Use of xenogeneic vaccine modified with embryonal nervous tissue antigens in the treatment of B16‑melanoma-bearing mice
The aim of the work was experimental study of anticancer efficacy of xenogeneic cancer vaccine (XCV) developed on the basis of rat embryonic nervous tissue and protein-containing metabolite of Bacillus subtilis В-7015 (70 kDa), in В-16 melanoma-bearing С57Bl/6 mice. Methods: Immunological methods an...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2014
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| Цитувати: | Use of xenogeneic vaccine modified with embryonal nervous tissue antigens in the treatment of B16-melanoma-bearing mice / І.М. Voeykova, N.І. Fedosova, О.М. Karaman, О.Y. Yudina, G.V. Didenko, G.S. Lisovenko, L.М. Evstratieva, G.P. Potebnya // Experimental Oncology. — 2014. — Т. 36, № 1. — С. 24-28. — Бібліогр.: 36 назв. — англ. |
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nasplib_isofts_kiev_ua-123456789-1453152025-02-09T09:44:25Z Use of xenogeneic vaccine modified with embryonal nervous tissue antigens in the treatment of B16‑melanoma-bearing mice Voeykova, І.М. Fedosova, N.І. Karaman, О.М. Yudina, О.Y. Didenko, G.V. Lisovenko, G.S. Evstratieva, L.М. Potebnya, G.P. Original contributions The aim of the work was experimental study of anticancer efficacy of xenogeneic cancer vaccine (XCV) developed on the basis of rat embryonic nervous tissue and protein-containing metabolite of Bacillus subtilis В-7015 (70 kDa), in В-16 melanoma-bearing С57Bl/6 mice. Methods: Immunological methods and methods of experimental oncology were used. Effects of XCV on primary and secondary organs of immune system of experimental animals, its anticancer and antimetastatic efficacy were evaluated. Results: It has been shown that XCV did not induced toxic effects on organism, and did not caused inflammatory reactions. The relation between the degree of XCV anticancer efficacy with the regimen of its use and the presence of primary tumor has been analyzed. It has been demonstrated that the developed XCV possesses significant antimetastatic activity if it is used after surgical removal of the primary tumor: in this case lung metastasis inhibition index reached 97.4%. Conclusion: High immunogenecity of new XCV creates perspectives for detailed study of its mechanisms of action. Key Words: oncofetal antigens, xenogeneic cancer vaccine, В-16 melanoma, immunotoxicity, effectors of anticancer defence. 2014 Article Use of xenogeneic vaccine modified with embryonal nervous tissue antigens in the treatment of B16-melanoma-bearing mice / І.М. Voeykova, N.І. Fedosova, О.М. Karaman, О.Y. Yudina, G.V. Didenko, G.S. Lisovenko, L.М. Evstratieva, G.P. Potebnya // Experimental Oncology. — 2014. — Т. 36, № 1. — С. 24-28. — Бібліогр.: 36 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/145315 en Experimental Oncology application/pdf Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Voeykova, І.М. Fedosova, N.І. Karaman, О.М. Yudina, О.Y. Didenko, G.V. Lisovenko, G.S. Evstratieva, L.М. Potebnya, G.P. Use of xenogeneic vaccine modified with embryonal nervous tissue antigens in the treatment of B16‑melanoma-bearing mice Experimental Oncology |
| description |
The aim of the work was experimental study of anticancer efficacy of xenogeneic cancer vaccine (XCV) developed on the basis of rat embryonic nervous tissue and protein-containing metabolite of Bacillus subtilis В-7015 (70 kDa), in В-16 melanoma-bearing С57Bl/6 mice. Methods: Immunological methods and methods of experimental oncology were used. Effects of XCV on primary and secondary organs of immune system of experimental animals, its anticancer and antimetastatic efficacy were evaluated. Results: It has been shown that XCV did not induced toxic effects on organism, and did not caused inflammatory reactions. The relation between the degree of XCV anticancer efficacy with the regimen of its use and the presence of primary tumor has been analyzed. It has been demonstrated that the developed XCV possesses significant antimetastatic activity if it is used after surgical removal of the primary tumor: in this case lung metastasis inhibition index reached 97.4%. Conclusion: High immunogenecity of new XCV creates perspectives for detailed study of its mechanisms of action. Key Words: oncofetal antigens, xenogeneic cancer vaccine, В-16 melanoma, immunotoxicity, effectors of anticancer defence. |
