EFFICIENCY OF XENOGENEIC ANTITUMOR VACCINE IN VIVO

DOI: 10.32471/oncology.2663-7928.t-24-1-2022-g.10377
The limited success of the existing methods of cancer treatment (especially at the late stages) requires for the search and development of new more effective means of treatment. High hopes are put on biotherapy, which main objective is to develop methods for amplifying immune response of the body. To reach this goal, antitumor vaccines (AVs), co-stimulatory cytokines and molecules to enhance antitumor immune response are elaborating.

The autologous AVs which are constructed based on tumor materials of cancer patients are widely used in modern medicine. A common problem of such vaccines is that the tumor-associated antigens (TAAs) have low immunogenic activity [1]. The immunological potency of the vaccines can be enforced with the help of microbial-derived adjuvants. They can attract toll-like receptors on the surface of lymphocytes to enhance the immune response and thus they can stimulate the synthesis of cytokines.

Another approach to overcome immune tolerance and to induce immune response against endogenous proteins is the use of foreign counterparts of TAAs [2, 3]. As long as most TAAs are conserved, they bare high degree of homology between human proteins and their counterparts in different animal species [4]. Therefore, minor structural interspecies differences can be successfully used to induce an effective immune response to poorly immunogenic tumor antigens [5–7].

It is a well-known fact that cancer cells express oncofetal antigens. These include the proteins that are expressed at certain stages of embryonic development but are rarely detected on normal adult cells. The use of embryonic proteins as «universal» specific immunogens opens new possibilities for constructing AVs [8, 5].

In the R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology (IEPOR) elaboration of a new approach to cancer vaccines development based on extracts of embryonic xenogeneic proteins combined with microbially-derived adjuvants is going on [9, 10]. The constructed xenogeneic AV (XAV) consists of chicken embryo proteins of high molecular weight and cytotoxic metabolite of Bacillus subtilis B-7025. The scope of the research was to study the anticancer efficacy and some immunological effects of the XAV in mice bearing Lewis lung carcinoma (LLC) or Ehrlich carcinoma (EC).

OBJECT AND METHODS

Experimental animals and tumor models. Research was carried out on C57Bl (10 animals per each group, males 2.5 months, 20–22 g, vivarium R.E. Kavetsky IEPOR National Academy of Science of Ukraine) and Balb/c mice (10 animals per each group, males, 2.5–3 months, 20–22 g, IEPOR vivarium). The use and care of experimental animals have been performed in accordance with the standard international rules on biologic ethics and was approved by Institutional Animal Care and Use Committee [11].

LLC and EC were used as experimental models.

Balb/c mice were injected with EC cells (3•105 cells per animal) intramuscularly in the thigh hind limb.

C57Bl mice were transplanted with different doses of LLC depending on the further experiment they were planned to be used in.

In mice which were planned to be immunized after tumor challenge, LLC cells were injected intramuscularly in the thigh hind limb (3•105 cells per animal).

In mice which were planned to be immunized after the surgical removal of the primary tumor, LLC cells were injected in the hind foot at a dose of 1•105 cells/mouse. On day 13 of tumor growth, when the primary tumor reached 0.6±0.2 cm in diameter, mice underwent surgical removal of the tumor. In brief, mice were injected with etaminal-sodium anesthesia (40 mg/kg of body weight), ligature above the site of operation was imposed and the tumor-bearing foot was removed. The wound was treated with iodine infusion.

Animals transplanted with LLC cells were divided on four groups:

1 — control group of tumor-bearing animals without surgery (control tumor-bearing);

2 — tumor-bearing animals treated with XAV (tumor + XAV);

3 — animals with removed tumor (RT);

4 — animals with RT treated with XAV (RT + XAV).

Experimental schedule. In LLC-bearing mice, vaccination started on the second day after tumor transplantation and continued on day 5; 12 and 19 of tumor growth (0.3 mg/ml in 0.3 ml per animal per injection).

In mice which underwent LLC-tumor resection, vaccination started on the second day after tumor removal (day 15 of the tumor growth) and continued on day 17; 24 and 31 after tumor transplantation.

On day 34 after tumor transplantation, all the animals were devitalized and subjected to immunological investigation.

In mice transplanted with EC-tumor, immunization schedule was the same as for the LLC-bearing mice: it started on the second day after tumor transplantation and continued on day 5; 12 and 19 of tumor growth. The XAV was injected in a dose of 0.3 mg/ml per animal per injection. On day 24 of tumor growth, all experimental mice were devitalized and material for immunological studies was taken.

