In-vivo visualization of tumor microvessel density and response to anti-angiogenic treatment by high resolution MRI in mice

Purpose

Inhibition of angiogenesis has shown clinical success in patients with cancer. Thus, imaging approaches that allow for the identification of angiogenic tumors and the detection of response to anti-angiogenic treatment are of high clinical relevance.

Experimental Design

We established an in vivo magnetic resonance imaging (MRI) approach that allows us to simultaneously image tumor microvessel density and tumor vessel size in a NSCLC model in mice.

Results

Using microvessel density imaging we demonstrated an increase in microvessel density within 8 days after tumor implantation, while tumor vessel size decreased indicating a switch from macro- to microvessels during tumor growth. Moreover, we could monitor in vivo inhibition of angiogenesis induced by the angiogenesis inhibitor PTK787, resulting in a decrease of microvessel density and a slight increase in tumor vessel size.

Conclusions

We present an in vivo imaging approach that allows us to monitor both tumor microvessel density and tumor vessel size in the tumor. Moreover, this approach enables us to assess, early-on, treatment effects on tumor microvessel density as well as on tumor vessel size. Thus, this imaging-based strategy of validating anti-angiogenic treatment effects has high potential in applications to preclinical and clinical trials.     

Role of oxygenation and vascularization in drug resistance

Oxygenation status and tumor vascularization seem to be important factors in determining therapeutic effectiveness and patient prognosis. An abundance of data on tumor oxygenation and vascularization is available and it clearly shows that most human solid tumors are heterogeneously oxygenated and vascularized. They contain hypoxic regions. Such regions and areas of reduced vascularization can affect the response to a variety of drugs. Direct measurements of pO₂ and the vascular density in various types of tumors have, upon correlation of the data to therapeutic outcome, shown that low pO₂ values and low vascular density are associated with a decreased response to therapy. Therefore, oxygenation status and the extent of tumor vascularization may well be important factors contributing to the difficulty of successful therapy in certain types of tumors. This revised version was published online in August 2006 with corrections to the Cover Date.     

Tumor T1 Relaxation Time for Assessing Response to Bevacizumab Anti-Angiogenic Therapy in a Mouse Ovarian Cancer Model

Purpose

To assess whether T₁ relaxation time of tumors may be used to assess response to bevacizumab anti-angiogenic therapy. Procedures: 12 female nude mice bearing subcutaneous SKOV3ip1-LC ovarian tumors were administered bevacizumab (6.25ug/g, n=6) or PBS (control, n=6) therapy twice a week for two weeks. T₁ maps of tumors were generated before, two days, and 2 weeks after initiating therapy. Tumor weight was assessed by MR and at necropsy. Histology for microvessel density, proliferation, and apoptosis was performed.

Results

Bevacizumab treatment resulted in tumor growth inhibition (p<0.04, n=6), confirming therapeutic efficacy. Tumor T₁ relaxation times increased in bevacizumab treated mice 2 days and 2 weeks after initiating therapy (p<.05, n=6). Microvessel density decreased 59% and cell proliferation (Ki67+) decreased 50% in the bevacizumab treatment group (p<.001, n=6), but not apoptosis.

Conclusions

Findings suggest that increased tumor T₁ relaxation time is associated with response to bevacizumab therapy in ovarian cancer model and might serve as an early indicator of response.     

Antiangiogenic Therapy Using Sunitinib Combined with Rapamycin Retards Tumor Growth But Promotes Metastasis

