Linda Elin Birkhaug Stuhr

CD133 negative glioma cells form tumors in nude rats and give rise to CD133 positive cells

CD133 is a cell surface marker expressed on progenitors of haematopoietic and endothelial cell lineages. Moreover, several studies have identified CD133 as a marker of brain tumor‐initiating cells. In this study, human glioblastoma multiforme biopsies were engrafted intracerebrally into nude rats. The resulting tumors were serially passaged in vivo, and monitored by magnetic resonance imaging. CD133 expression was analyzed at various passages. Tumors initiated directly from the biopsies expressed little or no CD133, and showed no contrast enhancement suggesting an intact blood‐brain barrier. During passaging, the tumors gradually displayed more contrast enhancement, increased angiogenesis and a shorter survival. Real‐time qPCR and immunoblots showed that this was accompanied by increased CD133 expression. Primary biopsy spheroids and xenograft tumors were subsequently dissociated and flow sorted into CD133 negative and CD133 positive cell populations. Both populations incorporated BrdU in cell culture, and expressed the neural precursor marker nestin. Notably, CD133 negative cells derived from 6 different patients were tumorgenic when implanted into the rat brains. For 3 of these patients, analysis showed that the resulting tumors contained CD133 positive cells. In conclusion, we show that CD133 negative glioma cells are tumorgenic in nude rats, and that CD133 positive cells can be obtained from these tumors. Upon passaging of the tumors in vivo, CD133 expression is upregulated, coinciding with the onset of angiogenesis and a shorter survival. Thus, our findings do not suggest that CD133 expression is required for brain tumor initiation, but that it may be involved during brain tumor progression.

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.

Lowering of tumor interstitial fluid pressure specifically augments efficacy of chemotherapy

Chemotherapy of solid tumors is presently largely ineffective at dosage levels that are compatible with survival of the patient. Here, it is argued that a condition of raised interstitial fluid pressure (IFP) that can be observed in many tumors is a major factor in preventing optimal access of systemically administered chemotherapeutic agents. Using prostaglandin E1‐methyl ester (PGE1), which is known transiently to reduce IFP, it was shown that 5‐fluorouracil (5‐FU) caused significant growth inhibition on two experimental tumors in rats but only after administration of PGE1. Furthermore, timing experiments showed that only in the period in which IFP is reduced did 5‐FU have an antitumor effect. These experiments uniquely demonstrate a clear and, according to the starting hypothesis, logical, synergistic effect of PGE1 and 5‐FU that offers hope for better treatment of many tumors in which raised IFP is likely to be inhibiting optimal results with water‐soluble cancer chemotherapeutic agents.

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.

Collagen-binding proteoglycan fibromodulin can determine stroma matrix structure and fluid balance in experimental carcinoma

Research on the biology of the tumor stroma has the potential to lead to development of more effective treatment regimes enhancing the efficacy of drug-based treatment of solid malignancies. Tumor stroma is characterized by distorted blood vessels and activated connective tissue cells producing a collagen-rich matrix, which is accompanied by elevated interstitial fluid pressure (IFP), indicating a transport barrier between tumor tissue and blood. Here, we show that the collagen-binding proteoglycan fibromodulin controls stroma structure and fluid balance in experimental carcinoma. Gene ablation or inhibition of expression by anti-inflammatory agents showed that fibromodulin promoted the formation of a dense stroma and an elevated IFP. Fibromodulin-deficiency did not affect vasculature but increased the extracellular fluid volume and lowered IFP. Our data suggest that fibromodulin controls stroma matrix structure that in turn modulates fluid convection inside and out of the stroma. This finding is particularly important in relation to the demonstration that targeted modulations of the fluid balance in carcinoma can increase the response to cancer therapeutic agents.

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.

