EMIM 2018 ControlCenter

Online Program Overview Session: PW-10

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Develop: New Tools for Cancer Imaging I

Session chair: Alberto Schuhmacher - Zaragosa, Spain; Gabriele Kramer-Marek - London, UK
 
Shortcut: PW-10
Date: Thursday, 22 March, 2018, 11:30 AM
Room: Banquet Hall | level -1
Session type: Poster Session

Abstract

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# 107

CEST MRI to contrast chondrosarcoma tumors: two contrasts in one acquisition. (#522)

L. Mazuel1, 2, R. Autissier1, 2, E. Maubert1, A. Voissière1, V. Weber1, Y. Gérard1, S. Besse1, J. M. Bonny2, E. Miot-Noirault1, C. Peyrode1, G. Pages2

1 INSERM, UMR1240, Clermont-ferrand, France
2 INRA, AgroResonance, Saint Genès Champanelle, France

Introduction

Chondrosarcoma (a malignant tumor of cartilage) is poorly vascularized and rich in proteoglycans (PGs).1 If the hypoxic and proteoglycan status of the tumor can be assessed by TEP imaging2 and scintigaphy3,4,5 it requires however 2 separated acquisitions. In this context we propose to develop an MRI strategy based on Chemical Exchange Saturation Transfer (CEST) to simultaneously co-register both hypoxia (pH) and PGs content in vivo.

Methods

The work hypotheses were tested in phantoms containing chondroitin sulfate A or creatine to validate PGs and pH imaging, respectively. NUDE mice (n=6) were implanted with HEMC-SS cells (human chondrosarcoma) in contact to tibia 7 weeks before imaging. MRI images were acquired at 11.7 T using a 40-mm quadratic volume coil. DWI was first performed to localize the tumor, then WASSR (B1=0,1μT for 1.5s, Δωsat=±1000Hz in 20Hz steps) and CEST Z-spectra (B1=1.5μT for 4s, Δωsat=±3000Hz in 50Hz steps) were acquired based on a RARE protocol.31P MRS was performed thanks to a volume coil. 23Na imaging was recorded by using a 20-mm surface coil. Data were analyzed using an in-house program written in Matlab®R2017a. After correction for B0 inhomogeneity, the CEST maps were generated.

Results/Discussion

Variations in PGs concentration and in pH (fig. 1) were observed in vitro by CEST MRI by monitoring the magnetization transfer ratio asymmetry at 450 (-OH) and 1000Hz (-NH), respectively. These moieties allow extracting the PG concentration and the pH, respectively. In vivo (fig. 2) results showed an increase of asymmetry at 450Hz in the chondrosarcoma compare to the muscle. This increase of asymmetry is expected as during the pathology development the PG concentration increase. In vivo, we also observed an increase of asymmetry at 1800Hz in the chondrosarcoma associated with acidosis in the hypoxic core. The two CEST images are currently being validated by 23Na imaging (GAG concentration) and 31P MRS (pH).

Conclusions

CEST MRI can be used as a new strategy for non-invasive assessment of chondrosarcoma. Indeed, CEST MRI offers the possibility to image in the same exam the 2 mains characteristics of this tumor: pH and PGs contain. CEST MRI is able to identify and differentiate zones of hypoxia in-vivo.

References

  1. Angelini, A. et al. Clinical outcome of central conventional chondrosarcoma. J. Surg.Oncol. 106,929–937 (2012).
  2. Voissiere A et al.targeting applied to hypoxia-activated prodrug therapy in chondrosarcoma:first proof-of-concept. Oncotarget (2017) in press
  3. Cornelis, F., De Clermont, H., Bernhard, J. C., Ravaud, A. & Grenier, N. L’imagerie d’évaluation thérapeutique en pratique clinique d’oncologie urologique. Prog. En Urol. 24,399–413 (2014).
  4. Peyrode, C. et al. A‘Proteoglycan targeting strategy’ for the scintigraphic imaging and monitoring of the swarm rat chondrosarcoma orthotopic model. Sarcoma 2011,691608 (2011).
  5. Miot Noirault, E. et al. In vivo scintigraphic imaging of proteoglycans. Methods Mol. Biol. Clifton NJ 836,183–198 (2012).
In vitro study
CEST-MRI of a chondroitin sulfate A phantom at 4 different concentrations  a) Z-spectra, b) MTRasym and c) MTR asym. Contrast observed at around 450 Hz is associated to PGs –OH function. CEST-MRI of a 50 mM creatin phantom at 4 different pH d) Z-spectra, e) MTRasym and f) MTR asym as function of pH. Contrast observed at around 1000 Hz is associated to creatin –NH function and reflects pH changes.
In vivo study
a) Tumor and muscle ROI b) z-spectra resulting from analysis of ROIs. c) MTRasym of ROIs without baseline correction. Increase in asymmetry was observed at 500Hz (-OH) in chondrosarcoma vs. muscle suggesting higher PGs contain. d) MTRasym of ROIs with baseline correction. Increase in asymmetry was observed at 1800Hz (-NH2) in chondrosarcoma vs. muscle suggesting changes in pH inside the tumor.
# 108

