18F-RB390: innovative ligand for imaging the T877A androgen receptor mutant in prostate cancer via positron emission tomography (PET).

BACKGROUND Detecting prostate cancer before spreading or predicting a favorable therapy are challenging issues for impacting patient's survival. Presently, 2-[(18) F]-fluoro-2-deoxy-D-glucose ((18) F-FDG) and/or (18) F-fluorocholine ((18) F-FCH) are the generally used PET-tracers in oncology yet do not emphasize the T877A androgen receptor (AR) mutation being exclusively present in cancerous tissue and escaping androgen deprivation treatment. METHODS We designed and synthesized fluorinated 5α-dihydrotestosterone (DHT) derivatives to target T877A-AR. We performed binding assays to select suitable candidates using COS-7 cells transfected with wild-type or T877A AR (WT-AR, T877A-AR) expressing plasmids and investigated cellular uptake of candidate (18) F-RB390. Stability, biodistribution analyses and PET-Imaging were assessed by injecting (18) F-RB390 (10MBq), with and without co-injection of an excess of unlabeled DHT in C4-2 and PC-3 tumor bearing male SCID mice (n = 12). RESULTS RB390 presented a higher relative binding affinity (RBA) (28.1%, IC50  = 32 nM) for T877A-AR than for WT-AR (1.7%, IC50  = 357 nM) related to DHT (RBA = 100%). A small fraction of (18) F-RB390 was metabolized when incubated with murine liver homogenate or human blood for 3 hr. The metabolite of RB390, 3-hydroxysteroid RB448, presented similar binding characteristics as RB390. (18) F-RB390 but not (18) F-FDG or (18) F-FCH accumulated 2.5× more in COS-7 cells transfected with pSG5AR-T877A than with control plasmid. Accumulation was reduced with an excess of DHT. PET/CT imaging and biodistribution studies revealed a significantly higher uptake of (18) F-RB390 in T877A mutation positive xenografts compared to PC-3 control tumors. This effect was blunted with DHT. CONCLUSION Given the differential binding capacity and the favorable radioactivity pattern, (18) F-RB390 represents the portrayal of the first imaging ligand with predictive potential for mutant T877A-AR in prostate cancer for guiding therapy. Prostate 75:348-359, 2015. © 2014 Wiley Periodicals, Inc.

PEG spacers of different length influence the biological profile of bombesin-based radiolabeled antagonists

INTRODUCTION The gastrin-releasing peptide receptor (GRPR) was shown to be expressed with high density on several types of cancers. Radiolabeled peptides for imaging and targeted radionuclide therapy have been developed. In this study, we evaluated the potential of statine-based bombesin antagonists, conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) through oligoethyleneglycol spacers, labeled with (177)Lu and we determined the effect of polyethyleneglycol (PEG) spacer length on in vitro and in vivo properties. METHODS The bombesin antagonists were synthesized on solid phase using Fmoc chemistry; the spacers Fmoc-dPEGx-OH (x=2, 4, 6 and 12) and the DOTA(tBu)3 were coupled using a standard procedure. The peptides were labeled with (177)Lu and evaluated in vitro (lipophilicity, serum stability, internalization and binding affinity assays). Biodistribution studies were performed in PC-3 tumor-bearing nude mice. RESULTS The solid-phase synthesis was straightforward with an overall yield ranging from 30% to 35% based on the first Fmoc cleavage. The hydrophilicity increased with spacer length (logD: -1.95 vs -2.22 of PEG2 and PEG12 analogs, respectively). There is a tendency of increased serum stability by increasing the spacer length (T1/2=246±4 and 584±20 for PEG2 and PEG6 analogs, respectively) which seems to reverse with the PEG12 analog. The IC50 values are similar with the only significant difference of the PEG12 analog. The (177)Lu-labeled PEG4 and PEG6 conjugates showed similar pharmacokinetic with high tumor uptake and excellent tumor-to-kidney ratios (7.8 and 9.7 at 4h for the PEG4 and PEG6 derivatives, respectively). The pancreas uptake was relatively high at 1h but it shows fast washout (0.46%±0.02% IA/g and 0.29%±0.08% IA/g already at 4h). CONCLUSION Among all the studied analogs the PEG4 and PEG6 showed significantly better properties. The very high tumor-to-non-target organ ratios, in particular tumor-to-kidney ratios, already at early time point will be important in regard to safety concerning kidney toxicity.