BACKGROUND: This study investigated the synergistic effect of sunitinib and rapamycin on tumor growth and metastasis in murine breast cancer model. METHODS: The synergistic antitumor effect of sunitinib and rapamycin on tumor growth and metastasis was investigated. Myeloid-derived suppressor cells (MDSCs) in spleens and lungs were assessed. Tumor hypoxia, vessel density and micrometastasis were evaluated. Versican, indoleamine 2,3-dioxygenase (IDO), arginase 1, interleukin-6 (IL-6), IL-10, and transforming growth factor β (TGF-β) in the lungs and tumors were examined. IL-6 and TGF-β in the blood were evaluated. RESULTS: Synergism between sunitinib and rapamycin on tumor growth was observed. Sunitinib plus rapamycin reduced splenomegaly, MDSCs in spleens and lungs, and microvessel density in tumor microenvironment, while exacerbated hypoxia and promoted cancer lung metastasis. Sunitinib plus rapamycin markedly induced versican, IDO, arginase 1, IL-6, and TGF-β expression in the lungs, whereas it reduced IDO and IL-10 expression in the primary tumor tissues. IL-6 levels in the circulation were increased after rapamycin and combination therapies. CONCLUSIONS: The combination of sunitinib plus rapamycin reduced the tumor growth but promoted tumor metastasis. This study warrants that further mTOR inhibition treatment should be closely watched in clinical setting, especially combined with antiangiogenic therapy.     

Electron paramagnetic resonance highlights that the oxygen effect contributes to the radiosensitizing effect of paclitaxel

Background

Paclitaxel (PTX) is a potent anti-cancer chemotherapeutic agent and is widely used in the treatments of solid tumors, particularly of the breast and ovaries. An effective and safe micellar formulation of PTX was used to administer higher doses of PTX than Taxol® (the current commercialized drug). We hypothesize that PTX-loaded micelles (M-PTX) may enhance tumor radiosensitivity by increasing the tumor oxygenation (pO₂). Our goals were (i) to evaluate the contribution of the “oxygen effect” to the radiosensitizing effect of PTX; (ii) to demonstrate the therapeutic relevance of the combination of M-PTX and irradiation and (iii) to investigate the underlying mechanisms of the observed oxygen effect.

Methodology and Principal Findings

We used (PEG-p-(CL-co-TMC)) polymeric micelles to solubilize PTX. pO₂ was measured on TLT tumor-bearing mice treated with M-PTX (80 mg/kg) using electron paramagnetic resonance (EPR) oximetry. The regrowth delay following 10 Gy irradiation 24 h after M-PTX treatment was measured. The tumor perfusion was assessed by the patent blue staining. The oxygen consumption rate and the apoptosis were evaluated by EPR oximetry and the TUNEL assay, respectively. EPR oximetry experiments showed that M-PTX dramatically increases the pO₂ 24 h post treatment. Regrowth delay assays demonstrated a synergy between M-PTX and irradiation. M-PTX increased the tumor blood flow while cells treated with M-PTX consumed less oxygen and presented more apoptosis.

Conclusions

M-PTX improved the tumor oxygenation which leads to synergy between this treatment and irradiation. This increased pO₂ can be explained both by an increased blood flow and an inhibition of O₂ consumption.     

Pyruvate Induces Transient Tumor Hypoxia by Enhancing Mitochondrial Oxygen Consumption and Potentiates the Anti-Tumor Effect of a Hypoxia-Activated Prodrug TH-302

Background

TH-302 is a hypoxia-activated prodrug (HAP) of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302.

Methodology/Results

The hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR) oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O₂), with minimal effect under aerobic conditions (21% O₂). Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500–1500 mm³. Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼550 mm³), significantly delayed tumor growth.

Conclusions/Significance

Our in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the appropriate tumor size and oxygen concentration.     

Arterial embolization hyperthermia using As2O3 nanoparticles in VX2 carcinoma-induced liver tumors

Background

Combination therapy for arterial embolization hyperthermia (AEH) with arsenic trioxide (As₂O₃) nanoparticles (ATONs) is a novel treatment for solid malignancies. This study was performed to evaluate the feasibility and therapeutic effect of AEH with As₂O₃ nanoparticles in a rabbit liver cancer model. The protocol was approved by our institutional animal use committee.

