Introduction
Parasites are double-edged sword, with a noticeable negative impact on their hosts, while possessing a powerful advantageous immunomodulatory effect that can be exploited for the host’s benefit [
1,
2]. This immunomodulatory activity was verified against various immune-related diseases as allergies, autoimmune diseases, and others [
3,
4]. Cancer is an immune-related disease with obvious immunosuppression [
5]. A powerful T-helper 1 (TH1) immune response induced by some parasites as
Toxoplasma gondii (
T. gondii) can be employed to counteract cancer TH2 immunosuppressive dominance [
1].
Pathogen-based cancer immunotherapy is an axial tool to counteract cancer immunosuppressive dominance. Bacille Calmette–Guerin (BCG), a
Mycobacterium bovis live
-attenuated vaccine, is a cancer bladder therapeutic vaccine [
6]. Number of pathogens are now in the pipeline investigating their antineoplastic effectiveness [
1,
7].
Relying on data affirming that low-dose chronic asymptomatic
T. gondii infection provoked an antineoplastic effect [
7] and that the low titer anti-
Toxoplasma antibody is associated with cancer resistance [
8] enrich repositioning of
T. gondii as a vaccine candidate for cancer immunotherapy. Whereas acquiring infection appears non-realistic to seize the antineoplastic activity, parasitic vaccines can be exploited to pursuit this activity.
Autoclaved parasitic vaccines are special type of killed vaccines retaining the essential parasitic immunogenic components [
9] that proved to be safe, easy to prepare, stable, and cheap [
10]. They revealed high homologous protective immunity against corresponding infection as toxoplasmosis, schistosomiasis, and trichinellosis [
9,
11,
12]. Autoclaved cercarial vaccine was protective against experimental schistosomiasis and provoked therapeutic antineoplastic activity against experimental cancer colon in mice [
11,
13]. In experimental toxoplasmosis, autoclaved
Toxoplasma vaccine (ATV) reduced hepatic and splenic load of
T. gondii tachyzoites, superior to
T. gondii lysate antigen with rise in splenic CD8
+ T cells [
9]. Since, intratumorally injected attenuated
T. gondii tachyzoites provoked an antineoplastic role against melanoma model [
14], exploring ATV therapeutic antineoplastic activity may promote its enrollment to parasite-based cancer vaccines for immunotherapy.
Cyclophosphamide (CP) has a differential dose-dependent action, an immunosuppressive and immunomodulatory role. Its precise immunomodulatory mechanism is not entirely clear, yet studies suggested a role for selective T regulatory (Treg) cell depletion [
15,
16,
17]. Its unique low-dose immunomodulatory action modifies the immunosuppressive tumor microenvironment, which augments the response to main adjunctive therapies while minimizing risk for adverse drug reactions [
17]. Since immune-mediated therapies are becoming prevalent in cancer, we investigated ATV therapeutic antineoplastic activity in reference to and in combination with low-dose CP in Ehrlich solid carcinoma (ESC), a well-established murine cancer model.
Discussion
The dominance of cancer immunosuppression remarks the significant role of cancer immunotherapy. Aside from the immunomodulatory agents in cancer pipeline, pathogens have evolved as promising candidates. Moreover, low titer of
T. gondii antibodies was related to cancer resistance [
8] and anti-
Toxoplasma antibodies selectively attached to mouse cancer cell lines [
30]. These data justify the investigation of the antineoplastic potential of
Toxoplasma-derived vaccine.
In this study, induction of ESC in mice universally augmented liver enzymes and impaired hepatic structure evidenced by the observed diffuse fatty changes of hepatocytes compared to normal control, in line with the previous studies [
20]. In fact, impacting liver enzymes and architecture has been a confirmed criteria in almost all cancer models [
22,
31]. These hepatic deleterious changes induced by ESC were generally corrected by all adopted treatments denoting a positive influence of CP and ATV on hepatic functions being highest with CP/ ATV. This was evidenced by the significant improvement of hepatic enzymes by all treatments. However, from the histopathological background, a generalized hepatic hyperimmune state was detected in the treated mice, particularly in the ATV-treated and CP/ATV-treated group, evidenced by marked hypertrophy and hyperplasia of Kupffer cells, and sinusoidal lymphocytic infiltration. This could speculate an exceptional immune-mediated role of ATV.
