Développements et applications de l'imagerie ultrasonore haute fréquence et fonctionnelle chez la souris

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Abstract 2338: Aflibercept (VEGF Trap) impairs tumor growth and perfusion, and restrains metastasis invasion in orthotopic models

Abstract Aflibercept (VEGF Trap), a potent angiogenesis inhibitor that binds to all isoforms of circulating VEGF as well as to placental growth factor, is currently in Phase 3 clinical trials in combination with chemotherapy for several cancer indications. The VELOUR Phase 3 trial in oxaliplatin pre-treated metastatic colorectal cancer showed that aflibercept in combination with FOLFIRI significantly improved overall survival, progression-free survival and response rate (in comparison with FOLFIRI plus placebo), with some increases in adverse events. The aim of the current study was to explore the effect of aflibercept on tumor vessel function and determine the impact of a prolonged treatment regimen on the development of metastases in orthotopic preclinical models. For the functional evaluation of tumor perfusion by contrast enhanced ultrasound imaging (CE-US), SCID mice underwent renal subcapsular implantation of the human renal SK-NEP-1 Ewing sarcoma. The pattern of vascularization in these rapidly growing tumors was found to be heterogeneous: tumor areas in the proximal kidney remained highly perfused, while avascular areas appeared in the distal kidney region which is likely a consequence of spontaneous necrosis in large tumors. Aflibercept administered bi-weekly at 40mg/kg for two weeks showed a statistically significant effect on tumor growth (5% T/C). Aflibercept efficiently down-regulated tumor perfusion inducing a reduction of tumor blood volume immediately after the first injection, followed by subsequent stabilization and rebound two weeks after the end of treatment. The MDA-MB-231-D3H2LN-luc breast tumor model spontaneously induces lymph node metastasis when orthotopically implanted into the inguinal mammary fat pads of SCID mice. Aflibercept (at 25 mg/kg) or vehicle were administered bi-weekly for five weeks in mice pair-matched by bioluminescent photon emission rates into aflibercept treatment and control groups. This continuous aflibercept treatment induced antitumor activity on the primary tumors with 19% T/C based on bioluminescent signal at the end of treatment. Aflibercept also controlled metastatic dissemination to the axillary regions with a median 21-day delay for lymph node invasion in treated mice. Conclusion: In an orthotopic Ewing sarcoma model in mice, repeated administrations of aflibercept reduced tumor perfusion and stabilized tumor growth. In an orthotopic mammary carcinoma model, prolonged treatment with aflibercept inhibited tumor growth and delayed spontaneous lymph node metastasis. These outcomes are consistent with the mechanism of action postulated for aflibercept which, upon prolonged treatment is expected to reduce the density of tumor vasculature and the leakiness of tumor vessels, thereby controlling tumor growth and metastatic dissemination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2338. doi:1538-7445.AM2012-2338</jats:p

Abstract C162: Influence of tumor genotype on aflibercept (VEGF Trap) antitumor activity in preclinical tumors.