Methodology/Principal Findings

In total, 60 VX₂ liver-tumor-bearing rabbits were randomly assigned to five groups (n = 12/group) and received AEH with ATONs (Group 1), hepatic arterial embolization with ATONs (Group 2), lipiodol (Group 3), or saline (Group 4), on day 14 after tumor implantation. Twelve rabbits that received AEH with ATONs were prepared for temperature measurements, and were defined as Group 5. Computed tomography was used to measure the tumors'' longest dimension, and evaluation was performed according to the Response Evaluation Criteria in Solid Tumors. Hepatic toxicity, tumor necrosis rate, vascular endothelial growth factor level, and microvessel density were determined. Survival rates were measured using the Kaplan-Meier method. The therapeutic temperature (42.5°C) was obtained in Group 5. Hepatotoxicity reactions occurred but were transient in all groups. Tumor growth was delayed and survival was prolonged in Group 1 (treated with AEH and ATONs). Plasma and tumor vascular endothelial growth factor and microvessel density were significantly inhibited in Group 1, while tumor necrosis rates were markedly enhanced compared with those in the control groups.

Conclusions

ATON-based AEH is a safe and effective treatment that can be targeted at liver tumors using the dual effects of hyperthermia and chemotherapy. This therapy can delay tumor growth and noticeably inhibit tumor angiogenesis.     

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.     

Quantitative imaging of pO2 in orthotopic murine gliomas: hypoxia correlates with resistance to radiation

Hypoxia is considered one of the microenvironmental factors associated with the malignant nature of glioblastoma. Thus, evaluating intratumoural distribution of hypoxia would be useful for therapeutic planning as well as assessment of its effectiveness during the therapy. Electron paramagnetic resonance imaging (EPRI) is an imaging technique which can generate quantitative maps of oxygen in vivo using the exogenous paramagnetic compound, triarylmethyl and monitoring its line broadening caused by oxygen. In this study, the feasibility of EPRI for assessment of oxygen distribution in the glioblastoma using orthotopic U87 and U251 xenograft model is examined. Heterogeneous distribution of pO₂ between 0 and 50?mmHg was observed throughout the tumours except for the normal brain tissue. U251 glioblastoma was more likely to exhibit hypoxia than U87 for comparable tumour size (median pO₂; 29.7 and 18.2?mmHg, p?=?.028, in U87 and U251, respectively). The area with pO₂ under 10?mmHg on the EPR oximetry (HF10) showed a good correlation with pimonidazole staining among tumours with evaluated size. In subcutaneous xenograft model, irradiation was relatively less effective for U251 compared with U87. In conclusion, EPRI is a feasible method to evaluate oxygen distribution in the brain tumour.     

Quantitative Multi-Parametric Magnetic Resonance Imaging of Tumor Response to Photodynamic Therapy

ObjectiveThe aim of this study was to characterize response to photodynamic therapy (PDT) in a mouse cancer model using a multi-parametric quantitative MRI protocol and to identify MR parameters as potential biomarkers for early assessment of treatment outcome.MethodsCT26.WT colon carcinoma tumors were grown subcutaneously in the hind limb of BALB/c mice. Therapy consisted of intravenous injection of the photosensitizer Bremachlorin, followed by 10 min laser illumination (200 mW/cm²) of the tumor 6 h post injection. MRI at 7 T was performed at baseline, directly after PDT, as well as at 24 h, and 72 h. Tumor relaxation time constants (T₁ and T₂) and apparent diffusion coefficient (ADC) were quantified at each time point. Additionally, Gd-DOTA dynamic contrast-enhanced (DCE) MRI was performed to estimate transfer constants (K^trans) and volume fractions of the extravascular extracellular space (v_e) using standard Tofts-Kermode tracer kinetic modeling. At the end of the experiment, tumor viability was characterized by histology using NADH-diaphorase staining.ResultsThe therapy induced extensive cell death in the tumor and resulted in significant reduction in tumor growth, as compared to untreated controls. Tumor T₁ and T₂ relaxation times remained unchanged up to 24 h, but decreased at 72 h after treatment. Tumor ADC values significantly increased at 24 h and 72 h. DCE-MRI derived tracer kinetic parameters displayed an early response to the treatment. Directly after PDT complete vascular shutdown was observed in large parts of the tumors and reduced uptake (decreased K^trans) in remaining tumor tissue. At 24 h, contrast uptake in most tumors was essentially absent. Out of 5 animals that were monitored for 2 weeks after treatment, 3 had tumor recurrence, in locations that showed strong contrast uptake at 72 h.ConclusionDCE-MRI is an effective tool for visualization of vascular effects directly after PDT. Endogenous contrast parameters T₁, T₂, and ADC, measured at 24 to 72 h after PDT, are also potential biomarkers for evaluation of therapy outcome.     

