Experience in creating primary cultures of endometrial cancer and studying cells carrying phenotype of cancer stem cells

Svetlana Yu. Filippova; Irina V. Mezhevova; Tatiana V. Chembarova; Inna A. Novikova; Ekaterina V. Verenikina; Oksana E. Zhenilo; Veronika V. Polovodova; Alexander V. Shaposhnikov; Elena V. Shalashnaya; Andrey A. Maslov; Oksana G. Ishonina

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South Russian

Journal of Cancer..

Vol. 5

No. 3, 2024

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South Russian

Journal of Cancer..

Vol. 5

No. 3, 2024

South Russian Journal of Cancer. 2024. Vol. 5, No. 3. P. 16-30

https://doi.org/10.37748/2686-9039-2024-5-3-2

https://elibrary.ru/eyiruh

ORIGINAL ARTICLE

Experience in creating primary cultures of endometrial cancer and

studying cells carrying phenotype of cancer stem cells

S. Yu. Filippova, I. V. Mezhevova, T. V. Chembarova , I. A. Novikova, E. V. Verenikina, O. E. Zhenilo,

V. V. Polovodova, A. V. Shaposhnikov, E. V. Shalashnaya, A. A. Maslov, O. G. Ishonina

National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation

tanyshamova@mail.ru

ABSTRACT

Purpose of the study. Was to investigate the possibility of applying the method of spheroid formation in culture for assessment

of the endometrial cancer (EC) tumor stem cells (TSC) content in complex samples containing various tumor cells and

microenvironment.

Materials and methods. Primary cultures were obtained from fragments of tumors removed during surgery as a first stage

of treatment at the Department of Gynecological Oncology, the National Medical Research Center for Oncology. After enzymatic

disaggregation of tissue, cell suspension was passaged in DMEM medium containing 10 % fetal bovine serum and 1 %

gentamicin to obtain primary two-dimensional cultures. To study the ability of cells to form spheroids, the primary culture

was removed from the culture plate and passaged with 2.0 . 104 cells per well of a six-well plate (n = 6) in DMEM medium

containing 0.35 % agarose and growth factors EGF (20 ng/ml) and FGF (20 ng/ml). After two weeks of cultivation, the average

size, number of formed spheroids, and frequency of spheroid formation were determined. For those cultures that had formed

spheroids, immunofluorescent

staining of the two-dimensional

culture for the marker CD133

was

performed, after which the

frequency of CD133+ cells was determined.

Results. A total of nine primary cultures of EC were obtained, five of which formed spheroids within two weeks of cultivation

under non-adhesive conditions. In these cultures, small polygonal CD133+ cells showed the strongest association with spheroid

formation, which were associated with the largest spheroids (98�110 .m in diameter).

Conclusion. There is a large number of microenvironmental cells in mixed cultures of CSC, some of which may express CD133,

including healthy stem

cells that also form spheroids in soft agar. A more detailed study of CSC subpopulations compared to

normal endometrium is required to establish a link between the observed diversity of cells in culture and their ability to form

spheroids and other characteristics of tumor stem cells.

Keywords: endometrial cancer, primary cell cultures, cancer stem cells, cell spheroids

For citation: Filippova S. Yu., Mezhevova I. V., Chembarova T. V., Novikova I. A., Verenikina E. V., Zhenilo O. E., Polovodova V. V., Shaposhnikov A. V.,

Shalashnaya E. V., Maslov A. A., Ishonina O. G. Experience in creating primary cultures of endometrial cancer and studying cells carrying phenotype of cancer

stem cells. South Russian Journal of Cancer. 2024; 5(3): 16-30. https://doi.org/10.37748/2686-9039-2024-5-3-2, https://elibrary.ru/eyiruh

For correspondence: Tatiana V. Chembarova � junior research fellow at the laboratory of cellular technologies, National Medical Research Centre for

Oncology, Rostov-on-Don, Russian Federation

Address: 63 14 line str., Rostov-on-Don 344037, Russian Federation

E-mail: tanyshamova@mail.ru

ORCID: https://orcid.org/0000-0002-4555-8556

SPIN: 5426-1873, AuthorID: 1051985

ResearcherID: AAR-3198-2021

Scopus Author ID: 57221303597

Compliance with ethical standards: the work followed the ethical principles set forth by World Medical Association Declaration of Helsinki, 1964, ed. 2013.

The study was approved by the Committee on Biomedical Ethics at the National Medical Research Centre for Oncology (extract from the minutes of the

meeting No. 25 dated 09/08/2022). Informed consent has been obtained from all participants of the study

Funding: this work was not funded

Conflict of interest: the authors declare that there are no obvious and potential conflicts of interest associated with the publication of this article

The article was submitted 26.12.2023; approved after reviewing 28.06.2024; accepted for publication 24.07.2024

� Filippova S. Yu., Mezhevova I. V., Chembarova T. V., Novikova I. A., Verenikina E. V., Zhenilo O. E., Polovodova V. V., Shaposhnikov A. V., Shalashnaya E. V.,

Maslov A. A., Ishonina O. G., 2024

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South Russian Journal of Cancer 2024. Vol. 5, No. 3. P. 16-30

Filippova S. Yu., Mezhevova I. V., Chembarova T. V. , Novikova I. A., Verenikina E. V., Zhenilo O. E., Polovodova V. V., Shaposhnikov A. V., Shalashnaya E. V., Maslov A. A.,

Ishonina O. G. Experience in creating primary cultures of endometrial cancer and studying cells carrying phenotype of cancer stem cells

INTRODUCTION

Endometrial cancer (EC) is the sixth most common

type of cancer in women. Over the past 30

years, the overall incidence of EC has increased by

132

%,

reflecting an increase in the prevalence of

risk factors, in particular, obesity and aging of the

population

[1]. In

Russia, EC

occupies

the

2nd

rank

in

the structure of oncological diseases of the female

genital organs. The total number of patients with

EC reached 195.6 per 100,000 population in 2022,

which is 31.8

%

more than in 2012 [2].

