Parasites' immunomodulators: a breakthrough in immunotherapeutics displaying antineoplastic activity against human colorectal and hepatocellular carcinoma cells.

Introduction

Cancer is a global health concern with a dramatic influence on patients’ quality of life, productivity, and psychological aspects [ 1 ]. Gastrointestinal cancers (GICs) constitute a huge bulk of cancer cases, of which colorectal carcinoma (CRC) is the greatest burden. Gastrointestinal cancers account for 26% of cancer incidences (5.26 million cases) and 35% of cancer deaths (3.70 million fatalities) globally in 2021 [ 2 , 3 ], within which liver cancer, mainly hepatocellular carcinoma (HCC), is considered the third most common cause of cancer-related deaths globally [ 4 ]. While chemotherapeutics, radiotherapy, and surgery are the cornerstones of GICs’ treatment, significant drawbacks exist that hinder their full safe clinical applicability. Thus, immunotherapy has emerged, aiming to enhance the suppressed immune response [ 5 ]. In parallel, researchers are seeking alternative cancer candidates that could be more immunogenic, safe, and have homology to cancer antigens.

Long ago, researchers were skeptical regarding the relationship between parasites and cancer due to the association of some parasites with carcinogenesis as Clonorchis sinensis , Schistosoma haematobium , and Opisthorchis viverrini [ 6 ]. Nevertheless, other parasites have recently been certified experimentally as anti-cancerous agents via multiple mechanistic strategies [ 7 , 8 ]. This may be related to the hygiene theory, which postulates that the lack of exposure to pathogenic agents, such as parasites, may be related to an increased prevalence of immune-mediated illnesses and cancer [ 9 ]. Additionally, retrospective studies have demonstrated that the prevalence of cancer is notably lower in patients with parasitic diseases such as hydatid or Chagas’ disease compared to the general population [ 10 , 11 ]. Subsequently, the antineoplastic activities of some parasites were verified experimentally against different cancer models [ 7 , 12 ].

The cytotoxic effects of certain parasites and their derivatives on cancer cells may be valuable tools towards attacking cancer cells either directly or by manipulating molecular processes at the proteomic and genetic levels to induce cell apoptosis [ 7 , 8 ]. Although live and attenuated T. gondii were able to directly invade and replicate within cancer cells and lead to their lysis, safety concerns may restrict their clinical applicability [ 13 – 19 ]. Thus, using live parasites to promote anti-cancerous effects sounds unreasonable and impractical; consequently, parasite immunomodulators (PIMs) (antigens and antibodies) may hold an optimistic value, as they are highly immunogenic and display homology to cancer antigens [ 7 ].

Interestingly, certain parasitic antigens exhibited direct cytotoxic effects on cancer cells, as antigens from Echinococcus granulosus (E. granulosus) [ 20 ] and Toxoplasma. gondii (T. gondii) [ 21 ]. In particular, autoclaved parasitic antigens exhibited potent immunogenicity against their corresponding parasitic infections, as experimental schistosomiasis [ 22 ], trichinellosis [ 23 ], toxoplasmosis [ 24 ], among others [ 25 , 26 ]. Further data supported their safety [ 27 – 29 ] and their immunomodulatory efficacy against non-parasitic diseases as experimental arthritis [ 30 ], psoriasis [ 31 ], and cancer models [ 28 , 29 , 32 , 33 ]. Thus, investigating the potency of those antigens on human cancer cell lines in vitro deserves further investigation.

Therapeutic monoclonal antibodies have gained tremendous value as supplemental cancer immunotherapeutic agents [ 34 ]. Mechanistically, these antibodies can selectively target and destroy cancer cells while sparing healthy cells. These antibodies can activate the complement pathway, stimulate the direct antibody-dependent cell cytotoxicity, induce signalling perturbation, interfere with growth factor receptors, restrict tumor cell proliferation, and opsonize cancer cells, consequently flagging the tumor cells and inviting immune effector cells [ 34 ]. However, the role of parasitic antibodies on CRC and HCC cells hasn’t been investigated in depth.

Based on the aforementioned data and the highlighted areas of research gaps between parasites and cancer, we aimed to experimentally screen Schistosoma mansoni ( S. mansoni ), Trichinella spiralis ( T. spiralis ) and T. gondii immunomodulators against two human cancer cell lines: the HCT-29 human CRC and the HepG2 human HCC cell lines.

Materials and methods

Ethics statement

All experimental mice were handled following the ARRIVE guidelines for animal care [ 35 ]. For human sera collection, the study followed the principles of the Helsinki Declaration (DoH 2024) of 1964, and informed consent, either written or by thumb printing, was obtained from each participant [ 36 ]. The study protocol was approved by the Ethics Committee of the Faculty of Medicine, Alexandria University (Permit Number: 0306484).

Experimental animals

Swiss albino mice were used for maintaining parasitic life cycles. Mice were housed in appropriate cages under standard laboratory conditions (27 ± 2 °C; 70–80% humidity; 12 h light/dark cycle) with a standard pellet diet and water ad libitum .

