Heterotopic Model Research Articles

Long-Pentraxin 3 Derivative as a Small-Molecule FGF Trap for Cancer Therapy

The fibroblast growth factor (FGF)/FGF receptor (FGFR) system plays a crucial role in cancer by affecting tumor growth, angiogenesis, drug resistance, and escape from anti-angiogenic anti-vascular endothelial growth factor therapy. The soluble pattern recognition receptor long-pentraxin 3 (PTX3) acts as a multi-FGF antagonist. Here we demonstrate that human PTX3 overexpression in transgenic mice driven by the Tie2 promoter inhibits tumor growth, angiogenesis, and metastasis in heterotopic, orthotopic, and autochthonous FGF-dependent tumor models. Using pharmacophore modeling of the interaction of a minimal PTX3-derived FGF-binding pentapeptide with FGF2, we identified a small-molecule chemical (NSC12) that acts as an extracellular FGF trap with significant implications in cancer therapy.

Abstract 3491: Enhancing nab-paclitaxel antitumor activity through addition of BMS-754807, a small-molecule inhibitor of IGF-1R/IR, in experimental pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States, and is expected to become the second-leading cause of cancer death by 2030. Gemcitabine, the standard of care agent over the past 16 years, has limited clinical benefits. Recently, nab-paclitaxel has shown greater efficacy against advanced PDAC in combination with gemcitabine. Our lab has demonstrated superior antitumor activity of nab-paclitaxel compared with gemcitabine or docetaxel in preclinical PDAC studies. Insulin like growth factor (IGF) signaling proteins are frequently overexpressed in PDAC and correlate with aggressive tumor phenotype and poor prognosis indicating that the IGF system is an important therapeutic target in PDAC patients. BMS-754807, a small molecule inhibitor of IGF-type 1 receptor (IGF-1R) and insulin receptor (IR), has shown efficacy in PDAC. We evaluated combination treatment benefits of nab-paclitaxel with BMS-754807 in experimental PDAC. In vitro cell proliferation and protein expression were measured by WST-1 assay and immunoblotting. Heterotopic tumor growth and animal survival experiments were performed in murine xenografts. In vitro analysis of four PDAC cell lines (AsPC-1, BxPC-3, MIA PaCa-2, Panc-1) revealed that single agent nab-paclitaxel and BMS-754807 both inhibited cell proliferation in a dose-dependent manner. To evaluate the combination treatment benefits of nab-paclitaxel and BMS-754807, the IC25 dose of BMS-754807 was combined with increasing doses of nab-paclitaxel. Addition of BMS-754807 decreased the nab-paclitaxel IC50 from 7.2 μM to 490 nM for AsPC-1, 430 nM to 50 nM for BxPC-3, 740 nM to 540 nM for MIA PaCa-2, and 690 nM to 280 nM for Panc-1 cell lines. These data suggest that BMS-754807 combines well with nab-paclitaxel and efficiently decreases the nab-paclitaxel IC50 in PDAC cell lines. In a heterotopic AsPC-1 PDAC model, relative to controls (100±21.3), percent net local tumor growth was 17.1±21.1 with nab-paclitaxel (p = 0.002), 37.6±16.2 with BMS-754807 (p = 0.01) and 0.76±9.1 with nab-paclitaxel plus BMS-754807 (p = 0.0002). In Panc-1 subcutaneous xenografts, compared with controls (100±31.5), percent net tumor growth was 7.8±27.2 with nab-paclitaxel (p = 0.002), 40.2±24.1 with BMS-754807 (p = 0.02) and -29.7±16.8 with nab-paclitaxel plus BMS-754807 (p = 0.0001). In an animal survival study, median animal survival was increased compared to controls (21 days) after nab-paclitaxel therapy (40 days, a 90% increase, p = 0.002), BMS-754807 therapy (27 days, a 29% increase, p = 0.01), and nab-paclitaxel plus BMS-754807 therapy (47 days, a 124% increase, p = 0.005). These results highlight measurable antitumor activity of BMS-754807 in experimental PDAC and support the potential of BMS-754807 in combination with nab-paclitaxel as promising targeted therapy for clinical pancreatic cancer therapy. Citation Format: Niranjan Awasthi, Margaret A. Schwarz, Roderich E. Schwarz. Enhancing nab-paclitaxel antitumor activity through addition of BMS-754807, a small-molecule inhibitor of IGF-1R/IR, in experimental pancreatic 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 3491. doi:10.1158/1538-7445.AM2015-3491

