Fully automated radiolabeling of [68Ga]Ga-EMP100 targeting c-MET for PET-CT clinical imaging.

BACKGROUND: c-MET is a transmembrane receptor involved in many biological processes and contributes to cell proliferation and migration during cancer invasion process. Its expression is measured by immunehistochemistry on tissue biopsy in clinic, although this technique has its limitations. PET-CT could allow in vivo mapping of lesions expressing c-MET, providing whole-body detection. A number of radiopharmaceuticals are under development for this purpose but are not yet in routine clinical use. EMP100 is a cyclic oligopeptide bound to a DOTA chelator, with nanomolar affinity for c-MET. The aim of this project was to develop an automated method for radiolabelling the radiopharmaceutical [68Ga]Ga-EMP100. RESULTS: The main results showed an optimal pH range between 3.25 and 3.75 for the complexation reaction and a stabilisation of the temperature at 90 °C, resulting in an almost complete incorporation of gallium-68 after 10 min of heating. In these experiments, 90 µg of EMP-100 peptide were initially used and then lower amounts (30, 50, 75 µg) were explored to determine the minimum required for sufficient synthesis yield. Radiolysis impurities were identified by radio-HPLC and ascorbic acid and ethanol were used to improve the purity of the compound. Three batches of [68Ga]Ga-EMP100 were then prepared according to the optimised parameters and all met the established specifications. Finally, the stability of [68Ga]Ga-EMP100 was assessed at room temperature over 3 h with satisfactory results in terms of appearance, pH, radiochemical purity and sterility. CONCLUSIONS: For the automated synthesis of [68Ga]Ga-EMP100, the parameters of pH, temperature, precursor peptide content and the use of adjuvants for impurity management were efficiently optimised, resulting in the production of three compliant and stable batches according to the principles of good manufacturing practice. [68Ga]Ga-EMP100 was successfully synthesised and is now available for clinical development in PET-CT imaging.

Modular One-Pot Strategy for the Synthesis of Heterobivalent Tracers.

Bivalent ligands, i.e., molecules having two ligands covalently connected by a linker, have been gathering attention since the first description of their pharmacological potential in the early 80s. However, their synthesis, particularly of labeled heterobivalent ligands, can still be cumbersome and time-consuming. We herein report a straightforward procedure for the modular synthesis of labeled heterobivalent ligands (HBLs) using dual reactive 3,6-dichloro-1,2,4,5-tetrazine as a starting material and suitable partners for sequential SNAr and inverse electron-demand Diels-Alder (IEDDA) reactions. This assembly method conducted in a stepwise or in a sequential one-pot manner provides quick access to multiple HBLs. A conjugate combining ligands toward the prostate-specific membrane antigen (PSMA) and the gastrin-releasing peptide receptor (GRPR) was radiolabeled, and its biological activity was assessed in vitro and in vivo (receptor binding affinity, biodistribution, imaging) as an illustration that the assembly methodology preserves the tumor targeting properties of the ligands.

Positron Emission Tomography Imaging of Neurotensin Receptor-Positive Tumors with 68Ga-Labeled Antagonists: The Chelate Makes the Difference Again.

Neurotensin receptor 1 (NTS1) is involved in the development and progression of numerous cancers, which makes it an interesting target for the development of diagnostic and therapeutic agents. A small molecule NTS1 antagonist, named [177Lu]Lu-IPN01087, is currently evaluated in phase I/II clinical trials for the targeted therapy of neurotensin receptor-positive cancers. In this study, we synthesized seven compounds based on the structure of NTS1 antagonists, bearing different chelating agents, and radiolabeled them with gallium-68 for PET imaging. These compounds were evaluated in vitro and in vivo in mice bearing a HT-29 xenograft. The compound [68Ga]Ga-bisNODAGA-16 showed a promising biodistribution profile with mainly signal in tumor (4.917 ± 0.776%ID/g, 2 h post-injection). Its rapid clearance from healthy tissues led to high tumor-to-organ ratios, resulting in highly contrasted PET images. These results were confirmed on subcutaneous xenografts of AsPC-1 tumor cells, a model of NTS1-positive human pancreatic adenocarcinoma.

Gout and pseudo-gout-related crystals promote GLUT1-mediated glycolysis that governs NLRP3 and interleukin-1ß activation on macrophages.

