JAK-STAT core cancer pathway: An integrative cancer interactome analysis.

Through a comprehensive review and in silico analysis of reported data on STAT-linked diseases, we analysed the communication pathways and interactome of the seven STATs in major cancer categories and proposed rational targeting approaches for therapeutic intervention to disrupt critical pathways and addictions to hyperactive JAK/STAT in neoplastic states. Although all STATs follow a similar molecular activation pathway, STAT1, STAT2, STAT4 and STAT6 exert specific biological profiles associated with a more restricted pattern of activation by cytokines. STAT3 and STAT5A as well as STAT5B have pleiotropic roles in the body and can act as critical oncogenes that promote many processes involved in cancer development. STAT1, STAT3 and STAT5 also possess tumour suppressive action in certain mutational and cancer type context. Here, we demonstrated member-specific STAT activity in major cancer types. Through systems biology approaches, we found surprising roles for EGFR family members, sex steroid hormone receptor ESR1 interplay with oncogenic STAT function and proposed new drug targeting approaches of oncogenic STAT pathway addiction.

Feasibility of Imaging EpCAM Expression in Ovarian Cancer Using Radiolabeled DARPin Ec1.

Epithelial cell adhesion molecule (EpCAM) is overexpressed in 55%-75% of ovarian carcinomas (OC). EpCAM might be used as a target for a treatment of disseminated OC. Designed ankyrin repeats protein (DARPin) Ec1 is a small (18 kDa) protein, which binds to EpCAM with subnanomolar affinity. We tested a hypothesis that Ec1 labeled with a non-residualizing label might serve as a companion imaging diagnostic for stratification of patients for EpCAM-targeting therapy. Ec1 was labeled with 125I using N-succinimidyl-para-iodobenzoate. Binding affinity, specificity, and cellular processing of [125I]I-PIB-Ec1 were evaluated using SKOV-3 and OVCAR-3 ovarian carcinoma cell lines. Biodistribution and tumor-targeting properties of [125I]I-PIB-Ec1 were studied in Balb/c nu/nu mice bearing SKOV-3 and OVCAR-3 xenografts. EpCAM-negative Ramos lymphoma xenografts served as specificity control. Binding of [125I]I-PIB-Ec1 to ovarian carcinoma cell lines was highly specific and had affinity in picomolar range. Slow internalization of [125I]I-PIB-Ec1 by OC cells confirmed utility of non-residualizing label for in vivo imaging. [125I]I-PIB-Ec1 provided 6 h after injection tumor-to-blood ratios of 30 ± 11 and 48 ± 12 for OVCAR-3 and SKOV-3 xenografts, respectively, and high contrast to other organs. Tumor targeting was highly specific. Saturation of tumor uptake at a high dose of Ec1 in SKOV-3 model provided a rationale for dose selection in further studies using therapeutic conjugates of Ec1 for targeted therapy. In conclusion, [125I]I-PIB-Ec1 is a promising agent for visualizing EpCAM expression in OC.

188Re-ZHER2:V2, a promising affibody-based targeting agent against HER2-expressing tumors: preclinical assessment.

UNLABELLED: Affibody molecules are small (7 kDa) nonimmunoglobulin scaffold proteins with favorable tumor-targeting properties. Studies concerning the influence of chelators on biodistribution of (99m)Tc-labeled Affibody molecules demonstrated that the variant with a C-terminal glycyl-glycyl-glycyl-cysteine peptide-based chelator (designated ZHER2:V2) has the best biodistribution profile in vivo and the lowest renal retention of radioactivity. The aim of this study was to evaluate (188)Re-ZHER2:V2 as a potential candidate for radionuclide therapy of human epidermal growth factor receptor type 2 (HER2)-expressing tumors. METHODS: ZHER2:V2 was labeled with (188)Re using a gluconate-containing kit. Targeting of HER2-overexpressing SKOV-3 ovarian carcinoma xenografts in nude mice was studied for a dosimetry assessment. RESULTS: Binding of (188)Re-ZHER2:V2 to living SKOV-3 cells was demonstrated to be specific, with an affinity of 6.4 ± 0.4 pM. The biodistribution study showed a rapid blood clearance (1.4 ± 0.1 percentage injected activity per gram [%ID/g] at 1 h after injection). The tumor uptake was 14 ± 2, 12 ± 2, 5 ± 2, and 1.8 ± 0.5 %IA/g at 1, 4, 24, and 48 h after injection, respectively. The in vivo targeting of HER2-expressing xenografts was specific. Already at 4 h after injection, tumor uptake exceeded kidney uptake (2.1 ± 0.2 %IA/g). Scintillation-camera imaging showed that tumor xenografts were the only sites with prominent accumulation of radioactivity at 4 h after injection. Based on the biokinetics, a dosimetry evaluation for humans suggests that (188)Re-ZHER2:V2 would provide an absorbed dose to tumor of 79 Gy without exceeding absorbed doses of 23 Gy to kidneys and 2 Gy to bone marrow. This indicates that future human radiotherapy studies may be feasible. CONCLUSION: (188)Re-ZHER2:V2 can deliver high absorbed doses to tumors without exceeding kidney and bone marrow toxicity limits.