| format |
Article |
| author |
Voeykova, І.М. Fedosova, N.І. Karaman, О.М. Yudina, О.Y. Didenko, G.V. Lisovenko, G.S. Evstratieva, L.М. Potebnya, G.P. |
| author_facet |
Voeykova, І.М. Fedosova, N.І. Karaman, О.М. Yudina, О.Y. Didenko, G.V. Lisovenko, G.S. Evstratieva, L.М. Potebnya, G.P. |
| author_sort |
Voeykova, І.М. |
| title |
Use of xenogeneic vaccine modified with embryonal nervous tissue antigens in the treatment of B16‑melanoma-bearing mice |
| title_short |
Use of xenogeneic vaccine modified with embryonal nervous tissue antigens in the treatment of B16‑melanoma-bearing mice |
| title_full |
Use of xenogeneic vaccine modified with embryonal nervous tissue antigens in the treatment of B16‑melanoma-bearing mice |
| title_fullStr |
Use of xenogeneic vaccine modified with embryonal nervous tissue antigens in the treatment of B16‑melanoma-bearing mice |
| title_full_unstemmed |
Use of xenogeneic vaccine modified with embryonal nervous tissue antigens in the treatment of B16‑melanoma-bearing mice |
| title_sort |
use of xenogeneic vaccine modified with embryonal nervous tissue antigens in the treatment of b16‑melanoma-bearing mice |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2014 |
| topic_facet |
Original contributions |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/145315 |
| citation_txt |
Use of xenogeneic vaccine modified with embryonal nervous tissue antigens in the treatment of B16-melanoma-bearing mice / І.М. Voeykova, N.І. Fedosova, О.М. Karaman, О.Y. Yudina, G.V. Didenko, G.S. Lisovenko, L.М. Evstratieva, G.P. Potebnya // Experimental Oncology. — 2014. — Т. 36, № 1. — С. 24-28. — Бібліогр.: 36 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
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2025-11-25T10:17:00Z |
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2025-11-25T10:17:00Z |
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| fulltext |
24 Experimental Oncology 36, 24–28, 2014 (March)
USE OF XENOGENEIC VACCINE MODIFIED WITH EMBRYONAL
NERVOUS TISSUE ANTIGENS IN THE TREATMENT
OF B16-MELANOMA-BEARING MICE
І.М. Voeykova, N.І. Fedosova, О.М. Karaman, О.Yu. Yudina, G.V. Didenko, G.S. Lisovenko,
L.М. Evstratieva, G.P. Potebnya
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv, Ukraine
The aim of the work was experimental study of anticancer efficacy of xenogeneic cancer vaccine (XCV) developed on the basis of rat
embryonic nervous tissue and protein-containing metabolite of Bacillus subtilis В-7015 (70 kDa), in В-16 melanoma-bearing
С57Bl/6 mice. Methods: Immunological methods and methods of experimental oncology were used. Effects of XCV on primary
and secondary organs of immune system of experimental animals, its anticancer and antimetastatic efficacy were evaluated. Results:
It has been shown that XCV did not induced toxic effects on organism, and did not caused inflammatory reactions. The relation
between the degree of XCV anticancer efficacy with the regimen of its use and the presence of primary tumor has been analyzed.
It has been demonstrated that the developed XCV possesses significant antimetastatic activity if it is used after surgical removal
of the primary tumor: in this case lung metastasis inhibition index reached 97.4%. Conclusion: High immunogenecity of new XCV
creates perspectives for detailed study of its mechanisms of action.
