Search

The transfer of intact mitochondria between heterogeneous cell types has been confirmed in various settings, including cancer. However, the functional implications of mitochondria transfer on tumor biology are poorly understood. Here we show that mitochondria transfer is a prevalent phenomenon in glioblastoma (GBM), the most frequent and malignant primary brain tumor. We identified horizontal mitochondria transfer from astrocytes as a mechanism that enhances tumorigenesis in GBM. This transfer is dependent on network-forming intercellular connections between GBM cells and astrocytes, which are facilitated by growth-associated protein 43 (GAP43), a protein involved in neuron axon regeneration and astrocyte reactivity. The acquisition of astrocyte mitochondria drives an increase in mitochondrial respiration and upregulation of metabolic pathways linked to proliferation and tumorigenicity. Functionally, uptake of astrocyte mitochondria promotes cell cycle progression to proliferative G2/M phases and enhances self-renewal and tumorigenicity of GBM. Collectively, our findings reveal a host–tumor interaction that drives proliferation and self-renewal of cancer cells, providing opportunities for therapeutic development.

Glioma refers to a group of primary brain tumors which includes glioblastoma (GBM), astrocytoma and oligodendroglioma as major entities. Among these, GBM is the most frequent and most malignant one. The highly infiltrative nature of gliomas, and their intrinsic intra- and intertumoral heterogeneity, pose challenges towards developing effective treatments. The glioma microenvironment, in addition, is also thought to play a critical role during tumor development and treatment course. Unlike most other solid tumors, the glioma microenvironment is dominated by macrophages and microglia—collectively known as tumor-associated macrophages (TAMs). TAMs, like their homeostatic counterparts, are plastic in nature and can polarize to either pro-inflammatory or immunosuppressive states. Many lines of evidence suggest that immunosuppressive TAMs dominate the glioma microenvironment, which fosters tumor development, contributes to tumor aggressiveness and recurrence and, very importantly, impedes the therapeutic effect of various treatment regimens. However, through the development of new therapeutic strategies, TAMs can potentially be shifted towards a proinflammatory state which is of great therapeutic interest. In this review, we will discuss various aspects of TAMs in the context of glioma. The focus will be on the basic biology of TAMs in the central nervous system (CNS), potential biomarkers, critical evaluation of model systems for studying TAMs and finally, special attention will be given to the potential targeted therapeutic options that involve the TAM compartment in gliomas.

Background Microtubes (MTs), cytoplasmic extensions of glioma cells, are important cell communication structures promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular, glioblastomas (GBMs), while they are uncommon in chemosensitive IDH-mutant and 1p/19q co-deleted oligodendrogliomas. The aim of this study was to identify potential signaling pathways involved in MT formation. Methods Bioinformatics analysis of TCGA was performed to analyze differences between GBM and oligodendroglioma. Patient-derived GBM stem cell lines were used to investigate MT formation under transforming growth factor-beta (TGF-β) stimulation and inhibition in vitro and in vivo in an orthotopic xenograft model. RNA sequencing and proteomics were performed to detect commonalities and differences between GBM cell lines stimulated with TGF-β. Results Analysis of TCGA data showed that the TGF-β pathway is highly activated in GBMs compared to oligodendroglial tumors. We demonstrated that TGF-β1 stimulation of GBM cell lines promotes enhanced MT formation and communication via calcium signaling. Inhibition of the TGF-β pathway significantly reduced MT formation and its associated invasion in vitro and in vivo. Downstream of TGF-β, we identified thrombospondin 1 (TSP1) as a potential mediator of MT formation in GBM through SMAD activation. TSP1 was upregulated upon TGF-β stimulation and enhanced MT formation, which was inhibited by TSP1 shRNAs in vitro and in vivo. Conclusion TGF-β and its downstream mediator TSP1 are important mediators of the MT network in GBM and blocking this pathway could potentially help to break the complex MT-driven invasion/resistance network.

