Anuradha Soundararajan

Chemoradionuclide Therapy with 186Re-labeled Liposomal Doxorubicin in Combination with Radiofrequency Ablation for Effective Treatment of Head and Neck Cancer in a Nude Rat Tumor Xenograft Model

PURPOSE To determine the therapeutic efficacy of rhenium 186 ((186)Re)-labeled PEGylated liposomal doxorubicin ((186)Re-liposomal doxorubicin) in combination with radiofrequency (RF) ablation of human head and neck squamous cell carcinoma (HNSCC) xenograft in nude rats. MATERIALS AND METHODS This investigation was approved by the animal care committee. Sixty nude rats with subcutaneously implanted HNSCC xenografts (six per group) were treated with (a) RF ablation (70 °C for 5 minutes), (b) PEGylated liposomes, (c) liposomal doxorubicin, (d) (186)Re-PEGylated liposomes (1295 MBq/kg), (e) (186)Re-liposomal doxorubicin (555 MBq/kg), (f) PEGylated liposomes plus RF ablation, (g) liposomal doxorubicin plus RF ablation, (h) (186)Re-PEGylated liposomes plus RF ablation, or (i) (186)Re-liposomal doxorubicin plus RF ablation. Six rats did not receive any treatment (control group). Tumor uptake in (186)Re therapy groups was monitored with small-animal single photon emission computed tomography for 5 days. Therapeutic efficacy was monitored for 6 weeks with measurement of tumor volume, calculation of the percentage injected dose of fluorine 18 fluorodeoxyglucose (FDG) in tumor from small-animal positron emission tomography (PET) images, and determination of viable tumor volume at histopathologic examination. Significant differences between groups were determined with analysis of variance. RESULTS The average tumor volume (± standard deviation) on the day of therapy was 1.32 cm(3) ± 0.17. At 6 weeks after therapy, control of tumor growth was better with (186)Re-liposomal doxorubicin than with liposomal doxorubicin alone (tumor volume, 2.26 cm(3) ± 0.89 vs 5.43 cm(3) ± 0.93, respectively; P < .01). The use of RF ablation with liposomal doxorubicin and (186)Re-liposomal doxorubicin further improved tumor control (tumor volume, 2.05 cm(3) ± 1.36 and 1.49 cm(3) ± 1.47, respectively). The tumor growth trend correlated with change in percentage of injected dose of FDG in tumor for all groups (R(2) = 0.85, P < .001). Viable tumor volume was significantly decreased in the group treated with (186)Re-liposomal doxorubicin plus RF ablation (0.54 cm(3) ± 0.38; P < .001 vs all groups except (186)Re-liposomal doxorubicin alone). CONCLUSION Triple and dual therapies had an observable trend ((186)Re-liposomal doxorubicin plus RF ablation > (186)Re-liposomal doxorubicin > liposomal doxorubicin plus RF ablation > liposomal doxorubicin) of improved tumor growth control and decreased viable tumor compared with other therapies. FDG PET could be used as a noninvasive surrogate marker for tumor growth and viability in this tumor model.

Combination Radiofrequency Ablation and Intravenous Radiolabeled Liposomal Doxorubicin: Imaging and Quantification of Increased Drug Delivery to Tumors

