EMIM 2018 ControlCenter

Online Program Overview Session: PW-27

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Develop: New Tools for Cancer Imaging II

Session chair: Adam Schuhendler - Ottawa, Canada; Jeannine Missbach-Güntner - Göttingen, Germany
 
Shortcut: PW-27
Date: Friday, 23 March, 2018, 11:30 AM
Room: Banquet Hall | level -1
Session type: Poster Session

Abstract

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# 233

Radiosynthesis and in vivo evaluation of [18F] (S)-2-amino-4-((2-((3-fluorobenzyl)oxy)benzyl)(2-((3-(fluoromethyl)benzyl)oxy)benzyl)amino)butanoic acid, a new ASCT-2 inhibitor. (#559)

T. L. Baguet1, S. De Lombaerde1, J. Verhoeven1, B. Descamps2, I. Goethals3, C. Vanhove2, F. De Vos1

1 Ghent University, Laboratory of Radiopharmacy, Gent, Belgium
2 Ghent University, Institute Biomedical Technology - Medisip, Infinity, Gent, Belgium
3 Ghent University, Nuclear Medicine, Gent, Belgium

Introduction

Cancer is characterized by upregulation of nutrient transporters such as GLUT-1, LAT-1 and ASCT-2. [18F]-FDG is the golden standard in oncology metabolism imaging, however this tracer shows sub-optimal characteristics in several tumors (e.g. brain-, prostate-, colorectalcancer). In a search to address these drawbacks, a 18F labeled inhibitor ([18F] (S)-2-amino-4-((2-((3-fluorobenzyl)oxy)benzyl)(2-((3-(fluoromethyl)benzyl)oxy)benzyl)amino)butanoic acid) of ASCT-2 was developed.

Methods

In a first section of this project, the radiochemical labelling with 18F was optimized. Therefore, labelling yield was tested in function of temperature, mass of the precursor, and the solvent. In a second section the tracer was injected in swiss nu/nu mice which were inoculated with PC-3 cells (5 x 106 cells). The tracer was synthesized by reacting 4mg precursor with 18F- in 200µL acetonitrile at 120°C for 20 minutes. Deprotection occurred after adding 200µL 4M HCl (4M/dioxane) and reacting 5 minutes at 90°C. Purification was done by means of semi-prep HPLC , the final formulation was a 20% EtOH/80% BPS mixture. A dynamic µPET-scan was acquired for two hours after injection of 18.5 MBq. Tumor uptake was analyzed by drawing volumes of interest around the tumor with PMOD.

Results/Discussion

Altering the solvent to acetonitrile resulted in substantial increase in labelling yield (Fig 1). In the temperature-yield curve an inflection point is seen between 100°C and 120°C, suggesting tis accounts for the Ekin necessary for good labelling yields. Increasing the amount of precursor from 2mg to 8mg did not increase the labelling yield significantly. µPET images shows the tracer has in vivo uptake with clear delineation of the tumor (fig. 2).  The tracer reports an average tumor to background ratio of 1.85. 

Conclusions

With this study we report the first radiolabeled inhibitor of ASCT-2, which shows a good TBR. Further experiments are needed to explore the imaging potential of the ASCT-2 transporter. Optimal labelling conditions are also reported which suggest the labelling should occur in acetonitrile with a temperature of 120°C.

Figure 2: Pet-scan female mouse
Figure 1: Optimization of labelling yield in function of solvent (left); temperature (middle) and ma

Yield in function of solvent was done with 4mg precursor at 120°C for 20 min. Yield in function of temperature was done with 4 mg precursor in acetonitrile for 20 min and yield in function of mass was done in acetonitrile at 120°C for 20 min.

Keywords: ASCT-2; Prostate Cancer; PET
# 234

In vivo Cancer Detection with Magnetic Particle Imaging (#333)

E. Y. Yu1, M. Bishop1, B. Zheng1, R. M. Ferguson2, A. P. Khandhar2, S. J. Kemp2, K. M. Krishnan2, 4, P. W. Goodwill3, S. M. Conolly1, 5

1 University of California, Berkeley, Department of Bioengineering, Berkeley, California, United States of America
2 Lodespin Labs, LLC, Seattle, Washington, United States of America
3 Magnetic Insight, Inc., Alameda, California, United States of America
4 University of Washington, Department of Material Science and Engineering, Seattle, Washington, United States of America
5 University of California, Berkeley, Department of Electrical Engineering and Computer Science, Berkeley, California, United States of America

Introduction

Magnetic Particle Imaging (MPI)  is a high-contrast and quantitative new imaging modality with high tracer sensitivity—even 2 ng of Iron is detectable in an animal [1-3]. MPI also uses no ionizing radiation, and the superparamagnetic iron-oxide (SPIO) tracers are safe even for patients with Chronic Kidney Disease (CKD). Here we demonstrate, for the first time, in vivo cancer imaging with MPI. 

