Significant Difference In Tumor Volume Research Articles

Garlic extract in bladder cancer prevention: Evidence from T24 bladder cancer cell xenograft model, tissue microarray, and gene network analysis.

There is a growing interest in the use of naturally occurring agents in cancer prevention. This study investigated the garlic extract affects in bladder cancer(BC) prevention. The effect of garlic extract in cancer prevention was evaluated using the T24 BC BALB/C-nude mouse xenograft model. Microarray analysis of tissues was performed to identify differences in gene expression between garlic extract intake and control diet, and gene network analysis was performed to assess candidate mechanisms of action. Furthermore, we investigated the expression value of selected genes in the data of 165BCpatients. Compared to the control group, significant differences in tumor volume and tumor weight were observed in the groups fed 20mg/kg (p<0.05), 200mg/kg, and 1000mg/kg of garlic extract (p<0.01). Genes (645) were identified as cancer prevention-related genes (fold change >2 and p<0.05) by tissue microarray analysis. A gene network analysis of 279 of these genes (p<0.01) was performed using Cytoscape/ClueGo software: 36genes and 37gene ontologies were mapped to gene networks. Protein kinaseA (PKA) signaling pathway including AKAP12, RDX, and RAB13 genes were identified as potential mechanisms for the activity of garlic extract in cancer prevention. In BC patients, AKAP12 and RDX were decreased but, RAB13 was increased. Oral garlic extract has strong cancer prevention activity invivo and an acceptable safety profile. PKA signaling process, especially increasing AKAP12 and RDX and decreasing RAB13, are candidate pathways that may mediate this prevention effect.

Abstract No. 2 - Inhibition of the PI3K/AKT/mTOR pathway prevents thermal ablation induced accelerated intrahepatic tumor growth in an orthotopic model of hepatocellular carcinoma

Preliminary studies have shown that thermal ablation of liver accelerates local intrahepatic hepatocellular carcinoma (HCC) growth. Additionally, the PI3K/AKT/mTOR pathway has been shown to play a critical role in HCC cell survival, proliferation and resistance to heat stress in vitro. The aim of the present study was to test the hypothesis that inhibition of PI3K/AKT/mTOR pathway prevents thermal ablation induced accelerated local intrahepatic HCC progression. In an IACUC-approved study, Sprague-Dawley rats were randomized to receive the oral PI3K/mTOR inhibitor NVP-BEZ235 (25mg/kg; N = 13) or vehicle control (NMP/PEG; N = 13) on a 2-dose schedule: 1 hour prior to ablation and 24 hours post-ablation. 3x10^6 N1S1 HCC cells were inoculated into the median hepatic lobe of each rat. Rats were then randomized to receive immediate laser thermal ablation at 3W for 90s (N = 14) or sham ablation (N = 12) of normal liver in the same hepatic lobe. Tumor growth was monitored by T2-weighted magnetic resonance imaging (MRI) 18 days post-ablation. Tumor volumes calculated from MR images and compared between treatment groups (vehicle + sham, vehicle + ablation, BEZ235 + sham, BEZ235 + ablation) There was no significant difference in tumor volume among sham treated rats that received vehicle (vehicle + sham) or BEZ235 (BEZ235 + sham) (p>0.05). Thermal liver ablation induced accelerated HCC tumor growth in the vehicle + ablation compared to the vehicle + sham group by day 18 post-ablation (3843 ± 565 mm3 v. 2066 ± 611 mm3, respectively; p = 0.04). However, treatment with the dual PI3K/mTOR inhibitor prevented accelerated HCC tumor growth in the ablation group (BEZ235 + ablation) compared with the sham group (BEZ235 + sham) (1730 ± 565 mm3 v.1737 ± 611 mm3; p >.05). These data suggest that PI3K/AKT/mTOR signaling may mediate accelerated local HCC progression caused by thermal ablation of liver and that inhibition of this pathway may prevent thermal ablation induced accelerated tumor growth. Further pre-clinical studies in this model are needed to optimize the drug dosing and schedule for clinical translation.

Early Changes in Contrast Enhanced Ultrasound Predict for Pathologic Response to Chemotherapy in an Animal Tumor Model

