Showing papers by "Rakesh K. Jain published in 2013"

Challenges and Key Considerations of the Enhanced Permeability and Retention Effect for Nanomedicine Drug Delivery in Oncology

Abstract: Enhanced permeability of the tumor vasculature allows macromolecules to enter the tumor interstitial space, whereas the suppressed lymphatic filtration allows them to stay there. This phenomenon, enhanced permeability and retention (EPR), has been the basis of nanotechnology platforms to deliver drugs to tumors. However, progress in developing effective drugs using this approach has been hampered by heterogeneity of EPR effect in different tumors and limited experimental data from patients on effectiveness of this mechanism as related to enhanced drug accumulation. This report summarizes the workshop discussions on key issues of the EPR effect and major gaps that need to be addressed to effectively advance nanoparticle-based drug delivery.

Compact high-quality CdSe–CdS core–shell nanocrystals with narrow emission linewidths and suppressed blinking

Abstract: High particle uniformity, high photoluminescence quantum yields, narrow and symmetric emission spectral lineshapes and minimal single-dot emission intermittency (known as blinking) have been recognized as universal requirements for the successful use of colloidal quantum dots in nearly all optical applications. However, synthesizing samples that simultaneously meet all these four criteria has proven challenging. Here, we report the synthesis of such high-quality CdSe-CdS core-shell quantum dots in an optimized process that maintains a slow growth rate of the shell through the use of octanethiol and cadmium oleate as precursors. In contrast with previous observations, single-dot blinking is significantly suppressed with only a relatively thin shell. Furthermore, we demonstrate the elimination of the ensemble luminescence photodarkening that is an intrinsic consequence of quantum dot blinking statistical ageing. Furthermore, the small size and high photoluminescence quantum yields of these novel quantum dots render them superior in vivo imaging agents compared with conventional quantum dots. We anticipate these quantum dots will also result in significant improvement in the performance of quantum dots in other applications such as solid-state lighting and illumination.

Normalizing Tumor Microenvironment to Treat Cancer: Bench to Bedside to Biomarkers

Abstract: For almost four decades, my work has focused on one challenge: improving the delivery and efficacy of anticancer therapeutics. Working on the hypothesis that the abnormal tumor microenvironment—characterized by hypoxia and high interstitial fluid pressure—fuels tumor progression and treatment resistance, we developed an array of sophisticated imaging technologies and animal models as well as mathematic models to unravel the complex biology of tumors. Using these tools, we demonstrated that the blood and lymphatic vasculature, fibroblasts, immune cells, and extracellular matrix associated with tumors are abnormal, which together create a hostile tumor microenvironment. We next hypothesized that agents that induce normalization of the microenvironment can improve treatment outcome. Indeed, we demonstrated that judicious use of antiangiogenic agents—originally designed to starve tumors—could transiently normalize tumor vasculature, alleviate hypoxia, increase delivery of drugs and antitumor immune cells, and...

Strategies for advancing cancer nanomedicine.

Abstract: Cancer nanomedicines approved so far minimize toxicity, but their efficacy is often limited by physiological barriers posed by the tumour microenvironment. Here, we discuss how these barriers can be overcome through innovative nanomedicine design and through creative manipulation of the tumour microenvironment.

Angiotensin inhibition enhances drug delivery and potentiates chemotherapy by decompressing tumour blood vessels

Abstract: Cancer and stromal cells actively exert physical forces (solid stress) to compress tumour blood vessels, thus reducing vascular perfusion. Tumour interstitial matrix also contributes to solid stress, with hyaluronan implicated as the primary matrix molecule responsible for vessel compression because of its swelling behaviour. Here we show, unexpectedly, that hyaluronan compresses vessels only in collagen-rich tumours, suggesting that collagen and hyaluronan together are critical targets for decompressing tumour vessels. We demonstrate that the angiotensin inhibitor losartan reduces stromal collagen and hyaluronan production, associated with decreased expression of profibrotic signals TGF-b1, CCN2 and ET-1, downstream of angiotensin-II-receptor-1 inhibition. Consequently, losartan reduces solid stress in tumours resulting in increased vascular perfusion. Through this physical mechanism, losartan improves drug and oxygen delivery to tumours, thereby potentiating chemotherapy and reducing hypoxia in breast and pancreatic cancer models. Thus, angiotensin inhibitors — inexpensive drugs with decades of safe use — could be rapidly repurposed as cancer

Angiotensin inhibition enhances drug delivery and potentiates chemotherapy by decompressing tumour blood vessels