Thus,

there is

still a need to develop new therapeutic approaches

to significantly improve the prognosis

in women with

recurrent EC or in the later stages of the disease. In

this context, tumor stem cells (TSC) of endometrial

cancer, capable of self-renewal and differentiation

into mature tumor cells, as well as contributing to

tumor recurrence, metastasis, heterogeneity, multidrug

and radiation resistance, represent a potential

target for drug development [3].

TSC

were

first

identified

in

patients

with

acute

myeloid

leukemia in 1994, and have since been considered

potential therapeutic targets in the treatment of

oncological

diseases, including solid tumors

[4]. TSC

in

EC

were

first

described

by Hubbard

in

2009

[5]. The

discovery of endometrial OSCs has radically changed

the views on the study of the biology of EC and the

development of approaches to the treatment of this

disease. As a rule, EC TSC is identified by the expression

of specific antigens, by the weak accumulation

of the Hoechst 33342 nuclear dye (the so�called

side population � "side population"), by the ability

to form colonies under conditions of reduced adhesion

and initiate the growth of a tumor

containing

TSC and differentiated cells originating from them

with the same phenotype as "parental", in mice with

immunodeficiency [3].

A number of markers

associated with

the TSC of

solid tumors have been studied in EC. Aldehyde dehydrogenase

1 (ALDH1) is one of 19 different enzymes

involved in aldehyde oxidation. This enzyme is highly

active in the early stages of stem cell differentiation.

Atypical

EC cells

with

a high level

of ALDH1

expression

are more tumorogenic, invasive and resistant

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to cisplatin

than

cells

with a low level

of ALDH1

expression.

Also, a high

level

of ALDH1

expression

correlates

with a worse prognosis in patients with EC [6].

Receptor tyrosine

kinase

c-Kit

or CD117

is

a receptor

for the Stem Cell Factor (SCF) and, upon activation,

triggers a number of intracellular signaling cascades

regulating cell survival, migration and proliferation,

including TSC [7]. When studying the cellular

composition

of EC, it was shown that CD117+ cells isolated

from Ishikawa and MFE280 EC cultures exhibit greater

proliferative ability, as well as the ability to form

colonies

in soft

agar in

the

presence

of SCF. A high

level of CD117 expression was also recognized as an

independent prognostic factor correlating with the

progression of EC [8]. The CD55 antigen is a complement

decay acceleration factor and is expressed at

a high

level

in

the

TSC of endometrioid ovarian

cancer

and EC. It has been shown that CD55+ cells are able

to regulate cell self-renewal and their resistance to

cisplatin to a greater extent than CD55

cells

[9]. A link

with TSC has also been established for the transmembrane

glycoprotein CD44, which plays the role

of an adhesion molecule. Cells overexpressing CD44

possess such characteristics of TSC as the ability

to self-renew and epithelial-mesenchymal transition

(EMF), as well as resistance to chemotherapy and

radiation therapy [10]. Probably, this marker is also

related to TSC EC, since oncospheres obtained on

soft agar from cells of EC cultures with stem prop

erties

are CD44

positive

[3]. In

addition, a number of

studies have noted the co-expression of CD44 and

another TSC marker CD133

in

the EC tissue

[11, 12].

The transmembrane glycoprotein of the cell surface

prominin-1 or CD133 has attracted considerable attention

due to the fact that its expression is often

observed in various subpopulations of somatic stem

cells. Usually, this glycoprotein is observed in the area

of various microvilli and protrusions of the plasma

membrane,

where CD133 can act as a regulator

of the

lipid composition of membranes or participate in the

mechanisms of cellular polarity and migration [13].

In

a study by Rutella et

al. (2009)

subpopulations

of

cells with the CD133+/CD44+ phenotype isolated from

permanent endometrial cancer cell lines showed the

ability to form tumor spheres, increased chemoresistance

and were

able

to initiate the formation

of a tumor

with the same phenotype as the original tumor

when transplanted to

immunodeficient mice [14]. Gao

(2012) also investigated the AN 3CA line and showed

that CD133+ cells express stem markers, demonstrate

greater mobility and invasive ability than CD133

cells

[15]. In

Friel's

work, the expression

of CD133

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in

the cells of primary EC tumors and the mechanism for

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controlling the expression of this marker in them were

investigated [16]. The authors

showed that

CD133+

cells accounted for 5.7�27.4 % of the total population

of tumor cells in the analyzed primary tumors. These

cells had increased tumorigenicity in immunodeficient

mice, which suggests that these cells belong

to the TSC. Similar results were obtained in the work

of Sun (2017), where CD133+CD44+ cells showed

a stronger association with all the classical properties

of TSC than for other markers studied in the

work, such as CD24, CD47, CD29, CXCR4, SSEA3 and

SSEA4. The

efficiency of spheroid formation in soft

agar was 11.7 % for CD133+ cells and 1.7 % for CD133

cells

[17]. In addition, CD133+CD44+ cells showed

an increased expression of stem cell transcription

factors Myc, Sox-2, Nanog and Oct4 compared with

other subpopulations [16], for which a direct relationship

with the degree of malignancy of endometrioid

carcinoma was

established [18].

