CC BY
38Physics of the Alive, Т. 16, No2, 2008. С.105-111.
© Perepelitsyna O.M., Garmanchouk L.V., Sydorenko M.V., Ostapchenko L.I.
УДК 577.3
REACTIVITY AND SENSIBILITY OF BREAST ADENOCARCINOMA (MCF-7) IN COCULTURE DEPENDS ON ARCHITECTURE OF CELLS MODEL
Perepelitsyna O.M.1, Garmanchouk L.V.2, Sydorenko M. V.1, Ostapchenko2 L.I.
1 Biotechnical problems of diagnostic Dep.,
Institute cryobiology and criomedicine NAS Ukraine, Kiev, Ukraine.
2 Taras Shevchenko National University of Kyiv
Was reseived October 12, 2008
A co-culture system was developed to investigate the interactions between MCF-7 breast epithelial cells and MT-4 (line human T-cell leukaemia) by paracrine mechanism (no contact coculture), reactivity and sensibility cells in different cell populations. For spheroids generation was chosen breast adenocarcinoma cell line MCF-7. For coculture - line human T-cell leukaemia MT-4 as source of paracrine influence. MCF-7 viability, allocation and proliferation were determinated in two fractions of MCF -7 cells from spheroid culture and monolayer. In results MT-4 reduced number of cells MCF-7 in fraction of adhesive cells and didn't influence on suspension fraction. Cells viability was different in this two fraction, in monolayer and spheroids, with MT-4 and without MT-4. MT-4 stimulated proliferation in adhesive fraction and supported proliferation in suspension. Proliferation reduced in both fractions without MT-4. We think that, in dependence of cell interaction in population influence of MT-4 by paracrine signal may stimulate proliferation MCF-7 cells, reduce adhesive to the bottom and lead to formation MTS. In the same time MT-4 supported proliferation cells in suspension but didn't influence on growth and further evolution spheroids.
Key words: Multicellular tumor spheroids (MTS, 3-D culture ), monolayer (2-D culture), MCF-7, MT-4, coculturing, proliferation, viability.
monolayers (2D). As a result, the phenotypes, functions, and regulation of signaling pathways under 2D and 3D conditions are fundamentally different [1]. A lot of authors demonstrated in experiments that MTS clear illustrates the cell-cell interactions in tumor cell population, cytology, morphology and micro environmental conditions found in micro tumor on avascular stage of development [2-5]. In our investigation tumor spheroids are used for comparative reactivity and viability two model system multycellular aggregates and cell monolayer in condition of coculturing with cell of immune system - lymphocytes. It is known that tumor is a combination of immune cells (macrophages, lymphocytes and dendric cells), endothelial cells, fibroblasts and perivascular cells as well as malignant cells [6]. Present and interaction between this cell elements has prognostic matter. Resent publication demonstrated that important chains of interaction in tumor micropopulation are chemokines [7]. Active source of paracrine influence are immune cells. For example, tumor infiltrating lymphocytes (TILs) and macrophages, in different way support tumor growth, promote the proliferation, invasion, and metastasis of tumor cells, and tumor vascularization
INTRODUCTION
Formation of intercellular contacts essential influents on sensibility and reactivity cells in tumor culture. This process includes changes of intercellular contacts and cell aggregational behaviour with substrate, reducing role of basemen membrane and exstracellular matrix for development tissue. Multicellular microagrigates have been widely used for tissue formation modeling. Generation spheroids illustrate changing in cells adhesion, formation of extracellular matrix and microenvironment. That is why microaggregates, from transformed malignant cells, are convenient model system in vitro for study effect of tissue architecture on characteristics of tumor microaggregats in vivo. In this work we compare sensibility and reactivity cells in two model systems monolayer and spheroids.
