Immunotherapy combining tumor and endothelium cell lysis with immune enforcement by recombinant MIP-3α Newcastle disease virus in a vessel-targeting liposome enhances antitumor immunity

Abstract

Background: Several agents for oncolytic immunotherapy have been approved for clinical use, but monotherapy is modest for most oncolytic agents. The combination of several therapeutic strategies through recombinant and nanotechnology to engineer multifunctional oncolytic viruses for oncolytic immunotherapy is a promising strategy.

Methods: An endothelium-targeting iRGD-liposome encapsulating a recombinant Newcastle disease virus (NDV), which expresses the dendritic cell (DC) chemokine MIP-3α (iNDV3α-LP), and three control liposomes were constructed. MIP-3α, HMGB1, IgG, and ATP were detected by western blotting or ELISA. The chemotaxis of DCs was examined by Transwell chambers. The phenotypes of the immune cells were analyzed by flow cytometry. The antitumor efficiency was investigated in B16 and 4T1 tumor-bearing mice. Immunofluorescence and immunohistochemistry were used to observe the localization of liposomes, molecular expression and angiogenesis. Synergistic index was calculated using the data of tumor volume, tumor angiogenesis and tumor-infiltrating lymphocytes.

Results: Compared with NDV-LP, treatment with iNDV3α-LP and NDV3α-LP induced stronger virus replication and cell lysis in B16 and 4T1 tumor cells and human umbilical vein endothelial cells (HUVECs) with the best response observed following iNDV3α-LP treatment. B16 and 4T1 cells treated with iNDV3α-LP produced more damage-associated molecular pattern molecules, including secreted HMGB1, ATP, and calreticulin. Moreover, iNDV3α-LP specifically bound to αvβ3-expressing 4T1 cells and HUVECs and to tumor neovasculature. Tumor growth was significantly suppressed, and survival was longer in iNDV3α-LP-treated B16-bearing and 4T1-bearing mice. A mechanism study showed that iNDV3α-LP treatment initiated the strongest tumor-specific cellular and humoral immune response. Moreover, iNDV3α-LP treatment could significantly suppress tumor angiogenesis and reverse the tumor immune suppressive microenvironment in both B16-bearing and 4T1-bearing mice.

Conclusions: In this study, iNDV3α-LP had several functions, such as tumor and vessel lysis, MIP-3α immunotherapy, and binding to αvβ3-expressing tumor and its neovasculature. iNDV3α-LP treatment significantly suppressed tumor angiogenesis and reversed the tumor immunosuppressive microenvironment. These findings offer a strong rationale for further clinical investigation into a combination strategy for oncolytic immunotherapy, such as the formulation iNDV3α-LP in this study.

Keywords: combined modality therapy; immunogenicity, vaccine; immunotherapy; oncolytic virotherapy; tumor microenvironment.

Figure 1

Construction and characteristic evaluation of the liposome formulation NDV-MIP3α. (A) Schematic presentation of the construction of iNDV3α-LP and three control liposomes NDV3α-LP, NDV-LP and Lp. (B) TEM images of liposomes LP, NDV-LP, NDV3α-LP, and iNDV3α-LP. (C) Size distribution of LP, NDV-LP, NDV3α-LP, and iNDV3α-LP. (D) Basic characteristics of LP, NDV-LP, NDV3α-LP, and iNDV3α-LP. LP, empty liposome; MIP-3α, macrophage inflammatory protein-3α; NDV; Newcastle disease virus; NDV-LP, liposome encapsulated with wild-type LP NDV; NDV3α-LP, liposome encapsulated with the recombinant MIP-3α NDV; iNDV3α-LP, iRGD-directed liposome encapsulated with the recombinant MIP-3α NDV.

Figure 2

Evaluation on replication and tumor-lysis capabilities of the NDVs and MIP-3α activity in the liposomes. (A and C) vVrus replication of 1 MOI NDV or NDV3α at the indicated time points in B16 (A) and 4T1 (B) cells. (B and D) Lysis of B16 (B) or 4T1 (D) cells by indicated formulations at the indicated MOI and time points. (E) Expression of MIP-3α in B16 and 4T1 cells treated with indicated formulations. (F) In vitro chemotaxis of DCs induced by the supernatants from B16 cells infected with 1 MOI NDV or NDV3α in the indicated formulations. (G) In vivo chemotaxis for DCs in mouse abdomen injected with supernatants from B16 or 4 T1 cells treated with the indicated formulations. Data are plotted as mean±SD; two-way ANOVA with Tukey multiple comparisons: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; DCs dendritic cells; MOI, multiplicity of infection; NDV3α, recombinant NDV encoding MIP-3α.

Figure 3

iNDV3α-LP treatment induces the immunogenic tumor cell death. (A) HMGB1 in B16 and 4T1 cell supernatants (sHMGB1) and whole-cells (cHMGB1) treated with the indicated formulations by western blot. (B) The titers of sHMGB1 in B16 and 4T1 cell supernatants by ELISA. (C) ATP released in B16 or 4T1 cell supernatants by ELISA. (D) CRT translocation to B16 or 4T1 cell surfaces by two counterstains with an FITG-conjugate anti-CRT antibody (green) and DiI (red). (E) FCM analysis of the CRT intensity on the B16 and 4T1 cell surface, showing the representative images and MFI fold change. CRT, calreticulin; FCM, flow cytometry; HMGB1, high mobility group box 1; iNDV3α-LP, iRGD-liposome loaded with NDV expressing MIP-3α; sHMGB1, secreted HMGB1.