Methods

Two cohorts of athymic nude rats (Groups A & B) were injected with 7 million MDA-MB-231-luc tumor cells subcutaneously and monitored for 4 weeks. The animals were injected with long circulating MPI-tailored SPIOs (LS-008, Lodespin Labs). In Groups A (n=3) and B (n=3), LS-008 was intravenously administered at a dose of 15 mg/kg and 5 mg/kg, respectively. The animals were then scanned at various time-points post-injection. A Field Free Point MPI scanner (Figure 1(a)) was used (Resolution: 1.2 mm, Group A FOV: 4 cm × 4 cm × 8.5 cm (cropped 5.8 cm), Scan Time: 5 minutes; Group B FOV: 4 cm × 4 cm × 14.5 cm, Scan Time: 9 minutes). For anatomical reference, micro-CT (General Electric EVS RS-09) scans were acquired (Resolution: 93μm, FOV: of 4 cm × 4.7 cm × 16.5 cm, Scan Time: 25 minutes).

Results/Discussion

Group A tumors were highlighted with a peak tumor-to-background ratio of roughly 50-to-1 as seen in Figure 1(d). The tumor is well appreciated, with initial wash-in on the tumor rim, peak uptake at 6 hours, and washout beyond 48 hours. The whole body dynamics in Group B rats was captured via MPI over time. As seen in Fig 1(e), the injected SPIOs are first distributed uniformly in the intravascular system, with large blood volume organs such as the heart and lungs and large blood vessels distinguishable. The SPIOs are subsequently cleared from the blood by the RES, highlighting the liver and spleen. Despite the lack of active targeting moieties and the systemic tracer administration, the contrast and sensitivity inherent to MPI allowed for the tumor to simultaneously be clearly visible through time. In addition, MPI enables quantitative analysis of tracer dynamics – blood pool and tumor iron content was successfully fit to a two compartment model as shown in Figure 1(f) [4].

Conclusions

This study shows MPI’s potential for sensitive and quantitative cancer imaging. Bioluminescence imaging requires genetic modification and suffers signal attenuation with depth. In contrast, MPI is quantitative, does not require genetic modifications, and has no signal attenuation with depth. The high sensitivity and superb contrast produce images comparable with other molecular imaging modalities such as PET and SPECT. With continual development, MPI will emerge as a robust imaging platform for evaluating cancer targeting moieties, detecting small metastases, and monitoring cell migration.

References

[1] Gleich, B., & Weizenecker, J. (2005). Nature, 435(7046).

[2] Goodwill, P. W., & Conolly, S. M. (2010). IEEE TMI, 29(11).

[3] Zheng, B., Vazin, T., Goodwill, P. W., Conway, A., Verma, A., Ulku Saritas, E., … Conolly, S. M. (2015). Scientific Reports, 5, 14055.

[4] Yu, E. Y., Bishop, M., Zheng, B., Ferguson, R. M., Khandhar, A. P., Kemp, S. J., Krishnan, K. M., Goodwill, P. W., Conolly, S. M (2017). Nano Letters, 17 (3).

Acknowledgement

We would like to acknowledge funding support from NSF GRFP, NIH R01 EB013689, CIRM RT2-01893, Keck Foundation 009323, NIH 1R24 MH106053, NIH 1R01 EB019458, and ACTG 037829.

In vivo cancer detection with MPI
Figure 1: (a) Field Free Point MPI Imager. Selection field gradient strength: 7 × 3.5 × 3.5 T/m. (b) MPI signal is linear with SPIO concentration. (c)&(d) MPI and corresponding bioluminescence image of a rat implanted with MDA-MB-231-luc and systemically injected with LS-008. (e) MPI images over time with CT overlay. (f) MPI is enables pharmacokinetic compartment modeling of the tracer dynamics.
# 235

Site-specific Antibody Conjugation Improves In Vitro and In Vivo Imaging (#393)

H. Toftevall1, H. Nyhlén1, M. Nordgren1, C. Christensen3, L. K. Kristensen2, 3, C. H. Nielsen2, 3, A. Kjaer3, F. Olsson1

1 Genovis AB, Lund, Sweden
2 Minerva Imaging, Copenhagen, Denmark
3 Rigshospitalet and Univ of Copenhagen, Dept Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Copenhagen, Denmark

Introduction

Conjugation of functional groups to antibodies is an important technique for diagnostics, therapeutics and imaging. Traditional methods rely on unspecific conjugation giving variations in the degree of labeling (DOL) and may impact the conjugate’s affinity and stability. GlyCLICK is a site-specific method based on enzymatic remodeling of the Fc glycans resulting in homogenous conjugates with a DOL=2. Trastuzumab conjugates obtained by random conjugation or site-specific GlyCLICK conjugation were compared by imaging of tumor cells in vitro, and of tumors in vivo using small animal PET/CT.

Methods

Site-specific labeling of trastuzumab were performed using GlyCLICK with DIBO-modified labels. The random labeling was performed with p-SCN-Bn-DFO. DFO-conjugates were incubated with 89Zr-oxalate. Random conjugations of fluorophores to mAb were carried out using kits according to manufacturer’s protocol. The DOL of the conjugates were determined by LC-MS and by spectrophotometry. The affinity was assessed by SPR analysis. In vitro imaging was performed by fluorescence microscopy and in vivo experiment was carried out using 89Zr-DFO-trastuzumab (GlyCLICK or random). 89Zr-DFO-trastuzumab was injected into tumor bearing mice. At five times, 4-168h post injection the mice underwent PET/CT imaging. After the last time-point the mice were euthanized, organs were resected, weighed and counted.

