The gene expression profiles of canine mammary cancer cells grown with carcinoma-associated fibroblasts (CAFs) as a co-culture in vitro

  • Magdalena Król1Email author,

    Affiliated with

    • Karol M Pawłowski1, 2,

      Affiliated with

      • Katarzyna Szyszko1,

        Affiliated with

        • Henryk Maciejewski3,

          Affiliated with

          • Izabella Dolka4,

            Affiliated with

            • Elisabetta Manuali5,

              Affiliated with

              • Michał Jank1 and

                Affiliated with

                • Tomasz Motyl1

                  Affiliated with

                  BMC Veterinary Research20128:35

                  DOI: 10.1186/1746-6148-8-35

                  Received: 26 September 2011

                  Accepted: 27 March 2012

                  Published: 27 March 2012

                  Abstract

                  Background

                  It is supposed that fibroblasts present in tumour microenvironment increase cancer invasiveness and its ability to metastasize but the mechanisms have not been clearly defined yet. Thus, the current study was designed to assess changes in gene expression in five various cancer cell lines grown as a co-culture with the carcinoma-associated fibroblasts (CAFs) in vitro.

                  Results

                  A carcinoma-associated fibroblast cell line was isolated from a canine mammary cancer. Then, a co-culture of cancer cells with the CAFs was established and maintained for 72 hrs. Having sorted the cells, a global gene expression in cancer cells using DNA microarrays was examined. The analysis revealed an up-regulation of 100 genes and a down-regulation of 106 genes in the cancer cells grown as a co-culture with the CAFs in comparison to control conditions. The PANTHER binomial statistics tool was applied to determine statistically over-manifested pathways (p < 0.05). Bulk of the up-regulated genes are involved in the adhesion, the angiogenesis, the epithelial-mesenchymal transition (EMT) and generally take part in the developmental processes. These results were further confirmed using real-time qPCR. Moreover, a wound-healing assay and growth characteristics on Matrigel matrix showed that CAFs increase cancer cell migration and matrix invasion.

                  Conclusion

                  The results of the current study showed that the co-culturing of cancer cells and the CAFs caused significant changes to the cancer gene expression. The presence of the CAFs in a microenvironment of cancer cells promotes adhesion, angiogenesis and EMT.

                  Background

                  Since canine mammary tumours in bulk are of epithelial origin this kind of cells is subjected to many studies. Over the last few years it has also been pin-pointed that concomitant changes occur within stromal cells, which contribute to the tumour microenvironment as well [1, 2]. Tumour microenvironment embraces inflammatory, fibroblastic, endothelial cells, adipocytes and other. Changes within these stromal cells have been postulated to increase the tumorigenic phenotype of the epithelial cell, promote malignant transformation, induce epithelial-mesenchymal transition (EMT) and promote tumour spreading and metastasis [3]. It is worth noting however, that in almost all the tumours, the main cell type of cancer stromal compartment is fibroblast. These cells are usually atypical and are termed carcinoma-associated fibroblasts (CAFs). We assume there is a cross-talk between the tumour cells and the CAFs, which promotes migratory, and invasive properties of cancer cells [3] though their exact role within cancer microenvironment has not been fully defined yet. Thus, the study was conducted to assess the changes in gene expression in cancer cells grown as a co-culture with the CAFs in vitro. As far as we know the study presented hereby is a pioneering microarray experiment in this field. Despite that our study involved five various cell lines, only one CAFs cell line was used, thus the results may be limited to this particular CAF model. Further studies in this field are required.

                  The analysis revealed an up-regulation within a span of 100 genes and a down-regulation within 106 genes in cancer cells grown as a co-culture with the CAFs, comparing against set control conditions. In this manuscript we focused mainly on the gene sets involved in adhesion, developmental process and neurotransmissions.

                  The results of our study can be extrapolated on human research because canine mammary tumours are being considered a spontaneous animal model of human breast cancer [4]. There are many similarities between human and canine mammary cancers: in both species they represent a heterogeneous group in terms of morphology and biological behaviour [5], in both similar cancer-related pathways are activated [68] as much as both species live under similar environmental conditions.