Laboratory maintenance of the parasitic life cycle

The life cycles of all parasites were maintained at the Department of Medical Parasitology, Faculty of Medicine, Alexandria University. The S. mansoni life cycle was maintained by serial passage between Biomphalaria alexandrina snails [ 37 ] and Swiss albino mice [ 38 ]. The T. spiralis life cycle was maintained through serial passage in Swiss albino mice by ingestion of live purified T. spiralis larvae [ 39 ]. The T. gondii life cycle was maintained by serial intraperitoneal (ip) passages of live tachyzoites of the virulent RH HXGPRT (-) strain in Swiss albino mice [ 40 ].

Preparation of parasitic antigens

To prepare autoclaved antigens, cercariae of S. mansoni , larvae of T. spiralis , and tachyzoites of T. gondii were collected and autoclaved to generate A Sm A, A Ts A, and A Tg A, respectively. Cercariae of S. mansoni were collected by exposing infected snails for one hour to sunlight in dechlorinated water to allow the shedding of the cercariae [ 22 ]. Larvae of T. spiralis were collected by enzymatic digestion of skeletal muscles from T. spiralis- infected mice [ 23 ]. Tachyzoites of T. gondii were harvested from the peritoneal exudate of T. gondii -infected mice [ 24 ]. They were autoclaved separately in screw-capped glass vials at 121 °C, under pressure of 15 lb for 15 min. The antigens were kept at -20 °C for later usage [ 24 ]. Protein quantification was performed using a NanoDrop™ 2000 spectrophotometer (Thermo-scientific) and expressed in mg/ml.

Anti-parasitic antibodies

Human sample collection

Non-cancerous patients visiting the Internal Medicine/Hepatology or Obstetrics and Gynecology outpatient clinics at Alexandria University Main and El-Shatby Hospitals, respectively, were the subjects of the study. Blood samples were collected from patients who were suspected of having schistosomiasis or toxoplasmosis, as well as from healthy controls. Sera were separated and stored at -20 °C for later usage.

Screening of serum samples for IgG-positive sera for S. mansoni and T. gondii

Collected serum samples were screened using ELISA kits for S. mansoni- specific IgG antibodies and T. gondii- specific IgG antibodies (MyBioSource Inc., San Diego, USA), Catalogue #: MBS495074 and MBS494548, respectively, according to the manufacturer’s instructions. Using an automated ELISA reader (Benchmark, BIO-RAD, California, U.S.A., Catalogue # 170–6850 Microplate Reader), the optical density of each sample was read at 450 nm within 15 min [ 41 ]. Samples with the highest titer were selected for the in vitro experimental study.

Preparation of IgG-positive serum for T. spiralis

Due to the scarce human infection with T. spiralis in Egypt, sera of mice chronically infected with T. spiralis (35 days post-infection) were used. An ELISA test was performed to confirm the positivity of T. spiralis IgG in the mice sera, as previously described [ 42 ]. In brief, wells of a 96-well plate were coated with diluted A Ts A, followed by an overnight incubation at 4 °C. 1% bovine albumin, used as a blocking buffer, was added to the plate, followed by incubation at room temperature for two hours. The wells were washed, and 100 µl of diluted mouse sera was added to the wells for two hours at 37 °C, followed by washing four times with phosphate buffer saline. A diluted secondary antibody (goat anti-mouse; Sigma-Aldrich) was added for one hour. Lastly, the plate was washed and incubated with the chromogenic substrate. The optical densities were read via an ELISA reader (BIO-RAD) at 450 nm within 15 min. Samples with the highest titer were selected for the in vitro experimental study [ 42 ].

Cancer cells and culture

Frozen vials of different cancer cell lines (HT-29 human CRC cell line and HepG2 HCC cell line) were purchased from VACSERA Co., Cell Culture Unit, Dokky, Giza, Egypt, originally supplied from the American Type Culture Collection (ATCC). Mycoplasma -free cancer cells were cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM) (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 2 mM L-glutamine (Thermo Fisher Scientific), 1 mM sodium pyruvate (Fisher Scientific), Streptomycin (100 mg/ml) (Thermo Fisher Scientific) and Amphotericin-B (5 mg/ml) (Thermo Fisher Scientific) and maintained at the Tissue Culture Unit at Alexandria University.

In vitro study protocol

Cancer cell maintenance

Cancer cell lines were cultured as previously described [ 43 ]. Five days before the experiment, cells were seeded in a sterile 24-well plate in complete culture media and incubated under a humidified atmosphere of 5% CO 2 at 37 °C in an automated CO 2 incubator. Cells were regularly checked using an inverted microscope until confluent.

Experimental protocol

One day before the experiment, cells were trypsinized, washed, resuspended in complete media, and plated in 96 microtiter plates [ 44 ]. Four sets of 96-well plates were prepared: two for each cell line. For the four plates, cells (HT-29 & HepG2) were cultured separately for 24 h and allowed to get confluent in a final volume of 100 µl of complete media/well (10,000 cells/well). The next day, the cells that did not attach and the excess liquid were discarded. A total of 100 µl of aliquoted media containing diluted antigens or serum was added [ 45 ].