OBJECTIVE: Macrophage activation by monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals mediates an interleukin (IL)-1ß-dependent inflammation during gout and pseudo-gout flare, respectively. Since metabolic reprogramming of macrophages goes along with inflammatory responses dependently on stimuli and tissue environment, we aimed to decipher the role of glycolysis and oxidative phosphorylation in the IL-1ß-induced microcrystal response. METHODS: Briefly, an in vitro study (metabolomics and real-time extracellular flux analysis) on MSU and CPP crystal-stimulated macrophages was performed to demonstrate the metabolic phenotype of macrophages. Then, the role of aerobic glycolysis in IL-1ß production was evaluated, as well in vitro as in vivo using 18F-fluorodeoxyglucose positron emission tomography imaging and glucose uptake assay, and molecular approach of glucose transporter 1 (GLUT1) inhibition. RESULTS: We observed that MSU and CPP crystals led to a metabolic rewiring toward the aerobic glycolysis pathway explained by an increase in GLUT1 plasma membrane expression and glucose uptake on macrophages. Also, neutrophils isolated from human synovial fluid during gout flare expressed GLUT1 at their plasma membrane more frequently than neutrophils isolated from bloodstream. Both glucose deprivation and treatment with either 2-deoxyglucose or GLUT1 inhibitor suppressed crystal-induced NLRP3 activation and IL-1ß production, and microcrystal inflammation in vivo. CONCLUSION: In conclusion, we demonstrated that GLUT1-mediated glucose uptake is instrumental during the inflammatory IL-1ß response induced by MSU and CPP crystals. These findings open new therapeutic paths to modulate crystal-related inflammation.

A comparative study of peptide-based imaging agents [68Ga]Ga-PSMA-11, [68Ga]Ga-AMBA, [68Ga]Ga-NODAGA-RGD and [68Ga]Ga-DOTA-NT-20.3 in preclinical prostate tumour models.

INTRODUCTION: Peptide-based imaging agents targeting prostate-specific membrane antigen (PSMA) have revolutionized the evaluation of biochemical recurrence of prostate cancer (PCa) but lacks sensitivity at very low serum prostate specific antigen (PSA) levels. Once recurrence is suspected, other positron emission tomography (PET) radiotracers could be of interest to discriminate between local and distant relapse. We studied [18F]fluorodeoxyglucose ([18F]FDG) targeting glucose metabolism, [18F]fluorocholine ([18F]FCH) targeting membrane metabolism and peptide-based imaging agents [68Ga]Ga-PSMA-11, [68Ga]Ga-AMBA, [68Ga]Ga-NODAGA-RGD and [68Ga]Ga-DOTA-NT-20.3 targeting PSMA, gastrin releasing peptide receptor (GRPr), αvß3 integrin and neurotensin type 1 receptor (NTSR1) respectively, in different PCa tumour models. METHODS: Mice were xenografted with 22Rv1, an androgen-receptor (AR)-positive, PCa cell line that expresses PSMA and PC3, an AR-negative one that does not express PSMA. PET imaging using the different radiotracers was performed sequentially and the uptake characteristics compared to one other. NTSR1 and PSMA expression levels were analysed in tumours by immunohistochemistry. RESULTS: [18F]FDG displayed low but sufficient uptake to visualize PC3 and 22Rv1 derived tumours. We also observed a low efficacy of [18F]FCH PET imaging and a low [68Ga]Ga-NODAGA-RGD tumour uptake in those tumours. As expected, an elevated tumour uptake was obtained for [68Ga]Ga-PSMA-11 in 22Rv1 derived tumour although no uptake was measured in the androgen independent cell line PC3, derived from a bone metastasis of a high-grade PCa. Moreover, in PC3 cell line, we obtained good tumour uptake, high tumour-to-background contrast using [68Ga]Ga-AMBA and [68Ga]Ga-DOTA-NT-20.3. Immunohistochemistry analysis confirmed high NTSR1 expression in PC3 derived tumours and conversely high PSMA expression in 22Rv1 derived tumours. CONCLUSION: PET imaging using [68Ga]Ga-AMBA and [68Ga]Ga-DOTA-NT-20.3 demonstrates that GRPr and NTSR1 could represent viable alternative targets for diagnostic or therapeutic applications in PCa with limited PSMA expression levels. More preclinical and clinical studies will follow to explore this potential. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT: Peptide-based imaging agents targeting PSMA represent a major progress in the evaluation of biochemical recurrence of PCa but sometimes yield false negative results in some lesions. Continuing efforts have thus been made to evaluate other radiotracers. Our preclinical results suggest that [68Ga]labelled bombesin and neurotensin analogues could serve as alternative PET radiopharmaceuticals for diagnostic or therapy in cases of PSMA-negative PCa.

