Optimization of in vitro model for analysis of tumor cell migration dynamics
Migration ability is an important feature of tumor cells. There are several approaches to analyze the dynamics of cancer cell migration in vitro. One of the most perspective and closer to the in vivo conditions is the model of initiation of the cell migration from 3D multicellular spheroids onto gro...
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Інститут молекулярної біології і генетики НАН України
2018
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| Cite this: | Optimization of in vitro model for analysis of tumor cell migration dynamics / A.O. Kravchenko, V.R. Kosach, K.A. Shkarina, I.V. Zaiets, I.O. Tykhonkova, A.I. Khoruzhenko // Вiopolymers and Cell. — 2018. — Т. 34, № 6. — С. 476-486. — Бібліогр.: 20 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1860182993921900544 |
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| author | Kravchenko, A.O. Kosach, V.R. Shkarina, K.A. Zaiets, I.V. Tykhonkova, I.O. Khoruzhenko, A.I. |
| author_facet | Kravchenko, A.O. Kosach, V.R. Shkarina, K.A. Zaiets, I.V. Tykhonkova, I.O. Khoruzhenko, A.I. |
| citation_txt | Optimization of in vitro model for analysis of tumor cell migration dynamics / A.O. Kravchenko, V.R. Kosach, K.A. Shkarina, I.V. Zaiets, I.O. Tykhonkova, A.I. Khoruzhenko // Вiopolymers and Cell. — 2018. — Т. 34, № 6. — С. 476-486. — Бібліогр.: 20 назв. — англ. |
| collection | DSpace DC |
| container_title | Вiopolymers and Cell |
| description | Migration ability is an important feature of tumor cells. There are several approaches to analyze the dynamics of cancer cell migration in vitro. One of the most perspective and closer to the in vivo conditions is the model of initiation of the cell migration from 3D multicellular spheroids onto growth surface. Aim. Optimization of the model for adequate quantitative characteristics of the tumor cell locomotion during several days. Methods. 2D and 3D MCF-7 cell culture, immunofluorescence analysis, and image analysis using computer software Fiji. Results. Unification of spheroid size allowed avoiding a significant data deviation. The obtained spheroids spread completely for 3 days. The highest migration ratio was observed at the 2nd day. The proliferation level at each of 3-day experiment was the same and did not exceed 3%. The validity of the model was tested after migration inhibition by rapamycin (mTOR signaling inhibitor). Additionally, this model was successfully applied to immunofluorescence analysis, namely investigation of p85S6K1 subcellular localization in moving MCF-7 cells. Conclusions. Double filtration of multicellular spheroids allowed unification of their size, which promotes an adequate interpretation of the migration assay. This model enabled the study of tumor cells migration dynamics and can be further used for the development of anticancer drug.
Міграційна здатність є важливою ознакою пухлинних клітин. Існує кілька підходів до аналізу динаміки міграції ракових клітин in vitro. Однією з найбільш перспективних і близьких до умов in vivo є модель ініціювання міграції клітин з тривимірного багатоклітинного сфероїда на ростову поверхню. Мета. Оптимізація моделі для адекватної кількісної оцінки міграції пухлинних клітин. Методи. 2- та 3-вимірна культура клітин лінії MCF-7, імунофлюоресцентний аналіз, аналіз зображень з використанням комп'ютерної програми Фіджі. Результати. Уніфікація розміру сфероїдів дозволила уникнути значного розкиду даних. Отримані сфероїди повністю розпластувались протягом 3 днів. Найвищий показник міграції спостерігався на 2-гу добу розпластування сфероїда. Рівень проліферації клітин за кожну добу 3-денного експерименту був майже однаковим і не перевищував 3%. Валідність моделі була протестована після пригнічення міграційної активності клітин під впливом рапаміцину (інгібітор сигналізації mTOR). Крім того, запропонована модель була успішно застосована для дослідження субклітинної локалізації p85S6K1 в мігруючих клітинах лінії MCF-7 за допомогою імунофлюоресцентного аналізу. Висновки. Подвійне фільтрування багатоклітинних сфероїдів дозволяє уніфікувати їх розміри, що в подальшому сприяє адекватній оцінці міграційного потенціалу клітин. Запропонована модель дозволяє вивчати динаміку міграційних процесів пухлинних клітин і може бути використана для тестування протипухлинних препаратів in vitro.