In the first two plates, cells (HT-29 & HepG2) were treated with serial dilutions of parasitic antigens (A Sm A, A Ts A, and A Tg A) at 3.125, 6.25, 12.5, 25, 50, and 100 µg/ml concentrations [ 21 , 46 , 47 ]. In the other two plates, cells (HT-29 & HepG2) were exposed to serial dilutions of S. mansoni , T. spiralis , and T. gondii IgG-positive sera. Sera were used at a concentration of 2.5, 5, 10, 20, and 40% [ 45 ]. Negative control wells were included where cells were cultured either in media only or media treated with sera negative for parasite IgG antibodies. The experiment was performed in triplicate, and the data presented is the mean of the experimental replicates [ 21 ].

MTT protocol

For the assessment of cytotoxicity, the following protocol was applied. After 24 h incubation, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used as previously described [ 21 , 48 ]. Cells were incubated for the last two hours with 10 µl of MTT labelling agent (MTT formazan, 2 mg/ml; Sigma), added to each well according to the manufacturer’s instructions. One hundred µl of dimethyl sulfoxide (DMSO) (Sigma) was added to dissolve the formazan crystals, and the absorbance values were measured at 490 nm [ 49 ]. The proliferation was checked for each treatment, as well as the negative controls. To calculate the proliferation percentage, the absorbance of each treated group was divided by the absorbance of the media-only negative control wells and multiplied by 100. The following equation was applied: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:(Atc/Anc)\times\:100$$\end{document} , where Atc is the absorbance of each treatment concentration and Anc is the absorbance of the media-only negative control. For the calculation of the percentage of inhibition in the antigen-treated groups, the proliferation percentage of each treatment concentration was subtracted from the proliferation percentage of the cells cultured in the media-only negative control group. To calculate the percentage of inhibition in the serum-treated groups, the proliferation percentage of each treatment concentration was subtracted from the proliferation percentage of the cells exposed to serum-negative for parasitic antibodies.

Statistical analysis of the data

Data were analyzed using the IBM SPSS software package version 20.0. (Armonk, NY: IBM Corp). Quantitative data were expressed as mean and standard deviation. Student t-test was used to compare two groups for normally distributed quantitative variables, while the One-way ANOVA test was used for comparing the different studied groups, followed by the Post-Hoc test (Tukey) for pairwise comparison. Results were considered statistically significant at the 5% level.

Results

Effect of parasites’ immunomodulators on HT-29 colorectal cancer cell line

Effect of parasitic antigens on HT-29 colorectal cancer cell line

Exposure of HT-29 colorectal cancer cells to serial concentrations of parasitic antigens (A Sm A, A Ts A, and A Tg A) for 24 h induced a statistically significant reduction (P˂ 0.05) in colorectal cancer cell proliferation at all concentrations tested compared to untreated colorectal cancer cells. The maximum inhibitory percentage was achieved at 25 µg/ml for all parasitic antigens, A Sm A, A Ts A, and A Tg A, with percentages of inhibition of 31.72%, 36.65%, and 31.23% respectively. The highest significant inhibition percentage was demonstrated for A Ts A (36.65%), which was significantly higher than A Sm A and A Tg A. Additionally, no significant difference was observed between A Sm A and A Tg A. While the three antigens induced significant inhibitory effects at all concentrations, a noteworthy finding was that at higher concentrations (50 µg/ml and 100 µg/ml), a significant proliferative effect was observed compared to the 25 µg/ml concentration for all antigens, with no significant difference between these two higher concentrations for all tested antigens (Fig. 1 , Suppl. Table 1 ).

Fig. 1 Graphical representation of HT-29 colorectal cancer cell proliferation percentage upon incubation with different concentrations of parasitic antigens for 24 h, compared to cells cultured in media only. Control: Cells cultured in media only, A Sm A: Cells incubated with autoclaved Schistosoma mansoni antigen, A Ts A: Cells incubated with autoclaved Trichinella spiralis antigen, A Tg A: Cells incubated with autoclaved Toxoplasma gondii antigen. HT-29 colorectal carcinoma cells were incubated with serial concentrations of different parasitic antigens for 24 h, and the proliferation percentage was measured using the MTT assay

Effect of serum positive for parasitic IgG on HT-29 colorectal cancer cell line

Exposure of HT-29 colorectal cancer cells to serial concentrations of serum positive for parasitic antibodies for 24 h induced a statistically significant inhibition in cell proliferation in a concentration-dependent manner compared to both the untreated and the negative sera controls (P˂ 0.05). The inhibitory effect was significant starting from 2.5% for S. mansoni and T. spiralis IgG-positive sera, and 5% for T. gondii IgG-positive sera, up to 40% for all parasitic antibodies. The maximum inhibitory percentage was observed at 40% for both T. spiralis and T.gondii IgG-positive sera, which showed comparable efficacy with percentages of inhibition of 49.90% and 50.43%, respectively, significantly higher than that exposed to S. mansoni IgG-positive sera (37.17%) (Fig. 2 , Suppl Table 2 ).