Results/Discussion

MS analysis of DFO-GlyCLICK-trastuzumab showed DOL=2 verified by a complete shift in mass of the intact conjugate as well as a shift only of the Fc. Randomly conjugated DFO-trastuzumab showed a mean DOL=3 with a distribution 0-6, with conjugation on the Fc, Fd and LC. Binding studies of GlyCLICK conjugates displayed preserved antigen binding compared to naked antibody due to the specific conjugation on Fc whereas the random conjugate displayed a reduced binding. The conjugates were compared by in vitro imaging using fixed settings on the microscope. This revealed the strongest signal when using the GlyCLICK AlexaFluor488 conjugated antibody DOL=2, compared to using conjugates with higher DOL indicating that the seen effect is due to differences in antigen binding. Finally, the random and site-specific 89Zr-DFO-trastuzumab were compared for imaging in vivo. A five-fold higher tumor uptake and a significantly longer circulation time was observed using the site-specific conjugated tracer.

Conclusions

The use of homogenous conjugates obtained by GlyCLICK technology improves the performance for in vitro and in vivo imaging applications. Preservation of the antigen binding sites and a quantitative conjugation are advantageous properties for dosing the antibody conjugate for optimal imaging. The site-specific conjugation technology enables attachment of different imaging modalities to the antibody with preserved immunoreactivity and can be utilized for various applications in imaging involved in cancer research.

Keywords: in vivo imaging, in vitro imaging, site-specific conjugation, antibody conjugation, PET/CT
# 236

Imaging of cancer cells death by magnetic hyperthermia, in vitro and in vivo (#249)

P. Jeanjean1, C. Genevois1, O. Sandre2, S. Mornet3, F. Couillaud1

1 Bordeaux University, Molecular Imaging and Innovative Therapies in Oncology (IMOTION) - EA 7435, Bordeaux, France
2 UMR 5629 / Bordeaux University / CNRS / Bordeaux-INP, Chemistry of Organic Polymers Laboratory (LCPO), Pessac, France
3 UMR 9048 / CNRS / Bordeaux University, Institute for Condensed Matter Chelmistry of Bordeaux (ICMCB), Pessac, France

Introduction

Thermotherapies use heat as a therapeutic tool. Magnetic nanoparticles (MNPs) placed in an Alternative Magnetic Field (AMF) induce local hyperthermia that is currently proposed for cancer treatment 1; 2. They can be used for local drug delivery or to induce cancer cell death. The principal aim of those therapies is to increase the temperature in the tumor without side effects in the surrounding tissues. Our project aims to internalize a high MNPs concentration into cells, and then, to induce cancer cell death by local magnetic hyperthermia, in vitro and in vivo.

Methods

MNPs are composed of clusters of iron oxide particles stabilized by a thin silica layer within red and near infrared dyes (fig 1). MNPs were used alone or surrounded by protamine3.

For in vitro studies, mouse prostate cancer (RM1) and human glioblastoma (U87) cells3 modified for luciferase firefly (LucF) expression were used. For in vivo studies, cells were first loaded with MNPs, then implanted in mice by subcutaneous injection. AMF were generated using an in vivo setup working at 4 different frequencies (DM3, Nanoscale Biomagnetics, Zaragoza, Spain). During AMF application, temperature was monitored using thermal optical probes (in vitro) or infrared camera (in vivo). Cells viability was followed by bioluminescence imaging3 (BLI) and MNPs internalization by fluorescence imaging (fig 2).

Results/Discussion

MNPs in solution were characterized for temperature increase at different frequencies and magnetic fields. Heating properties were characterized for each couple of frequency / magnetic field at different exposition times. BLI revealed that cell viability was affected by AMF alone. Toxicity and internalization levels were studied with naked MNPs or protamine-coated MNPs. Protamine was not toxic for U87 and RM1 cancer cells and significantly improved MNPs internalization. Incubation time did not influence internalization levels.  Both the in vitro and in vivo studies showed that the viability of MNPs-loaded cells, measured by BLI, decreased when they were subjected to AMF.  Cell death level increased when the MNPs concentrations into cells increased as well.

Conclusions

Results showed that MNPs added in cell culture medium were internalized into cells and did not induce any toxic effect on both U87 and RM1 cell lines. Coating MNPs with protamine improved cell loading. MNPs loaded into cells increased cell death both in vitro and in vivo when subjected to AMF hyperthermia.