Figure 4

iNDV3α-LP targets to αvβ3-expressing tumor cells and HUVECs and to tumor vasculature. (A) Immunoblotting detection of integrin αvβ3 expression in B16, 4T1, and HUVECs. (B–D) confocal fluorescent images showing that FITC-conjugated iNDV3α-LP (green, see also online supplemental figure S1F) binds to 4T1 (C) and HUVEC (D) cell membranes (DiI strained, red) but not to B16 (B); scale bar=5 µm. (E) FCM analysis of the green fluorescence intensity on the cell surface, showing the representative images and MFI fold change of the B16 cells, 4T1 cells, and HUVECs treated with the indicated formulations. (F) Uptake analysis of 4T1 cells by flow cytometry. (F) Confocal fluorescent images of B16 and 4T1 tumor sections, showing that only DiI-labeled iNDV3α-LP (red) specifically binds to FITC-dextran-marked blood vessels (green), which are merged as yellow (arrow). HUVECs, human umbilical vein endothelial cells; iNDV3α-LP, iRGD-liposome loaded with NDV expressing MIP-3α.

Figure 5

iNDV3α-LP treatment induces specific antitumor effects. (A) Representative images of 4T1 tumor masses treated with the indicated formulations on day 28 after tumor cell injection. (B and C) Data of the tumor volumes and survival rates of the tumor-bearing B16 and 4T1 mice treated with the indicated formulations at the indicated time points. (D) The synergistic indexes (SIs) calculated with the mean tumor volumes at the indicated time points in (B) and (C). ORR=mean tumor volume in the NDV-LP, NDV3α-LP, or iNDV3α-LP group/the mean tumor volume in the LP group; ERR=ORR of the NDV-LP group × ORR of NDV3α-LP; SI=ERR/ORR (ORR <1) or ORR/ERR (ORR >1), where SI>1 indicates a synergistic effect. ERR, expected relative ratio; iNDV3α-LP, iRGD-liposome loaded with NDV expressing MIP-3α; NDV3α-LP, liposome loaded with NDV expressing MIP-3α; NDV-LP, liposome loaded with single NDV; ORR, observed relative ratio.

Figure 6

iNDV3α-LP treatment enhances antitumor immunity. (A and B) CTL response using B16 (A) and 4T1 (B) as target cells and the splenocytes from the mice treated with the indicated formulations as effector cells. (C and D) The in vivo CTL response was examined by injecting CFSE-labeled B16 (D) or 4T1 cells into the mouse abdomen treated with the indicated formulations to analyze the proliferation of CD45⁻ tumor cells by FCM and calculate the percentage of cell lysis (C, see also online supplemental figure S1). (E–G) The percentage of CD8⁺ splenocytes secreting IFN-γ (E), TNF-α (F), and IL-2 (G) from B16- or 4T1-bearing mice treated with the indicated formulations. (H) The synergistic indexes (SIs) calculated with the mean percentage of CD8⁺ cell subtypes in (E–G). (I) Western blot detection of the tumor-specific IgG in the serum from mice treated with the indicated formulations. (J and K) ELISPOT detection of the splenocytes secreting IgG specific to B16 or 4T1 cells (J) and the average number of IgG-secreting cells in 10⁵ splenocytes (K). (L and M) The IgG titer in the serum from B16-bearing or 4T1-bearing mice treated with the indicated formulations (L) and the IgG subtypes (M). ELISPOT, enzyme-linked immunospot; iNDV3α-LP, iRGD-liposome loaded with NDV expressing MIP-3α.

Figure 7

iNDV3α-LP treatment inhibits tumor angiogenesis and reverses tumor immunosuppressive microenvironment. (A and B) Sections of frozen B16 tumor tissues from mice treated with the indicated formulations were stained with anti-CD31 antibody to show the tumor microvessels (A) and determine the vessel density by counting the number of microvessels per high-power field (B). (C) FITC-dextran uptake in alginate beads encapsulated with 2×10⁵ B16 or 4T1 cells treated with the indicated formulations. (D–H) CD45⁺ immune cells were gated from the total cell population from the tumor tissues treated with the indicated formulations. Representative flow cytometry images (D, also see online supplemental figure S3), and the mean percentage of TILs with the following features in the B16-bearing or 4T1-bearing mice treated with the indicated formulations: IFN-γ-secreting CD4⁺ lymphocytes (E), CD11b and GR-1 double positive MDSCs (F), Poxp3 and CD25 double positive Tregs (G), and F4/80 and CD206 double positive M2-type tumor-associated macrophages (H). (I) The synergistic indexes (SIs) were calculated with the mean percentage of TIL cell subtypes in (E–H). iNDV3α-LP, iRGD-liposome loaded with NDV expressing MIP-3α; MDSCs, myeloid-derived suppressor cells; TILs, tumor-infiltrating lymphocytes; Tregs:, regulatory T cells.