A Comparative pO2 Probe and [18F]-Fluoro-Azomycinarabino-Furanoside ([18F]FAZA) PET Study Reveals Anesthesia-Induced Impairment of Oxygenation and Perfusion in Tumor and Muscle

Tumor hypoxia can be identified by [¹⁸F]FAZA positron emission tomography, or invasively using oxygen probes. The impact of anesthetics on tumor hypoxia remains controversial. The aim of this comprehensive study was to investigate the impact of isoflurane and ketamine/xylazine anesthesia on [¹⁸F]FAZA uptake and partial oxygen pressure (pO₂) in carcinoma and muscle tissue of air- and oxygen-breathing mice.

Methods

CT26 colon carcinoma-bearing mice were anesthetized with isoflurane (IF) or ketamine/xylazine (KX) while breathing air or oxygen (O₂). We performed 10 min static PET scans 1 h, 2 h and 3 h after [¹⁸F]FAZA injection and calculated the [¹⁸F]FAZA-uptake and tumor-to-muscle ratios (T/M). In another experimental group, we placed a pO₂ probe in the tumor as well as in the gastrocnemius muscle to measure the pO₂ and perfusion.

Results

Ketamine/xylazine-anesthetized mice yielded up to 3.5-fold higher T/M-ratios compared to their isoflurane-anesthetized littermates 1 h, 2 h and 3 h after [¹⁸F]FAZA injection regardless of whether the mice breathed air or oxygen (3 h, KX-air: 7.1 vs. IF-air: 1.8, p = 0.0001, KX-O₂: 4.4 vs. IF-O₂: 1.4, p < 0.0001). The enhanced T/M-ratios in ketamine/xylazine-anesthetized mice were mainly caused by an increased [¹⁸F]FAZA uptake in the carcinomas. Invasive pO₂ probe measurements yielded enhanced intra-tumoral pO₂ values in air- and oxygen-breathing ketamine/xylazine-anesthetized mice compared to isoflurane-anesthetized mice (KX-air: 1.01 mmHg, IF-air: 0.45 mmHg; KX-O₂ 9.73 mmHg, IF-O₂: 6.25 mmHg). Muscle oxygenation was significantly higher in air-breathing isoflurane-anesthetized (56.9 mmHg) than in ketamine/xylazine-anesthetized mice (33.8 mmHg, p = 0.0003).

Conclusion

[¹⁸F]FAZA tumor uptake was highest in ketamine/xylazine-anesthetized mice regardless of whether the mice breathed air or oxygen. The generally lower [¹⁸F]FAZA whole-body uptake in isoflurane-anesthetized mice could be due to the higher muscle pO₂-values in these mice compared to ketamine/xylazine-anesthetized mice. When performing preclinical in vivo hypoxia PET studies, oxygen should be avoided, and ketamine/xylazine-anesthesia might alleviate the identification of tumor hypoxia areals.     