Diagnostic Nanobodies for Brain Tumors and Metastases (#381)

J. Puttemans1, M. D'Huyvetter1, S. Muyldermans2, N. Devoogdt1

1 Vrije Universiteit Brussel, ICMI, Jette, Brussels Hoofdstedelijk Gewest, Belgium
2 Vrije Universiteit Brussel, CMIM, Elsene, Brussels Hoofdstedelijk Gewest, Belgium

Introduction

Despite the extensive research in the treatment of brain-localised tumors, only minor improvements in overall survival have been observed and a cure for these cancers is still not available, often due to the incapacity of targeting vehicles to cross the blood-brain-barrier. Nanobodies are small antibody fragments from camelidae heavy-chain-only antibodies. Due to their favorable pharmacokinetics, Nanobodies can be applied as targeting tools for the molecular imaging of several tumor subtypes. We aim to expand the use of Nanobodies as vehicles for imaging and therapy of brain-localised tumors.

Methods

The anti-HER2 Nanobody 2Rs15d was labeled with either technetium-99m (Tc-99m) via the C-terminal hexahistidine tail or with Indium-111 (In-111) using CHX-A''-DTPA. Trastuzumab, an anti-HER2 mAb was  also labeled with In-111 using the same bifuntional chelator. Tumors were inoculated through stereotactic intracranial injection of firefly luciferase-positive HER2-overexpressing cancer cells. Tumor growth was followed up in time using in vivo bioluminescent imaging (BLI). Biodistribution and tumor uptake of both Nanobody and mAb vehicles were evaluated in HER2-overexpressing orthotopic tumor-bearing nude mice through in vivo micro-SPECT/CT imaging and ex vivo gamma counting after dissection.

 

Results/Discussion

BLI-confirmed tumor-bearing mice showed specific uptake of Tc-99m- and In-111-labeled anti-HER2 Nanobodies in brain-localized tumors as soon as 1 hour after intravenous injection and were still detectable after 3 days for the In-111-labeled Nanobodies. Extremely low uptake values were observed in healthy tissues, except in the kidney and bladder, through which Nanobodies are excreted. The mice treated with In-111-labeled Trastuzumab showed long-lasting bloodpool activity, but no tumor-uptake.

 

Conclusions

The Tc-99m- and In-111-labeled anti-HER2 Nanobodies were able to cross the disrupted blood-brain-barrier and migrate through brain tissue to specifically bind HER2-positive cancer cells after intravenous administration, whereas the mAb was unable to reach the tumor site. This implies that Nanobodies might be more appropriate alternatives as targeting vehicles for tumors located in the central nervous system. Whether this accumulation is suffucient to deliver therapeutic doses is still under investigation.

 

References

 

 

Acknowledgement

The authors would like to thank Cindy Peleman for her help with the SPECT/CT imaging and image reconstructions.

 

Figure 1: Follow-up and nuclear imaging of brain-localised tumors
(A) In vivo BLI imaging of intracranial firefly luciferase-positive HER2-overexpressing cancer cells. (B,C) Specific diagnostic tumor imaging with Nanobodies at 1h post-injection. Mice were injected with (B) Tc-99m-  and (C) In-111-labeled anti-HER2 Nanobodies. Images were taken 30 days after tumor implantation. 
Keywords: Nanobodies, Brain tumor, nuclear imaging, bioluminescent imaging, probes
# 109

Imaging Activated Platelets- A Novel Approach for Cancer Detection (#329)

M. L. Yap1, 2, J. McFadyen1, 3, 4, X. Wang1, 3, N. Zia5, 6, J. D. Hohmann1, M. Ziegler1, Y. Yao1, A. Pham7, M. Harris5, P. Donnelly6, M. Hogarth2, 8, 9, G. Pietersz1, 8, 9, B. Lim1, K. Peter1, 3, 9

1 Baker Heart and Diabetes Institute, Melbourne, VIC, Australia
2 University of Melbourne, Department of Pathology, Melbourne, VIC, Australia
3 Monash University, Department of Medicine, Melbourne, VIC, Australia
4 Alfred Hospital, Department of Hematology, Melbourne, VIC, Australia
5 Clarity Pharmaceuticals, Sydney, NSW, Australia
6 University of Melbourne, School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, Melbourne, VIC, Australia
7 Alfred Hospital, Department of Anatomical Pathology, Melbourne, VIC, Australia
8 Burnet Institute, Melbourne, VIC, Australia
9 Monash University, Department of Immunology, Melbourne, VIC, Australia

Introduction

The early detection of primary tumours and metastatic disease is vital for successful therapy and is contingent upon highly specific molecular mar­kers and sensitive, non-invasive imaging techniques. The relationship between platelets and cancer, and the abundance of activated platelets in the tumour microenvironment has been well described. Here we investigate a single-chain antibody (scFv), which was generated to specifically target the activated form of the platelet integrin receptor GPIIb/IIIa on activated platelets, as a novel biotechnological tool for molecular imaging of cancer.

