The receptor profile of breast tumor cells and formationof tumor spheroids under influence of MSC.
1
Introduction MATERAIL AND METHODS RESULTS CONCLUSIONS The receptor profile of breast tumor cells and formationof tumor spheroids under influence of MSC. Interferons are bioactive peptide substances that are synthesized in the body in response to foreign antigens. These cytokines are mediators of cell-cell interactions and have a systemic effect on the body and immunomodulatory properties. Mesenchymal stem cells (MSC) are the resident stem cells which there are present in adults and necessary for the vital activity of host tissue in normal life. MSC are involved in tissue regeneration and repair in response to disease and injury. It is established the presence of a pool of stem cells in the rapidly proliferating tissues, such as bone marrow, gastrointestinal tract and skin. In addition, there is the presence of a pool of stem cells, which is responsible for the damage in a number of tissues in the form of satellite cells in muscle or liver ovalo-cells. Scientists have repeatedly observed positive effect of injection a suspension of stem cells to cancer patients. The MSC` influance was manifested as a decreasing in tumor size or dispersal of small metastases, and a quick recovery of fully blood characteristics and improvement of the overall patient condition. However, more and more often there are publications that focused on the study of the oncogenic potential of stem cells. Thus, it appears that may have the potential carcinogenicity and neural stem cells in the bone marrow. In addition, transplantation of any tissue-specific stem cells positive for the marker of early embryogenesis - Oct-4 can be potentially oncogenic dangerous. Also revealed that CD133 + stem cells that have been isolated from human neyroopuholey have obligate carcinogenic activity in vivo. The above data indicate the necessarily to study tumorigenic potential of CD133 + cells (and other adult stem cells) before the start of clinical trials and stem cell therapy. Our aim at the first step was to investigate the biologically active substances of cellular origin - namely, the conditioned medium from the MSC and its impact on the life of the tumor cells of breast cancer line MCF-7. We involved immunocytochemical, cytological, biochemical, and statistical analysis methods. According to the results was investigated the effect of conditioned medium from MSCs (c-medium) on the expression of estrogen receptor and epidermal growth factor receptor, cytokeratins (clone AE1 and AE3) and 5, 6, 8, 18, and E-cadherin breast cancer cells, MCF-7. Cell line MCF-7 (line of human breast adenocarcinoma) were cultured under standard conditions (370 C, 100% humidity, 10% CO2) in the culture medium DMEM. Cells were seeded in 6-well plates and formed three groups. Group 1 - control, Group 2 - incubation with IFN alpha in a concentration of 10 MO / ml, Group 3 - incubation in DMEM 1: 1 with c-medium from MSCs. Incubation period - 48 hours. Expression of estrogen receptor (ER) and epidermal growth factor receptor (EGF-R) and cytokeratins (clones AE1/AE3, 5D3, LP34) was determined by immunohistochemical staining (Dako, USA). Analysis was performed by calculating the ratio of cells carrying the marker to total number of cells (histologic index, HS). Identification of estrogen receptor and epidermal growth factor receptor was performed on coverslips, which were covered by a monolayer of MCF-7 cells. Visualization of the results was performed by microphotography microscope Stemy 2000C, Zeiss. To study the direct effect of IFN alpha and MSC` c-medium on proliferation, adhesion and survival of MCF-7, cells were seated in the well of a 24-well plates and cultured under normal conditions (370 C, 100% humidity, 10% CO2) in full medium DMEM. After days of cultivation wells were divided into 4 groups: control, IFN alpha, c - medium from MSC and c-medium from T-lymphocytes. We counted the number of live and dead cells in adhesion and suspension fractions. Counting was performed on the 24th, 48th, 72nd and 96th hours of cultivation. The data were processed statistically in the "Statistics 6.0" and presented in a diagram (Fig. 1-5). ER EGFR Clone AE1/AE3 Pan -Cytok cells "+", % cells "+", % cells "+", % cells "+", % Control 66,0 89,1 67,2 86,7 IFN ALFA 17,5 77,3 56,8 79,8 MSC-c-medium 59,0 94,4 100 100 Figure 4. Dependence of the number of alive cells MCF-7 by IFN alpha dose, adhesive fraction *-p<0,05, **-p<0,01. As a result, it was found that increasing the dose of IFN alpha lead to reducing the number of living cells in the adhesive and suspention fractions. This trend is particularly noticeable with long periods of cultivation (72, 96 hours). At the same time, the number of dead cells in adhesion, do not increase as much as reducing the number of living cells. It was indicated cytotoxic effect of the IFN alpha. In suspension fraction the number of live and dead tumor cells were correlated with IFN alpha concentrations. In these samples IFN alpha also demonstrated ability to inhibite proliferative activity of tumor cells. Because the MSC are sources of IFN alpha in the body, we decided to compare the effects of interferons and MSC and to formation tumor spheroids when cultured for more than 10 days. Tabl.2 Comparing the size of multicellular spheroids under the influence of the test substances. Figure 5. Dependence of the number of alive cells MCF-7 by IFN alpha dose, suspention fraction * – p<0,05, ** – p<0,01. Demianenko D.P.1, Perepelytsina E.M.1, Sidorenko M.V.1, Gergeliuk T.S.1, Goncharuk E.I. 2, Goltsev A.N.2 11Department of biotechnical problems of diagnostic IPCC, NAS of Ukraine, Kiev, Ukraine 2 Institute for problems of Cryobiology and Cryomedicine, NAS of Ukraine, Charkov, Ukraine In studies, it was found that the MSC` c-medium and IFN alpha to the greatest extent inhibited the proliferation of tumor cells, as well as in the adhesion and suspension fraction (Fig. 1, 2, 3). MSC` c-medium and IFN alpha supported the tumor cells transition in suspension fraction at low level (Fig.1B, 3). At the same time, T cells stimulated tumor cell proliferation, reducing the adhesion characteristics and stimulating cell yield in suspension fraction. That is contributed to the increasing activity of metastatic tumor cells. Thus, c- medium from MSC and IFN α have obvious cytostatic effect on tumor population holding the number of alive cells in the suspension, and in the adhesive below the level of control. On the contrary, samples with c- medium from T-lymphocytes stimulated tumor population to growth and migration. The study demonstrated that the c-medium from MSC and IFN α2β reduced the proliferative activity of tumor cells, inhibited the ability of tumor cells to migrate to the suspension fraction and reduced the expression of estrogen receptor and epidermal growth factor receptor while increasing the expression of differentiation markers - cytokeratins. C-medium from the T-cells, by contrast, generally had a pro-oncogenic effects on the population of tumor cells, it stimulated cell proliferation, adhesion and reduced expression of estrogen receptor and epidermal growth factor receptor. Photo 1. Immunocytochemical staining of estrogen receptor (ER), epidermal growth factor receptor (EGFR), cytokeratins (clone AE1AE3 and Pan-cytokeratine). Table 1. Calculation of histological index (stained cells / all cells). Control Clone AE1/AE3 Pan -ck EGFR ER IFN-ALFA ER MSC- C-medium ER Clone AE1/AE3 Clone AE1/AE3 Pan -ck Pan -ck EGFR EGFR 4 Culture terms Control MSC` C-medium IFN-α IFN-γ The third day The seventh day The tenth day Figure 1. Dependence of the live MCF-7 cells number from the microenvironment in 5 groups: 1) control, 2) DMEM 1:1 MSC` c-medium, 3) DMEM 1:1 T-cells c-medium, 4) IFN alpha 10 U/ml, 5) IFN gamma, 6-day incubation, the adhesive fraction (A), suspension fraction (B). 4 0 200 400 600 800 1000 1200 0 point Control INF-γ IFN-α MSC` medium T-cells 10 cells/ml alive cells dead cells А 3 0 100 200 300 400 500 600 700 0 point Control INF-γ IFN-α MSC` medium T-cells 10 cells/ml alive cells dead cells B 3 Figure 3. The number of alive cells MCF-7, suspension fraction in 4 groups: control, DMEM 1:1 MSC` c-medium, DMEM 1:1 T-cells c-medium, IFN alpha. Figure 2. The number of alive cells MCF-7, the adhesive fraction in 4 groups: control, DMEM 1:1 MSC` c-medium, DMEM 1:1 T-cells c-medium, IFN alpha. 