Global gene expression profiles of canine macrophages and canine mammary cancer cells grown as a co-culture in vitro
© Król et al; licensee BioMed Central Ltd. 2012
Received: 28 July 2011
Accepted: 21 February 2012
Published: 21 February 2012
Solid tumours comprise various cells, including cancer cells, resident stromal cells, migratory haemopoietic cells and other. These cells regulate tumour growth and metastasis. Macrophages constitute probably the most important element of all interactions within the tumour microenvironment. However, the molecular mechanism, that guides tumour environment, still remains unknown. Exploring the underlying molecular mechanisms that orchestrate these phenomena has been the aim of our study.
A co-culture of canine mammary cancer cells and macrophages was established and maintained for 72 hrs. Having sorted the cells, gene expression in cancer cells and macrophages, using DNA microarrays, was examined. The results were confirmed using real-time qPCR and confocal microscopy. Moreover, their ability for migration and invasion has been assessed.
Microarray analysis showed that the up-regulated genes in the cancer cell lines are involved in 15 highly over-manifested pathways. The pathways that drew our diligent attention included: the inflammation pathway mediated by chemokine and cytokine, the Toll receptor signalling pathway and the B cell activation. The up-regulated genes in the macrophages were involved in only 18 significantly over-manifested pathways: the angiogenesis, the p53 pathway feedback loops2 and the Wnt signalling pathway. The microarray analysis revealed that co-culturing of cancer cells with macrophages initiated the myeloid-specific antigen expression in cancer cells, as well as cytokine/chemokine genes expression. This finding was confirmed at mRNA and protein level. Moreover, we showed that macrophages increase cancer migration and invasion.
The presence of macrophages in the cancer environment induces acquisition of the macrophage phenotype (specific antigens and chemokines/cytokines expression) in cancer cells. We presumed that cancer cells also acquire other myeloid features, such as: capabilities of cell rolling, spreading, migration and matrix invasion (what has also been confirmed by our results). It may, perhaps, be the result of myeloid-cancer cell hybrid formation, or cancer cells mimicking macrophages phenotype, owing to various proteins secreted by macrophages.
Solid tumours comprise cancer cells, resident stromal cells, and migratory haemopoietic cells. Intricate interactions between the cell types regulate tumour growth, progression, metastasis, and angiogenesis. Macrophages are an important element of these microenvironment interactions . They may represent either M1 or M2 phenotype. The classical activation by microbial products is that of the M1 phenotype (also thought to have anti-tumour properties), whereas alternative activation (caused by cancer cells) drives macrophages conversion toward the M2 phenotype. Cancer cells are known to release various chemoattractants which recruit macrophages to colonize the tumour site . On the other hand, counter-activated tumour-associated macrophages (TAMs) produce chemokines, cytokines, growth and angiogenic factors [1, 3], thus they actively contribute to tumour progression and their transition to malignancy. Exploring the underlying molecular mechanisms of this phenomenon seems to be utterly important. Therefore, to view and explain the molecular interactions between the cancer cells and TAMs, we established an in vitro co-culture and conducted a global gene expression analysis using DNA microarrays of macrophages and cancer cells. Neither there is an abundance of microarray data on the global gene expression in TAMs [2, 4, 5] available, nor there is much information on the changes of cancer cells and their gene expression whilst cultured with macrophages.
The findings confirm that cancer cells under co-culture conditions acquired the macrophage-specific antigen expression. It could as well be indicative of these cells also having other phenotypic characteristics of macrophages, such as: capabilities of cell rolling, spreading, diapedesis, or migration, that allow the metastasis process. Our in vitro studies confirmed that macrophages enhance tumour migration and invasion.
The cell lines used for the study have previously been used in other published research [6–9]. Two canine mammary adenocarcinoma cell lines (CMT-W1, CMT-W2), anaplastic cancer cell line (P114), simple carcinoma cell line (CMT-U27) and spindle-cell mammary tumor cell line (CMT-U309) were examined. CMT-W1 and CMT-W2 cell lines were kindly donated by Prof. Dr. Maciej Ugorski and Dr. Joanna Polanska from Wroclaw University (Poland), CMT-U27 cell line was kindly donated by Dr. Eva Hellmen from Swedish Agricultural University (Sweden) and P114 cell line was kindly donated by Dr. Gerard Rutteman from Utrecht University (The Netherlands).
Cells were cultured under optimal conditions: a medium RPMI-1640 enriched with 10% (v/v) heat-inactivated fetal bovine serum (FBS), penicillin-streptomycin (50 iU mL-1), and fungizone (2.5 mg mL-1) (reagents obtained from Sigma Aldrich, USA), in an atmosphere of 5% CO2 and 95% humidified air at 37°C, and routinely subcultured every other day.
Canine blood mononuclear cell separation
The anticoagulated whole blood from healthy dogs (patients of the Department of Small Animal Diseases with Clinic, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Poland) was collected for routine diagnostic purposes (in that case the ethical committee permission is not required). The remaining volume of the blood samples was taken to our analyses (with the written permission of the dog's owners) and immediately subjected to a mononuclear cell separation using Accuspin System-Histopaque 1077 (Sigma Aldrich, USA) according to the manufacturer's protocol. The blood specimen was placed on a porous high-density polyethylene barrier, separating lower chamber containing the Histopaque-1077 solution, in a sterile centrifuge tube. The tube was centrifuged at 800 × g for 30 min at room temperature. On centrifugation, erythrocytes and granulocytes descend through the frit to pellet below the Histopaque-1077. Lymphocytes and monocytes remained above the frit on the plasma-Histopaque-1077 interface. This layer of cells was aseptically removed with a pipette and transferred to a sterile 15-ml centrifuge tube. Then, the cells were washed with PBS once and subjected to further procedures.
