EMT6 Tumors Research Articles

In Vitro and In Vivo Evaluation of [18F]F-GAZ, a Novel Oxygen-Mimetic Azomycin-Glucose Conjugate, for Imaging Hypoxic Tumor

Several F-18-labeled 2-nitroimidazole (azomycin) derivatives have been proposed for imaging hypoxia using positron emission tomography (PET). Their cell penetration is based on passive diffusion, which limits their intracellular concentration maxima. The purpose of this study was to investigate the uptake of N-(2-[(18)F]fluoro-3-(6-O-glucosyl)propyl-azomycin ([(18)F]F-GAZ), a new azomycin-glucose conjugate, in vitro and in vivo. [(18)F]F-GAZ was synthesized from its tetraacetyl nosylate precursor by nucleophilic radiofluorination. [(18)F]F-GAZ was evaluated in vivo in EMT-6 tumor-bearing Balb/C mice utilizing the PET and biodistribution analysis. In vitro uptake of [(18)F]FDG by EMT-6 cells was measured in the presence of unlabeled F-GAZ, 2-FDG, and D-glucose. [(18)F]F-GAZ was rapidly cleared from all tissues, including the blood pool and kidneys, with ultimate accumulation in the urinary bladder. Uptake of tracer doses of [(18)F]F-GAZ into EMT-6 tumors was fast, reaching a standardized uptake value of 0.66±0.05 within 5-6 minutes postinjection (p.i.), and decreased to 0.24±0.04 by 60 minutes p.i. (n=6). A tumor-muscle ratio of 1.87±0.18 was observed after 60 minutes. Total uptake of [(18)F]F-GAZ in tumors (60 minutes) amounted to 1.25%±0.15% ID/g versus 0.61%±0.14% ID/g (n=4) in muscle. Similar biodistribution and excretion were observed using carrier-added (100 mg/kg) doses of F-GAZ. In vitro, D-glucose and unlabeled 2-FDG were two orders of magnitude more potent than F-GAZ as competitive inhibitors of [(18)F]FDG uptake into EMT-6 cells. Besides its interaction with glucose transporters, F-GAZ seems to be not transported in the presence of glucose. Furthermore, [(18)F]F-GAZ is unlikely to be effective as a hypoxia imaging agent. The low in vivo toxicity and substantial retention in tumor observed at high doses of F-GAZ do provide rationale for further testing as a radiosensitizer for external beam radiation therapy of radioresistant, hypoxic tumors.

Fluence Rate‐Dependent Photobleaching of Intratumorally Administered Pc 4 Does not Predict Tumor Growth Delay

We examined effects of fluence rate on the photobleaching of the photosensitizer Pc 4 during photodynamic therapy (PDT) and the relationship between photobleaching and tumor response to PDT. BALB/c mice with intradermal EMT6 tumors were given 0.03 mg kg(-1) Pc 4 by intratumor injection and irradiated at 667 nm with an irradiance of 50 or 150 mW cm(-2) to a fluence of 100 J cm(-2). While no cures were attained, significant tumor growth delay was demonstrated at both irradiances compared with drug-only controls. There was no significant difference in tumor responses to these two irradiances (P = 0.857). Fluorescence spectroscopy was used to monitor the bleaching of Pc 4 during irradiation, with more rapid bleaching with respect to fluence shown at the higher irradiance. No significant correlation was found between fluorescence photobleaching and tumor regrowth for the data interpreted as a whole. Within each treatment group, weak associations between photobleaching and outcome were observed. In the 50 mW cm(-2) group, enhanced photobleaching was associated with prolonged growth delay (P = 0.188), while at 150 mW cm(-2) this trend was reversed (P = 0.308). Thus, it appears that Pc 4 photobleaching is not a strong predictor of individual tumor response to Pc 4-PDT under these treatment conditions.

Photodynamic therapy-mediated DC immunotherapy is highly effective for the inhibition of established solid tumors

Dendritic cell (DC)-mediated immunotherapy has not been as effective as expected for most solid tumors. This study demonstrates immune responses against solid tumors mediated by DCs charged with photodynamic therapy (PDT)-induced tumor lysates, which contain the heat shock protein (HSP) 70. PDT tumor lysate-pulsed DC (PDT-DC) inhibited the growth of mammary EMT6 tumors to a greater extent than freeze/thawed tumor lysate-pulsed DC (FT-DC) or PDT tumor lysates. PDT-DC also showed significant anti-tumor effects against fully-established (i.e., late-stage) solid tumors. Taken together, these results suggest that PDT-DC vaccination is more effective than conventional DC therapy for the treatment of cancers.