Fig. 2 Graphical representation of HT-29 colorectal cancer cell proliferation percentage upon incubation with different concentrations of sera positive/ negative for parasitic IgG for 24 h, compared to cells cultured in media only. Control: Cells cultured in media only, Sera negative for parasitic IgG: Cells incubated with sera negative for parasitic IgG, S. mansoni IgG + sera: Cells incubated with sera positive for Schistosoma mansoni IgG, T. spiralis IgG + sera: Cells incubated with sera positive for Trichinella spiralis IgG, T. gondii IgG + sera: Cells incubated with sera positive for Toxoplasma gondii IgG. HT-29 colorectal cancer cells were incubated with serial concentrations of different sera positive for parasitic antibodies for 24 h, and the proliferation percentage was measured using the MTT assay

Notably, T. gondii IgG-positive sera at a low concentration of 2.5% had a biphasic effect on HT-29 colorectal cancer cells, promoting cell proliferation compared to cells exposed to media only and cells exposed to negative sera controls. Additionally, negative sera for parasite IgG antibodies promoted significant proliferation of HT-29 colorectal cancer cells compared to the untreated control at a concentration ranging from 20% to 40% (Fig. 2 , Suppl Table 2 ).

Effect of parasites’ immunomodulators on HepG2 hepatocellular carcinoma cell line

Effect of parasitic antigens on HepG2 hepatocellular carcinoma cell line

Exposure of HepG2 hepatocellular carcinoma cells to serial concentrations of parasitic antigens (A Sm A, A Ts A, and A Tg A) for 24 h resulted in a concentration-dependent decrease in cell proliferation. The inhibitory effect was observed at concentrations starting from 25 µg/ml for A sm A, 6.25 µg/ml for A Ts A, and 6.25 µg/ml for A Tg A up to 100 µg/ml for all antigens. The maximum inhibitory percentage was achieved at 100 µg/ml for all antigens, with A Ts A showing a 38.27%, A Tg A with a 34.68%, and A Sm A with an 8.03% inhibition percentage. A Ts A and A Tg A had similar inhibitory effects, both significantly higher than A Sm A. Remarkably, A Sm A and A Tg A at a low concentration of 3.125 µg/ml were noticed to have a biphasic effect on HepG2 hepatocellular carcinoma cells, as they induced significant cell proliferation (Fig. 3 , Suppl. Table 3 ).

Fig. 3 Graphical representation of HepG2 hepatocellular carcinoma cell proliferation percentage upon incubation with different concentrations of parasitic antigens for 24 h, compared to cells cultured in media only. Control: Cells cultured in media only, A Sm A: Cells incubated with autoclaved Schistosoma mansoni antigen, A Ts A: Cells incubated with autoclaved Trichinella spiralis antigen, A Tg A: Cells incubated with autoclaved Toxoplasma gondii antigen. HepG2 hepatocellular carcinoma cells were incubated with serial concentrations of different parasitic antigens for 24 h, and the proliferation percentage was measured using the MTT assay

Effect of serum positive for parasitic IgG on HepG2 hepatocellular carcinoma cell line

Exposure of HepG2 hepatocellular carcinoma cells to serial concentrations of sera positive for S. mansoni , T. spiralis , and T. gondii IgG for 24 h significantly inhibited cell proliferation in a concentration-dependent manner. The inhibition started at 2.5% for T. gondii IgG-positive sera, 5% for T. spiralis IgG-positive sera, and 10% for S. mansoni IgG-positive sera, up to 40% compared to negative sera control.

The maximum inhibitory percentage was observed at a concentration of 40% for T. spiralis IgG-positive sera (48.25%), which was statistically significantly higher than cells exposed to both T.gondii IgG-positive (41.98%) and S.mansoni IgG-positive sera (4.54%). Although S. mansoni IgG-positive sera at a concentration of 40% showed inhibition in cancer cell proliferation as compared to negative sera control, this value was significantly higher than that of the untreated cells cultured in media alone. Remarkably, negative sera for parasite IgG antibodies induced significant cell proliferation compared to the untreated control, starting from 20% to 40%. (Fig. 4 , Suppl. Table 4 ). Notably, among all tested parasites’ immunomodulators, anti- T. spiralis and T. gondii antibodies exhibited the maximum inhibitory effect on both cancer cell lines (Fig. 5 , Suppl. Table 5 ).