Rapamycin Inhibition of Polyposis and Progression to Dysplasia in a Mouse Model

Familial adenomatous polyposis (FAP) is often due to adenomatous polyposis coli (APC) gene germline mutations. Somatic APC defects are found in about 80% of colorectal cancers (CRCs) and adenomas. Rapamycin inhibits mammalian target of rapamycin (mTOR) protein, which is often expressed in human adenomas and CRCs. We sought to assess the effects of rapamycin in a mouse polyposis model in which both Apc alleles were conditionally inactivated in colon epithelium. Two days after inactivating Apc, mice were given rapamycin or vehicle in cycles of two weeks on and two weeks off. Polyps were scored endoscopically. Mice were euthanized at time points or when moribund, and tissue analyses were performed. In other studies, mice with demonstrable Apc-defective colon polyps were given rapamycin, followed by analysis of their colon tissues. The median survival of mice receiving rapamycin treatment cycles was 21.5 versus 6.5 weeks in control mice (p = 0.03), and rapamycin-treated mice had a significantly lower percentage of their colon covered with polyps (4.3+/− 2 vs 56.5+/− 10.8 percent, p = 0.001). Mice with Apc-deficient colon tissues that developed high grade dysplasia treated with rapamycin underwent treatment for significantly longer than mice treated with vehicle (15.8 vs 5.1 weeks, p = 0.003). In Apc-defective colon tissues, rapamycin treatment was linked to decreased levels of β-catenin and Sox9 at 7 weeks. Other effects of rapamycin in Apc-defectivecolon tissues included decreased proliferation and increased numbers of differentiated goblet cells at 7 weeks. Rapamycin did not affect β-catenin-regulated gene expression in cultured intestinal epithelial cells. Rapamycin has potent inhibitory effects in a mouse colon polyposis model, and mTOR inhibition is linked to decreased proliferation and increased expression of differentiation markers in Apc-mutant colon epithelium and delays development of dysplasia. Our findings highlight the possibility that mTOR inhibitors may have relevance for polyposis inhibition approaches in FAP patients.     

Betamethasone inhibits tumor development, microvessel density and prolongs survival in mice with a multiresistant adenocarcinoma TA3

Tumor resistance to traditional cancer treatments poses an important challenge to modern science. Thus, angiogenesis inhibition is an important emerging cancer treatment. Many drugs are tested and corticosteroids have shown interesting results. Herein we investigate the effect on microvessel density, survival time and tumoral volume of mice with TA3-MTX-R tumors. Twenty six mice were inoculated with l x l0? tumor cells; 4-5 days after injection, six mice were injected with PBS (group A) and twenty mice were treated with β-met (group B). All animals from Group A died on day 22. Group B was divided into Bl (treated discontinued) and B2 (treated daily) and observed until day 88. All mice were processed for histo-immunohistochemical analysis and the blood vessels were counted. A decrease in microvessel density and tumoral volume and longer survival times were observed in the treated group. We propose that the antiangiogenic β-met effect explains, at least partially, its tumor inhibitory properties. As an important perspective, we will experimentally combine these strategies with those recently described by us with regard to the important antiangiogenic-antitumor effects of Trypanosoma cruzi calreticulin. Since the molecular targets of these strategies are most likely different, additive or synergic effects are envisaged.     

Assessment of an scFv Antibody Fragment Against ELTD1 in a G55 Glioblastoma Xenograft Model

Glioblastoma (GBM), the most common primary brain tumor found in adults, is extremely aggressive. These high-grade gliomas, which are very diffuse, highly vascular, and invasive, undergo unregulated vascular angiogenesis. Despite available treatments, the median survival for patients is dismal. ELTD1 (EGF, latrophilin, and 7 transmembrane domain containing protein 1) is an angiogenic biomarker highly expressed in human high-grade gliomas. Recent studies have demonstrated that the blood-brain barrier, as well as the blood-tumor barrier, is not equally disrupted in GBM patients. This study therefore aimed to optimize an antibody treatment against ELTD1 using a smaller scFv fragment of a monoclonal antibody that binds against the external region of ELTD1 in a G55 glioma xenograft glioma preclinical model. Morphological magnetic resonance imaging (MRI) was used to determine tumor volumes and quantify perfusion rates. We also assessed percent survival following tumor postdetection. Tumor tissue was also assessed to confirm and quantify the presence of the ELTD1 scFv molecular targeted MRI probe, as well as microvessel density and Notch1 levels. In addition, we used molecular-targeted MRI to localize our antibodies in vivo. This approach showed that our scFv antibody attached-molecular MRI probe was effective in targeting and localizing diffuse tumor regions. Through this analysis, we determined that our anti-ELTD1 scFv antibody treatments were successful in increasing survival, decreasing tumor volumes, and normalizing vascular perfusion and Notch1 levels within tumor regions. This study demonstrates that our scFv fragment antibody against ELTD1 may be useful and potential antiangiogenic treatments against GBM.     