Abstract 3539: Anti-CSF-1R antibodies reduce tumor-associated macrophages and inhibit tumor growth in preclinical models

Abstract In cancer, increased infiltration of macrophages within and surrounding the tumor mass correlates with increased tumor invasiveness and growth. In addition, presence of tumor-associated macrophages (TAMs) has been shown to correlate with poor prognosis, particularly in breast, prostate, ovarian and cervical cancer. TAM proliferation, differentiation and survival is dependent on Colony Stimulating Factor - 1 Receptor (CSF-1R) activation, a type III integral membrane tyrosine kinase receptor selectively expressed on cells of the mononuclear phagocyte lineage. Given that TAMs enhance tumor growth and that activation of the CSF-1R pathway is required for TAM function, an antibody against mouse CSF-1R was generated for proof-of-principle studies. CS7, a monoclonal anti-mouse CSF-1R antibody inhibited both CSF-1 and IL-34 binding to mouse CSF-1R, leading to inactivation of the receptor and downstream signaling molecules. In addition, CS7 prevented monocyte proliferation and macrophage differentiation with IC50s of 0.1 nM and 0.75 nM, respectively. In murine models of breast cancer using CSF-1-secreting MDA-MB-231, Hcc1954, 4T1 or EMT6 tumor cells, CS7 treatment led to a marked reduction in TAMs and an associated decrease in tumor growth. In contrast, breast tumor xenografts with CSF-1 non-secreting breast tumor cell lines JimT1 and MCF-7 had limited or no decrease in tumor volume following CS7 treatment. In prostate models, DU145 tumor cell line xenografts (CSF-1-secreting) but not PC3 (CSF-1 non-secreting) tumor growth was inhibited by CS7, recaptitulating the results seen with breast xenografts. Thus, in breast and prostate preclinical models, CSF-1 secretion by tumor cells is a prerequisite for sensitivity to anti-CSF-1R treatment. Taken together, targeting CSF-1R with a monoclonal antibody inhibits CSF-1R signaling via CSF-1 and IL-34, prevents monocytic to macrophage differentiation, and reduces tumor volume in preclinical models, validating CSF-1R as a target for therapeutic application in cancer. 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 3539. doi:1538-7445.AM2012-3539

Abstract 5246: Use of γH2aX and other biomarkers to quantify distribution of drug activity in solid tumors

Abstract Background: The disorganized vasculature within solid tumors leads to decreased drug delivery and poor penetration of drugs from blood vessels to the tumor cells. Our laboratory has used quantitative immunohistochemistry (IHC) to show limited intratumoral distribution of the auto-fluorescent drugs doxorubicin and mitoxantrone. Most anti-cancer drugs are not fluorescent and do not have antibodies that recognize them; there is a need for biomarkers that can be used to quantify their distribution and efficacy in different regions of solid tumors. The aim of the present study was to characterize the spatial distribution within solid tumors of biomarkers of drug effect that can be used to test drug efficacy. Methods: Cleaved caspase 3 (a marker of apoptosis), γH2aX (a marker of DNA damage) and Ki67 (a marker of cell proliferation) were quantified in tumor sections in relation to blood vessels (recognized by CD31) using monoclonal antibodies and IHC. To validate the use of these biomarkers we compared their time-dependent distribution with (i) the distribution of auto-fluorescent doxorubicin and (ii) the distribution of a fluorescent monoclonal antibody that detects melphalan-induced DNA adducts. The biomarkers were then used to study the distribution of docetaxel in relation to tumor blood vessels. Results: The distributions of doxorubicin in MCF7 & MDA-MB-231 xenografts and of melphalan-induced DNA adducts in MCF-7 & EMT-6 tumors decreased exponentially with increasing distance from tumor blood vessels; they were similar to the distribution of γH2aX at 10 minutes after injection of these drugs. The distributions of cleaved caspase 3 and of change in Ki-67 at 24 hours following treatment also mirrored the distribution of the drugs. The distribution of these biomarkers following injection of docetaxel also showed a sharp decrease in drug activity with increasing distance from tumor blood vessels in solid tumors (MCF-7, PC-3 & A431). Conclusions: Activation of γH2aX occurs within 10 minutes after injection of several drugs and is the preferred biomarker for studying distribution of drug activity in tumors. There is heterogeneity of activity of many anticancer drugs in solid tumors, with rapid decrease in activity with increasing distance from tumor blood vessels. Poor distribution of drugs to cells within tumors is an important cause of drug resistance. Supported by a research grant from the Canadian Institutes of Health Research 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 5246. doi:1538-7445.AM2012-5246