Fig. 4 Graphical representation of hepatocellular carcinoma cell proliferation percentage upon incubation with different concentrations of sera positive/ negative for parasitic IgG for 24 h, compared to cells cultured in media only. Control: Cells cultured in media only, Sera negative for parasitic IgG: Cells incubated with sera negative for parasitic IgG, S. mansoni IgG + sera: Cells incubated with sera positive for Schistosoma mansoni IgG, T. spiralis IgG + sera: Cells incubated with sera positive for Trichinella spiralis IgG, T. gondii IgG + sera: Cells incubated with sera positive for Toxoplasma gondii IgG. HepG2 hepatocellular carcinoma cells were incubated with serial concentrations of sera positive for different parasitic antibodies for 24 h, and the proliferation percentage was measured using the MTT assay

Fig. 5 Graphical representation of the maximal inhibitory percentage of HT-29 colorectal and HepG2 hepatocellular carcinoma cells upon incubation with parasitic antigens and their corresponding parasitic IgG-positive sera for 24 h. A Sm A: Cells incubated with autoclaved Schistosoma mansoni antigen, A Ts A: Cells incubated with autoclaved Trichinella spiralis antigen, A Tg A: Cells incubated with autoclaved Toxoplasma gondii antigen. S. mansoni IgG + sera: Cells incubated with sera positive for Schistosoma mansoni IgG, T. spiralis IgG + sera: Cells incubated with sera positive for Trichinella spiralis IgG, T. gondii IgG + sera: Cells incubated with sera positive for Toxoplasma gondii IgG. HT-29 colorectal cancer cells and HepG2 hepatocellular carcinoma cells were incubated with serial concentrations of different parasitic antigens/ parasitic IgG-positive sera for 24 h, and the inhibitory percentages were measured using the MTT assay. *: Represents significance between the inhibitory percentages of the examined concentrations of parasitic antigens and their corresponding IgG + sera

Discussion

Parasites and their derivatives were proven to alleviate cancer in experimental models via multiple strategies [ 7 , 29 , 32 , 33 ]. While the immunomodulatory mechanism constitutes a main strategy in fighting cancer, other verified mechanisms are also recognized, including their direct cytotoxic capacity, inhibition of neoangiogenesis, supplemented by the molecular mimicry theory [ 12 , 15 , 29 , 33 , 50 – 52 ]. However, rather than utilizing a systematic platform for the selection process, previous studies have utilized a random selection pattern to match parasites with different cancer types, except for some studies that screened for the molecular homology between cancer cells and parasites [ 28 , 42 , 53 , 54 ]. Therefore, there are gaps in configuring which parasites may be linked to specific cancer types. Our objective was to identify potentially potent PIMs that may have antineoplastic potency. This mix-and-match selective strategy can assist researchers in designing a long-term road map for the development of potent novel cancer therapeutics of parasitic origin. Several parasitic antigens were documented to have antineoplastic activity both in vitro and in vivo [ 7 , 12 ]. However, the role of parasitic antibodies in combating cancer remains underexplored. Enforced by the molecular mimicry theory between parasites and cancer cells, antibodies that develop against parasites can potentially attack cancer cells. This may explain the restricted cancer development in seropositive patients for parasitic antibodies [ 55 ].

In this context, the current study investigated the antiproliferative effect of six different parasites’ immunomodulators (antigens and antibodies) of S. mansoni , T. spiralis , and T. gondii on human cancer cell lines (HT-29 CRC and HepG2 HCC cells). These parasites were selectively chosen due to their previous records of antineoplastic activity experimentally [ 7 ]. Correspondingly, these cancer types were specifically selected due to their high incidence and severe health consequences [ 2 , 4 ].

In vitro antineoplastic potential of parasites’ immunomodulators on human HT-29 colorectal cancer cell line

Schistosoma mansoni immunomodulators

There is conflicting evidence regarding the link between S. mansoni and colon cancer in both human and experimental studies [ 56 ]. Interestingly, emerging reports have shown the antineoplastic properties of S. mansoni and S. japonicum in different experimental cancer models [ 32 , 33 , 52 , 57 , 58 ].

The results of our study showed that in vitro exposure of HT-29 colorectal cells to A Sm A induced a significant inhibition of colorectal cell proliferation at all tested concentrations compared to the untreated control, with the maximum inhibitory percentage of 31.72% achieved at a concentration of 25 µg/ml. Our results are in agreement with other in vitro results, which reported in vitro antineoplastic activity for S. mansoni egg and cercarial antigens on colorectal cancer cell lines (HCT-116 and DLD-1) and MCF-7 human breast cancer cell line [ 32 , 59 ]. Additionally, in vivo studies reported the antineoplastic activity of S. mansoni in a murine cancer model [ 60 ], transplanted sarcoma model [ 61 ], colon, colorectal, and breast cancer murine models [ 32 , 33 , 59 ]. The antineoplastic activity of Schistosoma is believed to involve an immunomodulatory mechanism, potentially through molecular mimicry between S. mansoni and cancer cells [ 33 ]. This mimicry has been observed between S. mansoni and MCF-7 human breast cancer and A549 lung cancer cells at an approximate molecular weight of 80 kDa [ 42 ].

Additionally, our study demonstrated that S. mansoni IgG-positive human sera inhibited colorectal cancer cell proliferation in a concentration-dependent manner, with the maximum significant inhibitory percentage of 37.17%, noticed at a concentration of 40%. To the best of our knowledge, this is the first study highlighting the antineoplastic activity of anti-S. mansoni antibodies against colorectal cancer. Notably, negative sera for parasitic IgG antibodies induced significant proliferation of HT-29 colorectal cancer cells compared to untreated cells. This aligns with previous research showing that human serum promotes cell growth and migration [ 62 , 63 ].

