Comparison of three 18F-labeled 2-nitroimidazoles for imaging hypoxia in breast cancer xenografts: [18F]FBNA, [18F]FAZA and [18F]FMISO.

BACKGROUND: Tumour hypoxia is associated with increased metastasis, invasion, poor therapy response and prognosis. Most PET radiotracers developed and used for clinical hypoxia imaging belong to the 2-nitroimidazole family. Recently we have developed novel 2-nitroimidazole-derived PET radiotracer [18F]FBNA (N-(4-[18F]fluoro-benzyl)-2-(2-nitro-1H-imidazol-1-yl)-acet-amide), an 18F-labeled analogue of antiparasitic drug benznidazole. The present study aimed to analyze its radio-pharmacological properties and systematically compare its PET imaging profiles with [18F]FMISO and [18F]FAZA in preclinical triple-negative (MDA-MB231) and estrogen receptor-positive (MCF-7) breast cancer models. METHODS: In vitro cellular uptake experiments were carried out in MDA-MB321 and MCF-7 cells under normoxic and hypoxic conditions. Metabolic stability in vivo was determined in BALB/c mice using radio-TLC analysis. Dynamic PET experiments over 3 h post-injection were performed in MDA-MB231 and MCF-7 tumour-bearing mice. Those PET data were used for kinetic modelling analysis utilizing the reversible two-tissue-compartment model. Autoradiography was carried out in tumour tissue slices and compared to HIF-1α immunohistochemistry. Detailed ex vivo biodistribution was accomplished in BALB/c mice, and this biodistribution data were used for dosimetry calculation. RESULTS: Under hypoxic conditions in vitro cellular uptake was elevated in both cell lines, MCF-7 and MDA-MB231, for all three radiotracers. After intravenous injection, [18F]FBNA formed two radiometabolites, resulting in a final fraction of 65 ± 9 % intact [18F]FBNA after 60 min p.i. After 3 h p.i., [18F]FBNA tumour uptake reached SUV values of 0.78 ± 0.01 in MCF-7 and 0.61 ± 0.04 in MDA-MB231 tumours (both n = 3), representing tumour-to-muscle ratios of 2.19 ± 0.04 and 1.98 ± 0.15, respectively. [18F]FMISO resulted in higher tumour uptakes (SUV 1.36 ± 0.04 in MCF-7 and 1.23 ± 0.08 in MDA-MB231 (both n = 4; p < 0.05) than [18F]FAZA (0.66 ± 0.11 in MCF-7 and 0.63 ± 0.14 in MDA-MB231 (both n = 4; n.s.)), representing tumour-to-muscle ratios of 3.24 ± 0.30 and 3.32 ± 0.50 for [18F]FMISO, and 2.92 ± 0.74 and 3.00 ± 0.42 for [18F]FAZA, respectively. While the fraction per time of radiotracer entering the second compartment (k3) was similar within uncertainties for all three radiotracers in MDA-MB231 tumours, it was different in MCF-7 tumours. The ratios k3/(k3 + k2) and K1*k3/(k3 + k2) in MCF-7 tumours were also significantly different, indicating dissimilar fractions of radiotracer bound and trapped intracellularly: K1*k3/(k2 + k3) [18F]FMISO (0.0088 ± 0.001)/min, n = 4; p < 0.001) > [18F]FAZA (0.0052 ± 0.002)/min, n = 4; p < 0.01) > [18F]FBNA (0.003 ± 0.001)/min, n = 3). In contrast, in MDA-MB231 tumours, only K1 was significantly elevated for [18F]FMISO. However, this did not result in significant differences for K1*k3/(k2 + k3) for all three 2-nitroimidazoles in MDA-MB231 tumours. CONCLUSION: Novel 2-nitroimidazole PET radiotracer [18F]FBNA showed uptake into hypoxic breast cancer cells and tumour tissue presumably associated with elevated HIF1-α expression. Systematic comparison of PET imaging performance with [18F]FMISO and [18F]FAZA in different types of preclinical breast cancer models revealed a similar tumour uptake profile for [18F]FBNA with [18F]FAZA and, despite its higher lipophilicity, still a slightly higher muscle tissue clearance compared to [18F]FMISO.

N-Alkyl Carbamoylimidazoles as Versatile Synthons for the Synthesis of Urea-Based PSMA Inhibitors.

We describe N-alkyl carbamoylimidazoles as readily available and highly versatile synthons for synthesizing urea-based prostate-specific membrane antigen (PSMA) inhibitors. Urea formation proceeded in high yields (>80%) at room temperature under aqueous conditions. All novel compounds were tested for their PSMA inhibitory potency in a cell-based radiometric binding assay. Compound 17 was identified as a novel high-affinity PSMA inhibitor (IC50 = 0.013 µM) suitable for developing an 18F-labeled radioligand for PET imaging of PSMA in prostate cancer.