Influence of coupling bonds on the anti-tumor activity of polymer–pirarubicin conjugates

Pirarubicin (THP) was conjugated onto the pendant carboxyl groups of poly(ethylene glycol)-block-poly(l-lactide-co-2-methyl-2-carboxyl-propylene carbonate) [PEG-b-P(LA-co-MCC)] through hydrazone, ester, and amide bonds, respectively, and the conjugates were assembled into micelles with diameters between 30 and 60nm. The in vitro THP release of the three conjugate micelles was conducted in pH 7.4 and 5.0 buffer solutions, and conjugate micelles with hydrazone linkage had the fastest THP release rate. Their in vitro cytotoxicity was tested using mouse mammary adenocarcinoma EMT6 cells and in vivo anti-tumor activity in Balb/c mice models bearing EMT6 tumors were compared with free THP and with each other. The results showed that the polymer–THP conjugates displayed higher cell-uptakes and better anti-tumor activities than free THP at 4h, and among the three micelles, those with hydrazone linkage had the highest anti-tumor activity in vivo, while those with amide linkage were the lowest.

Tumor-targeted drug delivery using MR-contrasted docetaxel – Carboxymethylcellulose nanoparticles

A carboxymethylcellulose-based polymer conjugate with nanoparticle forming properties (Cellax) has been shown to enhance the pharmacokinetics, specificity of biodistribution, anti-tumor efficacy and safety of docetaxel (DTX) in comparison to the Taxotere™ formulation. We examined Cellax and Taxotere efficacy in four tumor models (EMT-6, B16F10, PC3, and MDA-MB-231), and observed variances in efficacy. To explore whether differences in tumor uptake of Cellax were responsible for these effects, we incorporated superparamagnetic iron oxide nanoparticles (SPIONs) into Cellax particles to enable magnetic resonance (MR) imaging (Cellax-MR). In the EMT-6 tumor model, Cellax-MR nanoparticles exhibited peak tumor accumulation 3–24 h post intravenous injection, and 3 days post-treatment, significant MR contrast was still detected. The amount of Cellax-MR deposited in the EMT-6 tumors was quantifiable as a hypointense volume fraction, a value positively correlated with drug content as determined by LC/MS analysis (R2 = 0.97). In the four tumor models, Cellax-MR uptake was linearly associated with anti-tumor efficacy (R2 > 0.9), and was correlated with blood vessel density (R2 > 0.9). We have affirmed that nanoparticle uptake is variable in tumor physiology, and that this efficacy-predictive parameter can be non-invasively estimated in real-time using a theranostic variant of Cellax.

Microfluidic technology: An economical and versatile approach for the synthesis of O-(2-[18F]fluoroethyl)-l-tyrosine ([18F]FET)

A new synthesis of O-(2-[18F]fluoroethyl)-l-tyrosine [18F]FET was developed using a NanoTek® microfluidic synthesis system (Advion BioSciences, Inc.). Optimal reaction conditions were studied through screening different reaction parameters like temperature, flow rate, reaction time, concentration of the labeling precursor, and the applied volume ratio between the labeling precursor and [18F]fluoride. [18F]FET was obtained after HPLC purification with 50% decay-corrected radiochemical yield starting from as little as 40μg of labeling precursor. Small animal PET studies in EMT-6 tumor bearing mice showed radioactivity accumulation in the tumor (SUV60min 1.21±0.2) resulting in an slightly increasing tumor-to-muscle ratio over time.

Confocal fluorescence imaging enables noninvasive quantitative assessment of host cell populations in vivo following photodynamic therapy.