Recombinant human endostatin normalizes tumor vasculature and enhances radiation response in xenografted human nasopharyngeal carcinoma models

Background

Hypoxic tumor cells can reduce the efficacy of radiation. Antiangiogenic therapy may transiently “normalize” the tumor vasculature to make it more efficient for oxygen delivery. The aim of this study is to investigate whether the recombinant human endostatin (endostar) can create a “vascular normalization window” to alleviate hypoxia and enhance the inhibitory effects of radiation therapy in human nasopharyngeal carcinoma (NPC) in mice.

Methodology/Principal Findings

Transient changes in morphology of tumor vasculature and hypoxic tumor cell fraction in response to endostar were detected in mice bearing CNE-2 and 5–8F human NPC xenografts. Various treatment schedules were tested to assess the influence of endostar on the effect of radiation therapy. Several important factors relevant to the angiogenesis were identified through immunohistochemical staining. During endostar treatment, tumor vascularity decreased, while the basement membrane and pericyte coverage associated with endothelial cells increased, which supported the idea of vessel normalization. Hypoxic tumor cell fraction also decreased after the treatment. The transient modulation of tumor physiology caused by endostar improved the effect of radiation treatment compared with other treatment schedules. The expressions of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2), MMP-9, and MMP-14 decreased, while the level of pigment epithelium-derived factor (PEDF) increased.

Conclusions

Endostar normalized tumor vasculature, which alleviated hypoxia and significantly sensitized the function of radiation in anti-tumor in human NPC. The results provide an important experimental basis for combining endostar with radiation therapy in human NPC.     

Antitumor activity of rapamycin in a Phase I trial for patients with recurrent PTEN-deficient glioblastoma

Background

There is much discussion in the cancer drug development community about how to incorporate molecular tools into early-stage clinical trials to assess target modulation, measure anti-tumor activity, and enrich the clinical trial population for patients who are more likely to benefit. Small, molecularly focused clinical studies offer the promise of the early definition of optimal biologic dose and patient population.

Methods and Findings

Based on preclinical evidence that phosphatase and tensin homolog deleted on Chromosome 10 (PTEN) loss sensitizes tumors to the inhibition of mammalian target of rapamycin (mTOR), we conducted a proof-of-concept Phase I neoadjuvant trial of rapamycin in patients with recurrent glioblastoma, whose tumors lacked expression of the tumor suppressor PTEN. We aimed to assess the safety profile of daily rapamycin in patients with glioma, define the dose of rapamycin required for mTOR inhibition in tumor tissue, and evaluate the antiproliferative activity of rapamycin in PTEN-deficient glioblastoma. Although intratumoral rapamycin concentrations that were sufficient to inhibit mTOR in vitro were achieved in all patients, the magnitude of mTOR inhibition in tumor cells (measured by reduced ribosomal S6 protein phosphorylation) varied substantially. Tumor cell proliferation (measured by Ki-67 staining) was dramatically reduced in seven of 14 patients after 1 wk of rapamycin treatment and was associated with the magnitude of mTOR inhibition (p = 0.0047, Fisher exact test) but not the intratumoral rapamycin concentration. Tumor cells harvested from the Ki-67 nonresponders retained sensitivity to rapamycin ex vivo, indicating that clinical resistance to biochemical mTOR inhibition was not cell-intrinsic. Rapamycin treatment led to Akt activation in seven patients, presumably due to loss of negative feedback, and this activation was associated with shorter time-to-progression during post-surgical maintenance rapamycin therapy (p < 0.05, Logrank test).

