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Int J Immunopathol PharmacolInt J Immunopathol PharmacolIJIspijiInternational Journal of Immunopathology and Pharmacology0394-63202058-7384SAGE Publications Sage UK: London, England 10.1177/205873841878799010.1177_2058738418787990Letter to the EditorVEGF-R2 and TNF-R1 expression and cytokine production by samples of
mammary adenocarcinomas and correlations with histopathological parameters of
these malignant tumors Autenshlyus Alexander 12Arkhipov Sergey 123Mikhailova Elena 12Arkhipova Valentina 1Varaksin Nikolay 41 Novosibirsk State Medical University,
Novosibirsk, Russia2 Institute of Molecular Biology and
Biophysics, Subdivision of Federal Research Center of Fundamental and Translational
Medicine, Novosibirsk, Russia3 Laboratory of Immunohistochemistry,
Biochemistry and Pharmacology, Central Research Laboratory, Novosibirsk State
Medical University4 Vector-Best Inc., Novosibirsk Region,
RussiaSergey Arkhipov, Laboratory of
Immunohistochemistry, Biochemistry and Pharmacology, Central Research
Laboratory, Novosibirsk State Medical University, Krasny Prospect 52,
Novosibirsk 630091, Russia. Email:
arhipowsergei@yandex.ru09 7 2018 Mar-Dec 2018 32 205873841878799022 12 2017 14 6 2018 © The Author(s) 20182018SAGE Publications Ltd unless otherwise noted.
Manuscript content on this site is licensed under Creative Commons
LicensesThis article is distributed under the terms of the Creative Commons
Attribution-NonCommercial 4.0 License (http://www.creativecommons.org/licenses/by-nc/4.0/) which
permits non-commercial use, reproduction and distribution of the work
without further permission provided the original work is attributed as
specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).Currently, the role of cytokines in the tumor progression, including breast
cancer, is universally recognized. At the same time, there are still many
questions concerning the role of individual cytokines and receptors for
cytokines in various morphogenetic processes underlying the tumor progression.
The objective of this work was to study cytokine production and vascular
endothelial growth factor (VEGF)-R2 and VEGF-R1 expression by mammary
adenocarcinoma (MAC) and the correlations with histopathological parameters of
malignant tumors. The object of the study was cultured tumor biopsy samples from
47 women aged 43–75 years with invasive ductal carcinoma, which was classified
as grade II–III adenocarcinoma. It was shown that the cytokine profiles of the
supernatants of MAC samples from patients differ greatly. A correlation between
the levels of VEGF-R2 and tumor necrosis factor (TNF)-R1 expression was
observed. Correlations were also revealed during analysis of the relations of
histopathological MAC indicators with KVEGF-R2/VEGF-A and
KTNF-R1/TNF-α coefficients, which are equal, respectively, to the
ratio of expression values of receptors VEGF-R2 and TNF-R1 to the concentrations
of the relevant cytokines (VEGF-A and TNF-α) in the culture supernatants of the
same MAC samples. A direct correlation was identified between
KVEGF-R/VEGF-A and some histopathological MAC characteristics:
proportion of cells undergoing mitosis or pathological mitosis in MAC and poorly
differentiated cells. KVEGF-R2/VEGF-A directly correlated with the
concentration in supernatant interleukin (IL)-18 and interferon (IFN)-γ.
KTNF-R1/TNF-α was inversely correlated with the concentration in
supernatant of IL-1Ra, IL-8, and granulocyte-macrophage colony-stimulating
factor (GM-CSF). The data obtained show that the high-level production of IL-18
and IL-1β by MAC, overexpression of VEGF-R2 in tumor (at relatively low VEGF-A
production), and the high level of IFN-γ production are attributed factors
contributing to the formation of a population of low-grade cells in the tumor.
The factors regulating the population of moderately differentiated cells in the
tumor are referred to as IL-1Ra, IL-8, and GM-CSF.

cytokines of tumor supernatantmammary adenocarcinomaTNF-R1TNF-αVEGF-AVEGF-R2cover-dateMarch-December 2018
==== Body
Introduction
Currently, the role of cytokines in the tumor progression, including breast cancer,
is universally recognized.1,2
At the same time, there are still many questions concerning the role of individual
cytokines and receptors for cytokines in various morphogenetic processes underlying
the tumor progression.

