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PDF file, 104K, SOX2 expression in primary human SOC.

PDF file, 99K, Includes additional information regarding Primer Sequences used for Real Time PCR analysis, Tissue Microarray Analysis, Lentiviral transduction, and PET/MRI analysis.

PDF file, 58K, Analysis of primary SOC cells transduced with SOX2-overexpressing and control lentiviruses.

PDF file, 127K, Treatment of OVCAR-3 cells with an alternative SOX2 shRNA construct.

PDF file, 1265K, Histological and immunohistochemical analysis of explanted tumors generated from 50.000 SOX2-overexpressing and control Caov-3 cells, respectively.

PDF file, 72K.

PDF file, 112K, Apoptotic effects of staurosporine (STS) and TRAIL treatment in SOX2-modified and control Caov-3 and OVCAR-3 cells.

PDF file, 1094K, Characterization of OVCAR-3 cells transduced with the SOX2 enhancer reporter construct.

Here, we describe immuno-Cerenkov luminescence imaging (immuno-CLI) with a specific monoclonal antibody-based tracer for the detection of prostate tumors, which is used in preclinical positron emission tomography (PET) imaging. As PET isotopes generate a continuous spectrum of light in the ultraviolet/visible (UV/vis) wavelength range (Cerenkov luminescence, CL) in dielectric materials and consequently inside living tissues, these isotopes can also be detected by luminescence imaging performed with optical imaging (OI) systems. Imaging tumors with tracers that are specifically binding to a tumor-associated antigen can increase diagnostic accuracy, enables monitoring of treatment efficacy, and can be advantageous compared to radiolabeled small molecules used in PET-oncology such as 2-deoxy-2-[18F]-fluoro-D-glucose ([18F]FDG; glucose metabolism) or [11C]choline (membrane synthesis) which was used to image prostate cancer. In this study, we compared on three consecutive days immuno-CLI and -PET of the applied 64Cu-labeled and well described monoclonal antibody 3/F11 in prostate-specific membrane antigen (PSMA)-positive (C4-2, PSMA+) and -negative (DU 145, PSMA-) prostate tumor xenografts, inoculated in SCID mice. In vivo immuno-CLI and -PET measurements demonstrated linear correlation of both modalities, in line with ex vivo analysis performed with CLI and γ-counting. As CLI is also able to trace radioisotopes used for theranostic approaches, immuno-CLI could be an interesting, low-cost imaging alternative to immuno-PET.

393 Objectives: The currently available PET tracers i.e. 18F-FET and 11C-methionine have proven high diagnostic efficacy in GBM. However, the development of SPECT based tracers is always viewed as a cost-effective alternate to PET imaging. The aim of the present study was to determine the diagnostic efficacy of 99mTc-labelled MDM (bis-methionine) SPECT in the diagnostic work up in glioma. Methods: The present study was conducted in 143 glioma patients (101M: 42F; mean age 41.97±11.9 years; range 18-71 years) who were newly diagnosed or previously treated or who were recruited for post-surgical radiotherapy/chemotherapy treatment from December 2014 to August 2018. Amongst, 143 patients, 29/143 (20.0%) were freshly diagnosed patients of glioma and the remaining 114/143 (80.0%) patients were on post-surgery follow-up (radiological/clinical) with chemotherapy/radiotherapy interventions. The patients were subjected to a detailed histopathological tumor analysis (including Ki-67 index), 99mTc-MDM-SPECT, conventional MRI, DSCE-MRI and MR spectroscopy for the disease evaluation at presentation and during the course of follow-up after surgery/chemo/radiotherapy. A total of 227 brain scans (99mTc-MDM-SPECT) and an equal number of conventional MRI scans were performed in 143 patients. The results of the two imaging modalities were compared and correlated with the clinical findings. In a sub-set of patients (n=43), a quantitative DSCE-MRI and MR spectroscopy analysis was done. The results of the later were compared with the 99mTc-MDM-SPECT quantitative results for validation of this technique for accuracy in the glioma detection and characterization. Results: On histopathological analysis, 26/29 patients (pre-surgery group) were diagnosed to have glioma ( G IV-13; G III-04; G II-09) and the remaining 3/29 patients were found to be meningioma. The mean target to non-target (T/NT) ratios of 99mTc-MDM in glioma grade II, grade III, and grade IV patients were estimated to be 2.46±2.3, 7.13±2.2 and 5.16±1.2 respectively. This ratio was much higher (15.9 ±6.8) in meningioma. The ROC curve analysis derived cut-off value of T/NT ratio of greater than 3.08 when used to discriminate low grade from high grade glioma provided 100% sensitivity, 87.5% specificity. In the post-surgery group, the final diagnosis could be made in 72/114 patients. Out of these, 47/72 showed tumor recurrent (Fig.1) or residual disease and the remaining 25 patients showed necrosis. The ROC curve analysis derived cut-off value of T/NT ratio of greater than 1.90 used to discriminate tumor recurrence from necrosis offered sensitivity and specificity of 97.9 % and 92.0% respectively. A similar analysis on DSCE-MRI quantitative data with derived nCBV cut of value of greater than 3.32 for discriminating tumor recurrence versus necrosis provided sensitivity and specificity of 84.6% and 93.0% respectively. MR spectroscopy data analysis estimated the cut-of ratios; sensitivity/specificity of different metabolites i.e. Cho/NAA, Cho/Cr, Cr/NAA, Cr/Cho and Cho/LL to be >1.57, 81.0%/73.0%; >1.64, 85.3%/73.7%; >1.06. 57.1%/ 63.6%; ≤ 0.60, 72.3%/81.0% and >0.90, 71.4%, /50% respectively. T/NT ratio showed a strongest linear correlation with nCBV(r = 0.775, P