H-1 parvovirus (H-1PV) is a promising anticancer therapy. However, in-depth understanding of its life cycle, including the host cell factors needed for infectivity and oncolysis, is lacking. This understanding may guide the rational design of combination strategies, aid development of more effective viruses, and help identify biomarkers of susceptibility to H-1PV treatment. To identify the host cell factors involved, we carry out siRNA library screening using a druggable genome library. We identify one crucial modulator of H-1PV infection: laminin γ1 (LAMC1). Using loss- and gain-of-function studies, competition experiments, and ELISA, we validate LAMC1 and laminin family members as being essential to H-1PV cell attachment and entry. H-1PV binding to laminins is dependent on their sialic acid moieties and is inhibited by heparin. We show that laminins are differentially expressed in various tumour entities, including glioblastoma. We confirm the expression pattern of laminin γ1 in glioblastoma biopsies by immunohistochemistry. We also provide evidence of a direct correlation between LAMC1 expression levels and H-1PV oncolytic activity in 59 cancer cell lines and in 3D organotypic spheroid cultures with different sensitivities to H-1PV infection. These results support the idea that tumours with elevated levels of γ1 containing laminins are more susceptible to H-1PV-based therapies., Rat H-1 parvovirus (H-1PV) is in clinical development for oncolytic therapy. Here, Kulkarni et al. identify LAMC1 as a modulator of H-1PV cell attachment and entry and find that LAMC1 levels and H-1PV oncolytic activity correlate in 59 tested cancer cell lines.

Background Suicide gene therapy for malignant gliomas has shown encouraging results in the latest clinical trials. However, prodrug application was most often restricted to short-term treatment (14 days), especially when replication-defective vectors were used. We previously showed that a substantial fraction of herpes simplex virus thymidine kinase (HSV-TK) transduced tumor cells survive ganciclovir (GCV) treatment in an orthotopic glioblastoma (GBM) xenograft model. Here we analyzed whether these TK+ tumor cells are still sensitive to prodrug treatment and whether prolonged prodrug treatment can enhance treatment efficacy. Methods Glioma cells positive for TK and green fluorescent protein (GFP) were sorted from xenograft tumors recurring after suicide gene therapy, and their sensitivity to GCV was tested in vitro. GBM xenografts were treated with HSV-TK/GCV, HSV-TK/valganciclovir (valGCV), or HSV-TK/valGCV + erlotinib. Tumor growth was analyzed by MRI, and survival as well as morphological and molecular changes were assessed. Results TK-GFP+ tumor cells from recurrent xenograft tumors retained sensitivity to GCV in vitro. Importantly, a prolonged period (3 mo) of prodrug administration with valganciclovir (valGCV) resulted in a significant survival advantage compared with short-term (3 wk) application of GCV. Recurrent tumors from the treatment groups were more invasive and less angiogenic compared with primary tumors and showed significant upregulation of epidermal growth factor receptor (EGFR) expression. However, double treatment with the EGFR inhibitor erlotinib did not increase therapeutic efficacy. Conclusion Long-term treatment with valGCV should be considered as a replacement for short-term treatment with GCV in clinical trials of HSV-TK mediated suicide gene therapy.

Stem cells play a pivotal role in the developmental stages of an organism and in adulthood as well. Therefore, it is not surprising that stem cells constitute a focus of extensive research. Indeed, several decades of stem cell research have tremendously increased our knowledge on the mechanistic understandings of stem cell biology. Interestingly, revealing the fundamental principles of stem cell biology has also fostered its application for therapeutic purposes. Many of the attributes that the stem cells possess, some of which are unique, allow multifaceted exploitation of stem cells in the treatment of various diseases. Cancer, the leading cause of mortality worldwide, is one of the disease groups that has been benefited by the potentials of therapeutic applications of the stem cells. While the modi operandi of how stem cells contribute to cancer treatment are many-sided, two major principles can be conceived. One mode involves harnessing the regenerative power of the stem cells to promote the generation of blood-forming cells in cancer patients after cytotoxic regimens. A totally different kind of utility of stem cells has been exercised in another mode where the stem cells can potentially deliver a plethora of anti-cancer therapeutics in a tumor-specific manner. While both these approaches can improve the treatment of cancer patients, there exist several issues that warrant further research. This review summarizes the basic principles of the utility of the stem cells in cancer treatment along with the current trends and pinpoints the major obstacles to focus on in the future for further improvement.