The purpose of this study was to examine CEUS whether could earlier predict pathologic response to chemotherapy than morphologic method in a MCF-7 tumor model. Twenty MCF-7 breast cancer bearing nude mice were included in this study. Animals received either adriamycin or a control substance were imaged before and after treatment with an ultrasound scanner after bolus injection of SonoVue. Dynamic contrast enhanced ultrasound was performed at pretherapy, Day 3, Day 5 and Day 7 after adriamycin administration. Regions of interest within the tumor were analyzed off-line to determine perfusion parameters including peak enhancement (PE), area under the curve of wash-in (WIAUC), wash-in rate (WIR), wash-in perfusion index (WIPI) and quality of fit (QOF). The section equivalent to ultrasound imaging plane was stained with Hematoxylin and Eosin to allow for assessment of tumor cell density and Ki67 for situation of tumor proliferation, CD34 for tumor micro-vascular density. There were no significant differences in tumors perfusion and tumor volumes before treatment. Treatment with adriamycin resulted into a significant decrease in PE, WIAUC, WIR and WIPI in comparison with control group (P < 0.05) at Day 3, when there was no significant difference in tumor volume. The volume of treated tumors was significant smaller than that of control tumors (P < 0.05) at day 7. The tumor cell density estimated by pathology slice was significantly lower in treated tumors than in control tumors at day 7 (P < 0.05). The positive Ki67 cell density and CD34 estimated by pathology slice were also significantly lower in treated tumors than in control tumors at day 7 (P < 0.05). The correlation between micro-vascular and perfusion parameters was obvious (P < 0.05). Changes in tumor micro-vessel functionality as depicted by CEUS early on after starting adriamycin -based chemotherapy can predict final pathologic response. Contrast enhanced ultrasound will be a new way for evaluating tumor response to chemoth.

Systemic administration of miRNA mimics by liposomal delivery system in animal model of colorectal carcinoma.

MiRNAs are important regulators of gene expression and changes in their levels are linked with various pathological states, including solid tumors. MiR-215 has been identified as a tumor suppressor in colorectal cancer (CRC). Following our previous in vitro and in vivo experiments, the aim of this project was to study the possibility of increasing the levels of miR-215 in tumor cells by systemic administration of miRNA mimics in liposomal delivery system in vivo. By subcutaneous xenotransplantation of human cancer cells to NSG mice, CRC model was established. The treatment (miR-215 mimics in liposomes [20 and 40 microg/mouse], control oligonucleotide in liposomes, or saline) was administered repeatedly by i.v. injection via tail-vein. Animals were sacrificed, tumor were dissected and measured by a caliper. Expression of miR-215 in tumors, lungs and liver was quantified by RT-PCR. There was no significant differences in tumor volume and miR-215 expression between all three treatment groups. Therefore, the decrease in tumor volume was not achieved. By comparing the levels of miR-215 in lungs, liver and tumors after the treatment, we suggest that the liposomes are accumulated in the lungs and do not concentrate sufficiently in the tumor site to exert significant tumor-suppressive effect.

Abstract 1334: Lipid-based nanoparticulate hydroxychloroquine (HCQ) formulations for use in combination with autophagy inducing drugs for treatment of breast cancer

Abstract Many targeted and broad spectrum anticancer drugs used to treat breast cancer trigger survival responses exemplified by the induction of cytoprotective macroautophagy (autophagy). Previously, we and others have shown that the anti-malarial agent hydroxychloroquine (HCQ) can improve the effects of anticancer drugs by inhibiting autophagy when used in high concentrations (1-20 μM). These levels are difficult to attain in vivo, thus, we developed novel liposomal formulations of HCQ (L-HCQ) designed to maintain therapeutic concentrations in plasma and tumor sites over extended periods of time. Liposomes (1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (CHOL) (55:45 molar ratio)) were prepared by extrusion to exhibit a mean particle size of 100 ± 20 nm. Copper HCQ complexation or ammnonium sulphate methods were used for loading HCQ into liposomes achieving &amp;gt;99% encapsulation efficiency (HCQ to lipid ratio: 0.22 ± 0.02 (mol:mol)). In vitro stability studies indicated that more than 80% of the liposomal associated HCQ was retained in the formulation for at least 24 h at 37 °C. In vivo pharmacokinetic studies, demonstrated that free HCQ was eliminated from the plasma compartment within 30 minutes following i.v. injection while the L-HCQ formulations maintained significantly higher plasma HCQ levels (&amp;gt;100 μM) over 24 h regardless of the loading method. Tolerability studies in non-tumor bearing CD1 mice showed no signs of toxicity following single and multiple doses (3 x week, i.v., 75 mg/kg). Inhibition of autophagy in vivo was examined in liver, heart and pancreas tissues of C57B1/6 mice 6 h after dosing with L-HCQ or free HCQ in combination with the autophagy inducing drug rapamycin. The results show that L-HCQ inhibited rapamycin-induced autophagy more effectively than free HCQ, as evident by a significant increase in LC3-II levels in all the examined tissue. Finally, the efficacy of L-HCQ alone (3 x week, i.v., 60 mg/kg) or in combination with the autophagy promoting drug gefitinib, an EGFR tyrosine kinase inhibitor (5 x week, oral gavage, 100 mg/kg), was tested in the JIMT-1 breast cancer xenograft model (s.c.) established in Rag2M mice. After four weeks of treatment, there were no significant differences in tumor volume between untreated and L-HCQ or gefitinib alone treated animals (p&amp;gt;0.05). In contrast, the gefitinib and L-HCQ combination engendered a significant inhibition of tumor growth compared to untreated controls (p&amp;lt;0.05). Moreover, molecular analysis confirmed inhibition of gefitinib-induced autophagy in vivo by L-HCQ, as judged by increased LC3-II and p62 protein levels in tumor tissue. In summary, this study established that L-HCQ was able to inhibit autophagy and improved sensitivity in an in vivo model of breast cancer treated with gefitinib. Citation Format: Jagbir Singh, Wieslawa H. Dragowska, Malathi Anantha, Ashleen S. Prasad, Jenna S. Rawji, Norman S. Chow, Marcel B. Bally. Lipid-based nanoparticulate hydroxychloroquine (HCQ) formulations for use in combination with autophagy inducing drugs for treatment of breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1334.