Abstract: Cancer and stromal cells actively exert physical forces (solid stress) to compress tumour blood vessels, thus reducing vascular perfusion. Tumour interstitial matrix also contributes to solid stress, with hyaluronan implicated as the primary matrix molecule responsible for vessel compression because of its swelling behaviour. Here we show, unexpectedly, that hyaluronan compresses vessels only in collagen-rich tumours, suggesting that collagen and hyaluronan together are critical targets for decompressing tumour vessels. We demonstrate that the angiotensin inhibitor losartan reduces stromal collagen and hyaluronan production, associated with decreased expression of profibrotic signals TGF-β1, CCN2 and ET-1, downstream of angiotensin-II-receptor-1 inhibition. Consequently, losartan reduces solid stress in tumours resulting in increased vascular perfusion. Through this physical mechanism, losartan improves drug and oxygen delivery to tumours, thereby potentiating chemotherapy and reducing hypoxia in breast and pancreatic cancer models. Thus, angiotensin inhibitors —inexpensive drugs with decades of safe use — could be rapidly repurposed as cancer therapeutics.

Vascular normalization as an emerging strategy to enhance cancer immunotherapy.

Abstract: The recent approval of Provenge has brought new hope for anticancer vaccine therapies. However, the immunosuppressive tumor microenvironment seems to impair the efficacy of vaccine therapies. The abnormal tumor vasculature creates a hypoxic microenvironment that polarizes inflammatory cells toward immune suppression. Moreover, tumors systemically alter immune cells' proliferation, differentiation, and function via secretion of growth factors and cytokines. For example, VEGF, a major proangiogenic cytokine induced by hypoxia, plays a critical role in immunosuppression via these mechanisms. Hence, antiangiogenic treatment may be an effective modality to potentiate immunotherapy. Here, we discuss the local and systemic effects of VEGF on tumor immunity and propose a potentially translatable strategy to re-engineer the tumor-immune microenvironment and improve cancer immunotherapy by using lower "vascular normalizing" doses of antiangiogenic agents.

Phase III Randomized Trial Comparing the Efficacy of Cediranib As Monotherapy, and in Combination With Lomustine, Versus Lomustine Alone in Patients With Recurrent Glioblastoma

Abstract: Purpose A randomized, phase III, placebo-controlled, partially blinded clinical trial (REGAL [Recentin in Glioblastoma Alone and With Lomustine]) was conducted to determine the efficacy of cediranib, an oral pan–vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor, either as monotherapy or in combination with lomustine versus lomustine in patients with recurrent glioblastoma. Patients and Methods Patients (N = 325) with recurrent glioblastoma who previously received radiation and temozolomide were randomly assigned 2:2:1 to receive (1) cediranib (30 mg) monotherapy; (2) cediranib (20 mg) plus lomustine (110 mg/m2); (3) lomustine (110 mg/m2) plus a placebo. The primary end point was progression-free survival based on blinded, independent radiographic assessment of postcontrast T1-weighted and noncontrast T2-weighted magnetic resonance imaging (MRI) brain scans. Results The primary end point of progression-free survival (PFS) was not significantly different for either cediranib alone...

Combining two strategies to improve perfusion and drug delivery in solid tumors

Abstract: Blood perfusion in tumors can be significantly lower than that in the surrounding normal tissue owing to the leakiness and/or compression of tumor blood vessels. Impaired perfusion reduces oxygen supply and results in a hypoxic microenvironment. Hypoxia promotes tumor progression and immunosuppression, and enhances the invasive and metastatic potential of cancer cells. Furthermore, poor perfusion lowers the delivery of systemically administered drugs. Therapeutic strategies to improve perfusion include reduction in vascular permeability by vascular normalization and vascular decompression by alleviating physical forces (solid stress) inside tumors. Both strategies have shown promise, but guidelines on how to use these strategies optimally are lacking. To this end, we developed a mathematical model to guide the optimal use of these strategies. The model accounts for vascular, transvascular, and interstitial fluid and drug transport as well as the diameter and permeability of tumor vessels. Model simulations reveal an optimal perfusion region when vessels are uncompressed, but not very leaky. Within this region, intratumoral distribution of drugs is optimized, particularly for drugs 10 nm in diameter or smaller and of low binding affinity. Therefore, treatments should modify vessel diameter and/or permeability such that perfusion is optimal. Vascular normalization is more effective for hyperpermeable but largely uncompressed vessels (e.g., glioblastomas), whereas solid stress alleviation is more beneficial for compressed but less-permeable vessels (e.g., pancreatic ductal adenocarcinomas). In the case of tumors with hyperpermeable and compressed vessels (e.g., subset of mammary carcinomas), the two strategies need to be combined for improved treatment outcomes.