Monocyte sorting and culturing
The CD64-positive monocytes were then separated and grown as a co-culture with cancer cells, as well as a mono-culture. The culturing conditions were the same as those for cancer cells. According to the subject data, culturing of monocytes for 72 hrs is sufficient for their differentiation towards macrophages [10, 11].
In this model, cancer cells (CMT-W1, CMT-W2, CMT-U27, CMT-U309, P114) were grown on separated flasks, and sorted monocytes were then layered on the top of each cell line. An Orange CellTracker fluorescent dye CMTMR (Invitrogen, USA) was used to stain the cancer cells population before the sorted monocyte population was added. Staining was accomplished by incubation in serum/antibiotics-free RPMI medium containing 5 μM CMTMR (10 mM stock in DMSO; Sigma Aldrich, USA) for 45 min at 37°C. Subsequently, the medium was aspirated, and the cancer cells were washed twice with PBS and incubated with complete RPMI for 1 hr. Sorted monocytes were placed on the CMTMR-stained cancer cells.
RNA isolation, validation, amplification, reverse transcription, labeling and hybridization
The sorted macrophages and cancer cells grown as the co-culture were centrifuged (2500 rpm for 5 min) in separated tubes, whereas cancer cells and macrophages grown as mono-cultures were washed with PBS and next scraped and centrifuged (2500 rpm for 5 min) in separated tubes. The total RNA from the samples was isolated using a Total RNA kit (A&A Biotechnology, Poland) according to the manufacturer's protocol. Isolated RNA samples were dissolved in RNase-free water. The quantity of RNA was measured using NanoDrop (NanoDrop Technologies, USA). The samples with adequate amounts of RNA were treated with DNaseI to eliminate a possibility of DNA contamination. The samples were subsequently purified using RNeasy MiniElute Cleanup Kit (Qiagen, Germany). Finally RNA samples were analyzed using BioAnalyzer (Agilent, USA) to measure the final RNA quality and integrity.
The Quick Amp Labeling Kit (Agilent, USA) was used to amplify and label target RNA to generate complementary RNA (cRNA) for oligo microarrays used in gene expression profiling and other downstream analyses. The gene expression of each cancer cell line, grown under co-culture conditions with macrophages, was compared against the gene expression of the same cancer cell line grown as a mono-culture (gene expression in CMT-U27 cell line grown as a co-culture with macrophages was compared to gene expression in CMT-U27 cell line grown as a mono-culture; gene expression in CMT-U309 cell line grown as a co-culture with macrophages was compared to gene expression in CMT-U309 cell line grown as a mono-culture; gene expression in P114 cell line grown as a co-culture with macrophages was compared to gene expression in P114 cell line grown as a mono-culture; gene expression in CMT-W1 cell line grown as a co-culture with macrophages was compared to gene expression in CMT-W1 cell line grown as a mono-culture; gene expression in CMT-W2 cell line grown as a co-culture with macrophages was compared to gene expression in CMT-W2 cell line grown as a mono-culture). The gene expression of macrophages grown as a co-culture with cancer cell lines was compared against the gene expression of macrophages grown as the mono-culture. Each sample was examined in a dye-swap to eliminate the effect of label factor. Thus, each biological condition was labelled once by Cy3 and once by Cy5. Taking the average of two labelled arrays, the dye effect on any particular gene was cancelled. The hybridization was performed with canine-specific AMADID Release GE 4 × 44 K microarrays (Agilent, USA) using Gene Expression Hybridization Kit (Agilent, USA) according to the manufacturer's protocol.
Signal detection, quantification and analysis
Acquisition and analysis of hybridization intensities were performed using DNA microarray scanner (Agilent, USA). Then, the results were extracted using Agilent's Feature Extraction Software with normalization and robust statistical analyses. Results were analyzed for statistical purposes using Feature Extraction and Gene Spring software (Agilent, USA). The unpaired t-test with Benjamin-Hochberg FDR < 5% (false discovery rate) correction was applied (with p value cut-off < 0.01). For further analysis we hierarchically clustered the genes and chose only those with values within upper and lower cut-off (100.00 and 20.00, respectively) in each of the slide. We analyzed only genes that were regulated in all the examined samples within the group (that is: in all the cell lines grown with macrophages and in all the macrophages samples grown with cell lines) whose expression changed at least 3-fold in each of examined slide. In this experimental model we examined each of the sample in duplicate (dye-swap), whereas significant genes were chosen from five biological repetitions (five various cell lines). The area of the analyses covered in this publication has been deposited in NCBI's Gene Expression Omnibus and is accessible via GEO Series accession number GSE29339.
Gene function was identified using the PANTHER pathway analysis software  and Pathway Studio software (Agilent, USA). PANTHER on-line platform allowed for wide analysis of the Canis familiaris regulated genes and also for statistical analysis of number of regulated genes involved in specific pathways or biological functions compared to the normal healthy cell of this specie.
Primers used for real-time qPCR
Optimum annealing temp. (°C)
Optimum annealing time (sec)
The cells were cultured on Lab-Tek (Nunc Inc., USA) 4-chamber culture slides and were fixed with ethanol after 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 rabbit polyclonal MCSF Receptor (other designations: MCSF-R or CSF-1R) obtained from Abcam (United Kingdom) primary antibodies were used (diluted in 1% bovine serum). According to the manufacturer's instructions the slides were incubated with antibodies at +4°C overnight. For the staining the EnVision kit (Labelled Polymers consist of secondary anti-rabbit antibodies conjugated with the HRP enzyme complex obtained from Dako) was used. To develop the coloured product, the 3,3'-Diaminobenzidine (DAB) substrate was used (Dako, Denmark). Finally, the haematoxylin was used for nuclei counterstaining.