Toward in vivo proof of binding of 18F-labeled inhibitor [18F]TRACK to peripheral tropomyosin receptor kinases.

BACKGROUND: Tropomyosin receptor kinases (TrkA, TrkB, TrkC) are a family of tyrosine kinases primarily expressed in neuronal cells of the brain. Identification of oncogenic alterations in Trk expression as a driver in multiple tumor types has increased interest in their role in human cancers. Recently, first- and second-generation 11C and 18F-labeled Trk inhibitors, e.g., [18F]TRACK, have been developed. The goal of the present study was to analyze the direct interaction of [18F]TRACK with peripheral Trk receptors in vivo to prove its specificity for use as a functional imaging probe. METHODS: In vitro uptake and competition experiments were carried out using the colorectal cancer cell line KM12. Dynamic PET experiments were performed with [18F]TRACK, either alone or in the presence of amitriptyline, an activator of Trk, entrectinib, a Trk inhibitor, or unlabeled reference compound TRACK in KM12 tumor-bearing athymic nude mice as well as B6129SF2/J and corresponding B6;129S2-Ntrk2tm1Bbd/J mice. Western blot and immunohistochemistry experiments were done with KM12 tumors, brown adipose tissue (BAT), and brain tissue samples. RESULTS: Uptake of [18F]TRACK was increasing over time reaching 208 ± 72% radioactivity per mg protein (n = 6/2) after 60 min incubation time. Entrectinib and TRACK competitively blocked [18F]TRACK uptake in vitro (IC50 30.9 ± 3.6 and 29.4 ± 9.4 nM; both n = 6/2). [18F]TRACK showed uptake into KM12 tumors (SUVmean,60 min 0.43 ± 0.03; n = 6). Tumor-to-muscle ratio reached 0.9 (60 min) and 1.2 (120 min). In TrkB expressing BAT, [18F]TRACK uptake reached SUVmean,60 min 1.32 ± 0.08 (n = 7). Activation of Trk through amitriptyline resulted in a significant radioactivity increase of 21% in KM12 tumor (SUVmean,60 min from 0.53 ± 0.01 to 0.43 ± 0.03; n = 6; p < 0.05) and of 21% in BAT (SUVmean,60 min from 1.32 ± 0.08; n = 5 to 1.59 ± 0.07; n = 6; p < 0.05) respectively. Immunohistochemistry showed TrkB > TrkA expression on BAT fat cells, but TrkA > TrkB in whole brain. WB analysis showed sevenfold higher TrkB expression in BAT versus KM12 tumor tissue. CONCLUSION: The present data show that radiotracer [18F]TRACK can target peripheral Trk receptors in human KM12 colon cancer as well as brown adipose tissue as confirmed through in vitro and in vivo blocking experiments. Higher TrkB versus TrkA protein expression was detected in brown adipose tissue of mice confirming a peripheral functional role of brain-derived neurotrophic factor in adipose tissue.

Towards Selective Binding to the GLUT5 Transporter: Synthesis, Molecular Dynamics and In Vitro Evaluation of Novel C-3-Modified 2,5-Anhydro-D-mannitol Analogs.

Deregulation and changes in energy metabolism are emergent and important biomarkers of cancer cells. The uptake of hexoses in cancer cells is mediated by a family of facilitative hexose membrane-transporter proteins known as Glucose Transporters (GLUTs). In the clinic, numerous breast cancers do not show elevated glucose metabolism (which is mediated mainly through the GLUT1 transporter) and may use fructose as an alternative energy source. The principal fructose transporter in most cancer cells is GLUT5, and its mRNA was shown to be elevated in human breast cancer. This offers an alternative strategy for early detection using fructose analogs. In order to selectively scout GLUT5 binding-pocket requirements, we designed, synthesized and screened a new class of fructose mimics based upon the 2,5-anhydromannitol scaffold. Several of these compounds display low millimolar IC50 values against the known high-affinity 18F-labeled fructose-based probe 6-deoxy-6-fluoro-D-fructose (6-FDF) in murine EMT6 breast cancer cells. In addition, this work used molecular docking and molecular dynamics simulations (MD) with previously reported GLUT5 structures to gain better insight into hexose-GLUT interactions with selected ligands governing their preference for GLUT5 compared to other GLUTs. The improved inhibition of these compounds, and the refined model for their binding, set the stage for the development of high-affinity molecular imaging probes targeting cancers that express the GLUT5 biomarker.

FOXM1 Inhibitors as Potential Diagnostic Agents: First Generation of a PET Probe Targeting FOXM1 To Detect Triple-Negative Breast Cancer in†vitro and in†vivo.

