Intratumoral delivery of RIG-I agonist SLR14 induces robust antitumor responses

Abstract

Cytosolic nucleic acid-sensing pathways can be triggered to enhance immune response to cancer. In this study, we tested the antitumor activity of a unique RIG-I agonist, stem loop RNA (SLR) 14. In the immunogenic tumor models, we observed significant tumor growth delay and an extended survival in SLR14-treated mice. SLR14 also greatly improved antitumor efficacy of anti-PD1 antibody over single-agent treatment. SLR14 was mainly taken up by CD11b⁺ myeloid cells in the tumor microenvironment, and many genes associated with immune defense were significantly up-regulated after treatment, accompanied by increase in the number of CD8⁺ T lymphocytes, NK cells, and CD11b⁺ cells in SLR14-treated tumors. Strikingly, SLR14 dramatically inhibited nonimmunogenic B16 tumor growth, and the cured mice developed an immune memory. Furthermore, a systemic antitumor response was observed in both bilateral and tumor metastasis models. Collectively, our results demonstrate that SLR14 is a promising therapeutic RIG-I agonist for cancer treatment, either alone or in combination with existing immunotherapies.

Graphical abstract

Figure 1.

I.t. injection of SLR14 results in significant antitumor effect. (A) Subcutaneous YMR1.7 melanoma model was established in the right flank of C57BL/6J mice. At day 5 after injection, mice with similar tumor volumes were randomly divided into four groups (8–10 mice per group). The first group of mice was i.t. injected with 50 µl of 5% glucose mixed with 1 mg/kg (25 µg) SLR14 and 4 µl jetPEI (SLR14). The second group of mice was i.t. treated with 50 µl of 5% glucose containing 4 µl jetPEI (vehicle). The third group of mice was i.t. treated with 50 µl PBS containing 25 µg CpG. The fourth group of mice was i.t. treated with 50 µl of 5% glucose (no treatment). The treatment was performed every 3 d for a total of five doses. (B) Average tumor volume (error bars = SD) for each group of YMR1.7-bearing mice. (C) The survival curve of YMR1.7-bearing mice after treatment. (D) Subcutaneous MC38 colon cancer model was established at the right flank of C57BL/6J mice. When tumor volume reached ≥100 mm³ (day 10), the mice with similar tumor volumes were i.t. treated with 1 mg/kg (25 µg) SLR14 or vehicle (four to five mice per group). The treatment was performed every 3 d for a total of four doses. (E) Average tumor volume (error bars = SD) for each group of MC38-bearing mice. (F) The survival curve of MC38-bearing mice after treatment. (G) Average tumor volume (error bars = SD) of MC38-bearing mice receiving different doses of SLR14 (1, 0.2, or 0.05 mg/kg) or jetPEI (vehicle). Five mice per group. (H) The survival curve of MC38-bearing mice receiving different doses of SLR14. Multivariate analysis of variance and multiple t tests were used for statistical analysis. *, P < 0.05; **, P < 0.01. Results are representative of at least two independent experiments.

Figure 2.

Combination treatment with SLR14 and anti-PD1 leads to better antitumor effects than single treatment. (A) Subcutaneous YMR1.7 melanoma model was established as described in Fig. 1 A. At day 5 after injection, the mice with similar tumor volumes were randomly divided into five groups (five mice per group) for i.t. treatment with vehicle, SLR14 (1 mg/kg) or no treatment, i.p. treatment with anti-PD1 (200 µg per mouse), or SLR14 (i.t.) plus anti-PD1 (i.p.). The treatment was performed every 3 d for a total of five doses. (B) Average tumor volume (error bars = SD) for each group of YMR1.7-bearing mice. (C) Subcutaneous MC38 colon cancer model was established as described in Fig. 1 D. When tumor volume reached ≥100 mm³ (day 10), the mice with similar tumor volumes were randomly divided into four groups (5–10 mice per group) for i.t. treatment with vehicle or SLR14 (1 mg/kg), i.p. treatment with anti-PD1 (5 µg per mouse), or SLR14 (i.t.) plus anti-PD1 (i.p.). The treatment was performed every 3 d for a total of five doses. (D) Average tumor volume of individual MC38-bearing mice in each group (error bars = SD). Multivariate analysis of variance was used for statistical analysis. *, P < 0.05; **, P < 0.01. Results are representative of two independent experiments.

