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Medulloblastoma (MB) is the most frequent solid tumor in children, localized in the brain’s posterior fossa. Its standard of care comprises maximal resection surgery followed by craniospinal irradiation and chemotherapy. Despite a long-term survival rate of 70%, wide disparities among patients have been observed. Relevant targets for naive and recurrent MB are urgently needed. Primary and recurrent MBs are characterized by aggressive invasion into surrounding brain tissue, active angiogenesis, and radioresistance. Integrin-αvβ3 was a major driver of these features in glioblastoma. Nevertheless, such observations have not yet been reported in MB. Integrin-αvβ3 was found to be expressed in a subset of MB patients. We investigated the role of integrin-αvβ3 using MB-derived cell lines with β3-subunit depletion or overexpression both in vitro and in vivo. Radioresistant MB cell lines were generated and showed increased integrin-αvβ3 expression, which correlated with increased susceptibility to pharmacological integrin-αvβ3 inhibition with cilengitide, a competitive ligand mimetic. Finally, we conducted single-photon emission computed tomography (SPECT)/magnetic resonance imaging (MRI) studies on orthotopic models using a radiolabeled integrin-αvβ3 ligand (99mTc-RAFT-RGD). This approach offers the prospect of a novel predictive imaging modality in MB. Altogether, our data pave the way for SPECT/MRI-based selection of a subpopulation of MB patients eligible for integrin-αvβ3-directed therapies.SIGNIFICANCEThis study demonstrates integrin-αvβ3’s fundamental role in MB tumorigenicity and radioresistance and the effect of its expression on cilengitide functional activity.

BackgroundSurgical resection followed by chemo-radiation postpones glioblastoma (GBM) progression and extends patient survival, but these tumours eventually recur. Multimodal treatment plans combining intraoperative techniques that maximise tumour excision with therapies aiming to remodel the immunologically cold GBM microenvironment could improve patients’ outcomes. Herein, we report that targeted photoimmunotherapy (PIT) not only helps to define tumour location and margins but additionally promotes activation of anti-GBM T cell response.MethodsEGFR-specific affibody molecule (ZEGFR:03115) was conjugated to IR700. The response to ZEGFR:03115-IR700-PIT was investigated in vitro and in vivo in GBM cell lines and xenograft model. To determine the tumour-specific immune response post-PIT, a syngeneic GBM model was used.ResultsIn vitro findings confirmed the ability of ZEGFR:03115-IR700 to produce reactive oxygen species upon light irradiation. ZEGFR:03115-IR700-PIT promoted immunogenic cell death that triggered the release of damage-associated molecular patterns (DAMPs) (calreticulin, ATP, HSP70/90, and HMGB1) into the medium, leading to dendritic cell maturation. In vivo, therapeutic response to light-activated conjugate was observed in brain tumours as early as 1 h post-irradiation. Staining of the brain sections showed reduced cell proliferation, tumour necrosis, and microhaemorrhage within PIT-treated tumours that corroborated MRI T2*w acquisitions. Additionally, enhanced immunological response post-PIT resulted in the attraction and activation of T cells in mice bearing murine GBM brain tumours.ConclusionsOur data underline the potential of ZEGFR:03115-IR700 to accurately visualise EGFR-positive brain tumours and to destroy tumour cells post-conjugate irradiation turning an immunosuppressive tumour environment into an immune-vulnerable one.

NeoB is a radiotracer targeting the gastrin-releasing peptide receptor (GRPR), a G-protein–coupled receptor expressed in various cancers. The aim of the present study was to evaluate the biodistribution and efficacy of this new therapeutic agent in Gastrointestinal Stromal Tumors (GIST). Eighty-two SCID mice bearing GIST-882 tumors were employed. [177Lu]Lu-NeoB biodistribution was evaluated up to seven days by organ sampling (200 pmol/0.8 MBq, i.v.). For efficacy evaluation, mice received either saline, 400 pmol or 800 pmol of [177Lu]Lu-NeoB (37MBq, 1/w, 3 w, i.v.). SPECT/CT imaging was performed at 24 h, and tumor volume was determined up to 100 days. Elevated and specific [177Lu]Lu-NeoB uptake was found in the GIST tumor, as demonstrated by in vivo competition (19.1 ± 3.9 %ID/g vs. 0.3 ± 0.1 %ID/g at 4h). [177Lu]Lu-NeoB tumor retention (half-life of 40.2 h) resulted in elevated tumor-to-background ratios. Tumor volumes were significantly reduced in both treated groups (p &lt, 0.01), even leading to complete tumor regression at the 400 pmol dose. [177Lu]Lu-NeoB exhibited excellent pharmacokinetics with elevated and prolonged tumor uptake and low uptake in non-target organs such as pancreas. The potential of this new theragnostic agent in different indications, including GIST, is under evaluation in the FIH [177Lu]Lu-NeoB clinical trial.

