Tomoko Ozawa

Pharmacologic Inhibition of Cyclin-Dependent Kinases 4 and 6 Arrests the Growth of Glioblastoma Multiforme Intracranial Xenografts

Activation of cyclin-dependent kinases 4 and 6 (cdk4/6) occurs in the majority of glioblastoma multiforme (GBM) tumors, and represents a promising molecular target for the development of small molecule inhibitors. In the current study, we investigated the molecular determinants and in vivo response of diverse GBM cell lines and xenografts to PD-0332991, a cdk4/6-specific inhibitor. In vitro testing of PD-0332991 against a panel of GBM cell lines revealed a potent G(1) cell cycle arrest and induction of senescence in each of 16 retinoblastoma protein (Rb)-proficient cell lines regardless of other genetic lesions, whereas 5 cell lines with homozygous inactivation of Rb were completely resistant to treatment. Short hairpin RNA depletion of Rb expression conferred resistance of GBM cells to PD-0332991, further demonstrating a requirement of Rb for sensitivity to cdk4/6 inhibition. PD-0332991 was found to efficiently cross the blood-brain barrier and proved highly effective in suppressing the growth of intracranial GBM xenograft tumors, including those that had recurred after initial therapy with temozolomide. Remarkably, no mice receiving PD-0332991 died as a result of disease progression while on therapy. Additionally, the combination of PD-0332991 and radiation therapy resulted in significantly increased survival benefit compared with either therapy alone. In total, our results support clinical trial evaluation of PD-0332991 against newly diagnosed as well as recurrent GBM, and indicate that Rb status is the primary determinant of potential benefit from this therapy.

Hyperpolarized 13C magnetic resonance metabolic imaging: application to brain tumors

In order to compare in vivo metabolism between malignant gliomas and normal brain, (13)C magnetic resonance (MR) spectroscopic imaging data were acquired from rats with human glioblastoma xenografts (U-251 MG and U-87 MG) and normal rats, following injection of hyperpolarized [1-(13)C]-pyruvate. The median signal-to-noise ratio (SNR) of lactate, pyruvate, and total observed carbon-13 resonances, as well as their relative ratios, were calculated from voxels containing Gadolinium-enhanced tissue in T(1) postcontrast images for rats with tumors and from normal brain tissue for control rats. [1-(13)C]-labeled pyruvate and its metabolic product, [1-(13)C]-lactate, demonstrated significantly higher SNR in the tumor compared with normal brain tissue. Statistical tests showed significant differences in all parameters (P < .0004) between the malignant glioma tissue and normal brain. The SNR of lactate, pyruvate, and total carbon was observed to be different between the U-251 MG and U-87 MG models, which is consistent with inherent differences in the molecular characteristics of these tumors. These results suggest that hyperpolarized MR metabolic imaging may be valuable for assessing prognosis and monitoring response to therapy for patients with brain tumors.

Inhibition of PI3K/mTOR pathways in glioblastoma and implications for combination therapy with temozolomide.

Due to its molecular heterogeneity and infiltrative nature, glioblastoma multiforme (GBM) is notoriously resistant to traditional and experimental therapeutics. To overcome these hurdles, targeted agents have been combined with conventional therapy. We evaluated the preclinical potential of a novel, orally bioavailable PI3K/mTOR dual inhibitor (XL765) in in vitro and in vivo studies. In vivo serially passaged human GBM xenografts that are more genetically stable than GBM cell lines in culture were used for all experiments. Biochemical downstream changes were evaluated by immunoblot and cytotoxicity by colorimetric ATP-based assay. For in vivo experiments, human xenograft GBM 39 grown intracranially in nude mice was altered to express luciferase to monitor tumor burden by optical imaging. XL765 resulted in concentration-dependent decreases in cell viability in vitro. Cytotoxic doses resulted in specific inhibition of PI3K signaling. Combining XL765 with temozolomide (TMZ) resulted in additive toxicity in 4 of 5 xenografts. In vivo, XL765 administered by oral gavage resulted in greater than 12-fold reduction in median tumor bioluminescence compared with control (Mann-Whitney test p = 0.001) and improvement in median survival (logrank p = 0.05). TMZ alone showed a 30-fold decrease in median bioluminescence, but the combination XL765 + TMZ yielded a 140-fold reduction in median bioluminescence (Mann-Whitney test p = 0.05) with a trend toward improvement in median survival (logrank p = 0.09) compared with TMZ alone. XL765 shows activity as monotherapy and in combination with conventional therapeutics in a range of genetically diverse GBM xenografts.

