Immunotherapy of hepatocellular carcinoma with small double-stranded RNA

Abstract

Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with limited therapeutic options. Since HCC has been shown to be immunogenic, immunotherapy is considered a promising therapeutic approach. Small interfering RNAs (siRNAs), depending on their structure and sequence, can trigger the innate immune system, which can potentially enhance the adaptive anticancer immune response in the tumor-bearing subjects. Immunostimulatory properties of nucleic acids can be applied to develop adjuvants for HCC treatment.

Methods: The transplantable HCC G-29 tumor in male CBA/LacSto (CBA) mice was used to study the effects of immunostimulatory RNA on tumor growth. Tumor size, metastases area in different organs of mice and mouse survival rate were analyzed. Furthermore the mouse serum IFN-α levels were measured using ELISA.

Results: In the present study, we found that a 19-bp RNA duplex (ImmunoStimulattory RNA or isRNA) with 3-nt overhangs at the 3'-ends of specific sequence displays immunostimulatory, antitumor, and antimetastatic activities in mice bearing HCC G-29. Our results demonstrate that isRNA strongly increases the level of interferon-α (IFN-α) by up to 25-fold relative to the level in mice injected with Lipofectamine alone (Mock), and to a lesser extent increases the level of proinflammatory cytokine interleukin-6 (IL-6) (by up to 5.5-fold relative to the Mock level), in mice blood serum. We showed that isRNA reliably (P < 0.05) inhibits primary tumor growth in mice compared to the mock group. Furthermore, injections of isRNA significantly enhanced necrotic processes in the center of the primary tumor, and decreased by twofold the width of the undifferentiated peripheral zone and the number of mitotic cells in this zone. The results showed that isRNA efficiently reduces the area of metastases in the liver, kidneys, and heart of CBA/LacSto mice with HCC.

Conclusions: The obtained results clearly demonstrate immunostimulatory and antimetastatic properties of the isRNAs in mice with HCC. Consequently, this short double-stranded RNA can be considered as a potential adjuvant for the therapy of HCC.

Figure 1

Stimulation of the innate immune response by isRNA and poly(I:C) in vivo. СBA/LacSto mice were intraperitoneally injected with isRNA (10 μg per mouse) or poly(I:C) (10 μg per mouse) in complex with Lipofectamine 2000. Serum IFN-a (A) and IL-6 (B) levels were measured by ELISA 6 h 1, 6, 10, and 16 hours after injection. Values for Lipofectamine-treated mice represent the Mock. The data represent means ± standard deviation (SD) calculated from measurements from at least three mice.

Figure 2

Influence of isRNA injections on HCC primary tumor growth. CBA/LacSto mice were intramuscularly injected with G-29 cells and treated with isRNA or poly(I:C) i.p. injections. The data represent means ± standard error of the mean (SEM) at day 22 (n = 8–10). P ≤ 0.05 according to Mann–Whitney test.

Figure 3

Cross-sections of HCC G-29 primary tumors 22 days after tumor inoculation. Peripheral zone of primary tumor in untreated (A) and isRNA-treated (B) mice: arrows show zone of undifferentiated cells. C, D - central area of primary tumor in untreated (C) and isRNA-treated (D) mice: necrosis and foci of cell death are indicated by arrows. Paraffin sections, Mallory’s trichrome staining.

Figure 4

The survival rate of mice after inoculation of HCC G-29 cells. Each group consisted of 7 mice.

Figure 5

Liver of CBA/LacSto mice 22 days after HCC G-29 cells inoculation showing internal and surface metastases. A. Untreated mice. Arrows show the typical metastasis. In the blood vessel (*) painted plasma and a deformed erythrocytes can be seen. B. Large surface metastasis in liver of poly(I:C) treated mice. C. Eosinophils (arrows) and D. mitoses (arrows) in liver of mice after isRNA injections. Paraffin sections, hematoxylin and eosin staining.

Figure 6

Treatment of HCC G-29-bearing mice with isRNA reduces the metastases area in different organs of mice. The ratio of the total metastases area to the total area of the organ was determined by microscopic analysis of organs sections, prepared at day 22. The data represent means ± SEM from five microscopic fields. Statistically significant differences between experimental groups and untreated group (control) are indicated by asterisks. **, P < 0.01; *, P < 0.05. Statistically significant differences between experimental groups and Lipofectamine-treated group (Mock) are indicated by triangles. ▲▲, P < 0.01; ▲, P < 0.05; Mann–Whitney U test. The area of metastases in the heart was evaluated using the average length of metastases and the mean number of metastases per mm² as described in the Methods section.