Guihua Zhai

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 18F-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. Mol Cancer Ther; 11(2); 405–15. ©2011 AACR.

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 mm3), which was significantly lower than that of the EMMPRIN knockdown group (80±15 mm3; P=0.001) or the control group (240±41 mm3; 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 &mgr;m2) was significantly larger than that of group 4 (113±26 &mgr;m2; P=0.049). These data show the high potential of anti-EMMPRIN therapy for pancreatic cancer and support its clinical translation.

Early therapy assessment of combined anti-DR5 antibody and carboplatin in triple-negative breast cancer xenografts in mice using diffusion-weighted imaging and 1H MR spectroscopy

To assess the early response of triple‐negative breast‐cancer (TNBC) following TRA‐8 and carboplatin therapy using DWI and MRS in 2LMP and SUM159 mouse models.

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).

Combination therapy with anti-DR5 antibody and tamoxifen for triple negative breast cancer.

TRA-8, a monoclonal antibody targeting death receptor, has demonstrated high therapeutic effect for triple negative breast cancer (TNBC) in preclinical models. Tamoxifen, the standard of care for ERα-positive breast cancer, induces apoptosis via ERβ, which commonly presents in TNBC cells. The current study investigates the combination effects of TRA-8 and tamoxifen for TNBC. In vitro assays were implemented with two ERβ-positive TNBC cell lines, SUM159 and 2LMP, and in vivo therapy studies were followed using orthotopic breast tumor mouse models. IC50 of tamoxifen for SUM159 and 2LMP were 29 μM and 38 μM, respectively. Synergy between TRA-8 (0–1000 ng/mL) and tamoxifen (20 μM) was observed for both the cell lines. Tamoxifen (400 mg/kg diet) markedly suppressed the growth of SUM159 tumors for 6 weeks after therapy initiation, but it did not induce antitumor effect for 2LMP tumors. TRA-8 (0.1 mg, weekly, i.p.) successfully arrested the growth of both SUM159 and 2LMP tumors during therapy, but an antagonistic effect was observed when tamoxifen was combined. TRA-8 uptake into tumors was not changed by tamoxifen treatment. Histological analysis confirmed that caspase-3 activation induced by TRA-8 was significantly decreased when tamoxifen was used in combination. In conclusion, our findings suggest that the combined use of TRA-8 and tamoxifen may cause antagonistic effects for TNBC patients.

Diffusion weighted imaging evaluated the early therapy effect of tamoxifen in an MNU-induced mammary cancer rat model.

Purpose To assess the optimal time point of diffusion-weighted imaging (DWI) for early prognosis of breast cancer following tamoxifen therapy using a methylnitrosourea (MNU)-induced ER-positive breast-cancer model. Methods Two groups of Sprague-Dawley rats (n = 15 for group 1; n = 10 for group 2) were used. All animals (50 days old) were intravenously injected with MNU (50 mg/kg body weight) to induce ER-positive mammary tumors. When tumors were approximately 2 cm in diameter, DWI was performed on days 0, 3, and 7, and intratumoral apparent diffusion coefficient (ADC) values were measured. Therapy started on day 0 with tamoxifen (10 mg/kg diet) and continued for 4 weeks for group 1, but only 1 week for group 2, while tumor volume was measured by caliper twice weekly. All animals of group 2 were euthanized on day 7 after imaging, and Ki-67, TUNEL, ERα, and ERβ staining were performed on tumor tissue. Results DW images of MNU-induced mammary tumors were successfully obtained with minimal motion artifact. For group 1, ADC change for 3 days after therapy initiation (ADC3D) was significantly correlated with tumor-volume change until day 11, but the significant correlation between ADC change for 7 days (ADC7D) and the tumor-volume change was observed until day 18. Similarly, for group 2, either ADC7D or ADC3D was significantly correlated with the tumor-volume change, but the higher significance was observed for ADC7D. Furthermore, ADC7D was significantly correlated with apoptotic (TUNEL stained), proliferative (Ki-67 stained), and ERβ-positive cell densities, but ADC3D was not significantly correlated with any of those. Conclusions ADC7D might be a more reliable surrogate imaging biomarker than ADC3D to assess effectiveness of tamoxifen therapy for ER-positive breast cancer, which may enable personalized treatment. The significant correlation between ADC7D and ERβ-positive cell density suggests that ERβ may play an important role as a therapeutic indicator of tamoxifen.

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

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 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<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<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<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

Abstract 4575: Novel monoclonal antibody targeting EMMPRIN for pancreatic cancer therapy

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 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<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