Multicellular tumor spheroids (MTS) is a intermediate model of the organization of solid tumors, between monolayer and organ culture. The 3D structures are characterized by establishment of
adhesion complexes, tissue polarity, cytoskeletal
structures, and cell volume that are significantly
different from those found in cells cultured as
1Q7
have complex receptor - ligand CXCL12-CXCR4. This system influences on development of breast cancer, takes part in organ-specific metastasing [17]. Interaction receptor CXCR4 with ligand CXCL12 plays important role in chemotacsis and migration of tumor cells to specific matastasing sites [13,14]. This ligand was found in lymphocytes from the local circulation, monocytes, subpopulation of cells-killers, dendric cells and endothelial [15,16]. In additional, T-lymphocytes products INF-3 too, in replay to mytogen stimulus such as TNF and phitohemagglutenine [ 18 ].
Spheroids generation. Multicellular tumor spheroids (MTS) are an intermediate model of the solid tumors organization, between monolayer and organ culture. The 3D structure is characterized by establishment of adhesion complexes, tissue polarity, cytoskeletal structures, and cell volume that are significantly different from those found in cells cultured as monolayers (2D). As a result, the phenotypes, functions, and regulation of signaling pathways under 2D and 3D conditions are fundamentally different [4]. A lot of authors demonstrated in experiments with MTS that clearly illustrated the cell-cell interactions in tumor cell population, cytology, morphology and micro environmental conditions found in micro tumor on avascular stage of development are differ than in monolayer culture[1-3]. Multicell tumor spheroids were generated by means standart method by Kelm JM [19]. We modificated this method and have License on useful model of generation MTS in Ukraine [20]. Cell confluent is trypsinized and single-cell suspension is seeded on low-adhesive substrate in density 5.0x104 cells/ml in growth medium with 0.24% of carboxy-methyl-cellulose (CMC). We generated spheroids in 6-wells test-plates (TPP, Switzerland) and Petry dishes. Dishes with cells are placed on shaker with low rotation (150 rmp) during one hour. Spheroids formation depends on the tipe of cells used, cells density at seeding the speed of rotation, the type of culture medium, concentration of the FTS and the incubation time.
Photo 5. MCF-7 spheroids are in culture.
Co-culturing. For monolayer growth MCF-7 cells were seeded on 6-wells in density 4.0x104 cells/ml in day before experiment for cells adaptation. For spheroids culture in 2 days before experiment generated spheroids and seeded in 6-wells at the same density. For coculturing MT-4 was seeded in diffusion chamber with nitrocellulose filters in concentration 5.0x105 cells/ml. Duration of coculturing was 24h and 48h. After that diffusion chambers were taken and analyzed MCF-7. Cells viability, proliferation and allocation in spheroids and monolayer of MCF-7 was determinated in two steps.
Proliferation activity assay. The first step is: suspension fraction was taken from culture. Spheroids were disaggregated. The second step is: adhesion fraction from the bottom of wells was taken with the help of trypsine. Cells were stained by tripan-blue and was calculated number of alive/died cells in all fractions. As a control samples were used spheroids and monolayer in standard condition without additional MT-4. Control was calculated at the same time and under the same scheme.
Allocation characteristics of culture. Allocation of MCF-7 was discovered by changing ratio cells in adhesive and suspension fraction during incubation of MCF-7 spheroids and monolayer with MT4 in comparison with control samples.
Cells viability by MTT colorimetric test. To investigate proliferation activity of cells MCF7 after coincubation with MT-4, we analysed it with 3-[4,5-dimetltiazol-2]-2,5-dipheniltetratetrazolium (MTT) by colorimetric assay [21]. In 96-well with 100 mkl of cells suspintion we added 20 mkl MTT solution (5mg/ml PBS, Sigma). After that cells were incubated with MTT during 4 h in standard conditions. Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, yielding purple MTT formazan crystals which are insoluble in aqueous solutions. The resulting purple solution is spectrophotometrically measured. An increase in cell number results in an increase in the amount of MTT formazan formed and an increase in absorbance. Then samples were centrifuged under 1500g, during 5 min and supernatant was extracted. In all wells were added 10mkl DMSO (Sigma) to MTT crystals dilution and 20mkl glycine. Optical absorption was detected on multi-well spectroscopy reader Multyscan (Labsystem, Finland) (OP540 nm).
