Density of Gr1-positive myeloid precursor cells, p-STAT3 expression and gene expression pattern in canine mammary cancer metastasis
Karol M. Pawowski
I. Dolka Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life SciencesWULS
, Nowoursynowska 159, 02-776 Warsaw,
K. M. Pawowski Department of Animal Environment Biology, Faculty of Animal Sciences, Warsaw University of Life SciencesWULS
, Ciszewskiego 8, 02-786 Warsaw,
The very recent studies on human and mice models have indicated an important role of myeloid precursor cells (progenitors or not fully differentiated cells that express the Gr1 antigen also called Gr1-positive myeloid suppressor cells) in the tumor progression and metastasis. They are thought to suppress the immune system and promote angiogenesis via Signal transducer and activator of transcription 3 (STAT3) activation. As of now there is no data available on the correlation of Gr1-positive cell number, phosphorylated STAT3 (p-STAT3) expression and cancer ability to metastasis. Thus, we counted the myeloid precursor cell number and analyzed p-STAT3 expression in 50 canine mammary tumors that gave local/distant metastases and did not metastasize. We showed that the number of Gr1-positive cells and p-STAT3 expression are significantly higher (p < 0.001) in the metastatic tumors than in the non-metastatic ones. We also observed higher expression of p-STAT3 in the canine mammary cancer cell lines with metastatic potential than in other cell lines (p < 0.001). Moreover, the number of myeloid precursors and p-STAT3 expression in metastatic tumors correlate strongly. The tumor infiltrating myeloid precursor cells may invigorate the STAT3 activity (probably via vascular endothelial growth factor - VEGF) that contributes to the tumor angiogenesis and furthermore tumor`s ability to metastasize. The analysis of gene expression in canine mammary cancer cell lines with metastatic potential indicated that semaphorin 3B (SEMA3B) and neuropilin receptors (NRP) may also be important elements in this process. Thus, we discuss the possible interactions within the tumor that may be required for cancer metastatis.
The fact that stromal cells within the tumor undergo various changes during tumor
initiation, progression, and metastasis is commonly known. The tumor microenvironment
may be composed of various cells, mainly of fibroblasts, newly formed blood vessels as
well as hematopoietic cells (Pollard 2008; Krl et al. 2011). The interactions between these
cells and cancer cells within the tumor lead to rapid and more invigorated tumor growth and
metastasis (Liang et al. 2010). How the tumor cells may revert the immune system from
performing its antitumor functions to promoting immunosuppression and tumorigenesis is
extremely interesting. The regulatory role of myeloid suppressor cells (the cells which
express the surface myeloid differentiation antigen 1 Gr1) on T cell activity has been only
indicated as important by very few recent studies (Dietlin et al. 2007; Kujawski et al. 2010;
Liang et al. 2010; Youn and Gabrilovich 2010). Gr1-positive cells from the blood are
actively recruited to the tumor microenvironment by various chemokines in the same
manner as other cells (for review, see: Gabrilovich and Nagraj 2009; Schmid and Varner
2010). The population comprises a heterogeneous population of bone-marrow-derived
myeloid progenitors and immature myeloid cells. Under normal conditions they
differentiate into granulocytes, macrophages, or dendritic cells, what helps to explain their
heterogeneity (Schmid and Varner 2010; Youn and Gabrilovich 2010). In cancer patients, a
partial differentiation block of hematopoietic cells may occur, which results in flourish of
these cells into cancer tissue.
The myeloid suppressor cells may also play another role in cancer progression. They
promote angiogenesis by production of angiogenic factors such as: vascular endothelial
growth factor (VEGF), matrix metalloproteinases (MMPs), and interleukins (ILs). These
factors additionally stimulate Gr1-positive cells accumulation in tumors (Voronov et al.
2003; Bunt et al. 2007; Melani et al. 2007; Allavena et al. 2008; Yang et al. 2008; Kujawski
et al. 2010).
The role of myeloid precursor cells in tumor angiogenesis was examined by Kujawski et al.