0 50 100 150 200 250 300 350 400 450 500 0 24 48 72 96 incubation time, h the cells number, x E03 cells/ml Control MSC` c-medium Т-cells IFNα2β 0 50 100 150 200 250 300 350 0 24 48 72 96 incubation time, h the cells number, x E03 cells/ml . Control MSC` c-medium Т-cells IFNα2β Photo 2. Formation of multicellular tumor spheroids during prolonged cultivation of cells under different conditions of the microenvironment. Analysis of the formation of multicellular spheroid carried out after ten day by of incubation by micriphotography and measuring the diameters of aggregates (Photo 2.) The smallest spheroid size was observed when added to cell cultures c-medium from MSC. Average tumor size unit in culture was in 16.5 times lower than the control parameters. It should be noted that the reduction in the size of tumor aggregates when incubated with MSC was observed with decreasing their number. It could be result of increasin adhesive properties of tumor cells under influence of stroma cells. Therefore, special attention is the data on the size of tumor spheroid by incubating cells with IFN alpha and gamma, as different origins cytokines. Reducing the size of tumor spheroid was observed in cultures that inkubuvalysya of interferons alpha and gamma. Moreover, dose-dependent reduction in the size of the spheroid was higher when adding IFN-α (at 3.67 and 1.68 times) for IFN-γ reduced the average size of the spheroid relative to control in 3.1 and 1.32 times. Thus according to the data we can draw conclusions about the cellular microenvironment creating conditions that would hold back the process of metastasis. Control MSC` c-medium IFN alpha, E03 U/ml IFN gamma, 10 U/ml Average Volume of spheroids, E-06 mm3 9,9±0,54 0,6 ±0,044 2,7 ±0,18 3,2 ±0,25
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Transcript of The receptor profile of breast tumor cells and formationof tumor spheroids under influence of MSC.
Introduction
MATERAIL AND METHODS
RESULTS
CONCLUSIONS
The receptor profile of breast tumor cells and formationof tumor spheroids under influence of MSC.
Interferons are bioactive peptide substances that are synthesized in the body in response to foreign antigens. These cytokines are mediators of cell-cell interactions and have a systemic effect on the body and immunomodulatory properties.Mesenchymal stem cells (MSC) are the resident stem cells which there are present in adults and necessary for the vital activity of host tissue in normal life. MSC are involved in tissue regeneration and repair in response to disease and injury. Itis established the presence of a pool of stem cells in the rapidly proliferating tissues, such as bone marrow, gastrointestinal tract and skin. In addition, there is the presence of a pool of stem cells, which is responsible for the damage in a numberof tissues in the form of satellite cells in muscle or liver ovalo-cells.
Scientists have repeatedly observed positive effect of injection a suspension of stem cells to cancer patients. The MSC` influance was manifested as a decreasing in tumor size or dispersal of small metastases, and a quick recovery of fullyblood characteristics and improvement of the overall patient condition. However, more and more often there are publications that focused on the study of the oncogenic potential of stem cells. Thus, it appears that may have the potentialcarcinogenicity and neural stem cells in the bone marrow. In addition, transplantation of any tissue-specific stem cells positive for the marker of early embryogenesis - Oct-4 can be potentially oncogenic dangerous. Also revealed that CD133 +stem cells that have been isolated from human neyroopuholey have obligate carcinogenic activity in vivo. The above data indicate the necessarily to study tumorigenic potential of CD133 + cells (and other adult stem cells) before the start ofclinical trials and stem cell therapy.
Our aim at the first step was to investigate the biologically active substances of cellular origin - namely, the conditioned medium from the MSC and its impact on the life of the tumor cells of breast cancer line MCF-7. We involvedimmunocytochemical, cytological, biochemical, and statistical analysis methods. According to the results was investigated the effect of conditioned medium from MSCs (c-medium) on the expression of estrogen receptor and epidermal growthfactor receptor, cytokeratins (clone AE1 and AE3) and 5, 6, 8, 18, and E-cadherin breast cancer cells, MCF-7.