Cilostazol Mediates Immune Responses and Affects Angiogenesis During the Acute Phase of Hind Limb Ischemia in a Mouse Model.

BACKGROUND: Cilostazol is a drug of choice for the treatment of intermittent claudication that also affects innate and adaptive immune cells. The purpose of our study was the evaluation of cilostazol's impact on the immune and angiogenic response in murine models of hind limb ischemia. METHODS: We used 108 immunodeficient NOD.CB17-Prkdcscid/J mice and 108 wild-type CB17 mice. At day 0 (D0), all animals underwent hind limb ischemia. Half of them in both groups received daily cilostazol starting at D0 and for the next 7 postoperative days, while the rest of them served as controls, receiving vehicle. Interleukin (IL) 2, IL-4, IL-6, IL-10, IL-17A, tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ) serum concentrations were measured by flow cytometry on postsurgery days D1, D3, D5, and D7. On D7, both groups underwent positron emission tomography scan with 68Ga-RGD. Mice were euthanatized and gastrocnemius muscles were obtained for histological evaluation. RESULTS: There was a statistically significant augmentation (P < .05) in IL-4, IL-10, IL-6, and IFN-γ concentrations in treated CB17 animals, while IL-2 was significantly suppressed. Significant difference was detected between the CiBisch and Bisch groups on D1 and D7 (P < .05) in CD31 staining. In treated NOD.CB17 animals, TNF-α, IL-6, and IFN-γ presented significant augmentation, while 68Ga-NODAGA-RGDfK uptake and CD31 expression were found significantly lower for both legs in comparison to the control. CONCLUSION: Cilostazol seems to significantly increase angiogenesis in wild-type animals during the first postoperational week. It also influences immune cells, altering the type of immune response by promoting anti-inflammatory cytokine production in wild-type animals, while it helps toward inflammation regression in immunodeficient animals.

Design of Bimodal Ligands of Neurotensin Receptor 1 for Positron Emission Tomography Imaging and Fluorescence-Guided Surgery of Pancreatic Cancer.

Neurotensin receptor 1 (NTSR1) is overexpressed in most human pancreatic ductal adenocarcinomas. It makes it an attractive target for the development of pancreatic cancer imaging agents. In this study, we sought to develop a bimodal positron emission tomography (PET)/fluorescent imaging agent capable of specifically targeting these receptors. Starting from the structure of a known NTSR1 agonist, a series of tracers were synthesized, radiometalated with gallium-68, and evaluated in vitro and in vivo, in mice bearing an AsPC-1 xenograft. PET imaging allowed us to identify the compound [68Ga]Ga-NODAGA-Lys(Cy5**)-AEEAc-[Me-Arg8,Tle12]-NT(7-13) as the one with the most promising biodistribution profile, characterized by high tumor uptake (2.56 ± 0.97%ID/g, 1 h post-injection) and rapid elimination from nontargeted organs, through urinary excretion. Fluorescence imaging gave similar results. On this basis, fluorescence-guided resection of tumor masses was successfully carried out on a preclinical model.

Hepatocellular Carcinomas With Mutational Activation of Beta-Catenin Require Choline and Can Be Detected by Positron Emission Tomography.