Evaluation of three different families of bombesin receptor radioantagonists for targeted imaging and therapy of gastrin releasing peptide receptor (GRP-R) positive tumors.

Two new classes of radiolabeled GRP receptor antagonists are studied and compared with the well-established statine-based receptor antagonist DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (RM2, 1; DOTA:1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; Sta:(3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid). The bombesin-based pseudopeptide DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leuψ(CHOH-CH2)-(CH2)2-CH3 (RM7, 2), and the methyl ester DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-OCH3 (ARBA05, 3) analogues are labeled with (111)In and evaluated in vitro in PC-3 cell line and in vivo in PC-3 tumor-bearing nude mice. Antagonist potency was assessed by immunofluorescence-based receptor internalization and Ca(2+) mobilization assays. The conjugates showed good binding affinity, the IC50 value of 2 (3.2 ± 1.8 nM) being 2 and 10 times lower than 1 and 3. Compared to (111)In-1, (111)In-2 showed higher uptake in target tissues such as pancreas (1.5 ± 0.5%IA/g and 39.8 ± 9.3%IA/g at 4 h, respectively), whereas the compounds had similar tumor uptake (11.5 ± 2.4%IA/g and 11.8 ± 3.9%IA/g at 4h, respectively). The displacement of the radioligand in vivo was different in different receptor positive organs and depended on the displacing peptide.

Intravital and whole-organ imaging reveals capture of melanoma-derived antigen by lymph node subcapsular macrophages leading to widespread deposition on follicular dendritic cells.

Aberrant antigens expressed by tumor cells, such as in melanoma, are often associated with humoral immune responses, which may in turn influence tumor progression. Despite recent data showing the central role of adaptive immune responses on cancer spread or control, it remains poorly understood where and how tumor-derived antigen (TDA) induces a humoral immune response in tumor-bearing hosts. Based on our observation of TDA accumulation in B cell areas of lymph nodes (LNs) from melanoma patients, we developed a pre-metastatic B16.F10 melanoma model expressing a fluorescent fusion protein, tandem dimer tomato, as a surrogate TDA. Using intravital two-photon microscopy (2PM) and whole-mount 3D LN imaging of tumor-draining LNs in immunocompetent mice, we report an unexpectedly widespread accumulation of TDA on follicular dendritic cells (FDCs), which were dynamically scanned by circulating B cells. Furthermore, 2PM imaging identified macrophages located in the subcapsular sinus of tumor-draining LNs to capture subcellular TDA-containing particles arriving in afferent lymph. As a consequence, depletion of macrophages or genetic ablation of B cells and FDCs resulted in dramatically reduced TDA capture in tumor-draining LNs. In sum, we identified a major pathway for the induction of humoral responses in a melanoma model, which may be exploitable to manipulate anti-TDA antibody production during cancer immunotherapy.

