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, 22 (12), 2046-55

Systemic Administration of Platelets Incorporating Inactivated Sendai Virus Eradicates Melanoma in Mice

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Systemic Administration of Platelets Incorporating Inactivated Sendai Virus Eradicates Melanoma in Mice

Tomoyuki Nishikawa et al. Mol Ther.

Abstract

Tumor microenvironments include a number of fibrin clots due to the microbleeding caused by cancer cell invasion into blood vessels, which suggests the potential utility of a platelet vector for systemic cancer treatment. We previously reported that inactivated Sendai virus (hemagglutinating virus of Japan; HVJ) envelope (HVJ-E) activates anti-tumor immunity and induces cancer cell-selective apoptosis. The hemagglutination activity that blocks the systemic administration of HVJ-E was dramatically attenuated by incorporation into platelets. Platelets incorporating HVJ-E (PH complex) were then injected into the tail veins of B16F10 melanoma-bearing mice. The PH complex primarily accumulated in tumor tissues and caused the significant accumulation of various immune cells in the tumor bed. Injections of the PH complex to the melanoma-bearing mouse significantly reduced the tumor size, and the tumor growth was ultimately arrested. Secretion of the chemokine regulated upon activation normal T-expressed and presumably secreted (RANTES) was upregulated following PH stimulation. The RANTES-depletion in melanoma-bearing mice significantly attenuated the cytotoxic T lymphocyte activity and led to a dramatic abrogation of the mouse melanoma suppression induced by the PH complex. Thus, a platelet vector incorporating viral particles, a Trojan horse for cancer treatment, will provide a new approach for cancer therapy using oncolytic viruses.

Figures

Figure 1
Figure 1
Infusion and release of HVJ-E or fluorescent particles from platelets. (a) Infusion of FITC-beads (green) into platelets (red). Platelet membrane was stained with DiI (di-alkyl indocarbocyanine). (b) Transmission electron microscopic analysis of HVJ-E-infused platelets (8,000×). (c) Fluorescence intensity measurements of SPHERO fluorescent particles released from thrombin-stimulated SPHERO/platelet complexes. The data are shown as the mean ± SEM of four independent experiments. **P < 0.01 (versus 0U/ml). (d) Western blot analysis of HVJ-E M (matrix protein of HVJ) protein after the thrombin stimulation of PH complexes. HVJ-E samples from 1 to 100 HAU (haemagglutinating units) were loaded as standard samples. The PH supernatant contained the released HVJ-E particles, and the PH pellet contained the HVJ-E particles remaining inside the platelets after thrombin stimulation. In sample P (platelets only), no thrombin was added.
Figure 2
Figure 2
HA assay of PH complexes. (a) The hemagglutinating activity of the PH complexes (containing 100 or 1,000 HAU of HVJ-E) was reduced by washing with buffer (washing once or twice). (b) Tumor tissue sections were stained with FITC-albumin (green) and anti-F protein to visualize the tumor blood vessels and HVJ-E, respectively, after the administration of PH complexes (PH), HVJ-E, platelets (Plt), or NaCl solution (NaCl).
Figure 3
Figure 3
The administration of PH complexes (PH), HVJ-E, platelets (Plt), or NaCl solution (NaCl) to B16F10 melanoma-bearing mice and the accumulation of delivered HVJ-E particles in tumor tissues. (a) Tumor sections were stained with anti-CD62P (red) and anti-F protein (green) to visualize the activated platelets and HVJ-E localization, respectively. (b) Tumor sections were stained with anti-CD62P (red) and anti-fibrin (green) to visualize the activated platelets and fibrin localization, respectively.
Figure 4
Figure 4
Suppression of tumor growth by PH complex treatment in B16F10 melanoma-bearing mice and the accumulation of immune cells in the tumor. (a) Tumor growth curve of B16F10 melanoma-bearing mice treated with PH complexes (PH), HVJ-E, platelets (Plt), or NaCl solution (NaCl). The data are shown as the mean ± SEM of four animals per group. The date when the number of mouse in the group became less than 2. (b,c) B16F10 tumor tissue sections from tumor-bearing mice treated with PH complexes (PH), HVJ-E, platelets (Plt) or NaCl solution were stained with anti-CD4 (red) and anti-CD8 (red) antibodies to visualize the localization of CD4+ and CD8+ T cells, respectively, in the tumor tissues.
Figure 5
Figure 5
Proliferation assay of B16F10 melanoma cells and isolated TECs from mouse F10 melanoma tumors. Different concentrations of HVJ-E (from 4 or 100 to 2,000 HAU) were infused into the same number of platelets (5.0 × 106 platelets) to construct various PH complexes. The PH complexes and different concentrations of HVJ-Es were added to (a) B16F10 or (b) TECs. The survival rates of the cells were measured. The data are shown as the mean ± SEM of four independent analyses.
Figure 6
Figure 6
HVJ-E-induced RANTES production contributed to tumor suppression. (a,b) Chemokine and cytokine arrays of PH-treated TECs isolated from (a) tumors and (b) B16F10 cells. (c) Tumor growth curves of B16F10 melanoma-bearing mice treated with PH complexes (PH), PH complexes + RANTES-neutralizing antibody (PH + RANTES Ab), PH complexes + control IgG (PH + Ctrl Ab) or NaCl solution (NaCl). The date when the number of mouse in the group became less than 2. The data are shown as the mean ± SEM of four animals per group. (d) RT-PCR of RANTES mRNA level in B16F10 tumor tissues. The F10 tissue samples were collected 48 hours after three injections. The data are shown as the mean ± SEM of four independent analyses. ‡‡P < 0.01 (versus PH + RANTES-neut ab); **P < 0.01 (versus NaCl).
Figure 7
Figure 7
Induction of the anti-tumor immune response by T cells and NK cells in PH-treated tumor-bearing mice. (a) ELISPOT assay. Induction of IFN-γ from splenocytes isolated from PH-treated F10 tumor-bearing mice after 48 hours of incubation with or without B16F10 cells. The data are shown as the mean ± SEM of six independent analyses. **P < 0.01. (bd) Tumor sizes of (b) CD4+ T cell-, (c) CD8+ T cell-, or (d) NK cell-depleted B16F10-bearing mice. The data are shown as the mean ± SEM of four animals per group. *P < 0.05 (versus PH), **P < 0.01 (versus PH).
Figure 8
Figure 8
Model depicting the mechanism by which the PH complex targets and induces chemokine production in F10 tumor tissues. The PH complex is inactive in normal blood vessels; however, when it interacts with fibrin clots in tumor vessels, the complex begins to release the HVJ-E particles (1), which induces TECs or cancer cells to secrete chemokines, mainly RANTES (2). The cytokines recruits immune cells to the tumor tissue (3), which enhance anti-tumor immunity.

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