References

 

  1. B. Sanz and al. Magnetic hyperthermia enhances cell toxicity with respect to exogenous heating. Biomaterials 114 (2017) 62-70
  2. C. Sanchez. Targeting a G-protein-coupled receptor overexpressed in endocrine tumors by magnetic nanoparticles to induce cell death. ACS Nano vol.8, no.2, 1350-1363 (2014).
  3. L. Adumeau. Impact of surface grafting density of PEG macromolecules on dually fluorescent silica nanoparticles used for the in vivo imaging of subcutaneous tumors. Biochimica et Biophysica Acta 1861 (2017) 1587 – 1596.
  4. H. Xia et al. Low molecular weight protamine-functionalized nanoparticles for drug delivery to the brain after intranasal administration. Biomaterials 32 (2011) 9888 -9898.

Acknowledgement

This work was supported in part by public Labex TRAIL (ANR-10-LABX-57) and in part by France Life Imaging (ANR-11-INBS-006). European COST RADIOMAG (Multifunctional Nanoparticles for Magnetic Hyperthermia and Indirect Radiation Therapy) network is acknowledged. Authors are grateful to Laetitia Medan from Bordeaux University animal’s facilities for the animals’ care.

Figure 1: MNPs picture by transmission electron microscopy
Clusters of iron oxide particles are stabilized by a thin silica layer.
Figure 2: Albinos C57bl6 mice implanted with RM1 cells.
RM1 cells were genetically modified for constitutive luciferase firefly expression. Mice bearing RM1 tumors received an intraperitoneal injection of luciferin (luciferase substrate) to measure the tumor size (A). The MNPs were injected and their emitted fluorescence was observed with Lumina apparatus (B).
Keywords: iron oxide nanoparticles, magnetic hyperthermia, cancer cell death, in vitro and in vivo studies, bioluminescence imaging
# 237

Raster-Scan Optoacoustic Mesoscopy furthers the mechanistic understanding of Vascular Targeted Photodynamic Therapy with WST-11 (#318)

K. Haedicke1, L. Agemy2, M. Omar3, 4, A. Berezhnoi3, 4, J. Coleman5, V. Ntziachristos3, 4, A. Scherz2, J. Grimm1, 6, 7

1 Memorial Sloan Kettering Cancer Center, Molecular Pharmacology Program, New York, New York, United States of America
2 Weizmann Institute of Science, Department of Plant Sciences, Rehovot, Israel
3 Technical University Munich, Chair for Biological Imaging, Munich, Germany
4 Helmholtz Zentrum Munich, Institute for Biological and Medical Imaging, Neuherberg, Germany
5 Memorial Sloan Kettering Cancer Center, Department of Surgery and Department of Urology, New York, New York, United States of America
6 Memorial Sloan Kettering Cancer Center, Department of Radiology, New York, New York, United States of America
7 Weill Cornell Medical College, Pharmacology Program and Department of Radiology, New York, New York, United States of America

Introduction

The tumor vascularization is the route for cancer proliferation. Thus, it is of great interest to treat the tumor by directly targeting vessels and monitor the tumor vascularization to evaluate treatment outcome. Vascular targeted photodynamic therapy (VTP) with WST-11 is an anti-angiogenic approach which occludes tumor vessels by the generation of reactive oxygen species [1,2,3]. Monitoring the efficacy, however, was so far only possible at low resolution or in a small field of view. We used the modality raster-scan optoacoustic mesoscopy (RSOM) [4] to better understand the mechanism of VTP.

Methods

Mice with CT26 tumors were treated with VTP by infusing WST-11 for 5 min and immediately illuminating the tumor with a 753 nm diode laser (120 mW/cm2) for 10 min. To investigate the role of NO species, one group was additionally injected with the NO scavenger hydroxocobalamin (HCA). Before and after VTP, the tumor area was imaged using RSOM. The mouse was placed into a warm water bath and the tumor illuminated in a 20 µm step-size raster with a nanosecond-pulsed monochromatic 532 nm laser. For the distinction of oxy- and deoxyhemoglobin, a second 515 nm laser was added. Generated ultrasound signals were detected with a spherically focused 50 MHz detector. Ultrasound frequencies were separated during reconstruction into 5-25 MHz and 25-80 MHz for larger and smaller vessels.

Results/Discussion

Already 5 min after VTP, a clear occlusion of vessels was observed using RSOM with increasing destruction of the tumor vascularization and the occurrence of hemorrhage over time. At 1 h after VTP, feeding arteries and draining veins were arrested. The addition of HCA inhibited the effect significantly. Histological staining confirmed these observations. Long-term imaging over 5 days revealed an initial vessel occlusion with a re-opening of the vascular network after 24 h. Subsequently, the whole tumor vascularization collapsed. The analysis of oxy- and deoxyhemoglobin illustrated increased oxygen right after VTP concomitant with the destruction of the first vessels and a decrease in signal over time.

Conclusions

Using RSOM, we were able to monitor the vascular effect of VTP by providing non-invasive high-resolution images in a label-free manner, based only on the optical properties of hemoglobin. Following the vascular occlusion over time and gaining new insights into the involvement of different radicals furthers our mechanistic understanding of VTP.

References

1. Gross et al. Nature Medicine 2003; 2. Madar-Balakirski et al. PLoS One 2010; 3. Kimm et al. Radiology 2016; 4. Omar et al. Neoplasia 2015

Acknowledgement

We thank the Thompson Family Foundation for supporting this study.