BACKGROUND & AIMS: In one-third of hepatocellular carcinomas (HCCs), cancer cells have mutations that activate ß-catenin pathway. These cells have alterations in glutamine, bile, and lipid metabolism. We investigated whether positron emission tomography (PET) imaging allows identification of altered metabolic pathways that might be targeted therapeutically. METHODS: We studied mice with activation of ß-catenin in liver (Apcko-liv mice) and male C57Bl/6 mice given injections of diethylnitrosamine, which each develop HCCs. Mice were fed a conventional or a methionine- and choline-deficient diet or a choline-deficient (CD) diet. Choline uptake and metabolism in HCCs were analyzed by micro-PET imaging of mice; livers were collected and analyzed by histologic, metabolomic, messenger RNA quantification, and RNA-sequencing analyses. Fifty-two patients with HCC underwent PET imaging with 18F-fluorodeoxyglucose, followed by 18F-fluorocholine tracer metabolites. Human HCC specimens were analyzed by immunohistochemistry, quantitative polymerase chain reaction, and DNA sequencing. We used hepatocytes and mouse tumor explants for studies of incorporation of radiolabeled choline into phospholipids and its contribution to DNA methylation. We analyzed HCC progression in mice fed a CD diet. RESULTS: Livers and tumors from Apcko-liv mice had increased uptake of dietary choline, which contributes to phospholipid formation and DNA methylation in hepatocytes. In patients and in mice, HCCs with activated ß-catenin were positive in 18F-fluorocholine PET, but not 18F-fluorodeoxyglucose PET, and they overexpressed the choline transporter organic cation transporter 3. The HCC cells from Apcko-liv mice incorporated radiolabeled methyl groups of choline into phospholipids and DNA. In Apcko-liv mice, the methionine- and choline-deficient diet reduced proliferation and DNA hypermethylation of hepatocytes and HCC cells, and the CD diet reduced long-term progression of tumors. CONCLUSIONS: In mice and humans, HCCs with mutations that activate ß-catenin are characterized by increased uptake of a fluorocholine tracer, but not 18F-fluorodeoxyglucose, revealed by PET. The increased uptake of choline by HCCs promotes phospholipid formation, DNA hypermethylation, and hepatocyte proliferation. In mice, the CD diet reverses these effects and promotes regression of HCCs that overexpress ß-catenin.

Preclinical Evaluation of 68Ga-DOTA-NT-20.3: A Promising PET Imaging Probe To Discriminate Human Pancreatic Ductal Adenocarcinoma from Pancreatitis.

Neurotensin receptor 1 (NTSR1) is overexpressed in human pancreatic ductal adenocarcinoma (PDAC). Specific noninvasive positron-emission tomography (PET) imaging probes may improve the diagnostic accuracy and the monitoring of therapy for patients with PDAC. Here, we report the use of the 68Ga-labeled neurotensin (NTS) analogue DOTA-NT-20.3 to image human PDAC in animal models and to discriminate tumors from pancreatitis. In addition to the preclinical study, two tissue microarray slides, constructed by small core biopsies (2-5) from standard paraffin-embedded tumor tissues, were used to confirm the high (78%) positivity rate of NTSR1 expression in human PDAC. PET imaging, biodistribution, blocking, and histology studies were performed in subcutaneous AsPC-1 pancreatic tumor-bearing mice. 68Ga-DOTA-NT-20.3 PET images showed rapid tumor uptake and high contrast between the tumor and background with a fast blood clearance and a moderate accumulation in the kidneys. Ex vivo biodistribution showed low uptake in normal pancreas (0.22% IA/g) and in the remaining organs at 1 h postinjection, kidney retention (5.38 ± 0.54% IA/g), and fast clearance from blood and confirmed high uptake in tumors (5.28 ± 0.93% IA/g), leading to a tumor-to-blood ratio value of 6 at 1 h postinjection. The significant decrease of tumor uptake in a blocking study demonstrated the specificity of 68Ga-DOTA-N-T20.3 to target NTSR1 in vivo. PET imaging was also conducted in an orthotopic xenograft model that allows tumors to grow in their native microenvironment and in an experimental pancreatitis model generated by caerulein injections. As opposed to 2-[18F]fluoro-deoxyglucose, 68Ga-DOTA-NT-20.3 distinguishes PDAC from pancreatitis. Thus, 68Ga-DOTA-NT-20.3 is a promising PET imaging probe for imaging PDAC in humans.

[68Ga]RGD Versus [18F]FDG PET Imaging in Monitoring Treatment Response of a Mouse Model of Human Glioblastoma Tumor with Bevacizumab and/or Temozolomide.