The FOXM1 protein controls the expression of essential genes related to cancer cell cycle progression, metastasis, and chemoresistance. We hypothesize that FOXM1 inhibitors could represent a novel approach to develop 18 F-based radiotracers for Positron Emission Tomography (PET). Therefore, in this report we describe the first attempt to use 18 F-labeled FOXM1 inhibitors to detect triple-negative breast cancer (TNBC). Briefly, we replaced the original amide group in the parent drug FDI-6 for a ketone group in the novel AF-FDI molecule, to carry out an aromatic nucleophilic (18 F)-fluorination. AF-FDI dissociated the FOXM1-DNA complex, decreased FOXM1 levels, and inhibited cell proliferation in a TNBC cell line (MDA-MB-231). [18 F]AF-FDI was internalized in MDA-MB-231 cells. Cell uptake inhibition experiments showed that AF-FDI and FDI-6 significantly decreased the maximum uptake of [18 F]AF-FDI, suggesting specificity towards FOXM1. [18 F]AF-FDI reached a tumor uptake of SUV=0.31 in MDA-MB-231 tumor-bearing mice and was metabolically stable 60 min post-injection. These results provide preliminary evidence supporting the potential role of FOXM1 to develop PET radiotracers.

Design, synthesis, and evaluation of positron emission tomography/fluorescence dual imaging probes for targeting facilitated glucose transporter 1 (GLUT1).

Increased energy metabolism followed by enhanced glucose consumption is a hallmark of cancer. Most cancer cells show overexpression of facilitated hexose transporter GLUT1, including breast cancer. GLUT1 is the main transporter for 2-deoxy-2-[18F]fluoro-d-glucose (2-[18F]FDG), the gold standard of positron emission tomography (PET) imaging in oncology. The present study's goal was to develop novel glucose-based dual imaging probes for their use in tandem PET and fluorescence (Fl) imaging. A glucosamine scaffold tagged with a fluorophore and an 18F-label should confer selectivity to GLUT1. Out of five different compounds, 2-deoxy-2-((7-sulfonylfluoro-2,1,3-benzoxadiazol-4-yl)amino)-d-glucose (2-FBDG) possessed favorable fluorescent properties and a similar potency as 2-deoxy-2-((7-nitro-2,1,3-benzoxadiazol-4-yl)amino)-d-glucose (2-NBDG) in competing for GLUT1 transport against 2-[18F]FDG in breast cancer cells. Radiolabeling with 18F was achieved through the synthesis of prosthetic group 7-fluoro-2,1,3-benzoxadiazole-4-sulfonyl [18F]fluoride ([18F]FBDF) followed by the reaction with glucosamine. The radiotracer was finally analyzed in vivo in a breast cancer xenograft model and compared to 2-[18F]FDG. Despite favourable in vitro fluorescence imaging properties, 2-[18F]FBDG was found to lack metabolic stability in vivo, resulting in radiodefluorination. Glucose-based 2-[18F]FBDG represents a novel dual-probe for GLUT1 imaging using FI and PET with the potential for further structural optimization for improved metabolic stability in vivo.

Enrichment and ratiometric detection of circulating tumor cells using PSMA- and folate receptor-targeted magnetic and surface-enhanced Raman scattering nanoparticles.

The presence of circulating tumor cells (CTCs) in a patient's bloodstream is a hallmark of metastatic cancer. The detection and analysis of CTCs is a promising diagnostic and prognostic strategy as they may carry useful genetic information from their derived primary tumor, and the enumeration of CTCs in the bloodstream has been known to scale with disease progression. However, the detection of CTCs is a highly challenging task owing to their sparse numbers in a background of billions of background blood cells. To effectively utilize CTCs, there is a need for an assay that can detect CTCs with high specificity and can locally enrich CTCs from a liquid biopsy. We demonstrate a versatile methodology that addresses these needs by utilizing a combination of nanoparticles. Enrichment is achieved using targeted magnetic nanoparticles and high specificity detection is achieved using a ratiometric detection approach utilizing multiplexed targeted and non-targeted surface-enhanced Raman Scattering Nanoparticles (SERS-NPs). We demonstrate this approach with model prostate and cervical circulating tumor cells and show the ex vivo utility of our methodology for the detection of PSMA or folate receptor over-expressing CTCs. Our approach allows for the mitigation of interference caused by the non-specific uptake of nanoparticles by other cells present in the bloodstream and our results from magnetically trapped CTCs reveal over a 2000% increase in targeted SERS-NP signal over non-specifically bound SERS-NPs.

A comparative PET imaging study of 44gSc- and 68Ga-labeled bombesin antagonist BBN2 derivatives in breast and prostate cancer models.