(2010). The mice studies have recently shown that this process may be driven by an active
Signal transducer and activator of transcription 3 (STAT3). The enhanced angiogenesis
correlates with the tumor aptitude to metastasize (Zetter 1998). Kujawski et al. (2010) also
showed that the phosphorylated STAT3 (p-STAT3) ablation in experimental mice reduced the
number in infiltrating myeloid precursor cells tumor. Although so far the relations between the
number of Gr1-positive cells, p-STAT3 expression and the tumor aptitude to metastasis has
not been assessed in vivo yet.
Therefore, we have decided to investigate whether the number of Gr1-positive cells and
expression of p-STAT3 are associated with the tumor aptitude to metastasize. We have
performed immunohistochemical analysis of 50 canine mammary tumors: 40 non-metastatic
tumors and 10 tumors that gave local or distant metastases. We also examined p-STAT3
expression in canine mammary cancer cell lines with metastatic potential and in non-metastatic
cells. To answer the question how the STAT3 may promote the angiogenesis, the supervised
analysis of gene expression in canine mammary cancer cell lines with high metastatic potential
was conducted (Krl et al. 2010b). The results of this microarray experiment have been
partially published (Krl et al. 2010b) but for the purposes of this manuscript we analyzed
only the unpublished data. We focused on the gene cluster involved in tumor angiogenesis
and interactions with hematopoietic cells.
Materials and methods
Tissue sections from canine mammary tumors were obtained from the archives of the
Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine,
Warsaw University of Life Sciences - WULS (Poland). The samples were surgically
obtained during the mastectomy from 50 female dogs of different breeds. Each tumor
sample was fixed in 8% neutral buffered formalin and routinely embedded in paraffin.
The 5 micrometer sticks were fixed on the slides and stained with hematoxylin and
eosin (HE) and subjected to the histological evaluation. A wide variety of the patient
cases including the information about the presence/absence of metastases is known. The
tumors that gave metastases were surgically removed together with the metastatic site.
The presence of neoplastic cells in the metastatic site was histologically confirmed. The
immunohistochemical examination of cytokeratin, vimentin, smooth muscle actin, s100
protein and p63 protein expression was performed (data not shown). The tumor types of
specimens were classified based on the World Health Organization (WHO) Histological
Classification and Mammary Tumors of the Dog and Cat classification (Misdorp et al.
1999). Histological tumor grading was conducted on HE-stained sections using a Misdorp
(2002) classification. The mammary carcinoma grading was assessed in respect to
tubule formation, degree of differentiation and mitotic index as: the 1st, 2nd and 3rd
grade of malignancy. All the tumors examined were classified as malignant carcinomas
(simple carcinomas or complex carcinomas) or benign adenomas. The types tumors
were split into three categories: benign (n = 10) and malignant (n = 30) non-metastatic
(which did not give metastases) and metastatic (n = 10) (which gave local or distant
metastases). Among the non-metastatic carcinomas 11 were considered as the 1st grade of
malignancy, 9 tumors as the 2nd grade of malignancy, 10 tumors as the 3rd grade of
malignancy; the examined 10 metastatic tumors were of the 2nd and 3rd grade of
The cell lines used for this study have previously been described in detail (Krl et al. 2009;
Krl et al. 2010a, b; Pawowski et al. 2011b). Three canine mammary adenocarcinoma cell
lines (non-metastatic PL-20, and metastatic CMT-W1, CMT-W2), non-metastatic anaplastic
cancer (P114), non-metastatic simple carcinoma (CMT-U27) and non-metastatic spindle-cell
mammary tumor (CMT-U309) were examined.
These cell lines were subjected to a detailed cytometric analysis (e.g. cell cycle analysis,
ploidy analysis, growth rate assessment and Ki67 expression, Bcl-2 expression, analysis of
cells susceptibility to campthotecin-induced apoptosis etc.) (Krl et al. 2010b). Based on
the results of these analyses they were classified into 3 various phenotypic groups: highly
proliferative (P114 and CMT-U27), highly apoptosis-resistant (PL20 and CMT-U27) and
metastatic (CMT-W1 and CMT-W2). The detailed results of these analyses have been
published (Krl et al. 2010b).
