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Figure S1. Myeloid cell distribution in naïve and LLC-bearing mice.

Supplemental figure legends

Overexpression of P-glycoprotein (P-gp) is associated with multidrug resistance. Since sorafenib (NEXAVAR®) is a P-gp (an efflux protein of ATP-binding cassette family) substrate, we tested whether bevacizumab (AVASTIN®), a monoclonal antibody directed toward VEGF (Vascular Endothelial Growth Factor) and sorafenib could modulate P-gp functionality. In vitro two human ovarian carcinoma cells (IGROV1) overexpressing or weakly expressing P-gp were used. Bevacizumab and sorafenib effects on P-gp functionality were evaluated by measuring doxorubicin intracellular accumulation. In vivo study was to document whether bevacizumab could modify sorafenib disposition in mice. Therefore, concentrations of sorafenib were determined by HPLC in plasma of mice bearing a human colorectal carcinoma xenograft when sorafenib is given orally (5 mg/kg) on day 4, alone or after a pretreatment with bevacizumab (5 mg/kg IP) on days 1 and 3. In vitro a significant doxorubicin accumulation and reversion of doxorubicin resistance in P-gp expressing cell lines were observed with bevacizumab or sorafenib pretreatment In vivo, sorafenib AUC was 1.44 fold significantly higher in bevacizumab pretreated group and Cmax was 1.35 fold higher in bevacizumab-pretreated group. Mean residence time of sorafenib increased in the presence of bevacizumab, this increase reflects an improvement of sorafenib bioavailability after bevacizumab pretreatment. We may conclude that bevacizumab pretreatment decreases P-gp functionality and increases doxorubicin intracellular accumulation in vitro and sorafenib plasma concentrations in vivo.

The metastatic progression of cancer remains a major issue in patient treatment. However, the molecular and cellular mechanisms underlying this process remain unclear. Here, we use primary explants and organoids from patients harboring mucinous colorectal carcinoma (MUC CRC), a poor-prognosis histological form of digestive cancer, to study the architecture, invasive behavior and chemoresistance of tumor cell intermediates. We report that these tumors maintain a robust apico-basolateral polarity as they spread in the peritumoral stroma or organotypic collagen-I gels. We identified two distinct topologies – MUC CRCs either display a conventional ‘apical-in’ polarity or, more frequently, harbor an inverted ‘apical-out’ topology. Transcriptomic analyses combined with interference experiments on organoids showed that TGFβ and focal adhesion signaling pathways are the main drivers of polarity orientation. Finally, we show that the apical-out topology is associated with increased resistance to chemotherapeutic treatments in organoids and decreased patient survival in the clinic. Thus, studies on patient-derived organoids have the potential to bridge histological, cellular and molecular analyses to decrypt onco-morphogenic programs and stratify cancer patients. This article has an associated First Person interview with the first author of the paper.

BackgroundIntratumorous immunotherapy for cancer is currently thriving. The aim of such local strategy is to improve the therapeutic index of these treatments, for higher on-target/on-tumor activity and less on-target/off-tumor adverse events. Strategies allowing for slow release of anti-CTLA4 in the tumor microenvironment could improve their clinical efficacy.The purpose of the study was to develop a radiopaque delivery platform to improve the targeting and exposure of intratumorous anti-CTLA4 antibodies for cancer immunotherapy.MethodsPickering emulsions of anti-CTLA4 antibodies were formulated with radiopaque ethiodized oil and poly-lactic-co-glycolic acid (PLGA) nanoparticles. We characterized the microscopic aspect and stability of such emulsions using Turbiscan. We monitored the release of anti-CTLA4 over time from these emulsions and evaluated their structure using mass spectrometry. We then tested the functionality of the released antibodies by preforming ex vivo competitive binding assays. Finally, we assessed the in vivo efficacy of intratumorous anti-CTLA4 Pickering emulsions.ResultsPickering emulsions of ethiodized oil and PLGA nanoparticles (PEEPs) resulted in a radiopaque water-in-oil emulsion with average internal phase droplet size of 42±5 µm at day 7. Confocal microscopy showed that anti-CTLA4 antibodies were effectively encapsulated by ethiodized oil with PLGA nanoparticles located at the interface between the aqueous and the oily phase. Turbiscan analysis showed that emulsions were stable with continuous and progressive release of anti-CTLA4 antibodies reaching 70% at 3 weeks. Structural and functional analysis of the released antibodies did not show significant differences with native anti-CTLA4 antibodies. Finally, intratumorous anti-CTLA4 PEEPs were able to eradicate tumors and cure mice in a syngeneic immunocompetent preclinical tumor model.ConclusionPickering emulsions of ethiodized oil and PLGA is an innovative radiopaque delivery platform that does not alter the functionality of anti-CTLA4 immune checkpoint antibodies. Beyond local anti-CTLA4 applications, these emulsions might be used with other therapeutic molecules for optimal intratumorous or intra-arterial delivery of novel cancer immunotherapies.

