Neospora caninum inhibits Lewis cancer and B16F10 melanoma lung metastasis development by activating the immune response in murine models

doi:10.21203/rs.3.rs-5445983/v1

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Neospora caninum inhibits Lewis cancer and B16F10 melanoma lung metastasis development by activating the immune response in murine models

https://doi.org/10.21203/rs.3.rs-5445983/v1

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published 07 Feb, 2025

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Malignant tumors are prevalent with high mortality rates in humans, dogs, and cats. Some microorganisms have been shown to inhibit cancer progression. The objective of this study is to evaluate the inhibitory effects of Neospora caninum, a livestock parasite, on three different tumor models in C57BL/6 mice, including Lewis subcutaneous tumors, Lewis and B16F10 melanoma lung metastasis. The results showed that a sufficient amount of N. caninum tachyzoites can significantly inhibit the development of subcutaneous tumors and lung metastasis (P < 0.001), and induce more than 50% tumor cell death in Lewis subcutaneous tumors. N. caninum treatment can significantly increases the infiltration of macrophages, NK cells, and CD8⁺ T cells (P < 0.0001) in Lewis subcutaneous tumors detected by immunohistochemistry, and the percentage of these immunocytes in the spleen (P < 0.05) of mice bearing B16F10 melanoma metastasis detected by flow cytometry. And with these changes, the mRNA expression levels of IL-12, IFN-γ, IL-2, IL-10, TNF-α and PD-L1 in tumor microenvironment and IL-12, IFN-γ, IL-2 in spleen were also significantly increased (P < 0.05). Altogether, our results indicate that a sufficient amount N. caninum tachyzoites not only inhibits the growth of Lewis subcutaneous tumors, but inhibits the development of Lewis and B16F10 melanomas lung metastatic in mice by activating potent immune responses. N. caninum and its anti-tumor properties may be an effective anti-tumor tool.

Neospora caninum

subcutaneous tumors

metastasis

cytokines

immune cells

Malignant tumor with a high degree of local invasiveness and high metastatic propensity has been identified as the primary contributor to mortality among individuals diagnosed with cancer [1]. Lung cancer is the second most prevalent form of cancer with high rates of occurrence and mortality in humans [2], and in dogs, lung cancer is a rare malignant tumor and exhibits poor prognosis [3]. Melanoma has a death rate of 65%, making it the worst type of skin cancer in humans [4], and it is the most common oral malignancy with a high degree of local invasiveness in the dog [5]. These extremely aggressive malignant tumors are challenging to treat with conventional therapy.

In recent years, with the deepening research on immunotherapy, it has been found that immunotherapy has become a powerful clinical strategy for treating cancer. Immunotherapy, aiming to boost natural defenses to eliminate malignant cells, can break immunosuppression, correct anti-tumor immune imbalance, and restore anti-tumor immune effect, thus delaying tumor recurrence and metastasis. Immunotherapy has been shown promising application prospects in the clinical treatment of specific malignant tumors and a monumental breakthrough for cancer treatment [6]. A growing number of living microorganisms have shown powerful anti-tumor effects, such as NDV [7], HSV-1 [8], Bifidobacterium bifidum [9], Listeria [10], Clostridium [11], and so on. Certain parasites have also exhibited potential anti-tumor effects. Plasmodium [12] and Trichinella spiralis [13] inhibit tumor growth by modifying the percentage of immune cells in the tumor microenvironment. Toxoplasma gondii could induce practical anti-tumor effects, and inhibit B16F10 melanomas and Lewis lung carcinoma growth in mice [14, 15].