Each slide was photographed 10 times using Olympus microscopy BX60. The colorimetric intensity of the CSF-1R expression reflected as IHC-stained antigen spots (brown colour) were counted by a computer-assisted image analyzer (Olympus Microimage™ Image Analysis, software version 4.0 for Windows, USA). The antigen spot colour intensity is expressed as a mean pixel optical density on a 1-256 scale.
The co-culture of cancer cells with macrophages was conducted as described above. The cancer cells grown as mono-culture and as a co-cultures were stained using Orange CellTracker fluorescent dye CMTMR, as described above. Cancer cells and macrophages grown as mono-cultures, as well as the co-culture were fixed with 70% ethanol (10 min) and washed three times in PBS. Cells were permeabilized with 0.5% Triton X-100/PBS (10 min), washed with PBS twice and incubated for 1 hr at room temperature with: mouse monoclonal anti-CD64 FITC-conjugated (Becton Dickinson, USA) antibodies (20 ul per 106 cells) and mouse monoclonal anti-CD14 FITC-conjugated (LifeSpan Biosciences, USA) antibodies (10 ul per 106 cells) according to the manufacturer's instructions. The cells were then washed three times with PBS and the coverslips were mounted on microscope slides using ICN mounting medium.
The cell imaging was performed by confocal laser scanning microscope FV-500 system (Olympus Optical Co, Germany). The combination of excitation/emission were: Argon 488 nm laser with 505-525 nm filter for FITC and HeNe 543 nm laser with 610 nm filter for CMTMR staining. The pictures were gathered separately for each fluorescence channel. The cells were examined using the Fluoview program (Olympus Optical Co., Germany).
To assess the ability to migration of cancer cells grown as a co-culture with macrophages, we applied a wound-healing test. The cancer cells (grown as the co-culture with macrophages and normal control cells) were separately seeded in multi-well plates and then (after 24 hrs when the cells were confluent), using a pipette tip (100 ul) a straight scratch has been made, simulating a wound. The images were captured at the beginning and at regular intervals (after 2, 4 and 6 hrs) during cell migration to close the wound. The images then were compared to quantify migration rate of the cells. This method is particularly suitable for studies of cell-cell interaction on cell migration . The pictures has been analyzed using a computer-assisted image analyzer (Olympus Microimage™ Image Analysis, software version 4.0 for Windows, USA).
BD BioCoat Matrigel™ invasion chambers (BD Biosciences, USA) pre-coated with BD Matrigel matrix were used according to the manufacturer's protocol. The assay insert plates were prepared by rehydrating the BD Matrigel Matrix coating with phosphate buffered saline for two hrs at 37°C. The rehydration solution was carefully removed, 2.5 × 105 of control cancer cells or cancer cells grown as co-culture with macrophages (at the ratio of 10:1) was added to each apical chamber and 0.75 ml RPMI-1640 containing chemoattractant (10% FBS) was added to the basal chamber. Uncoated insert plates, included as invasion controls, were used without rehydration. Assay plates were incubated for 22 hrs at standard culturing conditions. 2.5 μg/ml Calcein AM were added to 20 μl DMSO and then, 10 μl was transferred to 12 ml Hanks Buffered Saline Dispense. 0.5 ml Calcein solution was then transferred into each well of 24-well plate. The medium from insert was removed and multiwell inserts were transferred to the plate containing 0.5 ml/well calcein. Plates were incubated an hour at standard culture conditions. The fluorescence of invaded cells was measured with excitation wave length 485 nm and emission wave length Em 530 nm using Tecan Infinite 200 Reader (Tecan, Switzerland).
Cancer cells were treated with trypsin and resuspended in culture medium. 35 mm culture plates (Corning Inc., USA) were coated with 100 μl of growth factor reduced Matrigel (BD Biosciences, USA) and left to solidify for 30 min. at 37°C. The cells were then plated at a concentration of 104 cells/ml. The growth of cells on Matrigel was observed every day under phase-contrast microscope.
The analysis for statistical purposes was conducted using Prism version 5.00 software (GraphPad Software, USA). The two-way ANOVA, ANOVA + Tukey HSD (Honestly Significant Difference) post-hoc test and t-test were applied. The p-value < 0.05 was regarded as significant whereas p-value < 0.01 and p-value < 0.001 as highly significant.
Sorting of the co-cultured cells
Flow cytometry had easily distinguished the CMTMR-stained cells from the unstained macrophages (Figure 1D, E) and allowed a further proper sorting of each population (Figure 1F). The co-culture was maintained for at least 72 h. The differential staining prolonged for such period of time (Figure 1E, F and see also confocal microscopy results) showed no detrimental effect on proliferation and plating efficiency. The fluorescence intensity of the stained cancer cells after the 3-days co-culture with macrophages was the same as that of the control cancer cells grown as a mono-culture (Figure 1E, F; see also confocal microscopy results). Similar culture conditions had previously been described .
Our FACS sorting isolated a 97-99% pure population on postsort, with a positive result comparable to other reported data available . Sorting purity was also assessed using fluorescence microscopy (showing no stained cells in macrophages tube and no unstained cells in cancers tube).
