Lilia Ileva

Pazopanib reveals a role for tumor cell B-Raf in the prevention of HER2+ breast cancer brain metastasis.

Purpose: Brain metastases of breast cancer contribute significantly to patient morbidity and mortality. We have tested pazopanib, a recently approved antiangiogenic drug that targets VEGFR1, VEGFR2, VEGFR3, PDGFRβ, PDGFRα, and c-kit, for prevention of experimental brain metastases and mechanism of action. Experimental Design:In vitro assays included B-Raf enzymatic assays, Western blots, and angiogenesis assays. For in vivo assays, HER2 transfectants of the brain seeking sublines of MDA-MB-231 cells (231-BR-HER2) and MCF7 cells (MCF7-HER2-BR3, derived herein) were injected into the left cardiac ventricle of mice and treated with vehicle or pazopanib beginning on day 3 postinjection. Brain metastases were counted histologically, imaged, and immunostained. Results: Treatment with 100 mg/kg of pazopanib resulted in a 73% decline in large 231-BR-HER2 metastases (P < 0.0001) and a 39% decline in micrometastases (P = 0.004). In vitro, pazopanib was directly antiproliferative to 231-BR-HER2 breast cancer cells and inhibited MEK and ERK activation in vitro despite B-Raf and Ras mutations. Enzymatic assays demonstrated that pazopanib directly inhibited the wild type and exon 11 oncogenic mutant, but not the V600E mutant forms of B-Raf. Activation of the B-Raf targets pERK1/2 and pMEK1/2 was decreased in pazopanib-treated brain metastases whereas blood vessel density was unaltered. In the MCF7-HER2-BR3 experimental brain metastasis model, pazopanib reduced overall brain metastasis volume upon magnetic resonance imaging (MRI) by 55% (P = 0.067), without affecting brain metastasis vascular density. Conclusions: The data identify a new activity for pazopanib directly on tumor cells as a pan-Raf inhibitor and suggest its potential for prevention of brain metastatic colonization of HER2+ breast cancer. Clin Cancer Res; 17(1); 142–53. ©2010 AACR.

Chemopreventive activity of plant flavonoid isorhamnetin in colorectal cancer is mediated by oncogenic Src and β-catenin

Analysis of the Polyp Prevention Trial showed an association between an isorhamnetin-rich diet and a reduced risk of advanced adenoma recurrence; however, the mechanism behind the chemoprotective effects of isorhamnetin remains unclear. Here, we show that isorhamnetin prevents colorectal tumorigenesis of FVB/N mice treated with the chemical carcinogen azoxymethane and subsequently exposed to colonic irritant dextran sodium sulfate (DSS). Dietary isorhamnetin decreased mortality, tumor number, and tumor burden by 62%, 35%, and 59%, respectively. MRI, histopathology, and immunohistochemical analysis revealed that dietary isorhamnetin resolved the DSS-induced inflammatory response faster than the control diet. Isorhamnetin inhibited AOM/DSS-induced oncogenic c-Src activation and β-catenin nuclear translocation, while promoting the expression of C-terminal Src kinase (CSK), a negative regulator of Src family of tyrosine kinases. Similarly, in HT-29 colon cancer cells, isorhamnetin inhibited oncogenic Src activity and β-catenin nuclear translocation by inducing expression of csk, as verified by RNA interference knockdown of csk. Our observations suggest the chemoprotective effects of isorhamnetin in colon cancer are linked to its anti-inflammatory activities and its inhibition of oncogenic Src activity and consequential loss of nuclear β-catenin, activities that are dependent on CSK expression.

A New Approach in the Preparation of Dendrimer-Based Bifunctional Diethylenetriaminepentaacetic Acid MR Contrast Agent Derivatives

In this paper, we report a new method to prepare and characterize a contrast agent based on a fourth-generation (G4) polyamidoamine (PAMAM) dendrimer conjugated to the gadolinium complex of the bifunctional diethylenetriamine pentaacetic acid derivative (1B4M-DTPA). The method involves preforming the metal-ligand chelate in alcohol prior to conjugation to the dendrimer. The dendrimer-based agent was purified by a Sephadex G-25 column and characterized by elemental analysis. The analysis and SE-HPLC data gave a chelate to dendrimer ratio of 30:1 suggesting conjugation at approximately every other amine terminal on the dendrimer. Molar relaxivity of the agent measured at pH 7.4 displayed a higher value than that of the analogous G4 dendrimer based agent prepared by the postmetal incorporation method (r(1) = 26.9 vs 13.9 mM(-1) s(-1) at 3 T and 22 degrees C). This is hypothesized to be due to the higher hydrophobicity of this conjugate and the lack of available charged carboxylate groups from noncomplexed free ligands that might coordinate to the metal and thus also reduce water exchange sites. Additionally, the distribution populations of compounds that result from the postmetal incorporation route are eliminated from the current product simplifying characterization as quality control issues pertaining to the production of such agents for clinical use as MR contrast agents. In vivo imaging in mice showed a reasonably fast clearance (t(1/2) = 24 min) suggesting a viable agent for use in clinical application.

