Trypanosoma cruzi Antigenic Proteins Shared with Acute Lymphoblastic Leukemia and Neuroblastoma

Background. Research studies indicate that immunization with protein extracts of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, prevents the appearance of tumors in 60% of mice injected with the murine lung carcinoma tumor line. The molecular basis of this process is unknown, although the presence of specific antigens in tumor cells and on the surface of T. cruzi suggests an antiparasitic immune response, with an effective cross-reaction against cancer cells, hence the importance to identify the antigens involved and determine their potential as target cells in anticancer therapy. Aim. This study aimed to determine the presence of antigenic proteins of T. cruzi shared with acute lymphoblastic leukemia and neuroblastoma cells. Material and methods. To achieve this, polyclonal antibodies against T. cruzi were developed in rabbits, and reactivity was determined with protein extracts of acute lymphoblastic leukemia cells and neuroblastoma. The immunodetection of five different strains of T. cruzi against anti-T. cruzi polyclonal antibodies was also performed. Conclusion. The study allows the knowledge of the immunological interactions between cancer and parasites to be expanded and, therefore, contributes to the design of more and better projects that improve the therapeutic strategies applied in oncology.


Introduction
Cancer is a generic term that designates a wide group of diseases that can affect any part of the body. A defining characteristic of cancer is the rapid multiplication of abnormal cells that spread beyond their normal limits and can invade adjacent parts of the body or spread to other organs. This process, called metastasis, is the leading cause of cancer death [1]. Although incidence rates for all cancers combined are twice in more developed countries with respect to less developed ones, death rates are only 8% to 15% higher in developed countries due to risk factors, screening practices, and availability of treatment [2]. In Mexico, cancer is the third leading cause of death, 14/100 Mexicans die from this disease and 45.3% of deaths occur in the economically active population [3].
Acute lymphoblastic leukemia (ALL) is a malignant transformation and proliferation of lymphoid progenitor cells in the bone marrow, blood, and extramedullary sites. Overall, 80% occurs in children and it represents a devastating disease when it occurs in adults. The incidence of ALL follows a bimodal distribution, with the first peak in childhood and the second peak around 50 years old [4]. Symptoms of ALL include enlarged lymph nodes, bruising, fever, bone pain, frequent infections, and bleeding gums. Treatment may include chemotherapy or local-release drugs that kill cancer cells. Although dose escalation strategies have led to a significant improvement in outcomes for pediatric patients, the prognosis for the elderly remains extremely poor [5].
Neuroblastoma (NB) is an embryonic autonomic nervous system tumor usually found in the adrenal glands [6]. It can develop in the stomach, chest, neck, pelvis, and bones. It affects children under five years of age, and it is the most common cancer diagnosed during the first year of life. The most frequent symptoms are fatigue, loss of appetite, and fever. The clinical presentation is highly variable, from a mass that does not cause symptoms to a primary tumor that causes critical illness because of local invasion, widely disseminated disease, or both [7].
In this work, it was decided to work with ALL and NB because they are two of the most frequently diagnosed in children and adults, respectively, and because of the lack of studies to recognize antigens shared between these oncological cells and Trypanosoma. cruzi.
Parasitic infections caused by protozoa and helminths induce chronic inflammation that, added to cell divisions of the healing process, favors the appearance of cancer [8][9][10]. However, there is scientific evidence that some species could help in the fight against tumors. The design of effective vaccines for the treatment of cancer is one of the main challenges in cancer research. Several nonpathogenic parasites, such as Leishmania tarentolae, Toxoplasma gondii, and T. cruzi, have been used as candidates for designing cancer vaccines [11]. There is growing experimental and clinical evidence that the immune system is actively involved in the pathogenesis and control of tumor progression. An effective antitumor response depends on the correct interaction of various components of the immune system, such as antigen-presenting cells and different subpopulations of T cells. However, malignant tumors develop efficient mechanisms to evade their recognition and elimination. The beneficial effects reported for parasitic diseases in tumorigenesis range from the induction of apoptosis, the activation of the immune response, the avoidance of metastasis and angiogenesis, and the inhibition of proliferative signals to the regulation of inflammatory responses that promote cancer [12]. It has been reported for the parasitic malaria infection that inhibits cell proliferation in lung cancer [13] or by intratumoral injection of attenuated T. gondii for cases of melanoma [14]. It was also shown that, when administered to mice, three peptides from E. granulosus confer splenocytes with the ability to kill tumor cells. Furthermore, this cytotoxic activity was correlated with a higher number of activated NKs, which suggests that the immune response may be efficient to eliminate tumor cells in the presence of this infection [15]. E. granulosus is a cestode parasite that causes cystic echinococcosis disease. In a retrospective study, a significantly lower prevalence of cancer was seen in patients with hydatid disease [16]. Antigenic similarities were found between E. granulosus and some types of tumors [17]. As well as this, immunization with human hydatid cystic fluid (HCF) induces antibodies against CT26 colon carcinoma cells and protects against tumor growth in mice [18].
It has also been proven that T. cruzi, the protozoan parasite that causes Chagas disease, has mediated anticancer effects, with by-products derived from the parasite that inhibit the growth of tumor cells, involving both the cellular and humoral components of the immune response. Therefore, it is proposed to develop polyclonal antibodies against T. cruzi in rabbits and to determine the reactivity with NB tumor protein extracts and with protein extract of ALL cells in culture to determine the presence of the molecules involved in this process. The objective of this study was to determine the presence of antigenic proteins of T. cruzi shared with protein extract of cells in culture of ALL and extract protein of NB.