Key Words: oncofetal antigens, xenogeneic cancer vaccine, В-16 melanoma, immunotoxicity, effectors of anticancer defence.
Despite significant progress in the development
of modern methods of anticancer therapy, an efficacy
of treatment of cancer patients remains insufficient
therefore the development of new approaches in this
field is an actual task. Cancer biotherapy including
cancer vaccine therapy shows promising results [1–4].
In experimental and clinical studies aimed on the de-
velopment of immunotherapeutic means, an efficacy
of cancer autovaccine use has been demonstrated
[5, 6]. Also there were reported the data evidencing
on higher efficacy of immunization with xenogeneic
analogs of endogenous molecules, because such
immunization is capable to overcome immunologic
tolerance to tumor antigens and leads to significant
suppression of cancer development [7, 8].
A special place in the development of biotherapeu-
tical methods belongs to melanoma — a malignant
tumor of neuroectodermal origin which develops from
skin melanocytes. Melanoma cells are highly resistant
to chemo- and radiotherapy, while local surgical removal
of the tumor could not guaranty the development of re-
currence and distant metastases [9, 10]. At the same
time, it has been shown [9] that tumor antigens induce
immune reactions resulting in tumor cell destruction and
suppression of cancer development. So, one could con-
clude that immune therapy may be considered as an im-
portant element of systemic melanoma treatment.
An experimental model — В-16 melanoma — was
firstly created by Roscoe B. Jackson in 1954 from
ear skin region of С57Bl/6 mouse. This experimental
tumor is characterized by high immunogenicity and
significantly higher growth rate compared to other
solid tumors. В-16 melanoma model is appropriate
for the study of pathogenetic patterns and metasta-
sis of the disease and for evaluation of new methods
of melanoma treatment with CD40L/IFN-γ-matured,
IL-12p70-producing DCs [11].
The published results concerning melanoma therapy
are controversial and do not allow to chose the optimal
ways for prophylaxis and treatment of this pathology.
In regard to immune therapy, in clinical trials of xeno-
geneic vaccination of patients with uveal melanoma for
prevention of hematogenous metastases, in 27 patients
there has been recorded 49.7 months long recurrence-
free period. These data allow consider xenovaccina-
tion as an available method for metastasis prophylaxis
in uveal melanoma patients [12]. In patients with mela-
noma cross-immunization with vaccines comprising
mouse (xenogenic) or human gp100 plasmid DNA, sig-
nificantly improved patients survival, including at later
stages of the malignant process. The authors have
shown that the conjunction of vaccines with gp100 from
different species increased vaccination effectiveness,
also its biological safety was proved [13].
At present time there have been reported some
data on the efficacy of use of embryonic tissues for
therapy cancer patients. The use of embryonic tissues
for generation of xenogeneic cancer vaccines (XCV)
is based on antigenic similarity between tumor and
embryonic cells due to expression of a number of an-
tigens which play a key role in overcoming immune
tolerance [14]. The means of embryonic origin act
at epigenetic level through regulatory systems of an or-
ganism what could create grounds for the develop-
ment of new biotherapeutical approach — construction
of effective oncofetal cancer vaccines [14–16].
One of the promising approaches to their design
is the use of embryonic nerve tissue. Its choice was
Received: May 17, 2013.
*Correspondence: E-mail: irina_iepor@mail.ru
Abbreviations used: CIC — circulating immune complexes;
MII — metastasis inhibition index; PCM — protein-containing
metabolite of Bacillus subtilis В-7015 with molecular weight
of 70 kDa; PFENT — protein fraction of rat embryonic nervous
tissue; XCV — xenogeneic cancer vaccine.
Exp Oncol 2014
36, 1, 24–28
Experimental Oncology 36, 24–28, 2014 (March) 25
justified by the fact that tumor and embryonic cells are
similar to each other due to the expression of a number
of antigens that can be used to overcome immune
tolerance in induction of antitumor response [15–17].
