Ingrid Moen

Anti-VEGF treatment reduces blood supply and increases tumor cell invasion in glioblastoma

Bevacizumab, an antibody against vascular endothelial growth factor (VEGF), is a promising, yet controversial, drug in human glioblastoma treatment (GBM). Its effects on tumor burden, recurrence, and vascular physiology are unclear. We therefore determined the tumor response to bevacizumab at the phenotypic, physiological, and molecular level in a clinically relevant intracranial GBM xenograft model derived from patient tumor spheroids. Using anatomical and physiological magnetic resonance imaging (MRI), we show that bevacizumab causes a strong decrease in contrast enhancement while having only a marginal effect on tumor growth. Interestingly, dynamic contrast-enhanced MRI revealed a significant reduction of the vascular supply, as evidenced by a decrease in intratumoral blood flow and volume and, at the morphological level, by a strong reduction of large- and medium-sized blood vessels. Electron microscopy revealed fewer mitochondria in the treated tumor cells. Importantly, this was accompanied by a 68% increase in infiltrating tumor cells in the brain parenchyma. At the molecular level we observed an increase in lactate and alanine metabolites, together with an induction of hypoxia-inducible factor 1α and an activation of the phosphatidyl-inositol-3-kinase pathway. These data strongly suggest that vascular remodeling induced by anti-VEGF treatment leads to a more hypoxic tumor microenvironment. This favors a metabolic change in the tumor cells toward glycolysis, which leads to enhanced tumor cell invasion into the normal brain. The present work underlines the need to combine anti-angiogenic treatment in GBMs with drugs targeting specific signaling or metabolic pathways linked to the glycolytic phenotype.

Hyperbaric oxygen therapy and cancer—a review

Hypoxia is a critical hallmark of solid tumors and involves enhanced cell survival, angiogenesis, glycolytic metabolism, and metastasis. Hyperbaric oxygen (HBO) treatment has for centuries been used to improve or cure disorders involving hypoxia and ischemia, by enhancing the amount of dissolved oxygen in the plasma and thereby increasing O2 delivery to the tissue. Studies on HBO and cancer have up to recently focused on whether enhanced oxygen acts as a cancer promoter or not. As oxygen is believed to be required for all the major processes of wound healing, one feared that the effects of HBO would be applicable to cancer tissue as well and promote cancer growth. Furthermore, one also feared that exposing patients who had been treated for cancer, to HBO, would lead to recurrence. Nevertheless, two systematic reviews on HBO and cancer have concluded that the use of HBO in patients with malignancies is considered safe. To supplement the previous reviews, we have summarized the work performed on HBO and cancer in the period 2004–2012. Based on the present as well as previous reviews, there is no evidence indicating that HBO neither acts as a stimulator of tumor growth nor as an enhancer of recurrence. On the other hand, there is evidence that implies that HBO might have tumor-inhibitory effects in certain cancer subtypes, and we thus strongly believe that we need to expand our knowledge on the effect and the mechanisms behind tumor oxygenation.

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.

Hyperoxic Treatment Induces Mesenchymal-to-Epithelial Transition in a Rat Adenocarcinoma Model

Tumor hypoxia is relevant for tumor growth, metabolism and epithelial-to-mesenchymal transition (EMT). We report that hyperbaric oxygen (HBO) treatment induced mesenchymal-to-epithelial transition (MET) in a dimetyl-α-benzantracene induced mammary rat adenocarcinoma model, and the MET was associated with extensive coordinated gene expression changes and less aggressive tumors. One group of tumor bearing rats was exposed to HBO (2 bar, pO2 = 2 bar, 4 exposures à 90 minutes), whereas the control group was housed under normal atmosphere (1 bar, pO2 = 0.2 bar). Treatment effects were determined by assessment of tumor growth, tumor vascularisation, tumor cell proliferation, cell death, collagen fibrils and gene expression profile. Tumor growth was significantly reduced (∼16%) after HBO treatment compared to day 1 levels, whereas control tumors increased almost 100% in volume. Significant decreases in tumor cell proliferation, tumor blood vessels and collagen fibrils, together with an increase in cell death, are consistent with tumor growth reduction and tumor stroma influence after hyperoxic treatment. Gene expression profiling showed that HBO induced MET. In conclusion, hyperoxia induced MET with coordinated expression of gene modules involved in cell junctions and attachments together with a shift towards non-tumorigenic metabolism. This leads to more differentiated and less aggressive tumors, and indicates that oxygen per se might be an important factor in the “switches” of EMT and MET in vivo. HBO treatment also attenuated tumor growth and changed tumor stroma, by targeting the vascular system, having anti-proliferative and pro-apoptotic effects.