Results and Discussion
T. cruzi, the protozoan parasite that causes Chagas disease, shows anticancer effects through the presence of proteins that inhibit tumor cell growth [19]. There are scientific reports that show various molecular targets have the antitumor ability. However, it has not been determined which is the molecul with anticancer activity, nor the process that is carried out. The studies have been conducted in cell cultures and experimental animals and suggest the following facts: T. cruzi has antitumor effects by inducing host immunity against tumors. T. cruzi expresses calreticulin that can directly interact with endothelial cells and inhibit their proliferation, migration, and capillary morphogenesis, as well as inhibit tumor cells [20,21].

Determination of Antibodies by ELISA
The results obtained from ELISA immunodetection are shown in Figure 1. The protein extract of ALL culture was used as a target antigen. Subsequently, purified sera from hyperimmunized rabbits with the five different isolates of T. cruzi were used as the first antibody, and anti-rabbit IgG horseradish peroxidase (HRP) enzyme conjugate as a detection antibody. The substrate for the enzyme was added to quantify the primary antibody through a color change. The OD values were read at 450 nm with a reference filter of 620 nm in a spectrophotometer. The ALL control had an Ab titer of 2.674 and, for T. cruzi, it was from 1.228 (Ninoa) to 2.664 (Cuernavaca). and suggest the following facts: T. cruzi has antitumor effects by inducing host i against tumors. T. cruzi expresses calreticulin that can directly interact with en cells and inhibit their proliferation, migration, and capillary morphogenesis, a inhibit tumor cells [20,21].

Determination of Antibodies by ELISA
The results obtained from ELISA immunodetection are shown in Figure 1. tein extract of ALL culture was used as a target antigen. Subsequently, purified s hyperimmunized rabbits with the five different isolates of T. cruzi were used as antibody, and anti-rabbit IgG horseradish peroxidase (HRP) enzyme conjugate tection antibody. The substrate for the enzyme was added to quantify the prim body through a color change. The OD values were read at 450 nm with a refere of 620 nm in a spectrophotometer. The ALL control had an Ab titer of 2.674 an cruzi, it was from 1.228 (Ninoa) to 2.664 (Cuernavaca). The immunization of laboratory animals with total extracts of T. cruzi s lowed sera with a high antibody titer to be obtained, determined by ELISA (gre 1.6); however, it is observed that the immunogenicity is different between them Silvio and Ninoa isolates present a lower test titer ( Figure 1). A dot blot was perf ensure that there was an immunogenic reaction between the immunized serum protein extract of the ALL cells in culture and, although the detection signal was ensured the presence of polyclonal antibodies, which could be used in the Wes method. The immunization of laboratory animals with total extracts of T. cruzi strains allowed sera with a high antibody titer to be obtained, determined by ELISA (greater than 1.6); however, it is observed that the immunogenicity is different between them since the Silvio and Ninoa isolates present a lower test titer ( Figure 1). A dot blot was performed to ensure that there was an immunogenic reaction between the immunized serum and the protein extract of the ALL cells in culture and, although the detection signal was weak, it ensured the presence of polyclonal antibodies, which could be used in the Western blot method.