Synchrotron microbeam radiation therapy induces hypoxia in intracerebral gliosarcoma but not in the normal brain

PURPOSE Synchrotron microbeam radiation therapy (MRT) is an innovative irradiation modality based on spatial fractionation of a high-dose X-ray beam into lattices of microbeams. The increase in lifespan of brain tumor-bearing rats is associated with vascular damage but the physiological consequences of MRT on blood vessels have not been described. In this manuscript, we evaluate the oxygenation changes induced by MRT in an intracerebral 9L gliosarcoma model. METHODS Tissue responses to MRT (two orthogonal arrays (2 × 400Gy)) were studied using magnetic resonance-based measurements of local blood oxygen saturation (MR_SO2) and quantitative immunohistology of RECA-1, Type-IV collagen and GLUT-1, marker of hypoxia. RESULTS In tumors, MR_SO2 decreased by a factor of 2 in tumor between day 8 and day 45 after MRT. This correlated with tumor vascular remodeling, i.e. decrease in vessel density, increases in half-vessel distances (×5) and GLUT-1 immunoreactivity. Conversely, MRT did not change normal brain MR_SO2, although vessel inter-distances increased slightly. CONCLUSION We provide new evidence for the differential effect of MRT on tumor vasculature, an effect that leads to tumor hypoxia. As hypothesized formerly, the vasculature of the normal brain exposed to MRT remains sufficiently perfused to prevent any hypoxia.

Toward stable N4-modified neurotensins for NTS1-receptor-targeted tumor imaging with 99mTc

A series of Gly-neurotensin(8-13) analogues modified at the N-terminus by acyclic tetraamines (Demotensin 1-4) were obtained by solid-phase peptide synthesis techniques. Strategic replacement of amino acids and/or reduction of sensitive peptide bonds were performed to enhance conjugate resistance against proteolytic enzymes. During 99mTc-labeling, single species radiopeptides, [99mTc]Demotensin 1-4, were easily obtained in high yields and typical specific activities of 1 Ci/micromol. Peptide conjugates displayed a high affinity binding to the human neurotensin subtype 1 receptor (NTS1-R) expressed in colon adenocarcinoma HT-29 or WiDr cells and/or in human tumor sections. [99mTc]Demotensin 1-4 internalized very rapidly in HT-29 or WiDr cells by a NTS1-R-mediated process. [99mTc]Demotensin 3 and 4, which remained stable during 1 h incubation in murine plasma, were selectively studied in nude mice bearing human HT-29 and WiDr xenografts. After injection, [99mTc]Demotensin 3 and 4 effectively and specifically localized in the experimental tumors and were rapidly excreted via the kidneys into the urine, exhibiting overall biodistribution patterns favorable for NTS1-R-targeted tumor imaging in man.

New pansomatostatin ligands and their chelated versions: affinity profile, agonist activity, internalization, and tumor targeting

PURPOSE: Somatostatin receptor (sst) targeting is an established method to image and treat sst-positive tumors. Particularly, neuroendocrine tumors express the receptor subtype 2 in high density, but sst1, sst3, sst4, and sst5 are also expressed to some extent in different human tumors. Currently used targeting peptides mainly have sst2 affinity. We aimed at developing (radio)peptides that bind with high affinity to all receptor subtypes. EXPERIMENTAL DESIGN: Carbocyclic octapeptides were coupled with macrocyclic chelators for radiometal labeling. Affinity, internalization, and agonist potencies were determined on sst1- to sst5-expressing cell lines. Biodistribution was determined on nude mice bearing HEK-sst2 or AR4-2J and HEK-sst3 tumors. RESULTS: High affinity to all receptor subtypes was found. Y(III)-KE88 showed agonistic properties at all five sst receptor subtypes as it inhibits forskolin-stimulated cyclic AMP production. Surprisingly, very low or even absent sst2 receptor internalization was found compared with currently clinically established octapeptides, whereas the sst3 internalization was very efficient. Biodistribution studies of [(111)In]KE88 and [(67)Ga]KE88/[(68)Ga]KE88 reflected the in vitro data. In nude mice with s.c. implanted sst2 (HEK-sst2, AR4-2J)-expressing and sst3 (HEK-sst3)-expressing tumors, high and persistent uptake was found in sst3-expressing tumors, whereas the uptake in the sst2-expressing tumors was lower and showed fast washout. The kidney uptake was high but blockable by coinjection of lysine. CONCLUSION: This peptide family shows pansomatostatin potency. As radiopeptides, they are the first to show a full pansomatostatin profile. Despite some drawback, they should be useful for imaging sst2-expressing tumors with short-lived radiometals, such as (68)Ga, at early time points and for sst3-expressing tumors at later time points with longer-lived radiometals, such as (64)Cu or (86)Y.

