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, 12 (11), 906-14

RIG-I Helicase-Independent Pathway in Sendai Virus-Activated Dendritic Cells Is Critical for Preventing Lung Metastasis of AT6.3 Prostate Cancer

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RIG-I Helicase-Independent Pathway in Sendai Virus-Activated Dendritic Cells Is Critical for Preventing Lung Metastasis of AT6.3 Prostate Cancer

Tomonori Kato et al. Neoplasia.

Abstract

We recently demonstrated highly efficient antitumor immunity against dermal tumors of B16F10 murine melanoma with the use of dendritic cells (DCs) activated by replication-competent, as well as nontransmissible-type, recombinant Sendai viruses (rSeV), and proposed a new concept, "immunostimulatory virotherapy," for cancer immunotherapy. However, there has been little information on the efficacies of this method: 1) in more clinically relevant situations including metastatic diseases, 2) on other tumor types and other animal species, and 3) on the related molecular/cellular mechanisms. In this study, therefore, we investigated the efficacy of vaccinating DCs activated by fusion gene-deleted nontransmissible rSeV on a rat model of lung metastasis using a highly malignant subline of Dunning R-3327 prostate cancer, AT6.3. rSeV/dF-green fluorescent protein (GFP)-activated bone marrow-derived DCs (rSeV/dF-GFP-DC), consistent with results previously observed in murine DCs. Vaccination of rSeV/dF-GFP-DC was highly effective at preventing lung metastasis after intravenous loading of R-3327 tumor cells, compared with the effects observed with immature DCs or lipopolysaccharide-activated DCs. Interestingly, neither CTL activity nor DC trafficking showed any apparent difference among groups. Notably, rSeV/dF-DCs expressing a dominant-negative mutant of retinoic acid-inducible gene I (RIG-I) (rSeV/dF-RIGIC-DC), an RNA helicase that recognizes the rSeV genome for inducing type I interferons, largely lost the expression of proinflammatory cytokines without any impairment of antitumor activity. These results indicate the essential role of RIG-I-independent signaling on antimetastatic effect induced by rSeV-activated DCs and may provide important insights to DC-based immunotherapy for advanced malignancies.

Figures

Figure 1
Figure 1
Optimization of tumor lysate-pulsed rSeV/dF-GFP-DC vaccine on rat model of lung metastasis of AT6.3 prostate cancer. *P < .01. (A) Treatment regimen. (B) Optimization of dose-efficacy relationship. DCs were administered through the tail vein. The following numbers of animals were used: normal lung, n = 6; No Tx, n = 6; and rSeV/dF, n = 17 (3 x 104 cells, n = 6; 3 x 105 cells, n = 5; and 3 x 106 cells, n = 6). (C) Optimization of administration route. A total of 3 x 106 DCs were used per vaccine. The following numbers of animals were used: normal lung, n = 6; No Tx, n = 9; and rSeV/dF, n = 16 (subcutaneous [s.c.] n = 7 and intravenous [i.v.] n = 9). (D) Requirement of ex vivo pulsation of tumor lysate. A total of 3 x 106 DCs were used per vaccine. The following numbers of animals were used: normal lung, n = 6; No Tx, n = 7; and rSeV/dF, n = 16 (-lysate n = 8 and +lysate n = 8). N.S. indicates not significant.
Figure 2
Figure 2
Direct comparison of antimetastatic ability of iDC, LPS-DC, and rSeV/dF-GFP-DC on rat model of lung metastasis of AT6.3 prostate cancer. (A–C) Direct comparison of efficacy of iDC with or without tumor lysate pulsation (n = 10, respectively), LPS-DC with lysate (n = 11), and rSeV/dF-GFP-DC with lysate (n = 11): total wet lung weight (g) (A), number of metastatic nodules (B), and gross observation of left upper lobe (C). Normal lung, n = 6; and No Tx, n = 11. The treatment regimen was the same as that in panel A. All DCs were administered intravenously through the tail vein at 3 x 106 cells per vaccine. Note that the iDC group was included so as to exclude the effect of spontaneous activation through tumor antigen capture. *P < .01 and #P <.05. (D) CTL activity assessed in use of splenocytes. Each group contained n = 3.
Figure 3
Figure 3
Trafficking of DCs in vivo. At 3 and 24 hours after intravenous administration of 3 x 106 111In oxinate-labeled DCs, the whole body was screened by a γ-camera (data not shown). Twenty-four hours later, the tissue samples were harvested and subjected to a γ-counter. Each group contained n = 5. (A) Tissue distribution of radioactivity 48 hours after DC administration. No difference in accumulation was observed in any of the tissues sampled in this study. (B) Gene expression of ICAM-1/CD54 and CCR7 in DCs after treatment with LPS or rSeV/dF. Forty-eight hours after treatment, total RNA was extracted and subjected to quantitative real-time PCR. The GAPDH expression level was used for internal control of relative expression, and the data were expressed as percentages against relative expression of iDC.
Figure 4
Figure 4
Type I IFN (A), typical proinflammatory cytokine (B), and surface marker (C and D) expression profiles in iDC, LPS-DC, rSeV/dF-GFP-DC, and rSeV/dF-RIGIC-DC. Two days after treatment, gene or protein expression was determined by real-time quantitative RT-PCR or ELISA (A or B) or by FACS analyses (C and D). *P < .01. (A) Gene expression of type I IFNs. rSeV/dF stimulated IFN-β but not IFN-α expression, a finding diminished by rSeV/dF expressing RIG-IC, which is a dominant-negative inhibitor of a RNA helicase RIG-I. Each group contained n = 4. (B) Protein secretion (ELISA: TNF-α) and gene expression (real-time quantitative RT-PCR: others). Note that rSeV/dF-related up-regulation of IL-1β, IL-6, and IL-12/p35 was almost completely abolished by RIG-IC expression. Each group contained n = 3. (C and D) RIG-IC had no effect on surface markers of DCs, CD80/B7-1 and CD86/B7-2 (C), and αE2-integrin, a target antigen of OX62 (D). Each group contained n = 4 (C) and n = 12 (D, total of three independent experiments).
Figure 5
Figure 5
Antimetastatic activity of rSeV/dF-modified DC does not depend on RIG-I-related signaling. Comparison of the efficacy of rSeV/dF-modified DC expressing a reporter gene (GFP: n = 6) or RIG-IC (n = 6); total wet lung weight (g) (left graph) and the number of metastatic nodules (right graph) were demonstrated. Normal lung, n = 6; and No Tx, n = 11. Treatment regimen was followed as in Figure 1A. All DCs were administered intravenously through the tail vein at 3 x 106 cells per vaccine. *P < .01.

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