As is known, cytokines affect cells while interacting with the appropriate
receptors.3–5 For instance, it
has been shown that in a tumor, overexpression of such receptors as vascular
endothelial growth factor receptor 1 (VEGF-R1) and VEGF-R2 may indicate advanced
tumor progression in the case of breast cancer because of the important role they
play in the process of tumor angiogenesis.5,6 In addition, the production
level of the most important member of the VEGF family, VEGF-A, can vary in a tumor
and the organs of the immune system of a patient depending on the stage of tumor
development. This situation inevitably means ambiguity of the role of receptors for
VEGF-A in tumor progression in different patients. There is even less clarity in the
understanding of the function of the tumor necrosis factor (TNF) receptor family in
this process (TNF-R1 and others) because these receptors may be expressed on both
breast tumor cells and cells of a tumor microenvironment (lymphocytes, macrophages,
dendritic cells, and granulocytes).7,8 The ambiguous effect of many
cytokines on tumor growth in patients may be due to different ratios of cells
expressing receptors for mediators that regulate tumor growth within a tumor and
different levels of production of the cytokines that are complementary to them.
Thus, the research on the relations among cytokine production levels in a tumor,
expression of the relevant receptors on tumor cells (and on cells of the tumor
microenvironment), and the histopathological parameters of a tumor in each patient,
including the features of the formation of a population of low-differentiated forms
of tumor cells, characterized by a high degree of malignancy, is essential for
predicting the effectiveness of treatment and for evaluation of the risk of
recurrence and metastasis.

The objective of this work was to study VEGF-R2 and TNF-R1 expression and cytokine
production by mammary adenocarcinomas and their correlations with histopathological
parameters of these malignant tumors.

Materials and methods
Patients
The object of the study was cultured tumor biopsy samples from 47 women aged
43–77 years with invasive ductal breast cancer, treated at the Novosibirsk
regional oncological center, which was classified according to histological type
as mammary adenocarcinomas (MACs) grade II–III. The study and all study
protocols were approved by the Ethics Committee of the Institute of Molecular
Biology and Biophysics (Novosibirsk). All the studies were conducted in
accordance with the Helsinki Declaration (Brazil, Fortaleza, 2013), and all
recommendations of the International Committee of Medical Journal Editors
(ICMJE) were taken into account. At the time of registration, each patient is
assigned an individual identification number, which was then placed on the
results of his analyzes, as well as other documents relating to the data of this
study. Each patient was informed about the study conducted and its objectives
and methods. Written informed consent for participation in the study and for the
tumor biopsy procedure was signed by each patient and verified by a
physician.

Method of measurement of cytokine production
Each 8-mm3 tumor biopsy sample obtained by trepanobiopsy was placed in
a glass vial containing 1 ml of Dulbecco’s modified Eagle medium (DMEM)-F12 and
then incubated for 72 h. Before collection of the supernatant, the tumor biopsy
sample was taken out of the vial and placed in a solution of 10% neutral
formalin. The remaining cellular detritus were pelleted by centrifugation at
900 g for 15 min. Using enzyme-linked
immunosorbent assays, the concentrations of IL-2, IL-6, IL-8, IL-10, IL-17,
IL-18, IL-1β, IL-1Ra, TNF-α, interferon (IFN)-γ, granulocyte colony-stimulating
factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and
VEGF-A in the culture supernatants were determined. Enzyme-linked immunosorbent
assay was performed using enzyme immunoassay analyzer Multiskan spectrum (Thermo
Electron Corporation, Finland) with the kits manufactured by Vector-Best Inc.
(Russia).