There is an urgent need to develop therapeutic approaches that can increase the response rate to immuno-oncology agents. Photoimmunotherapy has recently been shown to generate anti-tumour immunological responses by releasing tumour-associated antigens from ablated tumour cell residues, thereby enhancing antigenicity and adjuvanticity. Here, we investigate the feasibility of a novel HER2-targeted affibody-based conjugate (ZHER2:2395-IR700) selectively to induce cancer cell death in vitro and in vivo. The studies in vitro confirmed the specificity of ZHER2:2395-IR700 binding to HER2-positive cells and its ability to produce reactive oxygen species upon light irradiation. A conjugate concentration- and light irradiation-dependent decrease in cell viability was also demonstrated. Furthermore, light-activated ZHER2:2395-IR700 triggered all hallmarks of immunogenic cell death, as defined by the translocation of calreticulin to the cell surface, and the secretion of ATP, HSP70/90 and HMGB1 from dying cancer cells into the medium. Irradiating a co-culture of immature dendritic cells (DCs) and cancer cells exposed to light-activated ZHER2:2395-IR700 enhanced DC maturation, as indicated by augmented expression of CD86 and HLA-DR. In SKOV-3 xenografts, the ZHER2:2395-IR700-based phototherapy delayed tumour growth and increased median overall survival. Collectively, our results strongly suggest that ZHER2:2395-IR700 is a promising new therapeutic conjugate that has great potential to be applicable for photoimmunotherapy-based regimens.

International audience; PURPOSE:This preclinical study aimed to evaluate placental oxygenation in pregnant rats by real-time photoacoustic (PA) imaging on different days of gestation and to specify variations in placental oxygen saturation under conditions of maternal hypoxia and hyperoxygenation.MATERIAL AND METHODS:Placentas of fifteen Sprague-Dawley rats were examined on days 14, 17, and 20 of pregnancy with a PA imaging system coupled to high-resolution ultrasound imaging. Pregnant rats were successively exposed to hyperoxygenated and hypoxic conditions by changing the oxygen concentration in inhaled gas. Tissue oxygen saturation was quantitatively analyzed by real-time PA imaging in the skin and 3 regions of the placenta. All procedures were performed in accordance with applicable ethical guidelines and approved by the animal care committee.RESULTS:Maternal hypoxia was associated with significantly greater decrease in blood oxygen saturation (ΔO2 Saturation) in the skin (70.74% ±7.65) than in the mesometrial triangle (32.66% ±5.75) or other placental areas (labyrinth: 18.58% ± 6.61; basal zone: 13.13% ±5.72) on different days of pregnancy (P

The development of novel therapeutic strategies allowing the destruction of tumour cells while sparing healthy tissues is one of the main challenges of cancer chemotherapy. Here, we report on the design and antitumour activity of a low-molecular-weight drug delivery system programmed for the selective release of the potent monomethylauristatin E in the tumour microenvironment of solid tumours. After intravenous administration, this compound binds covalently to plasmatic albumin through Michael addition, thereby enabling its passive accumulation in tumours where extracellular β-glucuronidase initiates the selective release of the drug. This targeting device produces outstanding therapeutic efficacy on orthotopic triple-negative mammary and pancreatic tumours in mice (50% and 33% of mice with the respective tumours cured), leading to impressive reduction or even disappearance of tumours without inducing side effects.

Objectives We have developed a relevant preclinical model associated with a specific imaging protocol dedicated to onco-pharmacology studies in mice. Materials and Methods We optimized both the animal model and an ultrasound imaging procedure to follow up longitudinally the lung tumor growth in mice. Moreover we proposed to measure by photoacoustic imaging the intratumoral hypoxia, which is a crucial parameter responsible for resistance to therapies. Finally, we compared ultrasound data to x-ray micro computed tomography and volumetric measurements to validate the relevance of this approach on the NCI-H460 human orthotopic lung tumor. Results This study demonstrates the ability of ultrasound imaging to detect and monitor the in vivo orthotopic lung tumor growth by high resolution ultrasound imaging. This approach enabled us to characterize key biological parameters such as oxygenation, perfusion status and vascularization of tumors. Conclusion Such an experimental approach has never been reported previously and it would provide a nonradiative tool for assessment of anticancer therapeutic efficacy in mice. Considering the absence of ultrasound propagation through the lung parenchyma, this strategy requires the implantation of tumors strictly located in the superficial posterior part of the lung.