International audience; Objective: The purpose of the present work is to assess the ability of sine waves to perform Electrochemotherapy (ECT) and to study the dependence of the frequency of the applied sine wave on the treatment efficacy. Methods: A subcutaneous tumor model in mice was used, and the electric field was delivered in combination with bleomycin. Sinusoidal electric fields of different frequencies, amplitudes, and durations were compared to square waves. Computer simulations were additionally performed. Results: The results confirmed the ability of a sinusoidal electric field to obtain successful ECT responses. A strong dependence with frequency was obtained. The efficacy of the treatment decreased when the frequency of the sine waves was increased. At low sinusoidal frequency, the efficacy of the treatment is very similar to that obtained with a square wave. The collateral effects such as skin burns and muscle contractions decreased for the highest frequency assayed. Conclusion: The use of sine wave burst represent a feasible option for the treatment of cancer by ECT. Significance: These results could have important implications for the treatment of cancer in the clinical world where ECT is performed with DC square pulses.

Osteosarcoma is the most prevalent primary bone malignancy in children and young adults. Resistance to chemotherapy remains a key challenge for effective treatment of patients with osteosarcoma. The aim of the present study was to investigate the preventive role of metallothionein-2A (MT2A) in response to cytotoxic effects of chemotherapy. A panel of human and murine osteosarcoma cell lines, modified for MT2A were evaluated for cell viability, and motility (wound healing assay). Cell-derived xenograft models were established in mice. FFPE tumour samples were assessed by IHC. In vitro experiments indicated a positive correlation between half-maximal inhibitory concentration (IC50) for drugs in clinical practice, and MT2A mRNA level. This reinforced our previously reported correlation between MT2A mRNA level in tumour samples at diagnosis and overall survival in patients with osteosarcoma. In addition, MT2A/MT2 silencing using shRNA strategy led to a marked reduction of IC50 values and to enhanced cytotoxic effect of chemotherapy on primary tumour. Our results show that MT2A level could be used as a predictive biomarker of resistance to chemotherapy, and provide with preclinical rational for MT2A targeting as a therapeutic strategy for enhancing anti-tumour treatment of innate chemo-resistant osteosarcoma cells.

International audience; Squalene-adenosine (SQAd) nanoparticles (NPs) were found to display promising pharmacological activity similar to many other nanomedicines, but their long-term stability was still limited, and their preparation required specific know-how and material. These drawbacks represented important restrictions for their potential use in the clinic. Freeze-drying nanoparticles is commonly presented as a solution to allow colloidal stability, but this process needs to be adapted to each nanoformulation. Hence, we aimed at developing a specific protocol for freeze-drying SQAd NPs while preserving their structural features. NPs were lyophilised, resuspended and analysed by dynamic light scattering, atomic force microscopy and small-angle scattering. Among four different cryoprotectants, trehalose was found to be the most efficient in preserving NPs physico-chemical characteristics. Interestingly, we identified residual ethanol in NP suspensions as a key parameter which could severely affect the freeze-drying outcome, leading to NPs aggregation. Long-term stability was also assessed. No significant change in size distribution or zeta potential could be detected after three-month storage at 4?°C. Finally, freeze-dried NPs innocuity was checked in vitro on cultured hepatocytes and in vivo on mice. In conclusion, optimisation of freeze-drying conditions resulted in safe lyophilised SQAd NPs that can be easily stored, shipped and simply reconstituted into an injectable form.