                  Methods

                  Cell lines

                  The cell lines used for this study have previously been given an account of [912]. Two canine mammary adenocarcinoma cell lines (CMT-W1, CMT-W2), an anaplastic cancer cell line (P114), a simple carcinoma cell line (CMT-U27) and a spindle-cell mammary tumour cell line (CMT-U309) were examined. The CMT-W1 and the CMT-W2 cell lines had kindly been donated by Prof. Dr. Maciej Ugorski and Dr. Joanna Polanska from Wroclaw University of Environmental and Life Sciences (Poland). The CMT-U27 cell line had kindly been donated by Dr. Eva Hellmen from Swedish University of Agricultural Sciences (Sweden) and the P114 cell line had kindly been donated by Dr. Gerard Rutteman from Utrecht University (The Netherlands).

                  The 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 sub-cultured every other day. The methods of canine mammary cancer cells culturing have previously been given an account of [912].

                  Tumour sample

                  A mammary tumour was surgically removed during mastectomy on a 12 years old mixed breeds female. The tumour then, was divided into equal halves, one of them was fixed in 10% neutral buffered formalin and routinely embedded in paraffin to perform histological assay. The other, was used to isolate and establish a carcinoma-associated fibroblast cell line.

                  Carcinoma-associated fibroblasts isolation

                  The cells isolation from cancer tissue has been described in our previous manuscript [13]. The tumor sample was collected into the medium RPMI 1640 (Sigma Aldrich, USA) containing flask immediately after mastectomy. The RPMI 1640 medium had been used to maintain the same culturing conditions for mono- and co-culture. The tumour sample was then sliced and cultured overnight in collagenase containing medium RPMI 1640 according to the Limon et al. [14] protocol (modified by Dr Eva Hellmen, Swedish University of Agricultural Sciences, Sweden). The following day, the medium was centrifuged and pellet was suspended in a fresh culture medium supplemented in FGF (10 nM/ml, obtained from Sigma Aldrich, USA), a medium that encourages preferential fibroblastic outgrowth.

                  Histopathological examination

                  The tissue sample embedded in paraffin block was cut into five μm sections and baked in 37°C 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 chamber. The tumor type was classified based on the World Health Organization (WHO) Histological Classification and Mammary Tumors of the Dog and Cat classification [15, 16]. The mammary carcinoma grading was assessed in respect to tubule formation, degree of differentiation and mitotic index.

                  The carcinoma-associated fibroblasts and the canine mammary cancer cells were cultured on Lab-Tek (Nunc Inc., USA) 4-chamber culture slides and were then fixed with ethanol after the 24 hrs.

                  The immunohistochemical examination of expression of Ki67, cytokeratin, vimentin, smooth muscle actin, s100 protein, p63 protein was performed on the tissue sample as well as on carcinoma-associated fibroblasts to confirm the origin of cell culture. The MUC1 expression was analyzed in the canine mammary cancer cell lines.

                  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): mouse monoclonal anti-Ki67 (Clone MIB-1) at the concentration 1:75; monoclonal mouse anti-human cytokeratin (Clone MNF116) at the concentration 1:50; monoclonal mouse anti-human vimentin (Clone Vim 3B4) at the concentration 1:50; monoclonal mouse anti-human actin (Clone HHF35) at the concentration 1:50; polyclonal rabbit anti-S100 (ready to use solution) all obtained from Dako (Denmark); monoclonal mouse anti-p63 protein (Santa Cruz Biotechnology, USA) and monoclonal mouse anti-MUC1 (Abcam, United Kindgdom) at the concentration 1:10. According to the manufacturer's instructions the slides were incubated with antibodies at +4°C overnight or 1 hr at room temperature. For the staining the anti-mouse or anti-rabbit EnVision kits (Labelled Polymers consist of secondary anti-rabbit antibodies conjugated with the HRP enzyme complex obtained from Dako) were used. To develop the coloured product, the 3,3'-Diaminobenzidine (DAB) substrate was used (Dako). Finally, the haematoxylin was used for nuclei counterstaining.

                  For each immunohistochemical analysis as the negative control, the staining without the use of primary antibodies was done. The pictures were taken using Olympus microscopy BX60 (Olympus, Germany).

                  Co-culture and sorting

                  The CAFs (105 cells) were grown on 75 cm2 culture flasks and the cancer cells (CMT-W1, CMT-W2, CMT-U27, CMT-U309, P114) were layered (5 × 105 cells) on the top of the CAFs (fibroblasts and cancer cells at 1:5 ratio [17]). An Orange CellTracker fluorescent dye CMTMR (Invitrogen, USA) was used to stain the CAFs' population before the cancer cell population was added. Initially, optimal staining conditions were determined by incubating CAFs in various concentrations of CMTMR (5-25 μM dye, according to the manufacturer's instructions) and checking the fluorescence signal after 72 hrs using FACS. The lowest concentration that gives positive results has been used in further experiments (5 μM). 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 CAFs were washed with PBS twice and incubated with complete RPMI for 1 hr and then again washed to remove any remnant non-metabolized CMTMR. The cancer cells were placed on the CMTMR-stained CAFs.