Despite the established connection between

CD133 and EC TSC in a number

of studies,

data

on the relationship between the expression of this

marker and the prognosis of the disease course

remain quite contradictory. Thus, the work of Elbasateeny

(2016) indicates a more pronounced association

of CD133 expression with the early stage of the

tumor (I�II) and a decrease in the expression of this

marker at later stages of the disease. The authors

suggested that CD44 and CD133 may be involved in

the development of endometrial cancer in the early

stages, and their overexpression may contribute

to

the early

diagnosis of

endometrial cancer

[11].

These results are supported by the data obtained

in Mancebo (2017), in which the authors found that

tumors in which CD133 expression was high were

less likely to have vascular invasion and more likely

to be highly differentiated, and were also associated

with

higher overall

and recurrence-free

survival

[19].

However, there is also the opposite data. Thus, Nakamura

(2010) showed a negative correlation between

CD133 expression in tumor tissue and life

expectancy without

recurrence [20]. The negative

prognosis of the course of EC with increased CD133

expression

in

tumor tissue

was

confirmed

in

the

work of Park in 2019 [12]. The observed discrepancies

in the data of different authors may be related

to the fact that CD133 expression is observed not

only in the EC TSC, but also in normal cells of the

glandular epithelium of the

endometrium [21]. Thus,

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CD133 in endometrial tissue can act simultaneous

ly as

a marker of epithelial differentiation and as

a marker

of OSC.

To study the dynamics, functioning and regulation

of stem cells, experimental methods are needed to

clearly distinguish between stem cells and their offspring.

Due to the lack of unique cell surface markers

specific

only to

stem

cells and a distinct morphological

phenotype,

stem

cells are usually identified

based on functional criteria. Stem cells from various

tissues are usually cultured in vitro in the form of

spheroids under

conditions excluding adhesion [22].

According to the literature data, the study of TSC in

EC by the method of counting spheroids in conditions

that do not support cell adhesion, both on simple

samples obtained after sorting by any marker [14,

17] and in whole samples of the primary culture of

EC [23]. In the latter case, the quantitative analysis

was reduced to measuring the size of spheroids,

while the assessment of the quantitative content of

TSC and the

establishment

of a link with

the

clinical

and pathological characteristics of the sample was

not carried out.

The purpose of the study: to study the possibility

of using the method of spheroid formation in culture

to assess the content of EC TSC in complex samples

containing various tumor cells and microenvironments.

To achieve this goal, we compared the

morphological characteristics of primary EC cultures

under conditions of adhesive growth and in soft agar,

and also studied the expression of the CD133 marker

in two-dimensional cultures.

MATERIALS AND METHODS

Nine primary cultures of EC were obtained from

fragments of tumors removed during surgery performed

as

the first

stage of the treatment

of EC.

Patients with EC were treated in the Oncological

Gynecology department of the National Medical Research

Centre for Oncology, in 2023. The histological

diagnosis was confirmed in the pathoanatomical

department of the of the National Medical Research

Centre for Oncology. The patients were aware of their

participation in the scientific study and signed an

informed consent for the collection of biological

material. The pathologist isolated a 0.5 cm3 fragment

corresponding to the malignant component

of the tumor within 20 minutes after extraction of

South Russian Journal of Cancer 2024. Vol. 5, No. 3. P. 16-30

Filippova S. Yu., Mezhevova I. V., Chembarova T. V. , Novikova I. A., Verenikina E. V., Zhenilo O. E., Polovodova V. V., Shaposhnikov A. V., Shalashnaya E. V., Maslov A. A.,

Ishonina O. G. Experience in creating primary cultures of endometrial cancer and studying cells carrying phenotype of cancer stem cells

the drug and placed it in a Hanks solution (Gibco,

USA) containing 1 % gentamicin (Biolot, Russia).

Next, the sample was fragmented with a scalpel to

a size of 1�2

mm3, after which 300 u/ml of collagenase

I (Thermo

Fisher

Scientific,

USA) was added

in a DMEM medium

(Biolot, Russia) and incubated

for an hour at a temperature of 37

�C with constant

stirring. At the end of fermentation, the sample was

additionally crushed by pipetting and passed through

a sterile nylon filter (d = 70

.m) (Beckton Dickinson,

USA). The resulting suspension was washed twice in

a phosphate buffer

and passed onto

a culture vial in

a DMEM medium containing 10

% FBS (Biolot, Russia)

and 1 % gentamicin and cultured under standard

conditions at 37 �C and 5.0

% CO2.

To study the ability of cells to spheroid formation,

a

two-dimensional

culture

was

removed

from culture

plastic

using a standard technique using a 0.1

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%

trypsin solution (Biolot, Russia). The resulting cell

suspension was mixed with 0.35 % agarose solution

in a serum-free DMEM medium with the addition of

insulin-transferin and growth factors EGF (20 ng/ml)

and FGF (20 ng/ml) and layered on a base of 0.7

%

agarose in the same medium. In total, 2.0 . 104 cells

were

added

in

this

way to the

well

of a

6-hole

tablet.

There were 6 repetitions for each culture. The plates

with cells were cultured for 2 weeks, during which

time the spheroids were photographed. At the end of

cultivation, the average size was determined and the

spheroids

in

the

well

were

counted

using

a

Lionheart

FX imager (BioTek, USA) using embedded software.