We report the use of optical imaging strategies to noninvasively examine photosensitizer distribution and physiological and host responses to 2-[1-hexyloxyethyl]-2 devinyl pyropheophorbide-a (HPPH)-mediated photodynamic therapy (PDT) of EMT6 tumors established in the ears of BALB/c mice. 24 h following intravenous (IV) administration of 1 μmol kg-1 HPPH, wide-field fluorescence imaging reveals tumor selectivity with an approximately 2-3-fold differential between tumor and adjacent normal tissue. Confocal microscopy demonstrates a relatively homogeneous intratumor HPPH distribution. Labeling of host cells using fluorophore-conjugated antibodies allowed the visualization of Gr1+/CD11b+ leukocytes and major histocompatibility complex class II (MHC-II)+ cells in vivo. Imaging of the treated site at different time-points following irradiation shows significant and rapid increases in Gr1+ cells in response to therapy. The maximum accumulation of Gr1+ cells is found at 24 h post-irradiation, followed by a decrease at the 48 h time-point. Using IV-injected FITC-conjugated dextran as a fluorescent perfusion marker, we imaged tissue perfusion at different times post-irradiation and found that the reduced Gr1+ cell density at 48 h correlated strongly with functional damage to the vasculature as reported via decreased perfusion status. Dual color confocal imaging experiments demonstrates that about 90% of the anti-Gr1 cell population co-localized with anti-CD11b labeling, thus indicating that majority of the Gr1-labeled cells were neutrophils. At 24 h post-PDT, an approximately 2-fold increase in MHC-II+ cells relative to untreated control is also observed. Co-localization analysis reveals an increase in the fraction of Gr1+ cells expressing MHC-II, suggesting that HPPH-PDT is stimulating neutrophils to express an antigen-presenting phenotype.

Preclinical evaluation of Laromustine for use in combination with radiation therapy in the treatment of solid tumors

Purpose: These studies explored questions related to the potential use of Laromustine in the treatment of solid tumors and in combination with radiotherapy.Materials and methods: The studies used mouse EMT6 cells (both parental and transfected with genes for O6-alkylguanine-DNA transferase [AGT]), repair-deficient human Fanconi Anemia C and Chinese hamster VC8 (BRCA2-/-) cells and corresponding control cells, and EMT6 tumors in mice assayed using cell survival and tumor growth assays.Results: Hypoxia during Laromustine treatment did not protect EMT6 cells or human fibroblasts from this agent. Rapidly proliferating EMT6 cells were more sensitive than quiescent cultures. EMT6 cells expressing mouse or human AGT, which removes O6-alkyl groups from DNA guanine, thereby protecting against G-C crosslink formation, increased resistance to Laromustine. Crosslink-repair-deficient Fanconi Anemia C and VC8 cells were hypersensitive to Laromustine, confirming the importance of crosslinks as lethal lesions. In vitro, Laromustine and radiation produced additive toxicities to EMT6 cells. Studies using tumor cell survival and tumor growth assays showed effects of regimens combining Laromustine and radiation that were compatible with additive or subadditive interactions. Conclusions: The effects of Laromustine on solid tumors and with radiation are complex and are influenced by microenvironmental and proliferative heterogeneity within these malignancies.

Long-Term Effect on EMT6 Tumors in Mice Induced by Combination of Laser Immunotherapy and Surgery

Laser immunotherapy (LI) has been demonstrated to be a promising modality for cancer treatment. The present study was designed to further investigate the impact of LI combined with surgery. LI consists of a near-infrared laser, a light-absorbing dye (indocyanine green, ICG), and an immunostimulant (glycated chitosan, GC). ICG and GC were intratumorally injected, followed by laser irradiation. Female BALB/c mice bearing EMT6 tumor cells were divided into 4 groups: control, LI, LI followed by immediate surgery resection of residual tumor (LI + S(0wk)), and LI followed by surgical removal of residual tumor after 1 week (LI + S(1wk)). Successfully treated mice from all treatment groups were rechallenged twice with 10(5) and 5 × 10(5) EMT6 cells, respectively. The LI + S(1wk) group had the highest survival rate (72%) after 90 days, whereas the mice survival rates of the LI + S(0wk), LI, and control groups were 50%, 46%, 0%, respectively. The median survival times of control, LI, LI + S(0wk), and LI + S(1wk) groups were 32, 66, 74, and 90 days, respectively. Survival rates of the treated mice after the first and second tumor rechallenges, ranging from 73% to 95%, were not significantly different among the 4 groups (P > .05). The results show that LI is a useful tool for the treatment of tumor-bearing mice. Long-term antitumor effect can be induced by LI. They also indicate that combination of LI with surgery can further improve the therapeutic efficiency of LI.