Cells were cultured in optimal conditions: a medium (RPMI-1640) enriched with 10%
(v/v) heat-inactivated fetal bovine serum (FBS), penicillin-streptomycin (50 iU mL1), and
fungizone (2.5 mg mL1), in an atmosphere of 5% CO2 and 95% humidified air at 37C,
and routinely subcultured every second day. The methods of canine mammary cancer cells
culturing have been previously described (Krl et al. 2009, 2010a, b).
Five m sections from paraffin blocks containing tumor tissue were baked in 37C
overnight. After dewaxing in xylene and rehydration in ethanol, for antigen retrieval,
the slides were placed in 0.02 M citrate buffer, pH 6.0 and boiled in the decloaking
The cell lines were cultured on Lab-Tek (Nunc Inc. USA) 4-chamber culture slides and
subjected to the immunohistochemical analysis were fixed with ethanol after the 24 hrs.
The samples were incubated in the Peroxidase Blocking Reagent (Dako, Denmark) for
10 min at room temperature prior to the antibody incubation. After 30 min incubation in 5%
bovine serum albumin (Sigma Aldrich, Germany), the following primary antibodies were
used (diluted in 1% bovine serum): rat monoclonal anti-Gr1 (in case of paraffin slides) and
rabbit polyclonal STAT3 (phospho S727) (for paraffin slides and cell lines) both obtained
from Abcam (United Kingdom). According to the manufacturer`s instructions the slides
were incubated with antibodies at +4C overnight. For the staining the anti-rat rabbit
polyclonal antibodies Horseradish peroxidase (HRP)-conjugated (Abcam) and EnVision kit
(Labelled Polymers consist of secondary anti-rabbit antibodies conjugated with the HRP
enzyme complex obtained from Dako) were used. To develop the colored product, the
3,3`Diaminobenzidine (DAB) substrate was used (Dako). Finally, the hematoxyline was used
for nuclei counterstaining.
For each immunohistochemical experiment of paraffin slides, two controls (negative and
positive) were used: as a positive control the tissues of canine lymph nodes (in case of Gr1
antibodies) and normal mammary tissue (in case of anti-STAT3 antibodies), whereas the
negative control were stained without use of primary antibodies. For each
immunohistochemical analysis of cell lines the negative control was used (the staining procedure without
use of primary antibodies).
Three consecutive tissue sections and four slides of each cell line were analyzed. The
1020 pictures of each slide were taken (depending on the sample size) using Olympus
microscopy BX60. In the case of the tissues only the regions of cancer cells and stroma
were analyzed, the tumor necrosis regions were avoided. The number of Gr1-positive cells
(brown color) and the colorimetric intensity of the p-STAT3 expression reflected as
IHCstained antigen spots (brown color) were counted by a computer-assisted image analyzer
(Olympus Microimage Image Analysis, software version 4.0 for Windows, USA). The
antigen spot color intensity is expressed as a mean pixel optical density on a 1256 scale.
Gene expression analysis
RNA from the cell suspension samples was isolated using a Total RNA kit (A&A
Biotechnology, Poland) according to the manufacturer s protocol. The quantity and
quality of isolated RNA was measured using BioAnalyzer (Agilent, USA). Equal
amounts of RNA isolated from all examined cell lines were pooled and used as the
For each microarray slide, total RNA (10 g) of each cell line was reverse-transcribed
using SuperScript Plus Direct cDNA Labeling System, (Invitrogen, USA) according to the
manufacturers protocol. Single-strand cDNAs were stained with Alexa 647 and Alexa 555
(Invitrogen). Dog-specific oligonucleotide microarray slides Canis familiaris V1.0.1 AROS
(Operon, USA) with 25,383 probes were used for the hybrydization. Hybridization was
performed using automatic hybridization station HybArray12 (PerkinElmer, USA). Two
replicates were performed (dye-swap).