Inhibition of carcinoma cell-derived VEGF reduces inflammatory characteristics in xenograft carcinoma

The stroma of carcinomas shares several characteristics with inflamed tissues including a distorted vasculature, active angiogenesis and macrophage infiltration. In addition, the tumor interstitial fluid pressure (PIF) of the stroma is pathologically elevated. We show here that bevacizumab [rhuMab vascular endothelial growth factor (VEGF), Avastin], a monoclonal antibody to VEGF, at a dose of 5 mg/kg modulated inflammation in KAT‐4 xenograft human anaplastic thyroid carcinoma tissue. At this dose, bevacizumab reduced the density of macrophages, MHC class II antigen expression by macrophages and IL‐1β mRNA expression. Furthermore, bevacizumab lowered tumor extracellular fluid volume, plasma protein leakage from tumor vessels, the number of CD31‐positive structures and tumor PIF. The tumor plasma volume and the number of α‐smooth muscle actin‐positive vessels, however, remained unchanged. Our data suggest that carcinoma cell‐derived VEGF either directly or indirectly participates in maintaining an inflammatory microenvironment in experimental KAT‐4 carcinoma. Furthermore, our data indicate that the reduction of inflammation resulting in reduced vascular permeability and decrease in the tumor extracellular fluid volume by bevacizumab contributes to reduced tumor PIF.

Bevacizumab treatment induces metabolic adaptation toward anaerobic metabolism in glioblastomas

Anti-angiogenic therapy in glioblastoma (GBM) has unfortunately not led to the anticipated improvement in patient prognosis. We here describe how human GBM adapts to bevacizumab treatment at the metabolic level. By performing 13C6-glucose metabolic flux analysis, we show for the first time that the tumors undergo metabolic re-programming toward anaerobic metabolism, thereby uncoupling glycolysis from oxidative phosphorylation. Following treatment, an increased influx of 13C6-glucose was observed into the tumors, concomitant to increased lactate levels and a reduction of metabolites associated with the tricarboxylic acid cycle. This was confirmed by increased expression of glycolytic enzymes including pyruvate dehydrogenase kinase in the treated tumors. Interestingly, l-glutamine levels were also reduced. These results were further confirmed by the assessment of in vivo metabolic data obtained by magnetic resonance spectroscopy and positron emission tomography. Moreover, bevacizumab led to a depletion in glutathione levels indicating that the treatment caused oxidative stress in the tumors. Confirming the metabolic flux results, immunohistochemical analysis showed an up-regulation of lactate dehydrogenase in the bevacizumab-treated tumor core as well as in single tumor cells infiltrating the brain, which may explain the increased invasion observed after bevacizumab treatment. These observations were further validated in a panel of eight human GBM patients in which paired biopsy samples were obtained before and after bevacizumab treatment. Importantly, we show that the GBM adaptation to bevacizumab therapy is not mediated by clonal selection mechanisms, but represents an adaptive response to therapy.

Hyperoxia retards growth and induces apoptosis and loss of glands and blood vessels in DMBA-induced rat mammary tumors

BackgroundThis study investigated the effects of hyperoxic treatment on growth, angiogenesis, apoptosis, general morphology and gene expression in DMBA-induced rat mammary tumors.MethodsOne group of animals was exposed to normobaric hyperoxia (1 bar, pO2 = 1.0 bar) and another group was exposed to hyperbaric hyperoxia (1.5 bar, pO2 = 1.5 bar). A third group was treated with the commonly used chemotherapeutic drug 5- Fluorouracil (5-FU), whereas animals housed under normal atmosphere (1 bar, pO2 = 0.2 bar) served as controls. All treatments were performed on day 1, 4, 7 and 10 for 90 min. Tumor growth was calculated from caliper measurements. Biological effects of the treatment, was determined by assessment of vascular morphology (immunostaining for von Willebrandt factor) and apoptosis (TUNEL staining). Detailed gene expression profiles were obtained and verified by quantitative rtPCR.ResultsTumor growth was significantly reduced (~57–66 %) after hyperoxic treatment compared to control and even more than 5-FU (~36 %). Light microscopic observations of the tumor tissue showed large empty spaces within the tissue after hyperoxic treatment, probably due to loss of glands as indicated by a strong down-regulation of glandular secretory proteins. A significant reduction in mean vascular density (30–50%) was found after hyperoxic treatment. Furthermore, increased apoptosis (18–21%) was found after hyperoxic treatment.ConclusionThus, by increasing the pO2 in mammary tumor tissue using normobaric and moderate hyperbaric oxygen therapy, a significant retardation in tumor growth is achieved, by loss of glands, reduction in vascular density and enhanced cell death. Hyperbaric oxygen should therefore be further evaluated as a tumor treatment.

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.