Dual combination therapy targeting DR5 and EMMPRIN in pancreatic adenocarcinoma

Abstract

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.

Figure 1

In vitro ATP lite assay (mean and SE) to measure killing of (A) MIA PaCa-2 or (B) PANC-1 human pancreatic-cancer cells at increasing concentrations of TRA-8 alone or in combination with anti-EMMPRIN antibody.

Figure 2

In vitro fluorescence imaging of antibodies and image analysis. (A,B) Representative fluorescence images of Cy5.5-TRA-8 and Cy3-anti-EMMPRIN antibody with MIA PaCa-2 and PANC-1 cells, when the two antibodies were used (A) separately or (B) combined. (C-F) The fluorescence signal intensities of Cy5.5-TRA-8 and Cy3-anti-EMMPRIN antibody along the yellow lines shown in the images of (C,E) MIA PaCa-2 or (D,F) PANC-1 cells, when the two antibodies were used (C,D) separately or (E,F) combined.

Figure 3

In vivo DCE-MRI and image analysis. (A) Representative contrast concentration maps acquired from dynamic contrast-enhanced MR images of two animals at 0 (baseline), 5, and 40 minutes after gadoteridol injection using the same intensity scale (from 0.4 to −0.1 mM), when the two animals were untreated (control) or treated with anti-EMMPRIN antibody at 3 days prior to imaging. (B) Contrast-enhancement curves averaged in the region of interest (ROI) indicated with the white square (2×1 window: 2 pixels) shown in Fig. 3A, together with the best-fitting 5^th order polynomial curves. The boundaries of the two tumor regions are indicated with dotted circles, in Fig. 3A. (C) K^trans and (D) k_ep maps of the tumors of the two animals untreated (control) or treated with anti-EMMPRIN antibody at 3 days prior to imaging, while the dark central area is necrosis caused by vascular insufficiency.

Figure 4

In vivo PET/CT imaging and image analysis. (A) Representative contrast enhanced (CE) CT image, ¹⁸F-FDG PET image, and fused PET/CT image of a mouse bearing a MIA PaCa-2 tumor orthotopically (axial view), while tumor location is indicated with a white arrow in each sub-figure. (B,D) Volume and (C,E) SUV_mean changes of (B,C) MIA PaCa-2 or (D,E) PANC-1 tumors after untreated (served as control) or treated with anti-EMMPRIN antibody, TRA-8, and combination, respectively (different Greek letters represent the statistical significance among the groups).

Figure 5

Histological analysis of tumor response. (A) Representative microphotographs of Ki67 and CD31 staining of MIA PaCa-2 or PANC-1 tumors after untreated (served as control) or treated with anti-EMMPRIN antibody, TRA-8, and combination for 2 weeks, respectively, while the proliferating and endothelial cells are indicated with black arrows in each sub-figure. (B) Proliferating (Ki67 positive) and (D) endothelial (CD31 positive) cells densities in the four groups, while statistical differences among groups are indicated by different Greek letters (MIA PaCa-2) or alphabets (PANC-1) above bars.