Миграционная способность является важной особенностью опухолевых клеток. Существует несколько подходов для анализа динамики миграции раковых клеток in vitro. Одной из наиболее перспективных и приближенных к условиям in vivo является модель инициации миграции клеток из трехмерных многоклеточных сфероидов на поверхность роста. Цель. Оптимизация модели для адекватных количественных характеристик локомоции опухолевых клеток в течение нескольких дней. Методы. 2D и 3D MCF-7 клеточная культура, иммунофлуоресцентный анализ и анализ изображений с использованием компьютерного программного обеспечения Фиджи. Результаты. Унификация размеров сфероидов позволила избежать значительного отклонения данных. Полученные сфероиды распространились полностью за 3 дня. Самый высокий коэффициент миграции наблюдался на 2-й день. Уровень пролиферации в каждом из 3-дневных экспериментов был одинаковым и не превышал 3%. Достоверность модели была проверена после ингибирования миграции рапамицином (ингибитор передачи сигналов mTOR). Кроме того, эта модель была успешно применена для иммунофлуоресцентного анализа, а именно для исследования субклеточной локализации p85S6K1 в движущихся клетках MCF-7. Выводы. Двойная фильтрация многоклеточных сфероидов позволила унифицировать их размер, что способствует адекватной интерпретации анализа миграции. Эта модель позволила изучить динамику миграции опухолевых клеток и может быть в дальнейшем использована для разработки противоопухолевого препарата.
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477
A. O. Kravchenko, V. R. Kosach, K. A. Shkarina
© 2018 A. O. Kravchenko et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf
of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any
medium, provided the original work is properly cited
UDC 576 + 577
Optimization of in vitro model for analysis
of tumor cell migration dynamics
A. O. Kravchenko1,2, V. R. Kosach1, K. A. Shkarina1, I. V. Zaiets1,2,
I. O. Tykhonkova1, A. I. Khoruzhenko1
1 Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143
2 ESC "Institute of Biology and Medicine", Taras Shevchenko National University of Kyiv
64/13, Volodymyrska Str., Kyiv, Ukraine, 01601
a.i.khoruzhenko@imbg.org.ua
Migration capacity is an important feature of tumor cells. There are several approaches to
analyze the dynamics of cancer cell migration in vitro. The model of initiation of cell migration
from 3D multicellular spheroids onto growth surface is one of the closest to the in vivo condi-
tions. Aim. Optimization of the model to study tumor cell mobility for several days. Methods. 2D
and 3D MCF-7 cell culture, immunofluorescence analysis and image analysis using the Fiji
computer software. Results. Unification of spheroid size allowed avoiding a significant data
deviation. The obtained spheroids spread completely for three days. The highest migration
ratio was observed on the second day. The proliferation level was similar during each day of
the three-day experiment; it did not exceed 3 %. The validity of the model was tested after
migration inhibition by a mTOR signaling inhibitor rapamycin. Additionally, this model was
successfully applied to immunofluorescence study of p85S6K1 subcellular localization in
moving MCF-7 cells. Conclusions. Double filtration of multicellular spheroids allowed uni-
fication of their size; this promotes an adequate interpretation of the migration assay. This
model allows to study tumor cell migration dynamics and can be further used for development
of anticancer drugs.
K e y w o r d s: Cancer cell migration assay, 2D and 3D culture, p85S6K1, multicellular spheroids
Introduction
The ability of malignant tumors to form metas-
tases is a critical step of the cancer progression
and distinguishes malignant tumor cells from
benign or normal ones [1].This feature of tumor
cells is determined by their ability to migrate
and penetrate surrounding tissues. Often the
metastases but not primary tumors lead to the
death of organism. That is why the processes
of cancer cell migration and invasion are the
most important targets of the anti-cancer basic
Methods ISSN 1993-6842 (on-line); ISSN 0233-7657 (print)
Biopolymers and Cell. 2018. Vol. 34. N 6. P 477–486
doi: http://dx.doi.org/10.7124/bc.000992
mailto:a.i.khoruzhenko@imbg.org.ua
478
A. O. Kravchenko, V. R. Kosach, K. A. Shkarina et al.
research and drug development. In normal adult
mammalian organism, only immune and pla-
centa cells are able to invade corresponding
tissues. However, an ability to migrate is not
limited to these cell types. There are also many
other cell populations that migrate to their final
niche in the course of embryogenesis and post-
embryonic development, including the develop-
ment of cardiovascular system, central nervous
system, and many others [2–6].
Nowadays, there are several approaches to
evaluate the cell migration and locomotor
properties in vitro. The most widely employed
methods are the transwell migration and inva-
sion assay, the “wound healing” assay and the
initiation of cell migration from multicellular
spheroids or tissue explants into matrigel or
onto growth surface. Each of them has the
advantages and disadvantages which leads to
the need of their improvement.
The first mentioned method is the transwell
migration and invasion assays. The basis of
this method is the initiation of directed cell
movement through the pores of the transwell
membrane toward the chemoattractant. This
approach is widely used in cancer research and
especially for test of antitumor properties of
corresponding drugs or their combination [1].
