anti-EMMPRIN Antibody Research Articles

Glucose Promotes EMMPRIN/CD147 and the Secretion of Pro-Angiogenic Factors in a Co-Culture System of Endothelial Cells and Monocytes.

Vascular complications in Type 2 diabetes mellitus (T2DM) patients increase morbidity and mortality. In T2DM, angiogenesis is impaired and can be enhanced or reduced in different tissues ("angiogenic paradox"). The present study aimed to delineate differences between macrovascular and microvascular endothelial cells that might explain this paradox. In a monoculture system of human macrovascular (EaHy926) or microvascular (HMEC-1) endothelial cell lines and a monocytic cell line (U937), high glucose concentrations (25 mmole/L) increased the secretion of the pro-angiogenic factors CD147/EMMPRIN, VEGF, and MMP-9 from both endothelial cells, but not from monocytes. Co-cultures of EaHy926/HMEC-1 with U937 enhanced EMMPRIN and MMP-9 secretion, even in low glucose concentrations (5.5 mmole/L), while in high glucose HMEC-1 co-cultures enhanced all three factors. EMMPRIN mediated these effects, as the addition of anti-EMMPRIN antibody decreased VEGF and MMP-9 secretion, and inhibited the angiogenic potential assessed through the wound assay. Thus, the minor differences between the macrovascular and microvascular endothelial cells cannot explain the angiogenic paradox. Metformin, a widely used drug for the treatment of T2DM, inhibited EMMPRIN, VEGF, and MMP-9 secretion in high glucose concentration, and the AMPK inhibitor dorsomorphin enhanced it. Thus, AMPK regulates EMMPRIN, a key factor in diabetic angiogenesis, suggesting that targeting EMMPRIN may help in the treatment of diabetic vascular complications.

EMMPRIN Promotes the Expression of MMP-9 and Exacerbates Neurological Dysfunction in a Mouse Model of Intracerebral Hemorrhage.

Extracellular matrix metalloproteinase inducer (EMMPRIN) has been shown to be a vital inflammatory mediator in several neurological and neurodegenerative diseases. However, the role of EMMPRIN in intracerebral hemorrhage (ICH) remains unexplored. In this study, we aimed to exploit a highly selective monoclonal anti-EMMPRIN antibody to functionally inhibit EMMPRIN activity and thus that of MMPs as the downstream effector. To induce ICH pathology, adult C57BL/6 male mice were injected with collagenase type VII or saline as control into the right basal ganglia and were euthanized at different time points. The anti-EMMPRIN monoclonal antibody was intravenously injected once daily for 3days to block the expression of EMMPRIN initiating at 4h post-ICH. Western blot and immunofluorescence analysis results revealed that EMMPRIN expression was significantly increased surrounding the hematoma at 3 and 7d time points after ICH when compared to the saline treated control group. EMMPRIN expression was co-localized with GFAP (astrocytes) and Iba1 (microglia) at 3d time point post-ICH, but not in the control group mice. The co-localization of EMMPRIN with CD31 in endothelial cells occurred in both groups and was higher in the ICH brain. However, EMMPRIN expression was not detected in neurons from either group. The inhibition of EMMPRIN reduced the expression of MMP-9, the number of infiltrated neutrophils, the degree of brain injury and promoted neurological recovery after ICH. In conclusion, EMMPRIN could mediate the upregulation of MMP-9 and exacerbate neurological dysfunction in a mouse model of experimental ICH. Furthermore, blocking EMMPRIN reduced brain injury and subsequently promoted neurological recovery in ICH mice brains. These outcomes highlight that inhibition of EMMPRIN can be a potential therapeutic intervention strategy to regulate MMP-9's pathological roles during ICH.

Inducing regulated necrosis and shifting macrophage polarization with anti-EMMPRIN antibody (161-pAb) and complement factors.