                  The co-culture was maintained for 72 hrs. Then, the cells were harvested by trypsynization, analyzed and sorted using FACS Aria II high speed cell sorter with Diva 5.0 software (Becton Dickinson, USA). Based on the FSC and SSC cytogram, live cells were gated to exclude all dead cells, cell debris and cell clumps. Within the gated cell populations, fluorescing cells were identified as CMTMR-labelled carcinoma-associated fibroblasts and non-fluorescent as cancer cells. Excitation wavelength used was 488 nm, whereas emission wavelength used was 578 nm. Cancer cells were sorted into RPMI 1640 medium in 15 ml polypropylene tubes (BD Biosciences).

                  Confocal microscopy

                  The CAFs grown as a mono-culture were stained using Orange CellTracker fluorescent dye CMTMR, as described above. The cells grown on plastic were fixed in 70% ethanol (10 min), washed in PBS three times and the coverslips were mounted on microscope slides using ICN mounting medium. The cell imaging was performed on confocal laser scanning microscope FV-500 system (Olympus Optical Co, Germany) after 1 hr, then after 72 hrs after the staining. The excitation/emission were: HeNe 543 nm laser with 610 nm filter for CMTMR staining. The cells were examined using the Fluoview program (Olympus Optical Co., Germany). The pictures have been analyzed using a computer-assisted image analyzer (Olympus Microimage™ Image Analysis, software version 4.0 for Windows, USA).

                  Wound-healing assay

                  To assess the migration ability of cancer cells grown as a co-culture with CAFs, we applied a wound-healing test. The cancer cells (grown as the co-culture with CAFs at the 5:1 ratio, and normal control cells) were separately seeded in multi-well plates and then, (after 72 hrs when the cells were confluent) using a pipette tip (100 ul) a straight scratch had been made, simulating a wound. The images were captured at the beginning and at regular intervals (after 2, 4 and 6 hours) during cell migration to close the wound. The images then were compared to quantify the cells' migration rate. This method is particularly suitable for studies of cell-cell interaction on cell migration [18]. The pictures have been analyzed using a computer-assisted image analyzer (Olympus Microimage™ Image Analysis, software version 4.0 for Windows, USA).

                  3D culture

                  Cancer cells were treated with trypsin and resuspended in culture medium. 35 mm culture plates (Corning Inc.) were coated with 100 μl of growth factor reduced Matrigel (BD Biosciences) and left to solidify for 30 min. at 37°C. The control cells were then plated at a concentration of 104 cells/ml. Co-cultured cells were plated at the same concentration (cancer cells and CAFs at 5:1 ratio). The growth of cells on Matrigel was observed everyday under phase-contrast microscope (Olympus).

                  Microarray analysis

                  The sorted cancer cells grown as a co-culture were centrifuged (2,500 rpm for 5 min), whereas cancer cells grown as mono-cultures were washed with PBS and harvested by trypsynization and centrifuged (2,500 rpm for 5 min). The total RNA from the samples was isolated using a Total RNA kit (A&A Biotechnology, Poland) according to the manufacturer's protocol. The isolated RNA samples were dissolved in RNase-free water. The quantity of the isolated RNA was measured using NanoDrop (NanoDrop Technologies, USA). The samples with adequate amounts of RNA were treated with DNaseI to eliminate DNA contamination. The samples were subsequently purified using RNeasy MiniElute Cleanup Kit (Qiagen, Germany). Finally the RNA samples were analyzed on a BioAnalyzer (Agilent, USA) to measure the final RNA quality and integrity.

                  The total RNA (10 μg) of each cell line was reverse-transcribed using SuperScript Plus Direct cDNA Labeling System, (Invitrogen, USA) according to the manufacturer's protocol for each microarray slide. 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 made (dye-swap).

                  The slides were analyzed using microarray scanner ScanArray HT and ScanExpress software (PerkinElmer, USA).

