NMRI Nude Mice Research Articles

Methionine tumor starvation by erythrocyte-encapsulated methionine gamma-lyase activity controlled with per os vitamin B6.

Erymet is a new therapy resulting from the encapsulation of a methionine gamma‐lyase (MGL; EC number 4.4.1.11) in red blood cells (RBC). The aim of this study was to evaluate erymet potential efficacy in methionine (Met)‐dependent cancers. We produced a highly purified MGL using a cGMP process, determined the pharmacokinetics/pharmacodynamics (PK/PD) properties of erymet in mice, and assessed its efficacy on tumor growth prevention. Cytotoxicity of purified MGL was tested in six cancer cell lines. CD1 mice were injected with single erymet product supplemented or not with vitamin B6 vitamer pyridoxine (PN; a precursor of PLP cofactor). NMRI nude mice were xenografted in the flank with U‐87 MG‐luc2 glioblastoma cells for tumor growth study following five intravenous (IV) injections of erymet with daily PN oral administration. Endpoints included efficacy and event‐free survival (EFS). Finally, a repeated dose toxicity study of erymet combined with PN cofactor was conducted in CD1 mice. Recombinant MGL was cytotoxic on 4/6 cell lines tested. MGL half‐life was increased from <24 h to 9–12 days when encapsulated in RBC. Conversion of PN into PLP by RBC was demonstrated. Combined erymet + PN treatment led to a sustained Met depletion in plasma for several days with a 85% reduction of tumor volume after 45 days following cells implantation, and a significant EFS prolongation for treated mice. Repeated injections in mice exhibited a very good tolerability with only minor impact on clinical state (piloerection, lean aspect) and a slight decrease in hemoglobin and triglyceride concentrations. This study demonstrated that encapsulation of methioninase inside erythrocyte greatly enhanced pharmacokinetics properties of the enzyme and is efficacy against tumor growth. The perspective on these results is the clinical evaluation of the erymet product in patients with Met starvation‐sensitive tumors.

Thrombospondin-targeting TAX2 peptide impairs tumor growth in preclinical mouse models of childhood neuroblastoma.

We have previously identified TAX2 peptide as an orthosteric antagonist for thrombospondin-1 (TSP-1) interaction with the cell-surface receptor CD47. TAX2 displays exciting antiangiogenic, antitumor, and antimetastatic properties in both allograft and xenograft models of melanoma as well as pancreatic carcinoma. Here, TAX2 therapeutic potential was investigated in two distinct preclinical mouse models of neuroblastoma. SK-N-BE(2) (MYCN-amplified) and SK-N-SH (MYCN-negative) human neuroblastoma cells have been implanted in outbred NMRI nude mice prior to systemic administrations of TAX2, and then tumor growth as well as intratumoral blood flow were longitudinally monitored. At study termination, subcutaneous xenografts were macroscopically and histopathologically examined. In both models, TAX2 induced a significant inhibition of tumor burden in mice engrafted with large pre-established neuroblastoma tumors. Indeed, TAX2 administered at biologically relevant doses sharply alters xenograft vascularization as well as multiple features of tumor progression. Altogether, our results present TAX2 peptide specifically targeting TSP-1:CD47 interaction as a new putative therapeutic approach for treating neuroblastoma, whether utilized alone or in combination with existing chemotherapy drugs.

OS1.6 Tumor Microtubes contribute to resistance against surgical lesions, and chemotherapy in malignant glioma

AbstractBackground:The current standard for the treatment of astrocytomas, including glioblastomas, consists of tumor resection, followed by radio- and chemotherapy. As we could recently demonstrate, a dense multicellular tumor network formed by intercellular tumor microtubes (TMs) greatly contributes to radioresistance. The neuronal growth-associated protein 43 (GAP-43) is involved in this mechanism. Here we asked whether similar resistance mechanisms can be found with respect to surgical lesions, and chemotherapy.Methods:Cranial windows were implanted into 8–10 weeks old male NMRI nude mice. Human glioblastoma stem-like cells (GBMSCs; stably transduced with GFP, RFP, YFP, shRNA for GAP-43, control vector, and/or H2B-GFP to label tumor cell nuclei) were stereotactically injected into the mouse’s brain. In established tumors, 100mg/kg temozolomide (TMZ) was applied to mice p.o. for three consecutive days (day 0,1,2). For surgical lesion experiments, a regional injury was set to the brain by a 26 gauge Hamilton syringe.Results:Similar to radiotherapy, GBMSCs that were not part of the TM-connected tumor cell network died in relevant numbers 7 days after chemotherapy, while TM-connected tumor cells survived. The number of TM-connected tumor cells even increased twofold after chemotherapy, which speaks for an adapative resistance mechanism. It is currently under investigation whether knock down of GAP-43 increases overall efficacy of TMZ therapy. After surgical resection, the lesioned area was rapidly re-colonized with TM-extending tumor cells, with a striking increase in tumor cells at the site of lesion that by far extended the tumor cell densitiy in unlesioned tumor areas. Qantification revealed that the tumor cell nuclear density increased to numbers five-fold above values before surfical intervention in this area, evident as early as five days (488.3%; P=0.033; 95% CI, 50.3–726.4), and extending to 14 days (523.9%; P=0.03; 95% CI, 66.7–781.1) after surgical procedure. This apparent,,repair response” gradually diminished with increasing distance to the surgical lesion site. Finally, while dexamethasone did not relevantly change these reponses, GAP-43 knock down reduced them significantly.Conclusion:TMs convey primary and adaptive resistance to TMZ chemotherapy, in accordance to what we have shown before for radiotherapy. They are also involved in a strong repair response at the very site of a surgical lesion. Reducing the ability of gliomas to repair themselves, and to resist genotoxic stress by interfering with TM formation and function appears as a promising avenue to improve treatment efficacies in these callenging diseases.