Coevolution of Solid Stress and Interstitial Fluid Pressure in Tumors During Progression: Implications for Vascular Collapse

Abstract: The stress harbored by the solid phase of tumors is known as solid stress. Solid stress can be either applied externally by the surrounding normal tissue or induced by the tumor itself due to its growth. Fluid pressure is the isotropic stress exerted by the fluid phase. We recently showed that growth-induced solid stress is on the order of 1.3 to 13.0 kPa (10-100 mmHg)--high enough to cause compression of fragile blood vessels, resulting in poor perfusion and hypoxia. However, the evolution of growth-induced stress with tumor progression and its effect on cancer cell proliferation in vivo is not understood. To this end, we developed a mathematical model for tumor growth that takes into account all three types of stresses: growth-induced stress, externally applied stress, and fluid pressure. First, we conducted in vivo experiments and found that growth-induced stress is related to tumor volume through a biexponential relationship. Then, we incorporated this information into our mathematical model and showed that due to the evolution of growth-induced stress, total solid stress levels are higher in the tumor interior and lower in the periphery. Elevated compressive solid stress in the interior of the tumor is sufficient to cause the collapse of blood vessels and results in a lower growth rate of cancer cells compared with the periphery, independently from that caused by the lack of nutrients due to vessel collapse. Furthermore, solid stress in the periphery of the tumor causes blood vessels in the surrounding normal tissue to deform to elliptical shapes. We present histologic sections of human cancers that show such vessel deformations. Finally, we found that fluid pressure increases with tumor growth due to increased vascular permeability and lymphatic impairment, and is governed by the microvascular pressure. Crucially, fluid pressure does not cause vessel compression of tumor vessels.

Improved tumor oxygenation and survival in glioblastoma patients who show increased blood perfusion after cediranib and chemoradiation

Abstract: Antiangiogenic therapy has shown clear activity and improved survival benefit for certain tumor types. However, an incomplete understanding of the mechanisms of action of antiangiogenic agents has hindered optimization and broader application of this new therapeutic modality. In particular, the impact of antiangiogenic therapy on tumor blood flow and oxygenation status (i.e., the role of vessel pruning versus normalization) remains controversial. This controversy has become critical as multiple phase III trials of anti-VEGF agents combined with cytotoxics failed to show overall survival benefit in newly diagnosed glioblastoma (nGBM) patients and several other cancers. Here, we shed light on mechanisms of nGBM response to cediranib, a pan-VEGF receptor tyrosine kinase inhibitor, using MRI techniques and blood biomarkers in prospective phase II clinical trials of cediranib with chemoradiation vs. chemoradiation alone in nGBM patients. We demonstrate that improved perfusion occurs only in a subset of patients in cediranib-containing regimens, and is associated with improved overall survival in these nGBM patients. Moreover, an increase in perfusion is associated with improved tumor oxygenation status as well as with pharmacodynamic biomarkers, such as changes in plasma placenta growth factor and sVEGFR2. Finally, treatment resistance was associated with elevated plasma IL-8 and sVEGFR1 posttherapy. In conclusion, tumor perfusion changes after antiangiogenic therapy may distinguish responders vs. nonresponders early in the course of this expensive and potentially toxic form of therapy, and these results may provide new insight into the selection of glioblastoma patients most likely to benefit from anti-VEGF treatments.

Vessel architectural imaging identifies cancer patient responders to anti-angiogenic therapy.

Abstract: Measurement of vessel caliber by magnetic resonance imaging (MRI) is a valuable technique for in vivo monitoring of hemodynamic status and vascular development, especially in the brain. Here, we introduce a new paradigm in MRI termed vessel architectural imaging (VAI) that exploits an overlooked temporal shift in the magnetic resonance signal, forming the basis for vessel caliber estimation, and show how this phenomenon can reveal new information on vessel type and function not assessed by any other noninvasive imaging technique. We also show how this biomarker can provide new biological insights into the treatment of patients with cancer. As an example, we demonstrate using VAI that anti-angiogenic therapy can improve microcirculation and oxygen saturation and reduce vessel calibers in patients with recurrent glioblastomas and, more crucially, that patients with these responses have prolonged survival. Thus, VAI has the potential to identify patients who would benefit from therapies.

Increase in tumor-associated macrophages after antiangiogenic therapy is associated with poor survival among patients with recurrent glioblastoma.