Abstract 4376: Characterization and targeting of TAF15, a radiation-inducible target in multiple cancer types

Abstract Currently, development of anti-cancer therapies is limited to antigens that are either overexpressed or that are specific to cancer. This limits both the number of available targets and their tumor selectivity. Using phage display technology, we identified a number of proteins in multiple cancer types that are induced by sub-lethal doses of ionizing radiation (IR), including TATA-box-binding protein associated factor 15 (TAF-15), a key transcriptional regulator. TAF15 presents a novel synergistic molecular target in cancer for both therapeutic and imaging purposes. In the first part of this study, we characterized TAF15 as a radiation-inducible molecular target both in vitro and in vivo using various analytical approaches. Surface expression analysis of TAF15 via flow cytometry showed that TAF15 is not only expressed on the surface of brain, lung and breast cancer, but is also induced by IR reproducibly on the surface of breast cancer and endothelial cells, and not in normal cells. Using a heterotopic tumor model in athymic nude mice, near-infrared (NIR) imaging analysis results showed that TAF15 can be successfully targeted in several different irradiated human tumor xenografts using both an anti-TAF15 antibody (Ab) and our in-house anti-TAF15 peptide. More importantly, we demonstrated that TAF15 can also be targeted in an orthotopic primary tumor model; NIR results showed high affinity binding of an anti-TAF15 Ab to irradiated primary breast tumors established in the mammary fat pads of nude mice as compared to isotype-matched controls. Furthermore, we have successfully genetically modified the knob domain of an adenovirus (Ad) type 5 fiber protein to contain our lead anti-TAF15 peptide and showed that this novel Ad-TAF15 targeting peptide binds strongly to irradiated breast tumor xenografts in a recent pilot study using bioluminescence imaging (BLI), a result that has major therapeutic and tumor imaging implications. We are currently evaluating the microscopic biodistribution of TAF15 in the primary tumor microenvironment using immunofluorescence and whether cell surface binding of TAF15 can activate an immune response, either via antibody-dependent cell mediated cytotoxicity (ADCC) or antibody dependent cell mediated phagocytosis (ADCP). In the second part of this study, we initiated anti-TAF15 monoclonal antibody (mAb) production against 3 different TAF15 epitopes using hybridoma technology. After screening for binding specificity to TAF15 binding via Western and dot blot analysis, we identified several lead anti-TAF15 antibody clones for further sub-cloning. We are currently evaluating the binding affinity to TAF15, in vivo efficacy and cancer cell specificity of these lead clones. Future studies include humanization of these candidate mAbs and evaluation of their cancer specificity and anti-cancer cytotoxicity with the goal of introducing the most efficacious and selective anti-TAF15 mAbs into clinical trials. Note: This abstract was not presented at the meeting. Citation Format: Lincoln Muhoro, Heping Yan, Sergey Kaliberov, Jerry Jaboin, David Curiel, Dennis Hallahan. Characterization and targeting of TAF15, a radiation-inducible target in multiple cancer types. [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 4376. doi:10.1158/1538-7445.AM2015-4376

Altered Biological Potential and Radioresponse of Murine Tumors in Different Microenvironments.