However, this approach has several disadvan-
tages, in particular time restrictions, which
limits the duration of the study to 24–48 hours;
relatively high costs, and the need of selecting
the optimal conditions for each particular cell
type and the type of attractant used. Another
widely applied method is the wound healing
assay. This method is based on estimation of
closure dynamics of the artificially formed free
space in the confluent cell monolayer. Alike
previous case, the main disadvantage of this
method is limitation of experiment duration,
usually up to 24 hours to exclude the effect of
cell proliferation in freed space. The third
experimental strategy of migration estimation
is based on the transformation of 3D multicel-
lular spheroid into 2D cellular monolayer
colony. This method provides several advan-
tages over above-mentioned approaches; the
main of them is that 3D culture of cancer cells
more closely reflects in vivo conditions [7].
Besides, it also enables easy detection of many
of biochemical and morphological properties
of moving cells. Depending on the studied cell
type, this model provides an opportunity to
characterize either collective or single-cell
migration. However, there are also several
known limitations to this model application.
The major difficulty is significant variation of
spheroid sizes. It, in turn, complicates the
comparison of cell migration kinetics between
spheroids with initially different size.
Therefore, there is a strong need for further
unification of the multicellular spheroid size.
There are several approaches to obtain the
spheroids of similar sizes including handling
drop method or application of special multi-
well plates, etc., but they require preliminary
preparation and are relatively expensive. From
our point of view the double filtration of spher-
oid suspension through nylon mesh filters
could provide strong unification of spheroid
size.
Another issue which should be regarded is
the input of proliferation in the value of dis-
tance or surfaces covered by the cells in migra-
tion tests. To compare the dynamics of cell
migration at regular terms, it is necessary to
ensure that the level of proliferation for the
same time period was also similar.
479
Optimization of in vitro model for analysis of tumor cell migration dynamics
To test the proposed approach, the inhibi-
tion of cell locomotion was applied.
The cell migration is strongly affected by a
variety of growth factors, hormones, cytokines
and other chemical cues, which induce the
activation of several signaling pathways with-
in migrating cell. The PI3K/mTOR/S6K cas-
cade has been previously described as an im-
portant regulator of mammalian cell migration
[8]. In normal tissue this pathway is involved
in the control of many intracellular events,
including protein synthesis regulation, the
G1/S phase cell cycle transition and many oth-
ers [9, 10]. mTOR/S6K signaling over activa-
tion and over expression has been observed in
many diseases including cancer, diabetes, and
other [11–13]. There are some significant dif-
ferences in PI3K/mTOR/S6K signaling in 3D
vs. a 2D cell culture system [14]. Earlier, we
confirmed that rapamycin, an inhibitor of
mTOR signaling, decreased the MCF-7 cell
locomotion in scratch test. So, it was used in
this study for validation of optimized locomo-
tion assay [15].
Additionally, the model of outspreading
spheroids can be applied for immunochemical
assay. Earlier we revealed the subcellular re-
localization of p70S6K1 from the cytoplasm
into the nuclei of MCF-7 cells in course of
migration from spheroid onto growth surface
by immunofluorescent analysis [16]. One of
the explanations of the kinase relocalization
was its association with the transcription factor
TBR2, expressed in actively migrating cells
like embryonic, cancer cells and lymphocytes.
Kinase of ribosomal protein S6 (S6K1) is one
of the key links of the mTOR signaling path-
way. S6K1 has several known isoforms:
p85(S6K1), the most highly expressed isoform
p70(S6K1), and additionally less studied iso-
forms p60(S6K1) and p31(S6K1). Both
p85(S6K1) and p70(S6K1) isoforms have been
previously shown to be regulated through
phosphorylation by the mTOR/S6K, PI3K/Akt
signaling pathway [9]. Initially p85S6K1 was
regarded as a nuclear isoform of S6K1, more-
over it contained the signal of nuclear localiza-
tion at N-terminus of molecule. But later it was
observed in cytoplasm as well. p70S6K1 and
p85S6K1 have both common and different
effectors and targets. To compare their subcel-
lular distribution in migrating cells, immuno-
fluorescent revealing of p85S6K1 in outspread-
ing spheroids of MCF-7 cells was performed.
So, the aim of this study was to improve the
method of initiation of cell migration from the
3D multicellular spheroids cells onto the
growth surface. Namely, the MCF-7 cell spher-
oid size unification was performed by double
filtration through nylon mesh filters with pore
diameter 30 and 100 mm; the index of MCF-7
cell proliferation at the 1st, 2nd, and 3rd days
in outspreaded spheroids was estimated; the
validity of quantitative migration assay was
tested using migration inhibition; availability
of the model for immunofluorescent analysis
was confirmed by corresponding assay of the
p85S6K1 relocalization in course of cell mi-
gration.The proposed approach could be useful
for basic cancer research and anticancer drug
development, as well as for other assay of the
cell locomotion dynamics.