The slides were analyzed using microarray scanner ScanArray HT and ScanExpress
software (PerkinElmer, USA). The data was automatically normalized (LOWESS method)
by ScanExpress software. The average ratio of 2 slides was calculated.
The part of these microarray results has been previously published, thus the detailed
methodology has been previously described (Krl et al. 2010b). The results of the
microarray experiment has been also validated using real-time qPCR (Krl et al. 2010b).
The IHC statistical analysis was conducted using Prism version 5.00 software (GraphPad
Software, USA). The ANOVA and Tukey Honestly Significant Difference (HSD) post-hoc
tests were applied to analyze the number of myeloid precursor cells and p-STAT3 density in
all the examined tumor sub-groups as well as in the examined cell lines. To assess the
correlation between the number of Gr1-positive cells and p-STAT3 expression in
nonmetastatic and metastatic groups the Pearson correlation coefficient was used and r2 was
calculated. The p < 0.05 was regarded as significant whereas p < 0.001 as highly significant.
The microarray data was analyzed with SAM software (Statistical Analysis of
Microarrays; Stanford University, USA). The list of genes differing significantly in
expression was generated with a false discovery rate (FDR) of <5%. Genes in which
expression changed at least 1.3-fold in each of 2 examined slides were subjected to further
analysis (this analysis has been previously accounted for, see: Krl et al. 2010b). The gene
function was identified using the NCBI database, PANTHER pathway analysis software
(Mi et al. 2005) and Architect Pathway software (Agilent, USA). The pathway analyses
were conducted using binominal statistic test (PANTHER) with the cut-off value p < 0.05.
Tumor samples analysis
The analysis showed that the number of Gr1-positive cells is significantly higher (p < 0.001)
in the tumors that gave local or distant metastases comparing to the non-metastatic
carcinomas or adenomas (68.58 30.96, 7.908 1.099 and 2.08 0.36, respectively)
(Fig. 1a, b). The malignancy-related to tendency increase regarding the number of myeloid
precursor cells was observed in the group of all non-metastatic tumors (increasing from
benign to the most malignant), however no significant differences between these groups
have been observed (Fig. 1a, b; Table 1). Gr1-positive cells constituted a heterogeneous
population observed rather as the cell niches than the single cells.
The expression of p-STAT3 was detected in cancer cells (cytoplasmic staining pattern)
and also in hematopoietic cells however not in the other stromal cells. Significantly higher
expression (p < 0.001) of this antigen was found in the metastatic tumors than in the
nonmetastatic carcinomas or adenomas (146.02.37, 115.61.726 and 118.14.0, respectively)
(Fig. 2a, b). The p-STAT3 expression was equally intense in the all non-metastatic tumors,
without any significant differences between benign adenomas and carcinomas of various
grade of malignancy (Fig. 2a, b; Table 1).
To assess the correlation between the number of Gr1-positive cells and p-STAT3 expression
in the metastatic and non-metastatic groups the Pearson correlation coefficient was used. The
high correlation between the number of Gr1-positive cells and p-STAT3 expression in metastatic
tumors was detected (r2=0.73, p<0.05). No significant relation between the number of
Gr1Fig. 1 a Pictures of Gr1-positive cells in canine mammary adenomas, carcinomas of the 1st, 2nd, 3rd grade
of malignancy and tumors that gave local/distal metastases (n= 50) obtained with Olympus BX60 microscope
(at the magnification of 200x). The Gr1 antigen is represented by brown colored structures. b The graph of
number of Gr1-positive cells (and SD) in canine mammary non-metastatic adenomas, carcinomas and
metastatic carcinomas. Ten to 20 pictures in each slide were analyzed (three consecutive sections). The
number of objects was counted by a computer-assisted image analyzer (Olympus Microimage Image
Analysis, software version 4.0 for Windows, USA). The statistical analysis was performed using Prism
version 5.00 software (GraphPad Software, USA). The ANOVA and Tukey HSD (Honestly Significant
Difference) post-hoc test were applied to analyze the number of myeloid precursor cells in all the examined
tumor groups. p< 0.001 was regarded as highly significant and marked as ***
positive cells and p-STAT3 expression was detected neither in the non-metastatic carcinomas
(r2 = 0.263, p < 0.186) nor in the non-metastatic adenomas (r2 = 0.197, p < 0.585).