PURPOSE To identify, with noninvasive imaging, the zone of radiopharmaceutical uptake after combination therapy with radiofrequency (RF) ablation and intravenous administration of technetium 99m ((99m)Tc) liposomal doxorubicin in a small-animal tumor model, and to quantify and correlate the uptake by using imaging and tissue counting of intratumoral doxorubicin accumulation. MATERIALS AND METHODS This study was approved by the animal care committee. Two phases of animal experiments were performed. In the first experiment, a single human head-and-neck squamous cell carcinoma tumor was grown in each of 10 male nude rats. Seven of these animals were treated with intravenous (99m)Tc-liposomal doxorubicin followed by RF tumor ablation at a mean temperature of 70 degrees C + or - 2 for 5 minutes, and three were treated with intravenous (99m)Tc-liposomal doxorubicin only. Combination single photon emission computed tomography-computed tomography (SPECT/CT) was performed at 15 minutes, 4 hours, and 20 hours after therapy. In the second experiment, two tumors each were grown in 11 rats, but only one of the tumors was ablated after intravenous administration of (99m)Tc-liposomal doxorubicin. SPECT/CT and planar scintigraphy were performed at the same posttreatment intervals applied in the first experiment, with additional planar imaging performed at 44 hours. After imaging, tissue counting in the excised tumors was performed. Radiotracer uptake, as determined with imaging and tissue counting, was quantified and compared. In a subset of three animals, intratumoral doxorubicin accumulation was determined with fluorimetry and correlated with the imaging and tissue-counting data. RESULTS At both SPECT/CT and planar scintigraphy, increased uptake of (99m)Tc-liposomal doxorubicin was visibly apparent in the ablated tumors. Results of quantitative analysis with both imaging and tissue counting confirmed significantly greater uptake in the RF ablation-treated tumors (P < .001). Intratumoral doxorubicin accumulation correlated closely with imaging (r = 0.9185-0.9871) and tissue-counting (r = 0.995) results. CONCLUSION Study results show that increased delivery of intravenous liposomal doxorubicin to tumors combined with RF ablation can be depicted and quantified with noninvasive imaging.

Protein kinase C iota as a therapeutic target in alveolar rhabdomyosarcoma

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer exhibiting skeletal-muscle differentiation. New therapeutic targets are required to improve the dismal prognosis for invasive or metastatic alveolar rhabdomyosarcoma. Protein kinase C iota (PKCι) has been shown to have an important role in tumorigenesis of many cancers, but little is known about its role in rhabdomyosarcoma. Our gene-expression studies in human tumor samples revealed overexpression of PRKCI. We confirmed overexpression of PKCι at the mRNA and protein levels using our conditional mouse model that authentically recapitulates the progression of rhabdomyosarcoma in humans. Inhibition of Prkci by RNA interference resulted in a dramatic decrease in anchorage-independent colony formation. Interestingly, treatment of primary cell cultures using aurothiomalate (ATM), which is a gold-containing classical anti-rheumatic agent and a PKCι-specific inhibitor, resulted in decreased interaction between PKCι and Par6, decreased Rac1 activity and reduced cell viability at clinically relevant concentrations. Moreover, co-treatment with ATM and vincristine (VCR), a microtubule inhibitor currently used in rhabdomyosarcoma treatment regimens, resulted in a combination index of 0.470–0.793 through cooperative accumulation of non-proliferative multinuclear cells in the G2/M phase, indicating that these two drugs synergize. For in vivo tumor growth inhibition studies, ATM demonstrated a trend toward enhanced VCR sensitivity. Overall, these results suggest that PKCι is functionally important in alveolar rhabdomyosarcoma anchorage-independent growth and tumor-cell proliferation and that combination therapy with ATM and microtubule inhibitors holds promise for the treatment of alveolar rhabdomyosarcoma.

Decreased in vitro mitochondrial function is associated with enhanced brain metabolism, blood flow, and memory in Surf1-deficient mice

Recent studies have challenged the prevailing view that reduced mitochondrial function and increased oxidative stress are correlated with reduced longevity. Mice carrying a homozygous knockout (KO) of the Surf1 gene showed a significant decrease in mitochondrial electron transport chain Complex IV activity, yet displayed increased lifespan and reduced brain damage after excitotoxic insults. In the present study, we examined brain metabolism, brain hemodynamics, and memory of Surf1 KO mice using in vitro measures of mitochondrial function, in vivo neuroimaging, and behavioral testing. We show that decreased respiration and increased generation of hydrogen peroxide in isolated Surf1 KO brain mitochondria are associated with increased brain glucose metabolism, cerebral blood flow, and lactate levels, and with enhanced memory in Surf1 KO mice. These metabolic and functional changes in Surf1 KO brains were accompanied by higher levels of hypoxia-inducible factor 1 alpha, and by increases in the activated form of cyclic AMP response element-binding factor, which is integral to memory formation. These findings suggest that Surf1 deficiency-induced metabolic alterations may have positive effects on brain function. Exploring the relationship between mitochondrial activity, oxidative stress, and brain function will enhance our understanding of cognitive aging and of age-related neurologic disorders.

