IN VIVO STUDY OF POTENTIAL MECHANISMS OF MACROPHAGE REPOLARIZATION ON THE BACKGROUND OF TUMOR GROWTH.

AIM: To study the activity of antitumor immunity effectors and to analyze possible mechanisms of peritoneal Mph M1/M2 repolarization of Balb/c mice under the influence of lectin from B. subtilis IMV B-7724 in the dynamics of the model tumor growth. MATERIALS AND METHODS: Studies were performed on Balb/c mice; Ehrlich adenocarcinoma (АСЕ) was used as an experimental tumor. Lectin from B. subtilis IMV B-7724 was administered to ACE-bearing mice at a dose of 1 mg/kg of body weight, 10 times. Immunological testing was performed on days 21 and 28 after tumor grafting. The functional activity of peritoneal macrophages (Mph), natural killer (NK) cells, cytotoxic lymphocytes (CTL), and cytokine levels (IFN-γ, IL-4) were studied by the standard methods. mRNA expression levels of transcription factors STAT-1, STAT-6, IRF5, and IRF4 in Mph were evaluated. RESULTS: The administration of lectin from B. subtilis IMV B-7724 to mice with solid ACE led to the preservation of the initial functional state of peritoneal Mph M1 during the experiment. The bacterial lectin ensured the preservation of the cytotoxic activity of CD8+ T-lymphocytes and a significant (p < 0.05) increase in the NK activity (by 2.7 times compared to the intact animals and by 12.9 times compared to the untreated mice). A strong positive correlation was noted between the levels of the functional activity of Mph and CD8+ T-lymphocytes of animals with tumors and the indices of the antitumor effectiveness of bacterial lectin. The indirect polarization of Mph was evidenced by a strong positive correlation between the level of the NO/Arg ratio (which characterizes the direction of Mph polarization) and the cytotoxic activity of CD8+ T-lymphocytes, NK cells, and the expression of STAT1/STAT6 (the 21st day) and IRF5/IRF4 (the 28th day). CONCLUSION: In ACE-bearing mice, repolarization of the peritoneal Mph toward M1 can occur not only due to the direct action of bacterial lectin on the cellular receptors but also with the involvement of other effectors of antitumor immunity (NK cells, T-lymphocytes). The transcription factors of the STAT and IRF signaling pathways are involved in the polarization process.

АNTIMETASTATIC EFFECT OF B. SUBTILIS IMV B-7724 LECTIN OBSERVED IN LEWIS LUNG CARCINOMA MODEL.

AIM: To study the antitumor and antimetastatic effects of B. subtilis IMV B-7724 lectin used in neoadjuvant and adjuvant settings in vivo. MATERIALS AND METHODS: Studies were performed on C57Bl/6J mice; Lewis lung carcinoma (LLC) was used as an experimental tumor. В. subtilis ІМV В-7724 lectin was administered to tumor-bearing mice or to mice which underwent surgical resection of the primary tumor. The lectin was injected subcutaneously, 10 times, at a single dose of 5 or 1 mg/kg of body weight. The standard indicators of tumor growth and metastasis were evaluated. RESULTS: Independently of the application settings, the lectin at a dose of 1 mg/kg of b.w. caused more pronounced effect than at a dose of 5 mg/kg of b.w. The administration of B. subtilis IMV B-7724 lectin to the mice with LLC in neoadjuvant setting did not cause notable antitumor effect but led to a significant decrease in the number and volume of lung metastases. The lectin administration in adjuvant setting significantly inhibited metastasis: the metastasis inhibition index reached 63.0% and 100% in the mice treated with the lectin at a dose of 5 mg/kg and 1 mg/kg respectively. The mean survival time of the treated animals significantly increased. CONCLUSION: A pronounced antimetastatic effect of B. subtilis IMV B-7724 lectin administered in an adjuvant setting was demonstrated.

Macrophage polarization in dynamics of Lewis lung carcinoma growth and metastasis.

AIM: To assess the functional state of macrophages based on various manifestations of their activity at the different stages of metastatic tumor growth in C57Bl mice. MATERIALS AND METHODS: On days 7, 14, 21 and 28 after Lewis lung carcinoma transplantation to C57Bl mice, macrophages from various anatomic sites were isolated and tested on their cytotoxicity, metabolic activity, NO production and arginase activity. RESULTS: In the populations of peritoneal and splenic macrophages, on days 7 and 21 of tumor growth antitumor (M1) cells prevailed while on days 14 and 28 tumor-promoting (M2) macrophages predominated. In the population of lung macrophages, cells with M1 phenotype were in the majority in the early stages of tumor growth. On days 21 and 28, M1 cells were gradually substituted by cells exhibiting M2 phenotype. This shift correlated with metastasis to lungs. CONCLUSION: Lewis lung carcinoma growth is accompanied by the gradual change in macrophage polarization from antitumor (M1) towards tumor-promoting (M2) type. These changes were more evident in population of lung macrophages and correlated with the parameters of metastasis.