p-STAT3 expression in the examined cell lines
The expression of p-STAT3 was detected in all the examined cell lines, they however
differed in the staining pattern. In the cell lines that did not give metastases the cytoplasmic
staining pattern was observed (Fig. 3a), whereas in metastatic CMT-W1 and CMT-W2 cell
lines the p-STAT3 expression was detected in cytoplasm and nuclei (Fig. 3b). Significantly
higher expression (p < 0.001) of this antigen was detected in the metastatic cell lines than in
the non-metastatic ones (142.2 2.43 and 110.9 2.73, respectively) (Fig. 3a, b, c). The
Table 1 The number of Gr1-positive cells and p-STAT3 expression in canine mammary non-metastatic
adenomas and adenocarcinomas of the 1st, 2nd and 3rd grade of malignancy and in the group of tumors that
gave local or distal metastases. Results are presented as meansSD
Fig. 2 a Pictures of p-STAT3 in canine mammary adenomas, carcinomas of the 1st, 2nd, 3rd grade of
malignancy and tumors that gave local/distal metastases (n =50) obtained using Olympus BX60 microscope
(at the magnification of 200x). The p-STAT3 is reflected as brown color. b The graph of mean optical density
of p-STAT3 (and SD) in canine mammary non-metastatic adenomas, carcinomas and metastatic carcinomas.
Ten to 20 pictures in each slide were analyzed (three consecutive sections). The colorimetric intensity of the
IHC-stained antigen spots was counted by a computer-assisted image analyzer (Olympus Microimage
Image Analysis, software version 4.0 for Windows, USA) and the antigen spot color intensity is expressed as
mean pixel optical density on a 1256 scale. The statistical analysis was performed using Prism version 5.00
software (GraphPad Software, USA). The ANOVA and Tukey HSD (Honestly Significant Difference)
posthoc tests were applied to analyze the optical density in all the examined tumor groups. p< 0.001 was regarded
as highly significant and marked as ***
Fig.3 a Representative picture of p-STAT3 in non-metastatic canine mammary cancer cell line (CMT-U309)
obtained using Olympus BX60 microscope (at the magnification of 200x). The p-STAT3 is reflected as brown
color. b Representative picture of p-STAT3 in canine mammary cell line (CMT-W1) with high metastatic
potential obtained using Olympus BX60 microscope (at the magnification of 200x). The p-STAT3 is reflected
as brown color. c The graph of mean optical density of p-STAT3 (and SD) in canine mammary non-metastatic
(CMT-U27, CMT-U309, P114 and PL20) and metastatic (CMT-W1 and CMT-W2) cancer cell lines. Ten
pictures in each slide were analyzed. The colorimetric intensity of the IHC-stained antigen spots was counted
by a computer-assisted image analyzer (Olympus Microimage Image Analysis, software version 4.0 for
Windows, USA) and the antigen spot color intensity is expressed as mean pixel optical density on a 1256 scale.
The statistical analysis was performed using Prism version 5.00 software (GraphPad Software, USA). The
unpaired t-test was applied to analyze the optical density in non-metastatic versus metastatic cell lines. p<0.001
was regarded as highly significant and marked as ***
p-STAT3 expression intensity was similar in all the non-metastatic cell lines, without any
significant differences between them (ranging from 107.6 4.39 to 117.0 3.77). The
values of optical density related to the p-STAT3 expression in the examined cell lines were
very similar to these reported on tumors.
Gene expression analysis
The microarray experiment revealed a group of significantly up-regulated genes (FDR < 5%,
fold change >1.3) in each of the cell line (Krl et al. 2010b).
For the purposes of this manuscript we run a supervised analysis of the unpublished
results of genes up-regulated in CMT-W1 and CMT-W2 metastatic cell lines to find genes
involved in angiogenesis and interactions with hematopoietic cells.