The Glucose-Dependent Insulinotropic Polypeptide Receptor: A Novel Target for Neuroendocrine Tumor Imaging-First Preclinical Studies

A new family of peptide receptors, the incretin receptor family, overexpressed on many neuroendocrine tumors (NETs) is of great importance because it may enable the in vivo peptide-based receptor targeting of a category of NETs that does not express the somatostatin receptor. Impressive in vivo diagnostic data were published for glucagonlike peptide 1 receptor-targeting radiopeptides. Recently, promising in vitro data have appeared for the second member of the incretin family, the glucose-dependent insulinotropic polypeptide (GIP) receptor. This prompted us to develop and evaluate a new class of radioligands with the potential to be used for the in vivo targeting of GIP receptor-positive tumors. METHODS GIP(1-42) was modified C-terminally, and the truncated peptides [Lys(30)(aminohexanoic acid [Ahx]-DOTA)]GIP(1-30)NH2 (EG1), [Lys(16)(Ahx-DOTA)]GIP(1-30)NH2 (EG2), and [Nle(14), Lys(30)(Ahx-DOTA)]GIP(1-30)NH2 (EG4) were conjugated with Ahx-DOTA via the Lys(16) and Lys(30) side chains. Their inhibitory concentration of 50% (IC50) was determined using [(125)I-Tyr(10)]GIP(1-30) as radioligand and GIP(1-30) as control peptide. The DOTA conjugates were labeled with (111)In and (68)Ga. In vitro evaluation included saturation and internalization studies using the pancreatic endocrine cell line INR1G9 transfected with the human GIP receptor (INR1G9-hGIPr). The in vivo evaluation consisted of biodistribution and PET imaging studies on nude mice bearing INR1G9-hGIPr tumors. RESULTS Binding studies (IC50 and saturation studies) showed high affinity toward GIP receptor for the GIP conjugates. Specific in vitro internalization was found, and almost the entire cell-associated activity was internalized (>90% of the cell-bound activity), supporting the agonist potency of the (111)In-vectors. (111)In-EG4 and (68)Ga-EG4 were shown to specifically target INR1G9-hGIPr xenografts, with tumor uptake of 10.4% ± 2.2% and 17.0% ± 4.4% injected activity/g, 1 h after injection, respectively. Kidneys showed the highest uptake, which could be reduced by approximately 40%-50% with a modified-fluid-gelatin plasma substitute or an inhibitor of the serine protease dipeptidyl peptidase 4. The PET images clearly visualized the tumor. CONCLUSION The evaluation of EG4 as a proof-of-principle radioligand indicated the feasibility of imaging GIP receptor-positive tumors. These results prompt us to continue the development of this family of radioligands for imaging of a broad spectrum of NETs.

Hepatic gene expression profile in mice perorally infected with Echinococcus multilocularis eggs

Alveolar echinococcosis (AE) is a severe chronic hepatic parasitic disease currently emerging in central and eastern Europe. Untreated AE presents a high mortality (>90%) due to a severe hepatic destruction as a result of parasitic metacestode proliferation which behaves like a malignant tumor. Despite this severe course and outcome of disease, the genetic program that regulates the host response leading to organ damage as a consequence of hepatic alveolar echinococcosis is largely unknown.