                  Real-time qPCR

                  The mRNA sequences of the key genes were obtained from NCBI database. Primers were designed using PRIMER3 software (free on-line access) and checked using Oligo Calculator (free on-line access) and Primer-Blast (NCBI database). Primers' sequences are listed in Table 1. HPRT and RPS19 genes were used as non-regulated reference genes for normalization of target gene expression [19, 20]. Quantitative RT-PCR was performed using fluorogenic Lightcycler Fast Strand DNA Sybr Green (Roche) and the Light Cycler (Roche). The results were analyzed using comparative Ct method [21]. Relative transcript abundance of the gene equals ΔCt values (ΔCt = Ctreference - Cttarget). Relative changes in transcript are expressed as ΔΔCt values (ΔΔCt = ΔCtco-culture - ΔCtcontrol). The experiment was conducted three times.
                  Table 1

                  Primers used for Real-time qPCR

                  Gene symbol

                  Forward primer

                  Reverse primer

                  Optimum annealing temp. (°C)

                  Optimum annealing time (sec)

                  DSP

                  CAGACTCACCGAAGAGGAAA

                  CTGCTGTGAAGTCTGGGAGT

                  61

                  7

                  MAG

                  TGCCATCGTCTGCTACATTA

                  CAGTCGCCTCTCACTCTCAT

                  60

                  6

                  PCDH19

                  CTTTCACATCACTGCACTCG

                  GTGTGTTGGGAGGTGAGTTC

                  61

                  6

                  HPRT

                  AGCTTGCTGGTGAAAAGGAC

                  TTATAGTCAAGGGCATATCC

                  59

                  6

                  RPS19

                  CCTTCCTCAAAAAGTCTGGG

                  GTTCTCATCGTAGGGAGCAAG

                  61

                  10

                  Primers sequences used in this study and their annealing optimal temperature and time. The mRNA sequences of key genes were obtained from NCBI database. Primers were designed using PRIMER3 software (free on-line access) and checked using Oligo Calculator (free on-line access) and Primer-Blast (NCBI database). HPRT and RPS19 genes were used as non-regulated reference genes for normalization of target gene expression [19, 20].

                  Statistical analysis

                  In the analysis of differential gene expression, background-corrected value of signal in each microarray channel was used. Prior to the analysis, non-specific filtering was performed, i.e. genes with small level of expression were removed (we set an arbitrary threshold according to which at least half of the samples' expression was to be at least 100). This reduced the number of genes down to 24 842. Then the log2 ratio of the sample vs control channels was calculated and the signal was loes normalized. Quality control, including MA analysis, and signal normalization were done with the Bioconductor software. The analysis of differential expression was performed using linear methods for microarrays (limma package in Bioconductor software) [22]. The method tests the null hypothesis of no differential expression between the sample and control groups using the moderated t-statistic [22], which has similar interpretation as the ordinary t-test statistic. We identified 206 genes with the p-value below 0.05 and fold change > 2.0.

                  The microarray data discussed in this publication has been deposited in NCBI's Gene Expression Omnibus and is freely accessible through GEO Series accession number GSE29601.

                  The gene function was identified using the NCBI database and PANTHER pathway analysis software [23]. The pathway analyses were conducted using binominal statistic test (PANTHER) with the cut-off value p < 0.05.

                  The statistical analysis of optical density, wound healing assay and Real-time qPCR was conducted using Prism version 5.00 software (GraphPad Software, USA). The one-way ANOVA, and ANOVA + Tukey HSD (Honestly Significant Difference) post-hoc test as well as 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.

                  Results

                  Tumor sample and CAFs examination

                  The histopathological and immunohistochemical assessment of the tissue sample, from which the carcinoma-associated fibroblasts were isolated, showed that the tumour type was a complex carcinoma of the 1st grade malignancy (Figure 1a). The histopathological and immunohistochemical analysis of the isolated cells (Figure 1b) revealed that the cell line did not express cytokeratin, S100 protein, p63 protein and actin, whereas a strong vimentin expression was detected (at the level of 3 in 0-3 scale) (Figure 1c). The analysis confirmed that the isolated cell line is an atypical colony of fibroblasts which are termed the CAFs.
                  http://static-content.springer.com/image/art%3A10.1186%2F1746-6148-8-35/MediaObjects/12917_2011_354_Fig1_HTML.jpg
                  Figure 1

                  Cancer associated fibroblasts isolated from canine mammary cancer. Representative pictures of A. Canine mammary complex carcinoma (HE staining) tissue from which the carcinoma-associated fibroblasts were isolated. B. The culture of carcinoma-associated fibroblasts (CAFs) isolated from the canine mammary complex carcinoma (HE staining) and C. Carcinoma-associated fibroblasts (CAFs) isolated from canine complex mammary carcinoma revealed a strong vimentin expression (brown color). The pictures were obtained using Olympus BX60 microscope (at the magnification of 200×).