Trichinella spiralis immunomodulators

Our study revealed an in vitro antineoplastic activity of T. spiralis immunomodulators against HT-29 colorectal cancer cells. Exposure of colorectal cancer cells to A Ts A and T. spiralis- IgG-positive mice sera induced a significant reduction in cell proliferation at all concentrations tested, with the maximum inhibitory percentage of 36.65% at 25 µg/ml and 49.90% at 40% for both A Ts A and T. spiralis IgG-positive sera, respectively. Our results align with a previous in vivo study showing a diminishing in colorectal carcinoma growth in mice infected with T. spiralis [ 64 ]. However, another in vivo study using a DMH-induced colon carcinogenesis murine model showed conflicting results [ 33 ]. Interestingly, that study reported a notable increase in the average lesion size compared to the control group. The authors suggested that the initial immune-stimulant response of T. spiralis antigen, including mucosal immune responses, may have interacted with the inflammatory effects induced by DMH, leading to the observed outcome [ 33 ]. Therefore, further research is necessary to investigate the in vivo anti-cancerous properties of T. spiralis antigens in colorectal cancer xenograft animal models.

Additionally, our finding of the significant inhibitory effect of T. spiralis IgG-positive sera on colorectal cancer cell proliferation further documents its antineoplastic activity. Previous studies have shown similar effects of anti- T. spiralis antiserum and antiserum prepared against TPD52, a shared antigen between T. spiralis and osteosarcoma, on osteosarcoma cells [ 54 ]. The inhibitory effect reported with T. spiralis IgG-positive on colorectal cells may postulate the existence of shared antigens between T. spiralis and colorectal cancer cells, thus warranting further investigation. Notably, shared antigens were demonstrated between T. spiralis and human cancer cell lines (MCF-7 breast and A549 lung cancer) at approximate molecular weights of 70 and 35 kDa [ 42 ], which could partially justify T. spiralis’ potent antineoplastic effects in breast cancer [ 65 , 66 ] and lung cancer murine models [ 67 , 68 ]. Our study is considered the first report of the antineoplastic activity of T. spiralis immunomodulators against colorectal cancer cells.

Toxoplasma gondii immunomodulators

In our in vitro study, A Tg A and T. gondii IgG-positive human sera significantly reduced the colorectal cancer cells’ proliferation at all concentrations tested, with the maximum inhibitory percentage observed at a concentration of 25 µg/ml and 40%, respectively. This is the first study demonstrating the antineoplastic activity of anti- T. gondii antibodies against colorectal cancer. These findings are consistent with previous studies that demonstrated apoptosis of HCT116 human CRC cells incubated in vitro with T. gondii recombinant proteins, such as GRA16 [ 69 ], and GRA8 [ 70 ].

Remarkably, T. gondii has shown promising anti-cancerous effects against nearly all types of cancer studied so far [ 7 ]. For instance, attenuated T. gondii strain (RH-ΔGRA17) significantly slowed tumor growth in a mouse model of colon adenocarcinoma (MC38) [ 71 ]. Toxoplasma gondii lysate antigens [ 72 ], derivatives such as T. gondii GRA8 [ 70 ], T. gondii profilin-like protein [ 73 ], and T. gondii rGRA6Nt protein [ 74 ], as well as exosomes derived from dendritic cells infected with T. gondii [ 75 , 76 ] significantly inhibited tumor growth in several murine models of CRC.

It is worth noting that in our study, the three examined antigens at higher concentrations of 50 µg/ml and 100 µg/ml induced a significant proliferative effect on HT-29 colorectal cancer cells compared to the 25 µg/ml concentration, with no significant difference between them. Despite this, the antineoplastic activity of the three parasitic antigens at these concentrations was still significantly lower compared to untreated cells. This relative increase within the inhibited range may be due to the heterogeneous nature of the HT-29 colorectal cell line, resulting in more proliferating cells compared to lower concentrations [ 77 ], although the proliferations are still inhibited compared to the untreated cells.

Collectively, our study is in agreement with other studies that demonstrated the in vitro antineoplastic activity of other parasites and their derivatives on colon cancer cells, both in vitro and in vivo, such as E. granulosus [ 78 – 80 ], Toxocara canis (T. canis) [ 47 ], Heligmosomoides polygyrus [ 81 ], Plasmodium [ 82 , 83 ] among others [ 84 – 87 ].

In vitro antineoplastic potential of parasites’ immunomodulators on human HepG2 hepatocellular carcinoma cell line

Schistosoma mansoni immunomodulators

A Sm A and S. mansoni IgG-positive human serum induced a significant inhibitory effect on HepG2 human HCC cell proliferation, with the highest inhibitory percentage at 100 µg/ml and 40% respectively. Our results align with previous studies demonstrating the in vitro antineoplastic potential of various parasites and their derivatives against different HCC cell lines, such as S. japonicum [ 52 , 88 – 90 ].