Development and preclinical characterisation of 99mTc-labelled Affibody molecules with reduced renal uptake.

PURPOSE: Affibody molecules are low molecular weight proteins (7 kDa), which can be selected to bind to tumour-associated target proteins with subnanomolar affinity. Because of rapid tumour localisation and clearance from nonspecific compartments, Affibody molecules are promising tracers for molecular imaging. Earlier, (99m)Tc-labelled Affibody molecules demonstrated specific targeting of tumour xenografts. However, the biodistribution was suboptimal either because of hepatobiliary excretion or high renal uptake of the radioactivity. The goal of this study was to optimise the biodistribution of Affibody molecules by chelator engineering. MATERIALS AND METHODS: Anti-HER2 Z(HER2:342) Affibody molecules, carrying the mercaptoacetyl-glutamyl-seryl-glutamyl (maESE), mercaptoacetyl-glutamyl-glutamyl-seryl (maEES) and mercaptoacetyl-seryl-glutamyl-glutamyl (maSEE) chelators, were prepared by peptide synthesis and labelled with (99m)Tc. The tumour-targeting capacity of these conjugates was compared with each other and with the best previously available conjugate, (99m)Tc-maEEE-Z(HER2:342,) in nude mice bearing SKOV-3 xenografts. The tumour-targeting capacity of the most promising conjugate, (99m)Tc-maESE-Z(HER2:342,) was compared with radioiodinated Z(HER2:342). RESULTS: All novel conjugates demonstrated successful tumour targeting and a low degree of hepatobiliary excretion. The renal uptakes of serine-containing conjugates, 33 +/- 5, 68 +/- 21 and 71 +/- 10%IA/g, for(99m)Tc-maESE-Z(HER2:342), (99m)Tc-maEES-Z(HER2:342) and (99m)Tc-maSEE-Z(HER2:342), respectively, were significantly reduced in comparison with (99m)Tc-maEEE-Z(HER2:342) (102 +/- 13%IA/g). For (99m)Tc-maESE-Z(HER2:342), a tumour uptake of 9.6 +/- 1.8%IA/g and a tumour-to-blood ratio of 58 +/- 6 were reached at 4 h p.i. CONCLUSIONS: A combination of serine and glutamic acid residues in the chelator sequence confers increased renal excretion and relatively low renal uptake of (99m)Tc-labelled Affibody molecules. In combination with preserved targeting capacity, this improved imaging of targets in abdominal area.

Pre-clinical evaluation of [111In]-benzyl-DOTA-Z(HER2:342), a potential agent for imaging of HER2 expression in malignant tumors.

Imaging of expression of human epidermal growth factor receptor type 2 (HER2) in breast carcinomas may help to select patients eligible for trastuzumab therapy. The Affibody molecule Z(HER2:342) is a small (7-kDa) non-immunoglobulin affinity protein, which binds to HER2 with a picomolar affinity. Previously, a benzyl-DTPA conjugate of Z(HER2:342) was labeled with 111In and demonstrated good targeting in murine xenografts. We considered that the use of the macrocyclic chelator DOTA could increase the label stability and enhance a choice of nuclides, which could be used as a label for Z(HER2:342). The goal of this study was the preparation and pre-clinical evaluation of the indium-111- labeled DOTA-derivative of Z(HER2:342). Isothiocyanate-benzyl-DOTA was coupled to recombinant Z(HER2:342), and the conjugate was efficiently labeled with 111In at 60 degrees C. The specificity of 111In-benzyl-DOTA-Z(HER2:342) binding to HER2 was confirmed in vitro using HER2-expressing breast carcinoma BT474 and ovarian carcinoma SKOV-3 cell lines. Biodistribution of 111In-benzyl-DOTA-Z(HER2:342) was performed in nude mice bearing LS174T xenografts and compared directly with the biodistribution of 111In-benzyl-DTPA-Z(HER2:342). In vivo, 111In-benzyl-DOTA-Z(HER2:342) demonstrated quick clearance from blood and non-specific organs except the kidneys. Four hours post injection (pi), the tumor uptake of 111In-benzyl-DOTA-Z(HER2:342) (4.4+/-1.0% IA/g) was specific and the tumor-to-blood ratio was 23. The use of benzyl-DTPA provided higher tumor-to-blood and tumor-to-liver ratios. gamma-camera imaging showed clear visualization of HER2-expressing xenografts using 111In-benzyl-DOTA-Z(HER2:342). 111In-benzyl-DOTA-Z(HER2:342) has a potential for imaging of HER2 expression in malignant tumors.