The microarray analysis revealed the significant up-regulation of 73 and 102 genes in
CMT-W1 and CMT-W2 cell lines, respectively (Krl et al. 2010b). The PANTHER
ontological analysis revealed significant up-regulation of 9 and 7 genes in CMT-W1 and
CMT-W2 cell lines, respectively, involved in angiogenesis and interactions with
hematopoietic cells (Tables 2 and 3). The PANTHER statistical pathway analysis revealed
up-regulation of significantly higher number (p < 0.05) of genes involved in angiogenesis,
cytokine/chemokine mediated signaling pathway, and interferon-gamma signaling pathway
(Table 4) in metastatic cell lines than in the other examined cell lines.
In both metastatic cell lines the up-regulation of semaphorin 3B (SEMA3B), which is
directly involved in angiogenesis, was observed. The neuropilin receptor 1 up-regulation in
CMT-W1, and neuropilin receptor 2 up-regulation in CMT-W2 cell line were also observed.
Relying on current and prior studies we suppose that, the activity of these genes may
correlate with the STAT3 regarding angiogenesis pathway.
As of now there are many papers available on canine mammary tumors examined at
different levels: from genes to proteins (Rao et al. 2009; Badowska-Kozakiewicz and
Malicka 2010; Krl et al. 2010a and b; Pawowski et al. 2009, 2011a and b) but there are
only few on the canine tumor microenvironment. The available literature focuses on the
angiogenesis in canine mammary tumors (Restucci et al. 2002; Jakab et al. 2008; Qui et al.
2008) or the role of MMPs in tumorigenesis (Hirayama et al. 2002; Papparella et al. 2002;
Kawai et al. 2006). Our previous study (Krl et al. 2011) was the first to give an account of
the relation between tumor-associated macrophages (TAMs) density and tumor ability to
metastasize. Although the role of macrophages, lymphocytes or other stromal cells in tumor
progression is commonly known, the possible impact of bone marrow derived suppressor
cells on tumor progression and metastasis is still unclear (Voronov et al. 2003; Yang et al.
2004 and 2008; Jodele et al. 2005; Bunt et al. 2007, Allavena et al. 2008) just as
mechanisms of increasing the rate of tumor angiogenesis.
Thus, we analyzed the number of myeloid suppressor cells (Gr1-positive) in 50 canine
mammary tumors. The myeloid differentiation antigen Gr1 is thought to be expressed on
the myeloid lineage precursor cells which was also confirmed by our results. This surface
marker used for the identification of myeloid suppressor cells is common for several
populations of myeloid precursors. There is no agreement between various investigators as
to which proteins should be treated as markers of different subsets of myeloid derived
suppressor cells in human and mice (Bronte et al. 2000; Almand et al. 2001; Mazzoni et al.
F L R R E la
G C L L L ir C R
SEMA3B sema domain,
(Ig), short basic domain,
secreted, (semaphorin) 3B
Other receptor;Other Other receptor mediated signaling 1,42
signaling molecule pathway;Neurogenesis;
Cytokine and chemokine
mediated signaling pathway;
Cell adhesion; Neurogenesis
signaling; Extracellular matrix
Biological process unclassified
Table 3 The list of up-regulated genes () involved in angiogenesis and interactions with hematopoietic cells
in CMT-W2 cell line compared with the genes pool. To classify the molecular function and biological process
in which the genes are involved Panther Database (www.pantherdb.org) was used. Data was analyzed using
SAM (Stanford University, USA) software, FDR <5%; Fold change >1,3
2002, Huang et al. 2006), thus it is probably not feasible to clearly determine their
subgroups using immunohistochemical staining of tumor tissues. But all the authors have
agreed that their immune suppressive functions (although at different level between the cell
subsets) can be identified by the level of Gr1 expression (Kusmartsev et al. 2000; Dietlin et
al. 2007; Youn and Gabrilovich 2010).
