Conjugation to a cell-penetrating peptide drives the tumour accumulation of the GLP1R antagonist exendin(9-39).

PURPOSE: Exendin, an analogue of the glucagon-like peptide 1 (GLP1), is an excellent tracer for molecular imaging of pancreatic beta cells and beta cell-derived tumours. The commonly used form, exendin-4, activates the GLP1 receptor and causes internalisation of the peptide-receptor complex. As a consequence, injection of exendin-4 can lead to adverse effects such as nausea, vomiting and hypoglycaemia and thus requires close monitoring during application. By comparison, the antagonist exendin(9-39) does not activate the receptor, but its lack of internalisation has precluded its use as a tracer. Improving the cellular uptake of exendin(9-39) could turn it into a useful alternative tracer with less side-effects than exendin-4. METHODS: We conjugated exendin-4 and exendin(9-39) to the well-known cell-penetrating peptide (CPP) penetratin. We evaluated cell binding and internalisation of the radiolabelled peptides in vitro and their biodistribution in vivo. RESULTS: Exendin-4 showed internalisation irrespective of the presence of the CPP, whereas for exendin(9-39) only the penetratin conjugate internalised. Conjugation to the CPP also enhanced the in vivo tumour uptake and retention of exendin(9-39). CONCLUSION: We demonstrate that penetratin robustly improves internalisation and tumour retention of exendin(9-39), opening new avenues for antagonist-based in vivo imaging of GLP1R.

New Radiolabeled Exendin Analogues Show Reduced Renal Retention.

PET imaging of the glucagon-like peptide-1 receptor (GLP-1R) using radiolabeled exendin is a promising imaging method to detect insulinomas. However, high renal accumulation of radiolabeled exendin could hamper the detection of small insulinomas in proximity to the kidneys and limit its use as a radiotherapeutic agent. Here, we report two new exendin analogues for GLP-1R imaging and therapy, designed to reduce renal retention by incorporating a cleavable methionine-isoleucine (Met-Ile) linker. We examined the renal retention and insulinoma targeting properties of these new exendin analogues in a nude mouse model bearing subcutaneous GLP-1R-expressing insulinomas. NOTA or DOTA was conjugated via a methionine-isoleucine linker to the C-terminus of exendin-4 (NOTA-MI-exendin-4 or DOTA-MI-exendin-4). NOTA- and DOTA-exendin-4 without the linker were used as references. The affinity for GLP-1R was determined in a competitive binding assay using GLP-1R transfected cells. Biodistribution of [68Ga]Ga-NOTA-exendin-4, [68Ga]Ga-NOTA-MI-exendin-4, [177Lu]Lu-DOTA-exendin-4, and [177Lu]Lu-DOTA-MI-exendin-4 was determined in INS-1 tumor-bearing BALB/c nude mice, and PET/CT was acquired to visualize renal retention and tumor targeting. For all tracers, dosimetric calculations were performed to determine the kidney self-dose. The affinity for GLP-1R was in the low nanomolar range (<11 nM) for all peptides. In vivo biodistribution revealed a significantly lower kidney uptake of [68Ga]Ga-NOTA-MI-exendin-4 at 4 h post-injection (p.i.) (34.2 ± 4.2 %IA/g), compared with [68Ga]Ga-NOTA-exendin-4 (128 ± 10 %IA/g). Accumulation of [68Ga]Ga-NOTA-MI-exendin-4 in the tumor was 25.0 ± 8.0 %IA/g 4 h p.i., which was similar to that of [68Ga]Ga-NOTA-exendin-4 (24.9 ± 9.3 %IA/g). This resulted in an improved tumor-to-kidney ratio from 0.2 ± 0.0 to 0.8 ± 0.3. PET/CT confirmed the findings in the biodistribution studies. The kidney uptake of [177Lu]Lu-DOTA-MI-exendin-4 was 39.4 ± 6.3 %IA/g at 24 h p.i. and 13.0 ± 2.5 %IA/g at 72 h p.i., which were significantly lower than those for [177Lu]Lu-DOTA-exendin-4 (99.3 ± 9.2 %IA/g 24 h p.i. and 45.8 ± 3.9 %IA/g 72 h p.i.). The uptake in the tumor was 7.8 ± 1.5 and 11.3 ± 2.0 %IA/g 24 h p.i. for [177Lu]Lu-DOTA-MI-exendin-4 and [177Lu]Lu-DOTA-exendin-4, respectively, resulting in improved tumor-to-kidney ratios for [177Lu]Lu-DOTA-MI-exendin-4. The new exendin analogues with a Met-Ile linker showed 2-3-fold reduced renal retention and improved tumor-to-kidney ratios compared with their reference without the Met-Ile linker. Future studies should demonstrate whether [68Ga]Ga-NOTA-MI-exendin-4 results in improved detection of small insulinomas in close proximity to the kidneys with PET/CT. [177Lu]Lu-DOTA-MI-exendin-4 might open a window of opportunity for exendin-based radionuclide therapy.