Materials and Methods
Cell culture.MCF-7 cell line derived from
metastatic site of malignant breast tumor was
used in this study [17]. MCF-7 cells were
cultured in Dulbecco’s Modified Eagle’s me-
480
A. O. Kravchenko, V. R. Kosach, K. A. Shkarina et al.
dium (DMEM) (Sigma, USA) supplemented
with 10 % fetal bovine serum (FBS, CellSera,
Australia), 4 mM glutamine (Sigma, USA),
50 U / ml penicillin and 50 g / ml streptomycin
(Sigma, USA) at 37 ° C in a 5 % CO2 hu-
midified incubator.
Generation of spheroids. For multicellular
spheroids generation, confluent monolayer of
MCF-7 cells was detached with 0.25 % tryp-
sin, 0.02 % EDTA in Hank′s Balanced Salt
Solution (Sigma, USA) to generate single cell
suspension, which was transferred into the
10-centimeter Petri dishes coated with 1 %
agarose (Serva, Heidelberg, Germany). Cells
were further cultivated for three days. The
resulting suspension of spheroids of different
size was subjected to the two-step filtration.
First, spheroids were passed through a sterile
nylon mesh (Spectrum, USA) with a pore di-
ameter of 100 μm to remove large cell aggre-
gates. The second step of the filtration was
carried using sterile filter mesh (Spectrum,
USA) with a pore diameter of 30μm for single
cell elimination.
The transformation of spheroids in a mono-
layer cell colony. The obtained spheroids of
uniform size were transferred into the 6-well
plates with a fresh complete medium. The
migration and proliferation processes were
analyzed at 0, 24, 48 and 72 hours post filtra-
tion. The images were acquired using bright
field and phase contrast microscopy (CETI
Versus inverted microscope, CETI, Belgium,
and Leica DM 1000, Leica Microsystems,
Germany, Magnification 25x, 100x). Only the
colonies of round shape were selected for fur-
ther analysis. A colony area was determined
using Fiji software, and an approximate colo-
ny radius was calculated using formula:
r=√S/π. The migration activity was expressed
as a difference of colonies radii at correspond-
ing time points. To alter the cell migration
dynamics the treatment of MCF-7 multicel-
lular spheroids with 10 nM rapamycin
(Calbiochem biochemicals, Los Angeles, USA)
was applied and then the cell locomotion was
estimated.
Proliferation activity analysis. The number
of mitotic cells was calculated at 24, 48 and
72 hours of spheroid outspreading. The im-
ages of spheroids were taken at indicated time
points, and then the number of proliferating
cells of corresponding morphology was calcu-
lated. Besides, cultured on cover glasses colo-
nies were fixed with 10 % formaldehyde solu-
tion (Thermo Scientific, USA) for 15 minutes
and afterwards stained with 2 % Hoechst
33342 (Molecular Probes, USA) in the dark
for 25 minutes. Samples were mounted on
slides into Mowiol mounting medium (Sigma-
Aldrich, St. Louis, USA) containing 2.5 %
DABCO (Sigma-Aldrich, St. Louis, USA), and
amount of mitotic cells were calculated using
fluorescent microscopy by morphological fea-
tures. Index of proliferation was calculated as
the per cent of mitotic cells in the population
of spreading spheroid.
Immunofluorescence analysis. Cultured on
cover glasses colonies at 0, 24, 48 and 72 hours
were fixed with 10 % formaldehyde solution
for 15 minutes, as described previously. For
membrane permeabilization, the cells were
treated with a 0.2 % Triton X-100 in PBS solu-
tion and afterwards incubated for 30 min at
room temperature in 10 mM cupric sulphate
and 50 mM ammonium acetate (pH 5.0) to
reduce autofluorescence. Non-specific antibody
binding was blocked with 10 % FCS in PBS for
481
Optimization of in vitro model for analysis of tumor cell migration dynamics
30 min at 37 °C. Anti-p85(S6K1) rabbit poly-
clonal antibodies were applied in dilution 1:100
overnight at 4 °C [18]. Secondary FITC conju-
gated anti-rabbit antibodies (Jackson Immuno
Research Laboratories, Pennsylvania, USA)
were applied in dilution 1:400 for 1h at 37 °C
in humidified chamber. Samples were mounted
into the Mowiol medium (Sigma-Aldrich, St.
Louis, USA) containing 2.5 % DABCO (Sigma-
Aldrich, St. Louis, USA). Fluorescent micros-
copy was performed using Leica DM 1000
fluorescent microscope (Leica Micro systems,
Wetzlar, Germany, Canon PowerShots70,
Magnification 100x, 400x).