It is worth to note that Kupffer cells are liver macrophages resident with antitumor and antimetastatic activity through interferon gamma, interleukin-12, and other inflammatory mediators production that have cytotoxic effect on cancer cells [
32]. Moreover, these mediators activate hepatic lymphocytes that migrate to cancerous tissues to interfere with their growth [
33]. Normally Kupffer cells can sample tumor cells, yet their efficacy to control tumor growth is limited and cancer immunotherapy can additively enhance Kupffer cell function [
34]. These data justify the speculated potent immune-mediated antineoplastic activity of ATV, since fascinating hyperplasia and hypertrophy of Kupffer cells were only detected upon ATV and CP/ATV treatment. However, immune-mediated hepatitis has been reported in patients with solid tumors receiving immunotherapy, while lacking signs of blood hepatotoxicity. This explains our findings of inflammatory hepatic reaction with all adopted treatments that was associated with improvement of hepatic transaminases [
35].
These findings match with the previous studies reporting mild elevation of liver transaminases induced by CP standard doses [
36], yet, this does not usually coincide with hepatic histopathological changes, as aforementioned [
35]. Indeed, treatment with CP showed mild central necrotic areas in liver sections that could be probably caused by CP hepatic metabolism [
15]. On the contrary, no hepatic focal necrotic areas were detected upon ATV, denoting a tolerable hepatic impact of ATV as previously reported [
9,
18].
Gross pathological examination of ESC excised from mice treated with all adopted treatments revealed significant reduction in both ESC weight and volume compared to ESC control with the highest reduction encountered in CP/ATV-treated mice. This was similarly encountered in the previous studies upon usage of
T. gondii in treatment of melanoma [
14], fibrosarcoma, and sarcoma in animal models [
26,
37]. Additionally, the combined CP/ATV inhibited ESC development by 13.3% denoting a synergistic antineoplastic potential of ATV while added to CP. This synergistic effect was more evident in ESC volume compared to both CP and ATV sole treatment. This is probably due to the significant difference in the degree of fibrosis encountered upon CP/ATV treatment compared to that in CP and ATV individual groups. In CP/ATV-treated ESC, excessive fibrosis was probably responsible for the detected markedly shrunken tumor volume.
Histopathological analysis of tumor sections from ESC control disclosed sheets of malignant cells synchronizing with other studies [
20]. Foci of central necrosis were noted probably due to hypoxia and nutrient deficiency [
38]. While the impact of necrosis on tumor prognosis is query, tumor-induced central necrosis is usually associated with bad prognosis as reported in gastrointestinal and liver tumors [
39,
40]. This negative impact is explained by the release of proinflammatory mediators promoting chronic inflammation, which invites immune cells including neutrophils that promote angiogenesis, tumor cell proliferation, and immunosuppression within the tumor [
38,
41].
On the contrary, tumor necrosis induced by treatment, chemotherapy, or immunotherapeutic agents as checkpoint inhibitors was correlated with better prognosis through decreasing viable tumor content. The released necrotic cell contents stimulate the immune system, promoting antigen presentation and cytotoxic T cell activity [
42,
43]. This fits within our results since all adopted treatments induced significantly more necrosis compared to ESC control. A cumulative effect of CP/ATV treatment promoted extensive necrosis in tumor cells compared to CP and ATV treatment alone. This synergistic effect of CP/ATV treatment indicates a better prognosis since more necrosis denotes better treatment response [
43].
