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.
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 1? 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.
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 x 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.
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.
Hypoxia 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.
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 dimethyl-alpha-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, pO(2) = 2 bar, 4 exposures à 90 minutes), whereas the control group was housed under normal atmosphere (1 bar, pO(2) = 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 (approximately 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.
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 O(2) 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.
The 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.
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