RSOM image of CT26 tumor

Figure 1: Non-invasive, high-resolution RSOM image of the vascularization of a subcutaneous CT26 colon carcinoma tumor. Red = Bigger vessels (5-25 MHz). Green = Smaller vessels (25-80 MHz). Yellow = Overlay. The image shows a maximum intensity projection (MIP) of the whole tumor.

Keywords: optoacoustic imaging, photodynamic therapy, wst-11, tumor vascularization
# 238

Evidence for the role of intracellular water lifetime as a tumour biomarker by “in vivo” Field-Cycling relaxometry (#288)

M. R. Ruggiero1, S. Baroni1, S. Pezzana1, G. Ferrante2, S. Geninatti Crich1, S. Aime1

1 University of Turin, Molecular biotechnologies and health sciences, Torino, Italy, Italy
2 Stelar S.r.l, Mede (PV), Italy, Italy

Introduction

The diagnostic power of MRI arises basically from the differences in the relaxation times(T1 and T2) between healthy and pathological tissues. However, at the high field strength of the currently available scanners, changes in T1 do not appear sensitive enough to report on peculiar aspects of the tumour stage. While, at low magnetic field strength, the marked increase of R1 observed in biological tissues might be beneficial to improve the MRI diagnostic potential in tumour phenotyping1-3. Herein it is shown that the in vivo acquisition of 1/T1 from 0.2 to 200mT fully supports this expectation.

Methods

Mouse mammary adenocarcinoma cells (4T1, TS/a, 168farn) were injected in murine muscle himdlimb. In vivo NMRD profiles were acquired over a continuum of magnetic field strength from 0.2-200mT on the Stelar SPINMASTER FFC NMR relaxometer (Fig.1) equipped with a 40mm 0.5 T FC magnet and a dedicated 11 mm solenoid detection coil. T1 was determined by the saturation recovery method and analysed as a mono-exponential decay and bi-exponetial , in order to sample better both fast and slow magnetization components. Immunofluorescence analysis of different transporters (GLUT-1, Na+/K+ ATPase) will be performed to better understand the biological mechanisms underlying T1 changes measured.

Results/Discussion

Longer T1 values for all adenocarcinoma cell lines were observed at any field when compared to the healthy tissue; significant variations among T1 values of the different implanted tumours were also observed (fig.2). Each R1 represents an average of values of water molecules in the extracellular (R1ex) and intracellular (R1in) compartments. According to bi-compartmental model, the evolution time of MZ is dependent on the relationship between |R1in - R1ex| and an “exchange” term |kin + kex| of the compartments. The fitting procedure of Mz shows large variation of kin values obtained for three breast cancer cell lines, inversely proportional to their metabolic activity (4T1>TSA>168Farn). Therefore, the expression of glucose transporter GLUT-1 and Na+/K+ ATPase were evaluated by immunofluorescence. The inhibition of those transporters confirmed the suggestion about the relevant role of the expression of this transporter in the modulation of transcytolemmal water exchange

Conclusions

Despite the FFC-NMR instrumentation is not endowed with spatial resolution, the herein reported results open new horizons for the non-invasive evaluation of tumour metabolic phenotypes,by providing useful information related to the tumour metastatic propensity.Cell water content and volume are related to the concentration of intracellular osmotic active compounds as well as to the extracellular tonicity. Ion pumps or active transporters up/down regulation occurring in the presence of a pathological state, can be exploited as a specific reporter of the cellular state.

References

1. Field-cycling NMR relaxometry with spatial selection. Pine KJ, Davies GR, Lurie DJ. 2010, Magn Reson Med., Vol. 63, pp. 1698-702.

2. Magnetic field dependence of 1/T1 of protons in tissue. Koenig SH, Brown RD 3rd, Adams D, Emerson D, Harrison CG. s.l. : Invest Radiol., 1984, Vol. 19, pp. 76-81.

3. Feasibility of high-resolution one-dimensional relaxation imaging at low magnetic field using a single-sided NMR scanner applied to articular cartilage. Rössler E, Mattea C, Stapf S. s.l. : J Magn Reson., 2015, Vol. 251, pp. 43-51.

4. Intratumor mapping of intracellular water lifetime: metabolic images of breast cancer? al, Springer CJ et. s.l. : NMR Biomed., 2014, Vol. 27, pp. 760-73.

Acknowledgement

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 668119 (project “IDentIFY”) and it was performed in the framework of the Consorzio Interuniversitario di Ricerca in Chimica dei Metalli dei Sistemi Biologici (CIRCMSB).

Figure 1.

Photographs of the Fast Field Cycling NMR Relaxometer showing the introduced modifications for the acquisition of in vivo NMRD profile of hind-limb tissue: a) the FFC magnet; b) the mouse holding system, c) the transmitter/receiver coil around the mouse’s leg.