Immunohistochemical analysis
Expression of receptors VEGF-R2 and TNF-R1 in the MAC biopsy samples was
determined by immunohistochemical analysis. The MAC biopsy samples, fixed in
neutral formalin, were dehydrated and embedded in paraffin. Dewaxing and
rehydration of the samples were performed according to the standard procedure by
means of a xylene and graded series of ethanol solutions. Antigen retrieval was
performed at: pH = 6 at 95°C for 20 min using the citrate-based antigen
retrievel solution (pH = 6.0) in the BioSan WB-4MS water bath (Latvia). To block
endogenous peroxidase, the slices were incubated in a 1% aqueous solution of
Н2О2 for 5 min. To reduce non-specific staining, the
slices were treated with normal blocking non-immune serum (from VECTASTAIN Elite
ABC Kit, Cat. No. PK-7200, Vector Laboratories, USA) for 10 min. Then the slices
were incubated for 1 h with primary antibodies specific for VEGF-R2 (rabbit
polyclonal, Cat. no. PAB367Hu01, Cloud-Clone Corp, USA) and TNF-R1 (TNFRSF1A;
rabbit polyclonal, Cat. no. PAB499Hu01, Cloud-Clone Corp.) at final
concentration of 5.0 µg/ml in an antibody dilution solution (Cat. no. 559148;
Becton Dickinson, USA). After a wash, they were incubated for 30 min with
secondary biotinylated antibodies (VECTASTAIN Elite ABC Kit, Vector
Laboratories). Then, the tissue slices were incubated with the avidin peroxidase
complex (VECTASTAIN Elite ABC Kit, Vector Laboratories) for 30 min and with a
chromogenic substrate containing diaminobenzidine (ImmPACT DAB, Vector
Laboratories, Cat. no. SK-4105) for 5 min. The slices were stained with
hematoxylin and eosin, then washed with water, and embedded in balsam after
dehydration.

Computer morphometric analysis
Histological samples stained for VEGF-R2 and TNF-R1 were photographed (at
magnification ×400) using an image analysis system based on a Micros MC 300A
microscope (Austria) and a digital CMOS camera based on the Aptina MT9J003
sensor-format 1/2.4-inch (China). Computer morphometric quantitative evaluation
of expression of receptors VEGF-R2 and TNF-R1 was performed in the ImageJ 1.50a
software (National Institute of Health, USA). Digital images of histological
sections stained on VEGF-R2 and TNF-R1 was decomposed into three color image
layers using “Colour Deconvolution” option in the “H & E DAB” mode. On a
digital section with a brown color corresponding to the DAB color, the colored
DAB structures were extracted using the “Threshold” option in the “MaxEntropy”
mode, which were marked binary. The resulting binary image of the memory was
measured in automatic mode. With this measurement, the percentage of the total
DAB color (corresponding to the VEGF-R2 or TNF-R1 color) was found relative to
the area of the entire digital image taken as 100% (3488 × 2616 pixels).
Immunohistochemical indicators of VEGF-R2 and TNF-R1 expression were as squares
of colored zones which were specific for VEGF-R2 or TNF-R1 expression (%,
percentage of colored area from a total area of tested image) in each digital
image of the tested section. KVEGF-R2/VEGF-A and
KTNF-R1/TNF-α coefficients were then determined, representing the
ratio of the expression value of receptors VEGF-R2 or TNF-R1 to the
concentration of the relevant cytokines (VEGF-A or TNF-α). The coefficients are
expressed in arbitrary units. Statistical analysis of expression of the
receptors under study was performed based on the average expression index
calculated during examination of eight digital photographs of each MAC
sample.

Histopathological analysis
Histopathological analysis of the tumors was performed by a pathologist using
histological sections of MAC samples stained with hematoxylin and eosin
according to the standard procedure. The average numbers of mitotic cells and
cells undergoing pathological mitosis were calculated after analyzing 10 fields
of view; also analyzed the shares (estimated percentage) of poorly
differentiated, moderately differentiated, and well-differentiated tumor
cells.

Statistical analysis
Histograms were created in the Microsoft Excel. Cluster analysis, determination
of Spearman’s rank correlation coefficient (r), assessment of differences
between groups by Mann–Whitney U-test, and identification of canonical
correlations (R) including their reliability (p)
were performed by means of software package Statistica 7.