N. caninum is a specialized intracellular protozoan, closely related to T. gondii. N. caninum primarily causes abortion or reproductive disorders in cattle, and neuromuscular disease in dogs [16]. Although antibodies against N. caninum have been detected in human serum, it has not been demonstrated that neosporosis is a zoonosis [17, 18]. Lantier et al. demonstrated for the first time that N. caninum can inhibit the development of murine subcutaneous thymoma EG7, and even causes tumor complete eradication [19]. And then, we found that N. caninum can inhibit the growth of subcutaneous B16F10 melanoma by significantly increasing the infiltration of CD8⁺ T cells and macrophages, and the mRNA expression levels of IL-12, IFN-γ, IL-2, IL-10, TNF-α in the TME [20]. In 2023, Battistoni et al. demonstrated N. caninum, engineered to secrete human IL-15, can inhibit the progression of melanoma lung metastasis [21]. In this study, different tumor models, including Lewis subcutaneous tumors, Lewis and B16F10 melanoma lung metastases in C57BL/6 mice were constructed to investigate the anti-tumor activity and immunological properties of N. caninum, providing the feasibility for future research on N. caninum as a potential agent for cancer immunotherapy.

2.1. Mice

Female C57BL/6 mice aged 6–8 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animals were maintained on a standard commercial diet and water throughout the experiments with a 12-hour/12-hour light–dark cycle. All animal experiments were approved by the Experimental Animal Commission of Henan University of Science and Technology (Permit No. SCXK [JING] 2021–0006). The experimental scheme conformed strictly to the guidelines of the Institutional Animal Care and Use Committee (No. 201) of Henan University of Science and Technology (Luoyang, Henan, China).

2.2. Tumor Cell

Murine Lewis lung carcinoma (LLC) cells and melanoma B16F10 cells were purchased from Procell Life Science & Technology (Wuhan, China). They were cultured in Dulbecco’s modified Eagle medium (DMEM; Servicebio, Wuhan, China) containing 10% fetal bovine serum (FBS; Clark Bio, Shanghai, China) in a humidified incubator at 37°C in 5% CO₂.

2.3. Neospora caninum

Neospora caninum (NC1 strain) tachyzoites were a gift from Prof. Qun Liu of China Agricultural University. As described previously [20], N. caninum were cultured in Vero cells in DMEM medium (Servicebio, Wuhan, China) containing 10% FBS (Clark Bio, Shanghai, China) in an incubator at 37°C with 5% CO₂. And the tachyzoites were purified by centrifugation at 750 g for 10 min, and washed twice in phosphate-buffered saline (PBS, pH = 7.2–7.5).

2.4. Murine models for subcutaneous tumors and lung metastases

For subcutaneous tumor models, 2×10⁵ LLC cells were subcutaneously inoculated into the left axilla of C57BL/6 mice. The mice's bodily condition and the size of the tumor were assessed on a regular basis. When the subcutaneous tumors reached 3–5 mm in diameter, N. caninum treatment was started in tumor-bearing mice. The termination of the experiment occurred when the tumors in mice of the control group reached a diameter of 1.5 cm. For lung metastasis, 2×10⁵ LLC cells or B16F10 cells were injected in mice by tail vein. Mice were euthanized and subjected to dissection on days 7, 8, 10, 12, 16, 18 and 20 following injections to examine the development of tumors in the lungs and other organs. When the metastatic melanomas were observed on the lungs, and Lewis metastatic foci or hemorrhage were observed on the lungs, N. caninum treatment was started in tumor-bearing mice. The experiment was terminated when an area of melanoma on the lung surface in mice of the control group appears more than 40% of lung.

2.5. Optimal dosing of N. caninum tachyzoites and sample collection

In order to explore the potential anti-tumor effects of different dose of N. caninum, 2×10⁵, 1×10⁶, 2×10⁶ N. caninum tachyzoites were used to treat Lewis subcutaneous tumor-bearing mice by intratumoral injection and subcutaneous injection respectively. And the daily monitoring of tumor progression and mouse condition was conducted. At 21 days post-tumor cells challenge, the tumors in mice of the control group reached a diameter of 1.5 cm, and all the mice were euthanized via carbon dioxide asphyxiation. The tumor volume was calculated using the formula: V = (ab²)/2, where 'a' represents the long axis and 'b' is the short axis of the tumor. The optimal treatment dose was determined based on the tumor volume and clinical manifestations of tumor-bearing mice.