Treatment of solid tumors is often hindered by an immunosuppressive tumor microenvironment (TME) that prevents effector immune cells from eradicating tumor cells and promotes tumor progression, angiogenesis, and metastasis. Therefore, targeting components of the TME to restore the ability of immune cells to drive anti‐tumoral responses has become an important goal. One option is to induce an immunogenic cell death (ICD) of tumor cells that would trigger an adaptive anti‐tumoral immune response. Here we show that incubating mouse renal cell carcinoma (RENCA) and colon carcinoma cell lines with an anti‐extracellular matrix metalloproteinase inducer polyclonal antibody (161‐pAb) together with complement factors can induce cell death that inhibits caspase‐8 activity and enhances the phosphorylation of receptor‐interacting protein kinase 3 (RIPK3) and mixed‐lineage kinase‐like domain (MLKL). This regulated necrotic death releases high levels of dsRNA molecules to the conditioned medium (CM) relative to the necrotic death of tumor cells induced by H2O2 or the apoptotic death induced by etoposide. RAW 264.7 macrophages incubated with the CM derived from these dying cells markedly enhanced the secretion of IFNβ, and enhanced their cytotoxicity. Furthermore, degradation of the dsRNA in the CM abolished the ability of RAW 264.7 macrophages to secrete IFNβ, IFNγ‐induced protein 10 (IP‐10), and TRAIL. When mice bearing RENCA tumors were immunized with the 161‐pAb, their lysates displayed elevated levels of phosphorylated RIPK3 and MLKL, as well as increased concentrations of dsRNA, IFNβ, IP‐10, and TRAIL. This shows that an antigen‐targeted therapy using an antibody and complement factors that triggers ICD can shift the mode of macrophage activation by triggering regulated necrotic death of tumor cells.

Interaction between the Wnt/β-catenin signaling pathway and the EMMPRIN/MMP-2, 9 route in periodontitis.

In periodontitis, the Wnt/β-catenin signaling pathway is related to the metabolism of the alveolar bone; further, extracellular matrix metalloproteinase inducer (EMMPRIN) expression is correlated with matrix metalloproteinases (MMPs) expression and inflammation severity. The aim of this study was to perform a preliminary investigation of the interaction between the Wnt/β-catenin signaling pathway and the EMMPRIN/MMPs route in periodontitis. Chronic periodontitis and healthy gingival tissues were obtained to detect the expression of Wnt3a, β-catenin, EMMPRIN and MMP-2, 9 by using immunohistochemical analysis. The human immortalized oral epithelial cell/human gingival fibroblast direct co-culture model was treated with 10μg/mL Porphyromonas gingivalis lipopolysaccharide (Pg. LPS). Anti-EMMPRIN antibody was used to block the effect of EMMPRIN. Dickkopf-1 (DKK-1) and Wnt3a were used as the inhibitor and activator of the Wnt/β-catenin signaling pathway, respectively. Immunofluorescence was performed to visualize the localization of β-catenin and EMMPRIN. Expression of the EMMPRIN, MMP-2, 9 and Wnt pathway's components was confirmed by western blotting and quantitative real-time polymerase chain reaction. Higher levels of Wnt3a, β-catenin, EMMPRIN and MMP-2, 9 were observed in chronic periodontitis gingival tissues compared with controls. Pg. LPS significantly enhanced β-catenin, p-GSK-3β, EMMPRIN and MMP-2, 9 inductions in the human immortalized oral epithelial cell/human gingival fibroblast co-culture model. Anti-EMMPRIN antibody markedly reduced the expression of MMP-2, 9 only in the presence of Pg. LPS. Co-expression of β-catenin and EMMPRIN was detected in the co-culture model. DKK-1 inhibited Wnt pathway, but upregulated the EMMPRIN/MMP-2, 9 routes. In contrast, activating Wnt pathway by Wnt3a repressed the EMMPRIN/MMP-2, 9 routes. The promotion effect of DKK-1 on MMP-2, 9 expressions was partially inhibited by the anti-EMMPRIN antibody. In addition, anti-EMMPRIN antibody led to a drastic decrease in β-catenin and p-GSK-3β. In periodontitis, EMMPRIN regulates MMP-2, 9 expressions, the activation of Wnt/β-catenin signaling pathway downregulates the EMMPRIN/MMP-2, 9 routes and the blockade of EMMPRIN attenuates Wnt/β-catenin signaling pathway.

