Orthotopic Pancreatic Tumor Xenografts Research Articles

Repurposing Pimavanserin, an Anti-Parkinson Drug for Pancreatic Cancer Therapy

Despite major advances in cancer treatment, pancreatic cancer is still incurable and the treatment outcomes are limited. The aggressive and therapy-resistant nature of pancreatic cancer warrants the need for novel treatment options for pancreatic cancer management. Drug repurposing is emerging as an effectual strategy in the treatment of various diseases, including cancer. In the present study, we evaluated the anticancer effects of pimavanserin tartrate (PVT), an antipsychotic drug used for the treatment of Parkinson disease psychosis. PVT significantly suppressed the proliferation and induced apoptosis in various pancreatic cancer cells and gemcitabine-resistant cells with minimal effects on normal pancreatic epithelial cells and lung fibroblasts. Growth-suppressive and apoptotic effects of PVT were mediated by the inhibition of the Akt/Gli1 signaling axis. The oral administration of PVT suppressed subcutaneous and orthotopic pancreatic tumor xenografts by 51%–77%. The chronic administration of PVT did not demonstrate any general signs of toxicity or change in behavioral activity of mice. Our results indicate that pancreatic tumor growth suppression by PVT was orchestrated by the inhibition of Akt/Gli1 signaling. Since PVT is already available in the clinic with an established safety profile, our results will accelerate its clinical development for the treatment of patients with pancreatic cancer.

Targeting the NRG1/HER3 pathway in tumor cells and cancer-associated fibroblasts with an anti-neuregulin 1 antibody inhibits tumor growth in pre-clinical models of pancreatic cancer

Neuregulin 1 (NRG1), a ligand for HER3 and HER4 receptors, is secreted by both pancreatic tumor cells (PC) and cancer-associated fibroblasts (CAFs), the latter representing the most abundant compound of pancreatic stroma. This desmoplastic stroma contributes to Pancreatic Ductal Adenocarcinoma (PDAC) aggressiveness and therapeutic failure by promoting tumor progression, invasion and resistance to chemotherapies. In the present work, we aimed at disrupting the complex crosstalk between PC and CAF in order to prevent tumor cell proliferation. To do so, we demonstrated the promising tumor growth inhibitory effect of the 7E3, an original antibody directed to NRG1. This antibody promotes antibody dependent cellular cytotoxicity in NRG1-positive PC and CAFs and inhibits NRG1-associated signaling pathway induction, by blocking NRG1-mediated HER3 activation. Moreover, 7E3 inhibits migration and growth of pancreatic cancer cells co-cultured with CAFs, both in vitro and in vivo using orthotopic pancreatic tumor xenografts. Our preclinical results demonstrate that the anti-NRG1 antibody 7E3 could represent a promising approach to target pancreatic stroma and cancer cells, thereby providing novel therapeutic options for PDAC.

Pharmacological Ascorbate Radiosensitizes Pancreatic Cancer.

The toxicity of pharmacologic ascorbate is mediated by the generation of H2O2 via the oxidation of ascorbate. Because pancreatic cancer cells are sensitive to H2O2 generated by ascorbate, they would also be expected to become sensitized to agents that increase oxidative damage such as ionizing radiation. The current study demonstrates that pharmacologic ascorbate enhances the cytotoxic effects of ionizing radiation as seen by decreased cell viability and clonogenic survival in all pancreatic cancer cell lines examined, but not in nontumorigenic pancreatic ductal epithelial cells. Ascorbate radiosensitization was associated with an increase in oxidative stress-induced DNA damage, which was reversed by catalase. In mice with established heterotopic and orthotopic pancreatic tumor xenografts, pharmacologic ascorbate combined with ionizing radiation decreased tumor growth and increased survival, without damaging the gastrointestinal tract or increasing systemic changes in parameters indicative of oxidative stress. Our results demonstrate the potential clinical utility of pharmacologic ascorbate as a radiosensitizer in the treatment of pancreatic cancer.

Dynamic contrast enhanced magnetic resonance imaging of an orthotopic pancreatic cancer mouse model.