Microtubes (MTs), cytoplasmic extensions of glioma cells, are important cell communication structures promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular glioblastomas (GBMs), while they are uncommon in chemosensitive IDH-mutant and 1p/19q co-deleted oligodendrogliomas. To identify potential signaling pathways involved in MT formation we performed a bioinformatics analysis of TCGA data showing that the TGF-β pathway is highly activated in GBMs compared to oligodendroglial tumors. In particular we observed that signaling pathways involved in extracellular matrix organization are differentially expressed between these tumor entities. Using patient-derived GBM stem cell lines, we demonstrated that TGF-β1 stimulation promotes enhanced MT formation and communication via Calcium signaling. Inhibition of the TGF-β pathway significantly reduced MT formation and its associated invasion in vitro and in vivo. Downstream of TGF-β, we identified thrombospondin 1 (TSP1) as a potential mediator of MT formation in GBM through SMAD activation. TSP1 was upregulated upon TGF-β stimulation and enhanced MT formation, which was inhibited by TSP1 shRNAs in vitro and in vivo. In conclusion, TGF-β and its downstream mediator TSP1 are important mediators of the MT network in GBM and blocking this pathway could potentially help to break the complex MT driven invasion/ resistance network.

Simple Summary We established a new minimally invasive mouse model for GBM relapse. For this, we utilized orthotopical implantation of HSVTK-transduced GBM cells and pharmacological treatment with GCV. In addition, we implanted patient-derived GBM cells of primary or recurrent tumors. We found that recurrent GBM were more aggressively invasive than primary GBM. Moreover, the recurring tumors had a higher ratio of monocyte-derived macrophages among the entire population of tumor associated myeloid cells. This shift in the composition of tumor-associated immune cells appeared to be independent from cell-death signaling or surgical intervention. This model provides the means to investigate the entire process of tumor relapse and test standard as well as experimental therapeutic strategies for relapsing GBM under defined conditions. Abstract Glioblastoma (GBM) recurrence after treatment is almost inevitable but addressing this issue with adequate preclinical models has remained challenging. Here, we introduce a GBM mouse model allowing non-invasive and scalable de-bulking of a tumor mass located deeply in the brain, which can be combined with conventional therapeutic approaches. Strong reduction of the GBM volume is achieved after pharmacologically inducing a tumor-specific cell death mechanism. This is followed by GBM re-growth over a predictable timeframe. Pharmacological de-bulking followed by tumor relapse was accomplished with an orthotopic mouse glioma model. Relapsing experimental tumors recapitulated pathological features often observed in recurrent human GBM, like increased invasiveness or altered immune cell composition. Orthotopic implantation of GBM cells originating from biopsies of one patient at initial or follow-up treatment reproduced these findings. Interestingly, relapsing GBM of both models contained a much higher ratio of monocyte-derived macrophages (MDM) versus microglia than primary GBM. This was not altered when combining pharmacological de-bulking with invasive surgery. We interpret that factors released from viable primary GBM cells preferentially attract microglia whereas relapsing tumors preponderantly release chemoattractants for MDM. All in all, this relapse model has the capacity to provide novel insights into clinically highly relevant aspects of GBM treatment.