The B-Raf Status of Tumor Cells May Be a Significant Determinant of Both Antitumor and Anti-Angiogenic Effects of Pazopanib in Xenograft Tumor Models

Pazopanib is an FDA approved Vascular Endothelial Growth Factor Receptor inhibitor. We previously reported that it also inhibits tumor cell B-Raf activity in an experimental brain metastatic setting. Here, we determine the effects of different B-Raf genotypes on pazopanib efficacy, in terms of primary tumor growth and anti-angiogenesis. A panel of seven human breast cancer and melanoma cell lines harboring different mutations in the Ras-Raf pathway was implanted orthotopically in mice, and tumor growth, ERK1/2, MEK1/2 and AKT activation, and blood vessel density and permeability were analyzed. Pazopanib was significantly inhibitory to xenografts expressing either exon 11 mutations of B-Raf, or HER2 activated wild type B-Raf; no significant inhibition of a xenograft expressing the common V600E B-Raf mutation was observed. Decreased pMEK staining in the responsive tumors confirmed that B-Raf was targeted by pazopanib. Interestingly, pazopanib inhibition of tumor cell B-Raf also correlated with its anti-angiogenic activity, as quantified by vessel density and area. In conclusion, using pazopanib, tumor B-Raf status was identified as a significant determinant of both tumor growth and angiogenesis.

Longitudinal imaging of cancer cell metastases in two preclinical models: a correlation of noninvasive imaging to histopathology.

Metastatic spread is the leading cause of death from cancer. Early detection of cancer at primary and metastatic sites by noninvasive imaging modalities would be beneficial for both therapeutic intervention and disease management. Noninvasive imaging modalities such as bioluminescence (optical), positron emission tomography (PET)/X-ray computed tomography (CT), and magnetic resonance imaging (MRI) can provide complementary information and accurately measure tumor growth as confirmed by histopathology. Methods. We validated two metastatic tumor models, MDA-MD-231-Luc and B16-F10-Luc intravenously injected, and 4T1-Luc cells orthotopically implanted into the mammary fat pad. Longitudinal whole body bioluminescence imaging (BLI) evaluated metastasis, and tumor burden of the melanoma cell line (B16-F10-Luc) was correlated with (PET)/CT and MRI. In addition, ex vivo imaging evaluated metastasis in relevant organs and histopathological analysis was used to confirm imaging. Results. BLI revealed successful colonization of cancer cells in both metastatic tumor models over a 4-week period. Furthermore, lung metastasis of B16-F10-Luc cells imaged by PET/CT at week four showed a strong correlation (R 2 = 0.9) with histopathology. The presence and degree of metastasis as determined by imaging correlated (R 2 = 0.7) well with histopathology findings. Conclusions. We validated two metastatic tumor models by longitudinal noninvasive imaging with good histopathology correlation.

In vivo MRI virtual colonography in a mouse model of colon cancer

We have developed a reliable noninvasive method for monitoring colonic tumors and mucosal inflammation in a mouse model of colon cancer using magnetic resonance colonography (MRC). After a mild cleansing enema, the colon is filled with Fluorinert, a perfluorinated liquid that does not produce a proton MR signal. The mouse is placed in a dedicated volume MR receiver coil, and high-resolution images are acquired in three planes. The Fluorinert enema distends the mouse colon, creating an artifact-free black homogeneous background, allowing clear delineation of the inflamed colonic wall and visualization of luminal tumors in various stages of development. A gadolinium-based contrast agent can be administered i.v. to the animal to detect mural inflammation or tumor vascularity. This technique is useful for serial monitoring of the effects of preventive or therapeutic strategies on tumor development without killing the animal or requiring invasive endoscopies. The animal preparation and imaging can be completed in ∼1.5 h.