RESULTS AND DISCUSSION
Proliferation. Proliferation calculated in monolayer and spheroids culture in control samples and with coincubation with MT-4. Start concentrations of cells was equal. For the first day number of cells increased in 50% in control samples and in spheroid
30% without MT-4.During that time, suspension fraction of 3-D culture increased (from 75 thous. to 225 thous.). In 3-D culture quantity cells in adhesion fraction on 48 h significantly decrease with MT-4 than in control (180 thous.-120 thous.cells).
Cell mass in suspension fraction 3-D culture stand constant with MT-4, and decrease in two times without MT-4. Such allocation during first 48 h may be results two process: one - stimulation/inhibition of
proliferation or cells change adhesive ability and as result migration in suspension/or making confluent. For differentiation this two mechanisms we investigate number alive and dead cells in adhesive and suspension fraction in 2-D and 3-D culture under influences of paracryne factors from MT-4 and in control, percent viability of cells.
Viability of MCF-7 cells in monolayer with MT-4 and in control according MTT-test.
According to results of MTT-test percent alive cells in adhesive fraction monolayer culture without MT-4 decreased from 98% to 50%, in that time with MT-4 that percent changed from 90% to 73%.
In suspension fraction number of alive cells without MT-4 didn't change, and with MT-4 decreased from 91% to 31% (Fig.4).
% Viability fracsions of 3-D culture of MCF-7
100
80
60
40
20
0 1 day 2day
H suspension with MT4 70 77,5
E adhesion with MT4 97 75
□ suspension control 80 73,5
□ adhesion control 78 85
Fig.4. Cell viability in monolayer culture of MCF-7.
Viability cells in adhesive fraction in coincubation 3-D culture with MT-4 decreased from 80% to 74%.In suspension fraction reverse process are demonstrated. Percent alive cells decreased from 98% to 75% in control and in presence MT-4 increased from 70% to 90% (Fig.5).
The observation described above demonstrated that influence of MT-4 cells reduced number of cells MCF-7 in fraction of adhesive cells and didn't influence on suspension fraction. Cells viability was different in
these two fractions: in monolayer and spheroids; with MT-4 and without MT-4. MT-4 stimulated proliferation in adhesive fraction of monolayer and supported proliferation in suspension fraction of spheroids. Proliferation was reduced in both fractions without MT4. The data obtained show that reactivity MCF-7 on paracrine sygnals from MT4 and proliferation of MCF7 cells depends on model of growth, characteristics of substrate, cell fraction, time of incubation. The data presented in this study support the hypothesis that lymphocytes take active part in tumor regulation by paracrine way. It is interesting that coculture lymphocytes with breast cancer cell line led to significant increase proliferation in adhesive fraction 2D culture MCF-7 and suspension fraction 3-D culture. It may be that paracrine factors from MT-4 in 2-D culture stimulate proliferation and promote making confluante. Then paracrine actions of MT-4 provoke active migration MCF-7 cells in suspension. However, after that paracrine signals from MT-4 are not enough for growth aggregates in Metacell-free culture and cells dead.
% Viability fracsions of 2-D culture of MCF-7
100
80
60
40
20
0 1 day 2day
Ei suspension with MT4 91 31
a adhesion with MT4 90 73
S suspension control 80 79,5
□ adhesion control 98 50
Fig. 5. Cell viability in spheroids culture of MCF-7.
Controversy in spheroid culture MT-4 stimulates proliferation and growth aggregates. This studies demonstrated that proliferation, viability and adhesive characteristics cells MCF-7 under coincubation with MT-4 depends on time of coculturing and model of growth. MT-4 by paracrine signal way stimulate proliferation cells but reduced number of cells MCF-7 in fraction of adhesive cells in 2-D culture. Because influence of MT-4 reduces adhesive cells MCF-7 to the bottom and lead to migration cells to suspension. MT-4 didn’t influence on proliferation in adhesive fraction in 3-D culture in that time stimulated proliferation in spheroids. Viability cells in this two fraction significantly different with MT-4 and without MT-4. In conclusion we can add that monolayer is
РЕАКТИВНІСТЬ ТА ЧУТЛИВІСТЬ АДЕНОКАРЦИНОМИ МОЛОЧНОЇ ЗАЛОЗИ (MCF-7) В КОКУЛЬТУРІ ЗАЛЕЖИТЬ ВІД АРХІТЕКТУРИ КЛІТИННОЇ МОДЕЛІ
Перепелиціна О.М.,Гарманчук Л.В., Сидоренко М.В., Остапченко Л.І.