Statistical analysis. All image analysis was
performed using the Fiji software [19]. Data
analysis was performed using Origin 9. All
data are expressed as median +/- SD. Each
experiment was repeated at least three times.
Results and Discussion
Unification of spheroid size. One of the main
drawbacks of the spheroid to monolayer tran-
sition model is the significant size variation of
multicellular spheroids generated by the stan-
dard liquid overlay method (Fig. 1a). To over-
come this obstacle, an additional step of dou-
ble filtration of generated spheroids suspension
was applied. It enabled to unify the size of the
colonies used for subsequent analysis, and, so,
to perform proliferation and migration mea-
surements more accurately and rapidly
(Fig. 1b).
At the first step of method optimization the
filtration of spheroid suspension through the
nylon mesh with a pore diameter of 100 μm
was applied to eliminate large cell clusters in
the resulting culture. Subsequent purification
from small cell aggregates and single cells was
performed using a 30-μm pore diameter nylon
mesh. For further analysis of purified colonies,
the value of median of spheroid size was de-
termined using image analysis. Average of
diameter median of MCF-7 cells spheroids in
5 experiments was 47.65 μm with standard
deviation ±21.79μm. It confirmed that filtration
A
B
Fig. 1. A — Suspension of MCF-7 multicellular spheroids before filtration. Black arrows and circles indicate spher-
oids of different size. Oc.10x, ob. 10x. B — Suspension of MCF-7 multicellular spheroids after double filtration. Blue
arrows indicate spheroids of similar size. Oc.10x, ob. 2,5x.
482
A. O. Kravchenko, V. R. Kosach, K. A. Shkarina et al.
was successful and the population of spheroids
of uniform size was generated.
Determination of proliferation activity.
Another major technical issue in multi-day
migration assays is the proliferation of studied
cells. Since proliferation has been shown to
affect other migration assays, it was important
to compare the proliferation activity of cells
in our model on the 1st, 2nd and 3rd days of
spheroid spreading. To analyze this parameter
in the proposed system, the cell proliferation
index after consecutive time periods was esti-
mated .We used two different approaches. In
the first case, the number of mitotic cells was
calculated directly in course of spheroid
spreading using transmitted light microscopy.
The mitotic cells in monolayer condition ac-
quire morphology distinct from the interphase
cells. Such cells become round, they exhibit
condensed chromatin, the morphology of
which reflects the corresponding stage of mi-
tosis (Fig 2). The per cent of such cells was
determined in each outspreading spheroid.
In the second case, the number of prolifer-
ating cells was detected at mentioned time
points using Hoechst 33342 staining and fluo-
rescent microscopy (Fig.3).
The proliferation index was expressed as the
percentage of dividing cells in each colony
analyzed. We detected that the level of cell
proliferation after 24, 48, and 72 hours of migra-
tion assay did not exceed 3 %. In particular, we
obtained the values for 24 hours of 2.54 %, for
48 — 2.6 %, for 72 — 2.94 %. This indicates
a similar effect of proliferative activity on the
spreading dynamics of the MCF-7 spheroids at
every time point, allowing us to neglect a po-
tential proliferation influence at comparison of
migration dynamics at neighboring time points.
Migration assay. In order to determine the
distance that cells passed during migration
process, the radii of outspreading spheroids
were measured after 0, 24, 48, and 72 h of
cultivation. The difference between the radii
values at neighboring time points was further
regarded as the length of the cell migration
track. From our point of view, the comparison
of linear parameters in migration assay is more
adequate than of squares since it is more valid
characteristic of the directed movement.
For this aim, the area of each spheroid was
quantified using Fiji software at all abovemen-
tioned time points, and radius was calculated
as described in Materials and Methods section.
We observed that a migration rate reached the
maximum at 48 hours post-filtration and de-
creased after 72 hours, which morphologi-
cally corresponded to the complete spreading
Fig. 2. Determination of mitotic cells in outspreading
MCF-7 cell spheroids at the 2nd day of migration initia-
tion. Arrows point out metaphase plates. Transmitted
light microscopy. Oc.10., ob.20x.
483
Optimization of in vitro model for analysis of tumor cell migration dynamics
of spheroids (Fig. 4, Fig. 5). Thus, the median
of migration distance after 24 hours was
7.64 mm, after 48 hours the cells passed an-
other 8.24 mm and after 72 hours another
6.8 mm.
For further validation of the proposed mod-
el, the analysis of locomotor properties of
MCF-7 cells under effect of Rapamycin
(mTOR inhibitor) was applied. Rapamycin and
its derivatives are the most well-known in-
hibitors of mTOR and are currently undergoing
clinical trials as novel anticancer agents. These
compounds have been shown to inhibit the
activity of mTOR complexes and significantly
decrease tumor cell motility in vitro [20].
