The neurofilament light chain protein (NfL) is a suggested general marker for neuronal loss. Its release from brain parenchyma into cerebral spinal fluid, and presumed detection in blood has seen it established as a first blood-based marker of disease activity and drug efficacy in multiple sclerosis (MS) and in the presymptomatic diagnosis and assessment of disease course for other neurodegenerative disorders. However, the lack of characterisation of its behaviour in circulation, largely due to its antibody-dependent measurement, have hampered the biological interpretation of these measurements, especially after acute injury such as in MS relapse or head trauma. Here, we describe a strategy for exploiting positron emission tomography (PET) imaging using isosteric protein mimics following the installation of a fluorine-18 label that is benign enough to provide sensitive, real-time information on the dynamics and trafficking of NfL protein. This circumvents the limits of current methods that integrate 18F into proteins through the bio-conjugation of bulky, unnatural groups, which we show perturb NfL's assembly and functional properties from those in the natural state. We use a visible-light-driven reaction to access radioactive isostere proteins that are unperturbed and so closely resemble their native form. In this way, generation of [18F]fluoroalkyl radicals that can be rapidly reacted at pre-defined sites on proteins creates mimics of proteinogenic side chains bearing near-zero-size labels to probe proteins in functionally 'true' form. These prosthetic-free, protein radiotracers can be generated in excellent radiochemical yield (up to 67%) via a semi-automated protocol in just 15 mins. High associated molar activities (precursor up to 102 GBq μmol-1) allowed high sensitivity dynamic observations in blood, brain and cerebrospinal fluid, enabling even the first unambiguous observations of spinal flow kinetics using proteins. These dynamics, including the high rate of spinal flow (on the order of mm per min) and drainage of NfL from CSF into sacral lymph nodes, now provides evidence that the slow fall rate of antibody-detected markers that is observed after acute neural insults is not due to a long half-life, but rather reflects sustained neuronal loss. This discovery will now help to better correlate clinical and radiological features of disease with NfL blood levels. Our methodology now demonstrates the broad potential of a near-zero-size labelling method for the functional study of proteins in whole organisms without interfering with their biological activity and native assembly. ### Competing Interest Statement A.W.J.P., B.J., V.G. and B.G.D. are listed on patents filed by Oxford University Innovations concerning protein editing.

In attempting to observe the behaviour of a protein of interest in vivo , akin to other observer effects, current techniques require modifications or interventions that inherently alter the protein or its host, leaving one uncertain as to whether natural behaviour remains unperturbed. The study of chromatin has mostly been restricted to defining its function in the nucleus, where histone proteins fulfil vital roles in packaging genomic DNA and regulating transcription. However, chromatin components can be released into the extracellular space, either intentionally via cellular secretion or during disease-induced cell death. These extracellular chromatin components, depending on the context, can consist of: free histones, free DNA, intact nucleosomes (histone octamers wrapped by DNA), or heterogeneous, higher order structures such as neutrophil extracellular traps (NETs). They have been associated with diverse pathologies such as inflammation, cancer, and sepsis, and distinct toxic effects. However, there is a widely acknowledged lack of methods that distinguish between them and between their unique functions. Here, we now address protein observer effects to explore the fate and function of extracellular free histones by utilizing FIRESCAPE ([18F]- F luorine I sotopic R adiolabeling E nabling S canning of C learance A fter P roteolytic E vents), a novel radiolabeling concept that leverages the unique, high-sensitivity properties of the radioisotope fluorine-18, 18F, and residue-specific protein editing chemistry. By installing close and then ‘true’ 18F-containing protein sidechain mimics site-specifically, FIRESCAPE enables the hierarchical in vivo scanning of the half-lives, proteolytic susceptibility and clearance of single residues in a protein of interest, and at microdoses far below toxic levels (low nanomole). These ‘radioequivalent’ proteins bearing near-zero-size, zero-background labels now precisely reveal the strikingly distinct distribution, half-lives, damage-inducing abilities and accumulation of free extracellular histones in cellulo and in vivo compared to intact nucleosomes. Free extracellular histone H3 is rapidly cleared from circulation, mediated first by proteolysis of the histone tail. By contrast, direct injection of free histones vs nucleosomes into tissue that is unprotected by such proteolysis (brain), provokes a starkly different response; free histones exhibited limited diffusion and swiftly promoted damage both in cell culture and in vivo , whilst intact nucleosomes were essentially passive and benign. Remarkably, synthetic extracellular histone H3 was observed to enter cells and integrate into chromatin, indistinguishable from native H3 in both localization and post-translational modification (PTM) accumulation, yet paired with cellular and tissue damage. The exploratory studies described here now provide much needed clarity to the distinct fates and effects of extracellular histones vs nucleosomes, in particular the strongly damaging effects of free histones, their rapid uptake into cells, and an associated histone-specific proteolysis pathway via the removal of the histone tail. ### Competing Interest Statement A.W.J.P., B.J., V.G. and B.G.D. are listed on patents filed by Oxford University Innovations concerning protein editing. University of Brunei Darussalam Chancellor Scholarship, - UKRI-BBSRC, BB/V010999/1 UKRI-EPSRC, EP/V011359/1, EP/T012021/1, EP/X527245/1