Methods

The scFvGPIIb/IIIa, which binds specifically to only the activated form of GPIIb/IIIa present on activated platelets, was conjugated to either Cy7, 64Cu or ultrasound-enhancing microbubbles. Additionally, we utilized three different conjugation methods for labelling of the contrast agents to the scFv. Molecular imaging via fluorescence imaging, PET and ultrasound was performed in mice bearing human cancer xenografts to confirm specific targeting of scFvGPIIb/IIIa to activated platelets in the tumour stroma.

Results/Discussion

Using the scFvGPIIb/IIIa we successfully showed specific targeting of activated platelets within the microenvironment of human breast adenocarcinoma, triple negative breast adenocarcinoma, fibrosarcoma and B cell lymphoma xenograft models via three different molecular imaging modalities. The presence of platelets within the tumour microenvironment, and as such their relevance as a molecular target epitope in cancer was further confirmed via immunofluorescence of human tumour sections of various cancer types, thus validating the translational importance of our novel approach to human disease.

Conclusions

Our study provides proof of concept for the early diagnosis and localisation of tumours by molecular imaging and targeting of activated platelets. These findings warrant further investigation of this activated platelet specific scFvGPIIb/IIIa, as a universal marker for cancer diagnosis and further on as a possible theranostic agent, by utilizing a single probe for cancer diagnosis, disease monitoring and targeted therapy.   

 

References

Yap ML, McFadyen JD, Wang X, Zia NA, Hohmann JD, Ziegler M, Yao Y, Pham A, Harris M, Donnelly PS, Hogarth PM, Pietersz GA, Lim B, Peter K. Targeting Activated Platelets: A Unique and Potentially Universal Approach for Cancer Imaging. Theranostics 2017; 7(10):2565-2574.

Acknowledgement

We would like to acknowledge and thank the Monash Micro Imaging team for their technical support and the Monash Biomedical Imaging for access to the PET/CT and FLECT used for small animal imaging.

Molecular Imaging of Activated Platelets in Cancer via Ultrasound, Fluorescence Imaging and PET/CT
Keywords: Cancer Imaging, FLECT, PET, Ultrasound, Platelets, GPIIb/IIIa
# 110

Identifying tumor markers in esophageal adenocarcinoma and lymph node metastases for targeted fluorescence imaging (#287)

D. de Gouw1, B. R. Klarenbeek1, M. Rijpkema2, K. Verrijp3, M. M. Rovers4, C. Rosman1, R. S. van der Post3

1 Radboud university medical center, department of surgery, Nijmegen, Netherlands
2 Radboud university medical center, department of radiology and nuclear medicine, Nijmegen, Netherlands
3 Radboud university medical center, department of pathology, Nijmegen, Netherlands
4 Radboud university medical center, department of health evidence, Nijmegen, Netherlands

Introduction

Neoadjuvant chemoradiotherapy (nCRT) followed by resection of the tumor with two field lymphadenectomy is a standard treatment for esophageal cancer. After nCRT, there are no lymph node metastases in more than 70% of patients, suggesting extensive overtreatment. Tumor-targeted fluorescence imaging is a promising technique to detect lymph node metastases intra-operatively by personalizing surgical treatment. The aim of this immunohistochemistry (IHC) study is to identify potential viable tumor markers for fluorescence imaging of lymph node metastases in esophageal adenocarcinoma (EAC).

Methods

IHC was performed on tissue microarrays from EAC’s patients that underwent surgical resection between 2007 and 2016. Patients were subdivided in five groups, non-pretreated patients with EAC with and without metastatic lymph nodes, complete responders, partial responders and non-responders after nCRT. Healthy lymph nodes and fibrotic tissue in complete responders served as negative control for marker expression. Five membranous markers, c-MET, CAIX, EGFR, EpCAM, HER2, and two cytoplasmic markers, VEGF-A and VEGF-A receptor were included. Tumor marker expression was scored on intensity (none (0), slight (1), moderate (2), strong (3)) and the percentage of positive cells (estimation). Threshold for positive detection rate was defined as an intensity of ≥2 in more than 10% of the cells.

Results/Discussion

An overview of the expression for each individual tumor marker is shown in graph 1 with an example in figure 1. EpCAM showed the highest expression in metastastic lymph nodes, with a median intensity of 3 (range 2-3) in >70% of the tumor cells. Expression was found in 37 out of 39 EAC’s (95%). VEGF-A and CAIX expression was observed in 28 of 33 (85%) and 10 of 33 (30%) of metastatic lymph nodes and 34 of 39 (87%) and 17 of 39 (44%) in the primary EAC’s, respectively. For the other tumor biomarkers the detection rate ranged between 0 and 11% for metastatic lymph nodes and primary EAC’s. Only EpCAM and VEGF-A showed weak, non-specific staining in the fibrotic tissue. There were no significant differences in expression between patients with and without nCRT.