Fibroblast Activation Protein-Targeted Photodynamic Therapy of Cancer-Associated Fibroblasts in Murine Models for Pancreatic Ductal Adenocarcinoma.

Patients with pancreatic ductal adenocarcinoma (PDAC) have a dismal 5 year survival of 9%. One important limiting factor for treatment efficacy is the dense tumor-supporting stroma. The cancer-associated fibroblasts in this stroma deposit excessive amounts of extracellular matrix components and anti-inflammatory mediators, which hampers the efficacy of chemo- and immunotherapies. Systemic depletion of all activated fibroblasts is, however, not feasible nor desirable and therefore a local approach should be pursued. Here, we provide a proof-of-principle of using fibroblast activation protein (FAP)-targeted photodynamic therapy (tPDT) to treat PDAC. FAP-targeting antibody 28H1 and irrelevant control antibody DP47GS were conjugated to the photosensitizer IRDye700DX (700DX) and the chelator diethylenetriaminepentaacetic acid. In vitro binding and cytotoxicity were evaluated using the fibroblast cell-line NIH-3T3 stably transfected with FAP. Biodistribution of 111In-labeled antibody-700DX constructs was determined in mice carrying syngeneic tumors of the murine PDAC cell line PDAC299, and in a genetically engineered PDAC mouse model (CKP). Then, tPDT was performed by exposing the subcutaneous or the spontaneous PDAC tumors to 690 nm light. Induction of apoptosis after treatment was assessed using automated analyses of immunohistochemistry for cleaved caspase-3. 28H1-700DX effectively bound to 3T3-FAP cells and induced cytotoxicity upon exposure to 690 nm light, whereas no binding or cytotoxic effects were observed for DP47GS-700DX. Although both 28H1-700DX and DP47GS-700DX accumulated in subcutaneous PDAC299 tumors, autoradiography demonstrated that only 28H1-700DX reached the tumor core. On the contrary, control antibody DP47GS-700DX was only present at the tumor rim. In CKP mice, both antibodies accumulated in the tumor, but tumor-to-blood ratios of 28H1-700DX were higher than that of the control. Notably, in vivo FAP-tPDT caused upregulation of cleaved caspase-3 staining in both subcutaneous and in spontaneous tumors. In conclusion, we have shown that tPDT is a feasible approach for local depletion of FAP-expressing stromal cells in murine models for PDAC.

Quantitative and longitudinal imaging of intramuscular transplanted islets of Langerhans with SPECT using [ 123 I]IBZM.

A non-invasive imaging method to monitor islet grafts could provide novel and improved insight into the fate of transplanted islets and, potentially, monitor the effect of therapeutic interventions. Therefore, such an imaging method could help improve long-term transplantation outcome. Here, we investigated the use of [ 123 I]IBZM for insulin positive graft volume quantification and longitudinal graft monitoring. SPECT images were acquired 6 weeks after islet transplantation in the calf muscle of rats. For longitudinal graft analysis, rats were monitored by SPECT for 10 weeks. After animals were euthanized, graft containing muscles were dissected for ex vivo analysis and insulin-positive graft volume determination. Six weeks after transplantation, a clear signal was observed in all grafts by SPECT imaging. Moreover, the intensity of the SPECT signal correlated linearly with insulin-positive graft volume, as determined histologically. Longitudinal graft follow-up showed a clear SPECT signal of the transplant from 3 until 10 weeks after transplantation. In this study, we demonstrate for the first time the successful application of a radiotracer, [ 123 I]IBZM, for non-invasive, in vivo graft volume quantification and longitudinal graft monitoring.

SPECT of Transplanted Islets of Langerhans by Dopamine 2 Receptor Targeting in a Rat Model.