INTRODUCTION: Radiolabeled peptides play a central role in nuclear medicine as radiotheranostics for targeted imaging and therapy of cancer. We have recently proposed the use of metabolically stabilized GRPR antagonist BBN2 for radiolabeling with 18F and 68Ga and subsequent PET imaging of GRPRs in prostate cancer. The present work studied the impact of 44gSc- and 68Ga-labeled DOTA complexes attached to GRPR antagonist BBN2 on the in vitro GRPR binding affinity, and their biodistribution and tumor uptake profiles in MCF7 breast and PC3 prostate cancer models. METHODS: DOTA-Ava-BBN2 was radiolabeled with radiometals 68Ga and 44gSc. Gastrin-releasing peptide receptor (GRPR) affinities of peptides were assessed in PC3 prostate cancer cells. GRPR expression profiles were studied in human breast cancer tissue samples and MCF7 breast cancer cells. PET imaging of 68Ga- and 44gSc-labeled peptides was performed in MCF7 and PC3 xenografts as breast and prostate cancer models. RESULTS: Radiopeptides [68Ga]Ga-DOTA-Ava-BBN2 and [44gSc]Sc-DOTA-Ava BBN2 were prepared in radiochemical yields of 70-80% (decay-corrected), respectively. High binding affinities were found for both peptides (IC50 = 15 nM (natGa) and 5 nM (natSc)). Gene expression microarray analysis revealed high GRPR mRNA expression levels in estrogen receptor (ER)-positive breast cancer, which was further confirmed with Western blot and immunohistochemistry. However, PET imaging showed only low tumor uptake of both radiotracers in MCF7 xenografts ([68Ga]Ga-DOTA-BBN2 (SUV60min 0.27 ± 0.06); [44gSc]Sc-DOTA-BBN2 (SUV60min 0.20 ± 0.03)). In contrast, high tumor uptake and retention were found for both radiopeptides in PC3 tumors ([68Ga]Ga-DOTA-BBN2 (SUV60min 0.46 ± 0.07); [44gSc]Sc-DOTA-BBN2 (SUV60min 0.51 ± 0.11)). CONCLUSIONS: Comparison of 68Ga- and 44gSc-labeled DOTA-Ava-BBN2 peptides revealed slight but noticeable differences of the radiometal with an impact on the in vitro GRPR receptor binding properties in PC3 cells. No differences were found in their in vivo biodistribution profiles in MCF7 and PC3 xenografts. Radiopeptides [68Ga]Ga-DOTA-Ava-BBN2 and [44gSc]Sc-DOTA-Ava-BBN2 displayed comparable tumor uptake and retention profiles with rapid blood and renal clearance profiles in both tumor models. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: The favorable PET imaging performance of [44gSc]Sc-DOTA-Ava-BBN2 in prostate cancer should warrant the development of an [43Sc]Sc-DOTA-Ava-BBN2 analog for clinical translation which comes with a main γ-line of much lower energy and intensity compared to 44gSc.

Tyrosine kinase inhibitor therapy and metabolic remodelling in papillary thyroid cancer.

Targeted therapy is increasingly used to manage metastatic papillary thyroid cancer. The focus of the present study was to examine glucose metabolism and tumor responses for thyroid cancer xenografts expressing the glycolytic pathway modulators platelet-derived growth factor receptor (PDGFR) and BRAFV600E. Radiolabelled glucose derivative [18F]FDG was used to analyze the effects of PDGFR blockade with imatinib, BRAF blockade with vemurafenib, as well as combined PDGFR and BRAF blockade in vitro and in vivo with PET. Dynamic PET data was correlated with immunohistochemistry staining and kinetic analysis for facilitative glucose transporter 1 (GLUT1) and hexokinase-II (HK2). Vemurafenib decreased [18F]FDG uptake in BCPAP cells in vitro; however, it was increased by ~70% with imatinib application to BCPAP cells. This metabolic response to tyrosine kinase inhibition required BRAFV600E as it was not seen in cell lines lacking mutated BRAF (TPC1). In xenografts, imatinib therapy in BCPAP thyroid tumour-bearing mice significantly increased [18F]FDG uptake and retention (>30%) in BCPAP tumours with PDGFRß or both (α+ß) isoforms. Kinetic analysis revealed that the increased glucose uptake is a consequence of increased phosphorylation and intracellular trapping of [18F]FDG confirmed by an increase in HK2 protein expression and activity, but not GLUT1 activity. BRAF inhibition alone, or combined PDGFR and BRAF inhibition, reduced (~60%) [18F]FDG uptake in both types of BCPAP (ß or α+ß) tumours. In terms of tumour growth, combination therapy with imatinib and vemurafenib led to a near abolition of the tumors (~90% reduction), but single therapy for BCPAP with PDGFRα expression was much less effective. In summary, imatinib led to a paradoxical increase of [18F]FDG uptake in xenografts that was reversed through BRAFV600E inhibition. The present data show that metabolic reprogramming in thyroid cancer occurs as a consequence of BRAF-mediated upregulation of HK2 expression that may permit tumour growth with isolated blockade of upstream tyrosine kinase receptors.