PurposeThis study was conducted to evaluate the biological features of murine hepatocarcinoma according to different tumor microenvironmental models and to determine the change in molecular and immunologic responses after radiation.Materials and MethodsTumor models were established in the liver (orthotopic) and thigh (heterotopic) of male C3H/HeN mice. Tumor growth and lung metastasis were assessed in these models. To evaluate the radiation effect, the tumors were irradiated with 10 Gy. Factors associated with tumor microenvironment including vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), transforming growth factor beta1 (TGF-β1), CD31, and serum interleukin-6 (IL-6) were evaluated. Tumor-infiltrating regulatory immune cells, regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs) were also analyzed.ResultsA higher number of lung metastases were observed in the orthotopic tumor model than in the heterotopic tumor model. VEGF, CD31, COX-2, and TGF-β1 expression was more prominent in the orthotopic tumor model than in the heterotopic tumor model. Expression of the angiogenic factor VEGF and key regulatory molecules (TGF-β1 and COX-2) decreased following radiation in the orthotopic tumor model, while the serum IL-6 level increased after radiation. In the orthotopic tumor model, the number of both Tregs and MDSCs in the tumor burden decreased after radiation.ConclusionThe orthotopic tumor model showed higher metastatic potential and more aggressive molecular features than the heterotopic tumor model. These findings suggest that the orthotopic tumor mouse model may be more reflective of the tumor microenvironment and suitable for use in the translational research of radiation treatment.

Molecular Imaging of Tumors Using a Quantitative T 1 Mapping Technique via Magnetic Resonance Imaging.

Magnetic resonance imaging (MRI) of glioblastoma multiforme (GBM) with molecular imaging agents would allow for the specific localization of brain tumors. Prior studies using T1-weighted MR imaging demonstrated that the SBK2-Tris-(Gd-DOTA)3 molecular imaging agent labeled heterotopic xenograft models of brain tumors more intensely than non-specific contrast agents using conventional T1-weighted imaging techniques. In this study, we used a dynamic quantitative T1 mapping strategy to more objectively compare intra-tumoral retention of the SBK2-Tris-(Gd-DOTA)3 agent over time in comparison to non-targeted control agents. Our results demonstrate that the targeted SBK2-Tris-(Gd-DOTA)3 agent, a scrambled-Tris-(Gd-DOTA)3 control agent, and the non-specific clinical contrast agent Optimark™ all enhanced flank tumors of human glioma cells with similar maximal changes on T1 mapping. However, the retention of the agents differs. The non-specific agents show significant recovery within 20 min by an increase in T1 while the specific agent SBK2-Tris-(Gd-DOTA)3 is retained in the tumors and shows little recovery over 60 min. The retention effect is demonstrated by percent change in T1 values and slope calculations as well as by calculations of gadolinium concentration in tumor compared to muscle. Quantitative T1 mapping demonstrates the superior binding and retention in tumors of the SBK2-Tris-(Gd-DOTA)3 agent over time compared to the non-specific contrast agent currently in clinical use.

Increased expression of Notch 1 in a rat liver transplantation model.

Liver transplantation is the standard treatment for end‑stage liver failure; however, rejection can result in allograft failure. In order to investigate the role of Notch 1 during rejection, the present study evaluated Notch 1 expression, as well as the levels of immune reactivity, in rat liver allografts. A heterotopic liver transplantation model was established using Dark Agouti (DA) rats as donors and Lewis rats as recipients (DA/Lewis), with DA recipient rats serving as controls (DA/DA). The concentration levels of immune reactivity markers and serum Notch 1 were measured on days 3, 5, and 7. The overall survival was significantly shorter (<10 days) in the DA/Lewis group, as compared with the DA/DA group (P<0.0001). The concentration levels of serum alanine aminotransferase and total bilirubin were significantly higher 5 and 7 days following transplantation in the DA/Lewis group, as compared with the DA/DA group (P<0.001). The concentration levels of serum Notch 1 were significantly higher in the DA/Lewis group, as compared with the DA/DA group on days 3, 5, and 7 following transplantation (P<0.0001). These results indicate that the expression levels of serum Notch 1 significantly increase during liver allograft rejection, suggesting that Notch 1 is involved in the mechanism underlying liver allograft rejection. Notch 1 may serve as a marker of acute rejection in a rat liver transplantation model.