Abstract 3840: The National Cancer Institute’s patient-derived models repository (PDMR)

The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR) comprised of quality-controlled, early-passage, clinically-annotated patient-derived xenografts (PDXs) to serve as a resource for public-private partnerships and academic drug discovery efforts. These models are offered to the extramural community for research use (https://pdmr.cancer.gov/), along with clinical annotation and molecular information (whole exome sequence, RNASeq), which is available in a publicly accessible database. The PDMR was established by NCI at the Frederick National Laboratory for Cancer Research (FNLCR) in direct response to discussions with academia and industry; the oncology community9s highest priority need was preclinical models that more faithfully reflect the patient9s tumor and are associated with the patient9s treatment history. NCI has focused on generating models to complement existing PDX collections and address unmet needs in the preclinical model space. The PDMR generates the majority of its PDXs by subcutaneous implantation except for those histologies having better success rates in either orthotopic or alternate implant sites. All SOPs and quality-control standards developed by the PDMR as well as those shared by collaborators are posted to a public web site that houses the PDMR database. In May 2017, the public website (https://pdmr.cancer.gov/) went live with its first 100 models from histologies including pancreatic, colorectal, renal, head and neck, and lung squamous cell cancers as well as melanoma and adult soft tissue sarcomas. In early 2018, the PDMR will begin releasing models from gynecological cancers, small cell lung cancer, chondro/osteo sarcomas, lung adenocarcinoma, and squamous cell skin and Merkel cell carcinomas. In addition, wherever available germline sequence and somatic variant calls will be added to the existing molecular characterization data for each model. NCI has also increased its focus on creating PDXs from racial and ethnic minorities through several funding opportunities. The overall goal of NCI is to create a long-term home for at least 1000 models such that sufficient biological and clinical diversity is represented to allow researchers to ask questions regarding the impact of tumor heterogeneity on target qualification or clinical response, whether PDXs more faithfully represent the human tumor for pharmacodynamic assay and predictive marker development, or if adequately powered preclinical PDX clinical trials can lead to better evaluation of therapies for future clinical use. Moving forward the PDMR plans to distribute in vitro, early-passage tumor cell cultures and cancer-associated fibroblasts as well as releasing PDX drug response data for a panel of FNA-approved therapeutic agents. Funded by NCI Contract No. HHSN261200800001E Citation Format: Yvonne A. Evrard, Michelle M. Gottholm Ahalt, Sergio . Y. Alcoser, Kaitlyn Arthur, Mariah Baldwin, Linda L. Blumenauer, Carrie Bonomi, Suzanne Borgel, Elizabeth Bradtke, Corinne Camalier, John Carter, Tiffanie Chase, Alice Chen, Lily Chen, Donna W. Coakley, Nicole E. Craig, Biswajit Das, Vivekananda Datta, Jordyn Davidson, Margaret R. DeFreytas, Emily Delaney, Michelle A. Eugeni, Raymond Divelbiss, Palmer Fliss, Thomas Forbes, Marion Gibson, Tara Grinnage-Pulley, Sierra Hoffman, Lilia Ileva, Paula Jacobs, Franklyn Jimenez, Joseph Kalen, Catherine Karangwa, Chris Karlovich, Candace Mallow, Chelsea McGlynn, Jenna E. Moyer, Michael Mullendore, Dianne L. Newton, Nimit Patel, Rajesh Patidar, Kevin Plater, Marianne Radzyminski, Lisa Riffle, Larry Rubinstein, Luke H. Stockwin, Mickey Williams, Melinda G. Hollingshead, James H. Doroshow. The National Cancer Institute9s patient-derived models repository (PDMR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 986.

Abstract LB-510: Longitudinal imaging of melanoma cancer cell metastasis in a preclinical model by bioluminescence, PET/CT, and MRI

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Metastasis of cancer cells from the primary tumor are a leading cause of poor prognosis and death from cancer. The lung is the most common organ for metastatic spread of cancer and leads to 20-50% of cancer-related deaths. Early detection of cancer metastasis to the lung and other organs could be beneficial in disease management. In animal studies, most secondary-site disseminated tumors are evaluated by optical imaging, and gross examination at necropsy and subsequent histopathological analysis. These techniques are limited in that they do not offer the ability to monitor animals during the course of tumor development, and optical microscopy may have inadequate field of view (3-D morphometrics) for thorough tumor evaluation. Noninvasive imaging allows for serial imaging of the same animal, with the ability to repeat measurements in real-time to enhance the observation of disease progression and therapeutic response. Here, we compared the multimodal noninvasive whole-body imaging techniques bioluminescence, 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) positron emission tomography (PET)/ computed tomography (CT), and magnetic resonance imaging (MRI) to image lung metastasis development following intravenous injection of melanoma cells (B16-F10 melanoma cell line expressing luciferase gene) in C57BL/6. By bioluminescence imaging, tumor cell localization in the lungs was evident as early as 3 days (1 x 105 photons/sec; total flux) post cancer cell inoculation and steadily increased through the end of week four (1 x 108 photons/sec; total flux). The increase in the lung tumor burden was confirmed by lung histopathology. As it was difficult to de-convolve the effects of photon flux spreading from lungs over other organs, other metastatic sites were not evaluated by bioluminescence. CT and 18F-FDG-PET are used clinically to image lung metastasis and offer excellent resolution of anatomical and metabolic details of cancer cells. We were able to image metastatic nodules in the lung by PET/CT at week four, which showed a strong correlation with histopathology analysis. [18F]FDG-PET/CT also revealed metastasis in the kidney and mesentery and were confirmed by histopathology. T2-weighted MRI at week 4 identified several lung tumors and metastatic cancers in the abdomen. A comparison of [18F]FDG/PET at week 4 to MRI and CT displayed several highly metabolic regions within the tumors. Histopathology confirmed the metastatic spread of the melanoma cells to the lung, mediastinum, kidney, and mesentery. Funded by NCI contract No. HHSN261200800001E. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-510. doi:1538-7445.AM2012-LB-510