PDF - 108KB, Supplementary methods.

Abstract [177Lu]Lu-PSMA improves progression-free and overall survival in metastatic castration resistant prostate cancer. However, the role of immune stimulation by molecularly targeted radionuclide therapy (MRT) is poorly understood. The aim of this study was to evaluate the anti-tumor immune response induced by [177Lu]Lu-PSMA. The markers of immunogenic cell death (ICD), translocation of calreticulin (CRT) to the cell membrane, HMGB1 release and ATP release, were evaluated in the RM1 cell line (PSMA-negative murine prostate carcinoma) and its derivative, RM1-PGLS (stably transduced with human PSMA and SFG-EGFp/Luc), using flow cytometry, ELISA and a luminescence detection assay respectively. The in vivo biodistribution of [177Lu]Lu-PSMA in RM1-PGLS tumor-bearing C57BL/6 mice was investigated using microSPECT-CT imaging, and the in vivo efficacy of [177Lu]Lu-PSMA was tested in RM1-PGLS tumor growth inhibition studies. Vaccination/rechallenge experiments were performed to elucidate whether [177Lu]Lu-PSMA elicits ICD in vivo. The Nanostring Pan Cancer Immune Profiling Platform was used to assess changes in the tumor immune microenvironment (TIME) in response to [177Lu]Lu-PSMA. External radiation (EBRT; 137Cs γ-radiation; 6-10 Gy) was used as a comparator treatment in most experiments. We observed a dose-dependent increase in cell surface CRT following [177Lu]Lu-PSMA and EBRT at 24 and 72 h and of HMGB1 release in response to [177Lu]Lu-PSMA and EBRT at 96 and 144 h. At 24 h following [177Lu]Lu-PSMA (60 MBq) there was a significant increase in ATP release compared to untreated samples. Collectively these data indicate [177Lu]Lu-PSMA causes ICD. A high RM1-PGLS tumor:muscle uptake ratio (210 ± 59) was noted in biodistribution experiments confirming highly specific tumor accumulation of [177Lu]Lu-PSMA. In tumor growth inhibition studies the time for tumors to reach a volume of 400 mm3 was delayed in mice treated with [177Lu]Lu-PSMA (60 MBq) compared to no treatment (30 versus 21 days; P<0.01). We showed that a subcutaneous inoculum of [177Lu]Lu-PSMA (60 MBq)-treated RM1-PGLS cells prevents tumor growth in approximately 50% of cases on rechallenge with viable RM1-PGLS cells one week later. This indicates that [177Lu]Lu-PSMA induces ICD in vivo. We observed a significant reduction in most tumor infiltrating lymphocyte (TIL) types at 2 days after in vivo exposure to [177Lu]Lu-PSMA (80 MBq), followed by an increase in TILs by Day 7. The observed pattern is similar to that after EBRT (6 Gy). There was overlap in the top 10 differentially expressed genes (treatment versus no treatment) for the [177Lu]Lu-PSMA and EBRT groups. The gene showing the greatest level of upregulation at day 7 in [177Lu]Lu-PSMA-treated tumors, IDO1, is known to be involved in immune tolerance and radioresistance. In conclusion, [177Lu]Lu-PSMA induces ICD in vitro and in vivo and modulates the TIME. Citation Format: Gemma Dias, Sarah Able, Irini Skaripa-Koukelli, Rachel Anderson, Gracie Wilson, Katherine A. Vallis. Evaluation of anti-tumor immunity in response to [177Lu]Lu-PSMA in a mouse model of prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5038.