Functions of tumor-associated macrophages and macrophages residing in remote anatomical niches in Ehrlich carcinoma bearing mice.

BACKGROUND: The impact of growing tumor on polarization and functions of tumor-associated macrophages is well known while its influence on residential macrophages occupying different anatomical niches reminds to be elucidated. AIM: To study changes in polarization and functions of macrophages isolated from discrete anatomical niches in tumor-bearing mice at different stages of tumor growth. MATERIALS AND METHODS: Ehrlich carcinoma was transplanted intramuscularly to Balb/c male mice. On days 7, 14, 21 and 28 after tumor transplantation, macrophages from tumor tissue, peritoneal cavity and spleen were isolated and analyzed. Nitric oxide production was measured by standard Griess reaction, arginase activity was determined by the measurement of urea, reactive oxygen species production was checked using NBT dye reduction assay and electron paramagnetic resonance spectroscopy, cytotoxic activity was estimated in MTT-assay. RESULTS: Independently of their localization in different anatomic niches, macrophages in mice with transplanted Ehrlich carcinoma gradually lose their tumoricidal activities while arginase activity is upregulated. This indicates the shift of polarization from M1-like towards M2-like phenotype. CONCLUSION: Our findings demonstrated that growing tumor could be able to subvert functioning of macrophages at the systemic level.

The effects of early postoperative immunization with xenogeneic embryo proteins on Lewis lung carcinoma model.

AIM: To investigate the effect of chicken embryo proteins (CEP) as a prototype of xenogeneic vaccine on immune reactions in mice immunized after Lewis lung carcinoma (LLC) surgical removal. MATERIALS AND METHODS: C57Bl male mice were immunized on days 1, 8, and 15 after surgical removal of LLC. The immune response was assessed on days 7, 14, 21 and 28 after tumor resection. Cytotoxic activity of natural killer cells (NK) and cytotoxic T-lymphocytes as well as antibody dependent cellular cytotoxicity was estimated in MTT-assay; specific antibodies were detected in ELISA; lymphocyte proliferation was tested in reaction of in vitro blast transformation. RESULTS: None of the immunized mice developed LLC metastases. Immunization with CEP seems to prevent the potential decrease in NK cell cytotoxic activity and spontaneous blast transformation activity of lymphocytes following the surgically induced stress. Further research on improving immunization schedule and elucidating the mechanisms of NK modulation with CEP is needed.

Anticancer effect and immunologic response to xenogeneic embryonic proteins in mice bearing Ehrlich solid carcinoma.

AIM: To investigate anticancer and immunologic effects of chicken embryonic proteins (CEP) in mice bearing Ehrlich solid carcinoma. MATERIALS AND METHODS: The study was carried out on male Balb/c mice bearing Ehrlich solid carcinoma. The immunizations were performed after the tumor transplantation. The immune status was assessed on days 7, 14, 21 and 28 after the tumor challenge. Cytotoxic activity (CAT) of macrophages (Mph), natural killer cells (NK), cytotoxic T-lymphocytes (CTL) and blood serum, as well as the influence of the blood serum on immune cells activity was checked in MTT-assay; Mph's cytochemical activity was tested in NBT-assay; Ehrlich antigen-specific or CEP-specific antibodies were detected in ELISA-assay; medium size circulating immune complexes (CIC) were detected in reaction of 4.5% polyethylene glycol precipitation. RESULTS: The immunization resulted in tumor growth suppression and significant 25.64% prolongation of the survival time. In both control and immunized mice with transplanted tumors antibodies specific to Ehrlich carcinoma antigens and to CEP were detected, but antibody response was more balanced in the treatment group. In the treatment group both cytochemical and CAT of Mph was moderately activated and well preserved until late stages of tumor development; CAT of NK and CTL remained in the range of the intact mice until day 28 after the tumor transplantation. The immunized mice were well protected from accumulation of CIC and suppressive activity of autologous blood serum. CONCLUSION: Collectively, our data indicate that CEP can elicit immunomodulating and immunoprotecting effects sufficient to provide tumor growth inhibition. The further elaboration of a xenogeneic anticancer vaccine based on CEP is warranted.

Anticancer effectiveness of vaccination based on xenogeneic embryo proteins applied in different schedules.