Cell line MCF-7 (line of human breast adenocarcinoma) were cultured under standard conditions (370 C, 100% humidity, 10% CO2) in the culture medium DMEM. Cells were seeded in 6-wellplates and formed three groups. Group 1 - control, Group 2 - incubation with IFN alpha in a concentration of 10 MO / ml, Group 3 - incubation in DMEM 1: 1 with c-medium from MSCs.Incubation period - 48 hours. Expression of estrogen receptor (ER) and epidermal growth factor receptor (EGF-R) and cytokeratins (clones AE1/AE3, 5D3, LP34) was determined byimmunohistochemical staining (Dako, USA). Analysis was performed by calculating the ratio of cells carrying the marker to total number of cells (histologic index, HS). Identification ofestrogen receptor and epidermal growth factor receptor was performed on coverslips, which were covered by a monolayer of MCF-7 cells. Visualization of the results was performed bymicrophotography microscope Stemy 2000C, Zeiss.To study the direct effect of IFN alpha and MSC` c-medium on proliferation, adhesion and survival of MCF-7, cells were seated in the well of a 24-well plates and cultured under normalconditions (370 C, 100% humidity, 10% CO2) in full medium DMEM. After days of cultivation wells were divided into 4 groups: control, IFN alpha, c - medium from MSC and c-medium fromT-lymphocytes. We counted the number of live and dead cells in adhesion and suspension fractions. Counting was performed on the 24th, 48th, 72nd and 96th hours of cultivation. The data wereprocessed statistically in the "Statistics 6.0" and presented in a diagram (Fig. 1-5).
ER EGFR Clone AE1/AE3 Pan -Cytokcells "+", % cells "+", % cells "+", % cells "+", %
Control 66,0 89,1 67,2 86,7IFN ALFA 17,5 77,3 56,8 79,8MSC-c-medium 59,0 94,4 100 100
Figure 4. Dependence of the number of alive cellsMCF-7 by IFN alpha dose, adhesive fraction*-p<0,05, **-p<0,01.
As a result, it was found that increasing the dose of IFN alpha lead to reducing the number of living cells in theadhesive and suspention fractions. This trend is particularly noticeable with long periods of cultivation (72, 96 hours).At the same time, the number of dead cells in adhesion, do not increase as much as reducing the number of livingcells. It was indicated cytotoxic effect of the IFN alpha. In suspension fraction the number of live and dead tumorcells were correlated with IFN alpha concentrations. In these samples IFN alpha also demonstrated ability to inhibiteproliferative activity of tumor cells.Because the MSC are sources of IFN alpha in the body, we decided to compare the effects of interferons and MSCand to formation tumor spheroids when cultured for more than 10 days.
Tabl.2Comparing the size of multicellular spheroids
under the influence of the test substances.
Figure 5. Dependence of the number of alive cellsMCF-7 by IFN alpha dose, suspention fraction* – p<0,05, ** – p<0,01.
Demianenko D.P.1, Perepelytsina E.M.1, Sidorenko M.V.1, Gergeliuk T.S.1, Goncharuk E.I. 2, Goltsev A.N.211Department of biotechnical problems of diagnostic IPCC, NAS of Ukraine, Kiev, Ukraine
2 Institute for problems of Cryobiology and Cryomedicine, NAS of Ukraine, Charkov, Ukraine
In studies, it was found that the MSC` c-medium and IFN alpha to the greatest extent inhibited theproliferation of tumor cells, as well as in the adhesion and suspension fraction (Fig. 1, 2, 3). MSC` c-mediumand IFN alpha supported the tumor cells transition in suspension fraction at low level (Fig.1B, 3). At the sametime, T cells stimulated tumor cell proliferation, reducing the adhesion characteristics and stimulating cellyield in suspension fraction. That is contributed to the increasing activity of metastatic tumor cells. Thus, c-medium from MSC and IFN α have obvious cytostatic effect on tumor population holding the number ofalive cells in the suspension, and in the adhesive below the level of control. On the contrary, samples with c-medium from T-lymphocytes stimulated tumor population to growth and migration.