Stem cells play a pivotal role in the developmental stages of an organism and in adulthood as well. Therefore, it is not surprising that stem cells constitute a focus of extensive research. Indeed, several decades of stem cell research have tremendously increased our knowledge on the mechanistic understandings of stem cell biology. Interestingly, revealing the fundamental principles of stem cell biology has also fostered its application for therapeutic purposes. Many of the attributes that the stem cells possess, some of which are unique, allow multifaceted exploitation of stem cells in the treatment of various diseases. Cancer, the leading cause of mortality worldwide, is one of the disease groups that has been benefited by the potentials of therapeutic applications of the stem cells. While the modi operandi of how stem cells contribute to cancer treatment are many-sided, two major principles can be conceived. One mode involves harnessing the regenerative power of the stem cells to promote the generation of blood-forming cells in cancer patients after cytotoxic regimens. A totally different kind of utility of stem cells has been exercised in another mode where the stem cells can potentially deliver a plethora of anti-cancer therapeutics in a tumor-specific manner. While both these approaches can improve the treatment of cancer patients, there exist several issues that warrant further research. This review summarizes the basic principles of the utility of the stem cells in cancer treatment along with the current trends and pinpoints the major obstacles to focus on in the future for further improvement.

Suicide gene therapy has represented an experimental cancer treatment modality for nearly 40 years. Among the various cancers experimentally treated by suicide gene therapy, high-grade gliomas have been the most prominent both in preclinical and clinical settings. Failure of a number of promising suicide gene therapy strategies in the clinic pointed toward a bleak future of this approach for the treatment of high-grade gliomas. Nevertheless, the development of new vectors and suicide genes, better prodrugs, more efficient delivery systems, and new combinatorial strategies represent active research areas that may eventually lead to better efficacy of suicide gene therapy. These trends are evident by the current increasing focus on suicide gene therapy for high-grade glioma treatment both in the laboratory and in the clinic. In this review, we give an overview of different suicide gene therapy approaches for glioma treatment and discuss clinical trials, delivery issues, and immune responses.

BACKGROUND Despite aggressive tumor behavior, extracranial metastases rarely develop in glioblastoma (GBM) patients. Two potential explanations have been suggested: 1) The blood-brain-barrier functions as a physical barrier that prevents the dissemination of GBM cells out of the central nervous system (CNS) or 2) that extracranial metastasis do occur, but the patients die before extracranial metastases manifest themselves. The first theory has been questioned based on the fact that circulating tumor cells (CTC) were found in blood samples of GBM patients without systemic metastases. To date it has not been proven if CTCs are able to reenter the brain and to what extent they are able to form systemic extracranial metastatic lesions. Therefore, the current study aimed at analyzing the dissemination patterns and the underlying mechanisms associated with the ability of GBM CTCs to form extracranial metastases. MATERIAL AND METHODS Five highly characterized human GBM stem cell (GSC) lines (P3, BG5, BG7, GG6, GG16), displaying GBM CNV patterns, were intracranially implanted in a first cohort, then transduced with a lentiviral Firefly Luciferase-eGFP vector and injected into the left cardiac ventricle of NOD/SCID mice in a second cohort. Mice were observed closely and tumor burden was assessed using in vivo as well as ex vivo bioluminescence imaging, MRI and PET. Mice were euthanized when the objective endpoint criteria (tumor burden) was met, then organs were harvested and fixed for further analysis. RESULTS First, a detailed characterization of the GSC line invasion patterns were assessed when grown as orthotopic xenografts in vivo dividing them into three categories: 1) Highly invasive without apparent angiogenesis (BG5) 2) Invasive with perivascular infiltration and angiogenesis (P3, BG7 and GG16) and 3) Angiogenic and highly circumscribed (GG6). Following intracardial injection, (7 out of 8) P3 animals developed extracranial and intracranial tumors with a distinctive pattern. Brain, adrenal gland, ovary and liver were amongst the organs most susceptible for tumor growth in the P3 group. For the BG5 and BG7 cell lines, no metastases were observed whereas only 1 animal out of 10 developed metastases in both groups GG16 and GG6. CONCLUSION Only one out of 5 GSC lines exhibited a strong metastatic potential when injected into the left cardiac ventricle. Compared to other tumors which exhibit a strong metastatic potential from the circulation, GSC lines do only to a very limited extent show this potential reflecting observations made in the clinic.