Tumors and tissues, including spleen, heart, liver, lungs, kidneys, and brain, in mice treated with PBS or 2×10⁶ N. caninum tachyzoites, were collected and preserved in 4% paraformaldehyde or stored at -80°C. And mouse tissues from all N. caninum treatment group were tested for the presence of N. caninum DNA based on the Nc5 gene [22]: F: 5′-CTC GCA GTC AAC CTA CGT CTTCT-3′; R: 5′-CCC AGT GCG TCC AAT CCT GTAAC-3′.

2.6. Experimental design of lung metastases and sample collection

For metastatic, mice received a tail vein injection of 2×10⁵ LLC cells or B16F10 cells suspended in PBS were randomly divided into two groups, respectively. Twelve and 8 days after inoculation of LLC and B16F10 cells respectively, 2×10⁶ N. caninum tachyzoites suspended in PBS or PBS (tumor control group) were injected subcutaneously in the neck of tumor-bearing mice. Lewis metastatic tumor-bearing mice were euthanized on day 21, and melanoma metastatic tumor-bearing mice were euthanized on day 18. Macroscopic scoring of the lung lobes from lung metastatic tumor animals was further quantified with Fiji software using an ImageJ [23]. Lung tissues and other mouse tissues were also collected and preserved in 4% paraformaldehyde or stored at -80°C for further analysis.

2.7. Histological analysis

Tumors of subcutaneous tumor-bearing mice, and lungs of Lewis and B16F10 lung metastasis mice were fixed in 4% paraformaldehyde, and embedded in paraffin and sectioned into 3 µm sections. Comprehensive pathological assessment on the samples was evaluated by HE staining with a light microscope. Infiltrating CD8⁺ T cells, macrophages and NK cells in tumor or lung were detected with anti-CD8 antibody (Servicebio, Wuhan, China), anti-CD68 antibody (Servicebio, Wuhan, China), and anti-NK antibody (Servicebio, Wuhan, China), and CD31 (Vascular endothelial cell markers associated with tumor angiogenesis)were detected with anti-CD31 antibody (Servicebio, Wuhan, China) according to the manufacturer’s instructions. The tumor sections were imaged with a digital scanner and tumor area, number of positive cell count, and total cell count were quantified by the Servicebio image analysis system.

2.8. Cytokine detection

The mRNA expression levels of IL-2, IL-10, IL-12, IL-15, TNF-α, IFN-γ, PD-L1, VEGF-A, and EGFR in tumor tissues or lungs, and spleens of mice was detected by RT-PCR as follows: total RNA was isolated from homogenized tissue with the FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme; Cat RC112) and reverse-transcribed with the EasyScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (Transgen; Cat AE341), following the manufacturer's instructions (Primer sequences are shown in Supplemental Table S1). Real-time quantitative PCR (RT-PCR) for these cytokines were performed using the PerfectStart™ Green qPCR SuperMix (Transgen; Cat AQ601) in a 7500 Fast Real-Time qPCR System (Bio-Rad, Hercules, CA, USA). Each sample was performed in triplicate. Relative gene expression was calculated with the 2^–△△Ct method, with β-actin used as the normalization control.

2.9. Cellular analysis

On days 18 after tumor inoculation, B16F10 melanoma metastatic mice were dissected, and spleen tissue was passed through a wire mesh to prepare single-cell suspensions, which were first lysed with a 10-fold dilution of RBC lysis (BOSTER; Clone AR1118) according to the manufacturer's instructions, and incubated for 5 min on ice. For each sample, 10⁶ spleen cells underwent Fc receptor blockade with TruStainFcX™ PLUS (anti-mouse CD16/32) antibody (BioLegend; Clone S17011E) on ice for 5–10 min. The samples were then stained with antibodies: CD45-PE (BioLegend; Clone 3.0-F11), CD3-cy7 (BioLegend; Clone 17A2), CD8-FITC (BioLegend; Clone 53 − 6.7), NK1.1-FITC (BioLegend; Clone S17016D), CD11c-FITC (BioLegend; Clone N418), and incubated for 20min on ice in the dark. All samples were analyzed on BD FACSVerse™ Flow Cytometer (BD Biosciences, Beijing, China) using the BD FACSuite software (BD Biosciences, Beijing, China). Flow cytometry staining scheme is as follows: CD8⁺ T cells (CD45⁺CD3⁺CD8⁺), NK cells (CD3⁺ NK1.1⁺), DC cells (CD3⁺CD11c⁺). The proportion of these immune cells in the spleens of mice were determined, and compared between the treated and untreated groups.