Quantitative CD3 PET Imaging Predicts Tumor Growth Response to Anti-CTLA-4 Therapy.

A CD3 PET imaging agent targeting T cells was synthesized to test the role of such imaging as a predictive marker. The 89Zr-p-isothiocyanatobenzyl-deferoxamine-CD3 PET probe was assessed in a murine tumor xenograft model of anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) immunotherapy of colon cancer. Imaging on day 14 revealed 2 distinct groups of mice stratified by PET signal intensity. Although there was no significant difference in tumor volume on the day of imaging, in the high-uptake group subsequent measurements revealed significantly smaller tumors than in either the low-uptake group or the untreated controls. In contrast, there was no significant difference in the size of tumors between the low-uptake and untreated control mice. These findings indicate that high CD3 PET uptake in the anti-CTLA-4-treated mice correlated with subsequent reduced tumor volume and was a predictive biomarker of response.

Development of Liposomal Formulation for Delivering Anticancer Drug to Breast Cancer Stem-Cell-Like Cells and its Pharmacokinetics in an Animal Model.

The objective of the present study is to develop a liposomal formulation for delivering anticancer drug to breast cancer stem-cell-like cells, ANV-1, and evaluate its pharmacokinetics in an animal model. The anticancer drug ESC8 was used in dexamethasone (Dex)-associated liposome (DX) to form ESC8-entrapped liposome named DXE. ANV-1 cells showed high-level expression of NRP-1. To enhance tumor regression, we additionally adapted to codeliver the NRP-1 shRNA-encoded plasmid using the established DXE liposome. In vivo efficacy of DXE-NRP-1 was carried out in mice bearing ANV-1 cells as xenograft tumors and the extent of tumor growth inhibition was evaluated by tumor-size measurement. A significant difference in tumor volume started to reveal between DXE-NRP-1 group and DXE-Control group. DXE-NRP-1 group showed ∼4 folds and ∼2.5 folds smaller tumor volume than exhibited by untreated and DXE-Control-treated groups, respectively. DXE disposition was evaluated in Sprague-Dawley rats following an intraperitoneal dose (3.67 mg/kg of ESC8 in DXE). The plasma concentrations of ESC8 in the DXE formulation were measured by liquid chromatography mass spectrometry and pharmacokinetic parameters were determined using a noncompartmental analysis. ESC8 had a half-life of 11.01 ± 0.29 h, clearance of 2.10 ± 3.63 L/kg/h, and volume of distribution of 33.42 ± 0.83 L/kg. This suggests that the DXE liposome formulation could be administered once or twice daily for therapeutic efficacy. In overall, we developed a potent liposomal formulation with favorable pharmacokinetic and tumor regressing profile that could sensitize and kill highly aggressive and drug-resistive cancer stem-cell-like cells.

Effect of Atmospheric-Pressure Plasmas on Drug Resistant Melanoma: The Challenges of Translating In vitro Outcomes into Animal Models

Atmospheric-pressure plasmas (APs) have been identified as a promising cancer therapy that is able to preferentially kill neoplastic cells through apoptosis. In vitro studies suggest that AP-generated reactive oxygen species (ROS) are the principal triggers of apoptosis-related signaling cascades via oxidative stress and direct interference with DNA, proteins, and other cellular components. The results of this study corroborate an ROS-mediated mechanism of cancer cell death, with apoptosis in AP-treated Mel-007 melanoma cells inhibited by pretreatment of cells with the ROS scavenger N-acetylcysteine or the caspase inhibitor zVAD-fmk. In an effort to compare apoptosis mechanisms and evaluate the in vitro−in vivo correlation for AP-induced apoptosis, Mel-007 cells were injected subcutaneously into mice to form solid tumors and were treated with AP Histological assessment of tumors from control and AP-treated animals showed no significant difference in tumor volume, mitotic rate, or percentage of necrosis or multinucleate cells. These results vary from those of other studies of AP treatment of xenograft tumors in murine models, in which a decrease in tumor size and tumor volume were observed. These findings focus our attention on challenges associated with translating the in vitro results to corresponding in vivo outcomes, and highlight concerns about the applicability of mechanisms established in vitro to an intrinsically dynamic in vivo environment.