Microtubes (MTs) are cytoplasmic extensions of glioma cells serving as important cell communication structures while also promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular glioblastomas, while they are uncommon in chemosensitive IDH mutated and 1p/19q co-deleted oligodendrogliomas. By performing a bioinformatics analysis on data from The Cancer Genome Atlas (TCGA) we identified the TGF-b pathway as being distinctly upregulated in glioblastomas compared to oligodendrogliomas, making this a signaling pathway potentially involved in MT formation. Based on patient-derived GBM stem cell line models we demonstrated that stimulation of TGF-b increased MT formation, while inhibition of TGF-b reduced MT formation. MT formation was verified by expression of GAP43 and nestin, which have previously been shown to be important structural proteins of MTs. Interestingly, we also observed a responder/non-responder relationship between GBM cell lines P3 and GG16/ GG6 regarding MT formation upon TGF-b stimulation. To determine downstream signaling mediators of the TGF-b pathway crucial for MT formation, we subsequently performed RNA sequencing of these cell lines. From the 34 initial candidates common to responders, but absent in non-responders, only 3 genes were left after filtering through TCGA data and in vivo RNA sequencing data of a GBM xenograft model derived from P3. Thrombospondin 1 (TSP1) emerged as the most interesting candidate as we have previously shown that transcription of this gene is activated by TGF-b/SMAD signaling and TSP1 also promotes invasiveness of GBM. TSP1 was upregulated by TGFB1 stimulation in responder cells and promoted MT formation. Transcriptional activation of TSP1 was absent in the non-responder cell line GG6 and could be reversed in the responder cell line P3 by TSP1 shRNAs in vitro and in vivo. Thus, TSP1 was experimentally verified as an important mediator of microtube formation downstream of TGF-b signaling.

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.

BACKGROUND: Progress in extending the survival of glioblastoma (GBM) patients has been slow. A better understanding of why patient survival remains poor is critical to developing new strategies. Postmortem studies on GBM can shed light on why patients are dying. METHODS: The brains of 33 GBM patients were autopsied and examined for gross and microscopic abnormalities. Clinical-pathologic correlations were accomplished through detailed chart reviews. Data were compared with older published autopsy GBM studies that predated newer treatment strategies, such as more extensive surgical resection and adjuvant temozolomide. RESULTS: In older GBM autopsy series, mass effect was observed in 72% of brains, with herniation in 50% of all cases. Infiltration of tumor into the brainstem was noted in only 21% of those older cases. In the current series, only 10 of 33 (30%) GBMs showed mass effect (P = 0.0003), and only 1 (3%) showed herniation (P < 0.0001). However, extensive GBM infiltration of the brainstem was present in 22 cases (67%, P < 0.0001), with accompanying destruction of the pons and white matter tracts. There was a direct correlation between longer median patient survival and the presence of brainstem infiltration (16.1 mo in brainstem-invaded cases vs 9.0 mo in cases lacking extensive brainstem involvement; P = 0.0003). CONCLUSIONS: With improving care, severe mass effect appears to be less common in GBM patients today, whereas dissemination, including life-threatening brainstem invasion, is now more pronounced. This has major implications regarding preclinical GBM models, as well as the design of clinical trials aimed at further improving patient survival.

Cancer is a devastating disease characterized by uncontrolled and aggressive cell growth. Suicide gene therapy (SGT) facilitating induction of malignancy-specific cell death represents a novel therapeutic approach to treat cancer, which has been investigated in several cancer types with very promising results. In addition, SGT has been suggested as a safeguard in adoptive immunotherapy and regenerative-medicine settings. Generally, SGT consists of two steps-vector-mediated delivery of suicide genes into tumors and subsequent activation of the suicide mechanism, e.g., by administration of a specific prodrug. This chapter provides a framework of protocols for basic and translational research using the Herpes-simplex-virus thymidine kinase (HSV-TK)/ganciclovir (GCV) system, the most widely used suicide gene approach. The protocols provide standard guidelines for the preparation of high-titer third-generation lentiviral vectors encoding a genetically improved HSV-TK version known as TK.007 and its application in in vitro and in vivo treatment setups.