Abstract: Antiangiogenic therapy is associated with increased radiographic responses in glioblastomas, but tumors invariably recur. Because tumor-associated macrophages have been shown to mediate escape from antiangiogenic therapy in preclinical models, we examined the role of macrophages in patients with recurrent glioblastoma. We compared autopsy brain specimens from 20 patients with recurrent glioblastoma who received antiangiogenic treatment and chemoradiation with 8 patients who received chemotherapy and/or radiotherapy without antiangiogenic therapy or no treatment. Tumor-associated macrophages were morphologically and phenotypically analyzed using flow cytometry and immunohistochemistry for CD68, CD14, CD163, and CD11b expression. Flow cytometry showed an increase in macrophages in the antiangiogenic-treated patients. Immunohistochemical analysis demonstrated an increase in CD68+ macrophages in the tumor bulk (P < .01) and infiltrative areas (P = .02) in antiangiogenic-treated patients. We also observed an increase in CD11b+ cells in the tumor bulk (P < .01) and an increase in CD163+ macrophages in infiltrative tumor (P = .02). Of note, an increased number of CD11b+ cells in bulk and infiltrative tumors (P = .05 and P = .05, respectively) correlated with poor overall survival among patients who first received antiangiogenic therapy at recurrence. In summary, recurrent glioblastomas showed an increased infiltration in myeloid populations in the tumor bulk and in the infiltrative regions after antiangiogenic therapy. Higher numbers of CD11b+ cells correlated with poor survival among these patients. These data suggest that tumor-associated macrophages may participate in escape from antiangiogenic therapy and may represent a potential biomarker of resistance and a potential therapeutic target in recurrent glioblastoma.

A phase II and biomarker study of ramucirumab, a human monoclonal antibody targeting the VEGF receptor-2, as first-line monotherapy in patients with advanced hepatocellular cancer.

Abstract: Purpose: To assess the efficacy and safety of the anti-VEGF receptor-2 (VEGFR-2) antibody ramucirumab as first-line therapy in patients with advanced hepatocellular carcinoma and explore potential circulating biomarkers. Experimental Design: Adults with advanced hepatocellular carcinoma and no prior systemic treatment received ramucirumab 8 mg/kg every two weeks until disease progression or limiting toxicity. The primary endpoint was progression-free survival (PFS); secondary endpoints included objective response rate (ORR) and overall survival (OS). Circulating biomarkers were evaluated before and after ramucirumab treatment in a subset of patients. Results: Forty-two patients received ramucirumab. Median PFS was 4.0 months [95% confidence interval (CI), 2.6–5.7], ORR was 9.5% (95% CI, 2.7–22.6; 4/42 patients had a partial response), and median OS was 12.0 months (95% CI, 6.1–19.7). For patients with Barcelona Clinic Liver Cancer (BCLC) stage C disease, median OS was 4.4 months (95% CI, 0.5–9.0) for patients with Child-Pugh B cirrhosis versus 18.0 months (95% CI, 6.1–23.5) for patients with Child-Pugh A cirrhosis. Treatment-related grade ≥3 toxicities included hypertension (14%), gastrointestinal hemorrhage and infusion-related reactions (7% each), and fatigue (5%). There was one treatment-related death (gastrointestinal hemorrhage). After treatment with ramucirumab, there was an increase in serum VEGF and placental growth factor (PlGF) and a transient decrease in soluble VEGFR-2. Conclusion: Ramucirumab monotherapy may confer anticancer activity in advanced hepatocellular carcinoma with an acceptable safety profile. Exploratory biomarker studies showed changes in circulating VEGF, PlGF, and sVEGFR-2 that are consistent with those seen with other anti-VEGF agents. Clin Cancer Res; 19(23); 6614–23. ©2013 AACR .

Generation of functionally competent and durable engineered blood vessels from human induced pluripotent stem cells