Improved Cuff Technique for Establishing a Mouse–Rat Heterotopic Cardiac Xenotransplantation Model

BackgroundThe small animal model of cardiac transplantation is the most common model in organ transplantation studies. The cervical heterotopic transplantation is widely performed because this allows for direct observation of the graft heartbeat and contributes to early prediction of graft rejection. ObjectiveA mouse–rat cervical heterotopic cardiac xenotransplantation model was modified with respect to the anesthesia method, cardiac graft harvesting method, and perioperative treatment. These improvements ensure the stability and reliability of xenotransplantation models for in vivo studies of immune-mediated graft rejection. MethodsAfter establishing isoflurane inhalation anesthesia, the donors' hearts were harvested. The experimental method involved separate ligation of the left and right superior venae cavae; the other blood vessels were ligated in a cluster. Both the donor and recipient animals were placed on a heating pad intraoperatively to maintain a body temperature of 37–40°C. The model establishment was divided into 3 stages: practice, stabilization, and stereotyping. The surgical success rate and operation time were recorded. Specimens were harvested at different time points for histopathological examination. ResultsThe anesthetic effect of isoflurane was well maintained, and no animals died of adverse anesthetic events. Body temperature was maintained at 37–40°C which effectively shortened the time to restoration. The modification of the cardiac graft harvesting method is conducive to rebeating of the donor heart. The success rates in the stabilization and stereotyping stages were significantly higher than that in the practice stage (P < .05). The operation time in the stabilization and stereotyping stages were significantly shorter than those in the practice stage (P < .05). Histopathological examination revealed thrombosis formation, interstitial hemorrhage, and inflammatory cell infiltration in the donor hearts. ConclusionOur findings suggest that the mouse–rat cervical heterotopic cardiac xenotransplantation model is the ideal animal model for studying xenograft rejection.

High intensity focused ultrasound-induced bubbles stimulate the release of nucleic acid cancer biomarkers

Prostate biopsy for prostate cancer (PCa) is invasive with associated morbidity and several diagnostic limitations, suggesting the need for a new approach. Recently, several nucleic acid cancer biomarkers (e.g., microRNA and mutant DNA) have been identified and shown promise for improving cancer diagnostics. However, the abundance of these biomarker classes in the circulation is low, impeding reliable detection and adoption into clinical practice. In order to stimulate the release of these intracellular biomarkers, the exposures optimized for mechanical disruption of cells may be desirable. Here, two approaches based on HIFU-induced bubble activity were tested for their ability to stimulate release of cancer-associated microRNAs in a heterotopic syngeneic rat prostate cancer model. In the first approach, tumor tissue was locally liquefied with boiling histotripsy (BH) - a HIFU technique utilizing millisecond-long pulses to create boiling bubbles via rapid shockwave heating. The interaction of shocks with the ensuing vapor cavity fractionates tissue with negligible thermal effect. In the second approach HIFU-induced inertial cavitation was used for permeabilization of tumor tissue and vasculature.

