Atelocollagen-mediated in vivo siRNA transfection in ovarian carcinoma is influenced by tumor site, siRNA target and administration route

Abstract

Ovarian cancer is the leading cause of death from gynecological malignancies worldwide, and innate or acquired chemoresistance of ovarian cancer cells is the major cause of therapeutic failure. It has been demonstrated that the concomitant inhibition of Bcl-xL and Mcl-1 anti-apoptotic activities is able to trigger apoptosis in chemoresistant ovarian cancer cells. In this context, siRNA-mediated Bcl‑xL and Mcl-1 inhibition constitutes an appealing strategy by which to eliminate chemoresistant cancer cells. However, the safest and most efficient way to vectorize siRNAs in vivo is still under debate. In the present study, using in vivo bioluminescence imaging, we evaluated the interest of atelocollagen to vectorize siRNAs by intraperitoneal (i.p.) or intravenous (i.v.) administration in 2 xenografted ovarian cancer models (peritoneal carcinomatosis and subcutaneous tumors in nude mice). Whereas i.p. administration of atelocollagen-vectorized siRNA in the peritoneal carcinomatosis model did not induce any gene downregulation, a 70% transient downregulation of luciferase expression was achieved after i.v. injection of atelocollagen-vectorized siRNA in the subcutaneous (s.c.) model. However, the use of siRNA targeting Bcl-xL or Mcl-1 did not induce target-specific downregulation in vivo in nude mice. Our results therefore show that atelocollagen complex formulation, the administration route, tumor site and the identity of the siRNA target influence the efficiency of atelocollagen‑mediated siRNA delivery.

Figure 1.

Schedules for in vivo siRNA treatments. (A) Mice were injected i.p. with 20×10⁶ SKOV3-Luc cells and tumors were allowed to develop for 4 weeks. Bioluminescence values were acquired at day 0 prior to treatment, day 3 and 7. Atelocollagen (0.5% final) in complex with 100 µg siRNA was injected i.p. at day 0 and 2. (B) Mice were injected bilaterally s.c. with 5×10⁶ SKOV3-Luc cells in each flank and tumors were allowed to develop for 4 weeks. Bioluminescence values were acquired at day 0 prior to treatment, day 3 and 7. Atelocollagen (0.05% final) in complex with 125 µg siRNA was injected i.p. at days 0, 1 and 2.

Figure 2.

i.p. injection for luciferase-targeted siRNA to i.p. carcinomatosis. (A) Evolution of the bioluminescent signal over time for NaCl- (4 mice), siGFP- (3 mice) and siLuc- (4 mice)-treated groups. Mean values ± SD for each group of animals relative to bioluminescence values acquired at day 0 for each individual. (B) Representative bioluminescence image for one animal from each group. (C) Bioluminescence values at day 3 for individual mice from each group relative to bioluminescence values acquired at day 0. (D) Box plot of bioluminescence values of each group of animals measured at day 3 relative to bioluminescence values acquired at day 0. No significant difference between groups.

Figure 3.

i.v. injection for luciferase-targeted siRNA to subcutaneous tumors. (A) Evolution of the bioluminescence signal over time for NaCl- (3 mice), siGFP- (3 mice) and siLuc (3 mice)-treated groups. Mean values ± SD for tumors from each group of animals relative to bioluminescence values acquired at day 0 for each tumor. (B) Representative bioluminescence image for one animal from each group. (C) Bioluminescence values at day 3 for individual tumors from each group relative to bioluminescence values acquired at day 0. (D) Box plot of bioluminescence values of each group of animals measured at day 3 relative to bioluminescence values acquired at day 0. The siLuc-treated group was significantly different from the NaCl- (p=0.004) and siGFP (p=0.015)-treated groups (Wilcoxon rank-sum test).

Figure 4.

In vitro efficiency of the siRNA batches used in vivo. (A) Luciferase-targeted siRNAs strongly decrease target expression in vitro. Transfection was performed with siLuc in SKOV3-Luc cells with final 20 nM of siRNAs, and target downregulation analyzed 72 h after transfection. Bioluminescent activity was measured in cell lysates. Results are relative to protein concentration in respective cell lysates ± SD. (B) Both Bcl-x_L- and Mcl-1-targeted siRNAs mediate strong downregulation in vitro on their respective targets. Transfection was performed with siGFP, siMcl1 or siBcl-x_L in SKOV3 cells with final 20 nM of siRNAs, and target downregulation analyzed 72 h after transfection. Mcl-1 and Bcl-x_L protein expression was assessed by western blotting. Actin was used as a loading control.

Figure 5.

i.v. injection for Mcl-1 or Bcl-x_L-targeted siRNA to subcutaneous tumors. (A and C) RT-qPCR analysis of Mcl-1 and Bcl-x_L mRNA expression level in tumor nodes harvested at day 3. Expression is relative to NaCl tumors for siGFP, siMcl-1 or siBcl-xL. Data is presented as mean values from 5 tumor nodes from 5 different animals for siMcl-1 and 4 tumor nodes from 4 different animals for siBcl-xL, ± SD. (B and D) IHC for Mcl-1 or Bcl-x_L on tumor nodes harvested at day 3. Images are representative of 6 different tumors in each group. Scale bar, 200 µm.