Results
Correlation between VEGF-R2 and TNF-R1 expression, KVEGF-R2/VEGF-A or
KTNF-R1/TNF-α coefficients and histopathological indicators
When we studied the receptor expression in MAC samples, a direct correlation
between immunohistochemical indicators of VEGF-R2 and TNF-R1 expression was
revealed (r = 0.57, p < 0.01). In contrast, no
significant correlation was found when we analyzed the relation of expression of
these receptors with histopathological MAC indicators.

However, correlations was found only between the KVEGF-R2/VEGF-A or
KTNF-R1/TNF-α coefficients and histopathological indicators.
KVEGF-R2/VEGF-A directly correlated with histopathological MAC
parameters that characterize tumor grade: the numbers of mitotic cells (
r = 0.47, p = 0.0426), cells undergoing
pathological mitosis (r = 0.51, p = 0.0246), and
the proportion of poorly differentiated cells (r = 0.54, p = 0.0174). KTNF-R1/TNF-α directly correlated only
with the number of moderately differentiated tumor cells in MAC ( r = 0.39,
p = 0.0499).

Correlation between VEGF-R2 and TNF-R1 expression, KVEGF-R2/VEGF-A
or KTNF-R1/TNF-α coefficients and with cytokine concentrations in the
MAC culture supernatants
When we studied the relation of the expression of receptor VEGF-R2 with cytokine
concentrations in the tumor culture supernatant, only two correlations were
identified: between VEGF-R2 and IL-18 as well as between VEGF-R2 and IFN-γ
(Table 1).
KVEGF-R2/VEGF-A also directly correlated with the concentration
of these cytokines in the supernatant. When we analyzed the relations between
the expression of receptor TNF-R1 in MAC and cytokine concentrations in the
tumor supernatant, no correlations were identified (Table 1).

Table 1. Coefficients of correlation (r) of the indicators of VEGF-R2 and TNF-R1
expression and of coefficients KVEGF-R2/VEGF-A and
KTNF-R1/TNF-α with cytokine concentrations in the MAC
culture supernatants (Spearman’s rank correlation).

Cytokines (pg/ml)	VEGF-R2 (colored
area, %)	TNF-R1 (colored area,
%)	KVEGF-R2/VEGF-A (AU)	KTNF-R1/TNF-α (AU)	
r (p-value)	r (p-value)	r (p-value)	r (p-value)	
IL-2	−0.11 (0.6322)	0.07 (0.7661)	−0.04 (0.8819)	0.23 (0.3359)	
IL-6	0.07 (0.7817)	−0.23 (0.3324)	−0.04 (0.8799)	−0.30 (0.2047)	
IL-8	−0.08 (0.7289)	−0.30 (0.2023)	−0.21 (0.3765)	−0.50 (0.0240)	
IL-10	−0.18 (0.442)	−0.21 (0.3765)	−0.15 (0.5185)	−0.39 (0.0909)	
IL-17	0.03 (0.8950)	−0.21 (0.3816)	0.15 (0.5238)	−0.31 (0.1814)	
IL-18	0.66 (0.0014)	0.29 (0.2023)	0.54 (0.0131)	0.36 (0.1179)	
IL-1b	−0.12 (0.6089)	0.06 (0.7865)	0.15 (0.5227)	0.27 (0.2567)	
IL-1Ra	0.33 (0.1503)	0.33 (0.1503)	0.35 (0.1317)	−0.54 (0.0169)	
TNF-α	−0.33 (0.1501)	−0.25 (0.2824)	−0.19 (0.4310)	−0.75 (0.0002)	
IFN-γ	0.49 (0.0304)	0.17 (0.4651)	0.56 (0.0103)	0.06 (0.7909)	
G-CSF	−0.07 (0.7768)	−0.27 (0.2537)	−0.01 (0.9824)	−0.25 (0.2944)	
GM-CSF	−0.14 (0.5457)	−0.15 (0.5329)	−0.07 (0.7743)	−0.51 (0.0269)	
VEGF-A	−0.33 (0.1542)	−0.36 (0.1147)	−0.76 (0.0001)	−0.35 (0.1264)	
VEGF: vascular endothelial growth factor; TNF: tumor necrosis factor;
G-CSF: granulocyte colony-stimulating factor; GM-CSF:
granulocyte-macrophage colony-stimulating factor; IFN: interferon;
IL: interleukin; AU: arbitrary unit.