Pancreatic islet transplantation can be a more permanent treatment for type 1 diabetes compared to daily insulin administration. Quantitative and longitudinal noninvasive imaging of viable transplanted islets might help to further improve this novel therapy. Since islets express dopamine 2 (D2) receptors, they could be visualized by targeting this receptor. Therefore, the D2 receptor antagonist based tracer [(125/123)I][IBZM] was selected to visualize transplanted islets in a rat model. BZM was radioiodinated, and the labeling was optimized for position 3 of the aromatic ring. [(125)I]-3-IBZM was characterized in vitro using INS-1 cells and isolated islets. Subsequently, 1,000 islets were transplanted in the calf muscle of WAG/Rij rats and SPECT/CT images were acquired 6 weeks after transplantation. Finally, the graft containing muscle was dissected and analyzed immunohistochemically. Oxidative radioiodination resulted in 3 IBZM isomers with different receptor affinities. The use of 0.6 mg/mL chloramine-T hydrate resulted in high yield formation of predominantly [(125)I]-3-IBZM, the isomer harboring the highest receptor affinity. The tracer showed D2 receptor mediated binding to isolated islets in vitro. The transplant could be visualized by SPECT 6 weeks after transplantation. The transplants could be localized in the calf muscle and showed insulin and glucagon expression, indicating targeting of viable and functional islets in the transplant. Radioiodination was optimized to produce high yields of [(125)I]-3-IBZM, the isomer showing optimal D2R binding. Furthermore, [(123)I]IBZM specifically targets the D2 receptors on transplanted islets. In conclusion, this tracer shows potential for noninvasive in vivo detection of islets grafted in the muscle by D2 receptor targeting.

Non-invasive quantification of the beta cell mass by SPECT with ¹¹¹In-labelled exendin.

AIMS/HYPOTHESIS: A reliable method for in vivo quantification of pancreatic beta cell mass (BCM) could lead to further insight into the pathophysiology of diabetes. The glucagon-like peptide 1 receptor, abundantly expressed on beta cells, may be a suitable target for imaging. We investigated the potential of radiotracer imaging with the GLP-1 analogue exendin labelled with indium-111 for determination of BCM in vivo in a rodent model of beta cell loss and in patients with type 1 diabetes and healthy individuals. METHODS: The targeting of (111)In-labelled exendin was examined in a rat model of alloxan-induced beta cell loss. Rats were injected with 15 MBq (111)In-labelled exendin and single photon emission computed tomography (SPECT) acquisition was performed 1 h post injection, followed by dissection, biodistribution and ex vivo autoradiography studies of pancreatic sections. BCM was determined by morphometric analysis after staining with an anti-insulin antibody. For clinical evaluation SPECT was acquired 4, 24 and 48 h after injection of 150 MBq (111)In-labelled exendin in five patients with type 1 diabetes and five healthy individuals. The tracer uptake was determined by quantitative analysis of the SPECT images. RESULTS: In rats, (111)In-labelled exendin specifically targets the beta cells and pancreatic uptake is highly correlated with BCM. In humans, the pancreas was visible in SPECT images and the pancreatic uptake showed high interindividual variation with a substantially lower uptake in patients with type 1 diabetes. CONCLUSIONS/INTERPRETATION: These studies indicate that (111)In-labelled exendin may be suitable for non-invasive quantification of BCM. TRIAL REGISTRATION: ClinicalTrials.gov NCT01825148, EudraCT: 2012-000619-10.

Towards effective CAIX-targeted radionuclide and checkpoint inhibition combination therapy for advanced clear cell renal cell carcinoma.

Background: Immune checkpoint inhibitors (ICI) are routinely used in advanced clear cell renal cell carcinoma (ccRCC). However, a substantial group of patients does not respond to ICI therapy. Radiation is a promising approach to increase ICI response rates since it can generate anti-tumor immunity. Targeted radionuclide therapy (TRT) is a systemic radiation treatment, ideally suited for precision irradiation of metastasized cancer. Therefore, the aim of this study is to explore the potential of combined TRT, targeting carbonic anhydrase IX (CAIX) which is overexpressed in ccRCC, using [177Lu]Lu-DOTA-hG250, and ICI for the treatment of ccRCC. Methods: In this study, we evaluated the therapeutic and immunological action of [177Lu]Lu-DOTA-hG250 combined with aPD-1/a-CTLA-4 ICI. First, the biodistribution of [177Lu]Lu-DOTA-hG250 was investigated in BALB/cAnNRj mice bearing Renca-CAIX or CT26-CAIX tumors. Renca-CAIX and CT26-CAIX tumors are characterized by poor versus extensive T-cell infiltration and homogeneous versus heterogeneous PD-L1 expression, respectively. Tumor-absorbed radiation doses were estimated through dosimetry. Subsequently, [177Lu]Lu-DOTA-hG250 TRT efficacy with and without ICI was evaluated by monitoring tumor growth and survival. Therapy-induced changes in the tumor microenvironment were studied by collection of tumor tissue before and 5 or 8 days after treatment and analyzed by immunohistochemistry, flow cytometry, and RNA profiling. Results: Biodistribution studies showed high tumor uptake of [177Lu]Lu-DOTA-hG250 in both tumor models. Dose escalation therapy studies in Renca-CAIX tumor-bearing mice demonstrated dose-dependent anti-tumor efficacy of [177Lu]Lu-DOTA-hG250 and remarkable therapeutic synergy including complete remissions when a presumed subtherapeutic TRT dose (4 MBq, which had no significant efficacy as monotherapy) was combined with aPD-1+aCTLA-4. Similar results were obtained in the CT26-CAIX model for 4 MBq [177Lu]Lu-DOTA-hG250 + a-PD1. Ex vivo analyses of treated tumors revealed DNA damage, T-cell infiltration, and modulated immune signaling pathways in the TME after combination treatment. Conclusions: Subtherapeutic [177Lu]Lu-DOTA-hG250 combined with ICI showed superior therapeutic outcome and significantly altered the TME. Our results underline the importance of investigating this combination treatment for patients with advanced ccRCC in a clinical setting. Further investigations should focus on how the combination therapy should be optimally applied in the future.

