The Radiotherapeutic Potential of the Epidermal Growth Factor Receptor

The epidermal growth factor receptor (EGFR) is overexpressed in a wide range of tumour types. Molecules which target and sterilise EGFR-overexpressing cells, or increase their sensitivity to conventional therapies may have a beneficial effect in the clinical management of these tumours. This study describes two approaches adopted to target EGFR-overexpressing cells. Firsdy, we examined the effect of exogenous epidermal growth factor (EGF), a polypeptide growth factor which binds to and activates EGFR, on the radiation sensitivity of a panel of cell lines with a wide range of EGFR expression. This effect was assessed by colony forming assay and the results obtained were analysed using the linear quadratic model of cell survival. Our results indicated that the presence of EGF did not alter the radiosensitivity of any of the cell lines examined. This suggests that the use of EGF as a radiosensitising agent is unlikely to be of benefit in a clinical setting. The second approach adopted was to investigate the possibility of using EGF as a means of delivering therapeutic ultra-short range radionuclides to EGFR- overexpressing cells. The short range of the particles emitted from these radionuclides (Auger electrons) requires that decay occurs in close proximity to cellular DNA for a radiobiological effect to be observed. For EGF to act as an effective delivery vehicle for these radionuclides, it must be capable of delivering the radionuclide to the nucleus of the target cell. Studies were carried out to assess the sub-cellular distribution of EGF after receptor binding and internalisation. Using both light and elctron microscopic techniques along with cell fractionation, we established that exogenous EGF does accumulate in the nucleus of some EGFR-overexpressing cell lines under the appropriate conditions. These results suggested that EGF may be suitable as a delivery vehicle for Auger-emitting radionuclides. Finally, we examined the effect of EGF conjugated to the Auger-emitting radionuclide 1231 on the clonogenic survival of a cell line panel with a wide range of EGFR expression. We observed that 123I-EGF was able to sterilise some EGFR-overexpressing cell lines, and that cell lines with a higher EGFR expression were more senitive tol23l-EGF than those with a lower EGFR expression. It was also observed that incubation conditions which abolished the nuclear uptake of EGF also abolished 1231-EGF-mediated cytotoxicity, suggesting that 123I-EGF sensitivity is dependent upon the nuclear uptake of the radioconjugate. The function of nuclear EGF remains to be established, as does the nuclear translocation mechanism. However, the possibility of EGFR-targeted therapies based upon this phenomenon, as outlined in this report, implies that further studies into the nuclear accumulation of EGF and other polypeptide growth factors are warranted

Molecular Targeting of Carbonic Anhydrase IX in Mice with Hypoxic HT29 Colorectal Tumor Xenografts

Background: Carbonic anhydrase IX (CAIX) is a membrane spanning protein involved in the enzymatic regulation of tumor acid-base balance. CAIX has been shown to be elevated in a number of hypoxic tumor types. The purpose of this study was to determine the efficiency of intact and IgG fragments of cG250 to target CAIX in vivo in a hypoxic tumor model. Methodology/Principal Findings: Conventional biodistribution studies were performed with 111 In-DO3A-cG250, 111 In-DO3A-F(ab’)2-cG250 and 111 In-DO3A-Fab-cG250. Additional ex vivo analysis of the tumor was performed with markers for tumor hypoxia, blood perfusion and endogenous CAIX expression. All four data sets were digitally correlated to determine the optimal agent for determining hypoxia in a HT29 colon cancer xenograft. The HT29 human colorectal tumor xenografts show strong CAIX expression in hypoxic areas of poor blood perfusion. The intact IgG had an initial high focal uptake at the periphery of these hypoxic regions and penetration into the areas of highest CAIX expression over the 7-day study period. The lower molecular weight antibody fragments had a faster uptake into areas of high CAIX expression, but had a much lower absolute uptake at the optimal imaging times. Conclusions/Significance: For the clinical detection of hypoxia induced CAIX using cG250 antibody based agents, imagin

