cytotoxic effect. Here, we evaluated the effects of As2O3 combined with stable Cathepsin L shRNA silencing on cell viability/metabolic activity, and apoptosis in primary cultures of recurrent malignantly transformed pilocytic astrocytoma (MPA). These cells expressed high Cathepsin L levels, and when grown as monolayers and spheroids, they were more resistant to As2O3 than the U87MG glioblastoma cell line. Caspases 3/7 activity in MPA58 spheroids was not significantly affected by As2O3, possibly due to higher chemoresistance of primary biopsy tissue of less malignant astrocytoma versus the malignant U87MG cell line. However, As2O3 treatment was cytotoxic to MPA spheroids after silencing of Cathepsin L expression. While Cathepsin L silencing only slightly decreased the live/dead cell ratio in As2O3‐treated MPA‐si spheroids under our experimental conditions, there was an increase in As2O3‐mediated apoptosis in MPA‐si spheroids, as indicated by elevated caspases 3/7 activity. Therefore, Cathepsin L silencing by gene manipulation can be applied when a more aggressive approach is needed in treatment of pilocytic astrocytomas with anaplastic features. HighlightsMalignantly transformed pilocytic astrocytomas express high levels of Cathepsin L.Primary malignant PA cells are more resistant to arsenic trioxide than GBM cells.Cathepsin L silencing increases As2O3‐mediated apoptosis in MPA58 spheroids.Cathepsin L silencing can improve therapy of anaplastic PAs with As2O3.

Tumour-associated glial host cells display a stem-like phenotype with a distinct gene expression profile and promote growth of GBM xenografts

BackgroundLittle is known about the role of glial host cells in brain tumours. However, supporting stromal cells have been shown to foster tumour growth in other cancers.MethodsWe isolated stromal cells from patient-derived glioblastoma (GBM) xenografts established in GFP-NOD/scid mice. With simultaneous removal of CD11b+ immune and CD31+ endothelial cells by fluorescence activated cell sorting (FACS), we obtained a population of tumour-associated glial cells, TAGs, expressing markers of terminally differentiaed glial cell types or glial progenitors. This cell population was subsequently characterised using gene expression analyses and immunocytochemistry. Furthermore, sphere formation was assessed in vitro and their glioma growth-promoting ability was examined in vivo. Finally, the expression of TAG related markers was validated in human GBMs.ResultsTAGs were highly enriched for the expression of glial cell proteins including GFAP and myelin basic protein (MBP), and immature markers such as Nestin and O4. A fraction of TAGs displayed sphere formation in stem cell medium. Moreover, TAGs promoted brain tumour growth in vivo when co-implanted with glioma cells, compared to implanting only glioma cells, or glioma cells and unconditioned glial cells from mice without tumours. Genome-wide microarray analysis of TAGs showed an expression profile distinct from glial cells from healthy mice brains. Notably, TAGs upregulated genes associated with immature cell types and self-renewal, including Pou3f2 and Sox2. In addition, TAGs from highly angiogenic tumours showed upregulation of angiogenic factors, including Vegf and Angiopoietin 2. Immunohistochemistry of three GBMs, two patient biopsies and one GBM xenograft, confirmed that the expression of these genes was mainly confined to TAGs in the tumour bed. Furthermore, their expression profiles displayed a significant overlap with gene clusters defining prognostic subclasses of human GBMs.ConclusionsOur data demonstrate that glial host cells in brain tumours are functionally distinct from glial cells of healthy mice brains. Furthermore, TAGs display a gene expression profile with enrichment for genes related to stem cells, immature cell types and developmental processes. Future studies are needed to delineate the biological mechanisms regulating the brain tumour-host interplay.

Abstract 5279: Over-expression of EGFRviii induces an angiogenic switch in infiltrative human glioblastomas

Glioblastoma multiforme (GBM) is the most common and most aggressive primary brain tumor where local cancer cell invasion into the normal brain represents a major obstacle preventing successful therapeutic interventions. A well known mediator of invasion, proliferation and survival, is the epidermal growth factor receptor (EGFR). Amplification and subsequent over-expression of EGFR protein is the most common genetic alteration in primary GBM, with a frequency of about 40%. Of the GBMs that have an amplification of EGFR, 63-75% also show genetic rearrangements of the gene, resulting in tumors that express both the wild type, as well as mutated forms of EGFR. The most common mutation in EGFR, is the EGFRviii, were the ligand binding part of the protein is missing (del exon 2-7). This mutation leads to a constitutive activation of the protein. In patients with amplified EGFR, 50-60% also express the EGFRviii. In this work we determined a functional role of EGFRviii within invasive human GBM cells in vivo. We cultured human GBM cells, lacking EGFR amplification and EGFRviii expression, in serum-free neuro-basal (NB) medium. By orthotopic transplantation into the brains of immunodeficient rats, tumors developed that showed a highly infiltrative non-angiogenic phenotype. By over-expressing EGFRviii in these tumors, we demonstrate a robust switch towards angiogenesis as shown by dynamic-contrast enhanced MRI and by histologic verification of angiogenic vessels. Molecular analysis confirmed an up-regulation of HIF1A as well as a panel of angiogenic factors. Our results demonstrate a distinct role of EGFRviii as a vital driver of angiogenic tumor growth within human GBMs in vivo. Thus, the presented observations should have important implications for therapeutic strategies targeting EGFRviii in patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5279. doi:1538-7445.AM2012-5279

Abstract LB-518: Amplification and activation of EGFR wild-type mediates invasion of human glioblastoma in vivo

Glioblastoma (GBM) is the most aggressive form of primary brain tumors with a median survival of 15 months. Although angiogenesis is one of the main features of GBMs, non-angiogenic tumor infiltration into brain parenchyma still is the major challenge for therapy. Tumor cells can migrate very far from the main tumor mass and the invasive pattern of tumor subpopulations has not been characterized properly. Epidermal growth factor receptor (EGFR) gene amplification is one of the major mutations of primary GBMs, where multiple copies of the wild-type EGFR gene are present as double minutes. Although studies have proposed a role for EGFR gene amplification in tumor development, the function of EGFR in vivo is not characterized properly mainly due to inefficient tumor models. Here, we report a key role for EGFR wild-type in tumor invasion. In a human GBM xenograft model, we show that tumor cells with EGFR amplification and expression are highly invasive and non-angiogenic. By blocking EGFR activation using Cetuximab and a dominant-negative approach, we show that maintenance of the non-angiogenic, invasive growth pattern is dependent on EGFR function and that downregulation of its activity leads to angiogenic tumor growth. As EGFR amplification and expression is present in 40-60% of GBMs, our results might implicate that activation of EGFR wild-type is one of the major mechanisms of glioblastoma invasion in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-518. doi:1538-7445.AM2012-LB-518