Figure 2.
A)T2-weighted MR image (1T) of the tumor bearing mouse(4T1); B)Observed relaxation rates (R1) as a function of the magnetic field strength before (black filled square) and after the intramuscular injection (red open triangle,day 4; red open circle,day 8; red open diamond,day 9; red open square,day 11); C)NMRD profiles of 4T1(open red triangle),TS/A(black open circle)and 168FARN (blue open circle).
Keywords: Fast Field Cycling relaxometry, intracellular water lifetime, T1 longitudinal relaxation time, breast tumour metabolic activity
# 239

Targeting CD20 with theranostic camelid single-domain antibody fragment (#204)

A. Krasniqi1, M. D'Huyvetter1, C. Xavier1, K. Van der Jeught2, S. Muyldermans3, J. Van Der Heyden5, T. Lahoutte1, 4, J. Tavernier5, N. Devoogdt1

1 Vrije Universiteit Brussel, In Vivo Cellular and Molecular Imaging, Brussels, Belgium
2 Vrije Universiteit Brussel, Laboratory of Molecular and Cellular Therapy, Brussels, Belgium
3 Vrije Universiteit Brussel, Laboratory of Cellular and Molecular Immunology, Brussels, Belgium
4 UZ Brussel, Nuclear Medicine, Brussels, Belgium
5 VIB and Ghent University, Cytokine Receptor Laboratory, Ghent, Belgium

Introduction

Anti-CD20 radioimmunotherapy is an effective approach for therapy of relapsed or refractory CD20pos lymphomas, but faces limitations due to poor tumor penetration and undesirable pharmacokinetics of full antibodies. Camelid single-domain Ab fragments (sdAbs) might circumvent some of the limitations of radiolabeled full antibodies.

Methods

In this study, a set of hCD20-targeting sdAbs was generated and their capacity to bind hCD20 was evaluated in vitro and in vivo. A lead sdAb, sdAb 9079, was selected based on its specific tumor targeting and significant lower kidney accumulation compared to other sdAbs. SdAb 9079 was then radiolabeled with 68Ga and 177Lu for PET imaging and targeted therapy. The therapeutic potential of 177Lu-DTPA-sdAb was compared to that of 177Lu-DTPA-Rituximab and unlabeled Rituximab in mice bearing hCD20pos tumors.

Results/Discussion

Radiolabeled with 68Ga, sdAb 9079 showed specific tumor uptake, with very low accumulation in non-target organs, except kidneys. The tumor uptake of 177Lu-DTPA-sdAb 9079 after 1.5 h was 3.4 ± 1.3% ID/g, with T/B and T/M ratios of 13.3 ± 4.6 and 32.9 ± 15.6. Peak tumor accumulation of 177Lu-DTPA-Rituximab was about 9 times higher, but concomitantly with high accumulation in non-target organs and very low T/B and T/M ratios (0.8 ± 0.1 and 7.1 ± 2.4). Treatment of mice with 177Lu-DTPA-sdAb 9079 significantly prolonged median survival compared to control groups and was as effective as treatment with Rituximab or its 177Lu-labeled variant.

Conclusions

Taken together, sdAb 9079 displays promising features as a theranostic drug to treat CD20pos lymphomas.

References

Mol Cancer Ther. 2017 Oct 20. doi: 10.1158/1535-7163.MCT-17-0554.

 

 

Keywords: CD20, Camelid single-domain Ab fragments, theranostic
# 240

Imaging prostate cancer using bi-modal fluorescent tomography /ultrasound imaging setup (#317)

C. Genevois1, C. Handschin1, 2, A. Koenig3, C. Vecco-Garda4, S. Mornet4, N. Grenier1, F. Couillaud1

1 IMOTION EA 7435 Université de Bordeaux, Molecular Imaging and Innovative Therapies in Oncology EA 7435, Bordeaux, Please select..., France
2 CEA Tech, Nouvelle-Aquitaine, Pessac, France
3 CEA, LETI-DTBS, Grenoble, France
4 CNRS, Institute for Condensed Matter Chemistry of Bordeaux, ICMCB, UPR 9048, CNRS, University of Bordeaux, Pessac, France

Introduction

To improve the biopsies guidance for prostate cancer, we propose to combine fluorescent tomography with ultrasound. This requires the development of both a dedicated imaging setup and fluorescent probes. Our study was dedicated to preclinical validations and is describing (1) an orthotopic prostate tumor mouse model, (2) in vivo prostate cancer labelling using silica-based fluorescent nanoparticles (3) and imaging prostate cancer using fluorescence tomography and bi-modal fluorescent tomography /ultrasound system.   

Methods

NIR/red dually fluorescent silica nanoparticles (NP) of 19 nm covered by a PEG layer was synthesized and characterized as reported1. Murine prostate cancer cell line RM1 engineered for constitutive expression of firefly luciferase (RM1-CMV/Fluc)1 were injected into prostate during open surgery2 (5 × 105/10 µL per lobe). Tumor growth was monitored by bioluminescence imaging (BLI). NP were injected into the mice via the tail vein and fluorescence was followed by Fluorescence Molecular Tomography (FMT®)3. Excised prostates were imaged by fluorescence reflectance imaging (FRI) and BLI3. The bi-modal fluorescent tomography /ultrasound system combined a commercial echographer (Aixplorer, SSI) with an home-made time resolved fluorescent tomograph4.