Compatibility of the Linear-Quadratic Formalism and Biologically Effective Dose Concept to High-Dose-Per-Fraction Irradiation in a Murine Tumor

To evaluate the compliance of linear-quadratic (LQ) model calculations in the high-dose range as used in stereotactic irradiation in a murine tumor model. Female 10-week-old Balb/c mice bearing 1-cm-diameter EMT6 tumors in the hind legs were used. Single doses of 10-25 Gy were compared with 2-5 fractions of 4-13 Gy given at 4-hour intervals. Cell survival after irradiation was determined by an invivo-invitro assay. Using an α/β ratio determined for invitro EMT6 cells and the LQ formalism, equivalent single doses for the hypofractionated doses were calculated. They were then compared with actually measured equivalent single doses for the hypofractionated doses. These fractionation schedules were also compared simultaneously to investigate the concordance/divergence of dose-survival curves plotted against actual radiation doses and biologically effective doses (BED). Equivalent single doses for hypofractionated doses calculated from LQ formalism were lower than actually measured doses by 21%-31% in the 2- or 3-fraction experiments and by 27%-42% in the 4- or 5-fraction experiments. The differences were all significant. When a higher α/β ratio was assumed, the discrepancy became smaller. In direct comparison of the 2- to 5-fraction schedules, respective dose-response curves almost overlapped when cell survival was plotted against actual radiation doses. However, the curves tended to shift downward by increasing the fraction number when cell survival was plotted against BED calculated using an α/β ratio of 3.5 Gy for invitro EMT6 cells. Conversion of hypofractionated radiation doses to single doses using the LQ formalism underestimated the invivo effect of hypofractionated radiation by approximately 20%-40%. The discrepancy appeared to be larger than that seen in the previous invitro study and tended to increase with the fraction number. BED appeared to be an unreliable measure of tumor response.

Enhancement of the radiation response of EMT-6 tumours by a copper octabromotetracarboranylphenylporphyrin

The carborane-containing porphyrin, copper (II) 2,3,7,8,12,13,17,18-octabromo-5,10,15,20-tetrakis(3-[1,2-dicarba-closo-dodecaboranyl]methoxyphenyl)-porphyrin (CuTCPBr), was investigated as a potential radiation enhancing agent for X-ray radiotherapy (XRT) in a subcutaneously implanted EMT-6 murine carcinoma. The biodistribution and toxicological profile of this porphyrin has been shown to be favourable for another bimodal radiotherapy technique, boron neutron-capture therapy. For the XRT studies, CuTCPBr was formulated in either 9% Cremophor (BASF Corporation, Ludwigschafen, Germany) EL and 18% propylene glycol (9% CRM) or a revised formulation comprising 1% Cremophor ELP, 2% Tween 80 (JT Baker, Mansfield, MA), 5% ethanol and 2.2% PEG 400 (CTEP formulation), which would be more clinically acceptable than the original 9% CRM formulation. Using the 9% CRM formulation of CuTCPBr, doses of 100, 210 or 400 mg kg(-1) of body weight were used in combination with single doses of 25-35 Gy 100 kVp X-rays. While doses of 100 mg kg(-1) and 210 mg kg(-1) did not result in any significant enhancement of tumour response, the 400 mg kg(-1) dose did. A dose modification factor of 1.20±0.10 was obtained based on the comparison of doses that produced a 50% local tumour control probability. With the CTEP formulation of CuTCPBr, doses of 83 and 170 mg kg(-1) produced significant radiation enhancement, with dose modification factors based on the TCP(50) of 1.29±0.15 and 1.84±0.24, respectively. CuTCPBr significantly enhanced the efficacy of XRT in the treatment of EMT-6 carcinomas in mice. The CTEP formulation showed a marked improvement, with over 9% CRM being associated with higher dose modification factors. Moreover, the radiation response in the skin was not enhanced.

A comparative small-animal PET evaluation of [11C]tariquidar, [11C]elacridar and (R)-[11C]verapamil for detection of P-glycoprotein-expressing murine breast cancer