The Fas receptor ligand FasL regulates immune cell levels by inducing apoptosis of Fas receptor–positive cells. Here, we studied the impact of host FasL on tumor development in mice. Genetically targeting FasL in naïve mice increased myeloid cell populations, but, in marked contrast, it reduced the levels of myeloid-derived suppressor cells (MDSC) in mice bearing Lewis lung carcinoma tumors. Analysis of the MDSC subset distribution revealed that FasL deficiency skewed cell populations toward the M-MDSC subset, which displays a highly immunosuppressive activity. Furthermore, tumor-bearing mice that were FasL-deficient displayed an enhanced proportion of tumor-associated macrophages and regulatory T cells. Overall, the immunosuppressive environment produced by FasL targeting correlated with reduced survival of tumor-bearing mice. These results disclose a new role for FasL in modulating immunosuppressive cells. Cancer Res; 75(20); 4292–301. ©2015 AACR.

Metastases account for 90% of cancer-related deaths; thus, it is vital to understand the biology of tumour dissemination. Here, we collected and monitored >50 patient specimens ex vivo to investigate the cell biology of colorectal cancer (CRC) metastatic spread to the peritoneum. This reveals an unpredicted mode of dissemination. Large clusters of cancer epithelial cells displaying a robust outward apical pole, which we termed tumour spheres with inverted polarity (TSIPs), were observed throughout the process of dissemination. TSIPs form and propagate through the collective apical budding of hypermethylated CRCs downstream of canonical and non-canonical transforming growth factor-β signalling. TSIPs maintain their apical-out topology and use actomyosin contractility to collectively invade three-dimensional extracellular matrices. TSIPs invade paired patient peritoneum explants, initiate metastases in mice xenograft models and correlate with adverse patient prognosis. Thus, despite their epithelial architecture and inverted topology TSIPs seem to drive the metastatic spread of hypermethylated CRCs.

Suppressor of cytokine signaling (SOCS) 1 is an inducible negative regulator of cytokine signaling but its role in human cancer is not completely established. Here we report that, while SOCS1 is expressed in normal colonic epithelium and colon adenocarcinomas, its level decreases during progression of colon adenocarcinomas, the lowest level being found in the most aggressive stage and least differentiated carcinomas. Forced expression of SOCS1 in metastatic colorectal SW620 cells reverses many characteristics of Epithelial–Mesenchymal Transition (EMT), as highlighted by the disappearance of the transcription factor ZEB1 and the mesenchymal form of p120ctn and the re-expression of E-cadherin. Furthermore, miRNA profiling indicated that SOCS1 also up-regulates the expression of the mir-200 family of miRNAs, which can promote the mesenchymal–epithelial transition and reduce tumor cell migration. Accordingly, overexpression of SOCS1 induced cell morphology changes and dramatically reduced tumor cell invasion in vitro . When injected in nude mice, SOCS1-expressing SW620 cells induced metastases in a smaller number of animals than parental SW620 cells, and did not generate any adrenal gland or bone metastasis. Overall, our results suggest that SOCS1 controls metastatic progression of colorectal tumors by preventing the mesenchymal–epithelial transition (MET), including E-cadherin expression. This pathway may be associated with survival to colorectal cancer by reducing the capacity of generating metastases.