PURPOSE: The aim of this study was to evaluate positron emission tomography (PET) imaging with [68Ga]NODAGA-c(RGDfK) ([68Ga]RGD), in comparison with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG), for early monitoring of the efficacy of an antiangiogenic agent associated or not with chemotherapy, in a mouse model of glioblastoma (GB). PROCEDURES: Mice bearing U87MG human GB cells line were parted into five groups of five mice each. One group was imaged at baseline before the treatment phase; another group was treated with bevacizumab (BVZ), another group with temozolomide (TMZ), another group with both agents, and the last one was the control group. Tumors growth and biological properties were evaluated by caliper measurements and PET imaging at three time points (baseline, during treatment t1 = 4-6 days and t2 = 10-12 days). At the end of the study, tumors were counted and analyzed by immunohistochemistry (CD31 to evaluate microvessel density). RESULTS: The tumor volume assessed by caliper measurements was significantly greater at t1 in the control group than in the TMZ + BVZ-treated group or in the BVZ-treated group. At t2, tumor volume of all treated groups was significantly smaller than that of the control group. [18F]FDG PET failed to reflect this efficacy of treatment. In contrast, at t1, the [68Ga]RGD tumor uptake was concordant with tumor growth in controls and in treated groups. At t2, a significant increase in tumor uptake of [68Ga]RGD vs. t1 was only observed in the TMZ-treated group, reflecting a lack of angiogenesis inhibition, whereas TMZ + BVZ resulted in a dramatic tumor arrest, reduction in microvessel density and stable tumor [68Ga]RGD uptake. CONCLUSIONS: [68Ga]RGD is a useful PET agent for in vivo angiogenesis imaging and can be useful for monitoring antiangiogenic treatment associated or not with chemotherapy.

Comparison and evaluation of two RGD peptides labelled with 68Ga or 18F for PET imaging of angiogenesis in animal models of human glioblastoma or lung carcinoma.

The aim of this study was to evaluate two RGD radiotracers radiolabelled with fluorine-18 or gallium-68, in detecting angiogenesis in grafted human tumours and monitoring their treatment with the anti-angiogenic agent bevacizumab. Sixteen mice bearing an U87MG tumour in one flank and a contralateral A549 tumour were treated with intravenous injections of bevacizumab twice a week for 3 weeks. PET images with 18F-RGD-K5 and 68Ga-RGD were acquired before treatment (baseline), after three bevacizumab injections (t1) and after seven bevacizumab injections (t2). In A549 tumours, the treatment stopped the tumour growth, with a tumour volume measured by calliper remaining between 0.28 and 0.40 cm3. The decrease in tumour uptake of both RGD tracers was non-significant. Therefore it was not possible to predict this efficacy on tumour growth based on RGD PET results, whereas ex vivo measurements showed a significantly lower tumour uptake of both tracers in mice sacrificed at t2 vs. at baseline. In U87MG tumours, the uptake measured on PET decreased during treatment, reflecting the partial therapeutic effect observed on tumour volume, consisting in a decrease in the slope of tumour growth. Using 18F-RGD-K5, this decrease in tumour SUVmax became significant at t1, whereas it was also observed with the 68Ga-RGD tracer, but only at t2. 18F-RGD-K5 appeared more efficient than 68Ga-RGD in the visualisation and follow-up of U87MG tumours. The comparison of those results with those of immunohistochemistry at baseline and at t2 favoured the hypothesis that tumour RGD uptake reflects other cancer properties than just its angiogenic capacity.

Liposomes for PET and MR Imaging and for Dual Targeting (Magnetic Field/Glucose Moiety): Synthesis, Properties, and in Vivo Studies.

We describe the potentiality of a new liposomal formulation enabling positron emission tomography (PET) and magnetic resonance MR() imaging. The bimodality is achieved by coupling a 68Ga-based radiotracer on the bilayer of magnetic liposomes. In order to enhance the targeting properties obtained under a permanent magnetic field, a sugar moiety was added in the lipid formulation. Two new phospholipids were synthesized, one with a specific chelator of 68Ga (DSPE-PEG-NODAGA) and one with a glucose moiety (DSPE-PEG-glucose). The liposomes were produced according to a fast and safe process, with a high radiolabeling yield. MR and PET imaging were performed on mice bearing human glioblastoma tumors (U87MG) after iv injection. The accumulation of the liposomes in solid tumor is evidenced by MR imaging and the amount is evaluated in vivo and ex vivo according to PET imaging. An efficient magnetic targeting is achieved with these new magnetic liposomes.

68Ga-DOTATOC and FDG PET Imaging of Preclinical Neuroblastoma Models.

