Fig 1.
Refractory state for interferon induction follows a single injection of isRNA in mice.
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.
Fig 2.
Treatment of melanoma B16-bearing mice with isRNA inhibits primary tumor growth.
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.
Fig 3.
isRNA potently reduces the number of surface B16 melanoma metastases in different organs of mice.
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.
Fig 4.
Treatment of melanoma B16-bearing mice with isRNA reduces the area of internal metastases in lungs and kidneys.
(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.
Fig 5.
The treatment of melanoma B16-bearing mice with isRNA increases the white pulp area in the spleen.
(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 Materials & Methods). 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.
Fig 6.
The effect of isRNA on the liver of mice with melanoma B16.
Representative histological images of liver tissues of healthy animals (A), untreated animals with subcutaneous melanoma (B) and B16-bearing animals treated with p.t. injections of isRNA/2X3-DOPE complexes (C). Insets show binuclear hepatocytes (left), necrosis in the liver parenchyma (center) and hepatocytes with dystrophy (right). Hematoxylin and eosin stain, original magnification x400.
Table 1.
The effect of isRNA treatment on the liver of B16 melanoma-bearing mice.