Cytostatic drugs used in cancer therapy were evaluated for their capacity to inhibit Echinococcus multilocularis metacestode growth and proliferation. Metacestode tissues were exposed in vitro to docetaxel, doxorubicin, navelbine, paclitaxel, and vorinostat for 1 week, then incubated in drug-free culture, and thereafter metacestodes were injected into the peritoneum of Meriones unguiculatus. Magnetic resonance imaging (MRI) and simultaneous positron emission tomography (PET) were applied to monitor in vivo growth of drug-exposed E. multilocularis in Meriones. At 3 month p.i., docetaxel (at 10 μM, 5 μM and 2 μM) inhibited in vivo growth and proliferation of E. multilocularis, and at 5 months p.i., only in the 2 μM docetaxel exposure group 0.3 cm 3 of parasite tissue was found. With paclitaxel and navelbine the in vivo growth of metacestodes was suppressed until 3 months p.i., thereafter, parasite tissues enlarged up to 3 cm 3 in both groups. E. multilocularis tissues of more than 10 g developed in Meriones injected with metacestodes which were previously exposed in vitro to doxorubicin, navelbine, paclitaxel or vorinostat. In Meriones infected with metacestodes previously exposed to docetaxel, the in vivo grown parasite tissues weighted 0.2 g. In vitro cultured E. multilocularis metacestodes exposed to docetaxel did not produce vesicles until 7 weeks post drug exposure, while metacestodes exposed to doxorubicin, navelbine and vorinostat proliferated continuously. In summary, docetaxel, and less efficaciously paclitaxel, inhibited in vivo and in vitro parasite growth and proliferation, and these observations suggest further experimental studies with selected drug combinations which may translate into new treatment options against alveolar echinococcosis.

Manganese in its divalent state (Mn2+) has features that make it a unique tool for tracing neuronal pathways. It is taken up and transported by neurons in an activity dependent manner and it can cross synapses. It also acts as a contrast agent for magnetic resonance imaging (MRI) enabling visualization of neuronal tracts. However, due to the limited sensitivity of MRI systems relatively high Mn2+ doses are required. This is undesirable, especially in long-term studies, because of the known toxicity of the metal. In order to overcome this limitation, we propose 52Mn as a positron emission tomography (PET) neuronal tract tracer. We used 52Mn for imaging dopaminergic pathways after a unilateral injection into the ventral tegmental area (VTA), as well as the striatonigral pathway after an injection into the dorsal striatum (STR) in rats. Furthermore, we tested potentially noxious effects of the radioactivity dose with a behavioral test and histological staining. 24 h after 52Mn administration, the neuronal tracts were clearly visible in PET images and statistical analysis confirmed the observed distribution of the tracer. We noticed a behavioral impairment in some animals treated with 170 kBq of 52Mn, most likely caused by dysfunction of dopaminergic cells. Moreover, there was a substantial DNA damage in the brain tissue after applying 150 kBq of the tracer. However, all those effects were completely eliminated by reducing the 52Mn dose to 20-30 kBq. Crucially, the reduced dose was still sufficient for PET imaging.

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease of hematological malignancy or bone marrow transplant patients caused by the ubiquitous environmental fungus Aspergillus fumigatus. Current diagnostic tests for the disease lack sensitivity as well as specificity, and culture of the fungus from invasive lung biopsy, considered the gold standard for IPA detection, is slow and often not possible in critically ill patients. In a previous study, we reported the development of a novel non-invasive procedure for IPA diagnosis based on antibody-guided positron emission tomography and magnetic resonance imaging (immunoPET/MRI) using a [64Cu] DOTA-labeled mouse monoclonal antibody (mAb), mJF5, specific to Aspergillus. To enable translation of the tracer to the clinical setting, we report here the development of a humanised version of the antibody (hJF5), and pre-clinical imaging of lung infection using a [64Cu] NODAGA-hJF5 tracer. The humanised antibody tracer shows a significant increase in in vivo biodistribution in A. fumigatus infected lungs compared to its radiolabeled murine counterpart [64Cu] NODAGA-mJF5. Using reverse genetics of the pathogen, we show that the antibody binds to the antigenic determinant beta 1,5-galactofuranose (Galf) present in a diagnostic mannoprotein antigen released by the pathogen during invasive growth in the lung. The absence of the epitope Galf in mammalian carbohydrates, coupled with the enhanced imaging capabilities of the hJF5 antibody, means that the [64Cu] NODAGA-hJF5 tracer developed here represents an ideal candidate for the diagnosis of IPA and translation to the clinical setting.

The specific and rapid detection of Enterobacteriaceae, the most frequent cause of gram-negative bacterial infections in humans, remains a major challenge. We developed a non-invasive method to rapidly detect systemic Yersinia enterocolitica infections using immunoPET (antibody-targeted positron emission tomography) with [64Cu]NODAGA-labeled Yersinia-specific polyclonal antibodies targeting the outer membrane protein YadA. In contrast to the tracer [18F]FDG, [64Cu]NODAGA-YadA uptake co-localized in a dose dependent manner with bacterial lesions of Yersinia-infected mice, as detected by magnetic resonance (MR) imaging. This was accompanied by elevated uptake of [64Cu]NODAGA-YadA in infected tissues, in ex vivo biodistribution studies, whereas reduced uptake was observed following blocking with unlabeled anti-YadA antibody. We show, for the first time, a bacteria-specific, antibody-based, in vivo imaging method for the diagnosis of a Gram-negative enterobacterial infection as a proof of concept, which may provide new insights into pathogen-host interactions.