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

Preclinical validation of AXL receptor as a target for antibody-based pancreatic cancer immunotherapy

AXL receptor tyrosine kinase (RTK) is implicated in proliferation and invasion of many cancers, particularly in pancreatic ductal adenocarcinoma (PDAC), for which new therapeutic options are urgently required. We investigated whether inhibition of AXL activity by specific monoclonal antibodies (mAbs) is efficient in limiting proliferation and migration of pancreatic cancer cells. Expression of AXL was evaluated by immunohistochemistry in 42 PDAC. The AXL role in oncogenesis was studied using the short hairpin RNA approach in a pancreatic carcinoma cell line. We further generated antihuman AXL mAbs and evaluated their inhibitory effects and the AXL downstream signaling pathways first in vitro, in a panel of pancreatic cancer cell lines and then in vivo, using subcutaneous or orthotopic pancreatic tumor xenografts. AXL receptor was found expressed in 76% (32/42) of PDAC and was predominantly present in invasive cells. The AXL-knockdown Panc-1 cells decreased in vitro cell migration, survival and proliferation, and reduced in vivo tumor growth. Two selected anti-AXL mAbs (D9 and E8), which inhibited phosphorylation of AXL and of its downstream target AKT without affecting growth arrest-specific factor 6 (GAS6) binding, induced downexpression of AXL by internalization, leading to an inhibition of proliferation and migration in the four pancreatic cancer cell lines studied. In vivo, treatment by anti-AXL mAbs significantly reduced growth of both subcutaneous and orthotopic pancreatic tumor xenografts independently of their KRAS mutation status. Our in vitro and preclinical in vivo data demonstrate that anti-human AXL mAbs could represent a new approach to the pancreatic cancer immunotherapy.

Early Therapy Evaluation of Combined Cetuximab and Irinotecan in Orthotopic Pancreatic Tumor Xenografts by Dynamic Contrast-Enhanced Magnetic Resonance Imaging

Early pancreatic cancer response following cetuximab and/or irinotecan therapies was measured by serial dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) before and during therapy. Groups 1 to 4 (n = 6/group) of SCID mice bearing orthotopic pancreatic adenocarcinoma xenografts expressing luciferase were treated with phosphate-buffered saline, cetuximab, irinotecan, or cetuximab combined with irinotecan, respectively, twice weekly for 3 weeks. DCE-MRI was performed on days 0, 1, 2, and 3 after therapy initiation, whereas anatomic magnetic resonance imaging was performed on days 0, 1, 2, 3, 6, and 13. Bioluminescence imaging was performed on days 0 and 21. At day 21, all tumors were collected for further histologic analyses (Ki-67 and CD31 staining), whereas tumor dimensions were measured by calipers. The Ktrans values in the 0.5 mm-thick peripheral tumor region were calculated, and the changes in Ktrans during the 3 days posttherapy were compared to tumor volume changes, bioluminescent signal changes, and histologic findings. The Ktrans changes in the peripheral tumor region after 3 days of therapy were linearly correlated with 21-day decreases in tumor volume (p < .001), bioluminescent signal (p = .050), microvessel densities (p = .002), and proliferating cell densities (p = .001). This study supports the clinical use of DCE-MRI for pancreatic cancer patients for early assessment of an anti-epidermal growth factor receptor therapy combined with chemotherapy.

In Vivo Targeted Contrast Enhanced Micro-Ultrasound to Measure Intratumor Perfusion and Vascular Endothelial Growth Factor Receptor 2 Expression in a Mouse Orthotopic Bladder Cancer Model

We evaluated the feasibility of using targeted contrast enhanced micro-ultrasound imaging to assess intratumor perfusion and vascular endothelial growth factor receptor 2 expressions in a mouse orthotopic bladder cancer model. We created an orthotopic mouse model by implanting MBT-2 murine bladder cancer cell lines in the bladder of syngeneic C3H/He mice (Jackson Laboratory, Bar Harbor, Maine). Successful tumor implantation was confirmed by transabdominal micro-ultrasound imaging on post-implantation day 11. Contrast enhanced micro-ultrasound imaging was done on days 14 and 21. Vascular endothelial growth factor receptor 2 targeted contrast agent was prepared by adding biotinylated anti-vascular endothelial growth factor receptor 2 monoclonal antibodies to streptavidin coated microbubbles. The targeted contrast agents were injected via the retro-orbital route. We quantified intratumor perfusion, vascular endothelial growth factor receptor 2 endothelial expression and blood volume in real time. In the initial study intratumor perfusion data and vascular endothelial growth factor receptor 2 expression could only be measured in 10 of 14 mice (71%) due to motion artifact. We modified our technique by applying an elastic band over the lower abdomen to minimize body wall movement. After the modification complete images were acquired in all mice at 2 consecutive imaging sessions. Measurements were made of intratumor perfusion and in vivo vascular endothelial growth factor receptor 2 expression. No adverse effects occurred due to anesthesia or the ultrasound contrast agent. Targeted contrast enhanced micro-ultrasound imaging enables investigators to detect and monitor vascular changes in orthotopic bladder tumors. It may be useful for direct, noninvasive, in vivo evaluation of novel anti-angiogenesis therapeutic agents. With the modified technique target enhanced contrast ultrasound can be applied in an orthotopic bladder cancer model.