Global gene expression analysis
Up/down-regulated genes in canine mammary cancer cell lines grown as co-culture with macrophages
V-type proton ATPase subunit e 1
C5a anaphylatoxin chemotactic receptor
C-C motif chemokine 2
C-C motif chemokine 3
C-C motif chemokine 4
C-C motif chemokine 5
C-C motif chemokine 8
C-C chemokine receptor type 5
Tumor necrosis factor receptor superfamily member 5
Cytochrome c oxidase copper chaperone
Granulocyte-macrophage colony-stimulating factor
Granulocyte colony-stimulating factor
Colony stimulating factor receptor 1
Dipeptidyl-peptidase 1 light chain
C-X-C chemokine receptor type 7
Ferritin heavy chain
Ferritin light chain
Interferon-induced GTP-binding protein Mx1
Epididymal secretory protein E1
Platelet-activating factor acetylhydrolase
Pulmonary surfactant-associated protein A
Proteasome subunit beta type-8
Proteasome subunit beta type
Prostaglandin E synthase
Signaling lymphocytic activation molecule
Toll-like receptor 2
Retinoic acid receptor RXR-beta
Homeobox protein MSX-2
Up/down-regulated genes in macrophages grown as a co-culture with canine mammary cancer cells
Abnormal spindle-like microcephaly-associated protein homolog
Collagen alpha-1(I) chain
Granulocyte-macrophage colony-stimulating factor
C-X-C motif chemokine 10
Interleukin-12 subunit alpha
Interleukin-13 receptor alpha-2 chain
Myc proto-oncogene protein
6-phosphofructokinase, muscle type
Protein-tyrosine phosphatase-like member A
DNA repair protein RAD51 homolog 1
60S ribosomal protein L23
40S ribosomal protein S17
40S ribosomal protein S18
Uveal autoantigen with coiled-coil domains and ankyrin repeats
wingless-type MMTV integration site family member 5b
wingless-type MMTV integration site family member 7a
wingless-type MMTV integration site family member 7b
Proto-oncogene tyrosine-protein kinase Yes
C5a anaphylatoxin chemotactic receptor
C-C motif chemokine 13
C-C motif chemokine 2
C-C chemokine receptor type 5
C-X-C chemokine receptor type 7
Endothelin B receptor
Inward rectifier potassium channel 2
Natural resistance-associated macrophage protein 1
Thymic stromal cotransporter homolog
Sodium/calcium exchanger 1
Toll-like receptor 2
Toll-like receptor 4
Over- representation of pathways in cells grown under co-culture conditions
The PANTHER binomial statistics tool allowed us to statistically determine over-manifestation of PANTHER pathways classification categories.
Genes involved in over-manfested cellular pathways in canine mammary cancer cells grown as a co-culture with macrophages
number of genes
Inflammation mediated by chemokine and cytokine signaling pathway
Toll receptor signaling pathway
B cell activation
T cell activation
Apoptosis signaling pathway
Adenine and hypoxanthine salvage pathway
Interleukin signaling pathway
Plasminogen activating cascade
Salvage pyrimidine deoxyribonucleotides
PDGF signaling pathway
Other important over-manifested pathways are apoptosis signaling pathway (6 genes, p = 3.86E-04), interleukin signaling pathway (6 genes, p = 8.86E-03) and PDGF signaling pathway (5 genes, p = 3.99E-02). The down-regulated genes in the cancer cells were not involved in any significantly over- manifested pathways.
Genes involved in over-manifested cellular pathways in macrophages grown as a co-culture with canine mammary cancer cells
number of genes
p53 pathway feedback loops 2
Wnt signaling pathway
Alzheimer disease-presenilin pathway
Cadherin signaling pathway
Plasminogen activating cascade
TGF-beta signaling pathway
Inflammation mediated by chemokine and cytokine signaling pathway
Alzheimer disease-amyloid secretase pathway
Alpha adrenergic receptor signaling pathway
Metabotropic glutamate receptor group I pathway
FGF signaling pathway
Succinate to proprionate conversion
Endothelin signaling pathway
Muscarinic acetylcholine receptor 1 and 3 signaling pathway
Over-representation of genes involved in particular biological processes in cells grown under co-culture conditions
The PANTHER binomial statistics tool allowed us to statistically determine over-manifestation of PANTHER biological processes classification categories. The most important biological processes in cancer cells grown as a co-culture were: macrophage activation (21 genes, p = 6.24E-11), cell motion (28 genes, p = 7.49E-06), mammary gland development (5 genes, p = 2.17E-03), cell-cell adhesion (19 genes, p = 5.06E-03), angiogenesis (11 genes, p = 9.51E-03).
The most important biological processes in macrophages grown as a co-culture were cell-matrix adhesion (10 genes, p = 3.92E-03) and cell-cell adhesion (28 genes, p = 5.79E-03).
The results were confirmed at mRNA level using real-time qPCR analysis
Fold change of genes randomly selected for confirmation of microarray results
MQ co-culture with CMT-U27 v.s. monoculture
MQ co-culture with CMT-U309 v.s. monoculture
MQ co-culture with P114 v.s. monoculture
MQ co-culture with CMT-W1 v.s. monoculture
MQ co-culture with CMT-W2 v.s. monoculture
CMT-U27 co-culture with MQ v.s. monoculture
CMT-U309 co-culture with MQ v.s. monoculture
P114 co-culture with MQ v.s. monoculture
CMT-W1 co-culture with MQ v.s. monoculture
CMT-W2 co-culture with MQ v.s. monoculture
Confocal microscopy and IHC analysis revealed myeloid-lineages markers expression in cancer cells following the co-culturing with macrophages
CSF1R expression at protein level in canine mammary cancer cell lines
Mean Optical Density (Arbitrary Units)
Invasion assay and 3D culture
To confirm the ability of these cell lines to matrix invasion, we have assessed their growth characteristics on Matrigel matrix (Figure 8C). After 22 hrs of culturing (similarly as in the invasion assay) on Matrigel CMT-U27, CMT-U309 and P114 cell lines formed colonies, whereas CMT-W1 and CMT-W2 cell lines formed branching structures (Figure 8C) what indicated their invasive phenotype. The culture was maintained for 1 week, showing that after 5 days also P114 cell line formed slight branches.
It has been evident over the last few years that macrophages play an important role (via various factors) in tumour cell invasion of the normal surrounding tissues, cancer proliferation and metastasis to local and distant sites . All the interactions within the tumour are intricately complex and a better understanding of them would require some further exploration of the underlying molecular processes.