At the same time, embryonic nerve cells possess
high adaptive capacity [18]. Therapeutic approaches
to the application of embryonic tissues, including
nervous, and switch on specific (substitute) and non-
specific mechanisms that are based on the modulation
process of regeneration, repair, proliferation and dif-
ferentiation [18–20]. Disclosure of these mechanisms
may be crucial for the development of new methods
of treatment of diseases, including cancer [9, 21–23].
However, presently the strict criteria for construc-
tion of cancer auto- and xenogeneic vaccines are
not worked out, and universal indications for vaccine
therapy are not formulated as well as the doses and
regimens of vaccination alone or in combination with
other treatment modalities.
The aim of the work was experimental study
of anticancer efficacy of XCV containing protein frac-
tion of rat embryonic nervous tissue (PFENT) and
protein-containing metabolite of Bacillus subtilis
В-7015 with molecular weight of 70 kDa (PCM) in mice
with В-16 melanoma dependent on schemes and
regimens of vaccination.
MATERIALS AND METHODS
The study has been carried out on male C57Bl/6 mice
2.5 months old weighting 18–19 g, bred in the vivarium
of R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobilogy of NAS of Ukraine (IEPOR).
The use and care of experimental animals have been
performed in accordance with standard international
rules on biologic ethics and was approved by Insti-
tutional Animal Care and Use Committee [24, 25].
In the work healthy laboratory animals were used;
prior to experiment animals were carried for 14 days
in quaran tine conditions. Later on, animals were
housed in a facility with constant temperature and
received balanced nutrition.
As experimental model, mouse melanoma
B-16 cells obtained from National Bank of Hu-
man and Animal Tissues of IEPOR, were used. This
strain is maintained by passages in C57Bl/6 mice.
For transplantation melanoma cell suspension pre-
pared by primary tumor trypsinization according
to the method [26], was injected i.m. in the right hind
leg at a dose of 4•105 cells/mouse in a volume of 0.2 ml
(for the study of vaccination at the background of tu-
mor growth) or in a foot of right hind leg at a dose
of 2.5•105 cells/mouse in a volume of 0.04 ml (for
the study of vaccination after surgical removal of pri-
mary tumor). The course of tumor development was
chara cterized by standard indices: frequency of tumor
development, latent period of tumor appearance,
number and volume of metastases.
In the study we used the XCV designed in the De-
partment of Construction of Biotherapeutical Means.
This vaccine contains protein fraction of nervous tis-
sue from rat embryo of late gestation period (PFENT)
and protein-containing metabolite (PCM) of B. subtilis
В-7025 with m.w. 70 kDa [27].
The studied vaccine was standardized by protein
content ([С] = 0.3 mg/ml in total). Vaccine or its sepa-
rate components (PFENT and/or PCМ) were injected
subcutaneously in the dorsum region in a volume
of 0.3 ml/mouse.
Three series of experiments have been performed.
In the first series intact mice were triply immunized
with vaccine with 3 day intervals for analysis of its
toxic, inflammatory and/or immunomodulating effects.
At the 7th day after the last vaccination, there have
been determined specific indices of weight and cel-
lularity of immunocompetent organs (spleen, thymus,
peripheral lymph nodes) by standard method of cell
staining using trypan blue. Cytotoxic activity of natu-
ral killer cells (NK) has been determined by MTT test
(K542 cells were used as targets in a ratio of 1:5 [28]);
cytotoxic activity of peritoneal macrophages (PMP)
was evaluated using NBT-test [29]. For analysis
of peripheral blood indices, blood samples were col-
lected in minitubes with EDTA solution and analyzed
in automatic hemoanalyzer РСЕ-210 (Erma. Inc.,
Japan). Absolute content of leukocytes, platelets,
erythrocytes, absolute and relative content of lympho-
cytes, monocytes, granulocytes, hemoglobin content
have been determined [30]. The level of circulating
immune complexes (CIC) in blood serum of mice was
determined in precipitation reaction: high molecular
weight CIC with the use of 3% polyethylene glycol
(PEG), medium molecular weight CIC — 4.5% PEG,
low molecular weight CIC — 6% PEG [30, 31].