Frequent truncating mutations of STAG2 in bladder cancer

Here we report the discovery of truncating mutations of the gene encoding the cohesin subunit STAG2, which regulates sister chromatid cohesion and segregation, in 36% of papillary non-invasive urothelial carcinomas and 16% of invasive urothelial carcinomas of the bladder. Our studies suggest that STAG2 has a role in controlling chromosome number but not the proliferation of bladder cancer cells. These findings identify STAG2 as one of the most commonly mutated genes in bladder cancer.

Detection of early response to temozolomide treatment in brain tumors using hyperpolarized 13C MR metabolic imaging

To demonstrate the feasibility of using DNP hyperpolarized [1‐13C]‐pyruvate to measure early response to temozolomide (TMZ) therapy using an orthotopic human glioblastoma xenograft model.

Hyperpolarized 13C MR spectroscopic imaging can be used to monitor Everolimus treatment in vivo in an orthotopic rodent model of glioblastoma.

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor in humans. Because the phosphatidylinositol-3-kinase (PI3K) signaling pathway is activated in more than 88% of GBM, new drugs which target this pathway, such as the mTOR inhibitor Everolimus, are currently in clinical trials. Early tumor response to molecularly targeted treatments remains challenging to assess non-invasively, because it is often associated with tumor stasis or slower tumor growth. Innovative neuroimaging methods are therefore critically needed to provide metabolic or functional information that is indicative of targeted therapeutic action at early time points during the course of treatment. In this study, we demonstrated for the first time that hyperpolarized (HP) 13C magnetic resonance spectroscopic imaging (MRSI) can be used on a clinical MR system to monitor early metabolic response of orthotopic GBM tumors to Everolimus treatment through measurement of the HP lactate-to-pyruvate ratios. The study was performed on a highly invasive non-enhancing orthotopic GBM tumor model in rats (GS-2 tumors), which replicates many fundamental features of human GBM tumors. Seven days after initiation of treatment there was a significant drop in the HP lactate-to-pyruvate ratio from the tumor tissue in treated animals relative to day 0 (67%±27% decrease). In the control group, no significant changes in the HP lactate-to-pyruvate ratios were observed. Importantly, at the 7 day time point, conventional MR imaging (MRI) was unable to detect a significant difference in tumor size between control and treated groups. Inhibition of tumor growth by conventional MRI was observed from day 15 of treatment. This implies that the decrease in the HP lactate-to-pyruvate ratio could be detected before any treatment-induced inhibition of tumor growth. Using immunohistochemical staining to further examine tumor response to treatment, we found that the decrease in the HP lactate-to-pyruvate ratio was associated with a drop in expression of lactate dehydrogenase, the enzyme that catalyzes pyruvate to lactate conversion. Also evident was decreased staining for carbonic anhydrase IX (CA-IX), an indicator of hypoxia-inducible factor 1α (HIF-1α) activity, which, in turn, regulates expression of lactate dehydrogenase. To our knowledge, this study is the first report of the use of HP 13C MRSI at a clinical field strength to monitor GBM response to molecularly targeted treatments. It highlights the potential of HP lactate-to-pyruvate ratio as an early biomarker of response, thereby supporting further investigation of this non-invasive imaging approach for eventual clinical application.

Establishing intracranial brain tumor xenografts with subsequent analysis of tumor growth and response to therapy using bioluminescence imaging.

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most commonly used procedure for human tumor xenograft establishment consisted of the collection of cells from culture flasks, followed by the subcutaneous injection of the collected cells in immunocompromised mice. Whereas this approach still sees frequent use in many laboratories, there has been a significant shift in emphasis over the past decade towards orthotopic xenograft establishment, which, in the instance of brain tumors, requires tumor cell injection into appropriate neuroanatomical structures. Because intracranial xenograft establishment eliminates the ability to monitor tumor growth through direct measurement, such as by use of calipers, the shift in emphasis towards orthotopic brain tumor xenograft models has necessitated the utilization of non-invasive imaging for assessing tumor burden in host animals. Of the currently available imaging methods, bioluminescence monitoring is generally considered to offer the best combination of sensitivity, expediency, and cost. Here, we will demonstrate procedures for orthotopic brain tumor establishment, and for monitoring tumor growth and response to treatment when testing experimental therapies.