Patients with metastatic medullary thyroid cancer (MTC) have limited systemic treatment options. The use of radiolabeled gastrin analogs targeting the cholecystokinin-2 receptor (CCK2R) is an attractive approach. However, their therapeutic efficacy is presumably decreased by their enzymatic degradation in vivo. We aimed to investigate whether the chemically stabilized analog

Preclinical studies involving 89 Zr often report significant bone accumulation, which is associated with dissociation of the radiometal from the tracer. However, experiments determining the uptake of unbound 89 Zr in disease models are not performed as routine controls. The purpose of the present study was to investigate the impact of free or weakly bound 89 Zr on PET quantifications in disease models, in order to determine if such control experiments are warranted. Methods Chemical studies were carried out to find a 89 Zr compound that would solubilize the 89 Zr as a weak chelate, thus mimicking free or weakly bound 89 Zr released in circulation. 89 Zr oxalate had the desired characteristics, and was injected into mice bearing FaDu and HT29 solid tumor xenografts, and mice infected in the lungs with the mold Aspergillus fumigatus , as well as in healthy controls (naive). PET/CT or PET/MR imaging followed to quantify the distribution of the radionuclide in the disease models. Results 89 Zr oxalate was found to have a plasma half-life of 5.1±2.3h, accumulating mainly in the bones of all animals. Both tumor types accumulated 89 Zr on the order of 2‐4 %ID/cm 3 , which is comparable to EPR-mediated accumulation of certain species. In the aspergillosis model, the concentration of 89 Zr in lung tissue of the naive animals was 6.0±1.1 %ID/g. This was significantly different from that of the animals with advanced disease, showing 11.6±1.8 %ID/g. Conclusions Given the high levels of 89 Zr accumulation in the disease sites in the present study, we recommend control experiments mapping the biodistribution of free 89 Zr in any preclinical study employing 89 Zr where bone uptake is observed. Aqueous 89 Zr oxalate appears to be a suitable compound for such studies. This is especially relevant in studies where the tracer accumulation is based upon passive targeting, such as EPR.

2-Deoxy-2-[18F]fluoro-d-glucose ([18F]FDG) has been used as a standard clinical positron emission tomography (PET) tracer for the follow-up of the rare but life-threatening parasitic disease alveolar echinococcosis (AE). Given that the disease is endemic in many countries in the northern hemisphere and the diagnosis is still challenging, the aim of our study was to evaluate further clinically relevant PET tracers as possible diagnostic tools for AE in vitro and in vivo. Various clinically used PET tracers were evaluated in vitro and assessed in an in vivo AE animal model based on PET/magnetic resonance (MR) measurements. In vitro binding assays displayed high uptake of [18F]FDG in a cell suspension of E. multilocularis tissue, whereas 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT) and [11C]choline were found to be taken up strongly by E. multilocularis vesicles. [18F]FDG and [18F]FLT displayed an elevated uptake in vivo, which appeared as several foci throughout the parasite tissue as opposed to [18F]fluoro-azomycinarabinofuranoside ([18F]FAZA) and [11C]choline. Our data clearly demonstrate that the clinically applied PET tracer [18F]FDG is useful for the diagnosis and disease staging of AE but also has drawbacks in the assessment of currently inactive or metabolically weak parasitic lesions. The different tested PET tracers do not show the potential for the replacement or supplementation of current diagnostic strategies. Hence, there is still the need for novel diagnostic tools.

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models. Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models. Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Manganese in its divalent state (Mn

Despite the success of therapeutics fighting against especially bacteria and fungi, infectious diseases still remain one of the main causes of death worldwide (WHO 2014). Besides effective medication, the early and reliable differential diagnosis of infectious diseases is of utmost importance; here noninvasive imaging can have a huge impact. The host defense against pathogens is dependent on an intact innate and adaptive immune response and effective communication between the immune cells. Dysfunction of one or more of these immune compartments leads to an open gateway for pathogens into the body of humans and animals. Infection is often accompanied with inflammation but both processes differ from each other. Inflammation is basically a nonspecific immune response which can have many reasons but does not necessarily require a microorganism in the inflamed site (Petruzzi et al. 2009). Very well-adapted pathogens have evolved strategies to act immunomodulatory to evade the immune response of the host and to finally enable their survival and transmission (Coombes and Robey 2010).

BACKGROUND Radioimmunoimaging with disease-specific tracers can be advantageous compared to that with nonspecific tracers for the imaging of glucose metabolism and cell proliferation. Monoclonal antibodies (mAbs) or their fragments are excellent tools for immuno-positron emission tomography (PET). In this study, PSMA-specific mAb 3/F11 and its recombinant fragments were compared for the imaging of prostate cancer in xenografts. METHODS Recombinant anti-PSMA antibody fragments D7-Fc and D7-CH3 were constructed by genetically fusing the binding domains of mAb 3/F11 (D7) to the human IgG3 CH3 or CH2–CH3 (Fc) domain. The fragments and the mAb 3/F11 were DOTA conjugated, tested in vitro, and radiolabeled with 64Cu. PSMA-positive C4-2 and PSMA-negative DU 145 prostate cancer xenografts were used for PET-MR imaging and for ex vivo biodistribution. RESULTS The constructs showed strong and specific binding to PSMA-positive C4-2 cells in vitro which did not decrease after DOTA conjugation. Both tested fragments showed stable accumulation in PSMA-positive C4-2 tumors at all measured time points but reduced uptake compared to the full-length antibody. Other organs and PSMA-negative tumors showed a very low tracer uptake only 3 hr after injection, with the exception of the kidneys, which demonstrated high radioactivity uptake due to rapid renal clearance of the mAb fragments. CONCLUSION Stable tumor uptake and fast serum clearance of the tested radiolabeled fragments was observed in this preclinical study compared to the full length mAb. Since the fragments show rapid and specific tumor uptake, the tested fragments might serve as tools for theranostic imaging with suitable isotopes for radioimmunotherapy. Prostate 74:743–755, 2014. © 2014 Wiley Periodicals, Inc.