A Pretargeted Approach for the Multimodal PET/NIRF Imaging of Colorectal Cancer

The complementary nature of positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging makes the development of strategies for the multimodal PET/NIRF imaging of cancer a very enticing prospect. Indeed, in the context of colorectal cancer, a single multimodal PET/NIRF imaging agent could be used to stage the disease, identify candidates for surgical intervention, and facilitate the image-guided resection of the disease. While antibodies have proven to be highly effective vectors for the delivery of radioisotopes and fluorophores to malignant tissues, the use of radioimmunoconjugates labeled with long-lived nuclides such as 89Zr poses two important clinical complications: high radiation doses to the patient and the need for significant lag time between imaging and surgery. In vivo pretargeting strategies that decouple the targeting vector from the radioactivity at the time of injection have the potential to circumvent these issues by facilitating the use of positron-emitting radioisotopes with far shorter half-lives. Here, we report the synthesis, characterization, and in vivo validation of a pretargeted strategy for the multimodal PET and NIRF imaging of colorectal carcinoma. This approach is based on the rapid and bioorthogonal ligation between a trans-cyclooctene- and fluorophore-bearing immunoconjugate of the huA33 antibody (huA33-Dye800-TCO) and a 64Cu-labeled tetrazine radioligand (64Cu-Tz-SarAr). In vivo imaging experiments in mice bearing A33 antigen-expressing SW1222 colorectal cancer xenografts clearly demonstrate that this approach enables the non-invasive visualization of tumors and the image-guided resection of malignant tissue, all at only a fraction of the radiation dose created by a directly labeled radioimmunoconjugate. Additional in vivo experiments in peritoneal and patient-derived xenograft models of colorectal carcinoma reinforce the efficacy of this methodology and underscore its potential as an innovative and useful clinical tool

PET Imaging of Extracellular pH in Tumors with \u3csup\u3e64\u3c/sup\u3eCu- and \u3csup\u3e18\u3c/sup\u3eF-Labeled pHLIP Peptides: A Structure–Activity Optimization Study

pH (low) insertion peptides (pHLIP peptides) target acidic extracellular environments in vivo due to pH-dependent cellular membrane insertion. Two variants (Var3 and Var7) and wild-type (WT) pHLIP peptides have shown promise for in vivo imaging of breast cancer. Two positron emitting radionuclides (64Cu and 18F) were used to label the NOTA- and NO2A-derivatized Var3, Var7, and WT peptides for in vivo biodistribution studies in 4T1 orthotopic tumor-bearing BALB/c mice. All of the constructs were radiolabeled with 64Cu or [18F]-AlF in good yield. The in vivo biodistribution of the 12 constructs in 4T1 orthotopic allografted female BALB/c mice indicated that NO2A-cysVar3, radiolabeled with either 18F (4T1 uptake; 8.9 ± 1.7%ID/g at 4 h p.i.) or 64Cu (4T1 uptake; 8.2 ± 0.9%ID/g at 4 h p.i. and 19.2 ± 1.8% ID/g at 24 h p.i.), shows the most promise for clinical translation. Additional studies to investigate other tumor models (melanoma, prostate, and brain tumor models) indicated the universality of tumor targeting of these tracers. From this study, future clinical translation will focus on 18F- or 64Cu-labeled NO2A-cysVar3

Polymeric nanocarriers co-encapsulating PET probes and protein therapeutics

Nanocarriers encapsulating nucleic acids or protein therapeutics are important tools for modulating biodistribution and enhancing intracellular delivery of biologics. We have recently developed inverse Flash NanoPrecipitation (iFNP), demonstrating its effectiveness in encapsulating biologics at high loadings and encapsulation efficiency. Here, we present the biodistribution of two iFNP nanocarriers using 64Cu positron emission tomography imaging in a murine adenocarcinoma xenograft model characterized by elevated macrophage content. Two nanocarriers with similar sizes and surfaces were prepared. iFNP produces core-shell-corona nanocarriers where the hydrophobic shell layer in one case was poly(lactic acid) (PLA), and the other nanocarrier shell was poly(styrene) (PS). While the expectation was that the biodistribution and clearance of both nanocarriers would be similar, it was found that the clearance of the PS nanocarrier oc-curred in less than 3 hours while the PLA nanocarrier exhibited sustained circulation times. The mechanism of nanocarrier instability for the PS shell nanocarrier manifests as the development of a negative surface charge due to the exposure of the anionic nanocarrier inner core. The stable PLA-based formulation exhibited circulation times greater than 24 hours and enhanced accumu-lation in the lymphatics and the tumor relative to the unstable formulation. The novel mecha-nism of encapsulation by iFNP motivates the fundamental studies on nanoparticle biodistribu-tion reported here