Figure 3.

SLR14 is mainly taken up by CD11b⁺ myeloid cells in the tumor microenvironment. Subcutaneous YMR1.7 melanoma model was established in C57BL/6J mice (six mice) as described in Fig. 1 or 2. At day 12 after injection, the mice were i.t. treated with 50 µl of 5% glucose containing 1 mg/kg (25 µg) AF647-conjugated SLR14 and 4 µl jetPEI (SLR14). 24 h later, tumors were harvested and digested to make single-cell suspensions for flow cytometry analysis. (A) Top: The percentage of SLR14⁺ cells in total CD11b⁺ cells within tumor; bottom, the percentage of CD11b⁺ cells in total SLR14⁺ cells within tumor. Error bars = SD. (B) The percentage of CD11b⁺ cells in total CD45⁺ cells and the percentage of SLR14⁺ cells in total CD11b⁺ cells in dLN (top) and ndLN (bottom). Error bars = SD. Unpaired t test was used for statistical analysis. *, P < 0.05; **, P < 0.01. Results are representative of two independent experiments.

Figure 4.

Transcriptomic analysis of tumor i.t. treated with SLR14 versus vehicle. Subcutaneous YMR1.7 melanoma model was established in C57BL/6J mice and i.t. treated with SLR14 (two mice) or vehicle (three mice). 24 h after the third treatment, tumors were harvested, and total RNAs were extracted for RNA-seq. (A) Volcano plot of differentially expressed genes between SLR14-treated versus vehicle-treated tumors. (B) Heat map of differentially expressed genes involved in type-I IFN-stimulated gene (ISG) between SLR14-treated versus vehicle-treated tumors. Data were generated from one experiment.

Figure 5.

I.t. SLR14 delivery enhances tumor infiltration of cytotoxic T lymphocytes and myeloid cells. Subcutaneous YMR1.7 melanoma was established in C57BL/6J mice and i.t. treated with vehicle, SLR14, or no treatment. 3 d after the fifth treatment, tumors were harvested and digested with 0.5 mg/ml Collagenase D and 40 µg/ml DNase I. Single-cell suspensions were prepared for flow cytometry analysis. (A) Percentages (top) and quantities (bottom) of tumor-infiltrating CD45⁺, CD11b⁺, CD8⁺, CD4⁺, FoxP3⁺CD4⁺, or NK1.1⁺ cells in each group. All T cells were CD44⁺. The cell numbers were normalized based on the tumor weight. Error bars = SD. 1, no treatment; 2, vehicle; 3, SLR14. (B) The ratio of tumor-infiltrating CD8⁺ T cells/CD4⁺ T cells or CD8⁺ T cells/CD4⁺FoxP3⁺ T reg cells in each group. Error bars = SD. (C) Subcutaneous YMR1.7 melanoma growth in RAG1^−/− mice treated with vehicle or SLR14. Treatment protocol was the same as described in Fig. 1 A. Left: Tumor growth curves (error bars = SD) for each group of mice. Right: Tumor growth curves of individual mice in each group. (D and E) IFNγ, TNFα, and GzmB productions of tumor-infiltrating CD8⁺ T lymphocytes after i.t. treatment (error bars = SD). Five mice per group. Unpaired t test was used for statistical analysis. *, P < 0.05; **, P < 0.01. N.S., no significance. Results are representative of two independent experiments.

Figure 6.

SLR14 exhibits robust antitumor effect in B16 melanoma. (A) Subcutaneous B16F10 or B16-ova model was established in C57BL/6 mice. At day 7 after injection, the mice with similar tumor volumes received no treatment or were i.t. treated with 25 µg SLR14 or vehicle. Treatment protocol was the same as that used in YMR1.7 or MC38 model. (B) Average tumor volume of B16F10-bearing mice (error bars = SD). (C) Average tumor volume of B16-ova–bearing mice (error bars = SD). Significance (**) was found between vehicle and SLR14 groups. (D) Tumor growth curves of individual B16-ova–bearing mice after treatment. (E) Survival curve of B16-ova–bearing mice after treatment. Five mice per group. Multivariate analysis of variance and multiple t test was used for statistical analysis. **, P < 0.01. Results are representative of at least two independent experiments.