Conclusions

Rapamycin has anticancer activity in PTEN-deficient glioblastoma and warrants further clinical study alone or in combination with PI3K pathway inhibitors. The short-term treatment endpoints used in this neoadjuvant trial design identified the importance of monitoring target inhibition and negative feedback to guide future clinical development.Trial registration: http://www.ClinicalTrials.gov (#{"type":"clinical-trial","attrs":{"text":"NCT00047073","term_id":"NCT00047073"}}NCT00047073).     

Cellular characterization of ultrasound-stimulated microbubble radiation enhancement in a prostate cancer xenograft model

Tumor radiation resistance poses a major obstacle in achieving an optimal outcome in radiation therapy. In the current study, we characterize a novel therapeutic approach that combines ultrasound-driven microbubbles with radiation to increase treatment responses in a prostate cancer xenograft model in mice. Tumor response to ultrasound-driven microbubbles and radiation was assessed 24 hours after treatment, which consisted of radiation treatments alone (2 Gy or 8 Gy) or ultrasound-stimulated microbubbles only, or a combination of radiation and ultrasound-stimulated microbubbles. Immunohistochemical analysis using in situ end labeling (ISEL) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) revealed increased cell death within tumors exposed to combined treatments compared with untreated tumors or tumors exposed to radiation alone. Several biomarkers were investigated to evaluate cell proliferation (Ki67), blood leakage (factor VIII), angiogenesis (cluster of differentiation molecule CD31), ceramide-formation, angiogenesis signaling [vascular endothelial growth factor (VEGF)], oxygen limitation (prolyl hydroxylase PHD2) and DNA damage/repair (γH2AX). Results demonstrated reduced vascularity due to vascular disruption by ultrasound-stimulated microbubbles, increased ceramide production and increased DNA damage of tumor cells, despite decreased tumor oxygenation with significantly less proliferating cells in the combined treatments. This combined approach could be a feasible option as a novel enhancing approach in radiation therapy.KEY WORDS: Angiogenesis, Microbubbles, Proliferation, Radiation, Ultrasound     

Noninvasive Monitoring of Small Intestinal Oxygen in a Rat Model of Chronic Mesenteric Ischemia

We noninvasively monitored the partial pressure of oxygen (pO₂) in rat’s small intestine using a model of chronic mesenteric ischemia by electron paramagnetic resonance oximetry over a 7-day period. The particulate probe lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) was embedded into the oxygen permeable material polydimethyl siloxane by cast-molding and polymerization (Oxy-Chip). A one-time surgical procedure was performed to place the Oxy-Chip on the outer wall of the small intestine (SI). The superior mesenteric artery (SMA) was banded to ~30 % of blood flow for experimental rats. Noninvasive measurement of pO₂ was performed at the baseline for control rats or immediate post-banding and on days 1, 3, and 7. The SI pO₂ for control rats remained stable over the 7-day period. The pO₂ on day-7 was 54.5 ± 0.9 mmHg (mean ± SE). SMA-banded rats were significantly different from controls with a noted reduction in pO₂ post banding with a progressive decline to a final pO₂ of 20.9 ± 4.5 mmHg (mean ± SE; p = 0.02). All SMA-banded rats developed adhesions around the Oxy-Chip, yet remained asymptomatic. The hypoxia marker Hypoxyprobe? was used to validate the low tissue pO₂. Brown cytoplasmic staining was consistent with hypoxia. Mild brown staining was noted predominantly on the villus tips in control animals. SMA-banded rats had an extended region of hypoxic involvement in the villus with a higher intensity of cytoplasmic staining. Deep brown stainings of the enteric nervous system neurons and connective tissue both within layers and in the mesentery were noted. SMA-banded rats with lower pO₂ values had a higher intensity of staining. Thus, monitoring SI pO₂ using the probe Oxy-Chip provides a valid measure of tissue oxygenation. Tracking pO₂ in conditions that produce chronic mesenteric ischemia will contribute to our understanding of intestinal tissue oxygenation and how changes impact symptom evolution and the trajectory of chronic disease.     