Contrary to our results, other studies linked liver cancer and S. mansoni infection, possibly exacerbated by hepatitis viruses B and C, which cumulatively may induce suppression in cell-mediated immunity and promote liver cancer [ 91 , 92 ]. Studies in liver cancer animal models have shown that S. mansoni infection can lead to liver dysplastic changes and an aggressive cancer pattern [ 93 ]. Furthermore, antigens secreted by S. mansoni have been found to activate human and hamster liver cells by inducing c-Jun and STAT3, crucial regulators of liver cancer [ 94 ].

Trichinella spiralis immunomodulators

In our study, a significant reduction in liver cancer cell (HepG2) proliferation was observed upon exposure to A Ts A and T. spiralis IgG-positive mice sera in a concentration-dependent manner, with the highest significant inhibition observed at a concentration of 100 µg/ml and 40%, respectively. This is the first report supporting the antineoplastic activity of anti- T. spiralis antibodies against HCC. This is consistent with previous in vitro studies that reported the inhibitory effect of T. spiralis antigens, derivatives, and proteins encoded by T. spiralis on various cancer cell lines, including ascitic hepatoma (H22), hepatoma (H7402) [ 46 , 95 ] and HepG2 cells [ 96 ].

Similarly, in vivo experimental studies have also shown that infecting mice with T. spiralis could inhibit the growth of ascitic hepatoma H22 [ 46 ] and Hepatoma Hep1-6 carcinoma growth [ 97 ], as well as slow tumor progression by upregulating the expression of the proapoptotic marker Bcl-2 and improving survival in mice [ 98 ]. Trichinella spiralis has also been shown to induce apoptosis in HCC by utilizing regulatory pathways similar to those involved in cancer cell apoptosis, offering a potential strategy for combating this malignancy [ 99 ].

Our results regarding the antineoplastic activity of T. spiralis immunomodulators against human cancer cell lines (HT-29 CRC and HepG2 HCC) are consistent with other in vitro studies on small and non-small lung cancer cells (H446, A549) [ 100 – 102 ], human acute and chronic myeloid leukemia (K562, U937, R1) [ 95 , 103 , 104 ], EL-4 lymphoma [ 105 ], allogenic P815 mastocytoma [ 105 ], S180 sarcoma [ 95 ], B16 melanoma [ 106 ], and human cervical carcinoma HeLa cells [ 107 ]. Our results are also consistent with the in vivo antineoplastic activity of T. spiralis infection and its derivatives in murine forestomach carcinoma [ 95 ], lung cancer [ 67 , 68 ], breast cancer [ 65 , 66 ], multiple myeloma [ 108 ], sarcoma [ 95 , 109 , 110 ], osteosarcoma [ 54 ], melanoma [ 106 , 111 – 113 ], and glioma cancer models [ 114 ]. To the best of our knowledge, our study is the first to report the in vitro antineoplastic activity of anti- T. spiralis antibodies against HCC.

Toxoplasma gondii immunomodulators

Similarly, A Tg A and T. gondii IgG-positive human sera induced a significant inhibition of HepG2 HCC cell proliferation in a concentration-dependent manner, with the maximum inhibitory percentage achieved at 100 µg/ml and 40% respectively. This synchronizes with previous studies utilizing T. gondii tachyzoites (RH strain) and derivatives on H7402 HCC cells [ 115 ], T. gondii GRA16 on HepG2 HCC cells [ 116 ] and T. gondii GRA15II-polarized macrophages on Hepa1-6 cells [ 117 ]. These findings were further supported by in vivo studies where T. gondii GRA16 and T. gondii GRA15II-polarized macrophages significantly reduced tumor size in HCC murine models [ 116 , 117 ].

Furthermore, our study demonstrated that T. gondii IgG-positive human sera induced a significant inhibition of HepG2 HCC cell proliferation across all tested concentrations in a concentration-dependent manner. To the best of our knowledge, this is the first report of the antineoplastic activity of anti- T. gondii antibodies against HCC. Our result aligns with and supports the study by Seyedeh et al., 2015, which concluded that individuals with low anti- Toxoplasma antibody levels exhibited resistance to cancer [ 55 ].

On the other hand, contradictory results have been reported regarding Toxoplasma seropositivity in cancer patients. It has been suggested that approximately 40% of cancer patients have Toxoplasma IgG antibodies in their sera [ 118 – 121 ]. Some studies reported significantly elevated Toxoplasma IgG levels in cancer patients, with higher titers associated with a more advanced cancer grade [ 122 – 124 ]. Moreover, toxoplasmosis has been linked to higher mortality rates in different types of cancers, including Hodgkin’s lymphoma, leukemia, melanoma, and brain cancers [ 118 , 121 , 125 ]. However, these associations may be attributed to the immunosuppressive effects of cancer and the intake of chemotherapy, which increase susceptibility to opportunistic infections such as toxoplasmosis [ 126 ].