Introduction Le traitement des cancers constitue un probleme de sante publique et fait appel differentes strategies therapeutiques, cependant l’intervention de transporteurs membranaires d’efflux, comme la P-glycoproteine (P-gp) joue un role important sur le plan pharmacocinetique, pharmacogenetique et dans la resistance aux medicaments anticancereux. Le blocage de l’angiogenese est une des strategies utilisees, pour restaurer la sensibilite de l’endothelium tumoral et des cellules tumorales. Ce travail avait pour objectif de documenter l’effet d’un pretraitement par les therapies ciblees anti-angiogeniques tels que le bevacizumab et le sorafenib sur l’activite des transporteurs d’efflux avec une reversion de la resistance des cellules tumorales et endotheliales surexprimant des transporteurs d’efflux et en particulier la P-gp. Materiels et methode In vitro, nous avons utilise deux lignees derivees d’un carcinome ovarien humain : – IGROV1-PAR, exprime peu la P-gp et sensible a la doxorubicine (lignee controle) ; – IGROV1-DXR (cellules de cancer ovarien), surexprimant la P-gp et resistante a la doxorubicine. Les concentrations intracellulaires de la doxorubucine ont ete mesurees par cytometrie de flux. In vivo pour l’etude du pretraitement par le bevacizumab sur les concentrations de l’irinotecan de son metabolite actif (SN-38) et du sorafenib. Les souris ont ete randomisees pour chaque medicament en deux groupes : groupe pretraite par le bevacizumab et groupe non petraite. Les dosages plasmatiques et tumoraux ont ete effectues par HPLC avec un detecteur a fluorescence (532 nm et 515 nm) pour l’irinotecan et son metabolite actif et un detecteur UV-Vis (255 nm) pour le sorafenib. Les pincipaux parametres pharmacocinetiques ont ete analyses a l’aide du logiciel Winonline : Cmax, Tmax, t1/2, AUC, MRT. Resultats et discussion Nos travaux ont montre in vitro qu’un pretraitement avec le bevacizumab et le sorafenib entrainait une reversion de la resistance a la doxorubicine (substrat de la P-gp) dans un modele de cellules IGR OV1-DXR (resistantes a la doxorubicine et surexprimant la P-gp) par modulation de la fonctionnalite de la P-glycoproteine. Cette reversion a ete objectivee par l’augmentation significative des concentrations intracellulaires de la doxorubicine dans les cellules IGR OV1-DXR. In vivo chez la souris xenogreffee de cancer colorectal humain, un pretraitement par le bevacizumab a entraine une augmentation significative des concentrations plasmatiques du SN38 (metabolite actif de l’irinotecan et substrat de la P-gp), de meme que de ĺAUC et de la Cmax plasmatique de l’irinotecan (substrat de la P-gp) apres administration orale de l’irinotecan. Une augmentation significative de ĺAUC plasmatique du sorafenib apres un pretraitement par le bevacizumab a ete egalement observee sur le meme modele experimental. Conclusion Ces resultats in vitro et in vivo s’expliquent par plusieurs mecanismes, notamment une inhibition ou une variation de la fonctionnalite des transporteurs d’efflux en particulier la P-gp, une reduction de ĺangiogenese, ainsi que d’une restauration de la sensibilite de l’endothelium tumoral et des cellules tumorales.

KRAS mutation is a negative predictive prognostic factor during metastatic colorectal cancer treatment with antiepidermal growth factor receptor antibodies. For affected patients, new therapeutics must be explored. Our objective was to study efficacy of two drugs with different mechanisms of action, everolimus (mTOR inhibitor) and lapatinib (tyrosine kinase inhibitor), in a mouse xenograft model. We chose a model obtained after engraftment of a tumor originating from a human tumor collection. The patient was affected by a metastasis colorectal carcinoma resistant to cetuximab with KRAS mutation. From a previous study in mice, we know that everolimus is a P-glycoprotein (P-gp) substrate and that a lapatinib pretreatment increases significantly (2.6-fold) everolimus AUC by inhibiting its intestinal P-gp efflux. We hence tested the effect of these drugs alone or combined. Mice bearing the xenografts were divided in four groups: control, lapatinib, everolimus, and L/E group (L/E: 2 days of lapatinib 200 mg/kg and then 3 days of everolimus 1 mg/kg). Tumor volumes and treatment toxicities were evaluated. Sixteen days after treatment initiation, the group L/E was the first one in which tumor volume average was significantly lower than the one of control group (193 ± 90 vs. 395 ± 171 mm(3) ; P = 0.0025). After 4 weeks of treatment, inhibition of tumor growth in lapatinib, everolimus, and L/E groups reached, respectively, 49, 53, and 57%. Each drug showed significant antitumor activity. Only moderate hematologic toxicity signs were observed. These results lead to new perspectives for new oral drugs in metastatic KRAS-mutated colorectal cancer resistant to standard chemotherapy.