Revised Arterial Anastomosis for Improving Murine Kidney Transplant Outcomes

ABSTRACTAim: One of the most challenging research microsurgical techniques is the mouse kidney transplant however, very few laboratories have made use of this important model due to its difficulty. One of the main obstacles to utilizing this procedure is the high incidence of post-operative arterial thrombosis. We believe this is caused by the path in which blood is required to flow from the recipient abdominal aorta, via the donor recipient aorta and on into the renal artery creating a tortuous route and areas of turbulence, which are prone to thrombus formation and failure of the graft. Methods: We describe revised methods of donor artery recovery, whereby the traditional transection of the donor aorta is replaced with a heel and toe cuff, which is created by dividing the donor abdominal aorta obliquely across the face of the renal arterial ostium, which then provides for an arterial end-to-side anastomosis of a scale similar to that used for the heterotopic heart model. This technique produces an anastomosis that facilitates free blood flow from the recipient abdominal aorta at less than 90° thereby reducing the likelihood of thrombus formation. Results: Utilizing this new technique the incidence of arterial thrombosis has decreased from 35% to 0% (n = 20 and 24, respectively) with no change in ischemia times. Conclusion: We describe a revised method of performing the arterial anastomosis during mouse kidney transplantation, which facilitates improved fluid dynamics by straightening the flow path for blood to the graft resulting in significantly reduced thrombus formation, excellent graft function, histology, and post-transplant survival.

Mesenchymal stromal cells improve cardiac function and left ventricular remodeling in a heart transplantation model

Ischemia/reperfusion (I/R) injury is an inevitable consequence of organ transplantation and a major determinant of patient and graft survival in heart transplantation. Bone marrow-mesenchymal stromal cell (BM-MSC) treatment is a potentially effective cell therapy for cardiac disease. We investigated the effects of intravenous delivery of BM-MSCs in the acute phase post-transplant in a heterotopic heart transplantation (HHT) model associated with I/R injury. Hearts of wild-type Lewis (WT LEW) rats were harvested and transplanted heterotopically into the necks of recipient WT LEW rats. Forty-eight hours after HHT, BM-MSCs were injected intravenously into animals in the experimental group, whereas controls received normal saline (NS). Eight days after BM-MSC injection, fractional shortening of transplanted hearts was significantly higher and left ventricular systolic diameter was lower in the BM-MSC group compared with controls, whereas no differences were found 28 days after infusion. A reduction in ventricular remodeling and cardiac fibrosis was observed by histochemical analysis and confirmed by cardiac magnetic resonance imaging in the BM-MSC group. The perivascular stromal cells' density and the number of capillaries were increased whereas the number of apoptotic cells decreased significantly in transplanted hearts in the BM-MSC group compared with the NS group. We showed early improvement in cardiac function and subsequent enhanced ventricular remodeling, reduced cardiac fibrosis, augmented neo-vascularization and decreased cardiomyocyte apoptosis of the transplanted heart in a heterotopic transplantation model after intravenous infusion of BM-derived MSCs. Our data suggest that clinical studies with BM-MSCs are warranted to understand their effects on cardiac graft and transplant recipient survival.

Fabrication method, structure, mechanical, and biological properties of decellularized extracellular matrix for replacement of wide bone tissue defects

The present paper was focused on the development of a new method of decellularized extracellular matrix (DECM) fabrication via a chemical treatment of a native bone tissue. Particular attention was paid to the influence of chemical treatment on the mechanical properties of native bones, sterility, and biological performance in vivo using the syngeneic heterotopic and orthotopic implantation models. The obtained data indicated that after a chemical decellularization treatment in 4% aqueous sodium chlorite, no noticeable signs of the erosion of compact cortical bone surface or destruction of trabeculae of spongy bone in spinal channel were observed. The histological studies showed that the chemical treatment resulted in the decellularization of both bone and cartilage tissues. The DECM samples demonstrated no signs of chemical and biological degradation in vivo. Thorough structural characterization revealed that after decellularization, the mineral frame retained its integrity with the organic phase; however clotting and destruction of organic molecules and fibers were observed. FTIR studies revealed several structural changes associated with the destruction of organic molecules, although all organic components typical of intact bone were preserved. The decellularization-induced structural changes in the collagen constituent resulted changed the deformation under compression mechanism: from the major fracture by crack propagation throughout the sample to the predominantly brittle fracture. Although the mechanical properties of radius bones subjected to decellularization were observed to degrade, the mechanical properties of ulna bones in compression and humerus bones in bending remained unchanged. The compressive strength of both the intact and decellularized ulna bones was 125–130MPa and the flexural strength of humerus bones was 156 and 145MPa for the intact and decellularized samples, respectively. These results open new avenues for the use of DECM samples as the replacement of wide bone tissue defects.