The present microarray analysis of five various canine mammary cancer cell lines and canine macrophages grown together, revealed significant changes in genes expression in comparison to the same cells grown as mono-cultures.
Under co-culture conditions the cancer cells express the macrophages-specific antigens, e.g. CD14, CD64, CD163, CSF1R (Table 3, Figures 5, 6 and 9). According to subject literature, CD163 is expressed not only by normal monocytes/macrophages but also by neoplasms [26, 27]. CD163-positive cancers had more severe histological aberrations due to genomic instability. Thus, it is hypothesized  that tumour cells express atypical genes (specific for macrophages) due to genomic instability. This may be caused by the presence of various proteins secreted by macrophages and changes induced by them on the microenvironment. Another theory explaining why cancer cells may exhibit myeloid cells-specific antigens is that the cells fuse, forming hybrids that adopt phenotypic features of both parental cells . Cancer cells may fuse spontaneously with several types of somatic cells [29–32]. Some authors suggest that the result of cancer-myeloid cell fusion is the hybrid with a metastatic phenotype [33–39]. The tumour cells that express myeloid antigens may also exhibit other phenotypic characteristics of macrophages, such as capabilities of cell rolling, spreading, dissociation, diapedesis, migration and matrix invasion. The metastatic cancer cells have all these capacities because the process of metastasis requires a coordinated steps promoting angiogenesis, controlling adhesion, proteolysis and motility. Our confocal imaging and IHC examination has proven that under co-culture conditions expression of macrophages markers (CD14, CD64, CSF1R) in almost all of the cancer cells was initiated. Moreover, migration and invasion assays showed that the presence of macrophages in cancer microenvironment triggers migration in all of the cancer cell lines. The co-culturing of macrophages increased invasion in the cell lines that show high invasive abilities in control conditions. The ability of CD163 receptor (and perhaps other macrophage-specific antigens) to trigger the production of pro-inflammatory mediators [40, 41] may be a key factor that brings changes to the cancer cell biology and stimulates it to cytokines/chemokines/growth factors production for further myeloid cell attraction.
Our microarray analysis of cancer cells grown with macrophages revealed the over-manifestation of genes activity which are involved in macrophages-cancer cells 'conversation'. We found up-regulation of the highly potent macrophage attracting factors: CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β) and CCL5 (RANTES) as well as CCR5 in cancer cells grown as co-culture with macrophages. Recent studies have demonstrated that CCL2 acts directly (via CCR5) in an autocrine manner on several human carcinomas regulating the migration and invasive properties of tumour cells [42, 43]. Furthermore, CCL3-CCR5 axis can increase the MMP-9 expression contributing to angiogenesis, ECM degradation and metastasis. Our gene expression analysis seems to support both hypotheses, as besides CCL2, CCL3 and CCR5 up-regulation, we also observed increased expression of MMP-9 in cancer cells grown under co-culture conditions with macrophages.
We found down-regulation of the pro-inflammatory CD163 in macrophages grown as co-culture with cancer cells. We also found down-regulation of several key inflammation cytokines and macrophage activators, such as: CCL2, CCL13, CCR1, and CCR5; whereas up-regulation of other inflammation cytokines: CXCL10, CSF-2. Decrease of the expression of these genes in macrophages grown with cancer cells as co-cultures may be induced by hypoxia. Down-regulation of CCL2, CCR1 and CCR5 genes in macrophages under hypoxic conditions has been demonstrated by various authors [44–46]. This phenomenon has a biological explanation, as decrease of CCL2, CCR1 and CCR5 expression inhibits chemotaxis signalling what in turn prevents TAMs from leaving hypoxic areas  and triggers their angiogenic effect (e.g. by CXCL10 chemokine). Up-regulation of HIF-1 in cancer cells may indicate hypoxic conditions in co-culture (similarly as in tumour). Hypoxia additionally regulates angiogenesis by up-regulating VEGF-C in cancer cells and it initiates their epithelial-mesenchymal transition (EMT) .
All genes given above are important to entrap macrophages in malignant tumours .
In macrophages grown as co-culture with cancer cells expression of three ligands of Wnt pathway increased significantly: Wnt5b, Wnt7a and Wnt7b. Although the Wnt activation has been described by many authors in various cancer cells [57, 58], only one study, so far , described the Wnt activation in macrophages taken from mice tumours. The authors described mechanism, by which macrophages stimulate Wnt signalling pathway in vascular endothelial cells by Wnt7b. This signalling cascade eventually results in the vascular remodelling. Thus, the new hypothesis is proposed, that the subpopulation of macrophages, modulating Wnt-signalling is located along the tumour vasculature to regulate endothelial cells proliferation and apoptosis .
This work was supported by grant no N N308012939 from the Ministry of Sciences and Higher Education. This work was performed owing to financial support of the Foundation for Polish Science (Start stipendium and Parent-Bridge program).