The frequency of spheroid formation was calculated

as the ratio of the number of spheroids larger than

40 .m in diameter to the total number of cells passioned

into the well of the tablet.

To carry out immunophenotyping on CD133, primary

cultures were planted on cover glasses. After

the formation of the cellular monolayer, the glasses

were

fixed in a 4

% paraformaldehyde

solution for

15 minutes at room temperature, after which permeabilization

was

carried out

in

a

0.5

% Triton

X-100

solution on a phosphate buffer for 10

minutes. After

washing, the glass was blocked for an hour in

a solution of 5

%

normal goat serum

(Gibco, USA) in

a phosphate buffer, after which

it

was

kept

overnight

at 4

�C in a solution of primary polyclonal rabbit antibody

to CD133

(ab19898, Abcam, USA) in a phosphate

buffer (1/100 dilution) with the addition of 1 %

goat serum. After washing the glass three times, it

was kept at room temperature for an hour in a solution

of secondary goat antibody conjugated with

Alexa Flour�594 (ab150080, Abcam, USA) (1/500

dilution), after which they were washed with a phosphate

buffer,

repainted in a solution of

the nuclear

dye DAPI (ab228549, Abcam, USA) and mounted

on slides in an Anti-Fade Fluorescence Mounting

Medium (ab104135, Abcam, USA). The proportion

of CD133 positive cells was determined on the Lionheart

FX imager (BioTek, USA) using embedded

software. 3 glasses were examined for each culture.

The values of the diameter of the spheroids, the

frequency of spheroid formation and the proportion

of CD133+ cells are given as a sample mean � standard

deviation.

STUDY RESULTS

We received 9 primary cultures of EC from 9 patients

who were treated in the Department of Oncological

Gynecology at the of the National Medical

Research Centre for Oncology in 2023. The results

of the pathological examination showed that 2 tumors

belonged to the histological type of highly

differentiated (G1), 6 to moderately differentiated

(G2) and one tumor to the low�differentiated type

G3 of endometrioid adenocarcinomas. At the same

time, lympho-vascular invasion was detected in three

cases, but none of the patients had metastases in

regional lymph nodes. All cases were attributed to

stage I�II of the disease.

All primary cell cultures formed a monolayer

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on

day 3 of cultivation under conditions that support

adhesion. The EC cells in the monolayer culture were

relatively small in size and polygonal in shape, often

forming "islands" and "rosettes" that were located

among the more elongated stroma cells, presumably

fibroblasts. When cultured in conditions that do not

support cell adhesion on agarose in the medium for

TSCs, the formation of spheroids was noted in 5 out

of 9 cultures, starting from the 5th day of cultivation.

In four other cultures, spheroids did not form

even after 2 weeks of cultivation. 1 culture obtained

from a highly differentiated tumor and 3 cultures

from moderately differentiated tumors did not form

spheroids. Thus, there was no obvious relationship

between the frequency of spheroid formation and

the degree of tumor differentiation. For cultures that

formed spheroids in soft agar, additional staining of

��-�� 2024. �. 5, � 3. �. 16-30

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the two-dimensional culture on the CD133 marker

was performed.

EC cell culture No. 1. Highly differentiated G1 endometrioid

adenocarcinoma without signs of vascular

invasion. In the primary culture, single large cells

of polygonal or rounded shape were observed among

numerous elongated cells, presumably fibroblasts

(Fig. 1A).

CD133 expression is weak, and rare marker

granules are observed in all cells (Fig. 1b). The frequency

of CD133+ cells ranged from 0.2 to 1.0 %

(0.8

� 0.15

%). In

the

culture in

agarose, a very slow

growth of cellular spheroids from single large cells

was

observed. On

the

fifth

day of cultivation, spheroids

had not yet been detected (Fig. 1B), and two

weeks later small spheroids of 4�8 cells were formed

(Fig. 1G). The frequency of spheroid formation on

the 14th day ranged from 0.1

to 0.7

% (0.5

� 0.1

%).

EC cell culture No. 2. Low-grade G3 endometrioid

adenocarcinoma without signs of vascular invasion.

Under cultivation conditions supporting cell adhesion,

the cells of

the primary culture formed a monolayer

of two types of cells, similar in appearance to

the culture of EC No. 3 (Fig. 3A): small polygonal

cells united in islands among larger and elongated

cells similar to fibroblasts (Fig. 2A).

At the same time, islands of polygonal cells could

form spheroid-like structures, within which increased

CD133 expression was observed. Also, among the

monolayer, there were individual large cells expressing

CD133 above the general level (Fig. 2B). The

frequency of CD133+ cells ranged from 2.7 to 8.0 %

(4.8

� 1.5

%). On

the

fifth day of cultivation in

conditions

that do not support adhesion, in the medium

�C

B

D

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Fig. 1. Primary culture of endometrial cancer No. 1. A � general view of monolayer culture; B � staining of monolayer culture on CD133;

C � view of cellular spheroids in agarose on the 5th day of cultivation. G � a type of cellular spheroids in agarose on the 14th day of

cultivation. The size of the scale ruler is 200

.m

South Russian Journal of Cancer 2024. Vol. 5, No. 3. P. 16-30

Filippova S. Yu., Mezhevova I. V., Chembarova T. V. , Novikova I. A., Verenikina E. V., Zhenilo O. E., Polovodova V. V., Shaposhnikov A. V., Shalashnaya E. V., Maslov A. A.,

Ishonina O. G. Experience in creating primary cultures of endometrial cancer and studying cells carrying phenotype of cancer stem cells

for TSC, the culture of EC No. 3 formed small cellu

lar spheroids

of 4�8

cells

with a frequency of about

15 % (Fig. 2B). Further, some of the cell spheroids

showed rapid growth and after two weeks of cultiva

tion reached 80�150 .m

in diameter (110.3 � 32.7

.m). The frequency of spheroid formation on the 14th

day ranged from 0.3

to 2.5

% (1.5

� 0.8

%)

(Fig. 2G).