Longitudinal tumor hypoxia imaging with [18F]FAZA-PET provides early prediction of nanoliposomal irinotecan (nal-IRI) treatment activity

BackgroundNon-invasive measurement of tumor hypoxia has demonstrated potential for the evaluation of disease progression, as well as prediction and assessment of treatment outcome. [18F]fluoroazomycin arabinoside (FAZA) positron emission tomography (PET) has been identified as a robust method for quantification of hypoxia both preclinically and clinically. The goal of this investigation was to evaluate the feasibility and value of repeated FAZA-PET imaging to quantify hypoxia in tumors that received multi-dose chemotherapy.MethodsFAZA-PET imaging was conducted over a 21-day period in a mouse xenograft model of HT-29 human colorectal carcinoma, following multi-dose chemotherapy treatment with irinotecan (CPT-11) or nanoliposomal irinotecan (nal-IRI, MM-398).ResultsTumors treated with 10 mg/kg nal-IRI maintained significantly lower levels of hypoxia and smaller hypoxic fractions compared to tumors that received 50 mg/kg CPT-11. Specifically, differences in FAZA uptake were detectable 9 days before any significant differences in tumor volume were observed between the treatment groups.ConclusionsThese findings highlight the potential use of FAZA-PET as an early marker of treatment response following multi-dose chemotherapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-015-0135-x) contains supplementary material, which is available to authorized users.

Chemotherapy for rat glioma using adriamycin-N-succinyl chitosan sustained release nanometer particles

Objective To observe the in vitro and in vivo releasing effects of adriamycin-N-succinyl chitosan(SCS)sustained release nanometer particles following implantation,and evaluate the local chemotherapeutic effect on rats bearing C6 gliomas. Methods The matrix with adriamycin was prepared.Gelatin and agar were used as major materials.The releasing effects of adriamycin-SCSsustained release nanometer particles were determined in artificial cerebrospinal fluid or in 20 normal rats brains by the method of fluorometry.The median survival time(MST)was measured in 20 SD rats bearing intracranial C6 gliomas, and the inhibitory effect of regional administration of adriamycin-SCS on growth of subcutaneous tumors was evaluated in 20 rats. Results Constant-release of adriamycin-SCS lasted over 30 days in artificial cerebro-spinal fluid or normal rabbit brains.Local controlled release of adriamycin could prolong the survival of rats bearing C6 gliomas with the MST being 35 days. Local controlled release of adriamycin could significant inhibit the growth of the subcutaneous tumors. As compared with control group, the growth of the subcutaneous tumors was slowed down or ceased in experimental group.There was statistically significant difference in tumor volume between the two groups(χ2= 7. 68,P<0. 05).There was significant difference in apoptosis index between control group(6. 4%) and experimental group(59. 3%, χ2= 23. 59,P<0. 05). Conclusion With high regional concentration and avoidance of toxicity of systemic chemotherapy, control released chemotherapy of adriamycin-SCS sustained nanometer particles remarkably inhibited the growth of C6 gliomas. Key words: Glioma; Adriamycin; N-succinyl chitosan; Sustained release particles

Endometrial Cancer: Combined MR Volumetry and Diffusion-weighted Imaging for Assessment of Myometrial and Lymphovascular Invasion and Tumor Grade

To investigate magnetic resonance (MR) volumetry of endometrial tumors and its association with deep myometrial invasion, tumor grade, and lymphovascular invasion and to assess the value of apparent diffusion coefficient (ADC) histographic analysis of the whole tumor volume for prediction of tumor grade and lymphovascular invasion. The institutional review board approved this retrospective study; patient consent was not required. Between May 2010 and May 2012, 70 women (mean age, 64 years; range, 24-91 years) with endometrial cancer underwent preoperative MR imaging, including axial oblique and sagittal T2-weighted, dynamic contrast material-enhanced, and diffusion-weighted imaging. Volumetry of the tumor and uterus was performed during the six sequences, with manual tracing of each section, and the tumor volume ratio (TVR) was calculated. ADC histograms were generated from pixel ADCs from the whole tumor volume. The threshold of TVR associated with myometrial invasion was assessed by using receiver operating characteristic curves. An independent sample Mann Whitney U test was used to compare differences in ADCs, skewness, and kurtosis between tumor grade and the presence of lymphovascular invasion. No significant difference in tumor volume and TVR was found among the six MR imaging sequences (P = .95 and .86, respectively). A TVR greater than or equal to 25% allowed prediction of deep myometrial invasion with sensitivity of 100% and specificity of 93% (area under the curve, 0.96; 95% confidence interval: 0.86, 0.99) at axial oblique diffusion-weighted imaging. A TVR of greater than or equal to 25% was associated with grade 3 tumors (P = .0007) and with lymphovascular invasion (P < .0001). There was no significant difference in the ADCs between grades 1 and 2 tumors (P > .05). The minimum, 10th, 25th, 50th, 75th, and 90th percentile ADCs were significantly lower in grade 3 tumors than in grades 1 and 2 tumors (P < .02). The combination of whole tumor volume and ADC can be used for prediction of tumor grade, lymphovascular invasion, and depth of myometrial invasion.