BACKGROUND/AIM: Somatostatine receptors subtype 2 (SSTR2) are regarded as a potential target in neuroblastoma (NB) for imaging and promising therapeutic approaches. The purpose of this study was to evaluate and compare the SSTR2 status by (68)Ga-[tetraxetan-D-Phe1, Tyr3]-octreotide ((68)Ga-DOTATOC) positron-emission tomography (PET) and the tumour metabolic activity by (18)F-fluorodeoxyglucose (FDG) PET in different experimental models of NB. MATERIALS AND METHODS: Three cell lines of human NB with different levels of expression of SSTR2 were grafted into nude mice. Animals were imaged with FDG and (68)Ga-DOTATOC and the maximum standardized uptake value (SUVmax) was determined to quantify tracer uptake. Ex vivo biodistribution of (68)Ga-DOTATOC and immunohistochemical analysis of NB xenografts were performed. RESULTS: Compared with FDG, the SUVmax of (68)Ga-DOTATOC uptake by the tumour was lower but the ratio to background was higher; there was a strong positive correlation between SUVmax values observed with the two tracers (r(2)=0.65). Sorting the cell lines according to uptake of FDG or (68)Ga-DOTATOC, injected activity per gram of tissue, Ki67 index or expression of SSTR2 assessed visually led to the same classification. CONCLUSION: (68)Ga-DOTATOC allows preclinical imaging of NB according to the intensity of the expression of SSTR2. In contrast with what has been reported for neuroendocrine tumours, in this NB model, the (68)Ga-DOTATOC uptake was positively correlated with FDG uptake and with Ki67 index, usual markers of tumour aggressiveness. If confirmed in humans, this result would favour a theranostic application of (68)Ga-DOTATOC in NB, even in advanced stages.

Apoptosis induction by combination of drugs or a conjugated molecule associating non-steroidal anti-inflammatory and nitric oxide donor effects in medullary thyroid cancer models: implication of the tumor suppressor p73.

BACKGROUND: Medullary thyroid cancer (MTC) is a C-cell neoplasm. Surgery remains its main treatment. Promising therapies based on tyrosine kinase inhibitors demand careful patient selection. We previously observed that two non-steroidal anti-inflammatory drugs (NSAID), indomethacin, celecoxib, and nitric oxide (NO) prevented tumor growth in a model of human MTC cell line (TT) in nude mice. METHODS: In the present study, we tested the NO donor: glyceryl trinitrate (GTN), at pharmacological dose, alone and in combination with each of the two NSAIDs on TT cells. We also assessed the anti-proliferative potential of NO-indomethacin, an indomethacin molecule chemically conjugated with a NO moiety (NCX 530, Nicox SA) on TT cells and indomethacin/GTN association in rMTC 6-23 cells. The anti-tumoral action of the combined sc. injections of GTN with oral delivery of indomethacin was also studied on subcutaneous TT tumors in nude mice. Apoptosis mechanisms were assessed by expression of caspase-3, TAp73α, TAp73α inhibition by siRNA or Annexin V externalisation. RESULTS: The two NSAIDs and GTN reduced mitotic activity in TT cells versus control (cell number and PCNA protein expression). The combined treatments amplified the anti-tumor effect of single agents in the two tested cell lines and promoted cell death. Moreover, indomethacin/GTN association stopped the growth of established TT tumors in nude mice. We observed a significant cleavage of full length PARP, a caspase-3 substrate. The cell death appearance was correlated with a two-fold increase in TAp73α expression, with inhibition of apoptosis after TAp73α siRNA addition, demonstrating its crucial role in apoptosis. CONCLUSION: Association of NO with NSAID exhibited amplified anti-tumoral effects on in vitro and in vivo MTC models by inducing p73-dependent apoptotic cell death.

Activated Alk triggers prolonged neurogenesis and Ret upregulation providing a therapeutic target in ALK-mutated neuroblastoma.

Activating mutations of the ALK (Anaplastic lymphoma Kinase) gene have been identified in sporadic and familial cases of neuroblastoma, a cancer of early childhood arising from the sympathetic nervous system (SNS). To decipher ALK function in neuroblastoma predisposition and oncogenesis, we have characterized knock-in (KI) mice bearing the two most frequent mutations observed in neuroblastoma patients. A dramatic enlargement of sympathetic ganglia is observed in AlkF1178L mice from embryonic to adult stages associated with an increased proliferation of sympathetic neuroblasts from E14.5 to birth. In a MYCN transgenic context, the F1178L mutation displays a higher oncogenic potential than the R1279Q mutation as evident from a shorter latency of tumor onset. We show that tumors expressing the R1279Q mutation are sensitive to ALK inhibition upon crizotinib treatment. Furthermore, our data provide evidence that activated ALK triggers RET upregulation in mouse sympathetic ganglia at birth as well as in murine and human neuroblastoma. Using vandetanib, we show that RET inhibition strongly impairs tumor growth in vivo in both MYCN/KI AlkR1279Q and MYCN/KI AlkF1178L mice. Altogether, our findings demonstrate the critical role of activated ALK in SNS development and pathogenesis and identify RET as a therapeutic target in ALK mutated neuroblastoma.

