The fibrous shape of carbon nanotubes (CNTs) raises concern that they may pose an asbestos-like inhalation hazard, leading to the development of diseases, especially mesothelioma. Direct instillation of long and short CNTs into the pleural cavity, the site of mesothelioma development, produced asbestos-like length-dependent responses. The response to long CNTs and long asbestos was characterized by acute inflammation, leading to progressive fibrosis on the parietal pleura, where stomata of strictly defined size limit the egress of long, but not short, fibers. This was confirmed by demonstrating clearance of short, but not long, CNT and nickel nanowires and by visualizing the migration of short CNTs from the pleural space by single-photon emission computed tomographic imaging. Our data confirm the hypothesis that, although a proportion of all deposited particles passes through the pleura, the pathogenicity of long CNTs and other fibers arises as a result of length-dependent retention at the stomata on the parietal pleura.

MUC1 is a highly attractive immunotherapeutic target owing to increased expression, altered glycosylation, and loss of polarity in >80% of human cancers. To exploit this, we have constructed a panel of chimeric Ag receptors (CAR) that bind selectively to tumor-associated MUC1. Two parameters proved crucial in optimizing the CAR ectodomain. First, we observed that the binding of CAR-grafted T cells to anchored MUC1 is subject to steric hindrance, independent of glycosylation status. This was overcome by insertion of the flexible and elongated hinge found in immunoglobulins of the IgD isotype. Second, CAR function was highly dependent upon strong binding capacity across a broad range of tumor-associated MUC1 glycoforms. This was realized by using an Ab-derived single-chain variable fragment (scFv) cloned from the HMFG2 hybridoma. To optimize CAR signaling, tripartite endodomains were constructed. Ultimately, this iterative design process yielded a potent receptor termed HOX that contains a fused CD28/OX40/CD3ζ endodomain. HOX-expressing T cells proliferate vigorously upon repeated encounter with soluble or membrane-associated MUC1, mediate production of proinflammatory cytokines (IFN-γ and IL-17), and elicit brisk killing of MUC1+ tumor cells. To test function in vivo, a tumor xenograft model was derived using MDA-MB-435 cells engineered to coexpress MUC1 and luciferase. Mice bearing an established tumor were treated i.p. with a single dose of engineered T cells. Compared with control mice, this treatment resulted in a significant delay in tumor growth as measured by serial bioluminescence imaging. Together, these data demonstrate for the first time that the near-ubiquitous MUC1 tumor Ag can be targeted using CAR-grafted T cells.

Functionalization of nanomaterials for precise biomedical function is an emerging trend in nanotechnology. Carbon nanotubes are attractive as multifunctional carrier systems because payload can be encapsulated in internal space whilst outer surfaces can be chemically modified. Yet, despite potential as drug delivery systems and radiotracers, such filled-and-functionalized carbon nanotubes have not been previously investigated in vivo. Here we report covalent functionalization of radionuclide-filled single-walled carbon nanotubes and their use as radioprobes. Metal halides, including Na(125)I, were sealed inside single-walled carbon nanotubes to create high-density radioemitting crystals and then surfaces of these filled-sealed nanotubes were covalently modified with biantennary carbohydrates, improving dispersibility and biocompatibility. Intravenous administration of Na(125)I-filled glyco-single-walled carbon nanotubes in mice was tracked in vivo using single-photon emission computed tomography. Specific tissue accumulation (here lung) coupled with high in vivo stability prevented leakage of radionuclide to high-affinity organs (thyroid/stomach) or excretion, and resulted in ultrasensitive imaging and delivery of unprecedented radiodose density. Nanoencapsulation of iodide within single-walled carbon nanotubes enabled its biodistribution to be completely redirected from tissue with innate affinity (thyroid) to lung. Surface functionalization of (125)I-filled single-walled carbon nanotubes offers versatility towards modulation of biodistribution of these radioemitting crystals in a manner determined by the capsule that delivers them. We envisage that organ-specific therapeutics and diagnostics can be developed on the basis of the nanocapsule model described here.

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Targeted therapies have yet to have significant impact on the survival of patients with bladder cancer. In this study, we focused on the urea cycle enzyme argininosuccinate synthetase 1 (ASS1) as a therapeutic target in bladder cancer, based on our discovery of the prognostic and functional import of ASS1 in this setting. ASS1 expression status in bladder tumors from 183 Caucasian and 295 Asian patients was analyzed, along with its hypothesized prognostic impact and association with clinicopathologic features, including tumor size and invasion. Furthermore, the genetics, biology, and therapeutic implications of ASS1 loss were investigated in urothelial cancer cells. We detected ASS1 negativity in 40% of bladder cancers, in which multivariate analysis indicated worse disease-specific and metastasis-free survival. ASS1 loss secondary to epigenetic silencing was accompanied by increased tumor cell proliferation and invasion, consistent with a tumor-suppressor role for ASS1. In developing a treatment approach, we identified a novel targeted antimetabolite strategy to exploit arginine deprivation with pegylated arginine deiminase (ADI-PEG20) as a therapeutic. ADI-PEG20 was synthetically lethal in ASS1-methylated bladder cells and its exposure was associated with a marked reduction in intracellular levels of thymidine, due to suppression of both uptake and de novo synthesis. We found that thymidine uptake correlated with thymidine kinase-1 protein levels and that thymidine levels were imageable with [ 18 F]-fluoro-L-thymidine (FLT)-positron emission tomography (PET). In contrast, inhibition of de novo synthesis was linked to decreased expression of thymidylate synthase and dihydrofolate reductase. Notably, inhibition of de novo synthesis was associated with potentiation of ADI-PEG20 activity by the antifolate drug pemetrexed. Taken together, our findings argue that arginine deprivation combined with antifolates warrants clinical investigation in ASS1-negative urothelial and related cancers, using FLT-PET as an early surrogate marker of response. Cancer Res; 74(3); 896-907. Ó2013 AACR.