Abstract 5197: Patient-derived xenograft (PDX) models of soft tissue sarcoma (STS): a preclinical platform for early drug testing

Abstract Background: STS constitutes a rare and very heterogeneous family of mesenchymal tumors. The limited treatment options available for advanced STS underline the need for reliable preclinical models to test novel therapeutic strategies. Methods: A panel of patient-derived xenografts (PDX) was established by subcutaneous implantation of fresh, surgically resected tumor specimens in immunodeficient athymic nude NMRI mice. Once tumor growth was observed, pieces of tumor were re-transplanted to next generations of mice. At each passage tumor fragments were collected for histolopathological and molecular characterization. A model was considered established after observing stable histological and molecular features for at least two passages. To evaluate the stability of the genomic profile we performed low-coverage whole-genome sequencing on DNA isolated from tumors obtained from at least two passages. In addition RNA-seq was used to better characterize the mutational profile of the ex-mouse tumors. Results: Until now 139 STS samples from consenting patients treated at the University Hospitals, Leuven, Belgium, have been transplanted. Twenty six well-characterized, stable STS PDX models have been established, maintaining the histopathological and molecular features of the original tumor. Seventeen models, analyzed with low-coverage DNA sequencing, showed a stable genomic profile in at least two different tumor passages. At this point the panel includes models of gastrointestinal stromal tumor (6 models), myxofibrosarcoma (5), dedifferentiated liposarcoma (3), malignant peripheral nerve sheath tumor (3), synovial sarcoma (2), leiomyosarcoma (3) epithelioid haemangioendothelioma (1), mesenchymal chondrosarcoma (1) and undifferentiated high grade sarcoma (2). Some of these models have already been successfully used for in vivo testing of novel agents, including both targeted and cytotoxic (pro-)drugs, and results served as a rationale for at least four prospective clinical trials. In addition 22 other xenografts are still in early stages of engraftment, not yet fulfilling our criteria of an “established model”. Conclusion: Our panel of mesenchymal PDX models is characterized by stable histological and molecular features. These clinically well-annotated models can contribute to reliable preclinical studies for new anticancer treatments for STS. The availability of unique, rare STS xenografts also allows to study the biology of these diseases. The platform is made available to collaborators from academia and industry. Citation Format: Agnieszka Wozniak, Jasmien Cornillie, Yemarshet K. Gebreyohannes, Bram Boeckx, Haifu Li, Thomas Van Looy, Giuseppe Floris, Jasmien Wellens, Lise Vreys, Daphne Hompes, Marguerite Stas, Friedl Sinnaeve, Diether Lambrechts, Maria Debiec-Rychter, Raf Sciot, Patrick Schoffski. Patient-derived xenograft (PDX) models of soft tissue sarcoma (STS): a preclinical platform for early drug testing. [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 5197.

The use of matrigel has no influence on tumor development or PET imaging in FaDu human head and neck cancer xenografts.

BackgroundIn preclinical research MatrixgelTM Basement Membrane Matrix (MG) is used frequently for the establishment of syngeneic and xenograft cancer models. Limited information on its influence on parameters including; tumor growth, vascularization, hypoxia and imaging characteristics is currently available. This study evaluates the potential effect of matrigel use in a human head and neck cancer xenograft model (FaDu; hypopharyngeal carcinoma) in NMRI nude mice. The FaDu cell line was chosen based on its frequent use in studies of cancer imaging and tumor microenvironment.MethodsNMRI nude mice (n = 34) were divided into two groups and subcutaneously injected with FaDu cells in medium either including (+MG) or excluding matrigel (−MG). In sub study I seven mice from each group (+MG, n = 7; −MG, n = 7) were 18F- fluorodeoxyglucose (18F-FDG) PET/CT scanned on Day 5, 8, 12, 15, and 19. In sub study II ten mice from each group (+MG, n = 10; −MG, n = 10) were included and tumors collected for immunohistochemistry (IHC) analysis of tumor microenvironment including; proliferation ratio, micro vessel density, average vessel area, hypoxia, nuclear density, and necrosis. Tumors for IHC were collected according to size (200–400 mm3, 500–700 mm3, 800–1100 mm3).ResultsFDG uptake and tumor growth was statistically compatible for the tumors established with or without MG. The IHC analysis on all parameters only identified a significantly higher micro vessel density for tumor size 500–700 mm3 and 800–1100 mm3 and average vessel area for tumor size 500–700 mm3 in the −MG group. Comparable variations were observed for tumors of both the +MG and −MG groups. No difference in tumor take rate was observed between groups in study.ConclusionsMatrigel did not affect tumor growth or tumor take for the FaDu xenograft model evaluated. Tumors in the -MG group displayed increased angiogenesis compared to the +MG tumors. No difference in 18F-FDG PET uptake for tumors of different groups was found. Based on these observations the influence of matrigel on tumor imaging and tumor microenvironment seems minor for this particular xenograft model.

Atorvastatin-Induced Inhibition of Human Melanoma In Vivo Development

Statins, 3-hydroxy 3-methylglutaryl CoA (HMG-CoA) reductase inhibitors, are pleiotropic pharmacological inhibitors, which block the mevalonate synthesis pathway. They are widely used as hypocholesterolemic agents and have shown protective effects against cancers. Our previous data showed that statin treatment induced MHC class I chain-related protein A (MICA) membrane overexpression in human melanoma cells, which increased their sensitivity to NK cell cytotoxicity and thus the inhibition of tumor development. MICA is a ligand of the NK cell activating receptor NKG2D. This receptor is essential for NK cell control of tumor development and it has the unique property of being able to recognize tumor cells of both murine and human origin. Here, using atorvastatin and the WM-266-4 melanoma cell line, we first confirmed that statin treatment enhances MICA membrane expression and decreases local tumor growth and pulmonary metastasis implantation in vivo. Furthermore our new experiments showed that atorvastatin intraperitoneal repeated injections induced a reduction of tumor growth following subcutaneous implantation of untreated WM 266-4 cells into NMRI nude mice and favored the innate anti-melanoma immune response by increasing splenic NK cell concentration and activation. This report confirms that statins could become effective pharmacological agents for melanoma immunotherapy.