PDF - 1380KB, Supplementary Figure S1. Schematic overview of the synthesis of (A) non-cleavable anti-gH2AX-PNE and (B) cleavable anti-gH2AX-N-SS-E. Supplementary Figure S2. (A) MDA-MB-468 cultures were exposed to 123I-EGF for 2 h plus increasing concentrations of EGF, anti-gH2AX-PNE or anti-gH2AX-N-SS-E. (B) Extracts from irradiated, gH2AX-containing 231-H2N cells were coated onto a RIA plate and exposed to 123I-anti-gH2AX for 2 h plus increasing amounts of anti-gH2AX, anti-gH2AX-PNE or anti-gH2AX-N-SS-E. For both (A) and (B) the relative amount of 123I that bound to plates was determined and LogIC50 values were calculated. Results are shown as the mean of three replicates plus or minus SD (C) PAGE analysis of 1. unmodified anti-gH2AX, 2. anti-gH2AX-PNE and 3. anti-gH2AX-N-SS-E. Supplementary Figure S3. (A) Anti-gH2AX-N-SS-(123I-EGF) was exposed to 0 or 1 mM glutathione. Size exclusion chromatography was performed using a G50 sephadex minicolumn. The amount of 123I in each fraction was measured. (B) 123I-EGF was synthesized as described in the methods section. G25 size exclusion chromatography was then performed on 123I-EGF and the cleaved product resulting from (A). (C) To demonstrate that EGF is released from rIgG-N-SS-E, rIgG-N-SS-E was exposed to glutathione and purified by G50 size exclusion chromatography. Then, EGFR positive MDA-MB-468 cells were exposed to 123I-EGF for 2 h at 4{degree sign}C with or without addition of the low molecular weight peak product. Blocking of 123I-EGF binding to MDA-MB-468 cells show that at least some of the low MW fraction binds to EGFR. Supplementary Figure S4: (A) 231-H2N cells were exposed for 4 h to AF555-labeled anti-γH2AX-PNE (red), fixed, and mounted with DAPI (blue). (B) 231-H2N cells were exposed for 4 h to AF555-labeled anti-γH2AX-N-SS-E (red), fixed, and mounted with DAPI (blue). Supplementary Figure S5: SQ20b cells were exposed for 1 h to AF555-labeled anti-gH2AX-N-SS-E or rIgG-N-SS-E, and irradiated (4 Gy) or sham irradiated. After 4 or 24 h, cells were fixed, stained for gH2AX (green) and mounted with DAPI (blue). Supplementary Figure S6: (A) MDA-MB-468, SQ20b or 231-H2N cells were exposed to IR (4 Gy) or sham-irradiated. After 1 or 4 h, cells were exposed for 2 h at 4{degree sign}C to increasing concentrations of 111In-EGF. To evaluate EGFR expression, the amount of membrane-bound 111In was determined. The number of EGFR/cell was unaltered by IR. (B) MDA-MB-468, SQ20b and 231-H2N cells were fixed, permeabilized and stained for EGFR (adapted from Cornelissen et al. J Nucl Med 2011; 52:776-783). Supplementary Figure S7. Proposed mechanism of action of 111In-anti-gH2AX-N-SS-E. (A) 111In-anti-gH2AX-N-SS-E binds to EGFR on the tumor cell membrane. (B) This binding triggers EGFR-mediated endocytosis and 111In-anti-gH2AX-N-SS-E becomes encapsulated in an endosome. (C) There, glutathione and reductive enzymes cleave the disulphide bond, to release 111In-anti-gH2AX-N, which (D) due to the NLS sequence, escapes from the endosome and localizes in the nucleus, through importin-NLS interaction. (E) gH2AX foci form following IR. (F) 111In-anti-gH2AX-N binds to gH2AX foci. (G) As 111In decays, it emits a shower of Auger electrons at the DSB site, which (H) causes more DSBs, and more gH2AX foci to be formed, eventually (I) leading to cell death. Supplementary Figure S8: 123I-labeled NLS shows a single peak on P4 SEC (black curve). When 123I-NLS was reacted with 0.8 mg/mL NaIO4 for 2 h there was no formation of higher molecular weight fragments (grey curve). However, after 24 h, there was evidence of dimerization (red curve).