[111In-diethylene triamine penta-acetic acid-D-Phe1]-octreotide (DTPA-octreotide) scintigraphy has gained widespread acceptance as a diagnostic clinical procedure in oncology for imaging somatostatin receptor-positive tumours. However, indium-111 as a radiolabel has several drawbacks, including limited availability, suboptimal gamma energy and high radiation burden to the patient. We have recently reported on the preclinical development of 99mTc-EDDA/HYNIC-TOC, a new octreotide derivative which showed promising results both in vitro and in vivo. We now report our initial clinical experiences with this new radiopharmaceutical in ten oncological patients. The clinical diagnoses were: carcinoid syndrome (n=5), thyroid cancer (n=3), pancreatic cancer (n=1) and pituitary tumour (n=1). The biodistribution and kinetics of 99mTc-EDDA/HYNIC-TOC were compared with those of 111In-DTPA-octreotide in six cases, and with those of 111In-DOTA-TOC in five cases. With the new tracer tumours were imaged within 15 min after injection and showed the highest target/non-target ratios 4 h after injection. Tumour uptake persisted up to 20 h p.i. The rate of blood clearance was similar to that of 111In-DTPA-octreotide but faster than that of 111In-DOTA-TOC, while urinary excretion was lower compared with the 111In derivatives. Semi-quantitative region of interest analysis showed that 99mTc-EDDA/HYNIC-TOC produced higher tumour/organ (target/non-target) ratios than the 111In derivatives, especially in relation to heart and muscle. Significantly more lesions could be detected in 99mTc images. We conclude that 99mTcEDDA/HYNIC-TOC shows better imaging properties for the identification of somatostatin receptor-positive tumour sites than currently available 111In-labelled octreotide derivatives.

Nuclear medicine imaging offers the possibility to study in vivo different aspects of inflammatory process by the use of radio-labeled molecules that bind to specific receptor targets on cells and tissues. This noninvasive technique, combined with pathogens engineered to express luciferase, allows quantification in the same animal of the spatial and temporal progression of the infection and identification of animal-to-animal variations in pathogen replication and dissemination. One strategy to use optical imaging in living animals is the use of luciferase reporter genes as internal sources of light called bioluminescence imaging (BLI). This enables real-time noninvasive imaging of infections and gene expression in living organisms. Nuclear medicine imaging is characterized by the use of radio pharmaceuticals (radiolabeled probes) that, administered in pico- and nanomolar amounts. These probes can also be used for early diagnosis of diseases, in susceptible subjects, for detection of disease relapse and radio-guided surgery

PurposeHuman epidermal growth factor receptor-2 (HER2) overexpression is a predictor of response to anti-HER2 therapy in breast and gastric cancer. Currently, HER2 status is assessed by tumour biopsy, but this may not be representative of the larger tumour mass or other metastatic sites, risking misclassification and selection of suboptimal therapy. The designed ankyrin repeat protein (DARPin) G3 binds HER2 with high affinity at an epitope that does not overlap with trastuzumab and is biologically inert. We hypothesized that radiolabelled DARPin G3 would be capable of selectively imaging HER2-positive tumours, and aimed to identify a suitable format for clinical application.MethodsG3 DARPins tagged with hexahistidine (His6) or with histidine glutamate (HE)3 and untagged G3 DARPins were manufactured using a GMP-compatible Pichia pastoris protocol and radiolabelled with 125I, or with 111In via DOTA linked to a C-terminal cysteine. BALB/c mice were injected with radiolabelled G3 and tissue biodistribution was evaluated by gamma counting. The lead construct ((HE)3-G3) was assessed in mice bearing HER2-positive human breast tumour (BT474) xenografts.ResultsFor both isotopes, (HE)3-G3 had significantly lower liver uptake than His6-G3 and untagged G3 counterparts in non-tumour-bearing mice, and there was no significantly different liver uptake between His6-G3 and untagged G3. (HE)3-G3 was taken forward for evaluation in mice bearing HER2-positive tumour xenografts. The results demonstrated that radioactivity from 111In-(HE)3-G3 was better maintained in tumours and cleared faster from serum than radioactivity from 125I-(HE)3-G3, achieving superior tumour-to-blood ratios (343.7 ± 161.3 vs. 22.0 ± 11.3 at 24 h, respectively). On microSPECT/CT, 111In-labelled and 125I-labelled (HE)3-G3 could image HER2-positive tumours at 4 h after administration, but there was less normal tissue uptake of radioactivity with 111In-(HE)3-G3. Preadministration of trastuzumab did not affect the uptake of (HE)3-G3 by HER2-positive tumours.ConclusionRadiolabelled DARPin (HE)3-G3 is a versatile radioligand with potential to allow the acquisition of whole-body HER2 scans on the day of administration.Electronic supplementary materialThe online version of this article (doi:10.1007/s00259-014-2940-2) contains supplementary material, which is available to authorized users.