Intrapleural Adenoviral-mediated Endothelial Cell Protein C Receptor Gene Transfer Suppresses the Progression of Malignant Pleural Mesothelioma in a Mouse Model

Abstract

Malignant pleural mesothelioma (MPM) is an aggressive thoracic cancer with a high mortality rate as it responds poorly to standard therapeutic interventions. Our recent studies showed that expression of endothelial cell protein C receptor (EPCR) in MPM cells suppresses tumorigenicity. The present study was aimed to investigate the mechanism by which EPCR suppresses MPM tumor growth and evaluate whether EPCR gene therapy could suppress the progression of MPM in a mouse model of MPM. Measurement of cytokines from the pleural lavage showed that mice implanted with MPM cells expressing EPCR had elevated levels of IFNγ and TNFα compared to mice implanted with MPM cells lacking EPCR. In vitro studies demonstrated that EPCR expression renders MPM cells highly susceptible to IFNγ + TNFα-induced apoptosis. Intrapleural injection of Ad.EPCR into mice with an established MPM originating from MPM cells lacking EPCR reduced the progression of tumor growth. Ad.EPCR treatment elicited recruitment of macrophages and NK cells into the tumor microenvironment and increased IFNγ and TNFα levels in the pleural space. Ad.EPCR treatment resulted in a marked increase in tumor cell apoptosis. In summary, our data show that EPCR expression in MPM cells promotes tumor cell apoptosis, and intrapleural EPCR gene therapy suppresses MPM progression.

Figure 1. IFNγ, TNFα and IL-6 levels in the pleural lavage of nude mice implanted with REN and REN(+EPCR) MPM cells in the pleural cavity.

REN(z) or REN(+EPCR) MPM cells (1 × 10⁶ cells) were injected into the thoracic cavity of nude mice (BALB/c, NU/J). At the end of 30 days following tumor cell implantation, mice were euthanized, tumor tissues and pleural lavages were collected. Cytokine levels in the pleural lavage were quantified by ELISA. (a) TNFα; (b) IFNγ; (c) IL-6. *p < 0.05; **p < 0.01 (n = 11 to 15 mice/group).

Figure 2. Transduction of EPCR expression in MPM cells promotes apoptosis.

Monolayers of REN MPM cells that lack EPCR expression were stably transfected with empty vector or EPCR expression vector. REN and REN(+EPCR) cells were treated with TNFα (10 ng/ml), IFNγ (100 U/ml) or TNFα + IFNγ (10 ng and 100 U/ml) or none (untreated/control) in 10% v/v serum-containing medium. After 72 h, cells were harvested and processed for TUNEL assay. The samples were analyzed using BD FACS Calibur. (a) Flow cytometry data from a typical experiment. (b) Mean data from three independent experiments. ***p < 0.001. (c) REN and REN(+EPCR) cells were treated with TNFα (10 ng/ml), IFNγ (100 U/ml) or TNFα + IFNγ (10 ng and 100 U/ml) for 24 h and cell extracts were subjected to western blot analysis and probed for apoptotic markers, cleaved caspase 3 (cl. Casp 3), cleaved poly (ADP-ribose) polymerase (cl. PARP), and p-BAD.

Figure 3. MPM cells constitutively expressing EPCR are susceptible to IFNγ/TNFα-induced apoptosis and the knock-down of EPCR suppresses apoptosis.

(a) MS-1 and M9K MPM cells, which constitutively express EPCR, were treated with a combination of TNFα 10 ng/ml) + IFNγ (100 U/ml) for 72 h in the serum-containing medium. At the end of 72 h, the cells were harvested and processed for measuring apoptosis by flow cytometry using TUNEL staining. (b) M9K MPM cells and M9K MPM cells that were transfected with shEPCR to knock-down the EPCR expression (M9K(-EPCR)) were treated with TNFα + IFNγ as described in Panel a and the extent of apoptosis was measured. (c) MS-1 cells expressing tissue factor (MS-1) and MS-1 cells transfected with shEPCR to knock-down the EPCR expression (MS-1(-EPCR)) were treated with TNFα + IFNγ as described in Panel a and the extent of apoptosis was measured. **p < 0.01.

Figure 4. Ad.EPCR treatment curtails the progression of MPM in the mouse model.