Therefore, we analyzed whether rapamycin
would affect the MCF-7 cell migration from
spheroids onto the growth surface. Noteworthy,
the effect of rapamycin on the rate of cell mi-
gration during the first day was minimal and
cells overcame 8.13 mm, whereas on the sec-
ond and third days of the experiment, a sig-
nificant decrease in the cell migration rate was
observed, 3.65 mm and 1.94 mm respectively
(Fig. 4). It could be potentially attributed to
the inhibition of mTORC2 complex involved
in the cytoskeleton regulation [19]. So, this
result confirmed the suitability of the proposed
model for the assessment of cell migration and
its inhibition.
Immunofluorescence analysis
The presented model allowed applying an im-
munofluorescence analysis of intracellular lo-
calization of variety antigens in the migrating
A
B
C
Fig. 4. Cultured multicellular spheroids of MCF-7 cells after 24 (a), 48 (b) and 72 (c) hours of spreading. Transmitted
microscopy. Oc.10x, ob.2,5x.
Fig. 3. Detection of mitotic cells in MCF-7 outspreaded
spheroid at the 2nd day of experiment. DNA counter-
stained with Hoechst 33342. Arrows point out proliferat-
ing cells. Oc.10x, ob.10x.
484
A. O. Kravchenko, V. R. Kosach, K. A. Shkarina et al.
cells of outspreading spheroids. Earlier we
revealed the subcellular relocalization of S6K1
from the cytoplasm into the nuclei of MCF-7
cells after initiation of migration [16]. The
presented model was used to determine the
distribution of one of S6K1 isoforms namely
p85S6K1 in the migrating MCF-7 cells. In 3D
conditions MCF-7 cells demonstrated pre-
dominantly cytoplasm localization of p85S6K1
(Fig. 6a, white arrows). After initiation of cell
migration a bright positive reaction in the nu-
clei (primarily on the leading edge) appeared
for p85S6K1 (Fig. 6a, green arrows). Note-
worthy, in 2D monolayer conditions the nuclei
of MCF-7 cells were strongly positive for
p85S6K1 (Fig. 6b). The obtained results could
suggest the important role of p85S6K1 real-
izing in the nuclei for cell spreading and mi-
gration. Besides, the applied model allowed
registration of the change of subcellular dis-
tribution of the intracellular protein in migra-
ting cell.
Proposed optimisation of cell migration
model namely spheroid size unification and
estimation of proliferation activity allow to
apply this model for detection of locomotor
properties of the breast cancer MCF-7 cells
during 3 days. Besides, the model is useful
for investigation of the subcellular localiza-
tion of proteins involved in the regulation of
cell locomotion. This approach will be help-
ful for anticancer drug test as well as for
study of the basic mechanisms of tumor pro-
gression.
Fig. 5. Dynamics of MCF-7 cell mi-
gration from 3D spheroid onto the
growth surface at 0, 24, 48, 72 hours
post-filtration in standard cell culture
conditions (blue line) or under the
Rapamycin treatment (10 nM)
(red line).
A
B
Fig. 6. Immunofluorescence detec-
tion of p85S6K1subcellular distribu-
tion in MCF-7 cell outspreading
spheroid, a — Oc.10x, ob. 10x, and
b–in monolayer culture, oc.10x, ob.
40x.White arrows pointed out nega-
tive nuclear reaction, green arrows
pointed out positive nuclear reaction.
485
Optimization of in vitro model for analysis of tumor cell migration dynamics
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Оптимізація моделі визначення динаміки
міграції пухлинних клітин in vitro
А. О. Кравченко, В. Р. Косач, АІ, К.А. Шкаріна,
І. В. Заєць, І. О. Тихонкова, А.І. Хоруженко
Міграційна здатність є важливою ознакою пухлинних
клітин. Існує кілька підходів до аналізу динаміки мі-
грації ракових клітин in vitro. Однією з найбільш пер-
спективних і близьких до умов in vivo є модель ініці-
ювання міграції клітин з тривимірного багатоклітин-
ного сфероїда на ростову поверхню. Мета. Оптимізація
моделі для адекватної кількісної оцінки міграції пух-
линних клітин. Методи. 2- та 3-вимірна культура клі-
тин лінії MCF-7, імунофлюоресцентний аналіз, аналіз
зображень з використанням комп’ютерної програми
Фіджі. Результати. Уніфікація розміру сфероїдів до-
зволила уникнути значного розкиду даних. Отримані
сфероїди повністю розпластувались протягом 3 днів.