Although T cells can be labeled for noninvasive in vivo imaging, little is known about the impact of such labeling on T-cell function, and most imaging methods do not provide holistic information about trafficking kinetics, homing sites, or quantification. Methods We developed protocols that minimize the inhibitory effects of (64)Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) ((64)Cu-PTSM) labeling on T-cell function and permit the homing patterns of T cells to be followed by PET. Thus, we labeled ovalbumin (OVA) T-cell receptor transgenic interferon (IFN)-γ-producing CD4(+) T (Th1) cells with 0.7-2.2 MBq of (64)Cu-PTSM and analyzed cell viability, IFN-γ production, proliferation, apoptosis, and DNA double-strand breaks and identified intracellular (64)Cu accumulation sites by energy dispersive x-ray analysis. To elucidate the fate of Th1 cell homing by PET, 10(7 64)Cu-OVA-Th1 cells were injected intraperitoneally or intravenously into healthy mice. To test the functional capacities of (64)Cu-OVA-Th1 cells during experimental OVA-induced airway hyperreactivity, we injected 10(7 64)Cu-OVA-Th1 cells intraperitoneally into OVA-immunized or nonimmunized healthy mice, which were challenged with OVA peptide or phosphate-buffered saline or remained untreated. In vivo PET investigations were followed by biodistribution, autoradiography, and fluorescence-activated cell sorting analysis. Results PET revealed unexpected homing patterns depending on the mode of T-cell administration. Within 20 min after intraperitoneal administration, (64)Cu-OVA-Th1 cells homed to the perithymic lymph nodes (LNs) of naive mice. Interestingly, intravenously administered (64)Cu-OVA-Th1 cells homed predominantly into the lung and spleen but not into the perithymic LNs. The accumulation of (64)Cu-OVA-Th1 cells in the pulmonary LNs (6.8 ± 1.1 percentage injected dose per cubic centimeter [%ID/cm(3)]) 24 h after injection was highest in the OVA-immunized and OVA-challenged OVA airway hyperreactivity-diseased littermates 24 h after intraperitoneal administration and lowest in the untreated littermates (3.7 ± 0.4 %ID/cm(3)). As expected, (64)Cu-OVA-Th1 cells also accumulated significantly in the pulmonary LNs of nonimmunized OVA-challenged animals (6.1 ± 0.5 %ID/cm(3)) when compared with phosphate-buffered saline-challenged animals (4.6 ± 0.5 %ID/cm(3)). Conclusion Our protocol permits the detection of Th1 cells in single LNs and enables temporal in vivo monitoring of T-cell homing over 48 h. This work enables future applications for (64)Cu-PTSM-labeled T cells in clinical trials and novel therapy concepts focusing on T-cell-based immunotherapies of autoimmune diseases or cancer.

The SRY-related HMG-box family of transcription factors member SOX2 regulates stemness and pluripotency in embryonic stem cells and plays important roles during early embryogenesis. More recently, SOX2 expression was documented in several tumor types including ovarian carcinoma, suggesting an involvement of SOX2 in regulation of cancer stem cells (CSC). Intriguingly, however, studies exploring the predictive value of SOX2 protein expression with respect to histopathologic and clinical parameters report contradictory results in individual tumors, indicating that SOX2 may play tumor-specific roles. In this report, we analyze the functional relevance of SOX2 expression in human ovarian carcinoma. We report that in human serous ovarian carcinoma (SOC) cells, SOX2 expression increases the expression of CSC markers, the potential to form tumor spheres, and the in vivo tumor-initiating capacity, while leaving cellular proliferation unaltered. Moreover, SOX2-expressing cells display enhanced apoptosis resistance in response to conventional chemotherapies and TRAIL. Hence, our data show that SOX2 associates with stem cell state in ovarian carcinoma and induction of SOX2 imposes CSC properties on SOC cells. We propose the existence of SOX2-expressing ovarian CSCs as a mechanism of tumor aggressiveness and therapy resistance in human SOC. Cancer Res; 73(17); 5544–55. ©2013 AACR.

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease caused by the fungus Aspergillus fumigatus, and is a leading cause of invasive fungal infection-related mortality and morbidity in patients with hematological malignancies and bone marrow transplants. We developed and tested a novel probe for noninvasive detection of A. fumigatus lung infection based on antibody-guided positron emission tomography and magnetic resonance (immunoPET/MR) imaging.Administration of a [64Cu]DOTA-labeled A. fumigatus-specific monoclonal antibody (mAb), JF5, to neutrophil-depleted A. fumigatus-infected mice allowed specific localization of lung infection when combined with PET. Optical imaging with a fluorochrome-labeled version of the mAb showed colocalization with invasive hyphae. The mAb-based newly developed PET tracer [64Cu]DOTA-JF5 distinguishedIPA from bacterial lung infections and, in contrast to [18F]FDG-PET, discriminated IPA from a general increase in metabolic activity associated with lung inflammation. To our knowledge, this is the first time that antibody-guided in vivo imaging has been used for non-invasive diagnosis of a fungal lung disease (IPA) of humans, an approach with enormous potential for diagnosis of infectious diseases and with potential for clinical translation.

Most frequently, gram-negative bacterial infections in humans are caused by Enterobacteriaceae and remain a major challenge in medical diagnostics. We non-invasively imaged moderate and severe systemic Yersinia enterocolitica infections in mice using the positron emission tomography (PET) tracer 3’-deoxy-3’-[18F]fluorothymidine ([18F]FLT), which is a marker of proliferation, and compared the in vivo results to the ex vivo biodistributions, bacterial loads, and histologies of the corresponding organs. Y. enterocolitica infection is detectable with histology using H&E staining and immunohistochemistry for Ki 67. [18F]FLT revealed only background uptake in the spleen, which is the main manifestation site of systemic Y. enterocolitica-infected mice. The uptake was independent of the infection dose. Antibody-based thymidine kinase 1 (Tk-1) staining confirmed the negative [18F]FLT-PET data. Histological alterations of spleen tissue, observed via Ki 67-antibody-based staining, can not be detected by [18F]FLT-PET in this model. Thus, the proliferation marker [18F]FLT is not a suitable tracer for the diagnosis of systemic Y. enterocolitica infection in the C57BL/6 animal model of yersiniosis.