Abstract 4476: Targeting Wnt5a and STAT3 pathways for the treatment of prostate cancer

Abstract Background and Aims Wnt5a protein levels are upregulated in prostate cancer (PCa) compared to benign tissue and patients with high Wnt5a protein levels have a better outcome after radical prostatectomy compared to patients with low Wnt5a levels (Syed Khaja AS, 2011; 2012). Thus, the reconstitution of the Wnt5a signaling pathway could be a promising therapeutic approach in PCa, as already shown in breast cancer (Säfholm A, 2008). Constitutively active STAT3 (pSTAT3) has been correlated to PCa progression and disruption of its signaling pathway could represent a promising strategy for the treatment of patients with advanced PCa (Hellsten R, 2008). We have analyzed the expression of Wnt5a and pSTAT3 in a cohort of patients with metastatic Castration Resistant Prostate Cancer (mCRPC). Using in vitro and in vivo techniques we have explored the effect of the Wnt5a-mimicking peptide FOXY5 for the reconstitution of the Wnt5a pathway in PCa, and we have studied the effect of the STAT3 inhibitor Galiellalactone (GL) on PCa cell viability, apoptosis, invasion, tumor growth and metastases. Methods Wnt5a and pSTAT3 protein expression was analyzed by IHC on a TMA construct with duplicate cores of malignant tissues from 38 patients with mCRPC. Statistical and correlation analyses were performed using the SPSS version 21. Cell viability was determined by MTT assay; apoptotic cells were visualized with M30 CytoDeath antibody 24h after treatment with FOXY5 or GL. Cell invasion was analyzed using matrigel pre-coated cell culture inserts in the presence or absence of FOXY5 or GL. For the animal study, NMRI nude mice were injected orthotopically with 1×106 DU145-Luc cells, and treated IP with GL every day or with FOXY5 every second day. Primary tumor growth and metastatic spread were evaluated weekly by in vivo luminescence using the IVIS Lumina II system. 9 weeks after injection animals were sacrificed and organs were analyzed for the presence of metastases. Results IHC analyses of TMA showed high pSTAT3 and low Wnt5a expression in CRPC patients, with a small but significant correlation (r = 0.4646; p = 0.0038). Our results show that GL inhibits viability and induces apoptosis of DU145-Luc cells in vitro, and that it reduces tumor growth and metastatic spread to lymph nodes in vivo. FOXY5 treatment has no effects on cell viability or on primary tumor growth, but it significantly reduces cell invasion in vitro and metastatic spread to lymph nodes in vivo. Conclusions Our results confirm that the reconstitution of the Wnt5a pathway and the inhibition of the STAT3 pathway are a promising therapeutic approach for the treatment of mCRPC. We found that GL and FOXY5 are good candidates for the treatment of mCRPC, as they inhibit tumor growth and metastatic spread to lymph nodes. Since these two compounds act through different mechanisms, our future studies will explore the possibility of a combination of GL and FOXY5 for the treatment of PCa. Citation Format: Giacomo Canesin, Susan Evans-Axelsson, Rebecka Hellsten, Nicholas Don-Doncow, Tommy Andersson, Anders Bjartell. Targeting Wnt5a and STAT3 pathways for the treatment of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4476. doi:10.1158/1538-7445.AM2015-4476

Abstract 689: Characterization of two novel oncogenic FGFR2 fusions sensitive to the FGFR-selective inhibitor Debio 1347 in cholangiocarcinoma

Abstract Dysregulation of the fibroblast growth factor receptor (FGFR) signaling pathway due to receptor overexpression, gene amplification, point mutations or fusions/chromosomal translocations is associated with cancer development and progression. FGFR gene fusions have recently been discovered in several indications including bladder cancer, glioblastoma, lung squamous cell cancer and cholangiocarcinoma. Predominant gene fusions have been identified in some indications such as FGFR3-TACC3 (with different genomic variations in the two genes) for bladder cancer whereas for other indications the genomic-altered landscape is a lot more heterogeneous, as seen in cholangiocarcinoma where at least ten FGFR2 fusions each with a different fusion partner have been identified. We herein report for the first time the discovery of two novel FGFR fusions in intra hepatic cholangiocarcinoma patient samples. These new fusions contain an intact FGFR2 tyrosine kinase domain and fused partner harbor oligomerization domains that suggest similar mechanism of constitutive kinase activation. Both fusions display oncogenic activities when introduced into Rat 2 cells in in vitro colony formation assays as compared to parental cells. The two fusions are also tumorigenic in vivo when implanted subcutaneously in NMRI nude mice, whereas parental cell do not grow in vivo. FGFR inhibition by the FGFR selective inhibitor Debio 1347 (CH5183284) reduced cell proliferation in vitro. In vivo sensitivity to FGFR inhibition could also be demonstrated for the two fusions. Altogether, these results suggest that these two novel oncogenic fusions are actionable targets in cholangiocarcinoma and that patients harboring these fusions could benefit from targeted FGFR inhibition, such as Debio 1347, currently investigated in a Phase I trial in selected patients harboring FGFR genetic alterations (NCT01948297). Citation Format: Anne Vaslin, Stefania Rigotti, Nathalie Lembrez, Grégoire Vuagniaux, Corinne Moulon, Hiroaki Tanaka. Characterization of two novel oncogenic FGFR2 fusions sensitive to the FGFR-selective inhibitor Debio 1347 in cholangiocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 689. doi:10.1158/1538-7445.AM2015-689