Nude mice (BALB/c, NU/J) were implanted with 1 × 10⁶ REN MPM cells in the thoracic cavity. On the 11th day (d 11) following the tumor cell implantation, a group of mice was killed to monitor the establishment of MPM tumors in the thoracic cavity. Then the remaining mice were divided randomly into three groups, and each group of mice was injected intrapleurally with either sterile PBS (100 μl), the control adenovirus (Ad.Con), or EPCR adenovirus (Ad.EPCR) (2 × 10⁹ pfu in 100 μl PBS, once every three days). At the end of 30 days (d 30), mice were euthanized, and tumor statistics were recorded. (a) tumor count; (b) tumor volume; (c) tumor burden; (d) a representative photograph showing differences in tumor growth in mice treated with PBS, control adenovirus or EPCR adenovirus following REN MPM cell implantation for 10 days. Arrows indicate tumors. A total of three independent experiments were conducted with a total of 16–18 mice/group. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5. EPCR expression in MPM tumors following the administration of Ad.EPCR.

NU/J mice were implanted with REN MPM cells and then treated with a control adenovirus or the adenovirus encoding EPCR as described in Fig. 4. At the end of the 30-day experimental period, tumors were excised and then processed for tissue sectioning and immunostaining with EPCR antibody. Red staining in the right panel of the micrograph indicates EPCR expression in tumor cells.

Figure 6. Transduction of EPCR expression in REN MPM tumors lacking EPCR induces tumor cell apoptosis.

NU/J nude mice were implanted with REN MPM cells and 10 days following the tumor cell implantation, mice were treated with PBS, control adenovirus or adenovirus encoding EPCR for 20 days as described in Fig. 4. At the end of the 30-day experimental period, tumors were removed and processed for tissue sectioning or preparing tissue extracts. Tissue sections were immunostained for TUNEL staining (panel a), and the percentage of apoptotic tumor cells was determined by counting tumor cells stained positive for TUNEL staining and the total number of tumor cells in that field (panel b). Tumor tissue extracts were subjected to western blot analysis and probed for apoptotic markers, Bax, p-BAD, cleaved caspase 3 (cl. Casp 3), and cleaved poly (ADP-ribose) polymerase (cl. PARP) (panel c). Panel (a,c) depict representative images. Cells stained with dark red color in panel a indicate apoptotic cells. Data shown in panel b were obtained by counting the number of apoptotic cells and the total number of tumor cells present in 20 to 25 randomly selected fields from 5 or more tumors derived from different mice. ***p < 0.001.

Figure 7. Transduction of EPCR expression in MPM recruits macrophages and NK cells into tumor environment.

Nu/J nude mice were implanted with REN MPM cells and 10 days following the tumor cell implantation, mice were treated with PBS, control adenovirus or adenovirus encoding EPCR for 20 days as described in Fig. 4. At the end of the 30-day experimental period, tumors were removed and processed for tissue sectioning. Tissue sections were stained for the expression of mouse macrophage marker F4/80 (a) or NK cell marker NK1.1 (c). The percentage of macrophages and NK cells in tumors was determined by counting the number of cells stained positive for either F4/80 or NK1.1 in a field and the number of tumor cells present in that field. Images shown in panels (a,c) are representative images. Cells stained with red color indicate macrophages (a) and NK cells (c; also identified by arrows). Data shown in panels (b,d) were obtained by counting the number of positively stained cells and the total number of tumor cells in 20 to 25 randomly selected fields from 5 or more tumors derived from different mice. *p < 0.05; **p < 0.01.

Figure 8. IFNγ and TNFα levels in the pleural lavage of nude mice implanted with REN and treated with PBS, control adenovirus or EPCR adenovirus.

REN MPM cells (1 × 10⁶ cells) were injected into the thoracic cavity of nude mice. After 10 days, mice were treated with PBS, control adenovirus or EPCR adenovirus as described in Fig. 4. At the end of 30 days following tumor cell implantation, mice were euthanized, and pleural lavages were collected. Cytokine levels in the pleural lavage were quantified using ELISA (a) TNFα; (b) IFNγ (n = 13 to 16 mice/group). *p < 0.05 in one-way ANOVA. Values in Ad.EPCR treated animals also differ in a statistically significant manner from PBS or Ad.Con treated animals.