Primarily, this necrosis could be due to the immunostimulatory activity of all treatments evidenced by the remarkable increase in lymphocytic aggregates around ESC and giant cell infiltrates by ATV and CP treatment, respectively, that was supported by the current IHC results. Also, necrosis could be due to blood supply deprivation of the ESC that was later justified by diminished VEGF expression. These results can correlate with the potential use of
T. gondii as checkpoint inhibitors after confirming inhibition of programed cell death and its ligand (PD-1/PDL-1) signaling pathway by
T. gondii [
44], in parallel to PD-1 blockers [
29]. While PD-1 blockers were effective only in early tumor stages, PD-1/PDL-1 pathway is inhibited by
T. gondii during both early and chronic infection stages, which potentiates its use in early and late tumor stages [
44].
Fibrosis is another tumor prognostic criteria since treatment with chemotherapeutic and immunotherapeutic agents promoted not only necrosis, but also fibrosis [
43]. Following chemotherapy, fibrosis enclosing tumor was associated with better pancreatic cancer prognosis [
45], as a sort of tissue healing following treatment-induced tumor necrosis [
43]. This matches with the present findings, where significantly more fibrosis was noted with all adopted treatments, most prominently with CP/ATV treatment, which justifies the noted difference in tumor weight and volume.
To thoroughly investigate the immune-mediated mechanism, IHC was performed on tumor sections from different groups. Analysis of immune cells, CD8
+ T and Treg cells, surrounding ESC showed a state of immunosuppressive dominance in ESC control, which coincides with cancer hallmarks [
5]. Upon CP treatment, a higher CD8
+ T cells and lower Treg cells with a higher CD8
+/Treg cell ratio compared to ESC control were shown surrounding the tumor. This matches with the previously investigated immunomodulatory role of low-dose CP and its influence on Treg cells depletion [
15,
16,
17]. Whereas treatment with ATV alone did not influence CD8
+ T cells surrounding ESC, while inducing a significant Treg cell depletion compared to both ESC control and CP-treated mice with a higher CD8
+/Treg cell ratio. Moreover, CP/ATV treatment promoted significantly higher CD8
+ T cells crawling around ESC with Treg cell depletion and a higher CD8
+/Treg cell ratio, adding more evidence to the speculated antitumoral immunostimulatory synergism between ATV and CP.
Since immune cells infiltrating tumor tissue and the effector T/Treg cells ratio shape and predict cancer outcome [
29], we explored the influence of treatments on the immune cells inside the ESC. Both ATV and CP treatments exhibited a significant Treg cell infiltrate depletion, while only ATV significantly induced CD8
+ T cells infiltration in ESC and increased CD8
+/Treg ratio inside the tumor. Again, CP/ ATV treatment promoted an antitumoral immunostimulatory synergistic effect with significantly higher CD8
+ T cells and lower Treg cells with a higher CD8
+/Treg cell ratio inside ESC compared to either treatment alone.
In fact, Treg cells are a well-established immunosuppressive T cell subtype that enable tolerance to self-antigens by suppressing, in particular, the high affinity antigen-specific cytotoxic T cells and memory cells. However, Treg cells have been linked to immune evasion, and cancer immune-tolerance and progression [
5]. Compared to other T lymphocytes, they are especially sensitive to low-dose CP, due to their low levels of intracellular ATP that impairs glutathione production necessary to neutralize CP toxic products. Moreover, low-dose CP has been reported to downregulate the expression of the glucocorticoid-induced TNFR family-related (GITR) gene that is a costimulatory molecule assisting in Treg proliferation. Also, Treg cells have an impaired DNA repair mechanism that cannot resist high-dose CP-mediated killing [
17].