Interleukin-5 facilitates lung metastasis by modulating the immune microenvironment.

Although the lung is the most common metastatic site for cancer cells, biologic mechanisms regulating lung metastasis are not fully understood. Using heterotopic and intravenous injection models of lung metastasis in mice, we found that IL5, a cytokine involved in allergic and infectious diseases, facilitates metastatic colonization through recruitment of sentinel eosinophils and regulation of other inflammatory/immune cells in the microenvironment of the distal lung. Genetic IL5 deficiency offered marked protection of the lungs from metastasis of different types of tumor cells, including lung cancer, melanoma, and colon cancer. IL5 neutralization protected subjects from metastasis, whereas IL5 reconstitution or adoptive transfer of eosinophils into IL5-deficient mice exerted prometastatic effects. However, IL5 deficiency did not affect the growth of the primary tumor or the size of metastatic lesions. Mechanistic investigations revealed that eosinophils produce CCL22, which recruits regulatory T cells to the lungs. During early stages of metastasis, Treg created a protumorigenic microenvironment, potentially by suppressing IFNγ-producing natural killer cells and M1-polarized macrophages. Together, our results establish a network of allergic inflammatory circuitry that can be co-opted by metastatic cancer cells to facilitate lung colonization, suggesting interventions to target this pathway may offer therapeutic benefits to prevent or treat lung metastasis.

Antibody mediated therapy targeting CD47 inhibits tumor progression of hepatocellular carcinoma

Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival times. The efficacy of current systemic therapies for HCC is limited. In this study, we used xenograft tumor models to investigate the use of antibodies that block CD47 and inhibit HCC tumor growth. Immunostaining of tumor tissue and HCC cell lines demonstrated CD47 over-expression in HCC as compared to normal hepatocytes. Macrophage phagocytosis of HCC cells was increased after treatment with CD47 antibodies (CD47mAbs) that block CD47 binding to SIRPα. Further, CD47 blockade inhibited tumor growth in both heterotopic and orthotopic models of HCC, and promoted the migration of macrophages into the tumor mass. Our results demonstrate that targeting CD47 by specific antibodies has potential immunotherapeutic efficacy in human HCC.

Irradiation-induced localization of IL-12-expressing mesenchymal stem cells to enhance the curative effect in murine metastatic hepatoma.

Irradiation in conjunction with gene therapy is considered for efficient cancer treatment. Mesenchymal stem cells (MSCs), due to their irradiation-promotable tumor tropism, are ideal delivery vehicles for gene therapy. In this study, we investigated whether treatment with radiation and interleukin (IL)-12-expressing MSCs (MSCs/IL-12) exerts improved antitumor effects on murine metastatic hepatoma. HCa-I and Hepa 1-6 cells were utilized to generate heterotopic murine hepatoma models. Tumor-bearing mice were treated with irradiation or MSCs/IL-12 alone, or a combination. Monocyte chemoattractant protein-1 (MCP-1/CCL2) expression was assessed in irradiated hepatoma tissues to confirm a chemotactic effect. Combination treatment strategies were established and their therapeutic efficacies were evaluated by monitoring tumor growth, metastasis and survival rate. IL-12 expression was assessed and the apoptotic activity and immunological alterations in the tumor microenvironment were examined. MCP-1/CCL2 expression and localization of MSCs/IL-12 increased in the irradiated murine hepatoma cells. The antitumor effects, including suppression of pulmonary metastasis and survival rate improvements, were increased by the combination treatment with irradiation and MSCs/IL-12. IL-12 expression was increased in tumor cells, causing proliferation of cluster of differentiation 8(+) T-lymphocytes and natural killer cells. The apoptotic activity increased, indicating that the cytotoxicity of immune cells was involved in the antitumor effect of the combined treatment. Treatment with irradiation and MSCs/IL-12 showed effectiveness in treating murine metastatic hepatoma. IL-12-induced proliferation of immune cells played an important role in apoptosis of tumor cells. Our results suggest that treatment with irradiation and MSCs/IL-12 may be a useful strategy for enhancing antitumor activity in metastatic hepatoma.