In the second series, immunization was performed
by therapeutic scheme at the background of В-16 me-
lanoma growth. Vaccination has been performed
by two schemes: at 1st, 7th, 14th days (scheme 1), and
at 7th, 14th, 28th days after tumor cell transplantation
(scheme 2). Antitumor effect has been evaluated
by the rate of tumor development, duration of latent
period, primary tumor growth dynamics, metastatic
volumes, and life span of mice.
The third series of the study included animal immu-
nization after surgical removal of primary tumor. Vac-
cination has been initiated in three days after radical
surgery by following schemes: triply with 3-day inter-
vals (sche me 3); triply with 7-day intervals (scheme 4).
Indices of В-16 melanoma lung metastasis were evalu-
ated at the 24th day after tumor removal (the 45th day
of tumor growth). Vaccination efficacy was evaluated
by metastasis inhibition index (MII). In all cases intact
animals or unvaccinated animals with transplanted
В-16 melanoma served a control. For comparison
of parameters obtained in experimental and control
groups the modulation indices were calculated [32].
Statistical analysis of the data was performed using
Student’s t-criterion. Values p < 0.05 were considered
statistically significant [33].
26 Experimental Oncology 36, 24–28, 2014 (March)
RESULTS AND DISCUSSION
Immunization of intact C57Bl/6 mice with XCV
or its separate components did not cause irritation
and / or inflammation in the place of preparation injec-
tion and had no influence of animal weight (Table 1).
Table 1. Weight and cellularity of immunocompetent organs of vaccinated
and not vaccinated С57Bl/6 mice at 7th day after last vaccination
Index
Group of animals (МІ, %)
Intact
animals
(n = 10)
PFENT
(n = 5)
PCM
(n = 5)
Vaccine
PFENT +
PCМ (n = 5)
Animal weight (g) 18.7±0.4
(17.8÷20.4)
18.7±0.4
(0.0)
19.1±0.9
(+2.1)
19.1±0.5
(+2.1)
Relative organ weight (× 10–3):
Spleen 6.1±0.9
(4.0÷8.6)
7.5±0.6
(+22.9)
8.8±1.5
(+44.2)
7.2±0.8
(+18.0)
Thymus 2.1±0.3
(0.9÷2.6)
1.6±0.2
(-23.8)
1.7±0.4
(-19.0)
1.8±0.2
(-14.2)
Peripheral lymph
nodes
3.3±0.2
(2.2÷3.7)
3.8±0.3
(+15.1)
3.2±0.3
(-3.0)
2.9±0.3
(-12.1)
Cellularity per 1 mg of organ
Spleen 1.10±0.10
(1.01÷1.32)
0.97±0.09
(-11.8)
1.13±0.09
(+2.7)
1.01±0.11
(-8.1)
Thymus 2.05±0.15
(1.78÷2.41)
1.93±0.24
(-5.8)
4.30±1.73**
(+109.7)
1.96±0.09
(-2.9)
Peripheral lymph
nodes
1.75±0.15
(1.50÷2.18)
1.40±0.30
(-20.0)
1.61±0.21
(-8.0)
1.40±0.12
(-20.0)
Viable cells (%)
Spleen 77.9±1.9
(75.0÷83.9)
75.8±1.7
(-2.6)
75.5±1.4
(-3.0)
73.3±0.6**
(-5.9)
Thymus 88.5±0.5
(87.8÷89.7)
88.6±1.4
(0.0)
91.3±2.3
(+3.1)
89.1±0.7
(+0.6)
Peripheral lymph
nodes
83.7±3.8
(76.1÷90.1)
86.9±1.9
(+3.8)
87.5±1.7
(+4.5)
88.0±1.6
(+5.1)
Notes: МІ — modulation index of value compared to intact control;
PFENT — protein fraction of rat embryonic nervous tissue; PCM — protein-
containing metabolite of Bacillus subtilis В-7015 with molecular weight
of 70 kDa. Values are mean ± SE. *Significant at р < 0.05, **Significant
at 0.1 < р < 0.05 compared to intact control.