EC cell culture No. 3. Moderately differentiated G2

endometrioid adenocarcinoma with signs of vascular

invasion. The

culture

in

the

monolayer had a

pronounced

division into small polygonal cells, united

into islands among larger and elongated cells similar

to fibroblasts (Fig. 3A).

Immunofluorescence staining showed an increased

content of the CD133 marker in small polygonal

cells, while individual cells showed a particularly

bright label (Fig. 3B). The frequency of CD133+

cells

ranged

from 5.8

to 16.9

% (11.2

� 5.2

%). When

grown

on

agarose

in

a

medium for TSC

on

the

5th

day in culture, about 15 % of the cells formed spher

oids 5�30 .m

in diameter (Fig. 3C). After

two weeks

of cultivation in agarose, individual cellular spheroids

increased in size to 30�150

.m in diameter, the remaining

spheroids degraded (98.3

� 51.4 .m). The

frequency of spheroid formation on day 14 ranged

from 0.3

to 10.1

% (5.7 � 4.1

%) (Fig. 3D).

EC cell culture No. 4. Moderately differentiated

G2 endometrioid adenocarcinoma without signs of

vascular invasion. In the monolayer culture, large polygonal

cells were observed separately or assembled

in small groups against the background of elongated

cells of various sizes (Fig. 4A).

When stained with the CD133 EC TSC marker,

a positive reaction was shown not only by large po

�C

B

D

Fig.

2.

Primary culture of

endometrial cancer

No.

2.

A

�

general view

of

the monolayer

culture;

B �

staining of

the monolayer

culture on

CD133; C � type of cellular spheroids in agarose on the 5th day of cultivation; D � type of cellular spheroids in agarose on the 14th day of

cultivation. The size of the scale ruler is 200

.m

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�.

�., ��

�.

�. , ��

�.

�., ��

�.

�., ��

�.

�., ��

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lygonal cells and their clusters, but also by numerous

small elongated cells. Large multinucleated cells

positive for CD133 were also found in the preparation

(Fig. 4B). The frequency of CD133+ cells ranged

from 40.1

to 65.2

% (average

52.2

� 10.2

%). After

five days of cultivation in agarose, about 5�10

% of

the culture cells formed small loose spheroids of

4�16 cells (Fig. 4C), two weeks later the spheroids

increased to

20�80 .m

in diameter

(54.7 � 28.4 .m),

the frequency of spheroid formation on the 14th day

ranged from 2.1 to 8.4

% (5.1 � 2.7

%) (Fig. 4D)

EC Culture No. 5. Moderately differentiated G2

endometrioid adenocarcinoma without signs of vascular

invasion. In the monolayer culture, islands of

rather large polygonal cells of epithelial morphology

were found among elongated cells of stromal origin

(Fig. 5A).

The reaction to CD133 was similar to the

EC culture No. 2, namely, positive staining was

demonstrated by individual large cells of elongated

or epithelial

morphology with a certain group

of small elongated cells, which, due to staining,

stand out against the background of larger elongated

cells negative for CD133 (Fig. 5B). The frequency

of CD133+ cells ranged from 22.4 to 51.2 %

(35.5

� 12.7

%). On

the

fifth

day of cultivation

in

conditions that do not support adhesion, EC culture

No. 5 formed small cellular spheroids of 2�4 cells

with

a frequency of about

5

% (Fig. 5C). Further,

some of the cell spheroids showed rapid growth

and reached 30�80 .m

in diameter

after

two weeks

of cultivation (average 55.4 � 25.1 .m). The frequency

of spheroid formation on day 14 ranged

from 0.5

to 2.8

% (1.8

� 0.9

%)

(Fig. 5D).

�C

B

D

Fig.

3.

Primary culture of

endometrial cancer

No.

3.

A

�

general view

of

iНе можете найти то, что вам нужно? Попробуйте сервис подбора литературы.

the monolayer

culture;

B �

staining of

the monolayer

culture on

CD133; C � type of cellular spheroids in agarose on the 5th day of cultivation; D � type of cellular spheroids in agarose on the 14th day of

cultivation. The size of the scale ruler is 200

.m

South Russian Journal of Cancer 2024. Vol. 5, No. 3. P. 16-30

Filippova S. Yu., Mezhevova I. V., Chembarova T. V. , Novikova I. A., Verenikina E. V., Zhenilo O. E., Polovodova V. V., Shaposhnikov A. V., Shalashnaya E. V., Maslov A. A.,

Ishonina O. G. Experience in creating primary cultures of endometrial cancer and studying cells carrying phenotype of cancer stem cells

The characteristics of primary EC cultures are

combined in the table (Table 1).

The lowest frequency of spheroid formation, as

well as the lowest frequency of CD133+ cells, were

observed in a culture obtained from a highly differentiated

tumor.