Abstract LB-383: In vitro and in vivo MRI evaluation of a novel metallofullerene nanoparticle

Some disadvantages of conventional small molecule contrast agents, such as Magnevist® (Gd - DTPA), are that they extravasate rapidly and are quickly eliminated from blood, limiting the viable MRI time. Macromolecular magnetic resonance imaging (MRI) contrast agents, on the other hand, are useful blood-pool agents and have found application in monitoring tumor vasculature and angiogenesis. There is an urgent need for new MRI contrast agents with higher relaxivity and longer blood half-life. Here, we report in vitro and in vivo characterization of a water-soluble derivatized fullerene nanoparticle contrast agent (NCL124) in comparison to the market standard, Magnevist. In comparison to Magnevist, NCL124 had higher R 1 and R2 relaxivities in human plasma at 22° C and 37°C. In vivo MRI imaging was performed on a LS174T dual-flank xenografts following a single intravenous injection of NCL 124 or Magnevist contrast agent (0.2 mmol Gd/Kg) using a 3.0 Tesla clinical whole-body MRI. Based on the relaxivity-time profiles, the Vss for NCL124 was similar to that of albumin (∼ 100 mL/Kg), suggesting high protein binding, while the Vss of Magnevist was similar to that of extracellular tissue water (∼200 mL/Kg), as expected for an extracellular diffusible tracer. NCL124 also had a longer blood half-life (∼ 100 min) than Magnevist (19 min). Taken together, the higher concentration (C max ) and exposure (AUCall) of NCL124 in the blood, coupled with slow elimination, supports the use of NCL124 as a blood-pool contrast agent. Intriguingly, the half-life of NCL124 varied by tumor size, while Magnevist showed similar half-life in both tumors. This suggests NCL124 may have significant utility in predicting differences in tumor vascular and lymphatic permeability, and potentially be useful as a marker for antiangiogenic therapy. Funded by NCI contract # HHSN261200800001E. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-383.

Abstract B59: Magnetic resonance imaging monitoring of tumor promotion and tumor progression in mouse model of inflammation-promoted colon cancer

B59 Purpose Early detection of pre-cancerous tissue has significantly increased survival for most cancers including colorectal cancer. Animal models that follow early stages of cancer are valuable for identifying molecular events and response indicators that correlate with the early stages of tumor development. The goal of this work was to generate new methods to monitor over time the early stages of colorectal cancer in mice using magnetic resonance imaging (MRI). Experimental Design Mice treated with azoxymethane and dextran sulafate sodium (DSS) were imaged by MRI throughout the tumor development. Early inflammation was imaged in T2-weighted MRI. Dark-lumen images were obtained in both T1 and T2-weighted images using a poly-fluorinated liquid enema. Individual tumor volumes were calculated and validated ex vivo. Results Using T2-weighted MRI, inflammation was detected 3 days after DSS exposure and subsided over the next week. The poly-fluorinated liquid enema distended the colon and provided a clear differentiation of the lumen of the colon from the mucosal lining. Tumors were detected as early as 29 days after initiation and as small as 1.2 mm3. Individual tumor growths were followed over time and tumor volumes measured by MRI correlated with volumes measured ex vivo. Conclusions Early detection of tumorigenesis is important for understanding tumor development. The use of MRI for detection of the initial stages of colon cancer allows real time evaluation of preclinical trials for prevention and intervention. The use of the poly-fluorinated liquid enema for Dark-lumen MRI in both T1 and T2-weighted imaging could simplify clinical MRI detection of colon cancer. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B59.