Abstract 5174: Imaging of tissue factor expression in an orthotopic pancreatic tumor mouse model using small animal PET/CT and MRI

Abstract Objectives: Tissue factor (TF) is up regulated in many solid tumors and its expression is linked to tumor angiogenesis, invasion, metastasis and prognosis. We have previously reported the use active site inhibited factor VII (FVIIai) labeled with 18F for specific in vivo imaging of TF expression. The aim of this study was to investigate the feasibility of non-invasive imaging of tumor TF expression in an orthotopic pancreatic tumor mouse model using 18F-FVIIai and small animal PET/CT and MR imaging. Methods: Female NMRI nude mice (N = 24) were inoculated orthotopically with 5×105 human pancreatic adenocarcinoma cells (BxPC-3.luc2). Tumor development was monitored by bioluminescence imaging. Non-invasive TF imaging was performed 13 days after tumor inoculation. Each mouse (N = 18) underwent small animal PET/CT imaging 4 hours after intravenous administration of 2.9-8.3 MBq 18F-FVIIai. Anatomical T2-weighted MR imaging was performed the day after followed by surgical removal of the primary tumors. Bioluminescence imaging was applied to monitor tumor regrowth and metastatic development. The primary endpoint was overall survival using humane endpoints. Results: The PET images showed high uptake of 18F-FVIIai in the orthotopic pancreatic tumors. A high tumor to normal tissue ratio clearly visualized the tumor margins on the combined PET/CT images. The pancreatic tumors had intense uptake in their outer margins and a low central uptake. This corresponded well with the findings on the T2-weigted MR images that showed central necrosis of the pancreatic tumors. Surgical removal of the primary orthotopic tumors reduced the bioluminescence signal more than 30-fold fold from 1.2×109 ±1.7×108 photons/s (mean ± SEM) to 3.4×107 ±1.7×107 photons/s. In comparison, the bioluminescence signal was 1.5×108 ±3.5×107 photons/s two days after inoculation of the BxPC-3.luc2 cells. All but one mouse had tumor re-growth and at autopsy, 5 animals had local disease, 5 animals had regional disease, and 7 animals had distant metastatic disease. Interestingly, 3 animals showed hematological spread with multiple metastatic foci found in the liver. The median overall survival was 30 days post surgery. Conclusion: PET/CT imaging with 18F-FVIIai is able to clearly visualize TF positive orthotopic pancreatic tumors. High intensity uptake of 18F-FVIIai in tumor margins corresponded to viable tumor visualized by MRI. Further quantitative image analysis of 18F-FVIIai uptake in tumor regions will reveal if TF expression measured by non-invasive PET imaging is able to predict prognosis and metastatic disease. Research Support: This work was supported by at grant from the Danish National Advanced Technology Foundation. Citation Format: Carsten H. Nielsen, Troels E. Jeppesen, Lotte K. Kristensen, Laura H. Larsen, Jacob Madsen, Bo Wiinberg, Lars C. Petersen, Andreas Kjaer. Imaging of tissue factor expression in an orthotopic pancreatic tumor mouse model using small animal PET/CT and MRI. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5174. doi:10.1158/1538-7445.AM2015-5174

Monitoring Combretastatin A4-induced tumor hypoxia and hemodynamic changes using endogenous MR contrast and DCE-MRI.

To benchmark MOBILE (Mapping of Oxygen By Imaging Lipid relaxation Enhancement), a recent noninvasive MR method of mapping changes in tumor hypoxia, electron paramagnetic resonance (EPR) oximetry, and dynamic contrast-enhanced MRI (DCE-MRI) as biomarkers of changes in tumor hemodynamics induced by the antivascular agent combretastatin A4 (CA4). NT2 and MDA-MB-231 mammary tumors were implanted subcutaneously in FVB/N and nude NMRI mice. Mice received 100 mg/kg of CA4 intraperitoneally 3 hr before imaging. The MOBILE sequence (assessing R1 of lipids) and the DCE sequence (assessing K(trans) hemodynamic parameter), were assessed on different cohorts. pO2 changes were confirmed on matching tumors using EPR oximetry consecutive to the MOBILE sequence. Changes in tumor vasculature were assessed using immunohistology consecutive to DCE-MRI studies. Administration of CA4 induced a significant decrease in lipids R1 (P = 0.0273) on pooled tumor models and a reduction in tumor pO2 measured by EPR oximetry. DCE-MRI also exhibited a significant drop of K(trans) (P < 0.01) that was confirmed by immunohistology. MOBILE was identified as a marker to follow a decrease in oxygenation induced by CA4. However, DCE-MRI showed a higher dynamic range to follow changes in tumor hemodynamics induced by CA4.

(99m) Tc radiolabeling and biological evaluation of nanoparticles functionalized with a versatile coating ligand.

Radiolabeling allows noninvasive imaging by single photon emission computed tomography (SPECT) or positron emission tomography (PET) for assessing the biodistribution of nanostructures. Herein, the synthesis of a new coating ligand for gold nanoparticles (AuNPs) and quantum dots (QDs) is reported. This ligand is multifunctional; it combines the metal chelate with conjugating functions to biological vectors. The concept allows the coupling of any targeting function to the chelator; an example for the prostate specific membrane antigen is given. Derivatized NPs can directly be labeled in one step with [(99m) Tc(OH2 )3 (CO)3 ](+) . AuNPs in particular are highly stable, a prerequisite for in vivo studies excluding misinterpretation of the biodistribution data. AuNPs with differing sizes (7 and 14 nm core diameter) were administered intravenously into nude NMRI mice bearing LNCaP xenografts. MicroSPECT images show for both probes rapid clearance from the blood pool through the hepatobiliary pathway. The 7 nm AuNPs revealed a significantly higher bone uptake than the 14 nm AuNPs. The high affinity towards bone mineral is further confirmed in vitro with hydroxyapatite.