Dexamethasone Attenuates X-Ray-Induced Activation of the Autotaxin-Lysophosphatidate-Inflammatory Cycle in Breast Tissue and Subsequent Breast Fibrosis.

We recently showed that radiation-induced DNA damage in breast adipose tissue increases autotaxin secretion, production of lysophosphatidate (LPA) and expression of LPA1/2 receptors. We also established that dexamethasone decreases autotaxin production and LPA signaling in non-irradiated adipose tissue. In the present study, we showed that dexamethasone attenuated the radiation-induced increases in autotaxin activity and the concentrations of inflammatory mediators in cultured human adipose tissue. We also exposed a breast fat pad in mice to three daily 7.5 Gy fractions of X-rays. Dexamethasone attenuated radiation-induced increases in autotaxin activity in plasma and mammary adipose tissue and LPA1 receptor levels in adipose tissue after 48 h. DEX treatment during five daily fractions of 7.5 Gy attenuated fibrosis by ~70% in the mammary fat pad and underlying lungs at 7 weeks after radiotherapy. This was accompanied by decreases in CXCL2, active TGF-ß1, CTGF and Nrf2 at 7 weeks in adipose tissue of dexamethasone-treated mice. Autotaxin was located at the sites of fibrosis in breast tissue and in the underlying lungs. Consequently, our work supports the premise that increased autotaxin production and lysophosphatidate signaling contribute to radiotherapy-induced breast fibrosis and that dexamethasone attenuated the development of fibrosis in part by blocking this process.

Inhibition of Autotaxin with GLPG1690 Increases the Efficacy of Radiotherapy and Chemotherapy in a Mouse Model of Breast Cancer.

Autotaxin catalyzes the formation of lysophosphatidic acid, which stimulates tumor growth and metastasis and decreases the effectiveness of cancer therapies. In breast cancer, autotaxin is secreted mainly by breast adipocytes, especially when stimulated by inflammatory cytokines produced by tumors. In this work, we studied the effects of an ATX inhibitor, GLPG1690, which is in phase III clinical trials for idiopathic pulmonary fibrosis, on responses to radiotherapy and chemotherapy in a syngeneic orthotopic mouse model of breast cancer. Tumors were treated with fractionated external beam irradiation, which was optimized to decrease tumor weight by approximately 80%. Mice were also dosed twice daily with GLPG1690 or vehicle beginning at 1 day before the radiation until 4 days after radiation was completed. GLPG1690 combined with irradiation did not decrease tumor growth further compared with radiation alone. However, GLPG1690 decreased the uptake of 3'-deoxy-3'-[18F]-fluorothymidine by tumors and the percentage of Ki67-positive cells. This was also associated with increased cleaved caspase-3 and decreased Bcl-2 levels in these tumors. GLPG1690 decreased irradiation-induced C-C motif chemokine ligand-11 in tumors and levels of IL9, IL12p40, macrophage colony-stimulating factor, and IFNγ in adipose tissue adjacent to the tumor. In other experiments, mice were treated with doxorubicin every 2 days after the tumors developed. GLPG1690 acted synergistically with doxorubicin to decrease tumor growth and the percentage of Ki67-positive cells. GLPG1690 also increased 4-hydroxynonenal-protein adducts in these tumors. These results indicate that inhibiting ATX provides a promising adjuvant to improve the outcomes of radiotherapy and chemotherapy for breast cancer.

Repeated Fractions of X-Radiation to the Breast Fat Pads of Mice Augment Activation of the Autotaxin-Lysophosphatidate-Inflammatory Cycle.

Breast cancer patients are usually treated with multiple fractions of radiotherapy (RT) to the whole breast after lumpectomy. We hypothesized that repeated fractions of RT would progressively activate the autotaxin-lysophosphatidate-inflammatory cycle. To test this, a normal breast fat pad and a fat pad containing a mouse 4T1 tumor were irradiated with X-rays using a small-animal "image-guided" RT platform. A single RT dose of 7.5 Gy and three daily doses of 7.5 Gy increased ATX activity and decreased plasma adiponectin concentrations. The concentrations of IL-6 and TNFα in plasma and of VEGF, G-CSF, CCL11 and CXCL10 in the irradiated fat pad were increased, but only after three fractions of RT. In 4T1 breast tumor-bearing mice, three fractions of 7.5 Gy augmented tumor-induced increases in plasma ATX activity and decreased adiponectin levels in the tumor-associated mammary fat pad. There were also increased expressions of multiple inflammatory mediators in the tumor-associated mammary fat pad and in tumors, which was accompanied by increased infiltration of CD45+ leukocytes into tumor-associated adipose tissue. This work provides novel evidence that increased ATX production is an early response to RT and that repeated fractions of RT activate the autotaxin-lysophosphatidate-inflammatory cycle. This wound healing response to RT-induced damage could decrease the efficacy of further fractions of RT.