Here, we describe the efficacy of the novel small molecule c-Met inhibitor BAY 853474 in reducing tumor growth in the Hs746T gastric cancer xenograft model and tested the suitability of 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) versus 3'-deoxy-3'-18F-fluorothymidine ([(18)F]FLT) for response monitoring in a gastric cancer xenograft mouse model using small animal PET.The c-Met inhibitor or vehicle control was administered orally at various doses in tumor-bearing mice. Glucose uptake and proliferation was measured using PET before, 48 and 96 h after the first treatment. The PET data were compared to data from tumor growth curves, autoradiography, Glut-1 and Ki-67 staining of tumor sections, and biochemical analysis of tissue probes, i.e., c-Met and ERK phosphorylation and cyclin D1 levels.BAY 853474 significantly reduces tumor growth. [(18)F]FDG uptake in Hs746T tumors was significantly reduced in the groups receiving the drug, compared with the control group. The [(18)F]FLT uptake in the tumor tissue was completely absent 96 h after treatment. Autoradiographic, immunohistochemical, and biochemical analyses confirmed the PET findings. Treatment with the c-Met inhibitor did not affect body weight or glucose levels, and no adverse effects were observed in the animals.These preclinical findings suggest that clinical PET imaging is a useful tool for early response monitoring in clinical studies.

Intracerebral experimental gliomas attract intravenously injected murine or human bone marrow–derived hematopoietic progenitor and stem cells (HPC) in vitro, ex vivo, and in vivo, indicating that these progenitor cells might be suitable vehicles for a cell-based delivery of therapeutic molecules to malignant gliomas. With regard to therapeutic application, it is important to investigate cell fates in vivo (i.e., the time-dependent intratumoral and systemic distribution after intravenously injection). Conventional histological analysis has limitations in this regard because longitudinal monitoring is precluded. Here, we used 2-photon laser scanning microscopy (2PLSM), positron emission tomography (PET), and MRI to study the fate of intravenously injected HPC carrying fluorescence, bioluminescence, and PET reporter genes in glioma-bearing mice. Our 2PLSM-based monitoring studies revealed that HPC homing to intracerebral experimental gliomas occurred already within the first 6 h and was most efficient within the first 24 h after intravenous injection. The highest PET signals were detected in intracerebral gliomas, whereas the tracer uptake in other organs, notably spleen, lung, liver, and muscle, remained at background levels. The results have important implications for designing schedules for therapeutic cell-based anti-glioma approaches. Moreover, the PET reporter-based imaging technique will allow noninvasive monitoring of cell fate in future cell-based therapeutic antiglioma approaches.

// Valerie S. Honndorf 1 , Stefan Wiehr 1 , Anna-Maria Rolle 1 , Julia Schmitt 1 , Luisa Kreft 1 , Letitia Quintanilla-Martinez 2 , Ursula Kohlhofer 2 , Gerald Reischl 1 , Andreas Maurer 1 , Karsten Boldt 3 , Michael Schwarz 4 , Holger Schmidt 5 , Bernd J. Pichler 1 1 Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Eberhard Karls University, Tuebingen, Germany 2 Institute of Pathology, University Hospital, Eberhard Karls University, Tuebingen, Germany 3 Medical Proteome Center, Institute for Ophthalmic Research, Eberhard Karls University, Tuebingen, Germany 4 Institute of Experimental and Clinical Pharmacology and Toxicology, Department of Toxicology, Eberhard Karls University, Tuebingen, Germany 5 Department of Radiology, Diagnostic and Interventional Radiology, Eberhard Karls University, Tuebingen, Germany Correspondence to: Valerie S. Honndorf, email: Valerie.Schmidt-Honndorf@med.uni-tuebingen.de Keywords: PET, genistein and cetuximab, A431 and Colo205 xenografts, molecular tumor imaging Received: December 21, 2015 Accepted: February 28, 2016 Published: April 07, 2016 ABSTRACT The natural phytoestrogen genistein is known as protein kinase inhibitor and tumor suppressor in various types of cancers. We studied its antitumor effect in two different xenograft models using positron emission tomography (PET) in vivo combined with ex vivo histology and nuclear magnetic resonance (NMR) metabolic fingerprinting. Procedures: A431 and Colo205 tumor-bearing mice were treated with vehicle or genistein (500 mg/kg/d) over a period of 12 days. Imaging was performed with 2-deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F]FDG) and 3′-deoxy-3′-[ 18 F]fluorothymidine ([ 18 F] FLT). In a second study A431 tumor-bearing mice were treated with vehicle, genistein (500 mg/kg/d), cetuximab (1 mg/3d) or a combination of the compounds and imaged using [ 18 F]FDG, [ 18 F]FLT and [ 64 Cu]NODAGA-cetuximab. Data were compared to histology and principal components analysis (PCA) of NMR fingerprinting data. Results: Genistein reduced tumor growth significantly in both xenografts. [ 18 F] FLT uptake was consistent in both models and corresponded to histological findings and also PCA whereas [ 18 F]FDG and [ 64 Cu]NODAGA-cetuximab were not suitable for therapy monitoring. Conclusions: As mono-therapy the natural isoflavone genistein has a powerful therapeutic effect in vivo on A431 and Colo205 tumors. [ 18 F]FLT has superior consistency compared to the other tested tracers in therapy monitoring, while the treatment effect could be shown on the molecular level by histology and metabolic fingerprinting.