An epitope-specific novel anti-EMMPRIN polyclonal antibody inhibits tumor progression

ABSTRACTExtracellular matrix metalloproteinase inducer (EMMPRIN/CD147) mediates tumor cell–macrophage interactions, and has been shown to induce both matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF). However, the epitope responsible for MMP induction is controversial, and the epitope responsible for VEGF induction is yet unknown. We generated a novel anti-EMMPRIN antibody directed against a specific epitope that successfully inhibited the production of both MMP-9 and VEGF in tumor cell–macrophage in vitro co-culture systems, exhibiting a U-shaped dose response. Furthermore, this antibody efficiently inhibited in vivo tumor progression in both the RENCA renal cell carcinoma and CT26 colon carcinoma subcutaneous tumor models, and reduced tumor size and number of metastatic foci in the 4T1 orthotopic model. This was achieved by inhibiting angiogenesis as assessed by immunohistochemical staining for the endothelial marker CD31, by inhibiting tumor cell proliferation as assessed by the staining for Ki-67, and by enhancing tumor cell apoptosis as assessed in the TUNEL assay. Moreover, administration of the antibody recruited more macrophages into the tumor, and skewed the tumor microenvironment for macrophages from TGFβ-dominated anti-inflammatory microenvironment, to a less immunosuppressive one. The antibody improved the ability of stimulated macrophages to perform antibody-dependent cell cytotoxicity (ADCC) and kill tumor cells. Thus, our new antibody maps the epitope capable of inducing both MMPs and VEGF, and places EMMPRIN as a good target for cancer therapy.

Homocysteine elicits an M1 phenotype in murine macrophages through an EMMPRIN-mediated pathway.

Hyperhomocysteinemia (HHcy) is associated with inflammatory diseases and is known to increase the production of reactive oxygen species (ROS), matrix metalloproteinase (MMP)-9, and inducible nitric oxide synthase, and to decrease endothelial nitric oxide production. However, the impact of HHcy on macrophage phenotype differentiation is not well-established. It has been documented that macrophages have 2 distinct phenotypes: the "classically activated/destructive" (M1), and the "alternatively activated/constructive" (M2) subtypes. We hypothesize that HHcy increases M1 macrophage differentiation through extracellular matrix metalloproteinase inducer (EMMPRIN), a known inducer of matrix metalloproteinases. murine J774A.1 and Raw 264.7 macrophages were treated with 100 and 500 μmol/L Hcy, respectively, for 24 h. Samples were analyzed using Western blotting and immunocytochemistry. Homocysteine treatment increased cluster of differentiation 40 (CD40; M1 marker) in J774A.1 and Raw 264.7 macrophages. MMP-9 was induced in both cell lines. EMMPRIN protein expression was also increased in both cell lines. Blocking EMMPRIN function by pre-treating cells with anti-EMMPRIN antibody, with or without Hcy, resulted in significantly lower expression of CD40 in both cell lines by comparison with the controls. A DCFDA assay demonstrated increased ROS production in both cell lines with Hcy treatment when compared with the controls. Our results suggest that HHcy results in an increase of the M1 macrophage phenotype. This effect seems to be at least partially mediated by EMMPRIN induction.

Dynamic contrast-enhanced MRI evaluates the early response of human head and neck tumor xenografts following anti-EMMPRIN therapy with cisplatin or irradiation.