LSST: from Science Drivers to Reference Design and Anticipated Data Products

(Abridged) We describe here the most ambitious survey currently planned in the optical, the Large Synoptic Survey Telescope (LSST). A vast array of science will be enabled by a single wide-deep-fast sky survey, and LSST will have unique survey capability in the faint time domain. The LSST design is driven by four main science themes: probing dark energy and dark matter, taking an inventory of the Solar System, exploring the transient optical sky, and mapping the Milky Way. LSST will be a wide-field ground-based system sited at Cerro Pach\'{o}n in northern Chile. The telescope will have an 8.4 m (6.5 m effective) primary mirror, a 9.6 deg$^2$ field of view, and a 3.2 Gigapixel camera. The standard observing sequence will consist of pairs of 15-second exposures in a given field, with two such visits in each pointing in a given night. With these repeats, the LSST system is capable of imaging about 10,000 square degrees of sky in a single filter in three nights. The typical 5$\sigma$ point-source depth in a single visit in $r$ will be $\sim 24.5$ (AB). The project is in the construction phase and will begin regular survey operations by 2022. The survey area will be contained within 30,000 deg$^2$ with $\delta<+34.5^\circ$, and will be imaged multiple times in six bands, $ugrizy$, covering the wavelength range 320--1050 nm. About 90\% of the observing time will be devoted to a deep-wide-fast survey mode which will uniformly observe a 18,000 deg$^2$ region about 800 times (summed over all six bands) during the anticipated 10 years of operations, and yield a coadded map to $r\sim27.5$. The remaining 10\% of the observing time will be allocated to projects such as a Very Deep and Fast time domain survey. The goal is to make LSST data products, including a relational database of about 32 trillion observations of 40 billion objects, available to the public and scientists around the world.Comment: 57 pages, 32 color figures, version with high-resolution figures available from https://www.lsst.org/overvie

Pilot study of PET imaging of 124I-iodoazomycin galactopyranoside (IAZGP), a putative hypoxia imaging agent, in patients with colorectal cancer and head and neck cancer

Background: Hypoxia within solid tumors confers radiation resistance and a poorer prognosis. 124I-iodoazomycin galactopyranoside (124I-IAZGP) has shown promise as a hypoxia radiotracer in animal models. We performed a clinical study to evaluate the safety, biodistribution, and imaging characteristics of 124I-IAZGP in patients with advanced colorectal cancer and head and neck cancer using serial positron emission tomography (PET) imaging. Methods: Ten patients underwent serial whole-torso (head/neck to pelvis) PET imaging together with multiple whole-body counts and blood sampling. These data were used to generate absorbed dose estimates to normal tissues for 124I-IAZGP. Tumors were scored as either positive or negative for 124I-IAZGP uptake. Results: There were no clinical toxicities or adverse effects associated with 124I-IAZGP administration. Clearance from the whole body and blood was rapid, primarily via the urinary tract, with no focal uptake in any parenchymal organ. The tissues receiving the highest absorbed doses were the mucosal walls of the urinary bladder and the intestinal tract, in particular the lower large intestine. All 124I-IAZGP PET scans were interpreted as negative for tumor uptake. Conclusions: It is safe to administer 124I-IAZGP to human subjects. However, there was insufficient tumor uptake to support a clinical role for 124I-IAZGP PET in colorectal cancer and head and neck cancer patients. Trial registration: ClinicalTrials.gov NCT0058827

Real-Time Imaging of HIF-1α Stabilization and Degradation

HIF-1α is overexpressed in many human cancers compared to normal tissues due to the interaction of a multiplicity of factors and pathways that reflect specific genetic alterations and extracellular stimuli. We developed two HIF-1α chimeric reporter systems, HIF-1α/FLuc and HIF-1α(ΔODDD)/FLuc, to investigate the tightly controlled level of HIF-1α protein in normal (NIH3T3 and HEK293) and glioma (U87) cells. These reporter systems provided an opportunity to investigate the degradation of HIF-1α in different cell lines, both in culture and in xenografts. Using immunofluorescence microscopy, we observed different patterns of subcellular localization of HIF-1α/FLuc fusion protein between normal cells and cancer cells; similar differences were observed for HIF-1α in non-transduced, wild-type cells. A dynamic cytoplasmic-nuclear exchange of the fusion protein and HIF-1α was observed in NIH3T3 and HEK293 cells under different conditions (normoxia, CoCl2 treatment and hypoxia). In contrast, U87 cells showed a more persistent nuclear localization pattern that was less affected by different growing conditions. Employing a kinetic model for protein degradation, we were able to distinguish two components of HIF-1α/FLuc protein degradation and quantify the half-life of HIF-1α fusion proteins. The rapid clearance component (t1/2 ∼4–6 min) was abolished by the hypoxia-mimetic CoCl2, MG132 treatment and deletion of ODD domain, and reflects the oxygen/VHL-dependent degradation pathway. The slow clearance component (t1/2 ∼200 min) is consistent with other unidentified non-oxygen/VHL-dependent degradation pathways. Overall, the continuous bioluminescence readout of HIF-1α/FLuc stabilization in vitro and in vivo will facilitate the development and validation of therapeutics that affect the stability and accumulation of HIF-1α