The Heterotopic Thoracic Cardiac Xenotransplantation Model (Pig-to-baboon) in Two Different Groups without and with an Additional Myelodepressive Regime

The Heterotopic Thoracic Cardiac Xenotransplantation Model (Pig-to-baboon) in Two Different Groups without and with an Additional Myelodepressive Regime

Morphine Promotes Tumor Angiogenesis and Increases Breast Cancer Progression.

Morphine is considered a highly potent analgesic agent used to relieve suffering of patients with cancer. Several in vitro and in vivo studies showed that morphine also modulates angiogenesis and regulates tumour cell growth. Unfortunately, the results obtained by these studies are still contradictory. In order to better dissect the role of morphine in cancer cell growth and angiogenesis we performed in vitro studies on ER-negative human breast carcinoma cells, MDA.MB231 and in vivo studies on heterotopic mouse model of human triple negative breast cancer, TNBC. We demonstrated that morphine in vitro enhanced the proliferation and inhibited the apoptosis of MDA.MB231 cells. In vivo studies performed on xenograft mouse model of TNBC revealed that tumours of mice treated with morphine were larger than those observed in other groups. Moreover, morphine was able to enhance the neoangiogenesis. Our data showed that morphine at clinical relevant doses promotes angiogenesis and increases breast cancer progression.

Pig BMSCs Transfected with Human TFPI Combat Species Incompatibility and Regulate the Human TF Pathway in Vitro and in a Rodent Model

Background: The activation of tissue factor (TF) is one of the major reasons for coagulation dysregulation after pig-to-primate xenotransplantation. Tissue factor pathway inhibitor (TFPI) is the most important inhibitor of TF. Studies have demonstrated species incompatibility between pig TFPI and human TF. Methods: A pig-to-macaque heterotopic auxiliary liver transplantation model was established to determine the origin of activated TF. Chimeric proteins of human and pig TFPI were constructed to assess the role of Kunitz domains in species incompatibility. Immortalised pig bone marrow mesenchymal stem cells transfected with human TFPI were tested for their ability to inhibit clotting in vitro. Results: TF from recipient was activated early after liver xenotransplantation. Pig TFPI Kunitz domain 2 bound human FXa, but Kunitz domain 1 did not effectively inhibit human TF/FVIIa. Immortalised pig bone marrow mesenchymal cells (BMSCs) transfected with human TFPI showed a prolonged recalcification time in vitro and in a rodent model. Conclusion: Recipient TF is relevant to dysregulated coagulation after xenotransplantation. Kunitz domain 1 plays the most important role in species incompatibility between pig TFPI and human TF, and clotting can be inhibited by human TFPI-transfected pig BMSCs. Our study shows a possible way to resolve the incompatibility of pig TFPI.

Fine grained osseointegrative coating improves biocompatibility of PEEK in heterotopic sheep model.

Polyetheretherketone (PEEK) materials already have been used successfully in orthopedic and especially spine surgery. PEEK is radiolucent and comparable with bone regarding elasticity. However, PEEK is inert and the adhesion of PEEK implants to bone tissue proceeds slowly because of their relatively low biocompatibility. The aim of the study is to evaluate the effect of titanium and CaP coating on the adhesion of bone tissue. Six adult sheep (body weight 57.6 ± 3.9 kg) were included in this study. Three different types of cylindrical dowels (12 mm length x 8 mm diameter) were implanted in long bones (tibia and femur): PEEK dowels without coating (the control group), and PEEK dowels with a nanocoating of calcium phosphate (CaP group) or titanium (titanium group). Animals were sacrificed after 6, 12 and 26 weeks. Dowels were explanted for micro CT and histology. Bone implant contact (BIC) ratio was significantly higher in the titanium versus control groups in the 6 to 12 weeks period (p = 0.03). The ratio between bone volume and tissue volume (BV/TV) was significantly higher in titanium versus control in the 6 to 12 weeks period (p = 0.02). A significant correlation between BIC and BV/TV was seen (r = 0.85, p < 0.05). Coating of PEEK dowels with a nanocoating of titanium has beneficial effects on adhesion of bone tissue.