Results/Discussion

Injection of RM1/CMV-Fluc cells in mouse prostate lobes resulted in rapid growth followed in vivo by BLI (Figure 1A). NP injected via the tail vein accumulated into tumor. FMT provided 3D images (Figure 1B) and allowed for quantification and kinetics. On excised prostate, the fluorescent signal from NP fit the BLI signal from RM1 cells (Figure 2).

This model was used to challenge the bi-modal fluorescent tomography /ultrasound imaging system. A specific optical module have been designed by fixing optical fibers on an ultrasound preclinical probe. Excitation at 780 nm (10 nm spectral range, 10 ps, 80 MHz, max 20 mW) is achieved towards 6 excitation point sources, and fluorescence is collected with 4 detection fibers. The data are analyzed with a numerical model to retrieve the location of the fluorescent emission. Optical location map is superposed to ultrasound (US) image. Fluorescent map superposed on US image were compared (sensibility and resolution) with images obtained by FMT.

Conclusions

RM1 tumors are very fast-growing, providing significant orthotopic prostate tumors in 4 days in immunocompetent mice. This model exhibited high levels of NP accumulation, allowing challenging bimodal home-made time resolved fluorescence tomograph coupled with US imaging with commercial equipment.

References

1.         Adumeau, L. et al. Impact of surface grafting density of PEG macromolecules on dually fluorescent silica nanoparticles used for the in vivo imaging of subcutaneous tumors. Biochim. Biophys. Acta 1861, 1587–1596 (2017).

2.         Mazzocco, C. et al. In vivo imaging of prostate cancer using an anti-PSMA scFv fragment as a probe. Sci. Rep. 6, 23314 (2016).

3.         Genevois, C. et al. In Vivo Imaging of Prostate Cancer Tumors and Metastasis Using Non-Specific Fluorescent Nanoparticles in Mice. Int. J. Mol. Sci. 18, (2017).

4.         Boutet, J. et al. Optical tomograph optimized for tumor detection inside highly absorbent organs. Opt. Eng. 50, 053203–053203 (2011).

Acknowledgement

This work was supported in part by public grants from Conseil Régional Nouvelle Aquitaine, Labex TRAIL (ANR-10-LABX-57) and France Life Imaging (ANR-11-INBS-006).

Figure 1. In vivo imaging of NP accumulation in orthotopic prostate tumors.

(A) Bioluminescence image of a representative mouse (B) FMT of a representative mouse 24 h after NP injection.

Ex vivo imaging of NP accumulation into prostate tumors.

Prostates were excised and imaged by BLI (A) and FRI (B).

Keywords: US imaging, Fluorescence Imaging, Prostate cancer, mouse model, Nanoparticles
# 241

Analysis of early PET-UUI biomarkers of anti-angiogenic treatment in a mouse model of paraganglioma (#394)

C. Facchin1, 2, A. Garofalakis1, 2, T. Viel1, 2, C. Lussey-Lepoutre2, 4, J. Favier1, 2, M. Tanter3, J. Provost3, B. Tavitian1, 2

1 Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, France
2 INSERM U970, Paris-Cardiovascular Research Center at HEGP, Paris, France
3 Institut Langevin, ESPCI Paris, PSL Research University, CNRS UMR 7587, INSERM U979, Paris, France
4 Pierre et Marie Curie University, faculty of medicine, Paris, France

Introduction

Paragangliomas (PGL) are tumors in which mutations of the gene coding for subunit B of succinate dehydrogenase (SDHB) is present in 10% and associated with a poor prognostic. Metastatic SDHB-associated PGL can be followed by positron emission tomography (PET/CT) with FDG (1) and SDHB-associated PLG have a high vessel density (2). Searching for early biomarkers of the response to sunitinib treatment, we imaged simultaneously the tumor metabolism using FDG-PET/CT and tumor vascularization using ultrafast ultrasound imaging (UUI).

Methods

We built a pre-clinical imaging instrument that acquires simultaneously coregistered image volumes of glucose metabolism using FDG-PET/CT and of vascularization using ultrasensitive Doppler UUI. A mouse tumor model of PGL was obtained from mouse cell lines knocked out for subunit B of the enzyme succinate dehydrogenase gene (Sdh) (2) and allografted in the fat pad of nude mice. Animals treated with the anti-angiogenic sunitinib (n=8) during 6 weeks or with vehicle (n=8) during 3 weeks were imaged every week with PET-UUI. Intergroup comparison for image-derived parameters was based on the Pearson correlation coefficient (R) for normal data distributions and on the Spearman R for non-normal distributions.