Система кокультивування була розроблена для вивчення взаємодії між клітинами МСБ-7 раку молочної залози та МТ-4 (линії Т-клітинної лейкемії) за допомогою паракрінних механізмів (без контактне культивування), реактивності т чутливості клітин у різних клітинних популяціях. Для генерації сфероїдів була обрана лінія аденокарциноми молочної залози МСБ-7. Для кокультури - лінія Т-клітинної лейкемії МТ-4 у якості джерела паракринного впливу. Виживаність, перерозподіл та проліферація клітин МСБ-7 були визначені в двох фракціях (суспензійній та адгезивній) двох модельних систем (моно шару та сфероїдів). В результаті було відмічено, що присутність МТ-4 знижує кількість клітин МСБ -7 в адгезивній фракції та не впливає на суспензійну. Виживаність клітин відрізнялася в двох фракціях клітин, в моношаровій і сфероїдній культурах, як в присутності МТ-4 так й без них. Клітини МТ-4 стимулюють проліферацію в адгезивній фракції та підтримують у суспензійній. У відсутності МТ-4 проліферація в обох фракціях знижується. Ми припускаємо, що клітини МТ-4 через паракринні механізми стимулюють проліферацію клітин МСБ-7, знижають адгезію клітин до субстрату що призводить до формування багатоклітинних пухлинних сфероїдів. Цікаво, що ефект от такого впливу суттєво залежить від взаємодії клітин та архітектури популяції.
Ключові слова: багатоклітинні пухлинні сфероїді (БПС, 3-Б культура), моношар (2-Б культура), МСБ-7, МТ-4, кокультивування, проліферація, виживаність, адгезія.
РЕАКТИВНОСТЬ И ЧУВСТВИТЕЛЬНОСТЬ АДЕНОКАРЦИНОМЫ МОЛОЧНОЙ ЖЕЛЕЗЫ (MCF-7) В КОКУЛЬТУРЕ ЗАВИСИТ ОТ АРХИТЕКТУРЫ КЛЕТОЧНОЙ МОДЕЛИ
Перепелицына Е.М., Гарманчук Л.В., Сидоренко М.В., Остапченко Л.И.
Система кокультивирования была разработана для исследования взаимодействия между клетками МСБ-7 рака молочной железы и МТ-4 ( линии Т-клеточной лейкемии человека) посредством паракринного механизма (без контактная кокультура), реактивности и чувствительности клеток в различных клеточных популяциях. Для генерации сфероидов была использована линия аденокарциномы молочной железы МСБ-7. Для кокультуры - линия Т-клеточной лейкемии МТ-4 в качестве источника паракринного влияния. Выживаемость, перераспределение и пролиферация клеток МСБ-7 определялась в двух фракциях (адгезионной и суспензионной) двух модельних систем (монослоя и сфероидов). В результате отмечено, что МТ-4 снижают количество клеток МСБ-7 во фракции адгезивных клеток и не влияют на суспензионную фракцию. Выживаемость клеток была разной в этих двух фракциях, как в монослойной так и в сфероидной культурах, как с МТ-4 так и без них. МТ-4 стимулирует пролиферацию в адгезионной фракции и поддерживает в суспензионной. В отсутствии МТ-4 пролиферация МСБ-7 снижается в обоих фракциях. Мы предполагаем, с помощью паракринных сигналов клетки МТ-4 могут стимулировать пролиферацию МСБ-7, снижать адгезию клеток к поверхности и активизировать формирование многоклеточных опухолевых сфероидов. Причем, эффект от этого влияния существенно зависит от взаимодействия клеток и архитектуры популяции.
Ключевые слова: многоклеточные опухолевые сфероиды (МОС, 3-Б культура), монослой (2-Б культура), МСБ-7, МТ-4, кокультивирование, пролиферация, выживаемость, адгезия.