Introduction Gene therapeutic strategies with suicide genes are currently investigated in clinical trials for brain tumors. Previously we have shown that lentiviral vectors delivering the suicide gene HSV-Tk to experimental brain tumors promote a highly significant treatment effect and are thus promising vectors for clinical translation. Aims Here, we tested lentiviral vectors delivering the suicide gene HSV-Tk.007, a highly active mutant of HSV-Tk, to rat brains as a preclinical toxicity study. We injected 106 VSV-G pseudotyped functional lentiviral particles harboring the suicide gene HSV-Tk.007 into the brain of healthy, immunocompetent rats. During prodrug treatment with Ganciclovir (GCV), we measured weight and assessed the behavior of the rats in an open field test. After 14 days of GCV treatment, we analyzed HSV-Tk.007 expression in different brain cell populations as well as inflammatory responses and apoptosis. Results During prodrug treatment with Ganciclovir, behavior experiments did not reveal differences between treated rats and the control groups. Analysis of HSV-Tk expression in different brain cell populations showed that transduced normal brain cells survived Ganciclovir treatment. There were no statistically significant differences in the number of transduced cells between treatment and control groups. Furthermore, inflammatory responses and apoptosis of brain cells were not observed. Conclusion Here we show that HSV-Tk.007 mediated suicide gene therapy is not toxic to normal brain cells. This observation is of high relevance for translation of lentivirus-mediated suicide gene therapies into the clinic to treat brain tumor patients.

Microtubes (MTs), which are cytoplasmic extensions of glioma cells, have recently been discovered as important cell communication structures. MTs are abundant in chemoresistant 1p/19q non-codeleted tumors, in particular glioblastomas, however are scarce in chemosensitive 1p/19q co-deleted oligodendrogliomas. Here we report that TGF-β is an important mediator of MT formation. TCGA data analysis revealed upregulation of TGF-β growth-factors and receptors in non-codeleted versus co-deleted tumors. TGF-β stimulation in vitro promotes enhanced MT formation in a panel of GBM stem cell lines which was blocked by a TGFBR2 inhibitor (Ly2157299). Analysis of RNA sequencing data comparing TGF-β stimulated versus unstimulated cells revealed extracellular matrix receptor interactions as a major regulated pathway. We identified Thrombospondin-1 (THBS1) as a major candidate of this pathway, which was upregulated upon TGF-β stimulation in GBM stem cell lines. Interestingly, one GBM stem cell line that did not respond to TGF-β stimulation with enhanced MT formation lacked also upregulation of THBS1. This non-responder cell line did not invade into fetal microbrains in vitro and xenografts in vivo, whereas responder cell lines showed a highly invasive and MT forming phenotype in both models. Knockdown of THBS1 in a responder cell line using shRNAs substantially reduced MT formation in vitro and in vivo. Thus, we identified THBS1 as an important mediator of MT formation downstream of TGF-β, which might play a role in therapy resistance of GBM.

Gene therapeutic strategies with suicide genes are currently investigated in clinical trials for brain tumors. Previously, we have shown that lentiviral vectors delivering the suicide gene HSV-Tk to experimental brain tumors promote a highly significant treatment effect and thus are promising vectors for clinical translation.In the present study, we tested lentiviral vectors delivering the suicide gene HSV-Tk.007, a highly active mutant of HSV-Tk, to rat brains as a preclinical toxicity study. We injected 10(6) vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped functional lentiviral particles harboring the suicide gene HSV-Tk.007 into the brain of healthy, immunocompetent rats. During prodrug treatment with ganciclovir (GCV), we measured weight and assessed the behavior of the rats in an open field test. After 14 days of GCV treatment, we analyzed HSV-Tk.007 expression in different brain cell populations, as well as inflammatory responses and apoptosis.During prodrug treatment with GCV, behavior experiments did not reveal differences between the treated rats and the control groups. Analysis of HSV-Tk expression in different brain cell populations showed that transduced normal brain cells survived GCV treatment. There were no statistically significant differences in the number of transduced cells between treatment and control groups. Furthermore, inflammatory responses and apoptosis of brain cells were not observed.We show that HSV-Tk.007-mediated suicide gene therapy is not toxic to normal brain cells. This observation is of high relevance for the translation of lentivirus-mediated suicide gene therapies into the clinic for the treatment of brain tumor patients. Copyright © 2016 John WileySons, Ltd.