Abstract Aflibercept (also called VEGF Trap) is a novel anti-angiogenic agent currently tested in combination with standard chemotherapy in several phase 3 clinical trials including second-line metastatic colorectal cancer. It is a recombinant fusion protein consisting of Vascular Endothelial Growth Factor (VEGF)-binding portions from the extracellular domains of human VEGF Receptors 1 and 2 fused to the Fc portion of the human IgG1. Aflibercept acts as a soluble decoy receptor that binds to VEGF-A, with high affinity, as well as the related ligands PIGF and VEGF-B. It neutralizes all isoforms of VEGF-A, blocks angiogenesis and effectively suppresses tumor growth in vivo. Aflibercept also binds Placental Growth Factor (P1GF), which has been involved in pathological angiogenesis and tumor progression. Preclinical studies were performed to assess the influence of somatic mutations on the activity of aflibercept against the growth of 37 human tumor xenografts in mice covering 17 different indications. These models were previously documented for KRAS, BRAF, and PIK3CA activating mutations and PTEN protein deficiency according to the Catalogue of Somatic Mutations in Cancer (COSMIC) database, literature, and internal studies. Aflibercept was administered twice weekly at 40 mg/kg. Aflibercept was found highly active in 6 models with tumor growth inhibition (ΔT/ΔC) ≤ 0%, very active in 9 models (ΔT/ΔC 0–10%), displayed some activity in 17 models (ΔT/ΔC 10–40%), and had no effect in 5 xenograft models (ΔT/ΔC &amp;gt; 40%). We stratified the models into two groups according to the tumor response: the “partially reponsive and non-responsive models” with detectable tumor growth under treatment (22/37 models), and the “responsive models” with no detectable tumor growth or regression under treatment (15/37 models). Mutations in KRAS (G12D/V/S, G13D, Q61H) were present in both responsive (3 models) and partially responsive/non-responsive models (4 models). Ten of 28 (36%) KRAS wild-type tumors and 3 of 7 (43%) KRAS mutated tumors were responsive to aflibercept indicating that KRAS status has no significant influence on aflibercept activity on tumors (p=1; Fisher exact test). Similarly, tumor models were also found sensitive to the anti-tumor effects of aflibercept whatever the BRAF and PIK3CA genotype status, and PTEN protein expression status. In summary, the status of KRAS, BRAF, PIK3CA and PTEN in these preclinical tumor models did not significantly influence the response to aflibercept. The impact of these genetic alterations on aflibercept activity will be further explored in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C162.</jats:p

High-throughput high-volume nuclear imaging for preclinical in vivo compound screening§

Abstract Background Preclinical single-photon emission computed tomography (SPECT)/CT imaging studies are hampered by low throughput, hence are found typically within small volume feasibility studies. Here, imaging and image analysis procedures are presented that allow profiling of a large volume of radiolabelled compounds within a reasonably short total study time. Particular emphasis was put on quality control (QC) and on fast and unbiased image analysis. Methods 2–3 His-tagged proteins were simultaneously radiolabelled by 99mTc-tricarbonyl methodology and injected intravenously (20 nmol/kg; 100 MBq; n = 3) into patient-derived xenograft (PDX) mouse models. Whole-body SPECT/CT images of 3 mice simultaneously were acquired 1, 4, and 24 h post-injection, extended to 48 h and/or by 0–2 h dynamic SPECT for pre-selected compounds. Organ uptake was quantified by automated multi-atlas and manual segmentations. Data were plotted automatically, quality controlled and stored on a collaborative image management platform. Ex vivo uptake data were collected semi-automatically and analysis performed as for imaging data. Results >500 single animal SPECT images were acquired for 25 proteins over 5 weeks, eventually generating >3500 ROI and >1000 items of tissue data. SPECT/CT images clearly visualized uptake in tumour and other tissues even at 48 h post-injection. Intersubject uptake variability was typically 13% (coefficient of variation, COV). Imaging results correlated well with ex vivo data. Conclusions The large data set of tumour, background and systemic uptake/clearance data from 75 mice for 25 compounds allows identification of compounds of interest. The number of animals required was reduced considerably by longitudinal imaging compared to dissection experiments. All experimental work and analyses were accomplished within 3 months expected to be compatible with drug development programmes. QC along all workflow steps, blinding of the imaging contract research organization to compound properties and automation provide confidence in the data set. Additional ex vivo data were useful as a control but could be omitted from future studies in the same centre. For even larger compound libraries, radiolabelling could be expedited and the number of imaging time points adapted to increase weekly throughput. Multi-atlas segmentation could be expanded via SPECT/MRI; however, this would require an MRI-compatible mouse hotel. Finally, analysis of nuclear images of radiopharmaceuticals in clinical trials may benefit from the automated analysis procedures developed

Investigation of 6-[¹⁸F]-Fluoromaltose as a Novel PET Tracer for Imaging Bacterial Infection