- Hagemann T, Wilson J, Burke F, Kulbe H, Li NF, Pluddemann A, Charles K, Gordon S, Balkwill FR: Ovarian cancer cells polarize macrophages toward a tumor-associated phenotype. J Immunol. 2006, 176: 5023-5032.View ArticlePubMedGoogle Scholar
- Pollard JW: Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer. 2004, 4: 71-78.View ArticlePubMedGoogle Scholar
- Smith HO, Anderson PS, Kuo DY, Goldberg GL, DeVictoria CL, Boocock CA, Jones JG, Runowicz CD, Stanley ER, Pollard JW: The role of colony-stimulating factor 1 and its receptor in the etiopathogenesis of endometrial adenocarcinoma. Clin Cancer Res. 1995, 1: 313-325.PubMedGoogle Scholar
- Duff MD, Mestre J, Maddali S, Yan ZP, Stapleton P, Daly JM: Analysis of gene expression in the tumor-associated macrophage. J Surg Res. 2007, 142 (1): 119-128. 10.1016/j.jss.2006.12.542.View ArticlePubMedGoogle Scholar
- Oljavo LS, Whittaker CA, Condeelis JS, Pollard JW: Gene expression analysis of macrophages that facilitate tumor invasion supports a role for Wnt-signaling in mediating their activity in primary mammary tumors. J Immunol. 2010, 184: 702-712. 10.4049/jimmunol.0902360.View ArticleGoogle Scholar
- Król M, Pawłowski KM, Skierski J, Rao NAS, Hellmen E, Mol JA, Motyl T: Transcriptomic profile of two canine mammary cancer cell lines with different proliferative and anti-apoptotic potential. J Physiol Pharmacol. 2009, 60: 95-106.PubMedGoogle Scholar
- Król M, Pawłowski KM, Skierski J, Turowski P, Majewska A, Polańska J, Ugorski M, Morty RE, Motyl T: Transcriptomic "portraits" of canine mammary cancer cell lines with various phenotype. J Appl Genet. 2010, 51: 169-183. 10.1007/BF03195725.View ArticlePubMedGoogle Scholar
- Król M, Polańska J, Pawłowski KM, Skierski J, Majewska A, Ugorski M, Motyl T: Molecular signature of cell lines isolated from mammary adenocarcinoma metastases to lungs. J Appl Genet. 2010, 51: 37-50. 10.1007/BF03195709.View ArticlePubMedGoogle Scholar
- Pawłowski KM, Popielarz D, Szyszko K, Motyl T, Król M: Growth Hormone Receptor RNA interference decreases proliferation and enhances apoptosis in canine mammary carcinoma cell line CMT-U27. Vet Comp Oncol. 2012, 10 (1): 2-15.View ArticlePubMedGoogle Scholar
- Hagemann T, Robinson SC, Schulz M, Trumper L, Balkwill FR, Binder C: Enhanced invasiveness of breast cancer cell lines upon co-cultivation with macrophages is due to TNF-α dependent up-regulation of matrix metalloproteases. Carcinogenesis. 2004, 28: 1543-1549.View ArticleGoogle Scholar
- Martinez FO, Gordon S, Locati M, Mantovani A: Transcriptional profiling of the human monocyte-to-macrophage differentiation and polatization: new moleculaes and patterns of gene expression. J Immunol. 2006, 177: 7303-7311.View ArticlePubMedGoogle Scholar
- Mi H, Lazareva-Ulitsky B, Loo R, Kejariwal A, Vandergriff J, Rabkin S, Guo N, Muruganujan A, Doremieux O, Campbell MJ, Kitano H, Thomas PD: The PANTHER database of protein families, subfamilies, functions and pathways. Nucleic Acids Res. 2005, 33: D284-D288.PubMed CentralPubMedGoogle Scholar
- Brinkhof B, Spee B, Rothuizen J, Penning LC: Development and evaluation of canine reference genes for accurate quantification of gene expression. Anal Biochem. 2006, 356: 36-43. 10.1016/j.ab.2006.06.001.View ArticlePubMedGoogle Scholar
- Etschmann B, Wilcken B, Stoevesand K, von der Schulenburg A, Sterner-Kock A: Selection of reference genes for quantitative real-time PCR analysis in canine mammary tumors using the GeNorm algorithm. Vet Pathol. 2006, 43: 934-942. 10.1354/vp.43-6-934.View ArticlePubMedGoogle Scholar
- Schmittgen TD, Livak KJ: Analyzing real-time PCR data by the comparative Ct method. Nat Prot. 2008, 3: 1101-1108. 10.1038/nprot.2008.73.View ArticleGoogle Scholar
- Rodriguez LG, Wu X, Guan JL: Wound-healing assay. Methods Mol Biol. 2005, 294: 23-29.PubMedGoogle Scholar
- Olumi AF, Grossfeld GD, Hayward SW, Carroll PR, Tlsty TD, Cunha GR: Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium. Cancer Res. 1999, 59: 5002-5011.PubMedGoogle Scholar
- Ohishi K, Vernum-Finney B, Serda RE, Anasetti C, Bernstein ID: The Notch ligand, Delta-1, inhibits the differentiation of monocytes into macrophages but permits their differentiation into dendritic cells. Blood. 2001, 98: 1401-1407.View ArticleGoogle Scholar
- Lewis CE, Pollard JW: Distinct role of macrophages in different tumor microenvionments. Cancer Res. 2006, 66 (2): 605-612. 10.1158/0008-5472.CAN-05-4005.View ArticlePubMedGoogle Scholar
- Green CE, Liu T, Montel V, Hsiao G, Lester RD, Subramaniam S, Gonias SL, Klemke RL: Chemoattractant signaling between tumor cells and macrophages regulates cancer cell migration, metastasis and neovascularization. PLoS One. 2009, 4: e6713-10.1371/journal.pone.0006713.PubMed CentralView ArticlePubMedGoogle Scholar