Conclusions

High expression rates in primary EAC and metastatic lymph nodes were observed using immunohistochemical antibodies for EpCAM, VEGF-A and CAIX, making these promising viable EAC tumor markers. VEGF-A has already proven to be a suitable target for molecular imaging; early results of phase 1 dose finding studies targeting VEGF-A in patient with EAC are in preparation.

Graph 1
Results of the expression for every tumor marker of the metastatic lymph node and primary EAC
Figure 1
Example of immunohistochemical staining of metastatic lymph nodes for each tumor marker. Scalebar equals 5 μm.
# 111

68Ga-NODAGA-exendin-4 PET/CT for the localization of insulinomas: preliminary data from a prospective multicenter imaging study. (#196)

M. Boss1, M. Buitinga1, M. Brom1, T. Jansen1, D. Wild2, V. Prasad3, P. Nuutila4, A. Brouwers5, F. Pattou6, M. Gotthardt1

1 Radboudumc, Nuclear medicine, Nijmegen, Netherlands
2 University of Basel Hospital, Nuclear medicine, Basel, Switzerland
3 Charite University Hospital, Nuclear medicine, Berlin, Germany
4 University of Turku, Nuclear medicine, Turku, Finland
5 University Medical Center Groningen, Nuclear medicine, Groningen, Netherlands
6 University Hospital Lille, General endocrine surgery, Lille, France

Introduction

Insulinomas are usually small, single tumors. Precise preoperative localization of the tumor is essential. Imaging techniques like CT, MRI and somatostatin receptor (SSTR) imaging have limited sensitivity (1,2). The stable glucagon like peptide-1 (GLP-1) analog exendin specifically binds the GLP-1 receptor (GLP-1R) (3), which is markedly upregulated in insulinomas. We propose 68Ga-NODAGA-exendin-4 PET/CT as a promising new method for localization of insulinomas. We here present the first results of a multi-center prospective imaging study to evaluate the effectiveness of 68Ga-exendin-4 PET/CT.

Methods

17 adults aged 24-65 with biochemically proven hyperinsulinemic hypoglycemia were included. PET/CT images were obtained one and two hours after injection of (5-7 µg) 95-105 MBq 68Ga-NODAGA-exendin-4. Within 8 weeks of the 68Ga-exendin PET/CT, current standard imaging was performed in all patients, consisting of CT or MRI and SSTR PET imaging.

Results/Discussion

In 4 patients, standard imaging as well as GLP-1R PET/CT were all negative. In 9 patients, insulinoma were identified using conventional imaging and SSTR PET/CT. All these lesions were also clearly visualized using GLP-1R PET/CT imaging, with low background uptake. In 1 patient an insulinoma was detected using SSTR PET/CT and GLP-1R PET/CT, which was missed using MRI. In 2 patients insulinoma were visualized using GLP-1R PET/CT, while all standard imaging modalities, including endoscopic ultrasound, were negative. Finally, 1 patient with MEN-1 syndrome presented with multiple lesions detected by MRI as well as SSTR PET/CT. In this patient only 1 of the lesions was found to be GLP-1R positive. Infusion of 68Ga-NODAGA-exendin-4 did not cause any relevant side-effects in the included patients.

These preliminary results indicate that 68Ga-NODAGA-exendin-4 PET/CT performs better than current standard imaging techniques for the detection of insulinoma.

 

Conclusions

68Ga-NODAGA-exendin-4 PET/CT detected insulinoma in more patients than standard imaging methods. Using this technique, insulinoma were clearly visualized with high tumor-to-background activities. This, combined with the absence of relevant side-effects suggests a possible valuable role for this new imaging method in future diagnosis of insulinoma.

References

  1. Nockel P, Babic B, Millo C, Herscovitch P, Patel D, Nilubol N, et al. Localization of Insulinoma Using 68Ga-DOTATATE PET/CT Scan. The Journal of clinical endocrinology and metabolism. 2017;102(1):195-9.
  2. McAuley G, Delaney H, Colville J, Lyburn I, Worsley D, Govender P, et al. Multimodality preoperative imaging of pancreatic insulinomas. Clinical radiology. 2005;60(10):1039-50.
  3. Brom M, Woliner-van der Weg W, Joosten L, Frielink C, Bouckenooghe T, Rijken P, et al. Non-invasive quantification of the beta cell mass by SPECT with (1)(1)(1)In-labelled exendin. Diabetologia. 2014;57(5):950-9.
Figure 1
SSTR PET/CT and GLP-1R PET/CT images of a patient with adult hyperinsulinemic hypoglycemia. SSTR PET/CT does not reveal any lesions in the pancreas. GLP-1R PET/CT shows a clear GLP-1 positive lesion in the pancreatic body.
Keywords: Insulinoma, GLP-1 receptor, Exendin, PET imaging
# 112

Development and evaluation of [11C]NMS-E973, a PET radiotracer for in vivo visualisation of HSP90 (#357)

K. Vermeulen1, E. Naus2, M. Ahamed3, B. Attili1, M. Siemons1, J. Schymkowitz2, F. Rousseau2, G. Bormans1

1 Laboratory for Radiopharmaceutical Research, KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, Leuven, Belgium
2 Switch Laboratory, VIB‐KU Leuven Center for Brain & Disease Research, Leuven, Belgium
3 School of Chemical and Physical Sciences, Flinders University, Adelaide, Australia

Introduction

PET visualisation of HSP90 and its extracellular variant eHSP90, important in metastasis and cell invasion enables the translational and functional study of this protein in vivo. Here we describe the in vitro and in vivo evaluation of [11C]NMS-E973, based on a specific HSP90 inhibitor 2, 3.