Atrial natriuretic peptide modulation of albumin clearance and contrast agent permeability in mouse skeletal muscle and skin: role in regulation of plasma volume

Atrial natriuretic peptide (ANP) via its guanylyl cyclase‐A (GC‐A) receptor participates in regulation of arterial blood pressure and vascular volume. Previous studies demonstrated that concerted renal diuretic/natriuretic and endothelial permeability effects of ANP cooperate in intravascular volume regulation. We show that the microvascular endothelial contribution to the hypovolaemic action of ANP can be measured by the magnitude of the ANP‐induced increase in blood‐to‐tissue albumin transport, measured as plasma albumin clearance corrected for intravascular volume change, relative to the corresponding increase in ANP‐induced renal water excretion. We used a two‐tracer method with isotopically labelled albumin to measure clearances in skin and skeletal muscle of: (i) C57BL6 mice; (ii) mice with endothelium‐restricted deletion of GC‐A (floxed GC‐A × tie2‐Cre: endothelial cell (EC) GC‐A knockout (KO)); and (iii) control littermates (floxed GC‐A mice with normal GC‐A expression levels). Comparison of albumin clearances in hypervolaemic EC GC‐A KO mice with normovolaemic littermates demonstrated that skeletal muscle albumin clearance with ANP treatment accounts for at most 30% of whole body clearance required for ANP to regulate plasma volume. Skin microcirculation responded to ANP similarly. Measurements of permeability to a high molecular mass contrast agent (35 kD Gadomer) by dynamic contrast‐enhanced magnetic resonance imaging (DCE‐MRI) enabled repeated measures in individual animals and confirmed small increases in muscle and skin microvascular permeability after ANP. These quantitative methods will enable further evaluation of the contribution of ANP‐dependent microvascular beds (such as gastro‐intestinal tract) to plasma volume regulation.

Combined Anti-Angiogenic Therapy Targeting PDGF and VEGF Receptors Lowers the Interstitial Fluid Pressure in a Murine Experimental Carcinoma

Elevation of the interstitial fluid pressure (IFP) of carcinoma is an obstacle in treatment of tumors by chemotherapy and correlates with poor drug uptake. Previous studies have shown that treatment with inhibitors of platelet-derived growth factor (PDGF) or vascular endothelial growth factor (VEGF) signaling lowers the IFP of tumors and improve chemotherapy. In this study, we investigated whether the combination of PDGFR and VEGFR inhibitors could further reduce the IFP of KAT-4 human carcinoma tumors. The tumor IFP was measured using the wick-in-needle technique. The combination of STI571 and PTK/ZK gave an additive effect on the lowering of the IFP of KAT-4 tumors, but the timing of the treatment was crucial. The lowering of IFP following combination therapy was accompanied by vascular remodeling and decreased vascular leakiness. The effects of the inhibitors on the therapeutic efficiency of Taxol were investigated. Whereas the anti-PDGF and anti-VEGF treatment did not significantly inhibit tumor growth, the inhibitors enhanced the effect of chemotherapy. Despite having an additive effect in decreasing tumor IFP, the combination therapy did not further enhance the effect of chemotherapy. Simultaneous targeting of VEGFR and PDGFR kinase activity may be a useful strategy to decrease tumor IFP, but the timing of the inhibitors should be carefully determined.

Hyperoxia increases the uptake of 5-fluorouracil in mammary tumors independently of changes in interstitial fluid pressure and tumor stroma.

BackgroundHypoxia is associated with increased resistance to chemo- and radiation-therapy. Hyperoxic treatment (hyperbaric oxygen) has previously been shown to potentiate the effect of some forms of chemotherapy, and this has been ascribed to enhanced cytotoxicity or neovascularisation. The aim of this study was to elucidate whether hyperoxia also enhances any actual uptake of 5FU (5-fluorouracil) into the tumor tissue and if this can be explained by changes in the interstitium and extracellular matrix.MethodsOne group of tumor bearing rats was exposed to repeated hyperbaric oxygen (HBO) treatment (2 bar, pO2 = 2 bar, 4 exposures à 90 min), whereas one group was exposed to one single identical HBO treatment. Animals housed under normal atmosphere (1 bar, pO2 = 0.2 bar) served as controls. Three doses of 5FU were tested for dose response. Uptake of [3H]-5FU in the tumor was assessed, with special reference to factors that might have contributed, such as interstitial fluid pressure (Pif), collagen content, oxygen stress (measured as malondialdehyd levels), lymphatics and transcapillary transport in the tumors.ResultsThe uptake of the cytostatic agent increases immediately after a single HBO treatment (more than 50%), but not 24 hours after the last repeated HBO treatment. Thus, the uptake is most likely related to the transient increase in oxygenation in the tumor tissue. Factors like tumor Pif and collagen content, which decreased significantly in the tumor interstitium after repeated HBO treatment, was without effect on the drug uptake.ConclusionWe showed that hyperoxia increases the uptake of [3H]-5FU in DMBA-induced mammary tumors per se, independently of changes in Pif, oxygen stress, collagen fibril density, or transendothelial transport alone. The mechanism by which such an uptake occur is still not elucidated, but it is clearly stimulated by elevated pO2.