2.10. Statistical analysis

Data were analyzed using GraphPad Prism 8.0 software [20]. The two-tailed Student t-test and the Mann–Whitney tests were used to evaluate parametric and non-parametric data respectively. Statistical significance was set at P < 0.05.

3.1. Three tumor-bearing murine models were successfully constructed

For Lewis subcutaneous tumors, tumors with 3–5 mm in diameter were observed at the site of inoculation in mice on day 9 after LLC challenge, and reached to 1.5 cm in diameter on day 21. For Lewis lung metastases, hemorrhagic spots were observed on the surface of the lungs on day 12 after LLC challenge, and HE staining confirmed the existence of lung metastases (Fig. 1a, b). For melanoma lung metastases, discrete black metastases were observed on the lung surface of mice on day 8 after B16F10 cells challenge (Fig. 1c). Hence, N. caninum treatment was initiated on day 12 and day 8 following the intravenous injection of LLC cells and B16F10 cells respectively in mice.

3.2. A sufficient amount of N. caninum tachyzoites can induce a stronger anti-tumor activity

On day 21 post tumor cells challenge, the tumor volume of mice in the control group reached to 1.5 cm in diameter. All mice in each group were euthanized (Fig. 2a). Measurement and analysis of tumor tissues showed a range of concentration dependence between the therapeutic effect and the number of N. caninum tachyzoites, with a better therapeutic effect at a dose of 2×10⁶ tachyzoites for both intratumoral and subcutaneous injections of N. caninum (Fig. 2b, c). The tumor volume in mice treated with N. caninum at the dose of 2×10⁶ tachyzoites was significantly lower than that of 2×10⁵ (P < 0.01) and 1×10⁶ (P < 0.05) (Fig. 2b, c).

3.3. N. caninum regresses Lewis subcutaneous tumors development

On day 21 after LLC challenge, the tumor volume in mice treated with PBS was significantly higher than that in mice treated with 2×10⁶ N. caninum tachyzoites by subcutaneous injection (P < 0.0001) and intratumoral injection (P < 0.0001) (Fig. 2b, c). HE staining of tumor tissue exhibited that cell death areas reached 75.3% and 50.9% in mice treated by intratumoral and subcutaneous injections of N. caninum respectively, significantly higher than 8.3% in the control group (P < 0.0001 and P < 0.0001) (Fig. 3a, b). N. caninum treatment leads to a significant decrease in the expression of platelet endothelial cell adhesion molecule-1 (CD31) in tumor tissues by immunohistochemistry (P < 0.05) (Fig. 3c, d). Both intratumoral and subcutaneous injections of N. caninum in tumor-bearing mice could significantly increase the number of CD8⁺ T cells, macrophages and NK cells in tumor tissue (P < 0.0001) (Fig. 4a-c). In addition, N. caninum could also significantly increase the expression levels of IL-2, IL-12, TNF-α and IFN-γ in tumor tissues, and IL-2, IL-12 and IFN-γ in spleen of Lewis subcutaneous tumor-bearing mice (P < 0.05), (Fig. 4d, e).

3.4. N. caninum regresses Lewis lung metastasis development

N. caninum treatment was initiated on day 12 after injection of LLC cells by tail vein injection in mice (Fig. 5a). On day 21, the tumor nodules were not observed, but lung hemorrhage was more pronounced in mice of the control group, compared to the mice in N. caninum treatment group (Fig. 5b). HE staining demonstrated that N. caninum treatment resulted in a decrease of tumor nodules in the lung tissue of Lewis lung metastasis-bearing mice (Fig. 5c). Moreover, N. caninum could significantly elevated the expression levels of IL-2, IL-12, IL-10, IFN-γ, TNF-α in lung tissues (P < 0.05), and IL-2, IL-12, IFN-γ in spleen (P < 0.05) (Fig. 5d, e).