Pretargeted immuno-PET and radioimmunotherapy of prostate cancer with an anti-TROP-2 x anti-HSG bispecific antibody.

PURPOSE: TF12 is a trivalent bispecific antibody that consists of two anti-TROP-2 Fab fragments and one anti-histamine-succinyl-glycine (HSG) Fab fragment. The TROP-2 antigen is found in many epithelial cancers, including prostate cancer (PC), and therefore this bispecific antibody could be suitable for pretargeting in this cancer. In this study, the characteristics and the potential for pretargeted radioimmunoimaging and radioimmunotherapy with TF12 and the radiolabeled di-HSG peptide IMP288 in mice with human PC were investigated. METHODS: The optimal TF12 protein dose, IMP288 peptide dose, and dose interval for PC targeting were assessed in nude mice with s.c. PC3 xenografts. Immuno-positron emission tomography (PET)/CT was performed using TF12/68Ga-IMP288 at optimized conditions. The potential of pretargeted radioimmunotherapy (PRIT) using the TF12 pretargeted ¹77Lu-IMP288 was determined. RESULTS: TF12 and ¹¹¹In-IMP288 showed high and fast accumulation in the tumor [20.4 ± 0.6%ID/g at 1 h post-injection (p.i.)] at optimized conditions, despite the internalizing properties of TF12. The potential for PRIT was shown by retention of 50% of the ¹¹¹In-IMP288 in the tumor at 48 h p.i. One cycle of treatment with TF12 and ¹77Lu-IMP288 showed significant improvement of survival compared to treatment with ¹77Lu-IMP288 alone (90 vs. 67 days, p<0.0001) with no renal or hematological toxicity. CONCLUSION: TROP-2-expressing PC can be pretargeted efficiently with TF12, with very rapid uptake of the radiolabeled hapten-peptide, IMP288, sensitive immuno-PET, and effective therapy.

[18F]FDG Uptake and Expression of Immunohistochemical Markers Related to Glycolysis, Hypoxia, and Proliferation in Indeterminate Thyroid Nodules.

PURPOSE: The current study explored the association between 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) uptake and the quantitative expression of immunohistochemical markers related to glucose metabolism, hypoxia, and cell proliferation in benign and malignant thyroid nodules of indeterminate cytology. PROCEDURES: Using a case-control design, 24 patients were selected from participants of a randomized controlled multicenter trial (NCT02208544) in which [18F]FDG-PET/CT and thyroid surgery were performed for Bethesda III and IV nodules. Three equally sized groups of [18F]FDG-positive malignant, [18F]FDG-positive benign, and [18F]FDG-negative benign nodules were included. Immunohistochemical staining was performed for glucose transporters (GLUT) 1, 3, and 4; hexokinases (HK) 1 and 2; hypoxia-inducible factor-1 alpha (HIF1α; monocarboxylate transporter 4 (MCT4); carbonic anhydrase IX (CA-IX); vascular endothelial growth factor (VEGF); sodium-iodide symporter (NIS); and Ki-67. Marker expression was scored using an immunoreactive score. Unsupervised cluster analysis was performed. The immunoreactive score was correlated to the maximum and peak standardized uptake values (SUVmax, SUVpeak) and SUVmax ratio (SUVmax of nodule/background SUVmax of contralateral, normal thyroid) of the [18F]FDG-PET/CT using the Spearman's rank correlation coefficient and compared between the three groups using Kruskal-Wallis tests. RESULTS: The expression of GLUT1, GLUT3, HK2, and MCT4 was strongly positively correlated with the SUVmax, SUVpeak, and SUVmax ratio. The expression of GLUT1 (p = 0.009), HK2 (p = 0.02), MCT4 (p = 0.01), and VEGF (p = 0.007) was statistically significantly different between [18F]FDG-positive benign nodules, [18F]FDG-positive thyroid carcinomas, and [18F]FDG-negative benign nodules. In both [18F]FDG-positive benign nodules and [18F]FDG-positive thyroid carcinomas, the expression of GLUT1, HK2, and MCT4 was increased as compared to [18F]FDG-negative benign nodules. VEGF expression was higher in [18F]FDG-positive thyroid carcinomas as compared to [18F]FDG-negative and [18F]FDG-positive benign nodules. CONCLUSIONS: Our results suggest that [18F]FDG-positive benign thyroid nodules undergo changes in protein expression similar to those in thyroid carcinomas. To expand the understanding of the metabolic changes in benign and malignant thyroid nodules, further research is required, including correlation with underlying genetic alterations.