Gene expression in tumor cells and stroma in dsRed 4T1 tumors in eGFP-expressing mice with and without enhanced oxygenation

BackgroundThe tumor microenvironment is pivotal in tumor progression. Thus, we aimed to develop a mammary tumor model to elucidate molecular characteristics in the stroma versus the tumor cell compartment by global gene expression. Secondly, since tumor hypoxia influences several aspects of tumor pathophysiology, we hypothesized that hyperoxia might have an inhibitory effect on tumor growth per se. Finally, we aimed to identify differences in gene expression and key molecular mechanisms, both in the native state and following treatment.Methods4T1 dsRed breast cancer cells were injected into eGFP expressing NOD/SCID mice. Group 1 was exposed to 3 intermittent HBO treatments (Day 1, 4 and 7), Group 2 to 7 daily HBO treatments (both 2.5bar, 100% O2, à 90 min), whereas the controls were exposed to a normal atmosphere. Tumor growth, histology, vascularisation, cell proliferation, cell death and metastasis were assessed. Fluorescence-activated cell sorting was used to separate tumor cells from stromal cells prior to gene expression analysis.ResultsThe purity of sorted cells was verified by fluorescence microscopy. Gene expression profiling demonstrated that highly expressed genes in the untreated tumor stroma included constituents of the extracellular matrix and matrix metalloproteinases. Tumor growth was significantly inhibited by HBO, and the MAPK pathway was found to be significantly reduced. Immunohistochemistry indicated a significantly reduced microvessel density after intermittent HBO, whereas daily HBO did not show a similar effect. The anti-angiogenic response was reflected in the expression trends of angiogenic factors.ConclusionsThe present in vivo mammary tumor model enabled us to separate tumor and stromal cells, and demonstrated that the two compartments are characterized by distinct gene expressions, both in the native state and following HBO treatments. Furthermore, hyperoxia induced a significant tumor growth-inhibitory effect, with significant down-regulation of the MAPK pathway. An anti-angiogenic effect after intermittent HBO was observed, and reflected in the gene expression profile.

Peritumoral TNFα administration influences tumour stroma structure and physiology independently of growth in DMBA-induced mammary tumours

Background. Systemic treatment of malignancies with high doses of tumour necrosis factor‐α (TNFα) has an anticancer effect, but also serious side effects. The aim of the present study was to elucidate the effects of local TNFα administration alone or in combination with chemotherapy on tumour stroma structure and physiology in di‐methyl‐benz‐anthracene (DMBA)‐induced mammary carcinomas in rats. Methods. TNFα (500 ng/mL in a volume of 5 µL) was given s.c. around the carcinoma and 5‐fluorouracil (5‐FU) (1.5 mg/kg, volume of 0.2 mL) was given i.p on days 1, 4, 7 and 10. Results. Treatment with TNFα resulted in a significant reduction of tumour interstitial fluid pressure (TIFP: 75–87 %, p<0.02–0.001), as well as in the number of tumour‐infiltrating macrophages, extracellular volume (ECV) and collagen fibril density in carcinoma. In addition, pH was lowered in tumours treated with TNFα, suggesting decreased aerobic metabolism. Treatment with TNFα, however, had no effect on tumour growth, arterial blood pressure, tumour vessel density, plasma volume or body weight. Concentrations of locally produced VEGF and IL‐1β in carcinoma interstitial fluid or in serum were not affected by TNFα. The study demonstrated that these cytokines are produced locally in the tumour. Furthermore, TNFα had no effect on efficacy of treatment with 5‐FU. Conclusions: Locally administered TNFα did not affect DMBA‐induced mammary tumour growth or vasculature, but reduced inflammation and ECM structure, suggesting the latter to be of importance in the observed reduction in TIFP.

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