Nonetheless, in the analysis of the associations of the KTNF-R1/TNF-α
coefficient, three negative correlations were identified: with IL-8, IL-1Ra, and
GM-CSF.

Peculiarities of distribution of VEGF-R2 and TNF-R1 expression in the samples
of MAC
Histopathological and immunohistochemical analyses suggested that in the tissue
samples tested, VEGF-R2 was predominantly expressed in endothelial cells of
mature and emerging blood vessels, as well as cells of duct epithelium (Figure 1(a) and (b)). VEGF-R2 was also
expressed by some tumor cells and by cells in the tumor microenvironment:
macrophages and fibroblast-like cells (myofibroblast and/or fibroblasts).
Indicators of VEGF-R2 expression (colored areas, %) varied in different patients
within the range: from 0.26 (weak expression) to 6.1 (overexpression). TNF-R1
was predominantly expressed by tumor cells (Figure 1(c) and (d)). Indicators of TNF-R1 expression
varied in different patients within the range of 0.27–5.6 (colored areas, %).
The results of the immunohistochemical analysis indicated that most of the tumor
cells expressed TNF-R1 in nucleus. An inverse correlation was identified between
the number of tumor cells expressing TNF-R1 and the number of poorly
differentiated cells in MAC (r = 0.53; p < 0.01).

Figure 1. Mammary gland adenocarcinoma (MAC). Images were captured at ×400
magnification. Immunohistochemical staining for VEGF-R2 and TNF-R1
(brown color). In addition, staining with hematoxylin & eosin: (a)
sample of MAC grade II with moderate VEGF-R2 expression, (b) sample of
MAC grade III with VEGF-R2 overexpression, (c) sample of MAC grade III
with moderate TNF-R1 expression, and (d) distribution histogram of the
proportion of cells expressing TNF-R1 when evaluating only those cells
that have expressed TNF-R1.

Canonical correlation between coefficients KVEGF-R2/VEGF-A and
KTNF-R1/TNF-α and histopathological MAC parameters
Using canonical correlation analysis, the relations between two groups of
variables were evaluated: (1) KTNF-R1/TNF-α and the percentage of
tumor cells expressing TNF-R1 (%) and (2) the number of tumor cells undergoing
mitosis, pathological mitosis, and the percentage of poorly differentiated
cells. A marked canonical correlation was revealed between these two groups of
indicators: R = 0.73 (p < 0.01). When we added
KVEGF-R2/VEGF-A to the first group of variables (to
KTNF-R1/TNF-α and percentage of tumor cells expressing TNF-R1),
the canonical correlation with histopathological parameters, characterized tumor
grade, increased: the canonical correlation coefficient reached 0.85 (p < 0.01).

Cytokine profiles of the supernatants of MAC samples and its relation to the
number of tumor cells of different degrees of differentiation
Using cluster analysis was constructed hierarchical dendrogram of the
distribution of patients depending on the ratio of poorly differentiated,
moderately differentiated, and well-differentiated tumor cells in the
supernatant samples MAC (Figure
2(a) and (b)). Depending on the ratio of tumor cells with different
differentiation in tumors, all patients were fairly clearly divided into two
clusters forming two groups, respectively, with a high (Group I: prevails poorly
differentiated tumor cells) and low (Group II: prevails well-differentiated
tumor cells) content of poorly differentiated tumor cells in MAC samples.

Figure 2. The results of formation of groups of patients with similar distribution
characteristics in tumor samples on ratio tumor cells with different
degrees of differentiation: (a) hierarchical dendrogram of the
distribution of 47 patients with mammary gland adenocarcinomas (MAC)
depending on the ratio of poorly differentiated, moderately
differentiated, and well-differentiated tumor cells in the samples MAC
formed by the method of cluster analysis and (b) histograms of the
distribution of tumor cells with different degrees of differentiation in
the groups of patients.