Effect of combined irradiation and EGFR/Erb-B inhibition with BIBW 2992 on proliferation and tumour cure in cell lines and xenografts.

Background and purposeIn previous experiments an enhanced anti-proliterative effect of the EGFR/ErbB tyrosine kinase inhibitor (TKI) BIBW 2992 with single dose irradiation was observed in FaDu tumour xenografts. Aim of the present experiment was to determine if this effect can also be seen in combination with a fractionated radiotherapy. Secondly we investigate the efficacy of BIBW 2992 on local tumour control for UT-SCC-15.Material and methodsTumour pieces of FaDu, UT-SCC-14, A431, UT-SCC-15 (squamous cell carcinomas) and A7 (glioma) tumour models were transplanted onto the right hind leg of NMRI (nu/nu) nude mice. For evaluation of tumour growth mice were either treated daily orally with BIBW 2992 (30 mg/kg body weight), or carrier up to a final tumour size of 15 mm or with a fractionated radiotherapy (15f/15d, 30 Gy) with simultaneous application of BIBW 2992 or carrier. For local tumour control UT-SCC-15 tumours were treated with a fractionated radiotherapy (30f/6weeks) or received 30f/6 weeks in combination with daily orally BIBW 2992 (22.5 mg/kg b.w.) during RT.ResultsA significant effect on tumour growth time was observed in all tumour models for BIBW 2992 application alone. However, substantial intertumoural heterogeneity could be seen. In the UT-SCC-14, UT-SCC-15 and A431 tumour models a total regression of the tumours and no recurrence during treatment time (73 days) were determined where as for the A7 tumour only a slight effect was noticeable. For the combined treatment of fractionated radiotherapy (15f/15d) and BIBW 2992 administration a significant effect on tumour growth time was seen compared to irradiation alone for A7, UT-SCC-15 and A431 (ER 1.2 – 3.7), this advantage could not be demonstrated for FaDu and UT-SCC-14. However, the local tumour control was not altered for the UT-SCC-15 tumour model when adding BIBW 2992 to fractionated irradiation (30f/6weeks).ConclusionA heterogeneous effect on tumour growth time of BIBW 2992 alone as well as in combination with fractionated irradiation could be demonstrated for all tumour models. However, the significant effect on tumour growth time did not translate into an improvement of local tumour control for the UT-SCC-15 tumour model.

Pazopanib, a Receptor Tyrosine Kinase Inhibitor, Suppresses Tumor Growth through Angiogenesis in Dedifferentiated Liposarcoma Xenograft Models

INTRODUCTION: The rarity of dedifferentiated liposarcoma (DDLPS) and the lack of experimental DDLPS models limit the development of novel therapeutic strategies. Pazopanib (PAZ) is a tyrosine kinase inhibitor that is approved for the treatment of non-adipocytic advanced soft tissue sarcoma. The activity of this agent has not yet been properly explored in preclinical liposarcoma models nor in a randomized phase Ш clinical trial in this entity. The aim of the present study was to investigate whether PAZ had antitumor activity in DDLPS models in vivo. MATERIAL AND METHODS: We established two patient-derived DDLPS xenograft models (UZLX-STS3 and UZLX-STS5) through implantation of tumor material from sarcoma patients in athymic nude NMRI mice. An animal model of the SW872 liposarcoma cell line was also used. To investigate the efficacy of PAZ in vivo, mice bearing tumors were treated for 2 weeks with sterile water, doxorubicin (1.2 mg/kg, intraperitoneally, twice per week), PAZ [40 mg/kg, orally (p.o.), twice per day], or PAZ plus doxorubicin (same schedules as for single treatments). RESULTS: Patient-derived xenografts retained the histologic and molecular features of DDLPS. PAZ significantly delayed tumor growth by decreasing proliferation and inhibited angiogenesis in all models tested. Combining the angiogenesis inhibitor with an anthracycline did not show superior efficacy. CONCLUSION: These results suggest that PAZ has potential antitumor activity in DDLPS primarily through antiangiogenic effects and therefore should be explored in clinical trials.

Abstract 855: Targeting STAT3 in vitro and in vivo reveals a novel therapeutic strategy to sensitize colorectal cancer cells to chemoradiotherapy

Abstract Introductory sentence: Increased activity of signal transducer and activator of transcription 3 (STAT3) is common in human malignancies, including colorectal cancers. Recently, we reported that STAT3 expression correlated with resistance to 5-fluorouracil (5-FU) based chemoradiotherapy. This is of considerable clinical relevance, because a large proportion of rectal cancers are resistant to preoperative multimodal treatment. We therefore examined whether STAT3 contributes to resistance to chemoradiotherapy. Experimental procedures: STAT3 mRNA and protein expression levels were determined in 12 colorectal cancers cell lines. STAT3 was inhibited using two different siRNAs and a small-molecular inhibitor (STATTIC) in the cell lines SW480 and SW837. Successful RNAi-mediated silencing of STAT3 or inhibition of phosphoSTAT3(Tyr705) was detected by Western blot and reduction of transcription factor activity was measured by a luciferase reporter assay. Additionally, we established doxycycline-inducible stable shRNA single cell populations and a non-silencing shRNA (shNEG) in SW480. To test the influence of STAT3 knock down or inhibition, clonogenic survival assays were performed. Therefore, RNAi or inhibitor treated cells were exposed to chemoradiotherapy using 3µM 5-FU and X-ray-irradiation at 1, 2, 4, 6, and 8 Gy. Finally, we tested the effect of a chemoradiotherapy combined with STATTIC treatment in a SW837 xenograft model in NMRI nude mice. To verify the sensitizing effect of STATTIC, tumor growth was recorded and growth delay assays were performed. Data: STAT3 was overexpressed in resistant cells at mRNA and protein level. siRNA transfected SW480, SW837, and SW480shRNA single cell clones showed a significant reduction of STAT3 protein and transcription factor activity after 96 hours. STATTIC inhibition led to a decreased phosphorylation of STAT3 after 1 hour. The silencing/inhibition resulted in a significantly increased chemoradiosensitivity with dose-reduction factors of 1.3 to 2.5 at a surviving fraction of 0.37. In vivo, additional STAT3 inhibition during chemoradiotherapy led to a profound chemoradiosensitization effect and a significant tumor growth delay in STATTIC treated mice. Survival of these mice was also enhanced, if compared to the control group. Conclusions: STAT3 is highly overexpressed in resistant colorectal cancer cells, and silencing or inhibition of STAT3 leads to a significantly increased chemoradiosensitivity in vitro and in vivo. This highlights the potential relevance of STAT3 for mediating treatment resistance and provides a first proof of concept that STAT3 represents a novel molecular target in rectal cancer to sensitize a priori resistant colorectal tumor cells to chemoradiotherapy. Citation Format: Melanie Spitzner, Birte Roesler, Christian Bielfeld, Carolin Herzberg, Georg Emons, Jochen Gaedcke, Margret Rave-Fränk, Tim Beißbarth, Thomas Ried, B. Michael Ghadimi, Marian Grade. Targeting STAT3 in vitro and in vivo reveals a novel therapeutic strategy to sensitize colorectal cancer cells to chemoradiotherapy. [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 855. doi:10.1158/1538-7445.AM2014-855