18F-FAZA PET imaging response tracks the reoxygenation of tumors in mice upon treatment with the mitochondrial complex I inhibitor BAY 87-2243.

We describe a noninvasive PET imaging method that monitors early therapeutic efficacy of BAY 87-2243, a novel small-molecule inhibitor of mitochondrial complex I as a function of hypoxia-inducible factor-1α (HIF1α) activity. Four PET tracers [(18)F-FDG, (18)F-Fpp(RGD)2, (18)F-FLT, and (18)F-FAZA] were assessed for uptake into tumor xenografts of drug-responsive (H460, PC3) or drug-resistant (786-0) carcinoma cells. Mice were treated with BAY 87-2243 or vehicle. At each point, RNA from treated and vehicle H460 tumor xenografts (n = 3 each) was isolated and analyzed for target genes. Significant changes in uptake of (18)F-FAZA, (18)F-FLT, and (18)F-Fpp(RGD)2 (P < 0.01) occurred with BAY 87-2243 treatment with (18)F-FAZA being the most prominent. (18)F-FDG uptake was unaffected. (18)F-FAZA tumor uptake declined by 55% to 70% (1.21% ± 0.10%ID/g to 0.35 ± 0.1%ID/g; n = 6, vehicle vs. treatment) in both H460 (P < 0.001) and PC3 (P < 0.05) xenografts 1 to 3 days after drug administration. (18)F-FAZA uptake in 786-0 xenografts was unaffected. Decline occurred before significant differences in tumor volume, thus suggesting (18)F-FAZA decrease reflected early changes in tumor metabolism. BAY 87-2243 reduced expression of hypoxia-regulated genes CA IX, ANGPTL4, and EGLN-3 by 99%, 93%, and 83%, respectively (P < 0.001 for all), which corresponds with reduced (18)F-FAZA uptake upon drug treatment. Heterogeneous expression of genes associated with glucose metabolism, vessel density, and proliferation was observed. Our studies suggest suitability of (18)F-FAZA-PET as an early pharmacodynamic monitor on the efficacy of anticancer agents that target the mitochondrial complex I and intratumor oxygen levels (e.g., BAY 87-2243).

NI-25 * PHYSIOLOGIC IMAGING IN PATIENTS WITH BRAIN METASTASES; HETEROGENEITY BASED UPON PRIMARY TUMOR SUBTYPE

BACKGROUND: Structural and biologic tissue characteristics prior to and following radiosurgery (SRS) can be quantified by analyzing physiologic imaging sequences including diffusion and perfusion weighted imaging (DWI, PWI) and considering their derived parameters including apparent diffusion coefficient (ADC) and relative cerebral blood volume (rCBV). Increasingly in neuro-oncology, these types of imaging sequences are being considered as prognostic and diagnostic markers. Patients' primary tumor type may impact these imaging values, and an understanding of how baseline DWI and PWI measures depend upon primary tumor type is necessary in order to consider how these markers may impact therapeutic response or when considering diagnostic possibilities such as radiation necrosis. METHODS: 78 patients with 187 brain metastases were identified retrospectively from a cohort of patients who underwent SRS between August, 2011 and March 2014 at two institutions. Included subjects had undergone MRI with physiologic (DWI and PWI) imaging prior to SRS. The physiologic sequences were processed and co-registered to anatomic sequences acquired on the day of treatment. Lesions were segmented and ROIs were generated for image analysis using the T1 + Gd sequence. Quantitative measures included rCBV, rCBF, ADC and tumor volume for each lesion. Tumor subtypes included breast, lung, colon, melanoma, renal cell, ovarian, and “other”. RESULTS AND CONCLUSIONS: There was no significant difference in tumor volume based upon tumor type, although there was a trend toward larger tumors at diagnosis for colon and ovarian primaries. There was no significant difference in ADC between tumor type; a significant elevation in rCBV was seen for renal cell and ovarian primary tumors (2.51 and 2.46) when compared with breast, colon, melanoma and lung primary tumors (1.18, 1.24, 1.38, 1.27, respectively). When considering questions of diagnostic specificity, it is important to recognize the relatively low rCBV in many active brain metastases, particularly as compared with primary brain tumors.