Abstract Suspensions of oxygen‐filled bubbles are under active investigation as potential means of relieving tissue hypoxia. Intravenous administration of large quantities of bubbles is, however, undesirable. Previous work by the authors has demonstrated that tumor oxygen levels can be increased following oral administration of phospholipid stabilized oxygen nanobubbles. The aim of this study was to determine whether this would enhance the efficacy of sonodynamic therapy (SDT), which is known to be inhibited in hypoxic tissue. Experiments were conducted in a murine model of pancreatic cancer. Animals were treated with SDT (intratumoural injection of 1 mM Rose Bengal followed by exposure to 1 MHz ultrasound, 0.1 kHz pulse repetition frequency, 30% duty cycle, 3.5 W cm−2 for 3.5 minutes) either with or without a prior gavage of oxygen bubbles. A statistically significant reduction in the rate of tumor growth was observed in the groups receiving oxygen nanobubbles either 5 or 20 minutes before SDT. Separate measurements of tumor oxygen using a fiber optic probe and expression of hypoxia inducible factor (HIF)1α following tumor excision, confirmed the change in tumor oxygen levels. These findings offer a potentially promising new approach to relieving tissue hypoxia in order to facilitate cancer therapy.

The intracellular environment hosts a large number of cancer- and other disease-relevant human proteins. Targeting these with internalized antibodies would allow therapeutic modulation of hitherto undruggable pathways, such as those mediated by protein–protein interactions. However, one of the major obstacles in intracellular targeting is the entrapment of biomacromolecules in the endosome. Here we report an approach to delivering antibodies and antibody fragments into the cytosol and nucleus of cells using trimeric cell-penetrating peptides (CPPs). Four trimers, based on linear and cyclic sequences of the archetypal CPP Tat, are significantly more potent than monomers and can be tuned to function by direct interaction with the plasma membrane or escape from vesicle-like bodies. These studies identify a tricyclic Tat construct that enables intracellular delivery of functional immunoglobulin-G antibodies and Fab fragments that bind intracellular targets in the cytosol and nuclei of live cells at effective concentrations as low as 1 μM.

Treatment options for patients with pancreatic cancer are limited and survival prospects have barely changed over the past 4 decades. Chemoradiation treatment (CRT) has been used as neoadjuvant therapy in patients with borderline resectable disease to reduce tumour burden and increase the proportion of patients eligible for surgery. Antimetabolite drugs such as gemcitabine and 5-fluorouracil are known to sensitise pancreatic tumours to radiation treatment. Likewise, photodynamic therapy (PDT) has also been shown to enhance the effect of radiation therapy. However, PDT is limited to treating superficial lesions due to the attenuation of light by tissue. The ability of the related technique, sonodynamic therapy (SDT), to enhance CRT was investigated in two murine models of pancreatic cancer (PSN-1 and BxPC-3) in this study. SDT uses low intensity ultrasound to activate an otherwise non-toxic sensitiser, generating toxic levels of reactive oxygen species (ROS) locally. It is applicable to greater target depths than PDT due to the ability of ultrasound to propagate further than light in tissue. Both CRT and the combination of CRT plus SDT delayed tumour growth in the two tumour models. In the PSN-1 model, but not the BxPC-3 model, the combination treatment caused an increase in survival relative to CRT alone (p = 0.038). The improvement in survival conferred by the addition of SDT in this model may be related to differences in tumour architecture between the two models. MRI and US images showed that PSN-1 tumours were less well perfused and vascularised than BxPC-3 tumours. This poor vascularisation may explain why PSN-1 tumours were more susceptible to the effects of vascular damage exerted by SDT treatment.