SDS Polyacrylamide Gel Electrophoresis
The recognition by Western blotting of the ALL and NB antigens (total extract) shows different specific bands for each isolate. Figure 2 shows the electrophoretic patterns of cell extract ALL and NB, respectively; both were run by 12% SDS PAGE under the same running conditions.

SDS Polyacrylamide Gel Electrophoresis
The recognition by Western blotting of the ALL and NB antigens (total shows different specific bands for each isolate. Figure 2 shows the electrophor terns of cell extract ALL and NB, respectively; both were run by 12% SDS PAG the same running conditions.

Immunodetection
The anti-T. cruzi antibodies were used to detect the presence of proteins sha the electrophoresed cells of ALL and NB. In the case of ALL, with the strai Cuernavaca, and Querétaro, while, with NB, antigenic proteins were immunodet all strains (Figure 3). The homologous immunodetection of Ab was made by Wes using the protein extract of T. cruzi strain as antigen and immunodetected with T. cruzi of each of five strains of T. cruzi ( Figure 4).

Immunodetection
The anti-T. cruzi antibodies were used to detect the presence of proteins shared with the electrophoresed cells of ALL and NB. In the case of ALL, with the strains CLB, Cuernavaca, and Querétaro, while, with NB, antigenic proteins were immunodetected in all strains (     Figure 2 shows the different electrophoretic patterns of a cell extract of ALL and NB, writing down the presence of proteins that vary in size and pH and are the basis for the later performance of Western blot. The results of the Western blot ( Figure 3) show that T. cruzi do share antigenic molecules with the ALL cells in culture and the NB extract developed in a mouse, which was recognized by specific antibodies present in serum. In the case of ALL, the recognition was with antibodies from CLB, Queretaro, and Cuernavaca strains, where the recognition of fragments of 95, 75, 70, 55, 50, and 43 KDa is seen. With the antibodies against Silvio and Ninoa strains, they did not show recognition, so they are not included in the figure. Regarding NB, recognition is observed with the immunized sera of the five strains, showing bands of 140, 90, 85, 80, 70, and 55 KDa. The bands recognized by the antibodies were observed with different intensities according to the heterogeneity of concentration in the cell extract. In this case, there was a reaction with the five strains, unlike ALL, which suggests that the genetic plasticity of T. cruzi also influences the recognition of shared antigenic proteins.
This plasticity is evidenced in Figure 4, where immunized serum against T. cruzi isolates recognized antigens in homologous protein extracts and, among them, the presence of different antigens is observed, which may be the origin of the differences between strains when recognizing antigens of ALL and NB. Reported research says that the antitumor capacity of T. cruzi is influenced by the discrete typing unit (DTU) to which the strain corresponds [22].
The heterologous immunodetection shows that there are shared antigens between the extracts of ALL and NB cells and T. cruzi cells and, on the other hand, the homologous immunodetection shows that the protein plasticity between strains of T. cruzi is remarkably high, since the antibodies obtained from each one by immunization in rabbits shows significant differences. Although the presence of T. cruzi and other infectious organisms have been reported to be involved in carcinogenesis, some of them with antitumor capacity have also been reported [23]. This study is a contribution to the knowledge of proteins involved in the mechanism that causes T. cruzi proteins to induce a decrease in oncological processes since the specific mechanisms of action are still not clear.   Figure 3) show that T. cruzi do share antigenic molecules with the ALL cells in culture and the NB extract developed in a mouse, which was recognized by specific antibodies present in serum. In the case of ALL, the recognition was with antibodies from CLB, Queretaro, and Cuernavaca strains, where the recognition of fragments of 95, 75, 70, 55, 50, and 43 KDa is seen. With the antibodies against Silvio and Ninoa strains, they did not show recognition, so they are not included in the figure. Regarding NB, recognition is observed with the immunized sera of the five strains, showing bands of 140, 90, 85, 80, 70, and 55 KDa. The bands recognized by the antibodies were observed with different intensities according to the heterogeneity of concentration in the cell extract. In this case, there was a reaction with the five strains, unlike ALL, which suggests that the genetic plasticity of T. cruzi also influences the recognition of shared antigenic proteins.