Abstract 4509: The fully human, proapoptotic fusion protein Granzyme B/VEGF121 targets tumor vasculature and inhibits tumor growth

Abstract Angiogenesis is a critical process in numerous diseases, and targeting neovascularization has therapeutic value in several disease states including tumor progression and metastatic spread. Vascular endothelial growth factor-A (VEGF-A) and its receptors play a central role in the control of neovascularization. Within the VEGF-A family, VEGF121 is a naturally-occurring splice variant that binds to receptors (VEGFR-1 and VEGFR-2) that are over-expressed on the endothelium of tumor vasculature but not normal vasculature. Granzyme B (GrB) is a serine protease that plays a critical role in the body's defense against viral infection and tumor development by initiating the apoptotic cascade via both caspase-dependent and -independent mechanisms. We expressed GrB/VEGF121 in HEK-293T cells and harvested the secreted fusion protein under serum-free conditions. Endothelial and tumor cells lines showed varying levels of sensitivity to GrB/VEGF121 that correlated closely to total VEGFR-2 expression. IC50 levels were found to be in the nanomolar range. GrB/VEGF121 internalized rapidly into VEGFR-2 expressing cells while the internalization into VEGFR-1 expressing cells was significantly reduced. Forty percent of VEGFR-2+ cells underwent mitochondrial depolarization within 24 h of exposure to GrB/VEGF121, compared to 13% of controls. Over this same time period, 35% of GrB/VEGF121-treated cells mobilized into early apoptosis, compared to 4% of control cells. In vivo, intravenous administration of GrB/VEGF121 to non-tumor bearing mice at doses up to 40 mg/kg had no observed toxicity. GrB/VEGF121 localized specifically in xenografts of non-VEGFR-expressing PC-3 and MDA-MB-231/Luc cells, primarily in perivascular regions and in other areas of the tumor less well-vascularized, whereas free GrB did not localize to tumor tissue. Treatment of mice bearing established prostate PC-3 tumor xenografts with GrB/VEGF121 at 27 mg/kg resulted in the regression of four of five tumors in this group. Tumors showed a two-fold lower Ki-67-labeling index compared with controls. Treatment of mice bearing orthotopic triple negative breast (MDA-MB-231/Luc) tumors with GrB/VEGF121 resulted in statistically significant differences in tumor volumes by Day 39 when compared to controls (saline vs 10 mg/kg GrB/VEGF121: P &amp;lt; 0.04; saline vs 25 mg/kg GrB/VEGF121: P &amp;lt; 0.03). In both tumor models, the vascular area as a percent of tumor area was significantly lower in tumor samples from mice treated with GrB/VEGF121 vs saline (PC-3: 14-fold, P &amp;lt; 0.0001; MDA-MB-231/Luc: 5-fold, P &amp;lt; 0.01) indicating that GrB/VEGF121 treatment directly impacted tumor vasculature. Our results show that targeted delivery of granzyme B to tumor vascular endothelial cells or to tumor cells activates apoptotic cascades and this completely human construct may have significant therapeutic potential. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Yu Cao, Walter N. Hittelman, Michael G. Rosenblum. The fully human, proapoptotic fusion protein Granzyme B/VEGF121 targets tumor vasculature and inhibits tumor growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4509. doi:10.1158/1538-7445.AM2014-4509

Conditional quantile regression models of melanoma tumor growth curves for assessing treatment effect in small sample studies.

Tumor growth curves provide a simple way to understand how tumors change over time. The traditional approach to fitting such curves to empirical data has been to estimate conditional mean regression functions, which describe the average effect of covariates on growth. However, this method ignores the possibility that tumor growth dynamics are different for different quantiles of the possible distribution of growth patterns. Furthermore, typical individual preclinical cancer drug study designs have very small sample sizes and can have lower power to detect a statistically significant difference in tumor volume between treatment groups. In our work, we begin to address these issues by combining several independent small sample studies of an experimental cancer treatment with differing study designs to construct quantile tumor growth curves. For modeling, we use a Penalized Fixed Effects Quantile Regression with added study effects to control for study differences. We demonstrate this approach using data from a series of small sample studies that investigated the effect of a naturally derived biological peptide, P28, on tumor volumes in mice grafted with human melanoma cells. We find a statistically significant quantile treatment effect on tumor volume trajectories and baseline values. In particular, the experimental treatment and a corresponding conventional chemotherapy had different effects on tumor growth by quantile. The conventional treatment, Dacarbazine (DTIC), tended to inhibit growth for smaller quantiles, while the experimental treatment P28 produced slower rates of growth in the upper quantiles, especially in the 95th quantile.

Comparison of biological characteristics between C6 glioma cells and a stable C6 glioma cell line expressing enhanced green fluorescent protein