Our results for the antineoplastic activity of T. gondii immunomodulators against human cancer cell lines (HT-29 CRC and HepG2 HCC) are in line with other in vitro studies on BGC-823 gastric and esophageal squamous cell carcinoma (EC109) [ 127 , 128 ], mammary cancer cell line (TUBO cells) [ 129 ], MCF-7 human breast cancer cells [ 48 ], and non-small lung cancer cells (A549) [ 128 ]. This is in addition to murine lymphoma and mastocytoma cell lines [ 130 ], fibrosarcoma WEHI-164 cells [ 131 ], ovarian cell lines (A2780 & A2780-CP) [ 132 ], DU-145 prostate cell line [ 128 ], and human glioma U373MG & U87MG cells [ 21 ]. In vivo studies have further confirmed the antineoplastic activity of T. gondii in pancreatic cancer [ 14 , 17 , 133 – 135 ], mammary tumors [ 136 – 138 ], lung cancer [ 71 , 139 , 140 ], sarcoma [ 136 , 141 – 144 ], melanoma [ 15 , 18 , 71 , 145 – 147 ], ovarian tumors [ 15 , 148 ], glioma [ 21 ], ependymoblastoma [ 149 ], and thymoma murine models [ 150 ].

Notably, the current study revealed a significant in vitro antineoplastic activity of S. mansoni , T. spiralis , and T. gondii IgG-positive sera against HT-29 CRC cells and HepG2 HCC cells. Our results align with previous in vitro experimental studies in the current era, where studies demonstrated that anti-hydatid cyst antibodies exhibited antineoplastic activity on NCI-H209/An1 human small-cell lung cancer cells [ 45 ] and 4T1 breast cancer cell line [ 151 ]. Parasitic antibodies have been found to selectively attach to various cancer cell lines, supporting the theory of molecular mimicry. For example, anti- T.cruzi cross-reacted with human mammary cancer cells (T47D and MCF-7 cell lines), human colon cells [ 85 ] and acute lymphoblastic leukemia cells [ 152 ]. Additionally, T. cruzi proteins, as well as antibodies against both T. canis and Acanthamoeba , cross-reacted with neuroblastoma cells [ 152 , 153 ]. Interestingly, both anti- T. cruzi and anti-hydatid cyst antibodies reacted with lung cancer cells [ 154 , 155 ]. Surprisingly, only the anti- T. gondii antiserum showed significant reactivity with mouse melanoma cells, while antibodies from other parasites did not [ 156 ]. However, it should be reported that various parasites have demonstrated in vivo antineoplastic activity in hepatoma and HCC, such as S. japonicum [ 52 , 88 – 90 ], Plasmodium berghei [ 157 ], Plasmodium yoelii [ 19 , 158 ], Angiostrongylus cantonensis [ 159 ] and Setaria equine [ 51 , 160 ].

Notably, in the current study, at a low concentration of 3.125 µg/ ml of A Sm A and A Tg A and a low concentration of 2.5% for T. gondii IgG-positive sera, a biphasic effect was demonstrated on HepG2 HCC cells and HT-29 CRC cells, respectively, where significant cellular proliferation was observed. This biphasic response may suggest interaction with multiple cellular targets or signalling pathways. It could also be attributed to hormesis phenomena, receptor dynamics, or disruption of cellular homeostasis [ 161 ]. Therefore, our study emphasizes the importance of mechanistic investigations of parasites’ immunomodulators’ antineoplastic activity in pharmacological and toxicological studies.

Additionally, there is an essential need for in-depth investigational studies to establish a concrete foundation for selective identification of parasite/cancer types. Further elaborative studies should focus on purification of parasitic antibodies for further identification, as well as molecular characterization and identification of the most immunogenic antigenic components of the autoclaved parasitic antigens for later clinical applicability. This could lead to the development of nature-based anti-cancer candidates of parasitic origin for targeting cancer.

Conclusion

The perception of the value of parasites has been entirely switched due to their potential anti-cancerous effects observed in experimental studies. Collectively, this study demonstrated that S. mansoni , T. spiralis , and T. gondii immunomodulators have significant antineoplastic effects on HT-29 colorectal and HepG2 hepatocellular carcinoma cells. The highest antineoplastic activity was reported upon using T. spiralis immunomodulators and anti- T. gondii antibodies for colorectal cancer cells. For hepatocellular carcinoma cells, T. spiralis immunomodulators and T.gondii antigen demonstrated the highest antineoplastic activity. These findings could enlighten the path for promising cancer-fighting strategies. This could lead to the development of nature-based candidates of parasitic origin targeting cancer for future research and clinical applications.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Author contributions

N.E.S., M.M.E; conceptualization, methodology, laboratory experiment, data analysis, original draft preparation, reviewing, and editing. A.S; data analysis, data curation.

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval

The study protocol was approved by the Ethics Committee of the Faculty of Medicine, Alexandria University (Permit Number: 0306454).

Consent to participate

The participants/ their legal guardians gave informed consent for participation in the current study.

Consent for publication

All authors agreed to the publication of the manuscript.

Competing interests

The authors declare no competing interests.

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