To assess the early therapeutic effects of anti-EMMPRIN (extracellular matrix metalloprotease inducer) antibody with/without cisplatin or X-ray radiation in head and neck cancer mouse models using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Mice bearing SCC1 (or OSC19) tumor xenografts were treated with anti-EMMPRIN antibody, radiation, cisplatin, or anti-EMMPRIN antibody plus cisplatin (or radiation) for a week (n = 4-5 per group). DCE-MRI was carried out on a 9.4T small animal MR scanner on days 0, 3, and 7, and K(trans) values were averaged in a 0.5-mm-thick peripheral tumor region. Ki67 and CD31 staining were implemented for all tumors after imaging. The K(trans) changes of SCC1 and OSC19 tumors treated with anti-EMMPRIN antibody for 3 days were -18 ± 8% and 4 ± 7%, respectively, which were significantly lower than those of control groups (39 ± 5% and 45 ± 7%; P = 0.0025 and 0.0220, respectively). When cisplatin was added, those were -42 ± 9% and -44 ± 9%, respectively, and with radiation, -45 ± 9% and -27 ± 10%, respectively, which were also significantly lower than those of control groups (P < 0.0001 for all four comparisons). In the eight groups untreated (served as control) or treated with anti-EMMPRIN antibody with/without cisplatin or radiation, the mean K(trans) change for 3 days was significantly correlated with the mean tumor volume change for 7 days (r = 0.74, P = 0.0346), Ki67-expressing cell density (r = 0.96, P = 0.0001), and CD31 density (r = 0.84, P = 0.0084). DCE-MRI might be utilized to assess the early therapeutic effects of anti-EMMPRIN antibody with/without chemotherapy or radiotherapy in head and neck cancer.

Functional role of EMMPRIN in the formation and mineralisation of dental matrix in mouse molars.

Our previous research has shown that the extracellular matrix metalloproteinase inducer (EMMPRIN) is expressed during and may function in the early development of tooth germs. In the present study, we observed the specific expression of EMMPRIN in ameloblasts and odontoblasts during the middle and late stages of tooth germ development using immunohistochemistry. Furthermore, to extend our understanding of the function of EMMPRIN in odontogenesis, we used an anti-EMMPRIN function-blocking antibody to remove EMMPRIN activity in tooth germ culture in vitro. Both the formation and mineralisation of dental hard tissues were suppressed in the tooth germ culture after the abrogation of EMMPRIN. Meanwhile, significant reductions in VEGF, MMP-9, ALPL, ameloblastin, amelogenin and enamelin expression were observed in antibody-treated tooth germ explants compared to control and normal serum-treated explants. The current results illustrate that EMMPRIN may play a critical role in the processing and maturation of the dental matrix.

Optimal Activation of Carboxyl-Superparamagnetic Iron Oxide Nanoparticles Bioconjugated with Antibody Using Orthogonal Array Design

This study aims to bioconjugate anti-EMMPRIN monoclonal antibody on the surface of carboxyl-SPIO nanoparticles and to optimize the activated conditions of bioconjugation. Anti-EMMPRIN monoclonal antibody bioconjugated carboxyl-SPIO nanoparticles were performed through a coupling strategy of EDC and sulfo-NHS. The procedure was comprised of two steps by activation of carboxyl-SPIO nanoparticles and conjugation with monoclonal antibody. The optimal activated parameters of bioconjugation were evaluated by single factor design and orthogonal array design. SDS-PAGE analysis and Bradford assay was used for testing and verifying the efficiency of activated conditions obtained from orthogonal array. The results show that pH value, temperature and reaction time were important factors that influence bioconjugated efficiency. The activated parameters with pH value 6.2, temperature 25 degrees C and reaction time 30 min were obviously optimal for activation of carboxyl-SPIO nanoparticles and conjugation with monoclonal EMMPEIN antibody. This coupling strategy for anti-EMMPRIN mAb bioconjugated on SPIO nanoparticles was efficient, and may be further applied in the fields of medical or biological practices.

Antagonistic Effects of Anti-EMMPRIN Antibody When Combined with Chemotherapy Against Hypovascular Pancreatic Cancers

To examine the antagonistic effects of anti-extracellular matrix metalloprotease inducer (anti-EMMPRIN) antibody when combined with chemotherapy using a hypovascular pancreatic tumor model. Severely compromised immunodeficient mice bearing orthotopic MIA PaCa-2 tumors were used (five to six animals per group). Dynamic contrast-enhanced magnetic resonance imaging was used to examine the relationship between tumor vascularity and size. Therapy was initiated when tumors were hypovascular. Treatments included: (1) gemcitabine alone, (2) anti-EMMPRIN antibody alone, and (3) combination, each for 2weeks. Additionally, another treatment arm included β-lapachone, an NAD(P)H/quinone 1 (NQO1) bioactivated agent. (18)F-fluoro-D-glucose-positron emission tomography/computed tomography imaging was used weekly to monitor therapeutic effects. Gemcitabine or anti-EMMPRIN monotherapy significantly delayed tumor growth, but the combination therapy showed an antagonistic effect. Similarly, tumor growth was significantly suppressed by β-lapachone alone, and additive effects were noted when combined with gemcitabine, but the therapeutic efficacy was reduced when anti-EMMPRIN antibody was added. Anti-EMMPRIN antibody with chemotherapy in hypovascular tumors results in antagonistic effects.