Objective To establish a stable C6 glioma line expressing enhanced green fluorescent protein (EGFP).Methods C6 glioma cells were transfected with the lentivirus vector enhancer promoter CMV and elongation factor-1α (EF-1α).C6 cells expressing EGFP were observed under the fluorescence microscopy and screened by flow cytometry.Tumorigenicity was evaluated by establishment of mouse skin and intracranial glioma model.Tumor volume was measured,and hematoxylin and eosin (HE) staining,immunohistochemical glial fibrillary acidic protein (GFAP) staining and MRI were used to monitor the tumor volume.Results Morphology observed under a fluorescence microscope and proliferation detected by methyl thiazol tetrazolium (MTT) of EGFP-C6 cells were similar to those of C6 cells (0.53 ±0.02 vs.0.51 ± 0.01).Subcutaneous model results showed there were no significant differences in tumor volume [C6:(2.20 ±0.91) cm3 and EGFP-C6:(2.10 ±0.84) cm3],HE staining and GFAP staining between EGFP-C6 and C6 cells.Intracranial model results showed there were no significant differences in tumor volume calculated by MRI [C6:(20.5±2.3) cm3 and EGFP-C6:(19.5±2.3) cm3],the survival of tumor-bearing rats,tumor formation rate,and the extracranial metastasis rate.Conclusion EGFP-C6 cell line expressing EGFP as a marker was established.Biological characteristics of EGFP-C6 cells were similar to those of C6 cells. Key words: Glioma; Biological characteristics; Enhanced green fluorescent protein

Stratification of 18F-Labeled PET Imaging Agents for the Assessment of Antiangiogenic Therapy Responses in Tumors

Successful antiangiogenic therapies have been developed for the treatment of various cancers, but not all patients respond. Therefore, the early determination of therapy efficacy is essential for patient management. This study was done to evaluate the utility of various PET imaging biomarkers for early determination of the response to therapy with the antiangiogenic agent axitinib, a multiple receptor tyrosine kinase inhibitor, in tumors with diverse biologic characteristics. Mice bearing U87-MG and MDA-MB-231 subcutaneous tumors were treated with axitinib (25 mg/kg intraperitoneally daily for 10 d), and tumor volumes were assessed with caliper measurements. The animals were concurrently imaged longitudinally with (18)F-FDG, 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT), and 2-(18)F-fluoroethyl-triazolyl conjugated c(RGDyK) peptide ((18)F-FtRGD) to determine the optimal radiopharmaceutical for measuring the early treatment response in the 2 tumor types. Daily administration of axitinib successfully retarded the growth of both U87-MG and MDA-MB-231 subcutaneous tumors, with significant differences in tumor volumes being observed from day 7 after therapy on. (18)F-FDG revealed a treatment efficacy response only at day 10 after treatment in both U87-MG tumor-bearing and MDA-MB-231 tumor-bearing animals. (18)F-FLT afforded earlier detection of the therapy response, revealing a significant difference between drug- and vehicle-treated animals at day 3 for animals bearing U87-MG tumors and at day 7 for animals bearing the more slowly growing MDA-MB-231 tumors. (18)F-FtRGD showed a rapid change in tumor retention that reached significance by day 7 in U87-MG tumor-bearing animals; in contrast, no significant difference in tumor retention was observed in MDA-MB-231 tumor-bearing animals. Longitudinal imaging with different radiopharmaceuticals displays various characteristics in different tumor types, depending on their biologic characteristics. Such studies may provide clinically important information to guide patient management and monitor the response to antiangiogenic therapy with the optimum noninvasive imaging agent in the relevant cancer type.

Monitoring Tumor Response to Linifanib Therapy with SPECT/CT Using the Integrinαvβ3–Targeted Radiotracer99mTc-3P-RGD2

The objective of this study was to determine the utility of (99m)Tc-3P-Arg-Gly-Asp (RGD2) single photon emission computed tomography (SPECT)/computed tomography (CT) for noninvasive monitoring of integrin αvβ3-expression response to antiangiogenic treatment with linifanib. Linifanib or vehicle therapy was carried out in female athymic nu/nu mice bearing U87MG glioma (high αvβ3 expression) or PC-3 prostate (low αvβ3 expression) tumors at 12.5 mg/kg twice daily. The average tumor volume was 180 ± 90 mm(3) the day prior to baseline SPECT/CT. Longitudinal (99m)Tc-3P-RGD2 SPECT/CT imaging was performed at baseline (-1 day) and days 1, 4, 11, and 18. Tumors were harvested at all imaging time points for histopathological analysis with H&E and immunohistochemistry. A significant difference in tumor volumes between vehicle- and linifanib-treated groups was observed after 4 days of linifanib therapy in the U87MG model. The percent injected dose (%ID) tumor uptake of (99m)Tc-3P-RGD2 peaked in the vehicle-treated group at day 11, while the %ID/cm(3) tumor uptake decreased slowly over the whole study period. During the first 2 days of linifanib treatment, a rapid decrease in both %ID/cm(3) tumor uptake and tumor/muscle ratios of (99m)Tc-3P-RGD2 was observed, followed by a slow decrease until day 18. No decrease in tumor uptake of (99m)Tc-3P-RGD2 or tumor volume was observed for either treatment group in the PC-3 model. Changes in tumor vasculature were confirmed by histopathological H&E analysis and immunohistochemistry. Longitudinal imaging using (99m)Tc-3P-RGD2 SPECT/CT may be a useful tool for monitoring the downstream biologic effects of linifanib therapy.

Abstract 948: In vivo evaluation of IGF2 RNA interference in a mouse xenograft model of paclitaxel-resistant human ovarian cancer.