3.5. N. caninum regresses B16F10 lung metastasis development

N. caninum treatment was initiated on day 8 after challenge with B16F10 cells by tail vein injection in mice(Fig. 6a). On day 18, the area of melanoma on the lung surface in mice of the control group reached to 41.3%, significantly higher than that (9.1%) in mice treated with N. caninum (P < 0.0001) (Fig. 6b), which is consistent with the results in lung tissue sections stained with hematoxylin and eosin (H&E) (Fig. 6c). Compared with the mice in the control group, the mRNA expression levels of IL-2, IL-12, IL-10, IFN-γ, IL-15 and TNF-α were significantly elevated in lung tissues of tumor-bearing mice treated with N. caninum (P < 0.05) (Fig. 6d). And IL-2, IFN-γ, IL-12 and IL-10 were also significantly elevated (P < 0.05) in the spleen of mice in N. caninum treatment group (Fig. 6e). Furthermore, the results of immunohistochemical analysis revealed that a large infiltration of CD8⁺ T cells, macrophages and NK cells in lung metastases were observed in N. caninum treatment groups (Fig. 6f). Flow cytometry analysis showed that the percentages of CD8⁺ T cells, NK cells, and DC cells in the spleens of tumor-bearing mice were significantly elevated following N. caninum treatment, compared to that in mice of the control group (P < 0.01, P < 0.05 or P < 0.001) (Fig. 7).

3.6. N. caninum significantly enhanced PD-L1 expression

We examined the mRNA expression of programmed death-ligand 1 (PD-L1), VEGF-A (from here referred to as VEGF), and EGFR in subcutaneous tumor tissues and metastatic lung tissues. The results indicate that N. caninum treatment resulted in a significantly increase of PD-L1 expression in both subcutaneous and lung metastases, compared to the control group (P < 0.01 or P < 0.001). The mRNA expression levels of VEGF and EGFR showed a different profile in the three tumor models, with significant reductions in Lewis lung metastases after N. caninum treatment (P < 0.01 or P < 0.05), but with no significant differences in Lewis subcutaneous tumors and B16F10 lung metastases (Fig. 8).

Microbial-mediated immunotherapy is a promising innovation in tumor therapy with broad application prospects. Its advantage lies in the ability of microorganisms to cope with tumor diversity as well as immune evasion mechanisms, activate the immune system, and enhance the anti-tumor immune activity. N. caninum is a specialized intracellular protozoan, of which there is no evidence of the zoonotic potential [8, 24]. Currently, there are only three reports on the successful inhibition of thymoma and melanoma by N. caninum [19–21]. In this study, we found that N. caninum can inhibit the development of tumors by activating the Th1-type immune response in three different murine tumor models, including Lewis subcutaneous tumors, Lewis and B16F10 melanoma lung metastasis.

Animal models with characteristics appropriate for studying tumors are essential in tumor research. The construction of a Lewis subcutaneous tumor model was successfully achieved in this investigation, with a previously established procedure [25]. The metastatic tumor models were constructed by tail vein injection method to simulate the naturally occurring metastasis of the tumor [26, 27]. Moreover, it has been shown that metastatic tumors formed by tail vein injection in mice are similar to metastatic tumors metastasized from in situ tumors to the lungs in terms of gene expression [28, 29]. In the present study, the occurrence of metastatic foci in the lung was observed on days 7 and 12 following the administration of B16F10 and LLC cells through the tail vein, and no anomalies were observed in any other tissues. On the 21st day after tumor cells injection, it was observed that only one out of the 30 melanoma mouse models exhibited a tumor with approximately 2 mm in diameter in the peritoneal mesentery. This occurrence may be ascribed to individual variations, which aligns the previous findings reported by Stackpole et al. [30]. In which melanoma metastasized first to the lungs, then from the lungs to other organs throughout the body, including the brain, adrenal glands, kidneys, ovaries, pancreas, and mesenteries.