Abstract: Efficient generation of competent vasculogenic cells is a critical challenge of human induced pluripotent stem (hiPS) cell-based regenerative medicine. Biologically relevant systems to assess functionality of the engineered vessels in vivo are equally important for such development. Here, we report a unique approach for the derivation of endothelial precursor cells from hiPS cells using a triple combination of selection markers—CD34, neuropilin 1, and human kinase insert domain-containing receptor—and an efficient 2D culture system for hiPS cell-derived endothelial precursor cell expansion. With these methods, we successfully generated endothelial cells (ECs) from hiPS cells obtained from healthy donors and formed stable functional blood vessels in vivo, lasting for 280 d in mice. In addition, we developed an approach to generate mesenchymal precursor cells (MPCs) from hiPS cells in parallel. Moreover, we successfully generated functional blood vessels in vivo using these ECs and MPCs derived from the same hiPS cell line. These data provide proof of the principle that autologous hiPS cell-derived vascular precursors can be used for in vivo applications, once safety and immunological issues of hiPS-based cellular therapy have been resolved. Additionally, the durability of hiPS-derived blood vessels in vivo demonstrates a potential translation of this approach in long-term vascularization for tissue engineering and treatment of vascular diseases. Of note, we have also successfully generated ECs and MPCs from type 1 diabetic patient-derived hiPS cell lines and use them to generate blood vessels in vivo, which is an important milestone toward clinical translation of this approach.

Cationic Nanoparticles Have Superior Transvascular Flux into Solid Tumors: Insights from a Mathematical Model

Abstract: Despite their great promise, only a few nanoparticle formulations have been approved for clinical use in oncology. The failure of nano-scale drugs to enhance cancer therapy is in large part due to inefficient delivery. To overcome this outstanding problem, a better understanding of how the physical properties (i.e., size, surface chemistry, and shape) of nanoparticles affect their transvascular transport in tumors is required. In this study, we developed a mathematical model for nanoparticle delivery to solid tumors taking into account electrostatic interactions between the particles and the negatively-charged pores of the vessel wall. The model predictions suggest that electrostatic repulsion has a minor effect on the transvascular transport of nanoparticles. On the contrary, electrostatic attraction, caused even by small cationic charges (surface charge density less than 3 × 10−3 C/m2) can lead to a twofold or more increase in the transvascular flux of nanoparticles into the tumor interstitial space. Importantly, for every nanoparticle size, there is a value of charge density above which a steep increase in transvascular transport is predicted. Our model provides important guidelines for the optimal design of nanoparticle formulation for delivery to solid tumors.

A randomized, double-blind, placebo-controlled 6-wk trial of the efficacy and tolerability of 5 mg vortioxetine in adults with major depressive disorder

Abstract: Vortioxetine (Lu AA21004) is a multi-modal antidepressant in clinical development for the treatment of major depressive disorder (MDD). The current study evaluated the efficacy and tolerability of 5 mg vortioxetine compared to placebo after 6 wk of treatment in adults with MDD in an out-patient setting. Adults aged 18-75 yr, with a diagnosis of MDD and a baseline Montgomery-Asberg Depression Rating Scale (MADRS) total score ≥30, were randomized to receive either 5 mg vortioxetine or placebo over 6 wk, followed by a 2-wk medication-free discontinuation period. The primary efficacy measure was change from baseline in Hamilton Rating Scale for Depression (HAMD)-24 total score at week 6 compared to placebo. Additional measures included response and remission rates, Clinical Global Impression Scale - Improvement scores, HAMD-24 total score in subjects with baseline Hamilton Anxiety Scale (HAMA) >19 and MADRS-S total score. Adverse events (AEs) were assessed throughout the study. A total of 600 adults were randomized. There were no significant differences in efficacy measures between subjects in the 5 mg vortioxetine and placebo groups at week 6. HAMD-24 total score in subjects with baseline HAMA >19 in the 5 mg vortioxetine group was improved at weeks 3-6 compared to the placebo group (nominal p value <0.05). The most common AEs for the vortioxetine and placebo groups were nausea (19.1 and 9.4%), headache (17.1 and 15.1%) and diarrhoea (11.4 and 7.0%), respectively. In this study of adults with MDD, 5 mg vortioxetine did not differ significantly from placebo in reducing depression symptoms after 6 wk of treatment.

Compact high-quality CdSe/CdS core/shell nanocrystals with narrow emission linewidths and suppressed blinking

Abstract: High particle uniformity, high photoluminescence quantum yields, narrow and symmetric emission spectral lineshapes and minimal single-dot emission intermittency (known as blinking) have been recognized as universal requirements for the successful use of colloidal quantum dots in nearly all optical applications. However, synthesizing samples that simultaneously meet all these four criteria has proven challenging. Here, we report the synthesis of such high-quality CdSe-CdS core-shell quantum dots in an optimized process that maintains a slow growth rate of the shell through the use of octanethiol and cadmium oleate as precursors. In contrast with previous observations, single-dot blinking is significantly suppressed with only a relatively thin shell. Furthermore, we demonstrate the elimination of the ensemble luminescence photodarkening that is an intrinsic consequence of quantum dot blinking statistical ageing. Furthermore, the small size and high photoluminescence quantum yields of these novel quantum dots render them superior in vivo imaging agents compared with conventional quantum dots. We anticipate these quantum dots will also result in significant improvement in the performance of quantum dots in other applications such as solid-state lighting and illumination.