Orthotopic Growth of Human Glioma Cells Quantitatively and Qualitatively Influences Radiation-Induced Changes in Gene Expression

The effect of radiation on gene expression has been most frequently studied using tissue culture models. To determine the influence of experimental growth condition on radiation-induced changes in gene expression, microarray analysis was done on two human glioma cell lines (U87 and U251) grown in tissue culture and as s.c. or i.c. xenografts. Compared with tissue culture, the number of genes, whose expression was affected by radiation in both cell lines, was increased in the s.c. xenografts and further increased in the orthotopic tumors. Furthermore, in each growth condition, radiation modulated the expression of a different set of genes. In addition, whereas there were few commonly affected genes after irradiation of U87 and U251 in tissue culture, there were 729 common changes after orthotopic irradiation. These results indicate that the influence of the orthotopic environment on radiation-induced modulation of gene expression in glioma cells was both quantitative and qualitative. Moreover, they suggest that investigations of the functional consequence of radiation-induced gene expression will require accounting for experimental growth conditions.

Comparing routes of delivery for nanoliposomal irinotecan shows superior anti-tumor activity of local administration in treating intracranial glioblastoma xenografts

BACKGROUND Liposomal drug packaging is well established as an effective means for increasing drug half-life, sustaining drug activity, and increasing drug efficacy, whether administered locally or distally to the site of disease. However, information regarding the relative effectiveness of peripheral (distal) versus local administration of liposomal therapeutics is limited. This issue is of importance with respect to the treatment of central nervous system cancer, for which the blood-brain barrier presents a significant challenge in achieving sufficient drug concentration in tumors to provide treatment benefit for patients. METHODS We compared the anti-tumor activity and efficacy of a nanoliposomal formulation of irinotecan when delivered peripherally by vascular route with intratumoral administration by convection-enhanced delivery (CED) for treating intracranial glioblastoma xenografts in athymic mice. RESULTS Our results show significantly greater anti-tumor activity and survival benefit from CED of nanoliposomal irinotecan. In 2 of 3 efficacy experiments, there were animal subjects that experienced apparent cure of tumor from local administration of therapy, as indicated by a lack of detectable intracranial tumor through bioluminescence imaging and histopathologic analysis. Results from investigating the effectiveness of combination therapy with nanoliposomal irinotecan plus radiation revealed that CED administration of irinotecan plus radiation conferred greater survival benefit than did irinotecan or radiation monotherapy and also when compared with radiation plus vascularly administered irinotecan. CONCLUSIONS Our results indicate that liposomal formulation plus direct intratumoral administration of therapeutic are important for maximizing the anti-tumor effects of irinotecan and support clinical trial evaluation of this therapeutic plus route of administration combination.

Bromophenol blue staining of tumors in a rat glioma model.

OBJECTIVE:For patients with gliomas, decreasing the tumor burden with macroscopic surgical resection may affect quality of life, time to tumor progression, and survival. Injection of bromophenol blue (BPB) may enhance intraoperative visualization of an infiltrating tumor and its margins and improve the extent of resection. In this study, we investigated the uptake of BPB in experimental rat brain tumors. METHODS:We first conducted a toxicity study with bolus intravenous injections of 5, 60, and 360 mg/kg doses of BPB in nontumor-bearing Fischer 344 rats. No adverse effects were observed in any of the animals during the 60 day observation period. We then injected 9L tumor cells intracerebrally into Fischer 344 rats and approximately 2 weeks later, administered a bolus intravenous injection of 5 to 360 mg/kg BPB. Fifteen minutes after BPB injection, we sacrificed the animals and removed their brains. In a subsequent study, we injected 180 mg/kg BPB and sacrificed animals at several time points to monitor tumor staining over time. RESULTS:The stain was clearly visible and localized to the tumor for all BPB concentrations 60 mg/kg or greater, and in an additional experiment, we found that tumor staining persisted for at least 8 hours after BPB injection. CONCLUSION:We conclude that BPB helped visualize experimental tumors at time points from a few minutes to several hours after injection. Because BPB also proved to be nontoxic to the animals at effective concentrations, we believe the compound may be potentially useful in helping neurosurgeons visualize brain tumors in humans.