DISCUSSION

In all the obtained primary cultures, the presence

of a stromal component is noted, represented by

elongated cells of different sizes, demonstrating

a negative (No. 1, No. 2, No. 3) up to medium and

high CD133 expression (No. 4, No. 5). In addition, polygonal

cells are noted in cultures, which occur as islands

among stromal cells. Their sizes vary between

cultures, and on this basis, cultures can be divided

into two groups � including small polygonal cells that

assemble into dense colonies-"domes" (cultures No.

1 and No. 2), and including larger polygonal cells that

can

form flat

islands

of different

sizes

(cultures

No.

1, No. 4 and No. 5). The expression of the CD133

marker in these cells is quite pronounced, especially

against

the

background

of a

weakly colored

stromal

component consisting of large cells (cultures No. 4

and No. 5).

The presence of elongated or process-shaped

cells

resembling fibroblasts

in

primary EC

cultures

and capable of forming spheroids under conditions

that do not support cell adhesion was also noted in

the work of Helweg (2022) [23]. The picture of the

composition of cell cultures obtained by us is also

similar in cell morphology to the results obtained in

the work of Chan

et

al. (2004)

[24]. In

this

study, the

�C

B

D

Fig.

4.

Primary culture of

endometrial cancer

No.

4.

A

�

general view

of

the monolayer

culture;

B �

staining of

the monolayer

culture on

CD133; C � type of cellular spheroids in agarose on the 5th day of cultivation; D � type of cellular spheroids in agarose on the 14th day of

cultivation. The size of the scale ruler is 200

.m

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B

D

Fig. 5. Primary culture of endometrial cancer No. 5. A � general view of monolayer culture; B � staining of monolayer culture on CD133;

C � type of cellular spheroids

in

agarose on

the

5th day of cultivation; D

� type

of cellular spheroids

in

agarose

on

the

14th

day of cultivation.

The size of the scale ruler is 200 .m

Table 1. Summary characteristics of primary endometrial cancer cultures

Culture

No. Grade

Lympho-

vascular

invasion

Reaction to CD133, proportion of

CD133+ cells, mean � SD %

The frequency of

spheroids . 40.m per 14 days,

average � SD, %

The diameter of

the spheroids for

14 days, average

� SD, .m

No. 1 G1 n/p

No. 2 G3 n/p

No. 3 G2 p

No. 4 G2 n/p

No. 5 G2 n/p

Note: not present � n/p, present � p

0.8 � 0.15 %, weak reaction in some

large stromal cells, single polygonal

cells with bright coloration

4.8 � 1.5 %, small polygonal cells in

dense colonies, individual stromal cells

11.2 � 5.2 %, small polygonal cells in

dense colonies

52.2 � 10.2 %, polygonal process large

multinucleated cells, stromal small

elongated cells

35.5 � 12.7 %, separate large and small

elongated stromal cells, separate large

polygonal cells

0.5 � 0.1

1.5 � 0.8 %

5.7 � 4.1 %

5.1 � 2.7 %

1.8 � 0.9 %

20 � 10

110.3 � 32.7

98.3 � 51.4

54.7 � 28.4

55.4 � 25.1

25

South Russian Journal of Cancer 2024. Vol. 5, No. 3. P. 16-30

Filippova S. Yu., Mezhevova I. V., Chembarova T. V. , Novikova I. A., Verenikina E. V., Zhenilo O. E., Polovodova V. V., Shaposhnikov A. V., Shalashnaya E. V., Maslov A. A.,

Ishonina O. G. Experience in creating primary cultures of endometrial cancer and studying cells carrying phenotype of cancer stem cells

authors studied the behavior in culture of various

cells of the normal human endometrium isolated

from the

epithelial

and basal

layers

[24]. According

to the authors, the epithelial layer gave two groups

of polygonal cells � small cells that gather in close

colonies

with a high ability to proliferate, and larger

cells that form looser colonies on cultural plastic.

The stromal component of endometrial tissue con

sisted of two types

of elongated cells: large

loosely

lying cells and small closely lying cells, giving curls in

a monolayer. In

addition, human

endometrial

stromal

cells were positively stained for

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fibroblast markers

(CD90, 5B5, type

I collagen), which

confirms

the

relationship

of these

cells

to fibroblasts

and, possibly,

myofibroblasts, which express

markers

of both fibroblasts

and smooth muscle cells. Thus, based on the

picture of the two-dimensional culture obtained in our

work, it is impossible to unambiguously identify which

cells belong to the TSC, since externally they are little

distinguishable from normal endometrial cells.

The expression of the TSC CD133 marker in the

cultures obtained by us ranged from 0.8 to 52.2 %

on average. The data obtained go beyond the range

of 5.7�27.4

% indicated by other authors [16, 17].

A nonlinear relationship

between

CD133

expression

and the degree of tumor differentiation was also

revealed � both high and low differentiation were

associated with reduced immunoreactivity on CD133

compared with average differentiation. No connection

was found between the level of CD133 expression,

the degree of differentiation and other clinical

and pathological characteristics of the tumor also

in Nakamura [20]. CD133

is known to occur in normal

differentiated endometrial cells. Thus, based on

existing data, it is impossible to determine to what

extent CD133

expression reflects the degree of cell

malignancy in primary EC cultures and the level of

their differentiation. To solve this problem, more extensive

studies

are

required, including a comparison

of normal endometrial tissue and RE.