<div><p></p><p>Despite advances in the field of nuclear medicine, the imaging of bacterial infections has remained a challenge. The existing reagents suffer from poor sensitivity and specificity. In this study we investigate the potential of a novel PET (positron emission tomography) tracer that overcomes these limitations.</p><p>Methods</p><p>6-[¹⁸F]-fluoromaltose was synthesized. Its behavior <i>in vitro</i> was evaluated in bacterial and mammalian cultures. Detailed pharmacokinetic and biodistribution profiles for the tracer were obtained from a murine model.</p><p>Results</p><p>6-[¹⁸F]-fluoromaltose is taken up by multiple strains of pathogenic bacteria. It is not taken up by mammalian cancer cell lines. 6-[¹⁸F]-fluoromaltose is retained in infected muscles in a murine model of bacterial myositis. It does not accumulate in inflamed tissue.</p><p>Conclusion</p><p>We have shown that 6-[¹⁸F]-fluoromaltose can be used to image bacterial infection <i>in vivo</i> with high specificity. We believe that this class of agents will have a significant impact on the clinical management of patients.</p></div

<i>In vivo</i> characterization of 6-[¹⁸F]-fluoromaltose.

<p>A) 3D color map from a PET/CT scan of a mouse bearing <i>E.coli</i> induced infection on the left thigh (red arrow) 1 hr after tail-vein injection of 7.4MBq of 6-[¹⁸F]-fluoromaltose. B) Region of interest analysis (ROIs) from PET/CT images at the indicated time points (n = 4 for each time point) * indicates statistical significance with p<0.05. C) Time activity curve showing accumulation of 6-[¹⁸F]-fluoromaltose in the infected muscle (n = 3).</p

Specificity of 6-[¹⁸F]-fluoromaltose for viable bacteria.

<p>A) A coronal slice from a PET/CT image of a mouse bearing 10⁸ CFU of viable bioluminescent <i>E.coli</i> on the right thigh (red arrow) and 10⁸ CFU of heat-inactivated <i>E.coli</i> on the left thigh, 1hr after tail-vein injection of 7.4MBq of 6-[¹⁸F]-fluoromaltose B) A transverse slice from the same mouse shown in A), with arrows indicating sites of viable and heat inactivated bacteria. C) Bioluminescent image of the mouse shown in A). D) ROI analysis from PET/CT scan of mice (n = 3). * indicates statistical significance.</p

<i>In vitro</i> characterization of 6-[¹⁸F]-fluoromaltose.

<p>A) Uptake of 6-[¹⁸F]-fluoromaltose in the indicated strains of bacteria for 60 minutes. B) 1 hour uptake of 6-[¹⁸F]-fluoromaltose in the mammalian cell lines, MDA MB231 and HeLa and its uptake in <i>E.coli</i> in the presence of 1 mM maltose. C) Bioluminescence imaging of a macrophage cell line J774 infected with a bioluminescent strain of <i>Listeria monocytogenes.</i> D) 1 hour uptake of 6-[¹⁸F]-fluoromaltose in the bioluminescent strain of <i>Listeria monocytogenes</i> and in macrophage cell line J774 with and without intracellular <i>Listeria</i> infections.</p

Uptake of 6-[¹⁸F]-fluoromaltose in infection versus inflammation.

<p>A) Ex-vivo biodistribution of 6-[¹⁸F]-fluoromaltose in mice bearing E.coli induced myositis, 2 h and 4 h after tail-vein injection of 7.4MBq of tracer. B) Ex-vivo biodistribution of 6-[¹⁸F]-fluoromaltose in mice bearing turpentine oil induced sterile abscess, 2 h after tail-vein injection of 7.4MBq of tracer. C) Representative gram stained muscle sections with a black arrow indicating presence of <i>E.coli</i> in the infected muscle section. D) Representative H&E stained muscle sections showing neutrophil infiltration in inflamed muscle.</p