Найвищий показник міграції спостерігався на 2-гу
добу розпластування сфероїда. Рівень проліферації
клітин за кожну добу 3-денного експерименту був
майже однаковим і не перевищував 3 %. Валідність
моделі була протестована після пригнічення міграцій-
ної активності клітин під впливом рапаміцину (інгібі-
тор сигналізації mTOR). Крім того, запропонована
модель була успішно застосована для дослідження
субклітинної локалізації p85S6K1 в мігруючих кліти-
нах лінії MCF-7 за допомогою імунофлюоресцентно-
го аналізу. Висновки. Подвійне фільтрування багато-
клітинних сфероїдів дозволяє уніфікувати їх розміри,
що в подальшому сприяє адекватній оцінці міграцій-
ного потенціалу клітин. Запропонована модель до-
зволяє вивчати динаміку міграційних процесів пух-
линних клітин і може бути використана для тестуван-
ня протипухлинних препаратів in vitro.
К л юч ов і с л ов а: Міграція ракових клітин, 2- та
тривимірна культура клітин, p85S6K1, сфероїди.
Оптимизация модели определения динамики
миграции опухолевых клеток in vitro
А. А. Кравченко, В. Р. Косач, К. А. Шкарина,
И. В. Заец, И. А Тихонкова, А. И. Хоруженко
Миграционная способность является важным призна-
ком опухолевых клеток. Существует несколько подхо-
дов к анализу динамики миграции ракових клеток in
vitro. Одним из наиболее перспективных и близких к
условиям in vivo является модель инициирования ми-
грации клеток из трехмерного многоклеточного сфе-
роида на ростовую поверхность. Цель. Оптимизация
модели для адекватной количественной характеристи-
ки миграции опухолевых клеток. Методы. 2- и 3-мер-
ная культура клеток линии MCF-7, иммунофлюорес-
центный анализ, анализ изображений с использовани-
ем компьютерной программы Фиджи. Результаты.
Унификация размеров сфероидов позволила избежать
значительного разброса данных. Полученные сферо-
иды полностью распластывались в течение 3 дней.
Самый високий показатель миграции наблюдался на
2-е сутки распластования сфероида. Уровень проли-
ферации клеток за каждые сутки 3-дневного экспери-
мента был близьким и не превышал 3 %. Валидность
модели была протестирована после подавления мигра-
ции под влиянием рапамицина (ингибитор сигнализа-
ции mTOR). Кроме того, предложенная модель была
успешно применена для исследования субклеточной
локализации p85S6K1 в мигрирующих клетках линии
MCF-7 с помощью иммунофлюоресцентного анализа.
Выводы. Двойная фильтрация генерируемых in vitro
многоклеточных сфероидов позволила унифицировать
их размер, что способствует адекватной оценке мигра-
ционного потенцала клеток. Предложенная модель
позволяет изучать динамику миграционных процессов
опухолевых клеток и может бать использована для
тестирования противоопухолевых препаратов in vitro.
К л юч е в ы е с л ов а: Миграция ракових клеток,
двух- и трехмерная культура клеток, p85S6K1, сферо-
иды.
Received 15.09.2018
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| id | nasplib_isofts_kiev_ua-123456789-154376 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0233-7657 |
| language | English |
| last_indexed | 2025-12-07T18:03:10Z |
| publishDate | 2018 |
| publisher | Інститут молекулярної біології і генетики НАН України |
| record_format | dspace |
| spelling | Kravchenko, A.O. Kosach, V.R. Shkarina, K.A. Zaiets, I.V. Tykhonkova, I.O. Khoruzhenko, A.I. 2019-06-15T14:41:10Z 2019-06-15T14:41:10Z 2018 Optimization of in vitro model for analysis of tumor cell migration dynamics / A.O. Kravchenko, V.R. Kosach, K.A. Shkarina, I.V. Zaiets, I.O. Tykhonkova, A.I. Khoruzhenko // Вiopolymers and Cell. — 2018. — Т. 34, № 6. — С. 476-486. — Бібліогр.: 20 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000992 https://nasplib.isofts.kiev.ua/handle/123456789/154376 576 + 577 Migration ability is an important feature of tumor cells. There are several approaches to analyze the dynamics of cancer cell migration in vitro. One of the most perspective and closer to the in vivo conditions is the model of initiation of the cell migration from 3D multicellular spheroids onto growth surface. Aim. Optimization of the model for adequate quantitative characteristics of the tumor cell locomotion during several days. Methods. 