Fibroblast Activation Protein-Targeting Minibody-IRDye700DX for Ablation of the Cancer-Associated Fibroblast with Photodynamic Therapy.

Fibroblast activation protein (FAP), expressed on cancer-associated fibroblasts, is a target for diagnosis and therapy in multiple tumour types. Strategies to systemically deplete FAP-expressing cells show efficacy; however, these induce toxicities, as FAP-expressing cells are found in normal tissues. FAP-targeted photodynamic therapy offers a solution, as it acts only locally and upon activation. Here, a FAP-binding minibody was conjugated to the chelator diethylenetriaminepentaacetic acid (DTPA) and the photosensitizer IRDye700DX (DTPA-700DX-MB). DTPA-700DX-MB showed efficient binding to FAP-overexpressing 3T3 murine fibroblasts (3T3-FAP) and induced the protein's dose-dependent cytotoxicity upon light exposure. Biodistribution of DTPA-700DX-MB in mice carrying either subcutaneous or orthotopic tumours of murine pancreatic ductal adenocarcinoma cells (PDAC299) showed maximal tumour uptake of 111In-labelled DTPA-700DX-MB at 24 h post injection. Co-injection with an excess DTPA-700DX-MB reduced uptake, and autoradiography correlated with FAP expression in the stromal tumour region. Finally, in vivo therapeutic efficacy was determined in two simultaneous subcutaneous PDAC299 tumours; only one was treated with 690 nm light. Upregulation of an apoptosis marker was only observed in the treated tumours. In conclusion, DTPA-700DX-MB binds to FAP-expressing cells and targets PDAC299 tumours in mice with good signal-to-background ratios. Furthermore, the induced apoptosis indicates the feasibility of targeted depletion of FAP-expressing cells with photodynamic therapy.

CPPs to the Test: Effects on Binding, Uptake and Biodistribution of a Tumor Targeting Nanobody.

Nanobodies are well-established targeting ligands for molecular imaging and therapy. Their short circulation time enables early imaging and reduces systemic radiation exposure. However, shorter circulation time leads to lower tracer accumulation in the target tissue. Cell-penetrating peptides (CPPs) improve cellular uptake of various cargoes, including nanobodies. CPPs could enhance tissue retention without compromising rapid clearance. However, systematic investigations on how the functionalities of nanobody and CPP combine with each other at the level of 2D and 3D cell cultures and in vivo are lacking. Here, we demonstrate that conjugates of the epidermal growth factor receptor (EGFR)-binding nanobody 7D12 with different CPPs (nonaarginine, penetratin, Tat and hLF) differ with respect to cell binding and induction of endocytosis. For nonaarginine and penetratin we compared the competition of EGF binding and performance of L- and D-peptide stereoisomers, and tested the D-peptide conjugates in tumor cell spheroids and in vivo. The D-peptide conjugates showed better penetration into spheroids than the unconjugated 7D12. Both in vivo and in vitro, the behavior of the agent reflects the combination of both functionalities. Although CPPs cause promising increases in in vitro uptake and 3D penetration, the dominant effect of the CPP in the control of biodistribution warrants further investigation.

Receptor-Targeted Photodynamic Therapy of Glucagon-Like Peptide 1 Receptor-Positive Lesions.

Treatment of hyperinsulinemic hypoglycemia is challenging. Surgical treatment of insulinomas and focal lesions in congenital hyperinsulinism is invasive and carries major risks of morbidity. Medication to treat nesidioblastosis and diffuse congenital hyperinsulinism has varying efficacy and causes significant side effects. Here, we describe a novel method for therapy of hyperinsulinemic hyperglycemia, highly selectively killing ß-cells by receptor-targeted photodynamic therapy (rtPDT) with exendin-4-IRDye700DX, targeting the glucagon-like peptide 1 receptor (GLP-1R). Methods: A competitive binding assay was performed using Chinese hamster lung (CHL) cells transfected with the GLP-1R. The efficacy and specificity of rtPDT with exendin-4-IRDye700DX were examined in vitro in cells with different levels of GLP-1R expression. Tracer biodistribution was determined in BALB/c nude mice bearing subcutaneous CHL-GLP-1R xenografts. Induction of cellular damage and the effect on tumor growth were analyzed to determine treatment efficacy. Results: Exendin-4-IRDye700DX has a high affinity for the GLP-1R, with a half-maximal inhibitory concentration of 6.3 nM. rtPDT caused significant specific phototoxicity in GLP-1R-positive cells (2.3% ± 0.8% and 2.7% ± 0.3% remaining cell viability in CHL-GLP-1R and INS-1 cells, respectively). The tracer accumulates dose-dependently in GLP-1R-positive tumors. In vivo, rtPDT induces cellular damage in tumors, shown by strong expression of cleaved caspase-3, and leads to a prolonged median survival of the mice (36.5 vs. 22.5 d, respectively; P < 0.05). Conclusion: These data show in vitro as well as in vivo evidence of the potency of rtPDT using exendin-4-IRDye700DX. This approach might in the future provide a new, minimally invasive, highly specific treatment method for hyperinsulinemic hypoglycemia.