- Guimaraes FSF, Abud APR, Oliveira SM, Oliveira CC, Cesar B, Andrade LF, Donatti L, Gabardo J, Trindade EST, Buchi DF: Stimulation of lymphocyte anti-melanoma activity by co-cultured macrophages activated by complex homeopathic mediciation. BMC Cancer. 2009, 9: 293-10.1186/1471-2407-9-293.PubMed CentralView ArticlePubMedGoogle Scholar
- Abraham D, Zins K, Sioud M, Lucas T, Schafer R, Stanley ER, Aharinejad S: Stromal cell-derived CSF-1 blockade prolongs xenograft survival of CSF-1-negative neuroblastoma. Int J Cancer. 2010, 126: 1339-1352.PubMed CentralPubMedGoogle Scholar
- Lethinen A, Aho S, Kulonen E: Penetration of various mononuclear ribonucleases into rat experimental granulation-tissue fibroblasts and their intracellular affects. Hoppe Saylers Z Physiol Chem. 1981, 362: 1575-1582. 10.1515/bchm2.1981.362.2.1575.View ArticleGoogle Scholar
- Bolpetti A, Silva JS, Villa LL, Lepique AP: Interleukin-10 production by tumor infiltrating macrophages plays a role in Human Papillomavirus 16 tumor growth. BMC Immunol. 2010, 11: 27.PubMed CentralView ArticlePubMedGoogle Scholar
- Oljavo LS, King W, Cox D, Pollard JW: High-density gene expression analysis of tumor-associated macrophages from mouse mammary tumors. Am J Pathol. 2009, 174: 1048-1064. 10.2353/ajpath.2009.080676.View ArticleGoogle Scholar
- Sabo I, Olsson H, Sun XF, Svanvik J: Expression of the macrophage antigen CD163 in rectal cancer cells is associated with early local recurrence and reduced survival time. Int J Canc. 2009, 125: 1826-1831. 10.1002/ijc.24506.View ArticleGoogle Scholar
- Sabo I, Stal O, Olsson H, Dore S, Svanvik J: Breast cancer expression of CD163, a macrophage scavenger receptor, is related to Elary distant recurrence and reduced patient survival. Int J Cancer. 2008, 123: 780-786. 10.1002/ijc.23527.View ArticleGoogle Scholar
- Powell AE, Anderson EC, Davies PS, Silk AD, Pelz C, Impey S, Wong MH: Fusion between intestinal epithelial cells and macrophages in a cancer context results in nuclear reprogramming. Cancer Res. 2011, 71 (4): 1497-1505. 10.1158/0008-5472.CAN-10-3223.PubMed CentralView ArticlePubMedGoogle Scholar
- Jacobsen BM, Harrell JC, Jedlicka P, Borges VF, Varella-Garcia M, Horwitz KB: Spontaneous fusion with, and transformation of mouse stroma by, malignant human breast cancer epithelium. Cancer Res. 2006, 66: 8274-8279. 10.1158/0008-5472.CAN-06-1456.View ArticlePubMedGoogle Scholar
- Liu C, Chen Z, Zhang T, Lu Y: Multiple tumor types may originate from bone marrow-derived cells. Neoplasia. 2006, 8: 716-724. 10.1593/neo.06253.PubMed CentralView ArticlePubMedGoogle Scholar
- Nygren JM, Jovinge S, Breitbach M, Sawen P, Roll W, Hescheler J, Taneera J, Fleischmann BK, Jacobsen SE: Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation. Nat Med. 2004, 10: 494-501. 10.1038/nm1040.View ArticlePubMedGoogle Scholar
- Su JL, Yen CJ, Chen PS, Chuang SE, Hong CC, Kuo IH, Chen HY, Hung MC, Kuo ML: The role of the VEGF-C/VEGFR-3 axis in cancer progression. Brit J Canc. 2007, 96: 541-545. 10.1038/sj.bjc.6603487.View ArticleGoogle Scholar
- Chakraborty AK, Pawelek J, Ikeda Y, Miyoshi E, Kolesnikova N, Funasaka Y, Ichihashi M, Taniguchi N: Fusion hybrids with macrophage and melanoma cells up-regulate N-acetylglucosaminyltransferase V, beta1-6 branching, and metastasis. Cell Growth Differ. 2001, 12: 623-630.PubMedGoogle Scholar
- Chakraborty AK, Pawelek JM: GnT-V, macrophage and cancer metastasis: a common link. Clin Exp Metastasis. 2003, 20: 365-373. 10.1023/A:1024007915938.View ArticlePubMedGoogle Scholar
- Chakraborty AK, Sodi S, Rachkovsky M, Kolesnikova N, Platt JT, Bolognia JL, Pawelek JM: A spontaneous murine melanoma lung metastasis comprised of host x tumor hybrids. Cancer Res. 2000, 60: 2512-2519.PubMedGoogle Scholar
- Pawelek JM, Chakraborty AK: Fusion of tumour cells with bone marrow-derived cells: a unifying explanation for metastasis. Nat Rev Cancer. 2008, 8: 377-386. 10.1038/nrc2371.View ArticlePubMedGoogle Scholar
- Pawelek JM, Chakraborty AK: The cancer cell--leukocyte fusion theory of metastasis. Adv Cancer Res. 2008, 101: 397-444.View ArticlePubMedGoogle Scholar
- Pawelek JM: Cancer-cell fusion with migratory bone-marrow-derived cells as an explanation for metastasis: new therapeutic paradigms. Future Oncol. 2008, 4: 449-452. 10.2217/147966126.96.36.1999.View ArticlePubMedGoogle Scholar
- Watkins SK, Li B, Richardson KS, Head K, Egilmez NK, Zeng Q, Suttles J, Stout RD: Rapid release of cytoplasmic IL-15 from tumor associated macrophages is an initial and critical event in IL-12 initiated tumor regression. Eur J Immunol. 2009, 39 (8): 2126-2135. 10.1002/eji.200839010.PubMed CentralView ArticlePubMedGoogle Scholar
