CCN-Based Therapeutic Peptides Modify Pancreatic Ductal Adenocarcinoma Microenvironment and Decrease Tumor Growth in Combination with Chemotherapy

Abstract

The prominent desmoplastic stroma of pancreatic ductal adenocarcinoma (PDAC) is a determinant factor in tumor progression and a major barrier to the access of chemotherapy. The PDAC microenvironment therefore appears to be a promising therapeutic target. CCN2/CTGF is a profibrotic matricellular protein, highly present in the PDAC microenvironment and associated with disease progression. Here we have investigated the therapeutic value of the CCN2-targeting BLR100 and BLR200, two modified synthetic peptides derived from active regions of CCN3, an endogenous inhibitor of CCN2. In a murine orthotopic PDAC model, the two peptides, administered as monotherapy at low doses (approximating physiological levels of CCN3), had tumor inhibitory activity that increased with the dose. The peptides affected the tumor microenvironment, inhibiting fibrosis and vessel formation and reducing necrosis. Both peptides were active in preventing ascites formation. An increased activity was obtained in combination regimens, administering BLR100 or BLR200 with the chemotherapeutic drug gemcitabine. Pharmacokinetic analysis indicated that the improved activity of the combination was not mainly determined by the substantial increase in gemcitabine delivery to tumors, suggesting other effects on the tumor microenvironment. The beneficial remodeling of the tumor stroma supports the potential value of these CCN3-derived peptides for targeting pathways regulated by CCN2 in PDAC.

Keywords: CCN2/CCN3; PDAC; matricellular proteins; tumor microenvironment.

Figure 1

CCN2 is expressed by FC1199 tumors. CCN2 was analyzed by immunohistochemistry (IHC) in healthy murine pancreas (A) and FC1199 orthotopic tumors (B,C). Black arrows: tubular structures of an adenocarcinoma with cellular atypia and differences in the thickness of the tubular wall. Red arrows: stromal areas. C is a higher magnification of the boxed area in B. CCN2 was expressed by both tumor cells (black arrows) and stroma cells (red arrow), scale bars: 50 µm. (D) CCN2 production by FC1199 cells in vitro. CCN2 in the cell lysate and conditioned media was measured by ELISA and expressed as µg/mL.

Figure 2

Antineoplastic activity of BLR100 or BLR200 on PDAC. (A) Schedule of treatment. The peptides were administered three times per week at 4.5 µg/kg, at the indicated day (black arrows). Dotted arrows indicate mice sacrifice and analysis on days 14 and 25. (B) Tumor in the pancreas of mice 4 days after tumor transplantation (H&E staining, scale bar 100 µm). (C) Tumor burden (evaluated as pancreas weight) in mice bearing FC1199 orthotopic tumors treated with vehicle, BLR100, or BLR 200 on day 14 (n = 5) or 25 (n = 7). Dotted line indicates the weight of pancreas in healthy mice. (D) Effect of treatments on ascites formation. Data are the percentage of mice presenting (black) or not (gray) ascites on day 25 (n = 7). (E) Tumor burden in mice treated with a scramble peptide, BLR100, or BLR200 (10 µg/kg) and sacrificed 25 days after tumor transplantation (n = 8). Dotted line indicates the weight of the pancreas in healthy mice. In C and E, data are mean ± SEM. One way ANOVA and Tukey’s, ** p < 0.005; * p < 0.05. (F) Effect of BLR100 or BLR200 on FC1199 cell proliferation in vitro. Data are the percentage of control proliferation, mean ± SEM of values from 2 independent experiments.

Figure 3

Microenvironmental changes induced by BLR100 or BLR200 on PDAC tumors. Representative images and relative quantification of (A) fibrosis (polarized light microscopy analysis of Sirius red stained sections; scale bars, 25 µm), (B) necrosis (asterisks, H&E staining; scale bars, 200 µm) and (C) tumor vessels (CD31 IHC; scale bars, 200 µm) of control and peptide-treated FC1199 orthotopic tumors. N ≥ 6. One way ANOVA and Dunnett’s: * p < 0.05.

Figure 4

Antineoplastic activity of BLR100 or BLR200 in combination with gemcitabine on PDAC. (A) Schedule of treatment. Mice received the peptides (black arrows) and gemcitabine (white arrows) at the indicated times. Dotted arrow indicates mice sacrifice and tumor analysis. (B) Effect of the indicated treatments on mouse body weight. Data are expressed as relative body weight, the percentage of mouse weight at the beginning of treatment, mean ± SEM. (C) Tumor burden (pancreas weight) in mice bearing FC1199 orthotopic tumors treated with vehicles, peptide BLR100, peptide BLR200, gemcitabine (gem), or gemcitabine in combination with the peptides. Dotted line: weight of pancreas in healthy mice (n = 7, mean ± SEM, One way ANOVA and Dunnett’s * p < 0.05, ** p < 0.005). (D) Effect of treatments on ascites formation. Data are the percentage of mice presenting (black) or not (gray) ascites (n = 7). (E,F) BLR100 and BLR200 did not affect FC1199 cell response to gemcitabine in vitro. (E) Proliferation of FC1199 cell exposed to increasing concentrations of gemcitabine alone or with the peptide (20 and 100 nM). (F) Inhibition of FC1199 cell proliferation by gemcitabine (15 nM) in the presence of BLR100 or BLR200 (100 nM). Data are percentage of control, from one experiment representative of two.

Figure 5

Effect of BLR200 on gemcitabine distribution. Gemcitabine (A) and the dFdU metabolite (B) were measured by HPLC–MS analysis in plasma and tumors of mice bearing FC1199 orthotopic tumors, treated or not with BLR200. Data are the ratio between tumor (T) and plasma (P) concentration, 30 min and 1 h after gemcitabine administration (mean ± SEM, n = 4).