Neospora Caninum Inhibits Tumor Development by Activating the Immune Response and Destroying Tumor Cells in a B16F10 Melanoma Model

Abstract Background Melanoma is a malignant tumor with a high mortality rate. Some microorganisms have been shown to activate the immune system and limit cancer progression. The objective of this study is to evaluate the anti-melanoma effect of Neospora caninum, a livestock pathogen with no pathogenic activity in humans. Methods Neospora caninum tachyzoites were inoculated into a C57BL/6 mouse melanoma model by intratumoral and distal subcutaneous injections. Tumor volumes were measured, and cell death areas were visualized by hematoxylin and eosin staining and quantified. Apoptosis in cell cultures and whole tumors was detected by propidium iodide (PI) and TUNEL staining, respectively. Cytokine and tumor-associated factor levels in tumors and spleens were detected by real-time quantitative polymerase chain reaction. Infiltration of macrophages and CD8+ T cells in the tumor microenvironment (TME) were detected by immunohistochemistry with anti-CD68 and anti-CD8 antibodies, respectively. Finally, 16S rRNA sequencing of mice cecal contents was performed to evaluate the effect of N. caninum on gut microbial diversity. Results Intratumoral and distal subcutaneous injections of N. caninum resulted in significant inhibition of tumor growth (P < 0.001), and more than 50% of tumor cells were dead without signs of apoptosis. Neospora caninum treatment significantly increased the mRNA expression levels of IL-12, IFN-γ, IL-2, IL-10, TNF-α, and PD-L1 in the TME, and IL-12 and IFN-γ in the spleen of tumor-bearing mice (P < 0.05). An increase in the infiltration of CD8+ T cells and macrophages in the TME was observed with these cytokine changes. Neospora caninum also restored the abundance of gut microbiota Lactobacillus, Lachnospiraceae, Adlercreutzia, and Prevotellaceae associated with tumor growth, but the changes were not significant. Conclusion Neospora caninum inhibits B16F10 melanoma by activating potent immune responses and directly destroying the cancer cells. The stable, non-toxic, and efficacious properties of N. caninum demonstrate the potential for its use as a cancer treatment. Graphical Abstract