Specimen quality evaluation in Canadian biobanks participating in the COEUR repository.

Human biological specimens are important for translational research programs such as the Canadian Ovarian Experimental Unified Resource (COEUR) funded by the Terry Fox Research Institute. Sample quality is an important consideration, as it directly impacts the quality of ensuing research. The aim of the present study was to determine the quality of tissues collected from different sites contributing to the COEUR cohort. Samples from high-grade serous ovarian tumors (fresh frozen and corresponding paraffin-embedded tissues) were provided by nine participating Canadian biobanks. All samples were shipped to a central site using a Standard Operating Protocol (SOP). DNA and RNA extraction was conducted by the quality control division of the Canadian Tumor Repository Network (CTRNet). DNA quality was determined by ß-globin gene PCR amplification, and RNA quality by the RNA integrity number (RIN), as measured by the Agilent BioAnalyzer. DNA of acceptable quality had at least three bands of ß-globin amplified from DNA (n=115/135), and a RIN number ≥7 was considered very good for RNA (n=80/135). Sample preparation and storage time had little effect on RNA or DNA quality. Protein expression was assessed on tissue microarray by immunohistochemistry with antibodies against p53, WT1, E-cadherin, CK-7, and Ki67 from formalin fixed-paraffin embedded (FFPE) tissues. As seen with a nonhierarchical clustering statistical method, there was no significant difference in immunostaining of paraffin tissues among specimens from different biobanks. Interestingly, patients with worse outcome were highly positive for p53 and weak for WT1. In conclusion, while there was no common SOP for retrospectively collected material across Canadian biobanks, these results indicate that specimens collected at these multiple sites are of comparable quality, and can serve as an adequate resource to create a national cohort for the validation of molecular biomarkers in ovarian cancer.

Evidence for copurification of micronuclei in sucrose density gradient-enriched plasma membranes from cell lines.

Sucrose density gradient-enriched membrane preparations and membrane fraction enrichment through affinity purification techniques are commonly used in proteomic analysis. However, published proteomic profiles characterized by the above methods show the presence of nuclear proteins in addition to membrane proteins. While shuttling of nuclear proteins across cellular compartments and their transient residency at membrane interfaces could explain some of these observations, the presence of nuclear proteins in proteomic profiles generated with crude and enriched membranes could be the result of nonspecific contamination of nuclear debris during cell fractionation procedures. We hypothesized that micronuclei arising from the genomic instability inherent to cancer cells may copurify with plasma membrane fractions on sucrose gradients. Using sucrose gradient-enriched plasma membranes from breast cancer cell lines derived from the MCF-7 cell line, we provide experimental evidence to indicate that micronuclei are present in fresh preparations of plasma membranes. The origin of these micronuclei was traced to budding of nuclei in intact cells. Furthermore, mass spectrometric analysis confirmed the presence of nuclear proteins as well as membrane and associated signaling proteins in sucrose gradient-enriched preparations.

Association of DNA repair and steroid metabolism gene polymorphisms with clinical late toxicity in patients treated with conformal radiotherapy for prostate cancer.

OBJECTIVE: To explore the possible relationship between single nucleotide polymorphisms (SNP) in candidate genes encoding DNA damage recognition/repair/response and steroid metabolism proteins with respect to clinical radiation toxicity in a retrospective cohort of patients previously treated with three-dimensional conformal radiotherapy (3-DCRT) for prostate cancer. EXPERIMENTAL DESIGN: One hundred twenty-four patients with prostate cancer underwent 3-DCRT at our institution between September 1996 and December 2000. Of these, 83 consented for follow-up of blood sampling and SNP analysis. Twenty-eight patients were documented as having experienced grade >/=2 late bladder or rectal toxicity (scoring system of Radiation Therapy Oncology Group) on at least one follow-up visit. We analyzed 49 SNPs in BRCA1, BRCA2, ESR1, XRCC1, XRCC2, XRCC3, NBN, RAD51, RAD52, LIG4, ATM, BCL2, TGFB1, MSH6, ERCC2, XPF, NR3C1, CYP1A1, CYP2C9, CYP2C19, CYP3A5, CYP2D6, CYP11B2, and CYP17A1 genes using the Pyrosequencing technique. RESULTS: Significant univariate associations with late rectal or bladder toxicity (grade >/=2) were found for XRCC3 (A>G 5' untranslated region NT 4541), LIG4 (T>C Asp(568)Asp), MLH1 (C>T, Val(219)Ile), CYP2D6*4 (G>A splicing defect), mean rectal and bladder dose, dose to 30% of rectum or bladder, and age <60 years. On Cox multivariate analysis, significant associations with toxicity were found for LIG4 (T>C, Asp(568)Asp), ERCC2 (G>A, Asp(711)Asp), CYP2D6*4 (G>A, splicing defect), mean bladder dose >60 Gy, and dose to 30% of rectal volume >75 Gy. CONCLUSIONS: In this study, we identified SNPs in LIG4, ERCC2, and CYP2D6 genes as putative markers to predict individuals at risk for complications arising from radiation therapy in prostate cancer.