UNLABELLED The pharmacokinetics of (18)F-fluorodeoxythymidine (FLT), (18)F-FDG, (11)C-choline, and (18)F-fluoroethylcholine (FEC) in 2 hormone-independent (PC-3, DU145) and 2 hormone-dependent (CWR22, PAC120) prostate cancer xenograft mouse models were evaluated by PET and compared by immunohistochemistry. Further investigation was performed to determine whether PET can detect early changes in tumor metabolism after androgen ablation therapy through surgical castration. METHODS PET was performed on 4 consecutive days. In addition, the CWR22 and PAC120 tumor models were surgically castrated after the baseline measurement and imaged again after castration. The tracer uptake was analyzed using time-activity curves, percentage injected dose per volume (%ID/cm(3)), and tumor-to-muscle ratio (T/M). RESULTS Regarding the hormone-independent prostate tumor models, (18)F-FLT showed the best T/M and highest %ID/cm(3) in PC-3 (2.97 ± 0.63 %ID/cm(3)) and DU145 (2.06 ± 0.75 %ID/cm(3)) tumors. (18)F-FDG seemed to be the tracer of choice for delineation of the PC-3 tumors but not for the DU145 tumors. Using (11)C-choline (PC-3: 1.33 ± 0.29 %ID/cm(3), DU145: 1.60 ± 0.27 %ID/cm(3)) and (18)F-FEC, we did not find any significant uptake in the tumors, compared with muscle tissue. Regarding the hormone-dependent prostate tumor models, the CWR22 model showed a highly significant (P < 0.01) decrease in tumor (18)F-FDG uptake from 4.11 ± 1.29 %ID/cm(3) to 2.19 ± 1.45 %ID/cm(3) after androgen ablation therapy. However, the (18)F-FLT, (11)C-choline, or (18)F-FEC tracers did not provide sufficient uptake or reliable information about therapy response in CWR22 tumors. The PAC120 model showed a significant increase in (18)F-FLT tumor uptake (P = 0.015) after androgen ablation therapy. The accumulation of (18)F-FEC (before: 2.32 ± 1.01 %ID/cm(3), after: 1.36 ± 0.39 %ID/cm(3)) was found to be the next highest after (18)F-FDG (before: 2.45 ± 0.93 %ID/cm(3), after: 2.18 ± 0.65 %ID/cm(3)) in PAC120 tumors before castration and is better suited for monitoring therapy response. CONCLUSION This comprehensive study in 2 hormone-dependent and 2 hormone-independent prostate tumor mouse models shows that (18)F-FLT and (18)F-FDG are the most appropriate tracers for delineation of PC-3, DU145 (except (18)F-FDG), and CWR22 tumors, but not for PAC120 tumors. (18)F-FEC and (11)C-choline, in particular, revealed insufficient T/M ratio in the prostate tumor models. The results may indicate that radiolabeled choline and choline derivatives compete with a high concentration of the precursor dimethylaminoethanol, resulting in reduced uptake in small-rodent tumor models, a hypothesis that is currently under investigation in our laboratory.

The cytostatic drugs Vincristine (VCR), Navelbine (NAV), and Methotrexate (MTX) were evaluated for their growth inhibitory potential against metacestodes of Echinococcus multilocularis (Em) by in vitro and in vivo assays. In vitro cultures of E. multilocularis were exposed to IC 90, IC 80, and IC 5 concentrations of VCR, NAV, or MTX for 1 week, then parasite tissue cultures were kept for 1 week without drug exposure in vitro, and thereafter, metacestode tissues were injected intra-peritoneally into Meriones unguiculatus. Metacestode growth was monitored for several months post-infection (p.i.) by body weight control, magnetic resonance imaging (MRI), and autopsy at 5 months p.i. Weight monitoring of infected M. unguiculatus did not provide conclusive evidence for Em-metacestode growth, while MRI could detect growing Em-metacestode in the MTX-treated group at 8 weeks (p.i.), whereas metacestodes exposed to VCR and NAV were at 17 weeks (p.i.) detectable. MRI disclosed progressive and massive growth of Em-metacestode in the VCR- and MTX-exposed groups, while the NAV-pretreated Em-metacestodes' volume did not exceed 4 cm(3). At autopsy, Em-metacestodes of less than 4 cm(3) were found in infected M. unguiculatus, which was not detected by MRI. In summary, the cytostatic drugs Methotrexate, Navelbine, and Vincristine--as applied in the present work--did not show parasitocidal or clear parasitostatic effects on metacestodes of E. multilocularis. While parasite growth in vivo was inhibited in NAV- and VCR-pretreated Em-metacestodes, MTX pretreatment seemed to enhance parasite proliferation. Magnetic resonance imaging appears suitable to monitor in vivo the effects of drugs on growth progression and regression only of larger Em-metacestode tissues.

Positron emission tomography (PET) and diffusion-weighted MRI (DW-MRI) were used to characterize the treatment effects of the MEK1/2 inhibitor selumetinib (AZD6244), docetaxel, and their combination in HCT116 tumor-bearing mice on the molecular level.Mice were treated with vehicle, selumetinib (25 mg/kg), docetaxel (15 mg/kg), or a combination of both drugs for 7 days and imaged at four time points with 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) or 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) followed by DW-MRI to calculate the apparent diffusion coefficient (ADC). Data was cross-validated using the Pearson correlation coefficient (PCC) and compared to histology (IHC).Each drug led to tumor growth inhibition but their combination resulted in regression. Separate analysis of PET or ADC could not provide significant differences between groups. Only PCC combined with IHC analysis revealed the highest therapeutic impact for combination therapy.Combination treatment of selumetinib/docetaxel was superior to the respective mono-therapies shown by PCC of PET and ADC in conjunction with histology.

Especially for neuroscience and the development of new biomarkers, a direct correlation between in vivo imaging and histology is essential. However, this comparison is hampered by deformation and shrinkage of tissue samples caused by fixation, dehydration and paraffin embedding. We used magnetic resonance (MR) imaging and computed tomography (CT) imaging to analyze the degree of shrinkage on murine brains for various fixatives. After in vivo imaging using 7 T MRI, animals were sacrificed and the brains were dissected and immediately placed in different fixatives, respectively: zinc-based fixative, neutral buffered formalin (NBF), paraformaldehyde (PFA), Bouin-Holland fixative and paraformaldehyde-lysine-periodate (PLP). The degree of shrinkage based on mouse brain volumes, radiodensity in Hounsfield units (HU), as well as non-linear deformations were obtained. The highest degree of shrinkage was observed for PLP (68.1%, P0.001), followed by PFA (60.2%, P0.001) and NBF (58.6%, P0.001). The zinc-based fixative revealed a low shrinkage with only 33.5% (P0.001). Compared to NBF, the zinc-based fixative shows a slightly higher degree of deformations, but is still more homogenous than PFA. Tissue shrinkage can be monitored non-invasively with CT and MR. Zinc-based fixative causes the smallest degree of brain shrinkage and only small deformations and is therefore recommended for in vivo ex vivo comparison studies.