As one may see in Table 1, immunization of experi-
mental animals has no significant influence on weight
and cellularity indices of immunocompetent organs
(spleen, thymus, peripheral lymph nodes) compared
to that in intact control. It evidenced on the absence
of toxic effects caused by vaccine and/or its compo-
nents on central and peripheral organs of immune sys-
tem of experimental animals. Introduction of the vaccine
and its separate protein components also did not affect
the studied indices of peripheral blood (Table 2).
We have observed just a decrease of leukocyte
counts in response on XCV administration, which oc-
curred possibly due to the decrease of total lympho-
cyte and monocyte counts; however, such event was
transitory and did not affect animal’s general state.
All mentioned above evidenced on an absence of im-
munotoxic and inflammatory reactions.
The study of vaccination impact on effector reac-
tions of unspecific immunity demonstrated the ab-
sence of significant alterations in intact and vaccinated
mice. PMP counts and their activi ty in the NBT test in in-
tact mice were 4.8 ± 0.7 • 106 cells and 0.57 ± 0.01 opti-
cal units, respectively. The correspon ding parameters
in all imm unized mice were at level of intact control.
Administration of separate components (PFENT
or PCM) led to suppression of cytotoxic activity of NK
(8.9 ± 1.7 and 4.2 ± 1.4 optical units, respective-
ly vs intact control: 18.8 ± 2.4 optical units, р < 0.05).
In the case of vaccination with XCV containing both
components such effect was not observed (15.4 ±
3.1 optical units). So, designed vaccine had no nega-
tive influence an activity of effector cellular pattern
of unspecific immunity — NK and PMP.
Table 2. Peripheral blood parameters of intact and vaccinated
C57Bl/6 mice (7th day after last vaccination)
Index Intact mice
(n = 10)
Experimental groups (МІ, %)
PFENT
(n = 5)
PCМ
(n = 5)
Vaccine
PFENT +
PCМ (n = 5)
Leucocytes
(×109/l)
8.95±0. 57
(7.7÷10.1)
7.08±0.86
(-20.9)
6.92±1.21
(-22.7)
5.00±0.72*
(-44.1)
Erythrocytes
(×1012/l)
10.04±0.26
(9.56÷10.56)
9.07±0.26*
(-9.6)
9.41±0.36
(-6.2)
9.65±0.35
(-3.8)
Platelets
(×109/l)
269.00±34.78
(216.0÷354.0)
290.25±33.45
(+7.9)
311.40±40.89
(+15.8)
254.80±21.03
(-5.3)
Hemoglobin
(g/l)
148.00±2.45
(143.0÷152.0)
130.75±3.63*
(-11.7)
129.00±7.07*
(-12.8)
138.40±6.73
(-6.5)
Leucogram
Lymphocytes
(×106/l)
6.28±0.37
(5.4÷6.9)
5.28±0.76
(-15.9)
4.96±0.97
(-21.0)
3.52±0.60*
(-43.9)
Monocytes
(×106/l)
0.98±0.12
(0.8÷1.2)
0.78±0.14
(-20.5)
0.66±0.12
(-32.3)
0.44±0.06*
(-54.9)
Granulocytes
(×106/l)
1.70±0.12
(1.5÷2.0)
1.03±0.12*
(-39.7)
1.30±0.28
(-23.5)
1.04±0.31
(-38.8)
Lymphocytes
(%)
70.10±0.80
(68.2÷71.5)
74.20±2.92
(+5.8)
71.32±3.16
(+1.7)
70.70±5.31
(+0.9)
Monocytes (%) 10.83±0.78
(9.1÷11.9)
11.10±1.89
(+2.5)
9.58±0.32
(-11.5)
8.98±0.80
(-17.0)
Granulocytes
(%)
19.08±0.62
(17.5÷19.9)
14.73±1.40*
(-22.8)
19.08±3.40
(0.0)
20.30±5.49
(+6.4)
Notes: МІ — modulation index of value compared to intact control;
PFENT — protein fraction of rat embryonic nervous tissue; PCM — protein-
containing metabolite of Bacillus subtilis В-7015 with molecular weight
of 70 kDa. Values are mean ± SE. *Significant at р < 0.05, **Significant
at 0.1 < р < 0.05 compared to intact control.