One important mechanism for chemoresistance of tumours is overexpression of the adenosine triphosphate-binding cassette transporter P-glycoprotein (Pgp). Pgp reduces intracellular concentrations of chemotherapeutic drugs. The aim of this study was to compare the suitability of the radiolabelled Pgp inhibitors [(11)C]tariquidar and [(11)C]elacridar with the Pgp substrate radiotracer (R)-[(11)C]verapamil for discriminating tumours expressing low and high levels of Pgp using small-animal PET imaging in a murine breast cancer model. Murine mammary carcinoma cells (EMT6) were continuously exposed to doxorubicin to generate a Pgp-overexpressing, doxorubicin-resistant cell line (EMT6AR1.0 cells). Both cell lines were subcutaneously injected into female athymic nude mice. One week after implantation, animals underwent PET scans with [(11)C]tariquidar (n = 7), [(11)C]elacridar (n = 6) and (R)-[(11)C]verapamil (n = 7), before and after administration of unlabelled tariquidar (15 mg/kg). Pgp expression in tumour grafts was evaluated by Western blotting. [(11)C]Tariquidar showed significantly higher retention in Pgp-overexpressing EMT6AR1.0 compared with EMT6 tumours: the mean ± SD areas under the time-activity curves in scan 1 from time 0 to 60 min (AUC(0-60)) were 38.8 ± 2.2 min and 25.0 ± 5.3 min (p = 0.016, Wilcoxon matched pairs test). [(11)C]Elacridar and (R)-[(11)C]verapamil were not able to discriminate Pgp expression in tumour models. Following administration of unlabelled tariquidar, both EMT6Ar1.0 and EMT6 tumours showed increases in uptake of [(11)C]tariquidar, [(11)C]elacridar and (R)-[(11)C]verapamil. Among the tested radiotracers, [(11)C]tariquidar performed best in discriminating tumours expressing high and low levels of Pgp. Therefore [(11)C]tariquidar merits further investigation as a PET tracer to assess Pgp expression levels in solid tumours.

Abstract 560: The use of molecular biomarkers to quantify drug distribution in solid tumors

Abstract The tumor microenvironment plays a critical role in the efficacy of chemotherapy. The disorganized vasculature within solid tumors leads to decreased drug delivery to the tumor and poor penetration of drugs from blood vessels to the tumor cells. Our laboratory has shown limited distribution of the auto-fluorescent drugs doxorubicin and mitoxantrone using quantitative immunohistochemistry. However, most anti-cancer drugs are not fluorescent and do not have antibodies that recognize them. The aim of the present study is to characterize the spatial distribution within solid tumors of biomarkers of drug effect: cleaved caspase 3 (a marker of apoptosis), γH2aX (a marker of DNA damage) and Ki67 (a marker of cell proliferation) in relation to blood vessels (recognized by an antibody to CD31). We have quantified the time-dependent distribution of these biomarkers in two models: human breast cancer MDA-MB-231 xenografts and murine mammary EMT6 syngeneic tumors following treatment with doxorubicin and melphalan respectively. Biomarker distributions have been compared to distributions of doxorubicin fluorescence. Melphalan's distribution within a solid tumor has been assessed for the first time with the use of a melphalan-DNA adduct recognizing monoclonal antibody. These studies are designed to provide a basis for selecting biomarkers to reflect the distribution of other non-fluorescent agents. The distribution of doxorubicin decreases exponentially with increasing distance from blood vessels, and is similar to the distribution of γH2aX at 10 minutes after drug injection. The activation of cleaved caspase 3 (indicating apoptosis) at 24 and 48 hours following treatment also mirrors the distribution of doxorubicin. The analysis of biomarker distributions in EMT6 tumors following treatment with melphalan in relation to tumor blood vessels is in progress. The results of these studies will be applied to quantify the intratumoral distribution of other non-fluorescent anticancer drugs (hypoxia-activated pro-drugs). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 560. doi:10.1158/1538-7445.AM2011-560

Abstract LB-164: B lymphocytes inhibit anti-tumor response in a murine EMT6 tumor model by facilitating expansion of T regulatory cells

Abstract B lymphocytes play a central role in inhibiting the immune response against certain tumors, but the underlying mechanisms by which B cells facilitate tumor growth are still poorly understood. Murine EMT6 mammary tumors grow readily in immune competent mice (ICM) but poorly in B-cell deficient μ−/− BALB/c mice (BCDM). Both decreased T regulatory cell (Treg) expansion and impaired Treg function were observed in BCDM compared to ICM. In this study, we investigated how the presence or absence of B cells affects expansion of Tregs in response to tumor implantation. We compared tumor growth, and the number of Treg cells, T cell function, number and character of tumor infiltrating cells, immune effector molecule, chemokine and cytokine expression in tumor tissue in ICM, compared to BCDM and B cell reconstituted BCDM (BCDM+B). Partial reconstitution of BCDM with adoptively transferred B cells restored tumor growth. Transfer of both normal and IL-10−/− B cells restored tumor growth in BCDM. Restored tumor growth correlated with an increase in CD4+FoxP3+ Treg cells in the spleen and draining lymph nodes and a decrease in interferon-γ secretion and cytotoxic T cell activity against EMT6 targets, in BCDM+B to BCDM. Tumor growth was completely inhibited in BCDM+B by depletion of Treg using PC61 antibody. Markedly increased levels of NK and CD8+ T cell infiltration into the tumor bed were seen in BCDM or BCDM+B treated with Treg depletion, compared to levels of infiltration seen in ICM or BCDM+B mice. PCR array analysis of mRNA expression in tumor tissue in BCDM, BCDM+B treated with PC61 antibody, or ICM following PC61 treatment, revealed down regulation of IL-17A, IL-17F, TGF-β and IL-23, CD73, VEGF-C, Rorγt and CCR4, when compared to either BCDM+B or BALB/c control mice. We observed similar changes in immune effector molecules/cytokines mRNA in all scenarios in which Treg levels were decreased. In conclusion, our studies indicate that the T cell anti-tumor response is enhanced in the absence of B cells, in part due to effects on Treg cell expansion and changes in immune effector molecule and cytokine profile in the tumor micro-environment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-164. doi:10.1158/1538-7445.AM2011-LB-164