THE AIM: To evaluate anticancer activity of vaccination with chicken embryo proteins (CEP) applied in different schedules. MATERIALS AND METHODS: C57Bl mice were vaccinated with CEP before (prophylactic schedule) or after (different therapeutic schedules with or without preliminary tumor removal) the Lewis lung carcinoma cells transplantation. The latent period of tumor development, tumor volume and metastasis rate were evaluated. RESULTS: Potent antimetastatic effect of CEP-based vaccination was seen in case of therapeutic regimen after primary tumor removal. The metastasis inhibition index (MII) reached 96.9 and 97.8% on 18(th) and 34(th) day after tumor removal, respectively. When CEP vaccination was performed in the settings of therapeutic regimen without primary tumor removal the anticancer effect was evident only if vaccinations started as early as 24 h after the cancer cells injections. The highest MII achieved in such condition was 77.6%, tumor volume in the group of vaccinated animals was by 53.1-42.1% lower than in the control tumor-bearing mice. CEP vaccination before tumor challenge (prophylactic immunization) led to a statistically significant prolongation of the latent period of tumor development, a reduction of tumor volume (35.8-48.8% compared to control unvaccinated mice) and a marked inhibition of metastasis (MII was 71.1%). CONCLUSION: Vaccination based on CEP exhibited both prophylactic and therapeutic anticancer effects. The last one is more pronounced when the vaccination starts shortly after the primary tumor resection.

Cytotoxic activity of immune cells following administration of xenogeneic cancer vaccine in mice with melanoma B-16.

AIM: To study the effects of xenogeneic cancer vaccine (XCV) developed on the basis of nervous tissue antigen from rat embryo of late gestation period and protein-containing metabolite of Bacillus subtilis with molecular weight of 70 kDa, on specific and unspecific antitumor reactions of cellular and humoral chains of immune system, and to analyze possible mechanisms of its antimetastatic action. MATERIALS AND METHODS: XCV was administered triply with 3-day intervals after surgical removal of experimental melanoma В-16 in C57Bl/6 mice. Cytotoxic activity (CTA) of splenocytes against target cells К-562 as well as CTA of splenocytes, peritoneal macrophages (PM) and blood serum against melanoma В-16 target cells were determined using МТТ test. The content of circulating immune complexes (CIC) in blood serum was evaluated by precipitation reaction. RESULTS: Immunologic effects of XCV vaccination in experimental animals with surgically removed melanoma B-16 in comparison with similarly treated unvaccinated mice were as follows: prevention of medium molecular weight CIC accumulation in blood serum during all observation period, significant increase (р < 0.05) of CTA of effectors of unspecific antitumor immunity (natural killer cells - NK - by 25.5 ± 1.7 vs 12.5 ± 5.4%, and PM - by 37.3 ± 0.6 vs 32.0 ± 0.9%, respectively) at 37(th) day after the surgery, and also preservation of functional activity of specific cytotoxic lymphocytes at the level of intact control. CONCLUSION: The results of the study allow propose that antimetastatic effect of XCV vaccination could be based on increased CTA of NK and PM, and preservation of CTL functional activity at late terms after surgical removal of B-16 primary tumors.

The anticancer efficiency of the xenogeneic vaccine and the indication for its use.

AIM: To investigate the anticancer efficiency of the xenogeneic vaccine in different tumor models and to assess the possibility whether level of antibodies (Ab) specific for vaccine's proteins can be used as an indication for its use. METHODS: Mice with Lewis lung carcinoma (LLC), Ehrlich carcinoma (EC) or Sarcoma 37 (S37) were immunized with a xenogeneic anticancer vaccine based on chicken embryo proteins (CEP) and its anticancer activity was examined. The level of specific Ab in the blood serum of non-immunized tumor-bearing mice was studied by ELISA. RESULTS: CEP application statically significantly inhibited the growth of LLC (the index of tumor growth inhibition was 42.10-53.13% depending on the day of tumor growth); vaccinated mice with EC showed significant tumor growth inhibition and life prolongation by 34.48%. Among mice with S37, there was noticed no antitumor effect. The number of tumor-bearing non-immunized mice which have had pre-existing CEP-specific Ab did not differ depending on the tumor model. The level of CEP-specific Ab among mice with LLC and EC increased with the growth of the tumor volume, but it decreased among mice be-aring S37. Probably, the low level of CEP-specific Ab alongside huge tumor burden shows it is futile to apply the CEP-based vaccine. CONCLUSION: Different tumor strains vary in their susceptibility to CEP-based vaccine. Probably, the low level of CEP-specific Ab when a tumor burden is huge shows it is futile to apply the CEP-based vaccine. Key Words: xenogeneic anticancer vaccine, chicken embryo proteins anticancer activity, Lewis lung carcinoma, Ehrlich carcinoma, Sarcoma 37, CEP-specific antibodies.

Xenogenic cancer vaccines.

In the current review the latest literature data on design of cancer vaccines on the basis of xenogenic tumor-associated antigens and proteins that play an important role in tumor progression have been summarized. The main benefits and disadvantages of xenogenic cancer vaccines, the results of experimental studies and clinical trials, and perspectives of use are analyzed.