This plasticity is evidenced in Figure 4, where immunized serum against T. cruzi isolates recognized antigens in homologous protein extracts and, among them, the presence of different antigens is observed, which may be the origin of the differences between strains when recognizing antigens of ALL and NB. Reported research says that the antitumor capacity of T. cruzi is influenced by the discrete typing unit (DTU) to which the strain corresponds [22].
The heterologous immunodetection shows that there are shared antigens between the extracts of ALL and NB cells and T. cruzi cells and, on the other hand, the homologous immunodetection shows that the protein plasticity between strains of T. cruzi is remarkably high, since the antibodies obtained from each one by immunization in rabbits shows significant differences. Although the presence of T. cruzi and other infectious organisms have been reported to be involved in carcinogenesis, some of them with antitumor capacity have also been reported [23]. This study is a contribution to the knowledge of proteins involved in the mechanism that causes T. cruzi proteins to induce a decrease in oncological processes since the specific mechanisms of action are still not clear.  Figure 5 shows the result of the confocal microscopy assay with ALL and NB cells against anti-T. cruzi antibodies, using DAPI and Alexa 488 as fluorogenic markers. Rabbit pre-immune serum was used as a negative control and T. cruzi cells were used to show the specificity of the antibodies.  Figure 5 shows the result of the confocal microscopy assay with ALL and NB cells against anti-T. cruzi antibodies, using DAPI and Alexa 488 as fluorogenic markers. Rabbit pre-immune serum was used as a negative control and T. cruzi cells were used to show the specificity of the antibodies. Confocal microscopy assay showed the presence of antigenic proteins by immunodetection in ALL and NB cells with antibodies against T. cruzi. Recognition is more intense in NB cells than in ALL cells. These results show the shared proteins of T. cruzi with ALL and NB cells. Therefore, it sets up the need for further research to find which are the most important and how they participate in the anticancer capacity of Trypanosoma. This study suggests that infection by T. cruzi generates a powerful immune response capable of enhancing and directing an efficient immune response against oncological cells. T. cruzi antigens have common epitopes with mammalian mucins [24] and with glycoproteins that share sialyl-Tn-like structures (specific human structures associated with cancer) [25]. We think all researchers are necessary to carry out studies of the antitumor action of T. cruzi and their products for the discovery and use of new molecules that may be proposed as therapeutic alternatives in the treatment of cancer. Confocal microscopy assay showed the presence of antigenic proteins by immunodetection in ALL and NB cells with antibodies against T. cruzi. Recognition is more intense in NB cells than in ALL cells. These results show the shared proteins of T. cruzi with ALL and NB cells. Therefore, it sets up the need for further research to find which are the most important and how they participate in the anticancer capacity of Trypanosoma. This study suggests that infection by T. cruzi generates a powerful immune response capable of enhancing and directing an efficient immune response against oncological cells. T. cruzi antigens have common epitopes with mammalian mucins [24] and with glycoproteins that share sialyl-Tn-like structures (specific human structures associated with cancer) [25]. We think all researchers are necessary to carry out studies of the antitumor action of T. cruzi and their products for the discovery and use of new molecules that may be proposed as therapeutic alternatives in the treatment of cancer.