Abstract 3971: PPM1D/WIP1, a potential target in neuroblastoma

Abstract Background: Gain of 17q is the most powerful genetic predictor of adverse clinical outcome in neuroblastoma (NB). 17q+ correlated with poor survival in our population-based material where we found aberrations of chromosome 17 in 85% of primary neuroblastoma tumors. Specifically, gain of WIP1 at 17q23 is frequently detected in NB. WIP1 is a serine/threonine phosphatase encoded by the gene PPM1D, member of the PP2C family known to be regulators of DNA repair pathways. Methods: A formazan-based spectrophotometric method was used to measure cell viability in several NB cell lines treated with different WIP1 inhibitors. Comparative genomic hybridization (CGH) was used to analyze primary NB tumors and cell lines. NB cells were examined for WIP1 expression using immunoblotting, immunohistochemistry and qPCR. Stable WIP1 knockdown NB cells were generated by shRNA transfections. Clonogenicity of WIP1 knockdown were examined by clonogenic assay. In vivo, WIP1 knocked- and non-silenced cells were injected in nude NMRI mice followed by daily evaluation of tumor formation. Results: Detailed CGH analysis revealed that genomic gain of WIP1 is evident with at least one extra copy in all tumor samples showing 17q gain. Results from our NB cell lines demonstrate 17q aberrations in all samples. These findings were corroborated by expression pattern of WIP1 mRNA and different immunostaining techniques. In a wide range of NB cell lines we found that all WIP1 inhibitors had good cytotoxic effect at higher concentrations (50µM) while some showed cytotoxic effects at lower concentrations. Transfection experiments with shRNA against WIP1 showed decreased cell viability, proliferation and colony formation in NB cells. Tumor xenograft development after WIP1 downregulation was significantly delayed with time to tumor development (0.10 mL) more than doubled compared to animals injected with cells transfected with the scrambled construct (15 days median, vs. 33 days). Conclusions: Our data suggest that WIP1 expression have significant oncogenic function in neuroblastoma development. Our results showed that WIP1 is present in at least one extra genomic copy in the majority of primary NB. WIP1 knocked cells exhibited decreased cell growth and clonogenic capacity compared to non-silenced cells. In addition, WIP1 inhibition could be a potential target in treating high-risk neuroblastoma but more studies investigating the effects of WIP1 inhibition in neuroblastoma are needed to evaluate its clinical significance. Citation Format: Jelena Milosevic, Malin Wickström, Diana Treis, Hjalmar Ståhlberg Nordegren, Lotta Elfman, Ninib Baryawno, Susanne Fransson, Baldur Sveinbjörnsson, Tommy Martinsson, John Inge Johnsen, Per Kogner. PPM1D/WIP1, a potential target in neuroblastoma. [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 3971. doi:10.1158/1538-7445.AM2014-3971

Preclinical imaging of kallikrein-related peptidase 2 (hK2) in prostate cancer with a (111)In-radiolabelled monoclonal antibody, 11B6.

BackgroundProstate cancer is a leading cause of death in the male population of the western world. Human kallikrein-related peptidase 2 (hK2) is abundantly expressed in malignant prostatic tissue, and its gene, KLK2, is regulated by the androgen receptor. 11B6 is a murine IgG1 monoclonal antibody directed against free human hK2. In this study, we performed a preclinical evaluation of 111In-labelled 11B6 in mouse xenografts to investigate its potential in the clinical staging and assessment of metastatic prostate cancer.Methods11B6 was radiolabelled with 111In through CHX-A″-DTPA chelation. In vivo biodistribution and uptake of 111In-DTPA-11B6 were measured until 168 h post-injection in NMRI nude mice bearing subcutaneous LNCaP xenografts. The binding specificity to hK2 was evaluated by both in vivo competitive binding assays with excess non-labelled 11B6 and hK2-negative DU145 xenografts. SPECT/CT imaging of subcutaneous and intra-tibial LNCaP xenografts was used to visualize the tumours.ResultsTumour uptake of 111In-DTPA-11B6 in LNCaP xenografts was 19% ± 0.78%IA/g at 48 h, giving a tumour-to-blood ratio of 1.6, which increases to 2.4 at 1 week post-injection. Accumulation was low in other organs except for the salivary glands, which is probably the result of cross-reactivity with mouse kallikreins. Significantly lower tumour accumulation was observed in competitive assays and DU145 xenografts. SPECT/CT imaging could clearly visualize the subcutaneous and intra-tibial LNCaP xenografts.ConclusionsOur study demonstrates the potential of 111In-DTPA-11B6 for the detection of metastatic prostate cancer and monitoring anti-androgen therapy, as it exhibits an increased uptake and accumulation in viable tumour when compared to normal tissue. A humanised version of the 11B6 monoclonal antibody is currently under evaluation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-014-0051-5) contains supplementary material, which is available to authorized users.