Figure 7.

Antitumor effect of SLR14 in B16-ova melanoma is partially mediated by T cells. (A) Subcutaneous B16-ova melanoma model was established in RAG1^−/− mice. At day 7 after injection, the mice with similar tumor volumes were i.t. treated with SLR14 or vehicle (five mice per group). The treatment protocol was the same as described in Fig. 6. (B) Average tumor volume for each group of mice (error bars = SD). (C) Subcutaneous B16-ova melanoma model was established in C57BL/6J mice. At day 7 after injection, the mice with similar tumor volumes were i.p. injected with T cell depletion antibodies (anti-CD4⁺, anti-CD8⁺, or both anti-CD4⁺ and anti-CD8⁺; five mice per group) at 200 µg/mouse, followed by i.t. injection of SLR14 or vehicle. In vivo T cell depletion was maintained every 3 d. (D) Average tumor volume for each group of mice (error bars = SD). Multiple t test was used for statistical analysis. *, P < 0.05. Results are representative of two independent experiments.

Figure 8.

SLR14 i.t. treatment induces an effective abscopal effect. (A) Bilateral B16-ova:B16-ova tumor model was established in both flank sides of C57BL/6J mice. At day 7 after injection, only one side of tumor was i.t. treated with SLR14 or vehicle (five mice per group). Treatment protocol was the same as described in Fig. 6. Tumor growth of both flank sides was monitored every 2 d. The average tumor volume (error bars = SD) of B16-ova at both treated and untreated (distant) flank sides is shown. Multivariate analysis of variance was used for statistical analysis. **, P < 0.01. Red double asterisks indicate comparison between treated (vehicle) and treated (SLR14). Black double asterisks indicate comparison between distant (vehicle) and distant (SLR14). (B) Bilateral MC38:B16-ova tumor model was established in both flank sides of C57BL/6J mice. Only MC38 tumor was treated with SLR14 or vehicle (five mice per group) when MC38 volume reached 100 mm³ (day 10–11). The treatment protocol was the same as described in Fig. 6. Tumor growth of both flank sides was monitored every day. The average tumor volume (error bars = SD) of MC38 or B16-ova at both treated and untreated (distant) flank sides is shown. Multiple t test was used for statistical analysis. *, P < 0.05. N.S., no significance. Results are representative of two independent experiments.

Figure 9.

I.t. SLR14 treatment significantly impedes tumor metastasis. Subcutaneous B16-ova melanoma model was established in C57BL/6J mice. At day 9 after injection (tumor volume ≥100 mm³), the mice were i.t. treated with 1 mg/kg (25 µg) SLR14 or vehicle. The treatment was performed every 3 d for a total of four doses. 24 h after the second i.t. treatment (D13), 10⁵ B16-Fluc cells were injected into the left ventricle of SLR14- or vehicle-treated mice. One group of naive C57BL/6J mice injected with the same numbers of B16-Fluc cells were used as control. Five to six mice per group. 1 wk later (D20), B16-Fluc cells were imaged for bioluminescence at a 10-s exposure setting on the IVIS Spectrum imager. Imaging was performed every other day for 2 wk. Results are representative of two independent experiments.

Figure 10.

B16-ova–cured mice after SLR14 treatment develop immune memory. (A) Subcutaneous B16-ova melanoma model was established in C57BL/6 mice. From day 7 after injection, the mice were i.t. treated with 1 mg/kg (25 µg) SLR14 every 3 d for a total of six doses. 10 d after last treatment, the cured mice (five mice) were challenged with B16-ova at the cured flank side. Age- and gender-matched naive mice injected with the same numbers of B16-ova were used as controls. Five mice per group. (B) Average tumor volume for each group of mice (error bars = SD). Multiple t test was used for statistical analysis. *, P < 0.05; **, P < 0.01. (C) Tumor growth curves of individual mice in each group. (D) Survival curve of tumor-challenged mice. Results are representative of two independent experiments.