Safety of XCV has been evidenced also by CIC
levels. As one may see in Figure, immunization did not
elevate the content of low, medium and high molecu-
lar weight CIC. Their content in blood serum of mice
from all experimental groups did not differ significantly
from respective indices in intact controls. An absence
of eleva ted level of medium molecular weight CIC after
administration of studied vaccine and/or its compo-
nents additionally evidenced on the absence of inflam-
matory processes caused by immunization. Level of low
molecular weight CIC was also in the margins of intact
control, i.e., immunization did not lead to synthesis
of incomplete or monovalent antibodies which, being
bound to antigen, do not eliminate it but, on the contrary,
mask it from attack of immune system [33, 34].
In the second series immunization was performed
by therapeutic scheme at the background of В-16 mela-
noma growth for determination of antitumor and anti-
metastatic activity of XCV. As it is presented in Table 3,
immunization of mice by both schemes (1 and 2) had
no effect on latent period of tumor development (vi-
sible tumors have developed on 9–12 days after trans-
plantation), and the tumor rates (tumors developed
in 80.0 ± 10.3% (12/15) and 86.7 ± 8.8% (13/15) of vac-
cinated animals respectively vs 94.1 ± 5.7% (16/17)
in control group). Tumor growth dynamics in vaccinated
animals did not differ from that in control group. Also
vaccination had no significant influence on the rate,
number and volume of metastases (Table 4).
Experimental Oncology 36, 24–28, 2014 (March) 27
0
5
10
15
20
25
30
35
High Medium Low
O
pt
ic
u
ni
t
Intact mice
PFENT
PCM
PFENT + PCM
Molecular weight
Figure. CIC content in blood serum of mice in response to vac-
cination (7th day after the last injection)
Table. 3. Growth of melanoma В-16 in unvaccinated and vaccinated
C57Bl/6 mice
Group of animals
Efficacy of tu-
mor transplanta-
tion (%)
Latent
period
of tumor
formation
(days)
Life du-
ration af-
ter tumor
trans-
plantation
(days)
Unvaccinated (n = 17) 94.1±5.7 (16/17) 11.6±1.9 31.2±1.2
Vaccinated by scheme 1 (n=15) 80.0±10.3 (12/15) 8.5±0.4 25.6±0.7*
Vaccinated by scheme 2 (n=15) 86.7±8.8 (13/15) 10.0±1.2 27.7±2.8
Notes: Scheme 1 — vaccination has been performed: at 1st, 7th, 14th days af-
ter tumor cell transplantation. Scheme 2 — vaccination has been performed:
at 7th, 14th, 28th days after tumor cell transplantation. Values are mean ± SE.
*Significant at р < 0.05 compared to unvaccinated mice.
Table 4. Metastasis of melanoma В-16 in unvaccinated and vaccinated
C57Bl/6 mice
Group of animals Metastases
rate (%)
Metastases
number
Volume of me-
tastases (mm3)
Unvaccinated (n=16) 92.3,0±7.1
(16/16)
6.6±1.1 8.1±5.4
Vaccinated by scheme 1 (n=12) 75.0±12.5
(9/12)
6.9±2.2 8.0±2.9
Vaccinated by scheme 2 (n=13) 91.7±7.7
(13/13)
13.7±5.2 11.3±5.2
Notes: Values are mean ± SE. Insignificant р > 0.05 compared to unvac-
cinated mice.