Methods

[11C]NMS-E973 was synthesized through methylation with [11C]MeI in DMSO using Cs2CO3 at 100 °C for 4 min. Biodistribution was studied in NMRI-mice. Autoradiography studies were performed on B16.F10 melanoma tumour slices. Slices were incubated with tracer with/without 100 µM of NMS-E973, PU-H71 or HSP70 inhibitor Pifithrin-µ. Dynamic 90-min µPET scans were acquired from B16.F10 inoculated C57BL/6 mice after injection of 11 MBq of tracer. [11C]NMS-E973 binding was challenged by pretreatment of the same mice with PU-H71 (50 mg/kg). To assess internalisation of [11C]NMS-E973, B16.F10 cells were incubated with tracer with/without 200 µM of NMS-E973, PU-H71 or Pifithrin-µ at 37 °C or 4 °C. HSP90 expression was biochemically characterized in B16.F10 melanoma and compared to non-malignant tissue.

Results/Discussion

Radiochemical yields were 30-40 % with a radiochemical purity of >97 % and a molar activity of 90 GBq/µmol. Biodistribution studies indicated low brain uptake and renal and hepatobiliary clearance. In vitro autoradiography experiments showed high specific binding. Similar results were observed in the internalisation study, where tracer uptake could be efficiently blocked by NMS-E973 and PU-H71. Internalisation studies performed at 4 °C showed a significant decrease (p ≤ 0.001) of the internalised fraction, suggesting active internalisation via eHSP90. The cell bound fraction can be an indication for the eHSP90 pool. µPET experiments showed high selective tumour uptake which could be significantly blocked by pretreatment with PU-H71 (SUV0-90min p ≤ 0.001). HSP90 upregulation was observed in B16.F10 tumour cell line and increased compared to non-malignant tissue.

Conclusions

We successfully radiolabeled and evaluated a carbon-11 labeled radiotracer for in vitro and in vivo visualization of HSP90. HSP90 PET can be used to determine expression and occupancy in a melanoma tumour model. Further translational evaluation is needed.

References

 

1. M.G. Brasca, et al., Bioorganic Med. Chem. 2013, 21, 7047–7063

2. G. Fogliatto, et al., Clin. Cancer Res. 2013, 19, 3520–3532

Specific tumour uptake of [11C]NMS-E973
A, C) Averaged baseline image of 90-min dynamic scan p.i. of 11 MBq of [11C]NMS-E973 without (A) or with (C) pretreatment of 50 mg/kg PU-H71 administered I.P. 45 min before tracer injection, in B16.F10 inoculated C57BL/6 mice. B) In vitro autoradiography experiment on B16.F10 slices incubated with tracer (7.4 kBq) without (left), with (right) 100 µM PU-H71.
Cell binding study of [11C]NMS-E973
Cell binding and internalisation of [11C]NMS-E973 in B16.F10 melanoma cells at 37 °C (A) and 4 °C (B). Specific cell uptake and cell surface binding was assessed by blocking with NMS-E973, PU-H71 or Pifithrin-µ (200 µM).
Keywords: HSP90, Carbon-11, Melanoma
# 114

Bimodal imaging system using ultrasound and fluorescence to localize prostate tumors. (#391)

C. Handschin1, 3, J. Boutet2, L. Herve2, A. Koenig2, O. Redon1, M. Perriollat2, C. Genevois3, F. Couillaud3, S. Morales2, J. - M. Dinten2, N. Grenier3, 4

1 CEA, CEA Tech Nouvelle Aquitaine, Pessac, France
2 CEA, Leti, Grenoble, France
3 IMOTION, EA 7435, Université de Bordeaux, Bordeaux, France
4 CHU Pellegrin, Service d'Imagerie Diagnostique et Interventionnelle de l'Adulte, Bordeaux, France

Introduction

Prostate cancer is the most common in the male population. The problem is especially critical as the methods of diagnosis are quite invasive. This is due to the lack of imaging technics permitting to localize the tumor inside the prostate. As a result, the most conventional methods consists in practicing a procedure of systematic biopsies, which is repeated until a cancerous region is detected. This procedure is obviously invasive and could be avoided with a more appropriate imaging system. We propose to use a combination system based on ultrasounds and fluorescence to localize the tumor.