The study demonstrated that the c-medium from MSC and IFN α2β reduced the proliferative activity of tumorcells, inhibited the ability of tumor cells to migrate to the suspension fraction and reduced the expression ofestrogen receptor and epidermal growth factor receptor while increasing the expression of differentiationmarkers - cytokeratins. C-medium from the T-cells, by contrast, generally had a pro-oncogenic effects on thepopulation of tumor cells, it stimulated cell proliferation, adhesion and reduced expression of estrogen receptorand epidermal growth factor receptor.
Photo 1. Immunocytochemical staining of estrogen receptor (ER), epidermal growth factor receptor (EGFR), cytokeratins (clone AE1AE3 and Pan-cytokeratine).
Table 1. Calculation of histological index (stained cells / all cells).
Control
Clone AE1/AE3 Pan -ck EGFR ER
IFN-ALFA
ER
MSC-C-medium
ER
Clone AE1/AE3
Clone AE1/AE3
Pan -ck
Pan -ck
EGFR
EGFR
4
Culture terms
Control MSC` C-medium IFN-α IFN-γ
The third day
The seventh
day
The tenth day
Figure 1. Dependence of the live MCF-7 cells number from the microenvironment in 5 groups: 1) control, 2)DMEM 1:1 MSC` c-medium, 3) DMEM 1:1 T-cells c-medium, 4) IFN alpha 10 U/ml, 5) IFN gamma, 6-dayincubation, the adhesive fraction (A), suspension fraction (B).
4
0 200 400 600 800 1000 1200
0 point
Control
INF-γ
IFN-α
MSC` medium
T-cells
10 cells/mlalive cells dead cells
А
30 100 200 300 400 500 600 700
0 point
Control
INF-γ
IFN-α
MSC` medium
T-cells
10 cells/mlalive cells dead cells
B
3
Figure 3. The number of alive cells MCF-7,suspension fraction in 4 groups: control, DMEM 1:1MSC` c-medium, DMEM 1:1 T-cells c-medium,IFN alpha.
Figure 2. The number of alive cells MCF-7, theadhesive fraction in 4 groups: control, DMEM 1:1MSC` c-medium, DMEM 1:1 T-cells c-medium,IFN alpha.
0
50
100
150
200
250
300
350
400
450
500
0 24 48 72 96incubation time, h
the
cells
num
ber,
x E
03 c
ells
/ml
Control MSC ̀c-medium Т-cells IFNα2β
0
50
100
150
200
250
300
350
0 24 48 72 96incubation time, h
the
cells
num
ber,
x E
03 c
ells
/ml
.
Control MSC ̀c-medium Т-cells IFNα2β
Photo 2. Formation of multicellular tumor spheroids during prolonged cultivation of cells under different conditions of the microenvironment.
Analysis of the formation of multicellular spheroid carried out after ten day by of incubation by micriphotography and measuring the diameters of aggregates (Photo 2.) The smallest spheroid size was observed when added to cell cultures c-medium from MSC. Average tumor size unit in culture was in 16.5 times lower than the control parameters. It should be noted that the reduction in the size of tumor aggregates when incubated with MSC was observed with decreasing their number. It could be result of increasin adhesive properties of tumor cells under influence of stroma cells. Therefore, special attention is the data on the size of tumor spheroid by incubating cells with IFN alpha and gamma, as different origins cytokines. Reducing the size of tumor spheroid was observed in cultures that inkubuvalysya of interferons alpha and gamma. Moreover, dose-dependent reduction in the size of the spheroid was higher when adding IFN-α (at 3.67 and 1.68 times) for IFN-γ reduced the average size of the spheroid relative to control in 3.1 and 1.32 times. Thus according to the data we can draw conclusions about the cellular microenvironment creating conditions that would hold back the process of metastasis.
Control MSC` c-medium IFN alpha, E03 U/ml IFN gamma, 10 U/ml
Average Volume of spheroids, E-06 mm3
9,9±0,54 0,6 ±0,044 2,7 ±0,18 3,2 ±0,25