The frequency of spheroid formation in our cultures

ranged from 0.5 to 5.7 % on average. It is

known that the frequency of cells with SC properties

in the normal endometrium is 0.02�0.1

% [24].

It

would not

be

too much

of an

assumption

to accept

these levels as indicative for estimating the frequency

of spheroid formation in cultures of normal endometrium.

In the scientific

literature, we have not

found data on the frequency of spheroid formation in

conditions that do not support cell adhesion in whole

EC

cultures, since

individual

subpopulations

purified

by marker expression or nuclear dye retention are

usually the object of research. However, taking into

account the fact that the frequency of spheroid formation

is the highest among CD133+ EC cells and

amounts

to 11.7

% in

this

subpopulation

[17], as

well

as the fact that the total content of these cells in EC

ranges from

5.7 to

27.4

%

[16],

we can multiply these

indicators to obtain an approximate the frequency of

spheroid formation in a mixed culture of EC, which

in this case will be 0.7�3.2 %. Thus, our results lie in

the range corresponding to the known data on EC,

therefore, the main part of the spheroids in our cultures

is presumably formed by malignant cells with

the TSC phenotype.

Our data show that in EC cultures, the overall re

sponse to CD133

does

not

show a clear relationship

with the frequency of spheroid formation. Thus, in

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cultures No. 4 and No. 5, the frequency of spheroid

formation is an order of magnitude lower than the

content of CD133+ cells. In this regard, the question

arises as to which cells are the sources of spheroids

in EC cultures? Comparison with the morphological

features and phenotype of the normal endometri

um [21, 24, 25] indicates

a

number of patterns. Thus,

in culture No. 1,

obtained from

a highly differentiated

tumor, in a monolayer we see large cells similar to

the limited dividing progenitor cells of the stromal

and epithelial components of the normal endome

trium

[24]. The expression of the CD133 marker

in

these cells is very weak, while this culture formed

small lymphoid (loose) spheroids on soft agar with

a frequency of less than 1

%. Lymphoid colonies in

conditions that do not support adhesion can form

immune cells, namely, T lymphocytes and NK in the

presence of specific cytokines such as IL-2, IL-15 or

IL-7

[26]. Nevertheless, their characteristic appearance

can be considered a reliable difference between

lymphocytes and other cells � even in a stimulated

state, these

are

small

cells

(6�15

.m)

with

a high

nuclear-cytoplasmic ratio [27]. In our case, the cells

in the colonies have larger

sizes (about 40 .m

in

diameter), which means that they are not lympho

cytes with a high probability. At the same time, the

spheroids of culture No. 1 are similar to the lymphoid

colonies that were obtained from the EC tissue in

Tabuchi's work [28].

Culture No.

2,

obtained from

a low-grade tumor (G3), also contains cd133

fibro

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blast-like

cells, however, a subpopulation

of small

polygonal cells stands out well against their background,

demonstrating bright immunoreactivity on

CD133, which morphologically most resembles cells

with stem

properties,

in the work of Chan et al. [24].

Also in this culture, when grown on soft agar, large

(about

90

.m in diameter)

spheroids

were observed,

albeit with a small frequency (about 1.5

%). Despite

the fact that no special marker selection was car

ried out, it would be a small assumption to assume

that these CD133+ cells form spheroids, since large

fibroblast-like cd133

cells did not produce similar

large

iНе можете найти то, что вам нужно? Попробуйте сервис подбора литературы.

spheroids

in

culture

No. 1. Confirmation

of this

assumption may also be the fact that culture No. 3,

similar

in phenotype to

culture No. 2,

with a subpopulation

of small CD133+ cells forming dense colonies

clearly standing out against the background of

a weakly colored stromal component,

also

formed

large (about 115 .m in diameter) spheroids in soft

agar with a frequency of about 10

%. Another source

of spheroids may be small spindle-shaped cells

brightly colored on CD133, found in cultures No. 5

and No. 4. The morphological features of these cells

correspond to tissue stem cells, of which CD133 express,

for example, epithelial or endothelial SC, which

are normally present in the endometrium [30, 31].

According to the literature data, the size and appearance

of colonies formed on soft agar by such normal

stem cells do not differ from spheroids obtained

from TSC [31]. Finally, for a subpopulation of relatively

large polygonal CD133+ cells that are present in

cultures No. 4 and No. 5, the connection with spheroids

cannot be traced, although these cells can be

confidently attributed to the malignant component,

since they sometimes show signs of multinucleation.

It can also not be argued that CD133-negative cells

do not produce spheroids on soft agar, since in the

work of Ding [29], in particular, they showed that both

CD133+ and CD133 cells sorted from endometrial

tumor tissue by this marker can form spheroids, but

in the second case less effectively.

Thus, it is impossible to unambiguously determine

which of the cells of the primary EC cultures obtained

by us took part in the formation of spheroids. Some

presumptive relationship can be established only

for small epithelial and fibroblast-like CD133+ cells,

of which

only the

first

can

be

conditionally classified

as

malignant. To identify exactly TSC

EC, it

is

necessary to

conduct a comparative study of cell

cultures on soft agar obtained from tumor tissue

and the corresponding normal tissue, in combination

with cell sorting using markers CD133, CD44, CD117,

CD24, CD47 and others, for which a connection with

TSC and normal stromal stem cells has been established

[32].

CONCLUSION

We were able to obtain and characterize the culture

of cellular spheroids from the postoperative EC

material. However, the indicators of the frequency

of spheroid formation and the average size of spheroids

in this culture cannot serve as a marker of the

amount of TSC in tumor tissue without comparing

these data for tumor tissue and normal endometrium.