Methods

No specific fluorescent marker is yet available to label tumors in human prostate. The method is then based on the ability of a fluorophores (ICG) to concentrate in the vicinity of the tumor due to the EPR effect. In order to propose real time anatomical imaging, we combine fluorescence measurements with ultrasound imaging thanks to a modified endorectal ultrasound probe including optical fibers [1]. An instrumentation constituted of a laser, the bimodal probe and a detection system has been built to excite the ICG at 780 nm and to collect fluorescence. A single photon counting system is used to measure the temporal distributions of the photons. Data are then analyzed with an appropriate numerical model. Finally a fluorescence location map is superposed to the ultrasound image.

Results/Discussion

As first result, we demonstrate the method on experimental phantoms. Intralipid (1 % dilution) has been used for its well-known optical scattering properties. Black Indian ink was added to reach an absorption of 0,45/cm at 780 nm to  mimic the prostate absorption. Fluorescent inclusion (capillary with 30 µL of fluorophore, 20 µmol/L) was placed at various locations in the phantom to generate the effect of an accumulation of fluorescence. An optical probe prototype was used to excite (5 mW) the fluorescent molecules and collect the temporal distribution of emitted photons. For each position we compare the theoretical location of the inclusion with the one retrieved from the measurement. The experiments show a correct localization (precision around 1mm) for positions until 15 mm depth (figure). These results demonstrate the feasibility of the proposed method for tumors located in the first 15 mm depths in front of the probe, which covers a non-negligible number of pathologies.

Conclusions

We demonstrated the ability of our instrumentation and method to probe an isolated fluorescent region located until 15 mm depth in an optical prostate phantom. In vivo experiments on mice with prostate tumor model will be conducted in the forthcoming weeks to validate the method in a more realistic situation [2-3]. It will be particularly interesting to test and study the sensibility of the instrumentation to specific fluorescence in provenance from tumor. Parallel developments are in progress to obtain an instrumentation with medical certifications and authorizations for use on patients.

References

[1] A. Laidevant, L. Hervé, M. Debourdeau, J. Boutet, N. Grenier, and J.-M. Dinten, « Fluorescence time-resolved imaging system embedded in an ultrasound prostate probe », Biomedical Optics Express, Volume 2, Issue 1, pages 194-206, (2011).

[2] C. Mazzocco, G. Fracasso, C. Genevois, N. Dugot-Senant, M. Figini, M. Colombatti, N. Grenier, F. Couillaud, « In Vivo Imaging of Prostate Cancer Using an Anti-PSMA scFv Fragment as a Probe» Scientific Reports, Volume 6, (2016).

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

Acknowledgement

This work is mainly supported by the French region “Nouvelle Aquitaine” for the instrumentation development. In vivo experiments on mice are supported by “Labex TRAIL” (ANR-10-LABX-57) and “France Life Imaging” (ANR-11-INBS-006). We would like also give a thanks to “CEATech Nouvelle Aquitaine” facilities for the technical support.

Tracking of a fluorescent inclusion in an optical prostate phantom.

Left (figure a): Example of a fluorescence localization map retrieved from the proposed method. Right: Comparison between retrieved position of the fluorescence region and effective one. Figure b: Study of the ability of the method to localize in the transverse direction of the probe (capillary located at 12 mm depth). Figure c: Study of the ability of the method to localize in depth.

Keywords: Prostate cancer, fluorescence imaging, ultrasound imaging, bimodal imaging, tumor localization, non-invasive technics
# 115

99mTc-labelled hydroxyapatite nanoparticles for tumor imaging and their in vitro/in vivo characterisation (#157)

Z. Novy1, V. Lobaz3, M. Vlk2, J. Kozempel2, S. Gurska1, M. Hrubý3, M. Hajduch1, M. Petrik1

1 Palacky University Olomouc, Institute of Translational Medicine, Olomouc, Czech Republic
2 Czech Technical University in Prague, Faculty of Nuclear Sciences and Physical Engineering, Praha, Czech Republic
3 Czech Academy of Science, Institute of Macromolecular Chemistry, Praha, Czech Republic

Introduction

Preclinical screening of selected hydroxyapatite (HAP) nanoparticles modified with various biocompatible polymers (PEG2000, PEG5000, POX5000, POX10000 and PHPMA12000). The goal of this work was indirect radiolabeling of newly prepared nanoparticles with radioisotope 99mTc via clinically used radiopharmaceutical 99mTc-HDP and subsequently to verify the stability of such labelled nanoparticles in vitro and then to reveal their in vivo biodistribution in normal mice using microSPECT/CT system. All studied nanoparticles were tested in means of their in vitro cytotoxicity on selected cell lines.

Methods

Radiolabeling: nanoparticles were incubated for 60 minutes with 99mTc-HPD in room temperature. Subsequently radiochemical purity control was employed using ITLC with two different mobile phases (ammonium acetate and acetone). Stability studies of labelled nanoparticles were performed in two environments - saline and human plasma (2h, 4h and 24h). In vivo biodistribution was monitored using microSPECT/CT and was registered 1h, 3h, 6h a 24 h after application of labelled compound for three different set-ups of experiement. The 99mTc-HDP imaging was also performed for comparison with labelled nanoparticles. Cytotoxicity: chosen nanoparticles (in unlabeled form) were tested using MTS method in six cell lines (B2, BJ, MRC5, HCT116, HCT116p53-/-, CCRF-CEM).