Effects of Vascular-Endothelial Protein Tyrosine Phosphatase Inhibition on Breast Cancer Vasculature and Metastatic Progression

Abstract: Unlike blood vessels in healthy tissue, the solid tumor microvasculature is dysfunctional and immature. Tumor vessels harbor structural and functional instability, which facilitates several distinct stages of tumor progression, including early sprouting angiogenesis, metastatic cell extravasation into distant organs, and the development of established tumor hypoxia, which fuels metastasis and impairs the efficacy of antitumor therapies (1–9). The endothelial cell (EC) receptor tyrosine kinase Tie-2/Tek is a critical regulator of vascular maturity. Tie-2 activation by its agonistic ligand angiopoietin 1 (Ang-1) increases perivascular cell (PVC) coverage, tightens EC junctions, reduces permeability, and increases vessel diameter (10–12). Angiopoeitin-2 (Ang-2) antagonizes the effects of Ang-1 in a context-dependent manner (6). Recent studies have shown a deleterious role for Ang-2 in mediating tumor vessel abnormalities and metastasis (13,14), suggesting that Tie-2 deactivation by Ang-2 in part mediates the unstable vessel phenotype seen within tumors. Current strategies targeting the Ang/Tie-2 axis in tumors have focused primarily on direct targeting of the angiopoietins, but such studies have not directly examined complex context-dependent interactions between Ang-1 and Ang-2 or the effects of angiopoietin blockade on Tie-2 activity (15–18). There are currently 10 agents targeting the Ang/Tie-2 axis under investigation in clinical trials (19). Vascular endothelial protein tyrosine phosphatase (VE-PTP) is an EC-specific receptor tyrosine phosphatase that dephosphorylates and consequently inactivates Tie-2 (20). The role of VE-PTP in the tumor vasculature is unknown. Recently, AKB-9778 has been developed as a novel, potent, and selective inhibitor of VE-PTP through structural modifications of 1,2,3,4-tetrahydroisoquinolinyl sulfamic acids (21). We hypothesized that pharmacological VE-PTP inhibition would activate Tie-2 signaling in ECs independent of Ang-1/Ang-2 ligands, stabilizing tumor vessels and mitigating the negative consequences of tumor vessel instability (20).

Spatial Charge Configuration Regulates Nanoparticle Transport and Binding Behavior In Vivo

Abstract: Detailed Charge arrangements: A new set of zwitterionic quantum dots were synthesized and used to study the influence of microscopic charge arrangements on the in vivo behavior of nanoparticles. Experiments using cultured cells and live mice demonstrate that the microscopic arrangement of surface charges strongly influence nonspecific binding, clearance behavior, and in vivo transport of nanoparticles.

Transgenic Mice for cGMP Imaging

Abstract: Rationale:Cyclic GMP (cGMP) is an important intracellular signaling molecule in the cardiovascular system, but its spatiotemporal dynamics in vivo is largely unknown. Objective:To generate and characterize transgenic mice expressing the fluorescence resonance energy transfer–based ratiometric cGMP sensor, cGMP indicator with an EC50 of 500 nmol/L (cGi500), in cardiovascular tissues. Methods and Results:Mouse lines with smooth muscle–specific or ubiquitous expression of cGi500 were generated by random transgenesis using an SM22α promoter fragment or by targeted integration of a Cre recombinase–activatable expression cassette driven by the cytomegalovirus early enhancer/chicken β-actin/β-globin promoter into the Rosa26 locus, respectively. Primary smooth muscle cells isolated from aorta, bladder, and colon of cGi500 mice showed strong sensor fluorescence. Basal cGMP concentrations were <100 nmol/L, whereas stimulation with cGMP-elevating agents such as 2-(N,N-diethylamino)-diazenolate-2-oxide diethylammoniu...

Abstract LB-344: Vascular normalization as an emerging strategy to enhance cancer immunotherapy.