Correlation of immunohistopathological expression of somatostatin receptor-2 in breast cancer and tumor detection with 68Ga-DOTATOC and 18F-FDG PET imaging in an animal model.

BACKGROUND: Fludeoxyglucose positron emission topography ((18)F-FDG PET) is insufficiently sensitive at detecting small or low-grade breast tumors. The characterization of somatostatin receptors (SSTR) in tumors and the development of (68)Ga-DOTATOC PET for imaging could be of interest. The aim of this study was to validate an animal model expressing SSTR2 and to correlate the immunohistochemical (IHC) analysis with (18)F-FDG and (68)Ga-DOTATOC uptake in vivo. MATERIALS AND METHODS: Ten nude mice were xenografted with the ZR-75-1 breast tumor cell line. Imaging was performed with (68)Ga-DOTATOC and (18)F-FDG and correlated to IHC analysis of SSTR2. RESULTS: IHC analyses showed that the tumors expressed SSTR2. On PET imaging, the tumors were barely visible with (18)F-FDG, whereas with (68)Ga-DOTATOC, specific two-fold higher uptake was observed (p<0.005). CONCLUSION: Our results suggest that (68)Ga-DOTATOC PET could be used for detection of breast tumors not detected with (18)F-FDG. SSTR2 status should be assessed to allow for individual treatment.

Hepatic myofibroblasts promote the progression of human cholangiocarcinoma through activation of epidermal growth factor receptor.

UNLABELLED: Intrahepatic cholangiocarcinoma (CCA) is characterized by an abundant desmoplastic environment. Poor prognosis of CCA has been associated with the presence of alpha-smooth muscle actin (α-SMA)-positive myofibroblasts (MFs) in the stroma and with the sustained activation of the epidermal growth factor receptor (EGFR) in tumor cells. Among EGFR ligands, heparin-binding epidermal growth factor (HB-EGF) has emerged as a paracrine factor that contributes to intercellular communications between MFs and tumor cells in several cancers. This study was designed to test whether hepatic MFs contributed to CCA progression through EGFR signaling. The interplay between CCA cells and hepatic MFs was examined first in vivo, using subcutaneous xenografts into immunocompromised mice. In these experiments, cotransplantation of CCA cells with human liver myofibroblasts (HLMFs) increased tumor incidence, size, and metastatic dissemination of tumors. These effects were abolished by gefitinib, an EGFR tyrosine kinase inhibitor. Immunohistochemical analyses of human CCA tissues showed that stromal MFs expressed HB-EGF, whereas EGFR was detected in cancer cells. In vitro, HLMFs produced HB-EGF and their conditioned media induced EGFR activation and promoted disruption of adherens junctions, migratory and invasive properties in CCA cells. These effects were abolished in the presence of gefitinib or HB-EGF-neutralizing antibody. We also showed that CCA cells produced transforming growth factor beta 1, which, in turn, induced HB-EGF expression in HLMFs. CONCLUSION: A reciprocal cross-talk between CCA cells and myofibroblasts through the HB-EGF/EGFR axis contributes to CCA progression.

The calpain/calpastatin system has opposing roles in growth and metastatic dissemination of melanoma.

Conventional calpains are ubiquitous cysteine proteases whose activity is promoted by calcium signaling and specifically limited by calpastatin. Calpain expression has been shown to be increased in human malignant cells, but the contribution of the calpain/calpastatin system in tumorigenesis remains unclear. It may play an important role in tumor cells themselves (cell growth, migration, and a contrario cell death) and/or in tumor niche (tissue infiltration by immune cells, neo-angiogenesis). In this study, we have used a mouse model of melanoma as a tool to gain further understanding of the role of calpains in tumor progression. To determine the respective importance of each target, we overexpressed calpastatin in tumor and/or host in isolation. Our data demonstrate that calpain inhibition in both tumor and host blunts tumor growth, while paradoxically increasing metastatic dissemination to regional lymph nodes. Specifically, calpain inhibition in melanoma cells limits tumor growth in vitro and in vivo but increases dissemination by amplifying cell resistance to apoptosis and accelerating migration process. Meanwhile, calpain inhibition restricted to host cells blunts tumor infiltration by immune cells and angiogenesis required for antitumor immunity, allowing tumor cells to escape tumor niche and disseminate. The development of highly specific calpain inhibitors with potential medical applications in cancer should take into account the opposing roles of the calpain/calpastatin system in initial tumor growth and subsequent metastatic dissemination.