Identification and functional characterization of variants in human concentrative nucleoside transporter 3, hCNT3 (SLC28A3), arising from single nucleotide polymorphisms in coding regions of the hCNT3 gene.

INTRODUCTION: Human concentrative nucleoside transporter 3, hCNT3 (SLC28A3), which mediates transport of purine and pyrimidine nucleosides and a variety of antiviral and anticancer nucleoside drugs, was investigated to determine if there are single nucleotide polymorphisms in the coding regions of the hCNT3 gene. METHODS AND RESULTS: Ninety-six DNA samples from Caucasians (Coriell Panel) were sequenced and sixteen variants in exons and flanking intronic regions were identified, of which five were coding variants; three of these were non-synonymous (S5N, L131F, Y513F) and were further investigated for functional alterations of the resulting recombinant proteins in Saccharomyces cerevisiae and Xenopus laevis oocytes. In yeast, immunostaining and fluorescence quantitation of the reference (wild-type) and variant CNT3 proteins showed similar levels of expression. Kinetic studies were undertaken in yeast with a high through-put semi-automated assay process; reference hCNT3 exhibited Km values of 1.7+/-0.3, 3.6+/-1.3, 2.2+/-0.7, and 2.1+/-0.6 muM and Vmax values of 1402+/-286, 1310+/-113, 1020+/-44, and 1740+/-114 pmol/mg/min, respectively, for uridine, cytidine, adenosine and inosine. Similar Km and Vmax values were obtained for the three variant proteins assayed in yeast under identical conditions. All of the characterized hCNT3 variants produced in oocytes retained sodium and proton dependence of uridine transport based on measurements of radioisotope flux and two-electrode voltage-clamp studies. CONCLUSION: These results suggested a high degree of conservation of function for hCNT3 in the Caucasian population.

Predictive models for breast cancer susceptibility from multiple single nucleotide polymorphisms.

Hereditary predisposition and causative environmental exposures have long been recognized in human malignancies. In most instances, cancer cases occur sporadically, suggesting that environmental influences are critical in determining cancer risk. To test the influence of genetic polymorphisms on breast cancer risk, we have measured 98 single nucleotide polymorphisms (SNPs) distributed over 45 genes of potential relevance to breast cancer etiology in 174 patients and have compared these with matched normal controls. Using machine learning techniques such as support vector machines (SVMs), decision trees, and naïve Bayes, we identified a subset of three SNPs as key discriminators between breast cancer and controls. The SVMs performed maximally among predictive models, achieving 69% predictive power in distinguishing between the two groups, compared with a 50% baseline predictive power obtained from the data after repeated random permutation of class labels (individuals with cancer or controls). However, the simpler naïve Bayes model as well as the decision tree model performed quite similarly to the SVM. The three SNP sites most useful in this model were (a) the +4536T/C site of the aldosterone synthase gene CYP11B2 at amino acid residue 386 Val/Ala (T/C) (rs4541); (b) the +4328C/G site of the aryl hydrocarbon hydroxylase CYP1B1 at amino acid residue 293 Leu/Val (C/G) (rs5292); and (c) the +4449C/T site of the transcription factor BCL6 at amino acid 387 Asp/Asp (rs1056932). No single SNP site on its own could achieve more than 60% in predictive accuracy. We have shown that multiple SNP sites from different genes over distant parts of the genome are better at identifying breast cancer patients than any one SNP alone. As high-throughput technology for SNPs improves and as more SNPs are identified, it is likely that much higher predictive accuracy will be achieved and a useful clinical tool developed.

IFN-gamma-inducible protein 10 (CXCL10) contributes to airway hyperreactivity and airway inflammation in a mouse model of asthma.