Noninvasive Monitoring of Glycemia-Induced Regulation of GLP-1R Expression in Murine and Human Islets of Langerhans.

Glucagon-like peptide 1 receptor (GLP-1R) imaging with radiolabeled exendin has proven to be a powerful tool to quantify ß-cell mass (BCM) in vivo. As GLP-1R expression is thought to be influenced by glycemic control, we examined the effect of blood glucose (BG) levels on GLP-1R-mediated exendin uptake in both murine and human islets and its implications for BCM quantification. Periods of hyperglycemia significantly reduced exendin uptake in murine and human islets, which was paralleled by a reduction in GLP-1R expression. Detailed mapping of the tracer uptake and insulin and GLP-1R expression conclusively demonstrated that the observed reduction in tracer uptake directly correlates to GLP-1R expression levels. Importantly, the linear correlation between tracer uptake and ß-cell area was maintained in spite of the reduced GLP-1R expression levels. Subsequent normalization of BG levels restored absolute tracer uptake and GLP-1R expression in ß-cells and the observed loss in islet volume was halted. This manuscript emphasizes the potency of nuclear imaging techniques to monitor receptor regulation noninvasively. Our findings have significant implications for clinical practice, indicating that BG levels should be near-normalized for at least 3 weeks prior to GLP-1R agonist treatment or quantitative radiolabeled exendin imaging for BCM analysis.

Immunohistochemical selection of biomarkers for tumor-targeted image-guided surgery of myxofibrosarcoma.

Myxofibrosarcoma(MFS) is the most common soft tissue sarcoma(STS) in elderly patients. Surgical resection remains the main treatment modality but tumor borders can be difficult to delineate with conventional clinical methods. Incomplete resections are a common problem and local recurrence remains a clinical issue. A technique that has shown great potential in improving surgical treatment of solid tumors is tumor targeted imaging and image-guided surgery with near-infrared fluorescence. To facilitate this technique, it is essential to identify a biomarker that is highly and homogenously expressed on tumor cells, while being absent on healthy non-malignant tissue. The purpose of this study was to identify suitable molecular targets for tumor-targeted imaging of myxofibrosarcoma. Ten potential molecular targets for tumor targeted imaging were investigated with immunohistochemical analysis in myxofibrosarcoma tissue (n = 34). Results were quantified according to the immunoreactive score(IRS). Moderate expression rates were found for uPAR, PDGFRa and EMA/MUC1. High expression rates of VEGF and TEM1 were seen. Strong expression was most common for TEM1 (88.2%). These results confirms that TEM1 is a suitable target for tumor-targeted imaging of myxofibrosarcoma. Keywords Image-guided surgery; Immunohistochemistry; Molecular imaging; Myxofibrosarcoma; Soft tissue sarcoma; Tumor endothelial marker 1(TEM1), Vascular endothelial growth factor (VEGF).

Targeted Optical Imaging of the Glucagonlike Peptide 1 Receptor Using Exendin-4-IRDye 800CW.

The treatment of choice for insulinomas and focal lesions in congenital hyperinsulinism (CHI) is surgery. However, intraoperative detection can be challenging. This challenge could be overcome with intraoperative fluorescence imaging, which provides real-time lesion detection with a high spatial resolution. Here, a novel method for targeted near-infrared (NIR) fluorescence imaging of glucagonlike peptide 1 receptor (GLP-1R)-positive lesions, using the GLP-1 agonist exendin-4 labeled with IRDye 800CW, was examined in vitro and in vivo. Methods: A competitive binding assay was performed using Chinese hamster lung (CHL) cells transfected with GLP-1R. Tracer biodistribution was determined in BALB/c nude mice bearing subcutaneous CHL-GLP-1R xenografts. In vivo NIR fluorescence imaging of CHL-GLP-1R xenografts was performed. Localization of the tracer in the pancreatic islets of BALB/c nude mice was examined using fluorescence microscopy. Laparoscopic imaging was performed to detect the fluorescent signal of the tracer in the pancreas of mini pigs. Results: Exendin-4-IRDye 800CW binds GLP-1R with a half-maximal inhibitory concentration of 3.96 nM. The tracer accumulates in CHL-GLP-1R xenografts. Subcutaneous CHL-GLP-1R xenografts were visualized using in vivo NIR fluorescence imaging. The tracer accumulates specifically in the pancreatic islets of mice, and a clear fluorescent signal was detected in the pancreas of mini pigs. Conclusion: These data provide the first in vivo evidence of the feasibility of targeted fluorescence imaging of GLP-1R-positive lesions. Intraoperative lesion delineation using exendin-4-IRDye 800CW could benefit open as well as laparoscopic surgical procedures for removal of insulinomas and focal lesions in CHI.