Background Triple negative breast cancer (TNBC) poses a serious clinical challenge as it is an aggressive form of the disease that lacks estrogen receptor, progesterone receptor, and ERBB2 (formerly HER2) gene amplification, which limits the treatment options. The Warburg phenotype of upregulated glycolysis in the presence of oxygen has been shown to be prevalent in TNBC. Elevated glycolysis satisfies the energy requirements of cancer cells, contributes to resistance to treatment by maintaining redox homeostasis and generating nucleotide precursors required for cell proliferation and DNA repair. Expression of the monocarboxylate transporter 1 (MCT1), which is responsible for the bidirectional transport of lactate, correlates with an aggressive phenotype and poor outcome in several cancer types, including breast cancer. In this study, 3-bromopyruvate (3BP), a lactate/pyruvate analog, was used to selectively target TNBC cells that express MCT1. Methods The cytotoxicity of 3BP was tested in MTT assays using human TNBC cell lines: BT20 (MCT1 + /MCT4 − ), MDA-MB-23 (MCT1 − /MCT4 + ), and BT20 in which MCT1 was knocked down (siMCT1-BT20). The metabolite profile of 3BP-treated and 3BP-untreated cells was investigated using LC-MS/MS. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of BT20 and MDA-MB-231 cells treated with 3BP were measured using a Seahorse XF96 extracellular flux analyzer. The impact of ionizing radiation on cell survival, alone or in combination with 3BP pre-treatment, was evaluated using clonogenic assays. Results Metabolomic analyses showed that 3BP causes inhibition of glycolysis, disturbance of redox homeostasis, decreased nucleotide synthesis, and was accompanied by a reduction in medium acidification. In addition, 3BP potentiated the cytotoxic effect of ionizing radiation, a treatment that is frequently used in the management of TNBC. Conclusions Overall, MCT1-mediated metabolic perturbation in combination with radiotherapy is shown to be a promising strategy for the treatment of glycolytic tumors such as TNBC, overcoming the selectivity challenges of targeting glycolysis with glucose analogs.

Telomerase represents an attractive target in oncology as it is expressed in cancer but not in normal tissues. The oligonucleotide inhibitors of telomerase represent a promising anticancer strategy, although poor cellular uptake can restrict their efficacy. In this study, gold nanoparticles (AuNPs) were used to enhance oligonucleotide uptake. “match” oligonucleotides complementary to the telomerase RNA template subunit (hTR) and “scramble” (control) oligonucleotides were conjugated to diethylenetriamine pentaacetate (DTPA) for 111In-labeling. AuNPs (15.5 nm) were decorated with a monofunctional layer of oligonucleotides (ON–AuNP) or a multifunctional layer of oligonucleotides, PEG(polethylene glycol)800-SH (to reduce AuNP aggregation) and the cell-penetrating peptide Tat (ON–AuNP–Tat). Match–AuNP enhanced the cellular uptake of radiolabeled oligonucleotides while retaining the ability to inhibit telomerase activity. The addition of Tat to AuNPs increased nuclear localization. 111In–Match–AuNP–Tat induced DNA double-strand breaks and caused a dose-dependent reduction in clonogenic survival of telomerase-positive cells but not telomerase-negative cells. hTR inhibition has been reported to sensitize cancer cells to ionizing radiation, and 111In–Match–AuNP–Tat therefore holds promise as a vector for delivery of radionuclides into cancer cells while simultaneously sensitizing them to the effects of the emitted radiation.