Malignant gliomas, the largest group of primary intracerebrial tumors, are one of the most-difficult-to-cure cancers. The outcome has not significantly improved in recent years, despite considerable advances in our understanding of the molecular pathogenesis and improvement of surgical techniques, radio- and chemo-therapy. For glioblastoma multiforme (GBM), the most malignant form of glioma, the median survival time is approximately 15 months after diagnosis. Although complete remission of experimental GBM on MRI has been reported by using a lentiviral vector based suicide gene therapy approach1, recurrence of tumors at distant sites is common which is mainly caused by invasive glioma cells that escape treatment. Thus, a better distribution of the suicide gene is needed in order to target and efficiently kill the infiltrative glioma cells to prolong recurrence-free time span and improve the therapeutic effect. By introducing EGFR, a gene that has been reported to promote invasion of glioma cells2 into our LCMV-pseudotyped lentiviral vector system, we want to enhance the distribution of the suicide gene HSV-TK. This might lead to a more efficient killing of glioma cells in both tumor core and invasive areas.

Malignant gliomas, the largest group of primary intracerebral tumours, are one of the most difficult-to-cure cancers. For glioblastoma, the most malignant form of glioma, the median survival time is generally less than one year. Lentiviral vector mediated suicide gene therapy has been reported to be a potential therapeutic option for glioma and has been validated in clinically relevant animal models. At this end, specific targeting of tumor cells sparing normal brain tissue is a significant concern. Although several studies may suggest that HSV-Tk mediated suicide gene therapy is not likely to be toxic for normal brain cells, it has never been investigated which effect lentiviral vector mediated HSV-Tk suicide gene therapy exerts on the normal brain in a non-tumor setting. We addressed this important question by delivering the suicide gene HSV-Tk. 007 to the brain of healthy, immunocompetent rats using lentiviral vectors pseudotyped with VSV-G. First, we studied behavior patterns and assessed any potential neurological symptoms in treatment and the control groups, during prodrug treatment with Ganciclovir. We did not observe any significant changes in their physical and behavioral patterns. Then, we carried detailed histological/immunohistochemical analyses of the brains to analyze potential differences in the number of transduced brain cells between control and treatment groups. There was no significant difference in numbers of neurons, astrocytes and oligodendrocyte progenitor cells among the groups, indicating successful survival of these cells after pro-drug treatment. Furthermore, potential inflammation and apoptosis of brain cells were also investigated. No significant infiltration of immune cells was detected across the groups. We could not detect any sign of apoptosis or necrosis across all groups. In conclusion, our study which suggests that lentiviral suicide gene therapy mediated by HSV-Tk. 007 is not toxic for normal brain cells.View Large Image | Download PowerPoint Slide

Reactive oxygen species (ROS) exert various biological effects and contribute to signaling events during physiological and pathological processes. Enhanced levels of ROS are highly associated with different tumors, a Western lifestyle, and a nutritional regime. The supplementation of food with traditional antioxidants was shown to be protective against cancer in a number of studies both in vitro and in vivo. However, recent large-scale human trials in well-nourished populations did not confirm the beneficial role of antioxidants in cancer, whereas there is a well-established connection between longevity of several human populations and increased amount of antioxidants in their diets. Although our knowledge about ROS generators, ROS scavengers, and ROS signaling has improved, the knowledge about the direct link between nutrition, ROS levels, and cancer is limited. These limitations are partly due to lack of standardized reliable ROS measurement methods, easily usable biomarkers, knowledge of ROS action in cellular compartments, and individual genetic predispositions. The current review summarizes ROS formation due to nutrition with respect to macronutrients and antioxidant micronutrients in the context of cancer and discusses signaling mechanisms, used biomarkers, and its limitations along with large-scale human trials. Antioxid. Redox Signal. 19, 2157–2196.