The Role of Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) in the Regulation of Bovine Endometrial Cell Functions1

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell surface glycoprotein that stimulates the production of several matrix metalloproteinases (MMPs) for tissue remodeling. Previously, we detected EMMPRIN in the bovine endometrium, and it is mainly expressed in the luminal and glandular epithelium whereas MMPs are expressed in the underlying stroma. From this expression pattern, we hypothesized that EMMPRIN may regulate stromal MMPs in endometrial cell functions. To test this hypothesis, a coculture of epithelial and stromal cells was performed using a transwell system. In the coculture, epithelial cells were cultured on the insert membrane and stromal cell on the surface of well plates. Expression of stromal MMP-2 and MMP-14 was significantly higher in coculture with epithelial cell. Further, with the addition of anti-EMMPRIN antibody into the epithelial cell compartment, the expression of stromal EMMPRIN and MMP-2 and MMP-14 was decreased. To identify the active site of EMMPRIN for the augmentation of MMPs, EMMPRIN synthetic peptides that correspond to the extracellular loop domain-I (EM1, EM2, EM3, and EM4) were added into the epithelial cell compartment, and only EM2 at a higher dose interfered with EMMPRIN-mediated expression of MMP-14. Next, we examined the effects of progesterone and/or estrogen on the expression of EMMPRIN, MMP-2, and MMP-14. Progesterone (300 nM) significantly stimulated the expression of EMMPRIN but had no effects on any of the MMPs. These results suggest that EMMPRIN derived from epithelial cells regulates MMPs in the endometrium under progesterone-rich conditions and may thereby modulate bovine endometrial cell functions during gestation.

A novel anti-EMMPRIN function-blocking antibody reduces T cell proliferation and neurotoxicity: relevance to multiple sclerosis

BackgroundExtracellular matrix metalloproteinase inducer (EMMPRIN; CD147, basigin) is an inducer of the expression of several matrix metalloproteinases (MMPs). We reported previously that blocking EMMPRIN activity reduced neuroinflammation and severity of disease in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE).MethodsTo improve upon EMMPRIN blockade, and to help unravel the biological functions of EMMPRIN in inflammatory disorders, we have developed several anti-EMMPRIN monoclonal antibodies.ResultsOf these monoclonal antibodies, a particular one, clone 10, was efficient in binding mouse and human cells using several methods of detection. The specificity of clone 10 was demonstrated by its lack of staining of EMMPRIN-null embryos compared to heterozygous and wild-type mouse samples. Functionally, human T cells activated with anti-CD3 and anti-CD28 elevated their expression of EMMPRIN and the treatment of these T cells with clone 10 resulted in decreased proliferation and matrix metalloproteinase- 9 (MMP-9) production. Activated human T cells were toxic to human neurons in culture and clone 10 pretreatment reduced T cell cytotoxicity correspondent with decrease of granzyme B levels within T cells. In vivo, EAE mice treated with clone 10 had a markedly reduced disease score compared to mice treated with IgM isotype control.ConclusionsWe have produced a novel anti-EMMPRIN monoclonal antibody that blocks several aspects of T cell activity, thus highlighting the multiple roles of EMMPRIN in T cell biology. Moreover, clone 10 reduces EAE scores in mice compared to controls, and has activity on human cells, potentially allowing for the testing of anti-EMMPRIN treatment not only in EAE, but conceivably also in MS.

