Alfalfa mosaic virus nanoparticles-based in situ vaccination induces antitumor immune responses in breast cancer model

Abstract

Background: Preclinical and clinical studies show that local and systemic antitumor efficacy is achievable by in situ vaccination (ISV) using plant virus nanoparticles in which immunostimulatory reagents are directly administered into the tumor rather than systemically. Aim: To investigate a minimally studied plant virus nanoparticle, alfalfa mosaic virus (AMV), for ISV treatment of 4T1, the very aggressive and metastatic murine triple-negative breast cancer model. Materials & methods: AMV nanoparticles were propagated and characterized. Their treatment impact on in vivo tumors were analyzed using determination of inherent immunogenicity, cytokine analysis, western blotting analysis and immunohistochemistry methodologies. Results: AMV used as an ISV significantly slowed down tumor progression and prolonged survival through immune mechanisms (p < 0.001). Conclusion: Mechanistic studies show that ISV with AMV increases costimulatory molecules, inflammatory cytokines and immune effector cell infiltration and downregulates immune-suppressive molecules.

Keywords: AMV; ISV; PVNP; TME.

Figure 1.. Alfalfa mosaic virus preparation has expected characteristics.

(A) UV/Vis spectroscopy (B) Dynamic light scattering was used to measure the zeta potential of alfalfa mosaic virus (AMV). (C) Transmission electron microscopy confirms the structural stability of AMV particles after extraction and that particles form of four different AMV shapes (form 1 [59 nm], 2 [48 nm], 3 [43 nm] and 4 [30 nm]).

Figure 2.. Alfalfa mosaic virus is recognized as immunogenic by human peripheral blood mononuclear cells, and its intratumoral injection reduces growth of 4T1 tumors.

(A) Human peripheral blood mononuclear cells exposed to AMV produce elevated levels of IL-6 pro-inflammatory cytokine in vitro. (B) Cell viability of 4T1 cancer cell line exposed to AMV. (C) The schedule for treatment of tumors with AMV in situ vaccine injections. (D) Tumor growth was monitored by caliper measurement. Therapeutic efficacy of AMV in a breast tumor model. Balb/C mice inoculated with 1 × 10⁶ 4T1 cells was followed by 100 μg of AMV, once tumors were approximately 100 >mm³, 10 days postinoculation. Tumor growth was traced by measuring volume (n = 4). (E) Tumor weight after sacrificing mice and tumor harvest on 20 day. Statistical significance was calculated by t-test (p < 0.05). AMV: Alfalfa mosaic virus.

Figure 3.. Alfalfa mosaic virus in situ vaccination induces dramatic increases in relevant cytokines and the protein expression alteration of apoptosis in 4T1 breast tumors (p < 0.05).

(A) IFN-α, (B) IFN-gamma, (C) IL-6, (D) IL-12. (E) Bax, Bcl2 and caspase-8 quantified expression level in the control and treated tumors with 100 μg of AMV. (F) Bax/Bcl2 protein expression ratio as an apoptotic index. AMV: Alfalfa mosaic virus.

Figure 4.. Immunohistochemistry reveals increases in relevant leukocyte populations associated with treatment, and H&E stained lung tissue indicating the prevention of lung metastasis.

(A–F) Tumor sections stained for PDL-1, CD4, CD8, CD11b, Ly6G and CD45 (magnification 40). (G) Hemotoxylin and eosin stained lung tissue post in situ vaccination with phosphate-buffered saline (control) and AMV indicating the greater efficacy of AMV treatment for the prevention of lung metastasis. (H) Metastasis area in the lung tissue were evaluated. Results are expressed as p < 0.05 (n = 4 animals per group). Quantitative analysis was performed using Fiji software to determine relative optical densities of the stained sections (p < 0.05). AMV: Alfalfa mosaic virus.