Previous studies indicated that the number of live N. caninum tachyzoites for the treatment of tumor is 2×10⁶ [19]. However, it was not clear whether a low dose of N. caninum would produce a sufficient anti-tumor immune response. Then N. caninum tachyzoites with 10 times and 5 times (2×10⁵ and 1×10⁶) lower than the previous therapeutic doses were used to assess the anti-tumor effects, respectively. And the results showed that the tumor size decreases with the therapeutic number of N. caninum tachyzoites increases, indicating that a sufficient number of N. caninum tachyzoites can stimulate the body to produce a stronger anti-tumor immune response within a safe range.

In our work, Lewis subcutaneous tumor volume in mice treated with N. caninum by intratumoral injection increased on the first few days after treatment, and then an abrupt shrinkage. Contrary to the hard texture of the tumor in mice of the control group and subcutaneous N. caninum treatment group, the touch of the tumor in mice treated with N. caninum by intratumoral injection is between soft and hard. It is speculated that a specific structure or antigen produced by the N. caninum is accountable for the active lysis of tumor cells. In addition, HE staining revealed that there were more cell deaths in tumor of mice treated with N. caninum by intratumoral injection, compared to the mice in the subcutaneous N. caninum treatment groups, which may also corroborate this result. Similar effects were also observed on subcutaneous B16F10 melanoma inhibited by N. caninum in mice [20].

Recent research conclusively demonstrated the significance of the tumor microenvironment (TME) for anti-tumor immune response [31]. Immunotherapy has the potential to reshape the TME effectively, enhancing the recognition and elimination of cancer cells by the host immune system [32]. The results of this study showed that N. caninum treatments increased the infiltration of CD8⁺ T cells, macrophages and NK cells in Lewis subcutaneous tumor, as well as increased the levels of Th1-type cytokines IL-12, IFN-γ, IL-10, TNF-α, and IL-2 in tumor tissues and lungs of lung metastatic mice, which transformed "cold tumor" into "hot tumors", similar to the report by [19]. IL-2 is a strong stimulator of proliferative and cytolytic activity, and combination therapy with IL-12 has been reported to synergistically induce anti-tumor responses [33]. IL-10, this complex cytokine has been shown to inhibit the expression of pro-inflammatory cytokines such as TNF-α and IFN-γ, it has also been investigated to increase NK cytolytic activity [34–36]. Our studies supported that IL-10 has an anti-tumor effect, and therefore the function of IL-10 must be reassessed on a case-by-case basis in the context of the TME. IL-15 promotes the regression of many types of tumors mainly by promoting the proliferation and activation of CD8⁺ T cells and NK cells [21, 37]. The upregulation of IL-15 may be a positive trend for the treatment of tumors in our study.

Several studies have revealed that intracellular protozoa can inhibit tumor growth and metastasis by inhibiting angiogenesis [38]. The vascular endothelial growth factor VEGF plays a critical mediator in angiogenesis, which can directly target tumor cells, promote tumor growth and metastasis, promote endothelial cell proliferation, migration, and invasion [39–41]. In our study, there was no statistically significant difference in mRNA expression of VEGF in Lewis subcutaneous tumors and B16F10 melanoma lung metastasis, but a significant decrease in the mRNA expression levels of VEGF was observed in the Lewis lung metastases after N. caninum treatment. Lantier et al. found that N. caninum could decrease the mRNA expression levels of VEGF in RG7 TME in mice treated by intratumoral injection, but not by subcutaneous injection [19]. However, Li et al. reported that N. caninum could not decrease the mRNA expression levels of VEGF in B16F10 melanoma tumor microenvironment in mice treated both by intratumoral injection and subcutaneous injection [20]. T. gondii, which is morphologically and structurally very similar to N. caninum, has been reported to inhibit the growth of melanoma and Lewis lung carcinoma tumors by inhibiting angiogenesis through induction of hypoxia and ischemic necrosis [42, 43], but not decrease the mRNA expression levels of VEGF in RG7 TME. Thus, we hypothesize that N. caninum possesses antiangiogenic capacity, but the manifestation of this potential may be related to the specific tumor type or manner of colonization.