Absence of somatostatin SST(2) receptor internalization in vivo after intravenous SOM230 application in the AR42J animal tumor model

Among clinically relevant somatostatin functions, agonist-induced somatostatin receptor subtype 2 (sst(2)) internalization is a potent mechanism for tumor targeting with sst(2) affine radioligands such as octreotide. Since, as opposed to octreotide, the second generation multi-somatostatin analog SOM230 (pasireotide) exhibits strong functional selectivity, it appeared of interest to evaluate its ability to affect sst(2) internalization in vivo. Rats bearing AR42J tumors endogenously expressing somatostatin sst(2) receptors were injected intravenously with SOM230 or with the [Tyr(3), Thr(8)]-octreotide (TATE) analog; they were euthanized at various time points; tumors and pancreas were analyzed by immunohistochemistry for the cellular localization of somatostatin sst(2) receptors. SOM230-induced sst(2) internalization was also evaluated in vitro by immunofluorescence microscopy in AR42J cells. At difference to the efficient in vivo sst(2) internalization triggered by intravenous [Tyr(3), Thr(8)]-octreotide, intravenous SOM230 did not elicit sst(2) internalization: immunohistochemically stained sst(2) in AR42J tumor cells and pancreatic cells were detectable at the cell surface at 2.5min, 10min, 1h, 6h, or 24h after SOM230 injection while sst(2) were found intracellularly after [Tyr(3), Thr(8)]-octreotide injection. The inability of stimulating sst(2) internalization by SOM230 was confirmed in vitro in AR42J cells by immunofluorescence microscopy. Furthermore, SOM230 was unable to antagonize agonist-induced sst(2) internalization, neither in vivo, nor in vitro. Therefore, SOM230 does not induce sst(2) internalization in vivo or in vitro in AR42J cells and pancreas, at difference to octreotide derivatives with comparable sst(2) binding affinities. These characteristics may point towards different tumor targeting but also to different desensitization properties of clinically applied SOM230.

BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination

The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.

Small-animal PET/CT for monitoring the development and response to chemotherapy of thymic lymphoma in Trp53-/- mice

Transgenic mouse models of human cancers represent one of the most promising approaches to elucidate clinically relevant mechanisms of action and provide insights into the treatment efficacy of new antitumor drugs. The use of Trp53 transgenic mice (Trp53 knockout [Trp53(-/-)] mice) for these kinds of studies is, so far, restricted by limitations in detecting developing tumors and the lack of noninvasive tools for monitoring tumor growth, progression, and treatment response.

Effect of electroporation-mediated diphtheria toxin A expression on PSA positive human prostate xenograft tumors in SCID mice

Current therapies to treat prostate cancer are often limited. Since it has been shown that very low concentrations of diphtheria toxin A (DT-A) result in abrogation of protein synthesis and apoptosis of cells, DT-A might serve as an efficient killer in cancer gene therapy. For this purpose we investigated in a quantitative manner using a stereological approach the apoptotic effect of DT-A in androgen receptor (AR) and prostate specific antigen (PSA) expressing cells after tumor formation in both flanks of SCID mice.

Self-sufficient control of urate homeostasis in mice by a synthetic circuit

Synthetic biology has shown that the metabolic behavior of mammalian cells can be altered by genetic devices such as epigenetic and hysteretic switches, timers and oscillators, biocomputers, hormone systems and heterologous metabolic shunts. To explore the potential of such devices for therapeutic strategies, we designed a synthetic mammalian circuit to maintain uric acid homeostasis in the bloodstream, disturbance of which is associated with tumor lysis syndrome and gout. This synthetic device consists of a modified Deinococcus radiodurans-derived protein that senses uric acids levels and triggers dose-dependent derepression of a secretion-engineered Aspergillus flavus urate oxidase that eliminates uric acid. In urate oxidase-deficient mice, which develop acute hyperuricemia, the synthetic circuit decreased blood urate concentration to stable sub-pathologic levels in a dose-dependent manner and reduced uric acid crystal deposits in the kidney. Synthetic gene-network devices providing self-sufficient control of pathologic metabolites represent molecular prostheses, which may foster advances in future gene- and cell-based therapies.