Dual Combination Therapy Targeting DR5 and EMMPRIN in Pancreatic Adenocarcinoma

The goal of the study was to assess the efficacy of combined extracellular matrix metalloprotease inducer (EMMPRIN)- and death receptor 5 (DR5)-targeted therapy for pancreatic adenocarcinoma in orthotopic mouse models with multimodal imaging. Cytotoxicity of anti-EMMPRIN antibody and anti-DR5 antibody (TRA-8) in MIA PaCa-2 and PANC-1 cell lines was measured by ATPlite assay in vitro. The distributions of Cy5.5-labeled TRA-8 and Cy3-labeled anti-EMMPRIN antibody in the 2 cell lines were analyzed by fluorescence imaging in vitro. Groups 1 to 12 of severe combined immunodeficient mice bearing orthotopic MIA PaCa-2 (groups 1-8) or PANC-1 (groups 9-12) tumors were used for in vivo studies. Dynamic contrast-enhanced-MRI was applied in group 1 (untreated) or group 2 (anti-EMMPRIN antibody). The tumor uptake of Tc-99m-labeled TRA-8 was measured in group 3 (untreated) and group 4 (anti-EMMPRIN antibody). Positron emission tomography/computed tomography imaging with (18)F-FDG was applied in groups 5 to 12. Groups 5 to 8 (or groups 9 to 12) were untreated or treated with anti-EMMPRIN antibody, TRA-8, and combination, respectively. TRA-8 showed high killing efficacy for both MIA PaCa-2 and PANC-1 cells in vitro, but additional anti-EMMPRIN treatment did not improve the cytotoxicity. Cy5.5-TRA-8 formed cellular caps in both the cell lines, whereas the maximum signal intensity was correlated with TRA-8 cytotoxicity. Anti-EMMPRIN therapy significantly enhanced the tumor delivery of the MR contrast agent, but not Tc-99m-TRA-8. Tumor growth was significantly suppressed by the combination therapy, and the additive effect of the combination was shown in both MIA PaCa-2 and PANC-1 tumor models.

Abstract 3630: Function-Blocking Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) Antibody Injection is Neuroprotective after Focal Cerebral Ischemia

Introduction: Extracellular metalloproteinase inducer, EMMPRIN, is a cell surface glycoprotein found on endothelial cells and astrocytes and has a key role in the expression of MMPs. It was reported that EMMPRIN expression is upregulated in a mouse model of permanent cerebral ischemia and that EMMPRIN co-localized with MMP-9 in infarcted tissue. There is evidence to support that elevation of MMP activity contributes to injury after stroke and that inhibition of MMPs can reduce infarct volume in rats. In this study we hypothesized that inhibition of EMMPRIN with a function-blocking antibody would reduce infarct volume and improve stroke outcome in mice subjected to a MCAO stroke through reduction in MMP activation. Methods: Transient 90-minute middle cerebral artery occlusion (MCAO) was performed on male wild-type mice treated with vehicle, rat IgG2a (eBioscience), or with function blocking anti-EMMPRIN antibody (eBioscience) injected via tail vein. A single injection was given at stroke onset. Mice were sacrificed at 48 hours post reperfusion. Brains were harvested for TTC staining (n=4/group; significance p&lt;0.05). A second cohort of vehicle and drug treated mice (n=3 vehicle, n=7 drug) were subjected to transient 90-minute middle cerebral artery occlusion and were sacrificed at 72 hours. These mice were given the initial dose at stroke onset and an additional dose at 48 hours. Both cohorts were subjected to behavioral analysis using a modified 4 point scoring system. Results: Mice treated with the function blocking EMMPRIN antibody had smaller infarct volumes when compared with vehicle treated animals. This reduction in damaged tissue was consistent in the cortex, striatum and total hemisphere of each group. Drug treated mice had improved behavior scores when compared to vehicle treated mice at both the 48hr and 72hrs time points. Furthermore, vehicle treated mice had higher mortality rates (20% at 48hrs and 66% at 72hrs) than drug mice (0% at 48hrs and 14% at 72hrs) at both time points. Discussion: Animals treated with a function-blocking antibody to EMMPRIN presented with less infarct volume and better behavior scores than similar animals treated with vehicle. Thus, EMMPRIN inhibition is neuroprotective. Considering monoclonal antibody therapy has been successful in the treatment of other diseases, this data suggests that inhibition of EMMPRIN via administration of an antibody could have therapeutic potential for stroke patients.