We aimed to quantitatively characterize the treatment effects of docetaxel in the HCT116 xenograft mouse model, applying diffusion-weighted magnetic resonance imaging (MRI) and positron emission tomography (PET) using 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]FDG) and 3'-deoxy-3'-[¹⁸F]-fluorothymidine ([¹⁸F]FLT). Mice were imaged at four time points over 8 days. Docetaxel (15 mg/kg) was administered after a baseline scan. Voxel-wise scatterplots of PET and apparent diffusion coefficient (ADC) data of tumor volumes were evaluated with a threshold cluster analysis and compared to histology (GLUT1, GLUT3, Ki67, activated caspase 3a). Compared to the extensive tumor growth observed in the vehicle-treated group (from 0.32 ± 0.21 cm³ to 0.69 ± 0.40 cm³), the administration of docetaxel led to tumor growth stasis (from 0.32 ± 0.20 cm³ to 0.45 ± 0.23 cm³). The [¹⁸F]FDG/ADC cluster analysis and the evaluation of peak histogram values revealed a significant treatment effect matching histology as opposed to [¹⁸F]FLT/ADC. [¹⁸F]FLT uptake and the Ki67 index were not in good agreement. Our voxel-based cluster analysis uncovered treatment effects not seen in the separate inspection of PET and MRI data and may be used as an independent analysis tool. [¹⁸F]FLT/ADC cluster analysis could still point out the treatment effect; however, [¹⁸F]FDG/ADC reflected the histology findings in higher agreement.

Preclinical studies involving (89)Zr often report significant bone accumulation, which is associated with dissociation of the radiometal from the tracer. However, experiments determining the uptake of unbound (89)Zr in disease models are not performed as routine controls. The purpose of the present study was to investigate the impact of free or weakly bound (89)Zr on PET quantifications in disease models, in order to determine if such control experiments are warranted.Chemical studies were carried out to find a (89)Zr compound that would solubilize the (89)Zr as a weak chelate, thus mimicking free or weakly bound (89)Zr released in circulation. (89)Zr oxalate had the desired characteristics, and was injected into mice bearing FaDu and HT29 solid tumor xenografts, and mice infected in the lungs with the mold Aspergillus fumigatus, as well as in healthy controls (naïve). PET/CT or PET/MR imaging followed to quantify the distribution of the radionuclide in the disease models.(89)Zr oxalate was found to have a plasma half-life of 5.1 ± 2.3 h, accumulating mainly in the bones of all animals. Both tumor types accumulated (89)Zr on the order of 2-4 %ID/cm(3), which is comparable to EPR-mediated accumulation of certain species. In the aspergillosis model, the concentration of (89)Zr in lung tissue of the naïve animals was 6.0 ± 1.1 %ID/g. This was significantly different from that of the animals with advanced disease, showing 11.6 ± 1.8 %ID/g.Given the high levels of (89)Zr accumulation in the disease sites in the present study, we recommend control experiments mapping the biodistribution of free (89)Zr in any preclinical study employing (89)Zr where bone uptake is observed. Aqueous (89)Zr oxalate appears to be a suitable compound for such studies. This is especially relevant in studies where the tracer accumulation is based upon passive targeting, such as EPR.

Combined PET and MR imaging (PET/MR imaging) has progressed tremendously in recent years. The focus of current research has shifted from technologic challenges to the application of this new multimodal imaging technology in the areas of oncology, cardiology, neurology, and infectious diseases. This article reviews studies in preclinical and clinical translation. The common theme of these initial results is the complementary nature of combined PET/MR imaging that often provides additional insights into biologic systems that were not clearly feasible with just one modality alone. However, in vivo findings require ex vivo validation. Combined PET/MR imaging also triggers a multitude of new developments in image analysis that are aimed at merging and using multimodal information that ranges from better tumor characterization to analysis of metabolic brain networks. The combination of connectomics information that maps brain networks derived from multiparametric MR data with metabolic information from PET can even lead to the formation of a new research field that we would call cometomics that would map functional and metabolic brain networks. These new methodologic developments also call for more multidisciplinarity in the field of molecular imaging, in which close interaction and training among clinicians and a variety of scientists is needed.

In this study, simultaneous positron emission tomography (PET)/magnetic resonance (MR) imaging was employed to evaluate the feasibility of the PET tracers 2-deoxy-2- 18 F-fluoro-D-glucose ( 18 F-FDG), 11 C-choline, and 18 F-fluorothymidine ( 18 F-FLT) to detect papillomavirus-induced tumors in an established rabbit model system. The combined PET/MR allowed the analysis of tracer uptake of the tumors using the morphologic information acquired by MR. New Zealand White rabbits were infected with cottontail rabbit papillomavirus genomes and were imaged for up to 10 months with a simultaneous PET/MR system during the course of infection. The uptake characteristics of the PET tracers 11 C-choline and 18 F-FLT of tumors and reference tissues were examined relative to the clinical standard, 18 F-FDG. Tracer biodistribution of various organs was measured by gamma-counting after the last PET scan and compared to the in vivo PET/MR 18 F-FDG uptake. Increased tracer uptake was found 2 months postinfection in primary tumors with 18 F-FDG and 11 C-choline, whereas 18 F-FLT failed to detect the tumors at all measured time points. Our data show that the PET tracer 18 F-FDG is superior for imaging papillomavirus-induced tumors in rabbits compared to 11 C-choline and 18 F-FLT. However, 11 C-choline imaging, which has previously been applied to detect various tumor entities in patients, appears to be an alternative to 18 F-FDG.