Results/Discussion

Radiolabeling of all studied nanoparticles with 99mTc-HDP resulted in radiochemical purity over 90%. Stability studies of such prepared radiopharmaceutical revealed decrease in radiochemical purity of maximum 10.7% in saline and 11.0% in human plasma respectively after 24 hours. SPECT/CT imaging showed very specific biodistribution of nanoparticles concentrated in liver and spleen even 24 p.i. for all tested compounds for in vitro labelled nanoparticles. The accumulation of activity was observed in heart and bladder in earlier time points p.i. (1h and 3h). Cytotoxicity: studied nanoparticles were considered nontoxic according to their IC50s obtained using above mention cell lines (IC50 ˃ 1000 µg/mL resp. ˃ 2000 µg/mL).

Conclusions

Tested nanoparticles show relatively facile radiolabeling with clinically used radiopharmaceutical 99mTc-HDP. Their stability in saline and human plasma is in highly satisfactory level. Biodistribution studies revealed specific accumulation in liver and spleen, where part of injected dose is excreted via kidneys. In vitro toxicity of the nanoparticles is relatively very low. All these parameters suggest the potential of these compounds for future studies aimed into tumor imaging based on EPR effect in vivo.

Acknowledgement

Acknowledgement to project of Czech Research Council No. 16-30544A.

SPECT/CT biodistribution of HAP nanoparticles coated with PEG200
MicroSPECT/CT imaging of biodistribution of 99mTc-HAP-PEG2000 nanoparticles for time 1 and 6 hours post injection time points and three different 99mTc-HDP application schemes.
Keywords: tumor imaging, nanoparticles, 99mTc-HDP, SPECT, CT
# 116

Optical in vivo imaging detection of preclinical models of intestinal tumors through the expression of Integrin aVb3 (#165)

G. Marelli1, R. Avigni1, P. Allavena1, 2, C. Garlanda1, 2, A. Mantovani1, 2, A. Doni1, M. Erreni1

1 IRCCS, Humanitas Clinical Institute, Dpt. of Immunology and Inflammation, Rozzano (Milan), Italy
2 Humanitas University, Pieve Emanuele (Milan), Italy

Introduction

Optical imaging techniques and, especially, Fluorescent Molecular Tomography (FMT) are becoming increasingly important for the study of different preclinical models of cancer, providing the possibility to evaluate tumor progression in a relatively simple and fast way 1. Intestinal tumors, in particular colorectal cancer (CRC), still represent a major cause of cancer-related death in Western countries: despite the presence of a number of preclinical model of intestinal carcinogenesis, few studies investigated the use of fluorescent probes for the early detection of tumors

Methods

A commercially available probe (IntegriSense) 2, specifically recognizing integrin αvβ3, was injected in APC+/min mice bearing small intestine adenomas or CRC. Mice were analyzed in vivo by FMT and obtained data were subsequently confirmed by 2D ex vivo imaging, conventional histology and confocal microscopy

Results/Discussion

FMT analysis revealed a clear accumulation of IntegriSense probe in the small intestine and in the colon of tumor bearing mice. These results were subsequently confirmed by 2D ex vivo imaging. Histological evaluation of tumor area significantly correlated with both in vivo FMT and 2D ex vivo imaging data, indicating the specificity of IntegriSense detected signal. In addition, integrin αvβ3 has been associated with cancer-related stroma and the early phase of tumor angiogenesis: confocal microscopy analysis indicated that IntegriSense probe colocalized with extracellular matrix proteins and CD31+-tumor associated vessels. Finally, IntegriSense imaging allowed the detection of early stage tumors and the monitoring of cancer progression

Conclusions

We demonstrated that the imaging of integrin αvβ3 by FMT can be used for the evaluation of intestinal carcinogenesis in genetic and chemically-induced mouse models. In particular, FMT technique increases the specificity of tumor detection compared to 2D optical imaging, ameliorating the anatomical resolution. Combining integrin αvβ3 FMT application with other imaging techniques (microCT or MRI), could improve the specificity of intestinal cancer visualization, laying the foundations for the use of integrin αvβ3 expression for the early detection of human intestinal tumors in the clinic

References

  1. Martelli, C., Lo Dico, A., Diceglie, C., Lucignani, G. & Ottobrini, L. Optical imaging probes in oncology. Oncotarget 7, 48753-48787 (2016).
  2. Kossodo, S. et al. Dual in vivo quantification of integrin-targeted and protease-activated agents in cancer using fluorescence molecular tomography (FMT). Mol Imaging Biol 12, 488-499 (2010).