Abstract Introduction. We previously showed that paclitaxel-resistance of human ovarian carcinoma cell lines was reverted by knockdown of IGF2 with RNA interference. The aim of this study was to determine if our paclitaxel-resistant cell line HEY-T30 was resistant to paclitaxel treatment in vivo and if this resistance could be reverted by IGF2 knockdown through short hairpin RNA. Methods. Paclitaxel-resistant HEY-T30 cells, derived in our laboratory from HEY ovarian carcinoma cells, were transfected with a plasmid containing shRNA to target IGF2 (shIGF2-p) or a nontargeting shRNA (shScrambled). Stably transfected clones were evaluated by reverse transcription real-time PCR for IGF2 mRNA expression and by sulforhodamine cytotoxicity assay for paclitaxel sensitivity. For in vivo experiments, HEY, HEY-T30, shIGF2-p or shScrambled cells were suspended in OptiMem and one million cells/animal injected subcutaneously into 4-week-old female nude mice. Tumors were allowed to grow to a mean volume of 125 mm3, prior to treatment initiation with paclitaxel (20 mg/kg administered intraperitoneally every 3 days x 5 doses) or vehicle (5% dextrose in water; D5W). Animals were weighed and tumors were measured with digital calipers three times per week. Effect of treatment was calculated as effect=1-(T/C) at the indicated time point, where T is the mean tumor volume of the paclitaxel treatment group and C is the mean tumor volume of the vehicle control group. Significance was calculated with a 2-way repeated measures ANOVA with a Bonferroni multiple comparisons post-test. Results. IGF2 mRNA levels of shIGF2-p cells were comparable to HEY, whereas shScrambled was similar to HEY-T30. The IC50s (concentration of 50% inhibition of proliferation) for paclitaxel in vitro were HEY: 2.3 nM; HEY-T30: 164.0 nM; shIGF2-p: 13.89 nM; shScrambled: 140.0 nM. HEY xenografts responded well to paclitaxel treatment, showing a significant growth retardation compared to the D5W group beginning at 9 days after treatment initiation (p=0.0005), with an effect of treatment of 83.2% at day 19. In contrast, HEY-T30 xenografts showed no significant difference in tumor volume between the paclitaxel-treated and the D5W group at any time point, confirming its resistance to paclitaxel (the effect of treatment was 5.5%). shScrambled xenografts were similarly resistant to paclitaxel as HEY-T30 xenografts. shIGF2-p xenografts responded well to paclitaxel treatment, showing a difference in tumor volume from day 15 onward (day 15, p=0.0457; day 19, p=0.0006) with an effect of treatment of 64.1% after 22 days. Discussion. HEY-T30 xenografts are resistant to paclitaxel treatment, whereas HEY xenografts respond well. IGF2 knockdown by shRNA restores paclitaxel sensitivity to the HEY-T30 xenografts. These in vivo results confirm our prior observations in cell lines and suggest a novel target for the treatment of paclitaxel-resistant disease. Citation Format: Jurriaan Brouwer-Visser, Jiyeon Lee, María J. Cossio, Gloria S. Huang. In vivo evaluation of IGF2 RNA interference in a mouse xenograft model of paclitaxel-resistant human ovarian cancer. [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 948. doi:10.1158/1538-7445.AM2013-948

Correlation between nasopharyngeal carcinoma tumor volume and the 2002 International Union Against Cancer tumor classification system

BackgroundThe correlation between primary tumor volume and nasopharyngeal carcinoma (NPC) UICC 2002 T classification, N classification and distant metastasis after radiation therapy was discussed to provide further evidence for the inclusion of tumor volume into the TNM classification staging system.MethodsBetween February 2001 and December 2008, 666 patients with NPC treated with intensity-modulated radiation therapy (IMRT) were analyzed retrospectively. Primary gross tumor volume was calculated from treatment planning computed tomography scans. The Kruskal-Wallis and Mann–Whitney tests were used for comparison of continuous variables and the chi-square test was used for categorical variables. A logistic regression model was used for multivariate analysis.ResultsMedian primary tumor volume of the 666 patients was 20.35 ml (range, 0.44 − 192.63 ml), and it gradually increased with T classification. Statistically significant differences in tumor volume were observed between patients with different T classifications (p < 0.001). The cervical lymph node metastasis rate was 64.7% (430/666); the differences in primary tumor volume between patients with or without lymph node metastasis were statistically significant (p < 0.001). Posttreatment distant metastasis occurred in 100 NPC patients, and the five-year distant metastasis-free survival was 84.2%. Univariate and multivariate analyses showed that N classification (p < 0.001) and tumor volume (p = 0.007) were the main factors influencing distant metastasis.ConclusionTumor volume was correlated with T classification, cervical lymph node mestastasis and distant metastasis after radiation therapy in nasopharyngeal carcinoma, suggesting that tumor volume should be included into the TNM staging system.