Labelling chemistry and characterization of [90Y/177Lu]-DOTA-ZHER2:342-3 Affibody molecule, a candidate agent for locoregional treatment of urinary bladder carcinoma.

The direct instillation of radiolabelled conjugates in the urinary bladder is a promising path for the treatment of bladder carcinoma. The targeting of HER2/neu receptors expressed on the surface of many bladder carcinoma cells shows potential to be developed as a therapeutic strategy, and patients identified with a high risk of progression may benefit from adjuvant targeted radionuclide therapy. A phage-display selected Affibody molecule (Z(HER2:342)) which binds to HER2/neu with picomolar affinity, can be used for targeting HER2/neu-expressing bladder carcinomas. A DOTA-derivative of Z(HER2:342), designated as DOTA-Z(HER2:342)-3, is considered as a suitable targeting agent for therapy. The DOTA chelator provides stable labelling with radiometals, and the low molecular weight (7.2 kDa) of the DOTA-Z(HER2:342)-3 compound is expected to enable efficient tumor penetration. DOTA-Z(HER2:342)-3 was radiolabelled with 90Y and 177Lu in 1 M ammonium acetate buffer, at pH 5.5, and in the presence of ascorbic acid. Nearly quantitative labelling yields were achieved for both nuclides after 15 min of incubation at 60 degrees C. After chelation, the conjugates retained their capacity to specifically bind to HER2/neu-expressing SKOV-3 cells. The radiolabelled affibody conjugate (DOTA-Z(HER2:342)-3) demonstrated high antigen-binding capacity and good cellular retention. Biodistribution in normal mice demonstrated low uptake in all organs and tissues except for kidneys.

[99mTc] HYNIC-hEGF, a potential agent for imaging of EGF receptors in vivo: preparation and pre-clinical evaluation.

Expression of epidermal growth factor receptors (EGFR) has prognostic and predictive value in many kinds of tumors. Imaging of expression of EGFR in vivo may give valuable diagnostic information. The epidermal growth factor (EGF), a natural ligand, is a possible candidate for the targeting of EGFR. The present study describes a method for preparation of (99m)Tc-EGF via the hydrazinopyridine-3-carboxylic acid (HYNIC) conjugation using tricine and ethylenediamine-N,N'-diacetic acid (EDDA) as co-ligands. Both conjugates bound EGFR expressing cells with nanomolar affinity, and demonstrated good intracellular retention. The complex with EDDA demonstrated much higher stability in blood serum and during cysteine challenge. Biodistribution of (99m)Tc-EDDA-HYNIC-EGF in normal mice demonstrated fast blood clearance of conjugate, and its ability to bind EGFR in vivo. (99m)Tc-EDDA-HYNIC-EGF is a promising candidate for visualization of EGFR expression in vivo.

Biosolids compost amendment for reducing soil lead hazards: a pilot study of Orgro amendment and grass seeding in urban yards.