Introduction The aim of this study is to develop new strategies for the exploration of relevant preclinical lung tumor models dedicated to onco-pharmacology studies in mice. Here we refine a translational approach while overcoming physical ultrasound (US) imaging limitations allowing lung tumors exploration. Our data were compared to Computed Tomography (CT) and Bioluminescence (BLI) aiming to validate this promising approach. Methods Human lung cancer cells NCI-H460-luc2 were orthotopically xenografted in Balb/c nude mice. BLI was performed 7 days after implantation then once a week until the end of the study (Day 28) using an IVIS-Lumina II (Perkin Elmer). Tumors were imaged with the Vevo®LAZR System (FUJIFILM VisualSonics Inc.), 3D scans of US, Contrast Enhanced Ultrasound (CEUS) (Vevo MicroMarkerTM) and photoacoustic (PA) image being recorded digitally. A comparison between in vivo US and CT was achieved with additional ex vivo weight and volumetric measurements. Results The control of the tumor cell implantation was determined by BLI allowing the early quantification of tumor burden. 3D US measurements made possible longitudinal tumor volumes assessment. Compared to the other in vivo measurements, results clearly indicated the gross correlation of volumes processed with US and CT (R2 = 0.75). Such a correlation is also observed with ex vivo US weight and volumetric measurements (R2 = 0.72, R2 = 0.75 and R2 = 0.65 respectively). PA imaging evidenced the hemoglobin saturation with oxygen inside tumors, and targeted CEUS imaging allowed assessing the relative VEGFR2 expression in lung tumors. Conclusions This study proved the ability of US imaging to detect and control the in vivo orthotopic lung tumor growth. This real-time approach is fast while being completely inert regarding the in situ proliferation, as compared to dosimetry related to repeated CT examinations. Furthermore it allows overcoming the limitation of BLI at the time tumors become hypoxic since the luciferase/luciferin reaction is strictly dependent upon O2 and ATP. We described here a PA and US imaging strategy to follow the orthotopic lung tumors growth and consider biomarkers such as VEGFR2 expression or hypoxia in vivo for the first time. This procedure would be of great interest to longitudinally investigate tumor proliferation in animals when evaluating new anticancer therapies efficacy, avoiding any perturbation of the tumor progression compared to other available imaging modalities. Citation Format: Florian Raes, Julien Sobilo, Sharuja Natkunarajah, Philippe Trochet, Dieter Fuchs, Stephanie Lerondel, Alain Le Pape. Novel imaging strategy to assess the antitumor efficacy of treatments in an orthotopic mouse lung cancer model using ultrasound and photoacoustic imaging. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4200.

Background Photoacoustic (PA) imaging is a recent promising modality combining advantages of ultrasounds (resolution) and photon imaging (sensitivity), thus providing anatomical and functional data. PA can be implemented by using the natural endogenous contrast agent hemoglobin, providing informations such as the oxygen saturation of hemoglobin (SO2). In the field of oncology, when tumors are growing, the central area becomes hypoxic. This phenomenon does not interfere with tumor growth because cancer cells can switch from aerobic metabolism (using O2) to anaerobic metabolism. Hypoxia is a crucial parameter that needs to be investigated and quantified as it is responsible for radio and chemoresistances. Among tumor cell lines, the MIA PaCa2 cell line (pancreatic adenocarcinoma) appeared quite relevant for a preclinical approach. The aim of this work was to assess the hypoxic status of tumors in a MIA PaCa-2 Luc+ orthotopic cancer model, and to compare bioluminescent signal and tumor volumes in a longitudinal study. Methods The tumor proliferation was monitored by bioluminescence imaging (BLI) and tumor volume with Ultrasound (US). BLI was performed using the IVIS-Lumina II imaging system (PerkinElmer) whereas US and PA with the VEVO LAZR system (VisualSonics). For the experiments, human ductal adenocarcinoma MIA PaCa-2-luc model was orthotopically implanted in nude mice. For the experiment, 7 days after engraftments, tumors were explored both by BLI and US 3 times a week during 12 weeks. Moreover, tumors were investigated by PA imaging so that average values of SO2 were calculated. Results and Discussion BLI was selected as it is one of the reference techniques to assess tumor growth in vivo. Overall, our data demonstrated that in our model there was a correlation between BLI and US measurements during the first four weeks of the study. But we observed a significant decrease in BLI signals whereas tumor volumes were continuously increasing. Bioluminescence reaction needs ATP and O2 to occur. In hypoxic situations, light emission becomes not proportional to tumor volume, due to the dependence of luciferin-luciferase reaction towards the metabolism. It appeared that BLI was no longer relevant in the study of advanced stages of MiaPaCa-2 tumors. PA imaging confirmed that there was hypoxia into tumors, approximately since the 5th week. So we were able, thanks to this approach, to accurately map the areas of hypoxia within the tumor. These results confirm that BLI should be used with caution, in particular during an evaluation of a therapeutic drug. The combination of these three methods (BLI, US, PA) should therefore allow to precisely characterize orthotopic tumor models including the control of the appearance or location of a hypoxic state within tumors, while specifying the limits of bioluminescence. Citation Format: Florian Raes, Thomas Barre, Jithin Jose, Philippe Trochet, Stéphanie Lerondel, Alain Le Pape. Characterization of tumor hypoxia by photoacoustic imaging and limitations of bioluminescence imaging in a Mia PaCa2-luc orthotopic model of pancreatic carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2048. doi:10.1158/1538-7445.AM2014-2048