Auger electron emitter indium-111 demonstrates cancer-selective radiotoxicity and SPECT imaging compatibility when conjugated to a ruthenium(ii) polypyridyl complex.

Approximately 50% of all colorectal cancer (CRC) patients will develop metastasis to the liver. 90 Y selective internal radiation therapy (SIRT) is an established treatment for metastatic CRC. There is still a fundamental lack of understanding regarding the radiobiology underlying the dose response. This study was designed to determine the radiosensitivity of two CRC cell lines (DLD-1 and HT-29) to 90 Y β − radiation exposure, and thus the relative effectiveness of 90 Y SIRT in relation to external beam radiotherapy (EBRT). A 90 Y-source dish was sandwiched between culture dishes to irradiate DLD-1 or HT-29 cells for a period of 6 d. Cell survival was determined by clonogenic assay. Dose absorbed per 90 Y disintegration was calculated using the PENELOPE Monte Carlo code. PENELOPE simulations were benchmarked against relative dose measurements using EBT3 GAFchromic ™ film. Statistical regression based on the linear-quadratic model was used to determine the radiosensitivity parameters and using R . These results were compared to radiosensitivity parameters determined for 6 MV clinical x-rays and 137 Cs γ -ray exposure. Equivalent dose of EBRT in 2 Gy ( ) and 10 Gy ( ) fractions were derived for 90 Y dose. HT-29 cells were more radioresistant than DLD-1 for all treatment modalities. Radiosensitivity parameters determined for 6 MV x-rays and 137 Cs γ -ray were equivalent for both cell lines. The ratio for 90 Y β − -particle exposure was over an order of magnitude higher than the other two modalities due to protraction of dose delivery. Consequently, an 90 Y SIRT absorbed dose of 60 Gy equates to an of 28.7 and 54.5 Gy and an of 17.6 and 19.3 Gy for DLD-1 and HT-29 cell lines, respectively. We derived radiosensitivity parameters for two CRC cell lines exposed to 90 Y β − -particles, 6 MV x-rays, and 137 Cs γ -ray irradiation. These radiobiological parameters are critical to understanding the dose response of CRC lesions and ultimately informs the efficacy of 90 Y SIRT relative to other radiation therapy modalities.

The spatial distribution of radiopharmaceuticals that emit short-range high linear-energy-transfer electrons greatly affects absorbed dose and biological effectiveness. The purpose of this study was to investigate the effect of heterogeneous radionuclide distribution on tumor control probability (TCP) in a micrometastasis model. Methods: The cancer cell lines MDA-MB-468, SQ20B, and 231-H2N were grown as spheroids to represent micrometastases. The intracellular distribution of a representative radiopeptide (111In-labeled epidermal growth factor) and radioimmunotherapeutic (111In-labeled trastuzumab) was determined in cell internalization experiments. The intratumoral distribution was evaluated by microautoradiography of spheroids. γH2AX staining was performed on spheroid sections to correlate DNA damage with radionuclide distribution. Experimental surviving fractions were obtained using clonogenic assays. A random close-packed algorithm, which models the random packing behavior of cells and reflects variation in the radii of cells and nuclei, was used to simulate 3-dimensional spheroids. Calculated survival fractions were generated using an iterative modeling method based on Monte Carlo–determined absorbed dose with the PENELOPE code and were compared with experimental surviving fraction. Radiobiologic parameters deduced from experimental results and Monte Carlo simulations were used to predict the TCP for a 3-dimensional spheroid model. Results: Calculated survival fractions agreed well with experimental data, particularly when an increased value for relative biological effectiveness was applied to self-dose deposited by sources located in the nucleus and when radiobiologic parameters were adjusted to account for dose protraction. Only in MDA-MB-468 spheroids treated with 111In-epidermal growth factor was a TCP of more than 0.5 achieved, indicating that for this cell type the radiopeptide would be curative when targeting micrometastases. This ability is attributed to the relative radiosensitivity of MDA-MB-468 cells, high nuclear uptake of the radiopeptide, and uniform distribution of radioactivity throughout the spheroid. Conclusion: It is imperative to include biologic endpoints when evaluating the distribution of radionuclides in models emulating micrometastatic disease. The spatial distribution of radioactivity is a clear determinant of biological effect and TCP as demonstrated in this study.