18F-FDG PET/CT Imaging Detects Therapy Efficacy of Anti-EMMPRIN Antibody and Gemcitabine in Orthotopic Pancreatic Tumor Xenografts

The objective of this study is to evaluate the therapeutic response to a novel monoclonal antibody targeting human extracellular matrix metalloproteinase inducer (EMMPRIN) in combination with gemcitabine in a pancreatic-tumor xenograft murine model by sequential 2-deoxy-2-[18F]fluoro-D-glucose ((18)F-FDG) positron emission tomography/computed tomgraphy (PET/CT) imaging. Four groups of SCID mice bearing orthotopic pancreatic tumor xenografts were injected with phosphate-buffered saline, gemcitabine (120 mg/kg BW), anti-EMMPRIN antibody (0.2 mg), or combination, respectively, twice weekly for 2 weeks, while (18)F-FDG PET/CT imaging was performed weekly for 3 weeks. Changes in mean standardized uptake value (SUV(mean)) of (18)F-FDG and volume of tumors were determined. The tumor SUV(mean) change in the group receiving combination therapy was significantly lower than those of the other groups. Tumor-volume changes of groups treated with anti-EMMPRIN monotherapy or combined therapy were significantly lower than that of the control group. These data provide support for clinical studies of anti-EMMPRIN therapy with gemcitabine for pancreatic cancer treatment.

Abstract 1795: Therapy assessment of a novel anti-EMMPRIN antibody and gemcitabine in an orthotopic pancreatic-tumor murine model by FDG PET/CT imaging

Abstract Purpose: To evaluate a novel monoclonal antibody targeting human EMMPRIN with/without gemcitabine in an orthotopic pancreatic-tumor model by sequential 18F-FDG PET/CT imaging. Methods: Cytotoxicity of anti-EMMPRIN mAb alone or in combination with gemcitabine was measured for human pancreatic (MIA PaCa-2) cells by ATPLite assay. For in vivo animal study, six groups of SCID mice bearing orthotopic MIA PaCa-2 tumors was used at 21 days after cell implantation; groups 1 and 2 (n=5 per group) were i.v. injected with Tc-99m labeled anti-EMMPRIN mAb and isotype control mAb respectively. SPECT/CT imaging was conducted at 4 hours after injection, while biodistribution analysis was followed at 24 hours after injection. Groups 3-6 (n=6 per group) were i.p. injected with PBS (served as control), gemcitabine (120mg/kg BW), anti-EMMPRIN mAb (0.2mg), and combination, respectively, twice weekly for 2 weeks, while 18F-FDG PET/CT imaging was applied weekly for 3 weeks. Intratumoral SUVmean and tumor-volume changes during therapy were quantified. All tumors of groups 3-6 were collected at day 35, and Ki-67 and TUNEL staining were implemented. Results: In vitro ATPLite assay demonstrated only modest killing efficacy by anti-EMMPRIN mAb alone or with gemcitabine. SPECT imaging showed the specific tumor uptake of Tc-99m-anti-EMMPRIN mAb. The %ID/g in liver, blood, and tumor of group 1 were 9.1±1.3, 13.4±3.0, and 27.6±3.2 respectively, while those of group 2 were 9.2±2.2, 10.8±1.3, and 5.8±1.7 respectively; tumor uptake of Tc-99m-anti-EMMPRIN mAb was significantly higher (p&amp;lt;0.001), while no differences were detected in liver and blood values. Intratumoral SUVmean changes of groups 3-6 relative to those at day 21 were 35±13, -19±19, 25±26, and -26±13% respectively at day 28, and 134±56, 60±32, 43±30, and -25±16% respectively at day 35; that of group 6 was significantly lower than those of the other groups (p&amp;lt;0.05), while no difference was detected among groups 3-5. Tumor-volume changes of groups 3-6 were 111±24, 72±26, 83±27, and 3±8% respectively at day 28, and 399±48, 275±57, 204±34, and 35±8% respectively at day 35; that of group 6 was significantly lower than those of groups 3-5 (p&amp;lt;0.05), and that of group 5 was also significantly lower than that of group 3 (p=0.042). The correlation between intratumoral SUVmean and tumor-volume changes was statistically significant (p=0.002). Proliferating cell density of group 6 was significantly lower than that of group 3 (p=0.008), while no difference was detected in apoptotic cell densities. Conclusions: SUVmean of orthotopic pancreatic tumor xenografts was significantly decreased by the combination therapy, consistent with reduced proliferating cell density. These data provide support for clinical studies of anti-EMMPRIN therapy with gemcitabine for pancreatic cancer treatment, and the application of PET/CT with FDG to monitor efficacy. 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 1795. doi:10.1158/1538-7445.AM2011-1795