XAV preparation. The XAV was constructed and studied at R.E. Kavetsky IEPOR, National Academy of Science of Ukraine. The XAV is composed of chicken embryonic proteins of high molecular weight extracted with EDTA (2% in Tris buffer, 1 h) [12], and cytotoxic metabolite of B. subtilis B-7025 [13]. The concentration of embryonic proteins and cytotoxic metabolite of B. subtilis B-7025 in the vaccine solution is 0.3 mg/ml for both components.

Methods of experimental oncology. In LLC-bearing mice, on day 34 of tumor growth the primary tumor was removed and weighted. The lungs of all LLC-transplanted mice were removed and checked for the metastases. The level of lung metastases was determined by counting the metastases number.

In EC-bearing mice the tumor diameter was measured by the calipers and the tumor volume was calculated by the formula: V=¹/₆•πd3, where d — the diameter of the tumor nodule.

The survival rate of animals was determined by the percentage of alive animals in the group to the total number of animals in the group at the beginning of the experiment.

The mean survival time of animals in each group was determined by the survival time of each mouse in a group.

Analysis of immune cells cytotoxic activity. The functional activity of immune cells was evaluated in MTT-assay [14, 15]. The target cells (either LLC or EC cells, in concentration of 3•105 cells in 100 µl of culture medium) and immune cells (either Lph or Mph in concentration of 1•106 in 100 µl of culture medium) were placed in 96-wells flat-bottomed plates and incubated for 18 h in 5% CO2, 100% of humidity, 37 °C. MTT (Sigma, USA) in concentration of 5 mg/ml was added in quantity of 0.02 ml per well and incubation continued for 4 hours more under similar conditions, after that plates were washed twice with NaCl. To resolve formazan granules, 0.12 ml (2 mol/L) of KOH and 0.14 ml of 50% DMSO solution were added to the sediment. Optical density was measured at λ = 545 nm vs λ = 630 nm with a micro ELISA reader (StatFax-2100, USA). All samples were in triplicate. Cytotoxic Activity Index (%) was calculated by the formula:

(((Аі+Аt)-Aі+t)/Aі+t)•100%, where

Ai — absorbance in the wells containing only immune cells (Lph or Mph);

At — absorbance in wells containing only target cells;

Ai+t — absorbance the in wells where immune cells and tumor target cells were added.

Analysis of blood serum cytotoxic activity. Cytotoxic activity of blood serum was studied in MTT-assay. To the preincubated target cells (1•106 cells in 100 µl of culture medium) 10 ml of autologous blood serum was added. Plates were incubated for 18 h in 5% CO2, 100% of humidity, 37 °C. All the next steps were the same as is described above.

Cooperative cytotoxic activity of lymphocytes and macrophages. The target cells (either LLC or EC cells, in concentration of 3•105 cells in 100 µl of culture medium) and both types of immune cells (Lph AND Mph in concentration of 1•106 in 100 µl of culture medium) were placed in 96-wells flat-bottomed plates and incubated for 18 h in 5% CO2, 100% of humidity, 37 °C. All the next steps were the same as is described above.

Zymozan induced respiratory burst of Mph was studied in NBT-assay [16]. In brief, Mph (1•106 cell/ml, 0.2 ml/well) were incubated with 0.02 ml/well of 0.2% NBT solution (Sigma, USA) and 0.02 ml/well of zymozan (100 µg/ml, Sigma, USA). After incubation (1 h, 5% СО2, 37 °С), the plates were washed for two times with 0.9% NaCl solution. The 2 mole/liter KOH solution (0.06 ml/well) and 50% DMSO solution (0.07 ml/well) were used to dissolve diformazan granules. Optical density was measured at λ = 630 nm with the use of micro ELISA reader (StatFax-2100, USA). Each sample was measured in triplicate. Index of potentiation (IP) was calculated as following:

IP = OU(induced test) / OU(spontaneous test), where

OU(induced test) and OU(spontaneous test) stand for optical units of wells with zymozan treated and untreated Mph respectively.

Statistical analysis. The statistical analysis was made using Student t-test. The difference was considered as significant when p<0.05. Calculations and graph plotting were performed with the OriginLab soft-ware.

RESULTS AND DISCUSSION

Study of the XAV efficiency in animals with LLC. Animals with LLC which were administrated XAV have smoler size of the primary tumor and significant decrease in metastases development. Data of the XAV anticancer efficiency in the groups are presented in Table 1.

Note: *p <0.05 comparing with the corresponding control group.