Abstract: Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The recent approval of a prostate cancer vaccine has renewed hope for anti-cancer immunotherapies. However, the immunosuppressive tumor microenvironment may limit the effectiveness of current immunotherapies. Antiangiogenic agents have the potential to modulate the tumor microenvironment and improve immunotherapy, but they are often used at high doses in the clinic to prune tumor vessels and may paradoxically compromise various therapies. Here, we demonstrate that targeting tumor vasculature with lower “vascular normalizing” doses, but not high “anti-vascular/anti-angiogenic” doses of an anti-vascular endothelial growth factor receptor 2 (VEGFR2) antibody results in a more homogeneous distribution of functional tumor vessels. Furthermore, lower doses are superior to the high doses at polarizing tumor-associated macrophages from immune inhibitory M2-like towards immune stimulatory M1-like phenotype, and in facilitating CD4+ and CD8+ T cell tumor infiltration. Based on this mechanism, scheduling lower dose anti-VEGFR2 therapy with T cell activation induced by a whole cancer cell vaccine therapy enhanced anti-cancer efficacy in a CD8+ T cell-dependent manner in both immune tolerant and immunogenic murine breast cancer models. These findings indicate that vascular normalizing lower doses of anti-VEGFR2 antibody can reprogram the tumor microenvironment away from immunosuppression toward potentiation of cancer vaccine therapies. Given that the combinations of high doses of bevacizumab with chemotherapy have not improved overall survival of breast cancer patients, our study suggests a new strategy to more effectively use antiangiogenic agents in breast cancer with active immunotherapy and potentially other anti-cancer therapies. Citation Format: Yuhui Huang, Jianping Yuan, Elda Righi, Dan G. Duda, Dai Fukumura, Mark C. Poznanasky, Rakesh K. Jain. Vascular normalization as an emerging strategy to enhance cancer immunotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-344. doi:10.1158/1538-7445.AM2013-LB-344 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Benefits of Vascular Normalization Are Dose and Time Dependent—Letter

Abstract: Arjaans and colleagues reported that treatment with the anti-VEGF antibody bevacizumab hampers antibody uptake in an ectopic xenograft model of human ovarian cancer in mice ([1][1]). They found that bevacizumab decreased vessel number and increased pericyte coverage and concluded that treatment-

Efficacy, Safety, Pharmacokinetics, and Biomarkers of Cediranib Monotherapy in Advanced Hepatocellular Carcinoma: A Phase II Study

Abstract: Purpose: We conducted a single-arm phase II study of cediranib, a pan-VEGFR tyrosine kinase inhibitor, in patients with advanced hepatocellular carcinoma (HCC). Experimental Design: Patients with histologically confirmed measurable advanced HCC and adequate hematologic, hepatic, and renal functions received cediranib 30-mg orally once daily (4 weeks/cycle). The primary endpoint was progression-free survival (PFS) rate at 3 months. Other endpoints included response rates, overall survival (OS), pharmacokinetics (PK), and biomarkers for cediranib. Results: Cediranib treatment resulted in an estimated 3-month PFS rate of 77% (60%, 99%). Median PFS was 5.3 (3.5,9.7) months, stable disease was seen in 5/17 patients (29%), and median OS was 11.7 (7.5–13.6) months. Grade 3 toxicities included hypertension (29%), hyponatremia (29%), and hyperbilirubinemia (18%). Cediranib PK were comparable to those seen in cancer patients with normal hepatic function. Plasma levels of VEGF and PlGF increased and sVEGFR1, sVEGFR2, and Ang-2 decreased after cediranib treatment. PFS was inversely correlated with baseline levels of VEGF, sVEGFR2, and bFGF and with on-treatment levels of bFGF and IGF-1, and directly associated with on-treatment levels of IFN-γ. OS was inversely correlated with baseline levels of sVEGFR1, Ang-2, TNF-α, CAIX, and CD34 + CD133 + CD45 dim circulating progenitor cells and on-treatment levels of sVEGFR2. Conclusions: Despite the limitations of primary endpoint selection, cediranib at 30-mg daily showed a high incidence of toxicity and preliminary evidence of antitumor activity in advanced HCC. Hepatic dysfunction did not seem to affect the steady-state PK of cediranib. Exploratory studies suggested proangiogenic and inflammatory factors as potential biomarkers of anti-VEGF therapy in HCC. Clin Cancer Res; 19(6); 1557–66. ©2013 AACR .

A randomised, double-blind, placebo-controlled phase 2 study of trebananib (AMG 386) in combination with FOLFIRI in patients with previously treated metastatic colorectal carcinoma

Abstract: A randomised, double-blind, placebo-controlled phase 2 study of trebananib (AMG 386) in combination with FOLFIRI in patients with previously treated metastatic colorectal carcinoma

Metabolic Tumor Profiling with pH, Oxygen, and Glucose Chemosensors on a Quantum Dot Scaffold

Abstract: Acidity, hypoxia, and glucose levels characterize the tumor microenvironment rendering pH, pO2, and pGlucose, respectively, important indicators of tumor health. To this end, understanding how these parameters change can be a powerful tool for the development of novel and effective therapeutics. We have designed optical chemosensors that feature a quantum dot and an analyte-responsive dye. These noninvasive chemosensors permit pH, oxygen, and glucose to be monitored dynamically within the tumor microenvironment by using multiphoton imaging.