In vivo high-resolution imaging of tumor development is possible through dorsal skinfold chamber implantable on mice model. However, current intravital imaging systems are weakly tolerated along time by mice and do not allow multimodality imaging. Our project aims to develop a new chamber for: 1- long-term micro/macroscopic visualization of tumor (vascular and cellular compartments) and tissue microenvironment; and 2- multimodality imaging (photonic, MRI and sonography). Our new experimental device was patented in March 2014 and was primarily assessed on 75 mouse engrafted with 4T1-Luc tumor cell line, and validated in confocal and multiphoton imaging after staining the mice vasculature using Dextran 155KDa-TRITC or Dextran 2000kDa-FITC. Simultaneously, a universal stage was designed for optimal removal of respiratory and cardiac artifacts during microscopy assays. Experimental results from optical, ultrasound (Bmode and pulse subtraction mode) and MRI imaging (anatomic sequences) showed that our patented design, unlike commercial devices, improves longitudinal monitoring over several weeks (35 days on average against 12 for the commercial chamber) and allows for a better characterization of the early and late tissue alterations due to tumour development. We also demonstrated the compatibility for multimodality imaging and the increase of mice survival was by a factor of 2.9, with our new skinfold chamber. Current developments include: 1- defining new procedures for multi-labelling of cells and tissue (screening of fluorescent molecules and imaging protocols); 2- developing ultrasound and MRI imaging procedures with specific probes; 3- correlating optical/ultrasound/MRI data for a complete mapping of tumour development and microenvironment.

International audience; In a previous study, we showed that cetuximab, a monoclonal antibody directed towards epidermal growth factor receptor, could inhibit P-glycoprotein (P-gp), an efflux protein of ATP-binding cassette family, and lead to an increased P-gp substrate intracellular concentration. Cetuximab is given with irinotecan to patients with metastasis colorectal cancer who did not respond to irinotecan-based therapy. The mechanism of this successful clinical reversion remains unknown. As irinotecan is a P-gp substrate, we tested here whether cetuximab could modify irinotecan concentration in mice. Therefore, concentrations of irinotecan and of its active metabolite SN-38 were measured by HPLC in plasma and tumour of mice bearing a human colorectal carcinoma xenograft when irinotecan is given orally alone or after a pretreatment with cetuximab. Pharmacokinetic analysis showed no significant modification of irinotecan concentrations but a significant increase (1.7-fold) in SN-38 AUCs in plasma and in tumour after a pretreatment with cetuximab. Those results suggest that cetuximab influence irinotecan distribution into tissues probably due to inhibition of P-gp. As SN-38 is 200-fold more potent than irinotecan, cetuximab could reverse irinotecan resistance by an effect on its active metabolite. Inhibiting SN-38 efflux by P-gp drug transporters in biliary system and tumour can lead to pharmacokinetic modification and a higher anticancer efficacy.

OBJECTIVES The purpose of this study was to investigate the impact of the arterial input on perfusion parameters measured using dynamic contrast-enhanced sonography combined with a deconvolution method after bolus injections of a contrast agent. METHODS The in vitro experiments were conducted using a custom-made setup consisting of pumping a fluid through a phantom made of 3 intertwined silicone pipes, mimicking a complex structure akin to that of vessels in a tumor, combined with their feeding pipe, mimicking the arterial input. In the in vivo experiments, B16F10 melanoma cells were xenografted to 5 nude mice. An ultrasound scanner combined with a linear transducer was used to perform pulse inversion imaging based on linear raw data throughout the experiments. A mathematical model developed by the Gustave Roussy Institute (patent WO/2008/053268) and based on the dye dilution theory was used to evaluate 7 semiquantitative perfusion parameters directly from time-intensity curves and 3 quantitative perfusion parameters from the residue function obtained after a deconvolution process developed in our laboratory based on the Tikhonov regularization method. We evaluated and compared the intraoperator variability values of perfusion parameters determined after these two signal-processing methods. RESULTS In vitro, semiquantitative perfusion parameters exhibited intraoperator variability values ranging from 3.39% to 13.60%. Quantitative parameters derived after the deconvolution process ranged from 4.46% to 11.82%. In vivo, tumors exhibited perfusion parameter intraoperator variability values ranging from 3.74% to 29.34%, whereas quantitative ones varied from 5.00% to 12.43%. CONCLUSIONS Taking into account the arterial input in evaluating perfusion parameters improves the intraoperator variability and may improve the dynamic contrast-enhanced sonographic technique.