P16.23 * A COMPARISON OF 18F-FET AND 18F-FLT MICROPET FOR THE ASSESSMENT OF ANTI-VEGF EFFICACY IN AN ORTHOTOPIC MURINE MODEL OF GLIOBLASTOMA

BACKGROUND: The radiolabeled amino acid O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET) and thymidine analog 3'-deoxy-3'-18F-fluorothimidine (18F-FLT) are widely used for positron emission tomography (PET) brain tumor imaging; however, results from comparative studies are scarce. Due to the limited transport of FLT across the intact blood-brain barrier (BBB) we hypothesize that anti-VEGF response assessment with FLT PET primarily reflects changes in the BBB, whereas FET PET is less affected by the tumor vasculature. The aim of this study was therefore to evaluate and compare the value of FLT and FET MicroPET for anti-VEGF response assessment in orthotopic human glioma xenografts. METHODS: Cells of human glioblasoma multiforme (GBM) neurosphere culture (NGBM_CPH048p6_LUC) were injected orthotopically in NMRI nude mice. At confirmed tumor take, mice were treated with anti-VEGF therapy (B20-4.1) or saline as control. Treatment response was followed by weekly bioluminescence, 18F-FLT and 18F-FET MicroPET/CT. The end-point was survival and brains from sacrificed mice were used for immunohistochemistry and subsequent quantification of the Ki67 proliferation index and micro-vessel density (MVD). RESULTS: The relative 18F-FET tumor-to-brain of (T/B) ratio of SUVmax was significantly decreased after one week (99 ± 6%, n = 6 vs. 124 ± 9%, n = 5; p = 0.04) and after two weeks (106 ± 10%, n = 5 vs. 156 ± 17%, n = 4; p = 0.03) in the B20-4.1 group as compared with the control group. However, with 18F-FLT MicroPET there was no significant difference in the T/B ratio of SUVmax neither after one week of treatment (134 ± 12%, n = 6 vs. 144 ± 6%, n = 6; p = 0.49) nor after two weeks of treatment (174 ± 11%, n = 5 vs. 208 ± 30%, n = 5; p = 0.34). We found a significant lower MVD in the B20-4.1 group as compared to the control group (66 ± 8%, n = 8 vs. 100 ± 10%, n = 7; p = 0.02). However, we found no difference in the Ki67 proliferation index in the treatment group as compared to the control group (18.9 ± 1%, n = 7 vs. 19.8 ± 2%; n = 6; p = 0.7). In addition, the median survival was not prolonged in the treatment group as compared to the control group (19 vs. 21 days, p = 0.34). CONCLUSION: In NGBM_CPH048p6_LUC xenografts B20-4.1 anti-VEGF treatment induced changes in the MVD and in 18F-FET uptake without affecting the Ki67 proliferation index. Surprisingly and in contradiction with the hypothesis, the 18F-FLT uptake was not affected by the changes in the MVD.

Molecular imaging of cell death in tumors. Increasing annexin A5 size reduces contribution of phosphatidylserine-targeting function to tumor uptake.

ObjectiveAnnexin A5 is a phosphatidylserine binding protein that binds dying cells in vivo. Annexin A5 is a potential molecular imaging agent to determine efficacy of anti-cancer therapy in patients. Its rapid clearance from circulation limits tumor uptake and, hence, its sensitivity. The aim of this study is to determine if non-invasive imaging of cell death in tumors will benefit from increasing circulation time of annexin A5 by increasing its size.ProceduresAnnexin A5 size was increased by complexation of biotinylated annexin A5 with Alexa-Fluor680-labeled streptavidin. The non-binding variant of annexin A5, M1234, was used as negative control. The HT29 colon carcinoma xenograft model in NMRI nude mice was used to measure tumor uptake in vivo. Tumor uptake of fluorescent annexin A5-variants was measured using non-invasive optical imaging.ResultsThe annexin A5-streptavidin complex (4∶1, moles:moles, Mw ∼200 kDa) binds phosphatidylserine-expressing membranes with a Hill-coefficient of 5.7±0.5 for Ca2+-binding and an EC50 of 0.9±0.1 mM Ca2+ (EC50 is the Ca2+ concentration required for half maximal binding)(annexin A5: Hill-coefficient 3.9±0.2, EC50 1.5±0.2 mM Ca2+). Circulation half-life of annexin A5-streptavidin is ±21 minutes (circulation half-life of annexin A5 is ±4 min.). Tumor uptake of annexin A5-streptavidin was higher and persisted longer than annexin A5-uptake but depended less on phosphatidylserine binding.ConclusionIncreasing annexin A5 size prolongs circulation times and increases tumor uptake, but decreases contribution of PS-targeting to tumor uptake and abolishes power to report efficacy of therapy.

Abstract B2: Switching to aflibercept treatment resulted in greater tumor responses than continuous bevacizumab treatment in patient-derived xenograft models of colorectal cancer.