Development and characterization of a theranostic multimodal anti-PSMA targeting agent for imaging, surgical guidance, and targeted photodynamic therapy of PSMA-expressing tumors.

Rationale: Prostate cancer (PCa) recurrences after surgery frequently occur. To improve the outcome after surgical resection of the tumor, the theranostic multimodal anti-PSMA targeting agent 111In-DTPA-D2B-IRDye700DX was developed and characterized for both pre- and intra-operative tumor localization and eradication of (residual) tumor tissue by PSMA-targeted photodynamic therapy (tPDT), which is a highly selective cancer treatment based on targeting molecules conjugated to photosensitizers that can induce cell destruction upon exposure to near-infrared (NIR) light. Methods: The anti-PSMA monoclonal antibody D2B was conjugated with IRDye700DX and DTPA and subsequently radiolabeled with 111In. To determine the optimal dose and time point for tPDT, BALB/c nude mice with PSMA-expressing (PSMA+) s.c. LS174T-PSMA xenografts received the conjugate (24-240 µg/mouse) intravenously (8 MBq/mouse) followed by µSPECT/CT, near-infrared fluorescence imaging, and ex vivo biodistribution at 24, 48, 72 and 168 h p.i. Tumor growth of LS174T-PSMA xenografts and overall survival of mice treated with 1-3 times of NIR light irradiation (50, 100, 150 J/cm2) 24 h after injection of 80 µg of DTPA-D2B-IRDye700DX was compared to control conditions. Results: Highest specific tumor uptake was observed at conjugate doses of 80 µg/mouse. Biodistribution revealed no significant difference in tumor uptake in mice at 24, 48, 72 and 168 h p.i. PSMA+ tumors were clearly visualized with both µSPECT/CT and NIR fluorescence imaging. Overall survival in mice treated with 80 µg of DTPA-D2B-IRDye700DX and 1x 150 J/cm2 of NIR light at 24 h p.i. was significantly improved compared to the control group receiving neither conjugate nor NIR light (73 days vs. 16 days, respectively, p=0.0453). Treatment with 3x 150 J/cm2 resulted in significantly prolonged survival compared to treatment with 3x 100 J/cm2 (p = 0.0067) and 3x 50 J/cm2 (p = 0.0338). Principal conclusions:111In-DTPA-D2B-IRDye700DX can be used for pre- and intra-operative detection of PSMA+ tumors with radionuclide and NIR fluorescence imaging and PSMA-targeted PDT. PSMA-tPDT using this multimodal agent resulted in significant prolongation of survival and shows great potential for treatment of (metastasized) prostate cancer.

Validation of 111In-Exendin SPECT for the Determination of the ß-Cell Mass in BioBreeding Diabetes-Prone Rats.

The changes in ß-cell mass (BCM) during the development and progression of diabetes could potentially be measured by radionuclide imaging using radiolabeled exendin. In this study, we investigated the potential of 111In-diethylenetriaminepentaacetic acid-exendin-3 (111In-exendin) in a rat model that closely mimics the development of type 1 diabetes (T1D) in humans: BioBreeding diabetes-prone (BBDP) rats. BBDP rats of 4-18 weeks of age were injected intravenously with 111In-exendin, and single-photon emission computed tomography (SPECT) images were acquired. The accumulation of the radiotracer was measured as well as the BCM and grade of insulitis by histology. 111In-exendin accumulated specifically in the islets, resulting in a linear correlation with the BCM (%) (Pearson r = 0.89, P < 0.0001, and r = 0.64 for SPECT). Insulitis did not have an influence on this correlation. These results indicate that 111In-exendin is a promising tracer to determine the BCM during the development of T1D, irrespective of the degree of insulitis.

Biological correlates of FDG uptake in non-small cell lung cancer.