Extracelluar matrix metalloproteinase as a novel target for pancreatic cancer therapy

The objective of this study was to evaluate extracellular matrix metalloproteinase (EMMPRIN) as a novel target in orthotopic pancreatic cancer murine models. MIA PaCa-2 human pancreatic tumor cells were implanted in groups 1 and 3-7, whereas MIA PaCa-2 EMMPRIN knockdown cells were implanted in group 2. Dosing with anti-EMMPRIN antibody started immediately after implantation for groups 1-3 (residual tumor model) and at 21 days after cell implantation for groups 4-7 (established tumor model). Groups 3, 5, and 7 were treated with anti-EMMRPIN antibody (0.2-1.0 mg) twice weekly for 2-3 weeks, whereas the other groups served as the control. In the residual tumor model, tumor growth of anti-EMMPRIN-treated group was successfully arrested for 21 days (15 ± 4 mm(3)), which was significantly lower than that of the EMMPRIN knockdown group (80 ± 15 mm(3); P=0.001) or the control group (240 ± 41 mm(3); P<0.001). In the established tumor model, anti-EMMPRIN therapy lowered tumor volume increase by approximately 40% compared with the control, regardless of the dose amount. Ki67-expressed cell density of group 5 was 939 ± 150 mm(-2), which was significantly lower than that of group 4 (1709 ± 145 mm(-2); P=0.006). Microvessel density of group 5 (30 ± 6 mm(-2)) was also significantly lower than that of group 4 (53 ± 5 mm(-2); P=0.014), whereas the microvessel size of group 5 (191 ± 22 μm(2)) was significantly larger than that of group 4 (113 ± 26 μm(2); P=0.049). These data show the high potential of anti-EMMPRIN therapy for pancreatic cancer and support its clinical translation.

The Role of Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) in the Regulation of Bovine Endometrial Cell Functions

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell surface glycoprotein that stimulates the production of several matrix metalloproteinases (MMPs) for tissue remodeling. Previously, we detected EMMPRIN in the bovine endometrium, and it is mainly expressed in the luminal and glandular epithelium whereas MMPs are expressed in the underlying stroma. From this expression pattern, we hypothesized that EMMPRIN may regulate stromal MMPs in endometrial cell functions. To test this hypothesis, a coculture of epithelial and stromal cells was performed using a transwell system. In the coculture, epithelial cells were cultured on the insert membrane and stromal cell on the surface of well plates. Expression of stromal MMP-2 and MMP-14 was significantly higher in coculture with epithelial cell. Further, with the addition of anti-EMMPRIN antibody into the epithelial cell compartment, the expression of stromal EMMPRIN and MMP-2 and MMP-14 was decreased. To identify the active site of EMMPRIN for the augmentation of MMPs, EMMPRIN synthetic peptides that correspond to the extracellular loop domain-I (EM1, EM2, EM3, and EM4) were added into the epithelial cell compartment, and only EM2 at a higher dose interfered with EMMPRIN-mediated expression of MMP-14. Next, we examined the effects of progesterone and/or estrogen on the expression of EMMPRIN, MMP-2, and MMP-14. Progesterone (300 nM) significantly stimulated the expression of EMMPRIN but had no effects on any of the MMPs. These results suggest that EMMPRIN derived from epithelial cells regulates MMPs in the endometrium under progesterone-rich conditions and may thereby modulate bovine endometrial cell functions during gestation.