A

more detailed study of the cellular

subpopulations

of EC in comparison with normal endometrium

is required to establish a link between the observed

diversity of cells in culture and their ability to spheroid

formation and other characteristics of TSCs.

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Information about authors:

Svetlana Yu. Filippova � research fellow at the Laboratory

of Cellular Technologies, National Medical Research Centre for Oncology, Rostov-on-Don,

Russian Federation

ORCID: https://orcid.org/0000-0002-4558-5896, SPIN: 9586-2785, AuthorID: 878784, ResearcherID: AAH-4408-2020, Scopus

Author ID: 57189618843

Irina V. Mezhevova � junior research fellow at the Laboratory of Cellular Technologies, National Medical Research Centre for Oncology,

Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0002-7902-7278, SPIN: 3367-1741, AuthorID: 1011695, ResearcherID: AAI-1860-2019

Tatiana V. Chembarova � junior research fellow at the Laboratory of Cellular Technologies, National Medical Research Centre for Oncology,

Rostov- on-Don, Russian Federation

ORCID: https://orcid.org/0000-0002-4555-8556, SPIN: 5426-1873, AuthorID: 1051985, ResearcherID: AAR-3198-2021, Scopus

Author ID: 57221303597

Inna A. Novikova �

Dr. Sci. (Med.),

Deputy General Director

for Science,

National Medical Research Centre

for Oncology,

Rostov-on-Don,

Russian Federation

ORCID: https://orcid.org/0000-0002-6496-9641, SPIN: 4810-2424, AuthorID: 726229, ResearcherID: E-7710-2018, Scopus Author ID: 57202252773

Ekaterina V. Verenikina � Dr. Sci. (Med.), Head of the Department of Oncological Gynecology, National Medical Research Centre for Oncology,

Rostov- on-Don, Russian Federation

ORCID: https://orcid.org/0000-0002-1084-5176, SPIN: 6610-7824, AuthorID: 734269, Scopus Author ID: 57194271506

Oksana E. Zhenilo � Cand. Sci. (Med.), MD, physician oncologist at the Department of Oncological Gynecology, National Medical Research Centre

for Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0002-9833-8530, SPIN: 4078-7080, AuthorID: 732220

Veronika V. Polovodova � PhD student, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0009-0004-8863-6229, SPIN: 3051-4734, AuthorID: 1214151

Alexander V. Shaposhnikov � Dr. Sci. (Med.), MD, professor, chief researcher at the Thoracoabdominal Department, National Medical Research

Centre for Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0001-6881-2281, SPIN: 8756-9438, AuthorID: 712823

South Russian Journal of Cancer 2024. Vol. 5, No. 3. P. 16-30

Filippova S. Yu., Mezhevova I. V., Chembarova T. V. , Novikova I. A., Verenikina E. V., Zhenilo O. E., Polovodova V. V., Shaposhnikov A. V., Shalashnaya E. V., Maslov A. A.,

Ishonina O. G. Experience in creating primary cultures of endometrial cancer and studying cells carrying phenotype of cancer stem cells

Elena V. Shalashnaya � Cand. Sci. (Biol.), senior researcher at the Laboratory

for the Study

of the Malignant Tumors

Pathogenesis, National Medical

Research Centre for Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0001-7742-4918, SPIN: 2752-0907, AuthorID: 476958, ResearcherID: AAE-4085-2022, Scopus

Author ID: 55144159900

Andrey

A. Maslov

� Dr. Sci. (Med.), MD, professor, chief physician, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0001-7328-8074, SPIN: 5963-5915, AuthorID: 817983

Oksana G. Ishonina � Cand. Sci. (Biol.), Head of the Department of Training and Retraining of Specialists, National Medical Research Centre for

Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0002-5300-1213, SPIN: 4051-5165, AuthorID: 612417, Scopus Author ID: 37115461900

Contribution of the authors:

Filippova S. Yu. � concept and study design, manuscript writing, interpretation of results;

iНе можете найти то, что вам нужно? Попробуйте сервис подбора литературы.

Mezhevova I. V. � conducting the experiment;

Chembarova T. V. � technical editing of the manuscript;

Novikova I. A. � scientific editing of the article;

Verenikina E. V. � provision of biological patient material;

Zhenilo O. E. � provision of biological patient material;

Polovodova V. V. � provision of biological patient material;

Shaposhnikov A. V. � scientific editing of the article;

Shalashnaya E. V. � scientific editing of the article;

Maslov A. A. � scientific editing of the article;

Ishonina O. G. � scientific editing of the article.

Experience in creating primary cultures of endometrial cancer and studying cells carrying phenotype of cancer stem cells Текст научной статьи по специальности «Клиническая медицина»

Аннотация научной статьи по клинической медицине, автор научной работы — Svetlana Yu. Filippova, Irina V. Mezhevova, Tatiana V. Chembarova, Inna A. Novikova, Ekaterina V. Verenikina

Похожие темы научных работ по клинической медицине , автор научной работы — Svetlana Yu. Filippova, Irina V. Mezhevova, Tatiana V. Chembarova, Inna A. Novikova, Ekaterina V. Verenikina

Опыт создания первичных культур рака эндометрия и исследования в них клеток, обладающих фенотипом опухолевых стволовых клеток

Текст научной работы на тему «Experience in creating primary cultures of endometrial cancer and studying cells carrying phenotype of cancer stem cells»