Efficacy of irreversible electroporation in human pancreatic adenocarcinoma: advanced murine model

Irreversible electroporation (IRE) is a promising cell membrane ablative modality for pancreatic cancer. There have been recent concerns regarding local recurrence and the potential use of IRE as a debulking (partial ablation) modality. We hypothesize that incomplete ablation leads to early recurrence and a more aggressive biology. We created the first ever heterotopic murine model by inoculating BALB/c nude mice in the hindlimb with a subcutaneous injection of Panc-1 cells, an immortalized human pancreatic adenocarcinoma cell line. Tumors were allowed to grow from 0.75 to 1.5 cm and then treated with the goal of complete ablation or partial ablation using standard IRE settings. Animals were recovered and survived for 2 days (n = 6), 7 (n = 6), 14 (n = 6), 21 (n = 6), 30 (n = 8), and 60 (n = 8) days. All 40 animals/tumors underwent successful IRE under general anesthesia with muscle paralysis. The mean tumor volume of the animals undergoing ablation was 1,447.6 mm3 ± 884). Histologically, in the 14-, 21-, 30-, and 60-day survival groups the entire tumor was nonviable, with a persistent tumor nodule completely replaced fibrosis. In the group treated with partial ablation, incomplete electroporation/recurrences (N = 10 animals) were seen, of which 66% had confluent tumors and this was a significant predictor of recurrence (P < 0.001). Recurrent tumors were also significantly larger (mean 4,578 mm3 ± SD 877 versus completed electroporated tumors 925.8 ± 277, P < 0.001). Recurrent tumors had a steeper growth curve (slope = 0.73) compared with primary tumors (0.60, P = 0.02). Recurrent tumors also had a significantly higher percentage of EpCAM expression, suggestive of stem cell activation. Tumors that recur after incomplete electroporation demonstrate a biologically aggressive tumor that could be more resistant to standard of care chemotherapy. Clinical correlation of this data is limited, but should be considered when IRE of pancreatic cancer is being considered.

Blockade of aquaporin 1 inhibits proliferation, motility, and metastatic potential of mesothelioma in vitro but not in an in vivo model.

Background. Malignant mesothelioma (MM) is an aggressive tumor of the serosal membranes, mostly the pleura. It is related to asbestos exposure and has a poor prognosis. MM has a long latency period, and incidence is predicted to remain stable or increase until 2020. Currently, no biomarkers for a specific targeted therapy are available. Previously, we observed that expression of aquaporin 1 (AQP1) was an indicator of prognosis in two independent cohorts. Here we determine whether AQP1 inhibition has therapeutic potential in the treatment of MM. Methods. Functional studies were performed with H226 cells and primary MM cells harvested from pleural effusions. AQP1 expression and mesothelial phenotype was determined by immunohistochemistry. AQP1 function was inhibited by a pharmacological blocker (AqB050) or AQP1-specific siRNA. Cell proliferation, migration, and anchorage-independent cell growth were assessed. A nude mouse heterotopic xenograft model of MM was utilised for the in vivo studies. Results. Inhibition of AQP1 significantly decreases cell proliferation, metastatic potential, and motility without inducing nonspecific cytotoxicity or increasing apoptosis. In vivo blockade of AQP1 had no biologically significant effect on growth of established tumours. Conclusions. Targeted blockade of AQP1 restricts MM growth and migration in vitro. Further work is warranted to fully evaluate treatment potential in vivo.