Targeted Delivery of an Antibody–Mutant Human Endostatin Fusion Protein Results in Enhanced Antitumor Efficacy

The antiangiogenic protein endostatin showed considerable preclinical antitumor activity, but limited efficacy in phase I/II trials. Prior studies using an anti-HER2 antibody-murine endostatin fusion showed enhanced antitumor activity compared to anti-HER2 antibody or endostatin given alone, or in combination. We have generated two anti-HER2 human endostatin fusion proteins by fusing either wild-type or a mutant human endostatin (huEndo-P125A) to the 3' end of a humanized anti-HER2 IgG3 antibody. Antitumor efficacy was examined in murine and human breast tumor models. HuEndo-P125A antibody fusion protein (αHER2-huEndo-P125A) inhibited VEGF and bFGF induced endothelial cell proliferation, and tube formation in vitro, more efficiently than endostatin alone, wild-type endostatin fusion protein (αHER2-huEndo), or parental anti-HER2 antibody (αHER2 IgG3). Wild-type and mutant human endostatin was rapidly cleared from serum in mice (T½(2) = 2.0-2.1 hours), whereas αHER2-huEndo fusion proteins had a significantly prolonged half-life (T½(2) = 40.7-57.5 hours). Treatment of SK-BR-3 breast cancer xenografts with anti-HER2 IgG3-huEndo-P125A fusion resulted in greater inhibition of tumor growth and improved survival, compared to treatment with either αHER2 IgG3 (P = 0.025), human endostatin (P = 0.034), or anti-HER2 IgG3-huEndo (P = 0.016). αHER2-huEndo-P125A specifically inhibited tumors expressing HER2 in mice simultaneously implanted with murine mammary tumor EMT6 cells and with EMT6 engineered to express HER2 antigen (EMT6-HER2). Targeting of endostatin using antibody fusion proteins could improve antitumor activity of either anti-HER2 antibody and/or endostatin and provides a versatile approach that could be applied to other tumor targets with alternative antibody specificities.

Radiopharmacological evaluation of 6-deoxy-6-[18F]fluoro-d-fructose as a radiotracer for PET imaging of GLUT5 in breast cancer

Several clinical studies have shown low or no expression of GLUT1 in breast cancer patients, which may account for the low clinical specificity and sensitivity of 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) used in positron emission tomography (PET). Therefore, it has been proposed that other tumor characteristics such as the high expression of GLUT2 and GLUT5 in many breast tumors could be used to develop alternative strategies to detect breast cancer. Here we have studied the in vitro and in vivo radiopharmacological profile of 6-deoxy-6-[(18)F]fluoro-D-fructose (6-[(18)F]FDF) as a potential PET radiotracer to image GLUT5 expression in breast cancers. Uptake of 6-[(18)F]FDF was studied in murine EMT-6 and human MCF-7 breast cancer cells over 60 min and compared to [(18)F]FDG. Biodistribution of 6-[(18)F]FDF was determined in BALB/c mice. Tumor uptake was studied with dynamic small animal PET in EMT-6 tumor-bearing BALB/c mice and human xenograft MCF-7 tumor-bearing NIH-III mice in comparison to [(18)F]FDG. 6-[(18)F]FDF metabolism was investigated in mouse blood and urine. 6-[(18)F]FDF is taken up by EMT-6 and MCF-7 breast tumor cells independent of extracellular glucose levels but dependent on the extracellular concentration of fructose. After 60 min, 30±4% (n=9) and 12±1% (n=7) ID/mg protein 6-[(18)F]FDF was found in EMT-6 and MCF-7 cells, respectively. 6-deoxy-6-fluoro-d-fructose had a 10-fold higher potency than fructose to inhibit 6-[(18)F]FDF uptake into EMT-6 cells. Biodistribution in normal mice revealed radioactivity uptake in bone and brain. Radioactivity was accumulated in EMT-6 tumors reaching 3.65±0.30% ID/g (n=3) at 5 min post injection and decreasing to 1.75±0.03% ID/g (n=3) at 120 min post injection. Dynamic small animal PET showed significantly lower radioactivity uptake after 15 min post injection in MCF-7 tumors [standard uptake value (SUV)=0.76±0.05; n=3] compared to EMT-6 tumors (SUV=1.23±0.09; n=3). Interestingly, [(18)F]FDG uptake was significantly different in MCF-7 tumors (SUV(15 min) 0.74±0.12 to SUV(120 min) 0.80±0.15; n=3) versus EMT-6 tumors (SUV(15 min) 1.01±0.33 to SUV(120 min) 1.80±0.25; n=3). 6-[(18)F]FDF was shown to be a substrate for recombinant human ketohexokinase, and it was metabolized rapidly in vivo. Based on the GLUT5 specific transport and phosphorylation by ketohexokinase, 6-[(18)F]FDF may represent a novel radiotracer for PET imaging of GLUT5 and ketohexokinase-expressing tumors.