2D and 3D MCF-7 cell culture, immunofluorescence analysis, and image analysis using computer software Fiji. Results. Unification of spheroid size allowed avoiding a significant data deviation. The obtained spheroids spread completely for 3 days. The highest migration ratio was observed at the 2nd day. The proliferation level at each of 3-day experiment was the same and did not exceed 3%. The validity of the model was tested after migration inhibition by rapamycin (mTOR signaling inhibitor). Additionally, this model was successfully applied to immunofluorescence analysis, namely investigation of p85S6K1 subcellular localization in moving MCF-7 cells. Conclusions. Double filtration of multicellular spheroids allowed unification of their size, which promotes an adequate interpretation of the migration assay. This model enabled the study of tumor cells migration dynamics and can be further used for the development of anticancer drug. Міграційна здатність є важливою ознакою пухлинних клітин. Існує кілька підходів до аналізу динаміки міграції ракових клітин in vitro. Однією з найбільш перспективних і близьких до умов in vivo є модель ініціювання міграції клітин з тривимірного багатоклітинного сфероїда на ростову поверхню. Мета. Оптимізація моделі для адекватної кількісної оцінки міграції пухлинних клітин. Методи. 2- та 3-вимірна культура клітин лінії MCF-7, імунофлюоресцентний аналіз, аналіз зображень з використанням комп'ютерної програми Фіджі. Результати. Уніфікація розміру сфероїдів дозволила уникнути значного розкиду даних. Отримані сфероїди повністю розпластувались протягом 3 днів. Найвищий показник міграції спостерігався на 2-гу добу розпластування сфероїда. Рівень проліферації клітин за кожну добу 3-денного експерименту був майже однаковим і не перевищував 3%. Валідність моделі була протестована після пригнічення міграційної активності клітин під впливом рапаміцину (інгібітор сигналізації mTOR). Крім того, запропонована модель була успішно застосована для дослідження субклітинної локалізації p85S6K1 в мігруючих клітинах лінії MCF-7 за допомогою імунофлюоресцентного аналізу. Висновки. Подвійне фільтрування багатоклітинних сфероїдів дозволяє уніфікувати їх розміри, що в подальшому сприяє адекватній оцінці міграційного потенціалу клітин. Запропонована модель дозволяє вивчати динаміку міграційних процесів пухлинних клітин і може бути використана для тестування протипухлинних препаратів in vitro. Миграционная способность является важной особенностью опухолевых клеток. Существует несколько подходов для анализа динамики миграции раковых клеток in vitro. Одной из наиболее перспективных и приближенных к условиям in vivo является модель инициации миграции клеток из трехмерных многоклеточных сфероидов на поверхность роста. Цель. Оптимизация модели для адекватных количественных характеристик локомоции опухолевых клеток в течение нескольких дней. Методы. 2D и 3D MCF-7 клеточная культура, иммунофлуоресцентный анализ и анализ изображений с использованием компьютерного программного обеспечения Фиджи. Результаты. Унификация размеров сфероидов позволила избежать значительного отклонения данных. Полученные сфероиды распространились полностью за 3 дня. Самый высокий коэффициент миграции наблюдался на 2-й день. Уровень пролиферации в каждом из 3-дневных экспериментов был одинаковым и не превышал 3%. Достоверность модели была проверена после ингибирования миграции рапамицином (ингибитор передачи сигналов mTOR). Кроме того, эта модель была успешно применена для иммунофлуоресцентного анализа, а именно для исследования субклеточной локализации p85S6K1 в движущихся клетках MCF-7. Выводы. Двойная фильтрация многоклеточных сфероидов позволила унифицировать их размер, что способствует адекватной интерпретации анализа миграции. Эта модель позволила изучить динамику миграции опухолевых клеток и может быть в дальнейшем использована для разработки противоопухолевого препарата. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Methods Optimization of in vitro model for analysis of tumor cell migration dynamics Оптимізація моделі in vitro для аналізу динаміки міграції пухлинних клітин Оптимизация in vitro модели для анализа динамики миграции опухолевых клеток Article published earlier |
| spellingShingle | Optimization of in vitro model for analysis of tumor cell migration dynamics Kravchenko, A.O. Kosach, V.R. Shkarina, K.A. Zaiets, I.V. Tykhonkova, I.O. Khoruzhenko, A.I. Methods |
| title | Optimization of in vitro model for analysis of tumor cell migration dynamics |
| title_alt | Оптимізація моделі in vitro для аналізу динаміки міграції пухлинних клітин Оптимизация in vitro модели для анализа динамики миграции опухолевых клеток |
| title_full | Optimization of in vitro model for analysis of tumor cell migration dynamics |
| title_fullStr | Optimization of in vitro model for analysis of tumor cell migration dynamics |
| title_full_unstemmed | Optimization of in vitro model for analysis of tumor cell migration dynamics |
| title_short | Optimization of in vitro model for analysis of tumor cell migration dynamics |
| title_sort | optimization of in vitro model for analysis of tumor cell migration dynamics |
| topic | Methods |
| topic_facet | Methods |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/154376 |
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