Abstract Background: The recombinant fusion protein aflibercept (afl) binds VEGF-A, -B, and placental growth factor (PlGF). Based on an OS benefit observed in the phase 3 VELOUR trial, aflibercept in combination with FOLFIRI was approved for patients with metastatic colorectal cancer (mCRC) following a prior oxaliplatin-containing regimen. In vitro, afl inhibited VEGFR signaling more potently and blocked cell migration more effectively vs. bevacizumab (bev) which only binds VEGF-A. Recent in vivo studies comparing the anti-tumor activity of afl vs. bev were conducted in patient-derived xenograft (PDX) CRC models. The PDX models are clinically relevant as they represent common genetic backgrounds in CRC and were derived from primary tumors or from metastases. A sequential treatment regimen of aflibercept post-bev vs. continuation of bev treatment was investigated. Methods: Forty eight different CRC adenocarcinoma tumors were engrafted subcutaneously (SC) into NMRI nude mice to generate 48 distinct PDX CRC models. ELISA and Luminex measured the expression of mouse and human VEGF-A and mouse and human PlGF in untreated tumors. Tumor-bearing mice (8 mice/group) were dosed with afl, bev, or placebo 2x/wk for 3 wks and SC tumor measurements were recorded 2x or 3x/wk. In 3 PDX models, mice that were treated with bev were randomized and either switched to afl treatment or continued to receive bev. Activity was expressed as relative change in tumor volume between treatment (ΔT) and control (ΔC) at 3 wks. A ΔT/ΔC ≤0% indicated complete tumor stasis. Student's t tests evaluated statistical differences in final tumor volumes. Results: Aflbercept induced complete tumor stasis in 31/48 PDX CRC models and bev in 2/48 PDX CRC models. The PDX models were characterized into 2 phenotypes based on statistical analysis: phenotype A, where afl activity was greater than bev activity (39 /48 PDX), and phenotype B, where there was no significant difference between the 2 drugs (9/48 PDX). Three models in which bev treatment resulted in eventual tumor escape vs. continuous inhibition by afl were selected for sequential treatment of bev followed by afl. Afl treatment began after bev treatment resulted in a greater than 2-fold increase in tumor volume. Greater anti-tumor activity occurred when afl was administered after bev vs. continuous bev treatment. At the end of the study, the tumor volumes were statistically significantly different between the afl and bev groups (T test p values &amp;lt; 0.0001). Analysis of angiogenic factors revealed that the tumor cells were the major source of VEGF whereas PlGF was primarily produced by the tumor stroma. Conclusions: Aflibercept induced complete tumor stasis more frequently in CRC PDX models than bevacizumab. As tumor levels of hVEGF-A were far greater than mVEGF-A, the inability of bevacizumab to bind mVEGF-A is unlikely to explain the higher and more consistent activity of aflibercept. In all 3 PDX models tested, switching to aflibercept treatment after bev treatment resulted in a greater tumor response than continuous bev treatment. The greater response to aflibercept than bev after an initial treatment with bev suggests that individual patients may derive more benefit from aflibercept than bev in second line treatment after an initial bev-based regimen. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B2. Citation Format: Marielle Chiron, Rebecca G. Bagley, Jack Pollard, Christophe Henry, Parminder Mankoo, Loic Vincent, Catherine Geslin, Tatjana Kloss, Donald A. Bergstrom. Switching to aflibercept treatment resulted in greater tumor responses than continuous bevacizumab treatment in patient-derived xenograft models of colorectal cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B2.

Synthesis and radiopharmacological evaluation of a high-affinity and metabolically stabilized 18F-labeled bombesin analogue for molecular imaging of gastrin-releasing peptide receptor-expressing prostate cancer

Bombesin (BBN) and BBN analogues have attracted much attention as high-affinity ligands for selective targeting of the gastrin-releasing peptide (GRP) receptor. GRP receptors are overexpressed in a variety of human cancers including prostate cancer. Radiolabeled BBN derivatives are promising diagnostic probes for molecular imaging of GRP receptor-expressing prostate cancer. This study describes the synthesis and radiopharmacological evaluation of various metabolically stabilized fluorobenzoylated bombesin analogues (BBN-1, BBN-2, BBN-3). Three fluorobenzoylated BBN analogues containing an aminovaleric (BBN-1, BBN-2), or an aminooctanoic acid linker (BBN-3) were tested in a competitive binding assay against (125)I-[Tyr(4)]-BBN for their binding potency to the GRP receptor. Intracellular calcium release in human prostate cancer cells (PC3) was measured to determine agonistic or antagonistic profiles of fluorobenzoylated BBN derivatives. Bombesin derivative BBN-2 displayed the highest inhibitory potency toward GRP receptor (IC50 = 8.7 ± 2.2 nM) and was subsequently selected for radiolabeling with fluorine-18 ((18)F) through acylation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). The radiopharmacological profile of (18)F-labeled bombesin [(18)F]BBN-2 was evaluated in PC3 tumor-bearing NMRI nude mice involving metabolic stability studies, biodistribution experiments and dynamic small-animal PET studies. All fluorobenzoylated BBN derivatives displayed high inhibitory potency toward the GRP receptor (IC50=8.7-16.7 nM), and all compounds exhibited antagonistic profiles as determined in an intracellular calcium release assay. The (18)F-labeled BBN analogue [(18)F]BBN-2 was obtained in 30% decay-corrected radiochemical yield with high radiochemical purity >95% after semi-preparative HPLC purification. [(18)F]BBN-2 showed high metabolic stability in vivo with 65% of the radiolabeled peptide remaining intact after 60 min p.i. in mouse plasma. Biodistribution experiments and dynamic small-animal PET studies demonstrated high tumor uptake of [(18)F]BBN-2 in PC3 xenografts (2.75 ± 1.82 %ID/g after 5 min and 2.45 ± 1.25 %ID/g after 60 min p.i.). Specificity of radiotracer uptake in PC3 tumors was confirmed by blocking experiments. The present study demonstrates that (18)F-labeled BBN analogue [(18)F]BBN-2 is a suitable PET radiotracer with favorable metabolic stability in vivo for molecular imaging of GRP receptor-positive prostate cancer.