Somatic point mutations in the active site of IDH (isocitrate dehydrogenase) 1 and IDH2 genes are observed in acute myeloid leukemia (AML). These mutations lead to the production and accumulation of R-2-hydroxyglutarate (2-HG) in the tumor blast cells as well as in the plasma of patients. High levels of 2-HG have been shown to inhibit alpha-ketoglutarate-dependent dioxygenases, including epigenetic regulators (i.e. histone or DNA demethylases), resulting in altered cellular differentiation. Ex vivo experiments in primary cells from AML IDH2 mutant patients have demonstrated that IDH2 mutant inhibitors are able to revert this phenotype. In order to test the biological activity of AG-221, an oral, reversible and selective inhibitor of mutated IDH2 currently in phase I trials, we developed primary human AML xenograft models. Primary blast cells from 3 AML patients with normal karyotype (n=3), NPM1 mutation (n=3) and FLT3ITD (n=1) were injected into immunodeficient NOD SCID IL2R gamma null (NSG) mice by intrafemoral injection after sub-lethal irradiation. Medullar and peripheral tumoral engraftment was monitored by flow cytometry (using species-specific antibodies) and serum 2-HG measurements. Engraftment of IDH2-R140Q human blast cells was not lethal, but these AML cells persisted over time. Twelve months after injection, IDH2-R140Q blast cell-engrafted mice were randomized into two groups, for treatment with AG-221 (30 mpk) or vehicle (0.5% methylcellulose / 0.2% Tween 80, in water) (n=10 mice; 5 mice per condition). Treatments were administered by oral gavage for 38 days twice per day (BID). Peripheral blood was collected at multiple time points for the determination of pharmacokinetics (PK) and pharmacodynamics (PD), and the assessment of the differentiation effects by AG-221 on human tumor cells. PK/PD analyses showed good plasma exposure of AG-221 and reduction of 2-HG. Furthermore, AG-221 administration also induced a burst of proliferation of human blasts followed by myeloid differentiation starting at day 20 in peripheral blood as measured by the expression of CD11b, CD14, CD15, CD24 and cell morphology. No effects on proliferation or differentiation were seen in the absence of AG-221 administration. Histological analyses of hematopoietic organs in treated animals showed a decrease of infiltrating human cells, as well as obvious morphological changes in the human cell population in AG-221-treated animals compared with vehicle-treated animals. Flow cytometry confirmed the differentiation of the human cells in the spleen and bone marrow of the AG-221-treated mice. Furthermore, cells undergoing differentiation retained the R140Q mutation, demonstrating the differentiating effect of the compound. In conclusion, AG-221 reduced serum 2-HG levels and triggered differentiation of leukemic blast cells engrafted in NSG mice. These results are consistent with what has been observed clinically in IDH2 mutant AML patients treated with AG-221 in a phase I dose escalation trial. Disclosures Quivoron: Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la recherche contre le Cancer (ARC): Grant, Grant Other; AGIOS: Grant Other. David:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la Recherche contre le Cancer (ARC): Grant, Grant Other; Association Laurette Fugain: Grant, Grant Other; AGIOS: Grant Other. Straley:Agios Pharmaceuticals: Employment, Stockholder Other. Travins:Agios Pharmaceuticals: Employment, Stockholder Other. Kim:Agios Pharmaceuticals: Employment, Stockholder Other. Chen:Agios Pharmaceuticals: Employment, Stockholder Other. Zhu:Agios Pharmaceuticals: Employment, Stockholder Other. Bawa:AGIOS: Grant Other. Bernard:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Ligue Nationale contre le cancer (LNCC): Grant, Grant Other; AGIOS: Grant Other. Yang:Agios Pharmaceuticals: Employment, Stockholder Other. Agresta:Agios Pharmaceuticals: Employment, Stockholder Other. de Botton:AGIOS: Grant Other. Yen:Agios: Employment. Penard-Lacronique:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la recherche contre le Cancer (ARC): Grant, Grant Other; AGIOS: Grant Other.