Production and Determination of Antibodies against T. cruzi in New Zealand Rabbits
A hyperimmune anti-T. cruzi serum was attempted by immunizing a male New Zealand rabbit (about 6 weeks old). Blood was drawn before the first immunization, and this was the pre-immune serum used as a control. It was administered subcutaneously with 300 µg of total extract of T. cruzi epimastigotes (axenic culture) strain CL Brener in 500 µL of complete Freund's adjuvant; 15 days later 300 µg of total extract was administered with 500 µL of incomplete Freund's adjuvant. Later, on days 29, 30, and 31, 100 µg of total extract dissolved in a saline solution were administered intravenously; finally, on day 39, venous blood was taken from the rabbit, which was centrifuged to separate the serum, which was collected, aliquoted, and stored at −20 • C. The total IgG antibodies present in the serum were purified from the polyclonal serum generated in a rabbit. For this, a protein A bound to agarose (22811, Thermo Fischer Scientific, Waltham, MA, USA) was used. The anti-T. cruzi antibody titer was obtained in the serum of the immunized rabbits by ELISA.
The OD values were read at 450 nm with a reference filter of 620 nm in an ELISA reader (ELISA LKB Wallac Olivetti AU). Each assay plate included negative and positive samples as controls [26].

Parasite Culture
Four different isolates of T. cruzi from infected humans in Mexico: Ninoa, Silvio, Queretaro, Cuernavaca, and strain CLB, were used to obtain specific antibodies of different isolates. Epimastigotes of four isolates and CLB control strain of T. cruzi were cultured and kept at 28 • C in 50 mL Nunc boxes of liver infusion tryptose (LIT) medium pH 7.2. After a period of 8 days, approximately 1 × 108 parasites/mL were obtained [27].

Antigen Preparation
The parasites of isolates of T. cruzi were harvested in 50 mL Falcon tubes, the medium was removed by centrifugation at 2000 rpm for 20 min, the supernatant was removed, and two washes were carried out with saline solution at 1500 rpm for 5 min. Parasites were counted in a Neubauer chamber. The button formed by parasites was resuspended in 1 mL of lysis buffer with protease inhibitors and cell disruption was performed by vortexing for 5 min, followed by three freeze/thaw cycles [30].

Cell Extract of ALL
The extract was prepared from cultures of ALL Line Sup B15 in the exponential growth phase, washed 2 times with PBS, and subsequently incubated in a hypotonic solution (10 mM Tris, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 ) supplemented with protease inhibitors at a final concentration of 500 mM EDTA, 20 mM PMSF, 10 mM leupeptin, and 1 mM pepstatin for 30 min on ice. The samples were sonicated with 6 pulses of 10 s, the material obtained was centrifuged at 4600 rpm for 10 min, and the supernatant was stored at −20 • C until use. The protein concentration of the parasitic extracts and the cell lines used was measured by spectrophotometry [31].

SDS Polyacrylamide Gel Electrophoresis
A dot blot method was conducted with 1 mg/mL of Ag from T. cruzi isolates and immunodetected with the first antibody (inoculated rabbit serum) diluted 1:50. Protein extract was treated with bromophenol blue and then heated to boiling for 15 min, thus achieving a Pharmaceuticals 2022, 15, 1421 8 of 10 more stable union between the SDS and the protein. Treated antigens (from T. cruzi or ALL proteins) were run in a polyacrylamide gel electrophoresis 12% for 40 min at 200 V.

Immunodetection
Electrophoresed proteins were transferred to a 0.2 µM nitrocellulose membrane (Trans-Blot-Transfer Medium Bio-Rad) for 3 h at 350 mA, 120 V in transfer buffer. The membrane was blocked overnight at 4 • C with 5% milk PBS, the strips of the membrane of approximately 1 cm were cut, the serum of the anti-T. cruzi rabbits diluted 1:50 in milk PBS was added to the 1% and incubated for 2 h at 37 • C, and positive controls were used to validate this test. After the incubation time, three washes with 0.1% PBS Tween were carried out, and the second anti-rabbit IgG antibody coupled to horseradish peroxidase was added in a 1:1000 dilution with 1% milk PBS, after which seven washes with PBS 0.1% Tween were performed. The strips were developed by adding the substrate 4 Chloro Naphthol (SIGMA); the reaction was carried out at room temperature for approximately 30 min and stopped with distilled water [32,33].

Confocal Microscopy
Sub15 ALL cells were cultured in RPMI 1640 medium (supplemented with 10% FBS and 1× antibiotic/antimycotic) and NB cells in DMEM medium. The axenic culture of T. cruzi was used as a positive control. Cells were harvested and washed with PBS. A total of 50,000 viable cells were fixed with 2% paraformaldehyde for 15 min. After 40 min of washing with PBS, it was blocked with 2% pig serum in PBS for 1 h and the permeabilized slides were incubated with 2% pig serum and 0.5% Triton in PBS for 20 min. They were incubated for 1 h with T. cruzi antibodies 1:300 with 2% pig serum in PBS. IgG purified from unimmunized New Zealand rabbit serum was used as a negative control. Reactivity was carried out with specific anti-rabbit IgG antibodies conjugated to Alexa-Fluor 488 (Invitrogen cat no. A-11034) diluted 1:300 with 2% pig serum in PBS for 1 h. Cell nuclei were stained with 20 µg/mL of 4,'6-diamidino-2-phenylindole dilactate (DAPI) (Invitrogen cat no. D1306). The images were taken with a Leica model TCS-5P8X confocal microscope, with a 63× immersion lens, 5× digital zoom, and analyzed with the LasX software.