EMMPRIN (CD147) regulation of MMP‐9 in bronchial epithelial cells in COPD

Extracellular matrix metalloproteinase inducer (EMMPRIN or CD147) induces the production of matrix metalloproteinases (MMP) such as MMP-9, which plays an important role in COPD. We determined its cellular origin and role in MMP-9 production in COPD. Bronchial biopsies, alveolar macrophages (AM) and blood monocytes (BM) from patients with COPD, healthy smokers and non-smokers, and bronchial epithelial cells (EC) from surgically resected airways from patients with COPD were stimulated with LPS or CRP in the presence and absence of an anti-EMMPRIN blocking antibody. EMMPRIN in BAL, plasma, conditioned media and cell lysates was quantified and immunohistochemical localization of EMMPRIN was determined in bronchial biopsies. MMP-9 activity and mRNA was also determined. EMMPRIN levels in BAL fluid were higher in patients with COPD compared with non-smokers and smokers. There was greater EMMPRIN expression in EC from patients with COPD compared with smokers and non-smokers. EC secreted and expressed more EMMPRIN protein than BM and AM. Blocking EMMPRIN decreased MMP-9 activity in supernatant of EC, but not in those from AM and BM, and decreased MMP-9 mRNA expression in EC. The increased EMMPRIN expression in COPD is reflected by an increased release from bronchial EC, which are one of the main source of EMMPRIN. EMMPRIN regulates MMP-9 expression in COPD.

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

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 4575: Novel monoclonal antibody targeting EMMPRIN for pancreatic cancer therapy

Abstract Purpose: To evaluate a novel monoclonal antibody targeting extracelluar matrix metalloproteinase (EMMPRIN) in orthotopic pancreatic-cancer murine models. Methods: In vitro cytotoxic assay of anti-EMMPRIN mAb was conducted with MIA PaCA-2 and PANC-1 human pancreatic-cancer cell lines. Binding assay of Tc-99m labeled anti-EMMPRIN mAb was repeated three times independently for MIA PaCa-2 cells or MIA PaCa-2 cells silenced for EMMPRIN gene expression by siRNA. For the in vivo animal study, MIA PaCa-2 cells were implanted in groups 1-6, while EMMPRIN silenced cells were implanted in group 7. For groups 1-4 (solid tumor model, n=5-7/group), dosing started at 21 days after cell implantation, while dosing started immediately after cell implantation for groups 5-7 (residual tumor model, n=10-12/group); groups 1 and 2 were injected with PBS and anti-EMMPRIN mAB (0.2mg) twice weekly for 3 weeks respectively; ultrasound imaging (USI) was applied at days 21, 28, 35, and 42 to determine tumor volume. Groups 3 and 4 were injected with PBS and anti-EMMPRIN mAb (1mg) twice weekly for 2 weeks, respectively; USI and diffusion-weighted MRI were applied at days 21, 28, and 35. Groups 5 and 6 were injected with PBS and anti-EMMPRIN mAB (0.2mg) twice weekly for 3 weeks respectively; group 7 was injected with PBS during the same time. For groups 5-7, USI was applied at 15 and 21 days post cell injection. Ki-67 and TUNEL staining were conducted on tumor sections from groups 1 and 2 at the study end. Results: In vitro treatment with antibody alone did not demonstrate significant cytotoxic effect in either cell line. The Kd in MIA PaCa-2 cells was 4.31±0.59 (mean ± SE) nM, which was not statistically different from that in EMMPRIN silenced cells. The average number of EMMPRIN per MIA PaCa-2 cell was 582,000±56,000, significantly higher than that per EMMPRIN silenced cells at 220,000±89,000 (p=0.03). Tumor-volume increases of group 1 relative to day 21 were 282±33, 495±107, and 725±166% at days 28, 35, and 42, while those of group 2 were 191±7, 263±27, and 429±39%; groups 1 and 2 were statistically different at day 28 (p=0.03), not at the other days. Group 3 showed an increase in tumor-volume of 317±55 and 505±96% at days 28 and 35 respectively, while those in group 4 were 189±27 and 244±41%; groups 3 and 4 were statistically different at day 35 (p=0.04), but not at day 28. Mean intra-tumoral ADC change of group 4 was not statistically significant from that of group 3. Tumor volumes of groups 5-7 were 66±17, 21±5, and 4±2mm3 at day 15, while those at day 21 were 240±41, 80±15, and 15±4mm3, which represent statistical differences among groups at each day (p&amp;lt;0.05). Proliferating cell density of group 2 was significantly lower than that of group 1 (p=0.01), while apoptotic cell densities were not. Conclusion: Tumor growth was successfully arrested following treatment with anti-EMMPRIN antibody for both solid- and residual-tumor models, consistent with reduced proliferating cell density. 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 4575. doi:10.1158/1538-7445.AM2011-4575