The aim of the study was to characterize the influence of biologically active substances of cell origin on proliferation, survival, receptor profile and ability to form multicellular spheroids tumor cells in vitro and in animal tumor models.

1. The object of research: human (hBM MSCs) and mouse bone marrow (mBM
MSCs) mesenchymal stem cells, breast cancer adenocarcinoma cell line - MCF-
7. The work involved biotechnology, immunohistochemical, cytofluorymetry,
molecular biology, biochemical methods and statistical analysis.
The aim of the study was to characterize the influence of biologically active
substances of cell origin on proliferation, survival, receptor profile and ability to
form multicellular spheroids tumor cells in vitro and in animal tumor models.
The study pro-and anti- oncogenic activity
of the cellular origin biological agents
2-D culture 3-D culture Tissue

2. Incubation of tumor cells MCF-7 in the presence of interferon alfa-2b (IFN-α-2b)
Fig.1. The dependence of the survival cell MCF-7 dose IFN-α-2b.
(A) adhesive fraction (B) suspension fraction.
А Б
Influence of IFN-α-2b in
survival of breast cancer cells
both in suspension and in
adheziy It factions manifested
on 3-4-th day of cultivation.
There was growth in the
number of dead cells in both
fractions when cultured with
IFN-α-2b at a concentration of
10 IU/ml compared to control
cells that were cultured
without cytokine. Thus, the
presence of culture medium
IFN-α-2b at a concentration of
10 IU/ml has cytostatic effect
on cells MCF-7 culture for 1-2
days and stimulates cancer
cell death 3-4-th day of
cultivation.
Fig.2. The density of MCF-7 adhesion cell culture at standard culture conditions (control) and in non-contact
co-culture with of hBM MSCs.
5
5

3. To compare the effects of bioactive substances on proliferative activity and adhesive
ability of MCF-7 cell populations it was incubated in conditioned medium fron MSCs, T-
lymphocytes, as well as the addition of IFN-α-2b at a concentration 2×104 IU/ml.
Fig. 3. The number of alive MCF-7 cells
(adhesive fraction) at incubation with C-medium
from MSCs, T-lymphocytes and with IFN-α-2b.
Figure 4. The number of alive MCF-7 cells
(suspension fraction) at incubation with C- medium
from MSCs, T-lymphocytes cells and with IFN-α-2b .
We demonstrated that T-lymphocytes stimulated the proliferation of tumor cells in the largest extent, IFN-α-
2b was less effective. Besides T-cells stimulated increasing metastatic properties of tumor cells and
contributed migration cancer cells to suspension fraction. Opposite, MSCs and IFN-α-2b have clearly
cytostatic effect on tumor populations. It decreased as the number of living tumor cells in suspension and
in adhesion lower than control.

4. Table 1. Influence of incubation conditions on
expression of EGFR and estrogen receptor in MCF-7 cells.
Incubation EGFR+ EGFR+/- EGFR- ER + ER +/- ER -
Control 66.6 16.6 16.6 32 50 50
+MSC 83.3 11.1 5.5 40,9 18,2 40,9
+Т-lymphocytes 88.8 7.4 3.7 85,7 0 14,3
+ IFN-α-2b 72.7 18.2 9 39,3 46,4 14,3
Incubation Е-cadherine + Е-cadherine +/- Е-cadherine - АE1/АE3 + АE1/АE3 +/- АE1/АE3-
Control 50 0 50 46.6 46.6 6.6
+MSC 17,4 43,5 39 100 0 0
+Т-lymphocytes 80,0 20,0 0 59,1 36,3 4,5
+ IFN-α-2b 11,1 3,7 92,6 77,7 11,1 11,1
Table 2. Influence of incubation conditions on the expression of E-catherine
and family of cytokeratines AE1/AE3 in MCF-7 cells.
control MSC
control
Т-lymphocytes
IFN-α-2b
MSC
IFN-α-2bТ-lymphocytes
AE1/AE3
ER

5. The influence of substances on the cellular origin of the
formation and growth of multicellular tumor spheroids
Figure 5. Multicellular tumor spheroids volume cell
line MCF-7 during incubation with IFN-α-2b, K-
environment of T cells and MSCs.
A - control,
B - IFN-α-2b at a concentration of 103 IU / ml
C - IFN-α-2b at a concentration of 104 IU / ml
D - IFN-α-2b at a concentration of 105 IU / ml
E – C medium from T-lymphocytes,
F – C medium from MSCs.
As a result, it was determined that T-
lymphocytes generally have a stimeluted
impact on the development of transformed
MCF-7 cells in vitro. It manifested in
activation of the expression of ER and R-
EGF, stimulation of cell proliferation,
decreasing adhesive properties and
stimulation of tumor spheroids growth. At
the same time, IFN-α-2b and MSCs C-
medium inhibited multicellular tumor
spheroid growth, by 50-60%, compared with
the control.

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Окружністьстегна,мм
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Fig.6. Cinetics of Earlich` adenocarcinoma growth in
mice hip muscle depending on various conditions on
injection EAC cells and adding mMSCs lysate.
It was determined the average increase the circumference of hip animals in all groups. In control EAC
(II group/intramuscular injection of 106 cells EAC) the figure was in 2.33 times higher that in intact
control (I group). In 4-th group (once intra abdominal injection mMSC lysate and intramuscular injection
of EAC) in 2.04 times, while in 6-th group (8x intra abdominal injection mMSC lysate and intramuscular
injection of EAC) in 1.96 times higher than in control. Thus, the single dose mMSC lysate leaded to
slowdown the growth of tumors in the muscle of the thigh in 17% of males and 9% females. Multiple
input of mMSC lysate inhibited tumor growth by 31% in males and stimulated by 5% in females.
Architecture muscle
is preserved,
ascites them with
individual cells.
The formation of
solid tumors
between muscle
fibers.
The remains of
muscle tissue
surrounded by AS
tumor cells and
tissue atrophy
zones.

7. Objects of our research in this project:
MCF-7 cell line in 2D culture 3D culture of MCF-7 MCS in culture
T-cells in suspension culture Balb2 miceHistological sections of MCF-7
MTS

8. http://morpho-cell.org
https://www.researchgate.net/profile/Olena_Perepelytsina