Introduction: •Malignant gliomas, the largest group of primary intracerebral tumours, are one of the most difficult-to-cure cancers. The outcome has not improved significantly in the past years, despite considerable advances in our understanding of the molecular pathogenesis and the improvement of surgical techniques, radio- and chemotherapy. For glioblastoma multiforme (GBM), the most malignant form of glioma, the median survival time is generally less than one year.•Invasive glioma cells escape current therapies and are initiating recurrent tumors. Although complete remission of experimental GBM on MRI was observed, recurrent tumors came up which were frequently observed at different sites compared to the primary tumor 1. Thus invasive cells migrated to thes areas and initiated recurrent tumors.•The distribution of the therapeutic gene needs to be enhanced in order to target the invasive glioma cells and thereby improve the therapeutic effect and prolong the recurrence-free time window.By introducing EGFR, a gene that enhances invasion2, in addition to the suicide gene HSV-tk into our vector system, we want to improve the distribution of the therapeutic gene and also track invasive tumor cells.Key findings: •Expression of EGFR initiates an invasive program in transduced glioblastoma cells in vitro.•EGFR mediated infiltration of glioblastoma cells is retained in vivo in a rat xenograft model. [figure3 a &b]•The bicistronic lentiviral vector containing HSV-TK and EGFR [figure4] shows satisfactory cytotoxic profile in vitro. [figure5a & b] View Large Image | Download PowerPoint SlideConclusion: Expression of EGFR can enhance infiltrative nature of glioblastoma cells both in vitro and in vivo. We are currently taking preparations for an in vivo therapeutic study of our bicistronic lentiviral construct containing a recombinant HSV-TK and EGFR in a clinically relevant GBM rat model.

EGFR gene amplification is observed in upwards of 40% of glioblastoma (GBM) biopsies, and genetic aberration to EGFR at large has been described to occur in excess of 50%. Our lab has developed a clinically relevant in vivo xenograft model derived from patient biopsies in which we are able to accurately study EGFR signaling in human GBM. We previously reported that wild-type EGFR amplification and activation promotes invasion and development of GBM independent of angiogenesis. Importantly, detected in half of GBMs harboring EGFR amplification, the most prominent EGFR mutation arises upon deletion of exons 2 through 7 to generate the constitutively active truncation mutant EGFRvIII. Here we report that while wild-type EGFR overexpression sustains invasion, EGFRvIII, in contrast, induces more aggressive and angiogenic tumor growth. We further performed signaling complex immunocapture mass spectrometry which revealed that EGFRvIII-mediated signaling is divergent from that of its wild-type counterpart. In particular the non-receptor tyrosine kinase c-SRC was highly upregulated and activated in EGFRvIII tumors as verified on western blots and by immunohistochemistry. Inhibition of c-SRC activity by shRNA and Src inhibitors decreased VEGF levels in EGFRvIII tumor cells. Furthermore, EGFRvIII+ xenograft tumors expressing c-SRC shRNA became less angiogenic and aggressive - indeed similar to control tumors lacking EGFRvIII. When overexpressing constitutively activated Src in control tumor cells, we could recapitulate the oncogenic phenotype of EGFRvIII as the tumor cells secreted higher levels of VEGF and became more aggressive and angiogenic in vivo. We hereby report evidence that the EGFRvIII-driven oncogenic activity and angiogenic tumor growth is mediated by c-SRC. In conclusion, our results suggest that the EGFRvIII-SRC-VEGF axis represents an important mechanism of tumor angiogenesis in GBM.