Thus, we examined the expression of the whole Gr1-positive myeloid suppressor cells
set in the canine mammary cancer tissues. We classified Gr1-positive cells as a wider group
of various myeloid precursor cells that shows the suppressive features. So far it was mainly
assessed in the experimental mice models as much as in various in vitro conditions but not
in domestic animals. This study showed that the Gr1-positive cells in canine mammary
tumors formed the morphologically heterogenous population. We showed that the number
of Gr1-positive cells is noticeably higher (p < 0.001) in the metastatic tumors, whereas no
significant differences were observed between neither non-metastatic adenomas and
carcinomas nor non-metastatic malignant tumors of the various grade of malignancy
(Fig. 1, Table 1).
Table 4 The list of over-represented cellular pathways of PANTHER classification categories in CMT-W1
and CMT-W2 cell lines in comparison to the other cells lines used in this study. The binomial test for each
PANTHER pathway was used (p<0.05)
Metastatic CMT-W1 and CMT-W2 cell lines
Non-metastatic cell lines used in this study
Based on Kujawski et al. (2010) results suggesting the possible crosslink between myeloid
suppressor cells and STAT3 and its contribution to angiogenesis we examined the p-STAT3
expression in canine mammary tumor tissues. We detected significantly higher (p<0.001)
expression of p-STAT3 in metastatic tumors than in non-metastatic tumors (Fig. 2, Table 1).
The p-STAT3 expression was observed not only in cancer cells, but also in the stromal
hematopoietic cells. Similar results were obtained by Kujawski et al. (2010) using mouse
systems and human breast cancer tissues. We also showed that the number of myeloid
precursor cells and p-STAT3 expression in metastatic tumors correlate highly (r2 = 0.73;
p < 0.05), whereas in non-metastatic carcinomas or adenomas the significant correlation
between Gr1-positive cells and p-STAT3 expression has not been observed. It means that the
level of p-STAT3 expression in metastatic tumors was not risen only because of the higher
number of myeloid precursor cells but also due to its higher expression in the cancer cells.
We also observed a significantly higher (p < 0.001) expression of p-STAT3 in metastatic
canine mammary cancer cell lines than in non-metastatic cell lines (Fig. 3).
The genesis of metastasis has not been fully accounted for. However the extensive
studies in this field provide ever more information about associated mechanisms. One of the
factors that contributes to tumor metastasis is the myeloid precursor cells infiltration. The
STAT3 is a negative regulator of hematopoiesis. Its ablation in hematopoietic cells results in
e.g. increased numbers of matured granulocytes (Lee et al. 2002; Kortylewski et al. 2005;
Panopoulous et al. 2006; Kujawski et al. 2010). Thus, its higher expression may be one of
the factors that partially block the bone-marrow-derived cells differentiation in patients with
cancer that leads to a spread myeloid precursor cells in cancer tissue (Schmid and Varner
2010). Since the p-STAT3 expression has also been observed in hematopoietic cells within
the tumor we assume the autocrine signaling may also occur. These tumor-infiltrating
myeloid precursors enhance angiogenesis by secretion of various angiogenic factors
(Voronov et al. 2003; Bunt et al. 2007; Melani et al. 2007; Allavena et al. 2008; Yang et al.
2008; Kujawski et al. 2010). On the other hand, STAT3 contributes to angiogenesis not only
by enhancing the Gr1-positive cells accumulation per se but also by increasing the
angiogenic factors secretion by Gr1-positive cells and tumor cells as well (Kujawski et al.
2010), being the most important angiogenesis inducers VEGF (Zhao et al. 2011). Some studies
give evidence of STAT3 and VEGF correlation in angiogenesis. Suppression of STAT3
reduces the VEGF expression in various cancers, identifying STAT3 as a target of
antiangiogenic therapy (Niu et al. 2002; Zhao et al. 2011). Bartoli et al. (2003) has showed that
VEGF promotes rapid phosphorylation of STAT3 which is then paired with VEGF receptor
(VEGFR). The STAT3/VEGFR complex stimulates the VEGF autocrine expression (Fig. 4).