Acknowledgement

Supported by IG grant from the Italian Association for Cancer Research (AIRC)

Keywords: Colorectal cancer, Intestine tumor, Fluorescent molecular tomography, optical in vivo imaging, integrin
# 117

A BON-SSTR2 chicken chorioallantoic membrane (CAM) model and imaging portfolio for the analysis of Lu-177-DOTATOC therapeutic effects and sensitizing agents (#461)

E. J. Koziolek3, 2, 1, F. Briest4, 6, S. Exner5, 2, D. Sedding4, C. Grötzinger5, 2, P. Grabowski4, 6, W. Brenner3, 2, 7

1 German Cancer Research Center (DKFZ), Heidelberg, Germany
2 German Cancer Consortium (DKTK), Heidelberg, Germany
3 Charite-Universitätsmedizin, Department of Nuclear Medicine, Berlin, Germany
4 Charite-Universitätsmedizin, Institute of Medical Immunology, Berlin, Germany
5 Charite-Universitätsmedizin, Department of Hepatology and Gastroenterology and Molecular Cancer Research Center, Tumor Targeting Laboratory, Berlin, Germany
6 CoE Zentralklinik Bad Berka GmbH, Department of Gastroenterology and Endocrinology, Bad Berka, Germany
7 Charite-Universitätsmedizin, Berlin Experimental Radionuclide Imaging Center (BERIC), Berlin, Germany

Introduction

Peptide receptor radionuclide therapy (PRRT) is a promising therapy option for SSTR2-positive pancreatic neuroendocrine neoplasms (NEN). However, therapeutic effects are not always satisfying concerning resistance to PRRT. Slow proliferation of NENs may provide sufficient time for the repair of beta-particle induced-DNA damage. The ubiquitin-proteasome-system is involved in DNA damage repair and affected by the proteasome inhibitor bortezomib. Here, we present a portfolio for imaging PRRT therapeutic effects in an in ovo xenograft model and first results of Bortezomib used as a sensitizer.

Methods

We established a PRRT in vivo model by inoculating SSTR2-transfected BON cells (~5x106) as tumor plaques (matrixgel) onto the chorioallantoic membrane (CAM) of fertilized chicken eggs. Established xenografted tumors were treated with 20 MBq Lu-177-DOTATOC with and without bortezomib and compared to untreated controls. Tumor plaques were imaged by SPECT/CT (Mediso) for Lu-177-DOTATOC uptake, F-18-FDG PET and a multiparametric MRI (1T PET/MRI, Mediso) portfolio to assess tumor growth, characterize tumor tissue morphology (T1w, T2w high resolution, DWI) and vasculature (angiography; GadospinP,nanoPET Pharma). Therapeutic effects were monitored for 7 days and validated by tumor size and vitality comparison, ADC values and appearance of protein rich fluid during PRRT.

Results/Discussion

First, multimodal imaging portfolio for in ovo xenograft model and therapy response validation will be presented, effects of matrixgel and appearance of protein rich fluid will be explained: following tumor plaque inoculation, matrixgel is present up to 5 days and needs to be considered when tumor size/ADC is used as a therapy response criteria. Due to the biology of the CAM system protein rich fluid may occur in strongly damaged tumor areas.

BON-SSTR2 tumors revealed an uptake of 1.4-2.2 %ID Lu-177-DOTATOC by SPECT/CT 20 h post injection. During PRRT tumor growth was first arrested before absolute tumor volume decreased by >50% in relation to saline controls (confirmed by F-18-FDG PET). ADC values reflected cell biology under PRRT very well, while ADC values of control tumors corresponded to normal tumor growth.

Angiography of plaque-supplying vasculature under different treatment regimes versus control and further data of the combined treatment will be presented in the meeting.

Conclusions

The BON-SSTR2 CAM xenograft model is a useful model for studying treatment effects such as PRRT or combined treatment regimens in vivo. In particular imaging of this model is easy to set up and allows a precise validation of therapeutic response by multiple parameters. However, effects of matrixgel and occurring protein rich fluid should be considered to correctly interpret and validate therapeutic effects by the different parameters.

The combinatory treatment of bortezomib plus Lu-177-DOTATOC should be considered for further preclinical testing in an appropriate mouse model.

 

 

Multiparametric MR imaging of tumor plaques innoculated on CAM of fertilized eggs:
Tumor plaques (red circle) were treated i.v. with PBS (upper panel) or Lu-177-DOTATOC (lower panel) and imaged 7 days post treatment by T2w (high resolution), T1w and DWI for ADC mapping for therapy response validation. In order to place ROIs for ADC values correctly, anatomical T2w images were fused to ADC maps. Imaging was performed with a 1T nanoscanPET/MRI( Mediso).
SPECT/CT of Lu-177-DOTATOC tumor uptake in ovo
Lu-177-DOTATOC (20 MBq) was injected i.v. and SPECT/CT performed 20h post injection to monitor SSTR2-specific tumor uptake (red circle, right panel) in ovo. Presented tumor showed an uptake of 2.2 %ID. CT confirms plaque position (red circle, left panel).
Keywords: PET/MRI, in ovo tumor model, PRRT, SPECT/CT, therapy response validation