Real-Time Iterative Monitoring of Radiofrequency Ablation Tumor Therapy with 15O-Water PET Imaging

A method that provides real-time image-based monitoring of solid tumor therapy to ensure complete tumor eradication during image-guided interventional therapy would be a valuable tool. The short, 2-min half-life of 15O makes it possible to perform repeated PET imaging at 20-min intervals at multiple time points before and after image-guided therapy. In this study, 15O-water PET was evaluated as a tool to provide real-time feedback and iterative image guidance to rapidly monitor the intratumoral coverage of radiofrequency (RF) ablation therapy. Methods: Tumor RF ablation therapy was performed on head and neck squamous cell carcinoma (SCC) xenograft tumors (length, ∼23 mm) in 6 nude rats. The tumor in each animal was ablated with RF (1-cm active size ablation catheter, 70°C for 5 min) twice in 2 separate tumor regions with a 20-min separation. The 15O-water PET images were acquired before RF ablation and after the first RF and second RF ablations using a small-animal PET scanner. In each PET session, approximately 100 MBq of 15O-water in 1.0 mL of saline were injected intravenously into each animal. List-mode PET images were acquired for 7 min starting 20 s before injection. PET images were reconstructed by 2-dimensional ordered-subset expectation maximization into single-frame images and dynamic images at 10 s/frame. PET images were displayed and analyzed with software. Results: Pre–RF ablation images demonstrate that 15O-water accumulates in tumors with 15O activity reaching peak levels immediately after administration. After RF ablation, the ablated region had almost zero activity, whereas the unablated tumor tissue continued to have a high 15O-water accumulation. Using image feedback, the RF probe was repositioned to a tumor region with residual 15O-water uptake and then ablated. The second RF ablation in this new region of the tumor resulted in additional ablation of the solid tumor, with a corresponding decrease in activity on the 15O-water PET image. Conclusion: 15O-water PET clearly demonstrated the ablated tumor region, whereas the unablated tumor continued to show high 15O-water accumulation. 15O-water imaging shows promise as a tool for on-site, real-time monitoring of image-guided interventional cancer therapy.

18F-FDG microPET imaging detects early transient response to an IGF1R inhibitor in genetically engineered rhabdomyosarcoma models

Alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS) are among the most common and most treatment resistant soft tissue sarcomas of childhood. Here, we evaluated the potential of 18F‐Fluorodeoxyglucose (FDG) as a marker of therapeutic response to picropodophyllin (PPP), an IGF1R inhibitor, in a conditional mouse model of ARMS and a conditional model of ERMS/undifferentiated pleomorphic sarcoma (UPS).