A comparative analysis of the differences between the two groups of patients by
the production of cytokines by MAC specimens was conducted, one of which was
dominated by low-grade tumor cells in MAC samples and the other by highly
differentiated tumor cells. Significant differences between groups were found
only in the production of IL-1β and IL-18 (Figure 3).

Figure 3. The results of analysis of the differences between two groups of patients
on the production of cytokines by MAC specimens in which prevails poorly
differentiated or well-differentiated tumor cells. Each 8-mm3
tumor biopsy MAC sample obtained by trepanobiopsy was placed in a glass
vial containing 1 ml of DMEM-F12 nutrient medium and then incubated for
72 h. Using enzyme-linked immunosorbent assays, the concentrations
(pg/ml) in the culture supernatants were determined. Bars correspond to
mean ± SE. *p < 0.05, **p < 0.01 (Mann–Whitney U-test).

Discussion
A number of studies have shown that different human cancer cell lines express
different amounts of TNF-R1.9,10 Therefore, the induction of apoptosis can depend to a large
extent on the concentration of death receptors on the tumor cell membrane.2,10 It was determined that TNF-R1
internalization into the nucleus may be is an indicator of irreversibility of the
apoptotic process in a cancer cell.9,10

The data we obtained on the relation between KTNF-R1/TNF-α and the number
of MAC tumor cells expressing TNF-R1 predominantly in the nucleus on one hand, and
the number of poorly differentiated cells and cells with pathological mitosis on the
other hand, indicate that the TNF-R1 overexpression in tumor cells and the process
of TNF-R1 internalization into the nucleus are important during formation of the
most malignant subpopulation of poorly differentiated cells.

As is known, the key growth regulators of vasculature in a tumor are VEGF-A and its
receptors VEGF-R1 (Flt-1) and VEGF-R2 (KDR).4,6,11 It is important to note,
however, that VEGF-R2 expression has been demonstrated in both neoplastic
endothelial cells and directly in tumor cells.12 The main recipient of the biological effect of VEGF-A is VEGF-R2.

Analyzing the tumor slices from the patients with a high KVEGF-R2 /VEGF-A
value, we detected not only a number of cells in the tumor microenvironment
(endotheliocytes, duct epithelial cells, and macrophages) expressing VEGF-R2 but
also some tumor cells with VEGF-R2. These data indicate that the activation of a
VEGF-R2–dependent autocrine mechanism that stimulates proliferation of tumor cells
should be regarded as one of the factors responsible for accumulation of poorly
differentiated tumor cells.

The direct correlations between VEGF-R and IL-18, between VEGF-R and IFN-γ, and the
inverse correlations of IL-8, GM-CSF, and IL-1Ra with the KTNF-R1/TNF-α
coefficient indirectly point to the role played by the cells of the tumor
microenvironment in the regulation of VEGF-R2/VEGF-A– and TNF-R1/TNF-α–dependent
mechanisms of tumor progression. We have been shown that in the group with a
predominance of low-differentiated tumor cells, MAC samples produced more IL-18 and
I L-1β than in the group with a predominance in the tumor of highly differentiated
tumor cells. Thus, the data obtained show that the high-level production of IL-18
and L-1β by tumor, overexpression in VEGF-R2 tumor (at relatively low VEGF-A
production), and the relatively high level of IFN-γ production are attributed
factors contributing to the formation of a population of low-grade cells in the
tumor. The factors regulating the population of moderately differentiated cells in
the tumor are referred to as IL-1Ra, IL-8, and GM-CSF.

It can be assumed, that if you change the state of TNF-R1/TNF-α; VEGF-R2/VEGF-A; and
the production of IL-18, IFN-γ, IL-1Ra, IL-8, and GM-CSF in a tumor, for example, by
cytokine-directed therapy, you can slow down the formation process of poorly
differentiated forms of tumor cells and, probably, stimulate the process of their
reverse differentiation. To check this assumption, it is necessary to conduct
additional researches.

Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the
research, authorship, and/or publication of this article.

Funding: The author(s) received no financial support for the research, authorship, and/or
publication of this article.
==== Refs
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