Allergic asthma is an inflammatory disease of the airways characterized by eosinophilic inflammation and airway hyper-reactivity. Cytokines and chemokines specific for Th2-type inflammation predominate in asthma and in animal models of this disease. The role of Th1-type inflammatory mediators in asthma remains controversial. IFN-gamma-inducible protein 10 (IP-10; CXCL10) is an IFN-gamma-inducible chemokine that preferentially attracts activated Th1 lymphocytes. IP-10 is up-regulated in the airways of asthmatics, but its function in asthma is unclear. To investigate the role of IP-10 in allergic airway disease, we examined the expression of IP-10 in a murine model of asthma and the effects of overexpression and deletion of IP-10 in this model using IP-10-transgenic and IP-10-deficient mice. Our experiments demonstrate that IP-10 is up-regulated in the lung after allergen challenge. Mice that overexpress IP-10 in the lung exhibited significantly increased airway hyperreactivity, eosinophilia, IL-4 levels, and CD8(+) lymphocyte recruitment compared with wild-type controls. In addition, there was an increase in the percentage of IL-4-secreting T lymphocytes in the lungs of IP-10-transgenic mice. In contrast, mice deficient in IP-10 demonstrated the opposite results compared with wild-type controls, with a significant reduction in these measures of Th2-type allergic airway inflammation. Our results demonstrate that IP-10, a Th1-type chemokine, is up-regulated in allergic pulmonary inflammation and that this contributes to the airway hyperreactivity and Th2-type inflammation seen in this model of asthma.

IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking.

IFN-gamma-inducible protein 10 (IP-10, CXCL10), a chemokine secreted from cells stimulated with type I and II IFNs and LPS, is a chemoattractant for activated T cells. Expression of IP-10 is seen in many Th1-type inflammatory diseases, where it is thought to play an important role in recruiting activated T cells into sites of tissue inflammation. To determine the in vivo function of IP-10, we constructed an IP-10-deficient mouse (IP-10(-/-)) by targeted gene disruption. Immunological analysis revealed that IP-10(-/-) mice had impaired T cell responses. T cell proliferation to allogeneic and antigenic stimulation and IFN-gamma secretion in response to antigenic challenge were impaired in IP-10(-/-) mice. In addition, IP-10(-/-) mice exhibited an impaired contact hypersensitivity response, characterized by decreased ear swelling and reduced inflammatory cell infiltrates. T cells recovered from draining lymph nodes also had a decreased proliferative response to Ag restimulation. Furthermore, IP-10(-/-) mice infected with a neurotropic mouse hepatitis virus had an impaired ability to control viral replication in the brain. This was associated with decreased recruitment of CD4(+) and CD8(+) lymphocytes into the brain, reduced levels of IFN-gamma and the IFN-gamma-induced chemokines monokine induced by IFN-gamma (Mig, CXCL9) and IFN-inducible T cell alpha chemoattractant (I-TAC, CXCL11) in the brain, decreased numbers of virus-specific IFN-gamma-secreting CD8(+) cells in the spleen, and reduced levels of demyelination in the CNS. Taken together, our data suggest a role for IP-10 in both effector T cell generation and trafficking in vivo.

CD36, a class B scavenger receptor, is expressed on microglia in Alzheimer's disease brains and can mediate production of reactive oxygen species in response to beta-amyloid fibrils.

A pathological hallmark of Alzheimer's disease is the senile plaque, composed of beta-amyloid fibrils, microglia, astrocytes, and dystrophic neurites. We reported previously that class A scavenger receptors mediate adhesion of microglia and macrophages to beta-amyloid fibrils and oxidized low-density lipoprotein (oxLDL)-coated surfaces. We also showed that CD36, a class B scavenger receptor and an oxLDL receptor, promotes H(2)O(2) secretion by macrophages adherent to oxLDL-coated surfaces. Whether CD36 is expressed on microglia, and whether it plays a role in secretion of H(2)O(2) by microglia interacting with fibrillar beta-amyloid is not known. Using fluorescence-activated cell sorting analysis and immunohistochemistry, we found that CD36 is expressed on human fetal microglia, and N9-immortalized mouse microglia. We also found that CD36 is expressed on microglia and on vascular endothelial cells in the brains of Alzheimer's disease patients. Bowes human melanoma cells, which normally do not express CD36, gained the ability to specifically bind to surfaces coated with fibrillar beta-amyloid when transfected with a cDNA encoding human CD36, suggesting that CD36 is a receptor for fibrillar beta-amyloid. Furthermore, two different monoclonal antibodies to CD36 inhibited H(2)O(2) production by N9 microglia and human macrophages adherent to fibrillar beta-amyloid by approximately 50%. Our data identify a role for CD36 in fibrillar beta-amyloid-induced H(2)O(2) production by microglia, and imply that CD36 can mediate binding to fibrillar beta-amyloid. We propose that similar to their role in the interaction of macrophages with oxLDL, class A scavenger receptors and CD36 play complimentary roles in the interactions of microglia with fibrillar beta-amyloid.