Studying the immunological parameters, it was found that at terminal stages of tumor growth the level of Lph and Mph cytotoxic activity in vaccinated animals was elevated comparing with unvaccinated animals (Table 2).

Note: *p <0.05 comparing with the corresponding control group.

Thus, application of XAV in operated and non-operated animals with LLC significantly inhibited tumor growth and reduces the level of lung metastasis. Possibly, the XAV antitumor effect is ensured by the elevated functional activity of lymphocytes and macrophages which lasted till the late stages of tumor development.

Study of the XAV efficiency in animals with EC. The vaccination scheme was the same as in the application of XAV in animals with LLC: it started on the second day after the tumor transplantation and continued on day 5, 12 and 19 of tumor growth.

According to the results of the experiment, the XAV has antitumor effect comparable with the vaccine prepared from extracts of Ehrlich cancer cells: tumor volume in mice of both groups was significantly smaller as compared with unvaccinated tumor-bearing control till the day 20 of tumor growth (Fig. 1a). Although tumor volume of mice immunized ether with XAV or Ehrlich-cells-based vaccine were almost similar, the survival rate of former group of mice was significantly higher as compared to the latter one (Fig. 1b). For example, on day 24 of tumor growth 90% of XAV-vaccinated mace were still alive while in the other vaccinated group this index made only 60%. This may point to the better life quality of the XAV-vaccinated mice.
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To address the mechanisms which may underlie XAV anticancer effect some immunological parameters of the XAV-vaccinated mice were analyzed. It was found that the antitumor efficiency of XAV may be related to activation of Lph and Mph cytotoxic activity. To illustrate, cooperative cytotoxic activity of Lph and Mph of immunized mice was approximately three times higher than in the group of control tumor-bearing mice (Fig. 2).

In XAV-vaccinated mice, blood serum cytotoxic activity was elevated too as compared to control tumor-bearing mice (Fig. 3).

Moreover, the respiratory burst of Mph in the vaccinated mice remained unaffected while in the group of control tumor-bearing mice it fallen almost 2 times beneath the level of the intact control mice (Fig. 4). This points to the Mph exhaustion or repolarization towards M2-like phenotype in the control tumor bearing mice.

So, the chicken embryo proteins-based vaccine administered together with Bacillus subtilis B-7025 cytotoxic metabolite demonstrated its significant antitumor efficacy. Probably, this efficiency relies on macrophages and lymphocytes stimulation.

The utilization of embryonic tissue homogenates in antitumor therapy began in the 30s of the XX century. G. Ficher, et al. demonstrated antitumor efficacy of extracts derived from fetal liver and thymus [17]. Also, it was shown that the administration of embryonic tissue to rats 2–3 weeks before Jensen sarcoma transplantation leads to tumor rejection. Nowadays the scientific society continues working on the use of drugs based on embryonic tissue in antitumor therapy and have received quite encouraging results. Animal embryos can be used for manufacturing such vaccines [18]. Preliminary works in our Institute demonstrated that chicken embryo proteins possess remarkable anticancer and immunomodulating activities and can be used in cancer vaccines engineering [19]. One of the possible ways to improve vaccine efficacy is to combine it with immunological adjuvant.

The products of Bacillus subtilis are under the study of R.E. Kavetsky Institute since first half of the XX century [20]. For example, some proteins of Bacillus subtilis B-7025 were shown to possess cytotoxic and immunomodulating activities and therefore are feasible to be used in the construction of cancer vaccines. These cytotoxic proteins already have been used for construction of autologous vaccines [21, 22]. In this study, the cytotoxic protein of B. subtilis B-7025 is used as the adjuvant in the AV based on xenogeneic embryo proteins.

Newly constructed embryonic vaccine was very effective in the treatment of animals with model tumors (LLC and EC), which was manifested in the delaying of primary tumor progression and in suppression of metastasis. Antitumor effect of the vaccine was shown to relay on activation and maintaining of immune cells (lymphocytes and macrophages) functional activity.

CONCLUSIONS

1. The AV based on the separate faction of embryo extract (proteins with molecular weight above 50 kD) and cytotoxic metabolite of B. subtilis B-7025 have an inhibitory effect on the progression of primary tumor node and the development of metastases that was demonstrated on different models of tumor growth.

2. Antitumor effect of the XAV was shown to be based on the elevated activity of immune cells (lymphocytes and macrophages).

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Адреса для листування:
Діденко Г.В.
03022, Київ, вул. Васильківська, 45
Інститут експериментальної патології,
онкології і радіобіології iм. Р.Є. Кавецького
НАН України
E-mail: gennadij_d@ukr.net
Одержано: 24.11.2021