We suppose that an absence of antitumor and
antimetastatic effects of vaccination could be related,
firstly, to insufficient activation of effector cells of anti-
cancer defense incapable to eliminate large amounts
of В-16 melanoma cells, and secondly, to an action
of immunosuppressive factors produced by tumor
cells or immune cells of animals [34, 35].
To support this hypothesis, in the third series of our
study we have analyzed effects of vaccination after sur-
gical removal of primary tumor for prevention of recur-
rence and metastases development. Therefore, surgical
removal of tumors was performed at 21st day of tumor
growth. Evaluation of the results was carried out at 24th
day after the surgery (45th day of tumor development).
As it could be seen in Table 5, immunization re-
sulted in significant decrease of metastasis rate (me-
tastases have developed in 41.7 ± 11.2% of control
mice and in 16.7 ± 10.8% (р < 0.05) and 18.2 ± 11.6%
(0.05 < p < 0.1) of animals immunized by schemes 3 and
4, respectively). The number and volumes of metastases
were significantly lower only in the group of animals im-
munized by scheme 3: 0.3 ± 0.2 vs 4.6 ± 1.1 (р < 0.05),
and 0.3 ± 0.2 mm3 vs 0.8 ± 0.1 mm3 (0.05 < p < 0.1),
respectively, compared to the control.
These results coincide with the results of other
authors [9, 36]. In particular, prophylactic use of xe-
nogeneic vaccines developed on the basis of human
melanoma cells with the following removal of tumor
nodule led to decreased lung metastasis rate in mice
with В-16 melanoma. According to author’s opinion,
such vaccines could be used with high efficacy for
prophylaxis of recurrence in melanoma patients after
surgical treatment [36].
Table 5. Metastasis in unvaccinated and vaccinated C57Bl/6 mice at 24th
day after surgical removal of В-16 melanoma (day 45 after tumor trans-
plantation)
Group of animals
Metasta-
ses rate
(%)
MII
(%)
Metasta-
ses num-
ber
Volume
of metasta-
ses (mm3)
Unvaccinated (n=12) 41.7±11.2
(5/12)
4.6±1.1 0.8±0.1
Vaccinated by scheme 3 (n=12) 16.7±10.8*
(2/12)
97.4 0.3±0.2* 0.3±0.2**
Vaccinated by scheme 4 (n=11) 18.2±11.6
(2/11)
60.1 4.2±3.0 10.3±8.3
Notes: Scheme 3 — vaccination has been initiated in 3 days after radical sur-
gery by following schemes: triply with 3-day intervals. Scheme 4 — vaccina-
tion has been initiated in 3 days after radical surgery by following schemes:
triply with 7-day intervals. МІI — index of metastasis inhibition relatively to un-
vaccinated mice. Values are mean ± SE. *Significant at р < 0.05 vs unvac-
cinated animals; **significant at 0.1 < р < 0.05 vs unvaccinated animals.
So, triple introduction of XCV or its separate com-
ponents to intact C57Bl/6 mice had no immunotoxic
effect on experimental animals and did not cause in-
flammatory reactions. In a model of В-16 melanoma
it has been shown that its use is accompanied with
antitumor and antimetastatic effect only in the case
of surgical removal of primary tumor. In mice that under-
went surgical removal of tumors and were immunized
by scheme 3 we have registered significantly decreased
metastasis rate (р < 0.05), 15.3-fold decrease of me-
tastases number (р < 0.05) and 2.7-fold decrease
of metastases volume (0.05 < p < 0.1) compared with
unvaccinated mice treated only by surgery. For ani-
mals from this group, MII achieved 97.4%. It has been
shown that antimetastatic efficacy of vaccination was
dependent on the regimen of immunization (the most
effective one was triple vaccination with 3 day intervals
after surgical treatment).
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