EGFR-targeted PLGA nanoparticles co-deliver the Auger electron emitter111In and a ruthenium(ii) radiosensitizer for combined therapeutic effects.

PURPOSE:To assess the efficacy of different schedules for combining external beam radiotherapy (EBRT) with molecular radiotherapy (MRT) using 131I-mIBG in the management of neuroblastoma. MATERIALS AND METHODS:BALB/c nu/nu mice bearing SK-N-SH neuroblastoma xenografts were assigned to five treatment groups: 131I-mIBG 24h after EBRT, EBRT 6days after 131I-mIBG, EBRT alone, 131I-mIBG alone and control (untreated). A total of 56 mice were assigned to 3 studies. Study 1: Vessel permeability was evaluated using dynamic contrast-enhanced (DCE)-MRI (n=3). Study 2: Tumour uptake of 131I-mIBG in excised lesions was evaluated by γ-counting and autoradiography (n=28). Study 3: Tumour volume was assessed by longitudinal MR imaging and survival was analysed (n=25). Tumour dosimetry was performed using Monte Carlo simulations of absorbed fractions with the radiation transport code PENELOPE. RESULTS:Given alone, both 131I-mIBG and EBRT resulted in a seven-day delay in tumour regrowth. Following EBRT, vessel permeability was evaluated by DCE-MRI and showed an increase at 24h post irradiation that correlated with an increase in 131I-mIBG tumour uptake, absorbed dose and overall survival in the case of combined treatment. Similarly, EBRT administered seven days after MRT to coincide with tumour regrowth, significantly decreased the tumour volume and increased overall survival. CONCLUSIONS:This study demonstrates that combining EBRT and MRT has an enhanced therapeutic effect and emphasizes the importance of treatment scheduling according to pathophysiological criteria such as tumour vessel permeability and tumour growth kinetics.

The ability to image vascular endothelial growth factor (VEGF) could enable prospective, non-invasive monitoring of patients receiving anti-angiogenic therapy. This study investigates the specificity and pharmacokinetics of 111In-bevacizumab binding to VEGF and its use for assessing response to anti-angiogenic therapy with rapamycin.

The successful use of targeted radionuclide therapy in the treatment of solid tumours may be limited by radioresistance, which necessitates delivery of a high dose of radioactivity. Nanoparticle (NP)-based delivery systems possess a large surface area for attachment of radioisotopes and so offer a solution to this challenge. However, tumour uptake may be limited by rapid hepatic clearance of NP via the mononuclear phagocyte system. Liver uptake is further compounded when epidermal growth factor (EGF) is used as a targeting ligand, as EGF-tagged NP bind the EGF receptor (EGFR), which is expressed to a moderate extent by hepatocytes. This report describes an indium-111 (111In)-labelled PEGylated EGF-tagged gold (Au) NP (111In-EGF-Au-PEG) and an effective strategy of coadministration of targeting ligand to address these issues. Direct attachment of EGF to the surface of Au NP did not compromise surface coating with long-chain PEG. In vitro experiments showed that 111In-EGF-Au-PEG targets EGFR-positive cancer cells (MDA-MB-468): >11% of radioactivity was internalised after incubation for 4 h. In in vivo studies accumulation of NP was observed in MDA-MB-468 xenografts and tumour uptake was enhanced by the coadministration of 15 µg of the unlabelled targeting ligand, EGF, to block hepatic EGFR. Uptake was 3.9% versus 2.8% injected dose/g (%ID/g) of tumour tissue with and without unlabelled EGF, respectively. Coadministration of EGF reduced liver uptake by 25.95% to 7.56 %ID/g. This suggests that the coadministration of unlabelled targeting ligand with radiolabelled PEGylated NP offers a promising strategy for targeting EGFR-positive cancer and for minimising liver uptake.