Novel DOTA-neurotensin analogues for 111In scintigraphy and 68Ga PET imaging of neurotensin receptor-positive tumors.

Overexpression of the high affinity neurotensin receptor 1 (NTSR1), demonstrated in several human cancers, has been proposed as a new marker for human ductal pancreatic carcinoma and as an independent factor for poor prognosis for ductal breast cancer, head and neck squamous cell carcinoma, and non-small cell lung cancer. The aim of the present study was to develop new DOTA-neurotensin analogues for positron emission tomography (PET) imaging with (68)Ga and for targeted radiotherapy with (90)Y or (177)Lu. We synthesized a DOTA-neurotensin analogue series. Two of these peptides bear two sequence modifications for metabolic stability: DOTA-NT-20.3 shares the same peptide sequence as the previously described DTPA-NT-20.3. In the sequence of DOTA-NT-20.4, the Arg(8)-Arg(9) bond was N-methylated instead of the Pro(7)-Arg(8) bond in DOTA-NT-20.3. An additional sequence modification was introduced in DOTA-LB119 to increase stability. A spacer was added between DOTA and the peptide sequence to increase affinity. Binding to HT29 cells, which express NTSR1, in vivo stability, and biodistribution of the various analogues were compared, and the best candidate was used to image tumors of various sizes with the microPET in mice. (111)In-DOTA-NT-20.3, in spite of a relatively high uptake in kidneys, showed specific tumor uptake and elevated tumor to other organ uptake ratios. High contrast images were obtained at early time points after injection that allowed tumor detection at a time interval postinjection appropriate for imaging with the short-lived radionuclide (68)Ga. (111)In-DOTA-NT-20.4 displayed inferior binding to HT29 cells and reduced tumor uptake. (111)In-DOTA-LB119 displayed at early time points a significantly lower renal uptake but also a lower tumor uptake than (111)In-DOTA-NT-20.3, although binding to HT29 cells was similar. (68)Ga-DOTA-NT-20.3 displayed higher tumor uptake than (68)Ga-DOTA-LB119 and allowed the detection of very small tumors by PET. In conclusion, DOTA-NT-20.3 is a promising candidate for (68)Ga-PET imaging of neurotensin receptor-positive tumors. DOTA-NT-20.3 may also be considered for therapy, as the yttrium-labeled peptide has higher affinity than that of the indium-labeled one. A prerequisite for therapeutic application of this neurotensin analogue would be to lower kidney uptake, for example, by infusion of basic amino acids, gelofusin, or albumin fragments, to prevent nephrotoxicity, as with radiolabeled somatostatin analogues.

Pregnancy promotes melanoma metastasis through enhanced lymphangiogenesis.

The relationships of pregnancy and melanoma have been debatable. Our aim was to assess the influence of gestation on the course of melanoma in a classic murine model of tumor progression and in women. B16 mouse melanoma cells were injected in nonpregnant or pregnant mice on day 5 of gestation. Animals were evaluated for tumor progression, metastases, and survival. Tumor sections were analyzed for lymphatic and blood vessel number and relative surface and expression of angiogenic growth factors. Finally, primary melanomas from pregnant and nonpregnant women, matched for age and tumor thickness, were also considered. Tumor growth, metastasis, and mortality were increased in B16-injected pregnant mice. Tumors displayed an increase in intratumoral lymphangiogenesis during gestation. This increased lymphatic angiogenesis was not observed in normal skin during gestation, showing its specificity to the tumor. An analysis of melanoma from pregnant and matched nonpregnant women showed a similar increase in lymphatic vessels. Tumors from pregnant mice had increased expression of vascular endothelial growth factor A at the RNA and protein levels. The increased vascular endothelial growth factor A production by melanoma cells could be reproduced in culture using pregnant mouse serum. In conclusion, pregnancy results in increased lymphangiogenesis and subsequent metastasis. Caution should be applied in the management of patients with advanced-stage melanoma during gestation.