Synthesis and Ex Vivo Autoradiographic Evaluation of Ethyl-β-d-galactopyranosyl-(1,4′)-2′-deoxy-2′-[18F]fluoro-β-d-glucopyranoside—A Novel Radioligand for Lactose-Binding Protein: Implications for Early Detection of Pancreatic Carcinomas with PET

Previous studies demonstrated that the lactose-binding protein (hepatocellular carcinoma-intestine-pancreas and pancreatitis-associated proteins (HIP/PAP)) is upregulated >130 times in peritumoral pancreatic tissue as compared to normal pancreatic tissue. Therefore, we developed a new radiolabeled ligand of HIP/PAP, the ethyl-β-D-galactopyranosyl-(1,4')-2'-deoxy-2'-[¹⁸F]fluoro-β-D-glucopyranoside (Et-[¹⁸F]FDL) for noninvasive imaging of pancreatic carcinoma using positron emission tomography and computerized tomography (PET/CT). The novel precursor and radiolabeling methods for synthesis of Et-[¹⁸F]FDL produced no isomers; the average decay-corrected radiochemical yield was 68%, radiochemical purity >99%, and specific activity >74 GBq/µmol. The radioligand properties of Et-[¹⁸F]FDL were evaluated using an ex vivo autoradiography and immunohistochemistry in pancreatic tissue sections obtained from mice-bearing orthotopic pancreatic tumor xenografts. Et-[¹⁸F]FDL binding to peritumoral pancreatic tissue sections strongly correlated with HIP/PAP expression (r = 0.81) and could be completely blocked by treatment with 1 mM lactose. These results suggest that Et-[¹⁸F]FDL is a promising agent which should be evaluated for detection of early pancreatic carcinomas by PET/CT imaging.

Abstract 641: Early therapy assessment of cetuximab and irinotecan in orthotopic pancreatic tumor xenografts by dynamic contrast-enhanced magnetic resonance imaging

Abstract Purpose: To evaluate DCE-MRI as an early prognostic tool for effective anti-EGFR therapy with concurrent chemotherapy in a murine orthotopic pancreatic-cancer model, and to develop a novel timing-independent DCE-MRI biomarker for early therapy assessment. Methods: Groups 1-4 (n=6/group) of SCID mice bearing orthotopic pancreatic adenocarcinoma (luciferase-positive MIA PaCa-2) were treated with PBS (control), cetuximab (1mg), irinotecan (25mg/kg), or cetuximab plus irinotecan respectively twice weekly for 3 weeks. DCE-MRI was performed on days 0, 1, 2, and 3 after therapy initiation, while anatomical MRI was performed once weekly for 3 weeks. Bioluminescence imaging was performed on days 0 and 21. At day 21, all tumors were collected for CD31 and Ki-67 staining. The averaged Ktrans values in the entire tumor region or within the 0.5-mm thick peripheral tumor region were calculated. The best-fitting 2nd-order polynomial curves for the Ktrans changes were retrieved, and the quadratic coefficient for each curve was proposed as the novel MRI biomarker. Results: The change in Ktrans values of groups 1-4 for 3 days after therapy initiation in the entire tumor region were not statistically different among any of the groups. However, when analyzed in peripheral tumor region, change in Ktrans values were 102±16%, 42±21%, 15±6%, and −19±5% respectively, and the significant suppression of Ktrans increase was detected after irinotecan (p=0.008) or combination therapy (p&amp;lt;0.001). During 21 days, the tumor-volume increase was significantly suppressed by either monotherapy or combined therapy (p&amp;lt;0.050). The Ktrans changes observed for 3 days in the peripheral region were significantly correlated with tumor-volume changes (p&amp;lt;0.001), bioluminescence-signal changes (p=0.050), microvessel densities (p=0.002) and proliferating-cell densities (p=0.001). The mean Ktrans changes of all 4 groups in peripheral tumor region followed second-order polynomial curves, and the quadratic coefficient of each curve was proposed as a novel DCE-MRI based biomarker; the mean value of novel biomarker of groups 1-4 were 0.050±0.026, −0.015±0.028, −0.071±0.021, and −0.082±0.013 respectively. The values of the novel biomarker were significantly correlated with tumor-volume changes (p&amp;lt;0.001), bioluminescence-signal changes (p=0.019), microvessel densities (p=0.002), and proliferating-cell densities (p=0.001). Conclusion: This study supports the clinical use of DCE-MRI to evaluate an anti-EGFR therapy combined with chemotherapy for pancreatic adenocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 641.