Abstract 1043: Role of protein kinase Cι in alveolar rhabdomyosarcoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer showing skeletal muscle differentiation. New therapeutic targets are required to improve the dismal cure rates for patients with metastatic ARMS. Protein Kinase C iota (PRKCI) has been shown to play an important role in many cancers but little to date is known of this kinases role in rhabdomyosarcoma. Materials and Methods: Human samples were obtained from the Pediatric Cooperative Human Tissue Network (Columbus, OH). Mouse samples were obtained from a Myf6Cre, Pax3: Fkhr, p53 conditional mouse model of ARMS and a Pax7CreER, Ptch1, p53 conditional model of embryonal rhabdomyosarcoma (ERMS). Expression of PKCι in tumors was evaluated by quantitative RT-PCR, western blotting and immunohistochemistry. To elucidate the functional role of PKCι in ARMS, we performed invasion assays, anchorage-independent colony formation assays and in vitro growth inhibition assays using primary cell lines obtained from mouse ARMS tumors, treating either with the PKCι specific inhibitor Aurothiomalate (ATM) or by PKCι RNA interference. Results: Over-expression of PKCι at the mRNA and protein level was confirmed in human and mouse ARMS and ERMS. Immunoblotting confirmed that PKCι and its downstream targets, mitogen-activated protein kinase and nuclear factor-kappa B were expressed and highly activated in primary cell cultures. Inhibition of PKCι by RNA interference resulted in a dramatic decrease in cell viability and colony formation. Treatment of primary cell culture using ATM resulted in decreased cell viability (IC50: 54.3 – 256.9 uM) and colony formation, and increased G2/M blockade. Co-treatment with ATM and vincristine, a microtubule inhibitor currently used in rhabdomyosarcoma treatment regimens, resulted in a Combination Index of 0.572 – 1.017, suggesting cooperativity between these agents. Conclusion: These results suggest that PKCι has an important function in proliferation of ARMS and that the PKCι specific inhibitor ATM shows activity in ARMS at clinically-feasible drug concentrations. Furthermore, these results suggest that combination therapy with vincristine and ATM should be investigated further in ARMS. Ken Kikuchi and Anuradha Soundararajan contributed this presentation equally. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1043. doi:10.1158/1538-7445.AM2011-1043

Abstract 851: Interactions among transcription factors as a potential mechanism for inhibiting castrate-resistant prostate cancer (CRPCa) in transgenic adenocarcinoma of mouse prostate (TRAMP) through regulation of FLIP

Androgens are critical regulators of prostate differentiation and function as well as prostate cancer growth and survival. Prostate tumors initially regress in response to androgen-ablation therapy. However, most cancers eventually relapse with an androgen-depletion-independent phenotype that is often more aggressive than the original androgen-dependent tumor. Studies from our laboratory demonstrated that intervention with 2-ME 2 [2-methoxyestradiol] prevents early stage prostate cancer development and causes regression of established prostate tumors in the transgenic adenocarcinoma of mouse prostate model. Although 2-ME 2 was found to be safe, well tolerated, reduce or stabilize PSA levels when given to hormone refractory prostate cancer patients who had failed other treatments including hormone therapy, it was found to be less bioavailable. Accordingly second generation 2-ME 2 was developed using nanocrystal colloidal dispersion technology and has increased its bioavailability by 5-10 fold (EntreMed, Inc, Rockville, MD). In the current study we tested the ability of this newly formulated 2-ME 2 on the development and progression of castrate-resistant prostate cancer in TRAMP mice. 10-12 week old TRAMP mice were castrated and fed water containing different amounts of 2-ME 2 for 6 weeks. Efficacy was evaluated by magnetic resonance imaging, determining the prostate seminal vesicle complex volume and histological analysis of prostate tumor or tissue. Our results also show that 2-ME 2 inhibited development of CRPCa in these mice. Further molecular analysis using cell lines demonstrated 2-ME 2 treatment significantly inhibited DHT-stimulated cell proliferation in androgen-responsive LNCaP cells (p 2 (p 2 treatment reduced such binding to the FLIP promoter as evidenced by ChIP. 2-ME 2 treatment also modulated the interactions among these transcription factors as evidenced by Re-ChIP assays. Interestingly transcription factor CREB but not Sp1 seems to play a predominant role in androgen regulation of FLIP expression. Our findings suggest that 2-ME 2 affects androgen induced survival of prostate cancer cells through regulation of FLIP involving potential interactions between multiple transcription factors. Supported by ACS RSG-04-169 and NIH CA 135451 (APK). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 851. doi:10.1158/1538-7445.AM2011-851