Antitumor and Antimetastatic Effect of Small Immunostimulatory RNA against B16 Melanoma in Mice.

Small interfering RNAs, depending on their structure, delivery system and sequence, can stimulate innate and adaptive immunity. The aim of this study was to investigate the antitumor and antimetastatic effects of immunostimulatory 19-bp dsRNA with 3'- trinucleotide overhangs (isRNA) on melanoma B16 in C57Bl/6 mice. Recently developed novel cationic liposomes 2X3-DOPE were used for the in vivo delivery of isRNA. Administration of isRNA/2X3-DOPE complexes significantly inhibits melanoma tumor growth and metastasis. Histopathological analysis of spleen cross sections showed hyperplasia of the lymphoid white pulp and formation of large germinal centers after isRNA/2X3-DOPE administration, indicating activation of the immune system. The treatment of melanoma-bearing mice with isRNA/2X3-DOPE decreases the destructive changes in the liver parenchyma. Thus, the developed isRNA displays pronounced immunostimulatory, antitumor and antimetastatic properties against melanoma B16 and may be considered a potential agent in the immunotherapy of melanoma

isRNA potently reduces the number of surface B16 melanoma metastases in different organs of mice.

<p>C57BL/6 mice were i.v. inoculated with melanoma B16 cells (2 × 105 cells per mouse) and treated with isRNA/2X3-DOPE complexes by i.v. administration on days 2, 6 and 10. (A) Images of lungs of representative mice from different groups on day 15 after tumor cell implantation. The numbers of macroscopically visible melanoma metastases in the lung (B), kidney (C) and liver (D), mean ± SEM, n = 9. Statistically significant differences between the experimental group and untreated group (control) are indicated by an asterisk (*, P<0.05); and between the experimental and Mock groups by a cross (†, P<0.05); Mann-Whitney U test.</p

Treatment of melanoma B16-bearing mice with isRNA inhibits primary tumor growth.

<p>C57BL/6 mice with subcutaneous melanoma (1.5 × 105 cells per mouse) were treated with isRNA/2X3-DOPE complexes (p.t. or i.v. injections). (A) Dynamics of tumor growth. Black arrows indicate days of isRNA administration. The data represent mean ± SEM (n = 8–10). Statistically significant differences between experimental groups and the untreated group (control) are indicated by asterisks (**, P<0.01; *, P<0.05); and between experimental groups and the 2X3-DOPE-treated group by crosses (††, P<0.01; †, P<0.05); Mann-Whitney U test. (B) MR images of the representative mice from different groups on day 17 after tumor initiation. Frontal longitudinal sections with the maximum size of tumors from each group are shown. White arrows indicate tumor nodes.</p

Treatment of melanoma B16-bearing mice with isRNA reduces the area of internal metastases in lungs and kidneys.

<p>(A) Images showing internal lung metastases (top row) and kidney metastases (lower row) of the representative mice from different groups on day 15 after intravenous B16 tumor cell implantation. Arrows show the metastases. Paraffin sections, hematoxylin and eosin stain. (B) The ratio of the total metastases area to the total area of the organ. The data represent mean ± SEM (n = 9). Statistically significant differences between experimental and control groups are indicated by asterisks (** P<0.01, * P<0.05); and differences between experimental and Mock groups are indicated by crosses (†† P<0.01, † P<0.05); Mann-Whitney U test.</p

The treatment of melanoma B16-bearing mice with isRNA increases the white pulp area in the spleen.

<p>(A) Cross sections of the spleens of the representative mice with subcutaneous melanoma on day 18 after tumor initiation. Arrows show follicles on each image. Paraffin sections, hematoxylin and eosin stain. (B, C) The ratio of the total follicles area to the total area of the spleen for subcutaneous (B) and metastatic (C) melanoma (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150751#sec002" target="_blank">Materials & Methods</a>). I. v. administration–black bars, p.t. administration–white bars. The data represent mean ± SEM (n = 9). Statistically significant differences between experimental and control groups are indicated by asterisks (**, P<0.01; *, P<0.05); and differences between experimental and Mock groups are indicated by crosses (††, P<0.01; †, P<0.05); Mann-Whitney U test.</p

Folate-Equipped Cationic Liposomes Deliver Anti-MDR1-siRNA to the Tumor and Increase the Efficiency of Chemotherapy

In this study, we examined the in vivo toxicity of the liposomes F consisting of 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20-tetraazahexacosan tetrahydrochloride, lipid-helper 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and folate lipoconjugate (O-{2-[rac-2,3-di(tetradecyloxy)prop-1-yloxycarbonyl]aminoethyl}-O’-[2-(pteroyl-L-glutam-5-yl)aminoethyl]octadecaethyleneglycol) and investigated the antitumor effect of combined antitumor therapy consisting of MDR1-targeted siMDR/F complexes and conventional polychemotherapy using tumor xenograft initiated in immunodeficient mice. Detailed analysis of acute and chronic toxicity of this liposomal formulation in healthy C57BL/6J mice demonstrated that formulation F and parent formulation L (without folate lipoconjugate) have no acute and chronic toxicity in mice. The study of the biodistribution of siMDR/F lipoplexes in SCID mice with xenograft tumors formed by tumor cells differing in the expression level of folate receptors showed that the accumulation in various types of tumors strongly depends on the abandons of folate receptors in tumor cells and effective accumulation occurs only in tumors formed by cells with the highest FR levels. Investigating the effects of combined therapy including anti-MDR1 siRNA/F complexes and polychemotherapy on a multidrug-resistant KB-8-5 tumor xenograft in SCID mice demonstrated that siMDR/F increases the efficiency of polychemotherapy: the treatment leads to pronounced inhibition of tumor growth, reduced necrosis and inflammation, and stimulates apoptosis in KB-8-5 tumor tissue. At the same time, it does not induce liver toxicity in tumor-bearing mice. These data confirm that folate-containing liposome F mediated the extremely efficient delivery of siRNA in FR-expressing tumors in vivo and ensured the safety and effectiveness of its action

Refractory state for interferon induction follows a single injection of isRNA in mice.

<p>C57BL/6 mice were intravenously injected once or twice with 2X3-DOPE (white bars) or with isRNA/2X3-DOPE (black bars). The time intervals between the injections varied from 16 to 96 h. Serum IFN-α levels were measured by ELISA 6 h after the last injection. The data represent mean ± standard deviation (SD) calculated from measurements from at least three mice. Statistically significant differences between experimental groups and the 2X3-DOPE-treated group (Mock) are indicated by crosses (††, P<0.01); Mann-Whitney U test.</p