The dynamics of β-amyloid deposition and related second-order physiological effects, such as regional cerebral blood flow (rCBF), are key factors for a deeper understanding of Alzheimer's disease (AD). We present longitudinal in vivo data on the dynamics of β-amyloid deposition and the decline of rCBF in two different amyloid precursor protein (APP) transgenic mouse models of AD. Using a multiparametric positron emission tomography and magnetic resonance imaging approach, we demonstrate that in the presence of cerebral β-amyloid angiopathy (CAA), β-amyloid deposition is accompanied by a decline of rCBF. Loss of perfusion correlates with the growth of β-amyloid plaque burden but is not related to the number of CAA-induced microhemorrhages. However, in a mouse model of parenchymal β-amyloidosis and negligible CAA, rCBF is unchanged. Because synaptically driven spontaneous network activity is similar in both transgenic mouse strains, we conclude that the disease-related decline of rCBF is caused by CAA.

Radioimmunoimaging with disease-specific tracers can be advantageous compared to that with nonspecific tracers for the imaging of glucose metabolism and cell proliferation. Monoclonal antibodies (mAbs) or their fragments are excellent tools for immuno-positron emission tomography (PET). In this study, PSMA-specific mAb 3/F11 and its recombinant fragments were compared for the imaging of prostate cancer in xenografts.Recombinant anti-PSMA antibody fragments D7-Fc and D7-CH3 were constructed by genetically fusing the binding domains of mAb 3/F11 (D7) to the human IgG3 CH3 or CH2-CH3 (Fc) domain. The fragments and the mAb 3/F11 were DOTA conjugated, tested in vitro, and radiolabeled with (64) Cu. PSMA-positive C4-2 and PSMA-negative DU 145 prostate cancer xenografts were used for PET-MR imaging and for ex vivo biodistribution.The constructs showed strong and specific binding to PSMA-positive C4-2 cells in vitro which did not decrease after DOTA conjugation. Both tested fragments showed stable accumulation in PSMA-positive C4-2 tumors at all measured time points but reduced uptake compared to the full-length antibody. Other organs and PSMA-negative tumors showed a very low tracer uptake only 3 hr after injection, with the exception of the kidneys, which demonstrated high radioactivity uptake due to rapid renal clearance of the mAb fragments.Stable tumor uptake and fast serum clearance of the tested radiolabeled fragments was observed in this preclinical study compared to the full length mAb. Since the fragments show rapid and specific tumor uptake, the tested fragments might serve as tools for theranostic imaging with suitable isotopes for radioimmunotherapy.

The strength of nuclear imaging is based on its ability to reveal quantitative information. Especially positron emission tomography (PET) offers, as long as all corrections, such as for photon attenuation, scatter and partial volume effect are applied, highly quantitative data. This chapter describes the different pitfalls in PET image analysis and necessary correction methods to yield quantitative images. Furthermore, it provides practical recipes for quantitative image analysis.

BACKGROUND The prostate specific membrane antigen (PSMA) is expressed by virtually all prostate cancers and represents an ideal target for diagnostic and therapeutic strategies. This article compares the in vivo behavior and tumor uptake of three different radiolabeled anti-PSMA monoclonal antibodies (mAbs) and corresponding F(ab)2 and Fab fragments thereof. METHODS The mAbs 3/A12, 3/F11, and 3/E7 and fragments of 3/A12 were conjugated with the chelating agent DOTA and radiolabeled with 64Cu. For the microPET imaging studies, SCID mice bearing PSMA-positive C4-2 and PSMA-negative DU 145 prostate cancer xenografts were used. Each animal received 20–30 µg radiolabeled mAb or fragment corresponding to an activity of 8–14 MBq. Imaging was performed 3, 24, and 48 hr post-injection. After the last scan, mice were sacrificed and tracer in vivo biodistribution was measured by gamma-counting. RESULTS Static microPET images of mice with PSMA-positive tumors revealed a high uptake of the mAbs in the C4-2 tumors at 24 and 48 hr after tracer injection and only a minimal distribution in the DU 145 tumors and other organs. In contrast, the F(ab)2 and Fab fragments of 3/A12 were detected at a high extend in the kidney but not in the C4-2 tumors. These results were confirmed by gamma counting of dissected organs after the final imaging. CONCLUSIONS Due to the high and specific uptake of the 64Cu-labeled mAbs in PSMA-positive tumors, these antibodies represent excellent tools for prostate cancer imaging. Prostate 70: 1413–1421, 2010. © 2010 Wiley-Liss, Inc.

Background/Aims: Tumor dissemination is frequent in gastric cancer and implies a poor prognosis. Cure is only achievable provided an accurate staging is performed at primary diagnosis. In previous studies we were able to show a relevant impact of increased phosphoglycerate kinase 1 expression (PGK1; a glycolytic enzyme) on invasive properties of gastric cancer in-vivo and in-vitro. Thus the aim of the present study was to evaluate the effect of enhanced PGK1 expression in gastric cancer employing magnetic resonance (MR)-imaging combined with positron emission tomography (PET), a recently emerging new high resolution imaging technique in a mouse model. Methods: A metastatic nude mouse model simulating human gastric cancer behavior by orthotopic tumor implantation was established. Mice were divided into one control group (n=5) and two experimental groups (n=30) divided by half in animals baring tumors from MKN45-cells and MKN45-cells with plasmid-mediated overexpression of PGK1. In the course of tumor growth MR-imaging and PET/MRI fusion was performed. Successively experimental animals were examined macroscopically and histopathologically regarding growth, metastasis and PGK1 expression. Results: Elevated PGK1 expression increased invasive and metastatic behavior of implanted gastric tumors significantly. MR/PET- imaging results in-vivoand subsequent ex-vivo findings concerning tumor growth and metastasis correlated excellently and could be underlined by concordant immuohistochemical PGK1 staining. Conclusion: Consistent in-vivo findings suggest that PGK1 might be crucially involved in gastric malignancy regarding growth and metastasis, which was also underlined by novel imaging techniques. Thus, PGK1 may be exploited as a prognostic marker and/or be of potential therapeutic value preventing malignant dissemination.