Partly as a consequence of CP inhibitory effect on Treg cells, T cell responses to T cell receptor stimulation and the production of tumor antigen-specific T cells are improved [
23]. Additionally, reduced Treg by low-dose CP skews T-helper cells from a TH2 to TH1 phenotype, increasing expression of IL-2 gene, which stimulates expansion of memory cytotoxic T lymphocytes [
46]. This could explain the currently observed increase in CD8
+ T cells number and function by low-dose CP that assisted in immunogenic cell death (ICD) of ESC. The ICD is documented by the increased necrosis and fibrosis and the reduction in both ESC weight and volume.
It is to be noted that in this study, a twice dose of CP (50 mg/kg) was adopted two weeks apart. This was based on the reported depletion of Treg cells and increase in CD8
+ T cells infiltration with a higher CD8
+/Treg ratio induced by a single dose of 50 mg/kg CP preceding immunotherapy in tumor mice model [
16]. Since, Treg cell depletion induced by a single dose of CP is transient and recovery usually follows [
15,
16], we adopted a second dose of CP to maintain its immunomodulatory action. Moreover, research conducted using both pathogen and CP as cancer immunomodulators concluded the influence of CP treatment timing in relation to pathogen-derived vaccination as a crucial factor affecting the outcome. If treatments are administered before vaccination, liberation from tumor-associated immune suppression takes place [
47]. This justifies our rational use of CP injection one day prior to ATV administration.
An additional cancer criterion is neo-angiogenesis, mediated via VEGF. Scarcity of blood supply flags tumor cell death [
5]. Interestingly, VEGF is a dual agent, a proangiogenic factor, and an immunosuppressive promoter. Thus, VEGF level correlates with Treg cell population, while inversely correlates with CD8
+ T cells within the tumor [
48]. This matches with our findings since ESC control showed high VEGF and Treg cell with low CD8
+ T cell within tumor. Upon CP treatment, significant reduction in tumor VEGF was noted compared to ESC control and ATV-treated mice, which matches with the previous studies [
49]. Likewise, ATV treatment significantly reduced VEGF expression in line with studies using different
T. gondii variants in cancer murine models [
50,
51,
52]. Most probably, the noted inhibitory effect of ATV and CP on neovascularization lead to marked tumor hypoxia and avascular necrosis that stunted progressive neoplastic growth.
In this context, ATV can be considered a dual immunotherapeutic agent via a direct immune stimulation by tumor infiltration with CD8+ T cells and depletion of the immune-suppressive Treg cells as well as an antiangiogenic action. Apparently, these observed ATV actions would interfere with tumor growth and thus inducing tumor shrinkage by promoting tumor necrosis and fibrosis with subsequent reduction in tumor weight and volume.
The molecular mimicry theory and sharing of glycoprotein antigens between parasites and cancer [
7] can be the main tactic by which ATV provoked its observed immunomodulatory antineoplastic activity against ESC. Acknowledging the abundance of proteins linked to N and O glycans in
Toxoplasma [
53], adds a privilege for investigating
Toxoplasma shared antigens with various cancer cell lines that will not only support its antineoplastic activity, but also pave the way for its involvement in an effective antineoplastic vaccine. Regarding
Toxoplasma antigen, it appears that its combination with low-dose CP had boosted its immunomodulatory action and assisted in a superior antineoplastic activity. It is well documented that the use of optimized drug combinations against cancer is of optimum benefit not only to synergistically attack different antitumoral pathways, but also to assist in reducing the chemotherapy-induced toxicity and cancer drug resistance. Herein, the addition of ATV to CP enabled a maximum benefit of its low-dose use, thus reducing its potential toxicity that results from the cellular DNA damage induced by its standard anticancer dose [
36]. Additionally, cancer immunomodulation helps to induce loads of activated immune cells that are capable of killing tumor cells specifically, thus avoiding major toxicities of traditional chemotherapy. Also, it can overcome cancer drug resistance by enabling a continued tumor immune surveillance [
15]. This matches with the previous studies highlighting the synergistic depletion of Treg cells and increased infiltration of CD8
+ T cells upon combined use of CP and various immunotherapeutic agents [
15,
16,
17].