Fig. 4 The scheme of possible interactions between the up-regulated genes in canine mammary metastatic cell
lines, STAT3 and hematopoietic cells. The names of the identified in microarray study genes and STAT3 were
bolded and written in italic. The arrows indicate the activation. The bar-headed line indicates inhibition. The
explanation of abbreviations: Gr1+ cells myeloid precursor cells that express Gr1 antigen; MQ macrophages;
NRP neuropilin receptor; p38MAPK p38 mitogen activated protein kinase; p53 p53 gene; SEMA3B
semaphorin 3B; STAT3 Signal transducer and activator of transcription 3; VEGF vascular endothelial growth
factor; VEGFR vascular endothelial growth factor receptor. The graph was made in Corel Photo Paint 3.0 (by
Corel Draw, USA)
Two of the up-regulated genes in metastatic cell lines: NRP and SEMA3B are also
directly involved in this process (Tables 2 and 3). SEMA3B binds to the NRP forming a
complex with VEGFR (as neurophilins are co-receptors for VEGF) and having positive
effect on VEGF/NRP-driven vascular development (Gu et al. 2003). NRP may also increase
VEGF affinity toward VEGFR (Potrion et al. 2003). The SEMA3B increases p38 mitogen
activated protein kinase (p38 MAPK) activity in the cell, which induces VEGF expression
via STAT3 (Wang et al. 2007). It also increases cytokines/chemokines secretion from tumor
cells (via STAT3) what leads to macrophage infiltration (Potrion et al. 2003) and
angiogenesis promotion (Fig. 4). Moreover, Jung et al. (2001) showed that cytokines may
increase VEGF synthesis by the same mechanism as SEMA3B (via p38 MAPK and STAT3)
(Fig. 4). SEMA3B is activated by the p53 protein during DNA damage or as a stress
response (e.g. hypoxia, the main factor leading to increased angiogenesis).
Our microarray study also showed a few chemokines, interferon and interleukin
receptors expression (chemokine C-C motif ligand 13 and 15, interleukin 1 receptor 2,
interleukin 1 receptor accessory protein-like 2, interferon-induced protein 44-like) in
metastatic cell lines in comparison to the non-metastatic cells (Tables 3 and 4). Both
interferon and interleukins also activate STAT3 (Yang et al. 1998; Lee et al. 2006).
The other mechanism by which myeloid suppressor cells contribute to cancer
progression is the immune system depletion via regulation of T cell activity (Kusmartsev
et al. 2000; Dietlin et al. 2007). Estrela-Lima et al. (2010) showed that in the canine
mammary metastatic tumors the infiltration of a whole population of T lymphocytes is
significantly lower than in non-metastatic tumors. Thus, it is possible that myeloid
suppressor cells accumulation in metastatic tumors may decrease the T lymphocytes
number and contribute to the tumor progression and metastasis by those means (Fig. 4). It is
possible that myeloid precursor cells can also activate other critical oncogenic pathways for
metastatic process henceforth further studies in this field are crucial.
Relying on presented results and literature available we conclude, that STAT3 seems to
play a crucial role in the angiogenic pathway (Fig. 4). Our results regarding p-STAT3
expression in canine mammary cancer cell lines cultured in vitro and depletion of all the
hematopoietic/stromal cell signals may indicate, that the individual predisposition of cancer
cell to STAT3 activation could begin the further cascade which leads to angiogenesis and
metastasis: block of myeloid-derived cells differentiation, their tumor infiltration,
lymphocyte T inhibition, angiogenesis promotion via VEGF, SEMA3B and cytokines
production what also leads to macrophage accumulation and metastasis (Fig. 4). This
immature and as of yet rather preliminary working hypothesis needs further investigation.
Acknowledgments This work was supported by grants no N N308012939 and N N308230536 from
Ministry of Sciences and Higher Education. This work was performed by financial support of the Foundation
for Polish Science (Start stipendium and Parent-Bridge program). The authors would like to thank Dr. Eva
Hellmen, Dr. Gerard Rutteman, Prof. Dr. Maciej Ugorski and Dr. Joanna Polaska for their kind donation of
the examined cell lines. The experiments comply with the current laws of Poland. The authors declare that
they have no conflict of interest.
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