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

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 БInfluence 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.

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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.

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

MSCIFN-α-2bТ-

lymphocytes

AE1/AE3

ER

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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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.

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

DEPARTMENT OF BIOTECHNICAL PROBLEMS OF DIAGNOSTIC OF INSTITUTE FOR PROBLEMS OF CRYOBIOLOGY AND CRYOMEDICINE NAS UKRAINE

http://morpho-cell.orghttps://www.researchgate.net/profile/Olena_Perepelytsina