COX-2 Inhibition Combined with Radiation Reduces Orthotopic Glioma Outgrowth by Targeting the Tumor Vasculature

Cyclooxygenase 2 (COX-2) inhibitors have been shown to enhance tumor''s response to radiation in several animal models. The strong association of COX-2 and angiogenesis suggests that the tumor vasculature may be involved in this process. The current study investigated whether treatment with the COX-2 inhibitor E-6087 could influence response to local radiation in orthotopically growing murine gliomas and aimed to analyze the involvement of the tumor vasculature. GL261 glioma cells were injected into the cerebrum of C57bl/6 mice. From day 7 after tumor cell injection, mice were treated with COX-2 inhibitor at 50 mg/kg i.p. every third day. Radiation consisted of three fractions of 2 Gy given daily from day 9 to day 11. Mice were killed at day 21. The COX-2 inhibitor significantly enhanced the response to radiation, reducing mean volume to 32% of tumors treated with radiation only. The combination treatment neither increased apoptosis of tumor cells or stromal cells nor affected tumor microvascular density. In vitro, E-6087 and its active metabolite did not affect clonogenic survival of GL261 cells or human umbilical vein endothelial cell after radiation. In vivo, however, there was a nonsignificant increase in Angiopoietin (Ang)-1 and Tie-2 mRNA levels and a decrease of Ang-2 mRNA levels after combination treatment. These changes coincided with a significant increase in α-smooth muscle actin-positive pericyte coverage of tumor vessels. In conclusion, the antitumor effect of radiation on murine intracranial glioma growth is augmented by combining with COX-2 inhibition. Our findings suggest an involvement of the tumor vasculature in the observed effects.     

The proangiogenic factor ephrin-A1 is up-regulated in radioresistant murine tumor by irradiation

While the pre-treatment status of cancer is generally correlated with outcome, little is known about microenvironmental change caused by anti-cancer treatment and how it may affect outcome. For example, treatment may lead to induction of gene expression that promotes resistance to therapy. In the present study, we attempted to find a gene that was both induced by irradiation and associated with radioresistance in tumors. Using single-color oligo-microarrays, we analyzed the gene expression profiles of two murine squamous cell carcinomas, NR-S1, which is highly radioresistant, and SCCVII, which is radiosensitive, after irradiation with 137-Cs gamma rays or carbon ions. Candidate genes were those differentially regulated between NR-S1 and SCCVII after any kind of irradiation. Four genes, Efna1 (Ephrin-A1), Sprr1a (small proline-rich protein 1A), Srgap3 (SLIT-ROBO Rho GTPase activating protein 3) and Xrra1 [RIKEN 2 days neonate thymus thymic cells (NOD) cDNA clone E430023D08 3'], were selected as candidate genes associated with radiotherapy-induced radioresistance. We focused on Efna1, which encodes a ligand for the Eph receptor tyrosine kinase known to be involved in the vascular endothelial growth factor (VEGF) pathway. We used immunohistochemical methods to detect expression of Ephrin-A1, VEGF, and the microvascular marker CD31 in radioresistant NR-S1 tumor cells. Ephrin-A1 was detected in the cytoplasm of NR-S1 tumor cells after irradiation, but not in SCCVII tumor cells. Irradiation of NR-S1 tumor cells also led to significant increases in microvascular density, and up-regulation of VEGF expression. Our results suggest that radiotherapy-induced changes in gene expression related with angiogenesis might also modulate microenvironment and influence responsiveness of tumors.