Magnetic resonance imaging biomarkers in hepatocellular carcinoma: association with response and circulating biomarkers after sunitinib therapy

Abstract: To investigate the hypothesis that MRI derived diffusion-weighted imaging (DWI) and perfusion (MRP) parameters are sensitive image biomarkers for monitoring early antiangiogenic effects and predicting progression free survival (PFS) in advanced hepatocellular carcinoma (HCC). In this phase II clinical trial, 23 of 34 patients were included in the imaging and circulating biomarker study. DWI and MRP were performed at the baseline and at 2-weeks after initiation of sunitinib. The imaging protocol included an axial DWI sequence using b values of 50, 400 and 800 sec/mm2, and MRP using a series of coronal 3D-VIBE following 20 ml of Gd-DTPA at 2 ml/sec. These parameters were compared with clinical outcome and PFS at 6-months. Correlation between changes in MRI parameters and plasma biomarkers was also evaluated. After 2-week of sunitinib, substantial Ktrans changes in HCC were observed from median baseline value 2.15 min−1 to 0.94 min−1 (P = 0.0001) with increases in median apparent diffusion coefficient (ADC) from 0.88 × 10-3 mm2/s to 0.98 × 10-3 mm2/s (P = 0.0001). Tumor size remained unchanged by RECIST and mRECIST (both P > 0.05). Patients who showed larger drop in Ktrans and Kep at 2 weeks correlated with favorable clinical outcome, and higher baseline Ktrans and larger drop in EVF correlated with longer PFS (all P < 0.05). There was a significant association between a decrease in sVEGFR2 and the drop in Ktrans and Kep (P = 0.044, P = 0.030), and a significant and borderline association between decrease in TNF-α and the drop in Ktrans and Kep, respectively (P = 0.051, P = 0.035). In HCC, MRP may be a more sensitive biomarker in predicting early response and PFS following sunitinib than RECIST and mRECIST. ClinicalTrials.gov: NCT00361309

Coat protein-mediated transgenic resistance of peanut (Arachis hypogaea L.) to peanut stem necrosis disease through Agrobacterium-mediated genetic transformation.

Abstract: The absence of resistance genes against biotic stresses like Tobacco streak virus (TSV) within compatible peanut germplasm necessitates the deployment of genetic engineering strategy to develop transgenic resistance. Transgenic resistance in peanut (Arachis hypogaea L.) to peanut stem necrosis disease caused by TSV was obtained by transferring coat protein (CP) gene of TSV through Agrobacterium-mediated transformation of de-embryonated cotyledons and immature leaves of peanut cultivars Kadiri 6 (K6) and Kadiri 134 (K134). Integration of the transgene in T1, T2 and T3 generations were confirmed by PCR with gene-specific primers. On the basis of segregation analysis of the PCR amplicons, homozygosity was confirmed in progeny from five transgenic lines. Six transgenic plants from three different single copy transgenic lines homozygous for the transgene were selected for challenge inoculation in T3 generations. The transgenic lines remained symptomless throughout and showed traces or no systemic accumulation of virus indicating the tolerance/resistance to the TSV infection. CP gene expression was observed in transgenic lines by RT-PCR, real-time PCR and ELISA. The findings provide an effective strategy for developing peanut with resistance to peanut stem necrosis disease.

Applications of Next Generation High Throughput Sequencing Technologies in Characterization, Discovery and Molecular Interaction of Plant Viruses

Abstract: Present era of molecular biology is witnessing revolutionary developments in sequencing technology. This advancement has considerably influenced plant virology in the field of diagnostics and host virus interaction. Next generation high-throughput sequencing technology has made it possible to directly detect, identify and discover novel viruses in several plants in an unbiased manner without antibodies or prior knowledge of the virus sequences. Entire viral genome could be sequenced from symptomatic or asymptomatic plants through next generation sequencing of total nucleic acids including small RNAs. It provides census of viral population in a particular ecosystem or cropping system. Viral genome variability, evolution within the host and virus defence mechanism in plants can also be easily understood by massive parallel sequencing. In this article, we provide an overview of the applications of next generation sequencing technology in characterization, discovery and molecular interaction of plant viruses.