Results/Discussion

Sunitinib treatment stopped tumor growth during the first three weeks of treatment, but tumor growth resumed afterwards. Mean and maximal Standard Uptake Values (SUV mean and max) and Metabolic rate of glucose (MRGlu) were not correlated to tumor volume (p>0.05). In contrast, the total length of vessels (TLV) was correlated to tumor volume (R=0.65, p=0.008), to metabolic volume (MV) (R=0.66, p= 0.007) and to total lesion glycolysis (MV*SUV mean) (R=0.59, p=0.022). SUV max increased by 31% between W1 and W6, and this increase was associated with an increase in total length of vessels of 59% between W3 and W6. SUV max at W1 was correlated to SUV max at W6. Interestingly, the decrease in SUV max after the first week of treatment (i.e. between W0 and W1) was inversely correlated to the SUV max value at W6 (R=0.73) and the decrease in MRGlu was inversely correlated with total Doppler signal at W6 (R=0.89).

Conclusions

Our results suggest that the initial decrease in SUV max and MRGlu observed after one week of treatment could represent an early biomarker predicting the response to sunitinib treatment. Simultaneous coregistered metabolic and vascular imaging is a promising approach for treatment follow-up of SDHB-mutated PGL tumors.

References

  1. Timmers HJ et al. Superiority of fluorodeoxyglucose positron emission tomography to other functional imaging techniques in the evaluation of metastatic SDHB-associated pheochromocytoma and paraganglioma. Journal of clinical oncology, 2007.
  2. Favier J et al., The Warburg effect is genetically determined in inherited pheochromocytomas. PloS one, 2009;

 

Acknowledgement

This project was funded by Plan Cancer (ASC16026HSA-C16026HS) and by LABEX WIFI (Laboratory of Excellence ANR-10-LABX-24) within the French program “Investments for the Future” ANR-10-IDEX-0001-02 PSL and support from the Inserm Technology Research Accelerator in Biomedical Ultrasound. In vivo imaging was performed at the Life Imaging Facility of Paris Descartes University (Plateforme Imageries du Vivant - PIV), supported by France Life Imaging (grant ANR-11-INBS-0006) and Infrastructures Biologie-Santé (IBISA).

Keywords: Anti-angiogenic treatment, biomarker, FDG-PET, UUI, paraganglioma
# 242

Evaluation of tracer dose on MCF7 tumor uptake using FDG-PET and Choline-PET (#36)

S. Mairinger1, M. Sauberer1, T. Filip1, J. Stanek1, 2, T. Wanek1, C. Kuntner1

1 AIT Austrian Institute of Technology GmbH, Center for Health & Bioresources, Seibersdorf, Nieder­österreich, Austria
2 Medical University of Vienna, Department of Clinical Pharmacology, Vienna, Wien, Austria

Introduction

Currently tracer based molecular imaging is not only used in the nuclear imaging area (like PET and SPECT) but has found its application in MRI-research too. Specifically, hyperpolarized small molecules such as [U-13C6, U-D7]D-glucose can be used to assess glucose metabolism [1]. However, in MRI a much higher amount of substance (mmol-µmol) has to be injected than in PET (nmol-pmol). So the aim of the study was to evaluate the effect of tracer dose on tumor uptake by performing dynamic [18F]FDG-PET and [11C]Choline-PET scans in a breast cancer xenograft model.

Methods

One week after implantation of an estrogen pellet 7 × 106 MCF7 cells were subcutaneously injected into the right upper shoulder of female athymic nude mice. Once tumors had grown to a volume of 100-200 mm3, the animals underwent PET imaging. Tumor-bearing animals were divided into two groups: in group 1 a tracer dose (<10 µg/kg defined as microdose) was administered whereas in group 2 a much higher dose (15 mg/kg defined as macrodose) was administered by co-injection with the unlabeled substance (glucose or choline chloride). Animals underwent a 90-min [18F]FDG-PET scan and on the consecutive day a 60-min [11C]Choline-PET scan.

Results/Discussion

The time-activity curves (TACs) in the tumor were similar between the micro- and macrodose, both for [18F]FDG and [11C]Choline. Moreover, also tumor/muscle ratio was nearly identical between the two dosing groups and also the two used radiotracers. In addition the calculated area-under-the curve (AUC) from the tumor TACs did not show any significant difference between the two used tracers or dosing groups. [11C]Choline blood and plasma concentration was also comparable between the two dosing groups.

Conclusions

Summarizing, [18F]FDG and [11C]choline are equal radiotracers in visualizing and quantifying MCF7 tumors. We found no difference between microdose and macrodose (AUCs and blood differences are statistically not significant). After already 2 min the tumor maximum uptake was reached. Translating these findings to the hyperpolarized experiments in the same animal model, there should be enough time for the hyperpolarized small molecules to reach the tumor leading to a good contrast image.

References

[1] Mishkovsky M, Anderson B, Karlsson M, Lerche MH, Sherry AD, Gruetter R, et al. Measuring glucose cerebral metabolism in the healthy mouse using hyperpolarized 13C magnetic resonance. Scientific reports 2017;7:11719.

Acknowledgement

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 667192 (Hyperdiamond project).

Figure 1: Comparison between micro- and macrodose
Uptake of [18F]FDG and [11C]Choline in MCF7 tumors when administered as a microdose (left side) or macrodose (right side). Tumor uptake is given as %ID/g tissue.
Keywords: small animal PET, FDG, Choline, breast cancer model