To evaluate the sources of variation influencing the microvascularization parameters measured by dynamic contrast-enhanced ultrasonography (DCE-US).Firstly, we evaluated, in vitro, the impact of the manual repositioning of the ultrasound probe and the variations in flow rates. Experiments were conducted using a custom-made phantom setup simulating a tumor and its associated arterial input. Secondly, we evaluated, in vivo, the impact of multiple contrast agent injections and of examination day, as well as the influence of the size of region of interest (ROI) associated with the arterial input function (AIF). Experiments were conducted on xenografted B16F10 female nude mice. For all of the experiments, an ultrasound scanner along with a linear transducer was used to perform pulse inversion imaging based on linear raw data throughout the experiments. Semi-quantitative and quantitative analyses were performed using two signal-processing methods.In vitro, no microvascularization parameters, whether semi-quantitative or quantitative, were significantly correlated (P values from 0.059 to 0.860) with the repositioning of the probe. In addition, all semi-quantitative microvascularization parameters were correlated with the flow variation while only one quantitative parameter, the tumor blood flow, exhibited P value lower than 0.05 (P = 0.004). In vivo, multiple contrast agent injections had no significant impact (P values from 0.060 to 0.885) on microvascularization parameters. In addition, it was demonstrated that semi-quantitative microvascularization parameters were correlated with the tumor growth while among the quantitative parameters, only the tissue blood flow exhibited P value lower than 0.05 (P = 0.015). Based on these results, it was demonstrated that the ROI size of the AIF had significant influence on microvascularization parameters: in the context of larger arterial ROI (from 1.17 ± 0.6 mm(3) to 3.65 ± 0.3 mm(3)), tumor blood flow and tumor blood volume were correlated with the tumor growth, exhibiting P values lower than 0.001.AIF selection is an essential aspect of the deconvolution process to validate the quantitative DCE-US method.

Local and systemic control of Soft Tissue Sarcoma (STS) remains a clinical challenge. Radiation therapy is part of the standard of care of STS. The narrowness of its therapeutic window represents the main concern for different clinical settings. Thus, local delivery of radiation doses is critical to ensure optimal benefit-risk ratio. NBTXR3, biocompatible hafnium oxide nanoparticles were designed as therapeutics to be activated by ionizing radiation to achieve tumor control by enhancement of local energy deposition. A global non clinical program was implemented in mesenchymal tumor models. In vitro clonogenic survival assays were performed in two human fibrosarcoma cell lines (HT1080 and Hs913t) and a human liposarcoma cell line (SW872) to evaluate the cellular response to radiation-activated NBTXR3 at increasing concentrations. A marked radio-enhancement was observed in human STS cell lines with dose enhancement factors estimated at 4 Gy ranging from 1.48 up to 5.36 according to NBTXR3 concentrations. Further, in human immortalized MRC5V1 fibroblasts, no significant decrease in clonogenic surviving fraction was observed upon activation of NBTXR3 under similar conditions suggesting a differential radiation response between fibroblasts and fibrosarcoma, liposarcoma cell lines. This differential radiation response was further confirmed between HT1080 fibrosarcoma cell line and MRC5V1 fibroblasts upon activation of NBTXR3 at increasing concentration using high energy gamma-rays. Mechanistic studies have demonstrated in HT1080 cells that NBTXR3 activation induces an increase of 53BP1 foci associated with more “complex” DNA damages than ionizing radiation alone. The level of unrepaired DNA damages was also estimated by flow cytometry analysis showing a higher level of gamma-H2AX at 48h following NBTXR3 activation than after radiotherapy alone. DNA fragmentation and Annexin V staining have demonstrated that these unrepaired DNA damages upon NBTXR3 activation induces apoptosis in HT1080 fibrosarcoma cells. In vivo, tolerance and tumor growth delay were investigated in two human xenografted tumor models, HT1080 fibrosarcoma and a patient derived liposarcoma LPS80T3 (poorly differentiated grade 3). No toxicity related to NBTXR3 was reported. In both models, a significant advantage was demonstrated in terms of tumor growth inhibition and survival when compared to radiation therapy alone. These findings establish a novel approach of treatment, which may be applied to a broad range of tumors. Therefore, incrementing the amount of energy deposited within the tumor through the activation of NBTXR3 crystalline nanoparticles constitutes a revolutionary approach for future clinical investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2665. doi:10.1158/1538-7445.AM2011-2665