Early Therapy Evaluation of Combined Anti–Death Receptor 5 Antibody and Gemcitabine in Orthotopic Pancreatic Tumor Xenografts by Diffusion-Weighted Magnetic Resonance Imaging

Early therapeutic efficacy of anti-death receptor 5 antibody (TRA-8) combined with gemcitabine was measured using diffusion-weighted magnetic resonance imaging (DWI) in an orthotopic pancreatic tumor model. Groups 1 to 4 of severe combined immunodeficient mice (n = 5-7 per group) bearing orthotopically implanted, luciferase-positive human pancreatic tumors (MIA PaCa-2) were subsequently (4-5 weeks thereafter) injected with saline (control), gemcitabine (120 mg/kg), TRA-8 (200 mug), or TRA-8 combined with gemcitabine, respectively, on day 0. DWI, anatomic magnetic resonance imaging, and bioluminescence imaging were done on days 0, 1, 2, and 3 after treatment. Three tumors from each group were collected randomly on day 3 after imaging, and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining was done to quantify apoptotic cellularity. At just 1 day after starting therapy, the changes of apparent diffusion coefficient (ADC) in tumor regions for group 3 (TRA-8) and group 4 (TRA-8/Gem) were 21 +/- 9% (mean +/- SE) and 27 +/- 3%, respectively, significantly higher (P < 0.05) than those of group 1 (-1 +/- 5%) and group 2 (-2 +/- 4%). There was no statistical difference in tumor volumes for the groups at this time. The mean ADC values of groups 2 to 4 gradually increased over 3 days, which were concurrent with tumor volume regressions and bioluminescence signal decreases. Apoptotic cell densities of tumors in groups 1 to 4 were 0.7 +/- 0.4%, 0.6 +/- 0.2%, 3.1 +/- 0.9%, and 4.7 +/- 1.0%, respectively, linearly proportional to the ADC changes on day 1. Further, the ADC changes were highly correlated with the previously reported mean survival times of animals treated with the same agents and doses. This study supports the clinical use of DWI for pancreatic tumor patients for early assessment of drug efficacy.

Improved lentiviral transduction of human mesenchymal stem cells for therapeutic intervention in pancreatic cancer

Genetic modification of human bone marrow mesenchymal stem cells (MSC) is highly valuable for their exploitation in basic science and therapeutic applications, for example in cancer. We present here a new, fast and easy-to-use method to enrich a functional population of lentiviral (LV)-transduced MSC expressing enhanced green fluorescent protein (eGFP). We replaced the eGFP gene by a fusion gene of puromycin acetyltransferase and eGFP. Upon LV gene transfer and puromycin selection, we quickly obtained a pure transduced MSC population, in which growth, differentiation capacity and migration preferences were not compromised. Furthermore, we are the first to report the migration velocity of MSC among which 30% were moving and velocity of about 15 mum h(-1) was not altered by LV transduction. Manipulated MSC underwent senescence one passage earlier than non-transduced cells, suggesting the use for therapeutic intervention in early passage numbers. Upon tail vein application in nude mice, the majority of LV-transduced MSC could be detected in human orthotopic pancreatic tumor xenografts and to a minor extent in mouse liver, kidney and lung. Together, LV transduction of genes to MSC followed by puromycin selection is a powerful tool for basic research and improves the therapeutic prospects of MSC as vehicles in gene therapy.