PD-L1 has been demonstrated in several tumor models as the factor associated with poor prognosis [44], PD-L1 ligand expressed on the surface of tumor cells could bind to the PD-1 receptor expressed on the surface of T cells, leading to inhibition of T cell proliferation, activation, cytokine production, and alter metabolism and cytotoxic T lymphocytes (CTLs) killer functions. Thus, the overexpression of PD-L1 inhibits T-lymphocyte activation in the TME, which leading to immune evasion [45]. However, in this study, N. caninum treatment significantly increased the PD-L1 expression in both subcutaneous and lung metastases, consistent with our previous report [20]. T. gondii could also increase the expression of PD-L1 in tumors significantly, and in combination with PD-L1 blocking therapies, T. gondii showed a synergistic effect in prolonging mouse survival and inhibiting tumor growth in several preclinical mouse models [14]. Hence, N. caninum may also increase the anti-tumor effect of PD-L1 blockers by increasing the expression of PD-L1 in tumors microenvironment.

Although the presence of N. caninum DNA was detected in the lungs and brains of mice with subcutaneous and metastatic tumors, mice did not show symptoms of neosporosis after N. caninum treatments, suggesting that N. caninum may be a safe anti-tumor agent. However, we found that the tumors may continue to grow after the N. caninum treatment was stopped for a period of 15–20 days, and. a higher dosing of N. caninum treatment may lead to neosporosis in mice. Therefore, more rational protocols are needed, including that N. caninum can be modified to reduce toxicity without compromising treatment efficacy, anti-tumor properties from N. caninum can be screened to make a tumor vaccine, and N. caninum can be combined with immune checkpoint inhibitors against cancer.

Altogether, our results indicate that N. caninum not only inhibits the growth of Lewis subcutaneous tumors, but also inhibits the development of Lewis and B16 melanomas lung metastatic in mice by stimulating the immune response and inhibiting angiogenesis. N. caninum and its anti-tumor properties may be a safe and effective anti-tumor tool.

Conflict of Interest Statement

The authors report no declarations of interest.

Ethics approval and consent to participate

All animal experiments were approved by the Experimental Animal Commission of Henan University of Science and Technology (Permit No. SCXK [JING] 2021–0006). The experimental scheme conformed strictly to the guidelines of the Institutional Animal Care and Use Committee (No. 201) of Henan University of Science and Technology (Luoyang, Henan, China).

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 31502053).

Author Contribution

Weifeng Qian-Methodology, Supervision, Writing – review & editing, Funding acquisition. Yaqi Chen: Methodology, Data curation, Writing – original draft. Chen Li: Investigation, Software, Visualization, Writing – original draft. Xiaojin Li: Investigation, Methodology, Formal analysis. Chaochao Lv: Writing – review & editing, Formal analysis. Yanyan Jia: Methodology. Suhui Hu: Data curation, Software. Min Zhang: Software. Tianqi Wang: Supervision, Validation, Visualization. Wenchao Yan: Validation, Visualization. Meng Qi: Supervision, Writing – review & editing.

Acknowledgement

Special thanks belong to Prof. Qun Liu for providing N. caninum in this study

Data Availability

The datasets generated during and analysed during the current study are available from the corresponding author on reasonable request.

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Neospora caninum inhibits Lewis cancer and B16F10 melanoma lung metastasis development by activating the immune response in murine models

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Mice

2.2. Tumor Cell

2.3. Neospora caninum

2.4. Murine models for subcutaneous tumors and lung metastases

2.5. Optimal dosing of N. caninum tachyzoites and sample collection

2.6. Experimental design of lung metastases and sample collection

2.7. Histological analysis

2.8. Cytokine detection

2.9. Cellular analysis

2.10. Statistical analysis

3. Results

3.1. Three tumor-bearing murine models were successfully constructed

3.2. A sufficient amount of N. caninum tachyzoites can induce a stronger anti-tumor activity

3.3. N. caninum regresses Lewis subcutaneous tumors development

3.4. N. caninum regresses Lewis lung metastasis development

3.5. N. caninum regresses B16F10 lung metastasis development

3.6. N. caninum significantly enhanced PD-L1 expression

4. Discussion

Declarations

Conflict of Interest Statement

Funding

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

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