- Fabriek BO, van Bruggen R, Deng DM, Ligtenberg AJM, Nazmi K, Schomagel K, Vloet RP, Dijkstra CD, van den Berg TK: The macrophage scavenger receptor CD163 functions as an innate immune sensor for bacteria. Blood. 2009, 113: 887-892. 10.1182/blood-2008-07-167064.View ArticlePubMedGoogle Scholar
- van den Heuvel MM, Tensen CP, van As JH, van den Berg TK, Fluitsma DM, Dijkstra CD, Dopp EA, Droste A, van Gaalen FA, Sorg C, Hogger P, Beelen RHJ: Regulation of CD163 on human macrophages: cross-linking of CD163 induces signaling and activation. J Leukoc Biol. 1999, 66: 858-866.PubMedGoogle Scholar
- Loberg RD, Day LL, Harwood J, Ying C, John LN, Giles R, Neeley CK, Pienta KJ: CCL2 is a potent regulator of prostate cancer cell migration and proliferation. Neoplasia. 2006, 8: 578-586. 10.1593/neo.06280.PubMed CentralView ArticlePubMedGoogle Scholar
- Wu Y, Li YY, Matsushima K, Baba T, Mukida N: CCL3-CCR5 axis regulates intratumoral accumulation of leukocytes and fibroblasts and promotes angiogenesis in murine lung metastasis process. J Immunol. 2008, 181: 6384-6393.View ArticlePubMedGoogle Scholar
- Tausendschon M, Dehne N, Brune B: Hypoxia causes epigenetic gene regulation in macrophages by attenuating Jumonji histone demethylase activity. Cytokine. 2011, 53: 256-262. 10.1016/j.cyto.2010.11.002.View ArticlePubMedGoogle Scholar
- Sica A, Saccani A, Bottazzi B, Bernasconi S, Allavena P, Gaetano B, Fei F, LaRosa G, Scotton C, Balkwill F, Mantovani A: Defective expression of the monocyte chemotactic protein-1 receptor CCR2 in macrophages associated with human ovarian carcinoma. J Immunol. 2000, 164: 733-738.View ArticlePubMedGoogle Scholar
- Bosco MC, Reffo G, Puppo M, Varesio L: Hypoxia inhibits the expression of the CCR5 chemokine receptor in macrophages. Cell Immunol. 2004, 228: 1-7. 10.1016/j.cellimm.2004.03.006.View ArticlePubMedGoogle Scholar
- Murdoch C, Lewis CE: Macrophage migration and gene expression in response to tumor hypoxia. Int J Cancer. 2005, 117: 701-708. 10.1002/ijc.21422.View ArticlePubMedGoogle Scholar
- Yang MH, Wu KJ: TWIST activation by hypoxia inducible factor-1 (HIF-1). Cell Cycle. 2008, 7: 2090-2096. 10.4161/cc.7.14.6324.View ArticlePubMedGoogle Scholar
- Pollard JW: Review of: Overexpression of the colony-stimulating factor (CSF-1) and/or its receptor c-fms in mammary glands of transgenic mice results in hyperplasia and tumor formation. BCO. 2005, 8: e10.View ArticleGoogle Scholar
- Scholl SM, Pallud C, Beuvon F, Hacene K, Stanley ER, Rohrschneider L, Tang R, Pouillart P, Lidereau R: Anti-colonystimulating factor-1 antibody staining in primary breast adenocarcinomas correlates with marked inflammatory cell infiltrates and prognosis. J Natl Cancer Inst. 1994, 86: 120-126. 10.1093/jnci/86.2.120.View ArticlePubMedGoogle Scholar
- Song MN, Cho SY: CD14 Acts as an angiogenic factor by inducing basic fibroblast growth factor (bFGF). Bull Korean Chem Soc. 2007, 28: 1613-1614.View ArticleGoogle Scholar
- Król M, Pawłowski KM, Majchrzak K, Dolka I, Abramowicz A, Szyszko K, Motyl T: Density of tumor-associated macrophages (TAMs) and expression of their growth factor receptor MCSF-R and CD14 in canine mammary adenocarcinomas of various grade of malignancy and metastasis. Pol J Vet Sci. 2011, 14: 3-10. 10.2478/v10181-011-0001-3.PubMedGoogle Scholar
- Kirma N, Luthra R, Jones J, Liu YG, Nair HB, Mandava U, Tekmal RR: Overexpression of the colony-stimulating factor (CSF-1) and/or its receptor c-fms in mammary glands of transgenic mice results in hyperplasia and tumor formation. Cancer Res. 2004, 64: 4162-4170. 10.1158/0008-5472.CAN-03-2971.View ArticlePubMedGoogle Scholar
- Filderman AE, Bruckner A, Kacinski BM, Deng BM, Remold HG: Macrophage colony-stimulating factor (CSF-1) enhances invasiveness in CSF-1 receptor-positive carcinoma cell lines. Cancer Res. 1992, 53: 3661-3666.Google Scholar
- Wrobel CN, Debnath J, Lin E, Beausoleil S, Roussel MF, Brugge JS: Autocrine CSF-1R activation promotes Src-dependent disruption of mammary epithelial architecture. J Cell Biol. 2004, 165: 263-273. 10.1083/jcb.200309102.PubMed CentralView ArticlePubMedGoogle Scholar
- Murdoch C, Giannoudis A, Lewis CE: Mechanisms regulating the recritment of macrophages into hypoxic areas of tumors and other ischemic tissues. Blood. 2004, 104: 2224-2234. 10.1182/blood-2004-03-1109.View ArticlePubMedGoogle Scholar
- Kaler P, Galea V, Augenlicht L, Klampfer L: Tumor associated macrophages protect colon cancer cells from TRAIL-induced apoptosis through IL-1β-dependent stabilization of snail in tumor cells. PLoS One. 2010, 5 (7): e11700-10.1371/journal.pone.0011700.PubMed CentralView ArticlePubMedGoogle Scholar
- Rao NA, van Wolferen ME, Gracanin A, Bhatti SF, Król M, Holstege FC, Mol JA: Gene expression profiles of progestin-induced canine mammary hyperplasia and spontaneus mammary tumors. J Physiol Pharmacol. 2009, 60: 73-84.PubMedGoogle Scholar