PURPOSE: Each pathological stage of non-small cell lung cancer (NSCLC) consists of a heterogeneous population containing patients at much higher risk than others. Noninvasive functional imaging modalities, such as 18F-fluorodeoxyglucose positron emission tomography (FDG-PET), could play a role in further characterization of NSCLCs. As many factors can influence the extent of FDG uptake, the underlying mechanisms for FDG accumulation in tumors, are still a matter of debate. The aim of the present study was to investigate these possible mechanisms in the primary site of early stage preoperatively untreated NSCLC. METHODS: 19 patients with early stage NSCLC, who had undergone both preoperative FDG-PET imaging and curative surgery, were enrolled in this study. Standardized uptake values (SUVs) were used for evaluation of primary tumor FDG uptake. Final diagnosis, tumor type, tumor cell differentiation and size of the primary tumors were confirmed histopathologically in resected specimens. Histologic sections were analyzed for amount of inflammation and necrosis. Expression of the glucose membrane transporters (GLUT-1 and GLUT-3); the isoforms of the glycolytic enzyme hexokinase (HK-I, HK-II and HK-III); and the cysteine protease caspase-3, was evaluated immunohistochemically. RESULTS: FDG uptake was significantly higher in squamous cell carcinomas (mean SUV 13.4+/-4.9, n=8) compared to adenocarcinomas (7.1+/-3.3, n=8, p=0.007), or large cell carcinomas (5.9+/-1.9, n=3, p=0.02). The degree of FDG accumulation seemed to depend especially on GLUT-1, GLUT-3 and tumor cell differentiation. The summed standardized values of these three parameters correlated significantly with the SUV (r=0.47, p=0.05). CONCLUSION: The present study supports the hypothesis that tumor cell differentiation in combination with overexpression of GLUT-1 and GLUT-3 determine the extent of FDG accumulation and that squamous cell carcinomas accumulate more FDG than adenocarcinomas or large cell carcinomas.

[(18)F]FDG accumulation in an experimental model of multistage progression of cholangiocarcinoma.

AIM: The diagnosis of cholangiocarcinoma (CCA) is difficult, and due to the insidious course of the disease, most cases present at a relatively late stage. Positron emission tomography (PET), using [(18)F]fluoro-2-deoxyglucose ([(18)F]FDG) as a tracer is one the most powerful molecular imaging techniques available. We hypothesized that [(18)F]FDG accumulates at sites of early CCA development and that FDG-PET may be of value for the early diagnosis of CCA. METHODS: We added 300 mg/L thioacetamide to the drinking water of rats who went on to develop CCA within 20 weeks. From eight weeks onwards, groups of three rats were injected with [(18)F]FDG, subsequently the liver was perfused, dissected and subjected to quantitative autoradiography using a phosphor imaging system. The liver sections were stained for histology, and glutathione S-transferase (GST) enzyme activity was determined. We correlated [(18)F]FDG uptake with pathological liver changes. RESULTS: The experiments demonstrate that thioacetamide causes atypical bile ducts and invasive CCA. Rat livers harvested early after the start of administration of thioacetamide contained only cirrhosis and/or atypical bile ducts, but CCA and FDG accumulation were absent. At 20 weeks, all rats had developed CCA and all, except two animals with a very small carcinoma, had strongly elevated focal FDG uptake. Quantitative autoradiography revealed tumor-to-normal-liver ratios as high as 5:4. In all rats with a carcinoma, there was a backdrop of cirrhosis, and interestingly cirrhotic areas did not show elevated FDG accumulation. CONCLUSION: [(18)F]FDG accumulates in CCA, is able to distinguish CCA from liver cirrhosis, but is probably unsuitable to detect very early CCA lesions.

Non-invasive in vivo determination of viable islet graft volume by 111In-exendin-3.

Pancreatic islet transplantation is a promising therapy for patients with type 1 diabetes. However, the duration of long-term graft survival is limited due to inflammatory as well as non-inflammatory processes and routine clinical tests are not suitable to monitor islet survival. 111In-exendin-SPECT (single photon emission computed tomography) is a promising method to non-invasively image islets after transplantation and has the potential to help improve the clinical outcome. Whether 111In-exendin-SPECT allows detecting small differences in beta-cell mass (BCM) and measuring the actual volume of islets that were successfully engrafted has yet to be demonstrated. Here, we evaluated the performance of 111In-exendin-SPECT using an intramuscular islet transplantation model in C3H mice. In vivo imaging of animals transplanted with 50, 100, 200, 400 and 800 islets revealed an excellent linear correlation between SPECT quantification of 111In-exendin uptake and insulin-positive area of islet transplants, demonstrating that 111In-exendin-SPECT specifically and accurately measures BCM. The high sensitivity of the method allowed measuring small differences in graft volumes, including grafts that contained less than 50 islets. The presented method is reliable, convenient and holds great potential for non-invasive monitoring of BCM after islet transplantation in humans.