In situ inactivation of soil Pb is an alternative to soil removal and replacement that has been demonstrated in recent years at industrial sites with hazardous soil Pb concentrations. Most children exposed to elevated soil Pb, however, reside in urban areas, and no government programs exist to remediate such soils unless an industrial source caused the contamination. Modern regulated biosolids composts have low Pb concentrations and low bioaccessible Pb fractions and can improve grass growth on urban soils. High Fe and P biosolids composts can reduce the bioavailability and bioaccessibility of soil Pb and can aid in establishing vegetation that would reduce soil transfer into homes. For these reasons, we conducted a field test of their use to reduce Pb bioaccessibility in urban soils in Baltimore, MD USA. We chose biosolids compost for its expected reduction in the bioaccessible Pb fraction of urban soils, ease of use by urban residents, and ability to beautify urban areas. Nine urban yards with mean soil Pb concentrations >800 mg Pb kg(-1) were selected and sampled at several distances from the house foundation before soil treatment. The soils were rototilled to 20 cm depth to prepare the sites, and resampled. The yards were then amended with 6-8 cm depth of Orgro biosolids compost (110-180 dry t/ha) rich in Fe and P, mixed well by rototilling, and resampled. Kentucky bluegrass (Poa pratensis) was seeded and became well established. Soils were resampled 1 year later. At each sampling time, total soil Pb was measured using a modified U.S. EPA nitric acid hotplate digestion method (SW 846 Method 3050) and bioaccessible Pb fraction was measured using the Solubility/Bioaccesibility Research Consortium standard operating procedure with modifications, including the use of glycine-buffered HCl at pH 2.2. Samples of untreated soils were collected from each yard and mixed well to serve as controls for the Pb bioaccessibility of field treated soils over time independent of positional variance within yards. At 1-year post-treatment, grass cover was healthy and reductions in bioaccessible Pb concentrations compared to pre-tillage were 64% (from 1655 to 595 mg kg(-1)) and 67% (from 1381 to 453 mg kg(-1)) at the sampling lines closest to the houses. Little or no reduction in bioaccessible Pb concentration was observed at sampling lines more remote from the house that also had the lowest bioaccessible Pb concentrations at pre-tillage (620 and 436 mg kg(-1), respectively). For the control soils, changes over time in total Pb and bioaccessible Pb concentrations and the bioaccessible Pb fraction were insignificant. This study confirms the viability of in situ remediation of soils in urban areas where children are at risk of high Pb exposure from lead in paint, dust and soil.

Targeting of a head and neck squamous cell carcinoma xenograft model using the chimeric monoclonal antibody U36 radioiodinated with a closo-dodecaborate-containing linker.

OBJECTIVE: High rates of local recurrence and distant metastases following surgery of high-grade head and neck squamous cell carcinoma (HNSCC) necessitate the use of adjuvant systemic treatment. Radioimmunotargeting might be a possible treatment modality in this case. The nuclear properties of 131I make it a suitable isotope for treatment of minimal residual disease and small metastases, but the conventional radioiodine label has poor cellular retention and its radiocatabolites accumulate in the thyroid. We attempted to overcome these problems by using closo-dodecaborate derivatives for attachment of radioiodine. MATERIAL AND METHODS: We investigated the feasibility of targeting an SCC25 HNSCC xenograft in vivo using a benzylisothiocyanate derivative of closo-dodecaborate (DABI) as radioiodine linker and the chimeric anti-CD44v6 antibody U36. 125I was used in biodistribution studies. RESULTS: The use of DABI enabled tumor targeting and decreased the radioactivity uptake of the thyroid. CONCLUSION: Tumor localization of DABI-labeled U36 was similar to its para-iodobenzoate-labeled counterpart, presumably due to the strong dependence of targeting efficiency on tumor size.

[177Lu]Bz-DTPA-EGF: Preclinical characterization of a potential radionuclide targeting agent against glioma.

Patients with glioblastoma multiforme have a poor prognosis due to recurrences originating from spread cells. The use of radionuclide targeting might increase the chance of inactivating single tumor cells with minimal damage to surrounding healthy tissue. As a target, overexpressed epidermal growth factor receptors (EGFR) may be used. A natural ligand to EGFR, the epidermal growth factor (EGF) is an attractive targeting agent due to its low molecular weight (6 kDa) and high affinity for EGFR. 177Lu (T(1/2) = 6.7 days) is a radionuclide well suited for treatment of small tumor cell clusters, since it emits relatively low-energy beta particles. The goal of this study was to prepare and preclinically evaluate both in vitro and in vivo the [177Lu]Bz-DTPA-EGF conjugate. The conjugate was characterized in vitro for its cell-binding properties, and in vivo for its pharmacokinetics and ability to target EGFR. [177Lu]Bz-DTPA-EGF bound to cultured U343 glioblastoma cells with an affinity of 1.9 nM. Interaction with EGFR led to rapid internalization, and more than 70% of the cell-associated radioactivity was internalized after 30 minutes of incubation. The retention of radioactivity was good, with more than 65% of the 177Lu still cell-associated after 2 days. Biodistribution studies of i.v. injected [177Lu]Bz-DTPA-EGF in NMRI mice demonstrated a rapid blood clearance. Most of the radioactivity was found in the liver and kidneys. The liver uptake was receptor-mediated, since it could be significantly reduced by preinjection of unlabeled EGF. In conclusion, [177Lu]Bz-DTPA-EGF seems to be a promising candidate for locoregional treatment of glioblastoma due to its high binding affinity, low molecular weight, and ability to target EGFR in vivo.