Role of CD200 expression in regulation of metastasis of EMT6 tumor cells in mice

Previous studies have confirmed that levels of CD200 expression on the cells of the transplantable EMT6 mouse breast cancer line are increased markedly during growth in immunocompetent mice, unlike the persistent low levels of expression observed in NOD-SCID.IL-2(γr-/-) mice or mice with generalized over-expression of a CD200 transgene (CD200(tg) mice). Faster tumor growth occurs in both of these latter mice, with decreased evidence for a host immune reaction in lymph nodes draining the tumor (DLN). We now report evidence for a role for CD200 expression (by the host and/or tumor cells) in increased seeding of tumor cells to DLN in immunocompromised (CD200(tg) or NOD-SCID.IL-2(γr-/-)) vs immunocompetent mice, by limiting dilution cloning of tumor cells from DLN (vs contralateral lymph nodes, CLN), using control and GFP-tagged EMT6 cells. Neutralization of expressed CD200 by anti-CD200mAbs decreased the tumor metastasis at the same time as increasing detection of cytotoxic anti-tumor immune cells in DLN. Infusion of either anti-CD4 to deplete T-effector cells, or anti-TGFβ antibody, increased metastasis to DLN, as did indeed the infusion of EMT6 cells selected for the loss of TGFβRII expression. It is concluded that the increased CD200 expression by breast cancer cells (and/or host tissue) may be an important variable involved in determining the risk of metastasis.

Delivery of NKG2D Ligand Using an Anti-HER2 Antibody-NKG2D Ligand Fusion Protein Results in an Enhanced Innate and Adaptive Antitumor Response

NKG2D ligands link the innate and adapative immune response by activating the receptors expressed on effector cells of both the innate (NK) and adaptive immune systems (CD8(+) T cells). In this study, we explored the potential therapeutic utility of this intersection by fusing the murine NKG2D ligand Rae-1β to the 3' end of an anti-HER2 IgG3 antibody containing an intact Fc domain (anti-HER2 IgG3-Rae-1β), thereby targeting an NK cell activation signal to HER2+ breast tumor cells. The antitumor efficacy of this anti-HER2-Rae-1β fusion protein was examined in a mouse mammary tumor model engineered to express HER2 (EMT6-HER2 cells). We observed an enhanced cytotoxic response of NK effectors against EMT-HER2 cells in vitro. Mice implanted on one flank with EMT6-HER2 cells and contralaterally with control EMT6 cells exhibited rapid regression of EMT6-HER2 tumors but delayed regression of contralateral EMT6 tumors. IFNγ was implicated, given a lack of antitumor efficacy in IFNγ(-/-) mice. Depletion of either NK cells or CD8(+) T cells abrogated tumor growth inhibition, suggesting essential roles for each in the observed antitumor activity. Mice rejecting EMT6-HER2 tumors after anti-HER2-Rae-1β treatment showed markedly decreased tumor growth when rechallenged with EMT6-HER2 or EMT6 cells, whereas both EMT6 and EMT6-HER2 cells grew in control mice, indicating the development of an adaptive memory response. Our findings demonstrate that administration of an antibody-NKG2D ligand fusion protein can enhance innate and adaptive immune antitumor responses, also evoking additional nontargeted antigens to enhance the potential clinical utility of this approach.