                  Sorting of the co-cultured cells

                  The Flow-Cytometry easily distinguished the CMTMR-stained cells from the unstained ones (Figure 2a) and allowed the proper further sorting of each population (Figure 2b, c). The co-culture was maintained for 72 hrs. According to the manufacturer's instruction, CMTMR probes remain vividly fluorescent for at least 72 hrs after incubation in fresh medium at 37°C and through at least four cell divisions. The confocal observations confirmed that after 72 hrs of the staining with the CMTMR all the cells showed red-staining cytoplasm pattern (Figure 2c, d, e). No detrimental effects on proliferation and plating efficiency was observed. Our analysis of optical density of the red dye in the cells measured 1 hr and 72 hrs after the staining showed very similar results (any statistically significant difference had not been found) (Figure 2f). It indicates that there wasn't any staining loss, thus we suppose that the dye wasn't leaking from CAFs to stain cancer cells. It had been also suggested in the subject literature [24]. Moreover, confocal microscopy analysis of the red dye fluorescence with Nomarski Interferenced Contrast showed that all of the stained CAFs maintained their staining pattern after the 72 hrs. Thus, the artificial sorting of unstained CAFs as cancer cells is not very probable. Our FACS sorting isolated a 97-99% pure population on postsort (assessed by BC FACS Diva 5.0 software) what was checked by FACS (Figure 2b, c) and fluorescence microscopy (data not shown). Previously published study of the human fibroblasts and epithelial cells sorting based on the cell tracker staining showed similar results [25].
                  http://static-content.springer.com/image/art%3A10.1186%2F1746-6148-8-35/MediaObjects/12917_2011_354_Fig2_HTML.jpg
                  Figure 2

                  Histogram and pictures of unstained cancer cells and stained CAFs. A. Representative histogram and sorting gates of unstained cancer cells and CMTMR-stained carcinoma-associated fibroblasts (CAFs) grown as the co-culture for 72 hrs. B. Histogram of cancer cells sorted from co-culture showing only CMTMR-negative cells. C. Histogram of CAFs sorted from co-culture showing only CMTMR-positive cells. D. and E. Representative pictures obtained using confocal microscopy showing red-colored stained cytoplasm (CMTMR staining) of CAFs growing as a single culture for 1 hr and 72 hrs, respectively. The imaging of cells was performed on confocal laser scanning microscope FV-500 system (Olympus Optical Co, Germany). The cells were examined using the Fluoview program (Olympus Optical Co., Germany). F. The graph showing mean optical density of the red (reflecting CMTMR staining) in CAFs at 1 hr and 72 hrs after staining. For statistical purposes the t-test has been applied (Graph Pad 5.0), no significant difference has been observed between these two values.

                  Migration assay and growth characteristics on Matrigel matrix

                  The wound healing assay showed that in all the cancer cell lines the co-culturing with CAFs increased their migratory abilities (Figure 3). CMT-U27 cells grown with CAFs almost completely closed the wound (99%) in 6 hours, whereas CMT-U27 control cells after 6 hrs closed 68% of the wound. Similarly, CMT-U309 and P114 cells (grown with CAFs) after 6 hrs completely closed the wound (100%), whereas control cells closed only 55% and 50% (respectively) of the wound. CMT-W1 cells grown with CAFs completely closed the wound after 4 hrs, whereas CMT-W1 control cells after 6 hrs (after 4 hrs 64% of the wound was closed). CMT-W2 cells grown with CAFs closed 93% of the wound after 6 hrs, whereas control cells closed only 52% of the wound.
                  http://static-content.springer.com/image/art%3A10.1186%2F1746-6148-8-35/MediaObjects/12917_2011_354_Fig3_HTML.jpg
                  Figure 3

                  Wound healing assay of canine mammary cancer cells grown in control conditions and as a co-culture with CAFs. A Representative pictures of migration (wound closing) of CMT-U309 cell line grown as a mono-culture and co-culture with CAFs at 0, 2, 4 and 6 hrs after the scratch was made. B The graphs of % of wound closure after the 2, 4 and 6 hrs of migration. The pictures were taken using phase-contrast microscopy (Olympus) at the magnification of 100×. The statistical analysis was performed using Prism version 5.00 software (GraphPad Software, USA). The one-way ANOVA was applied to analyze the results. p < 0.05 was regarded as significant and marked as *, whereas p < 0.01 and p < 0.001 were regarded as highly significant and marked as ** and ***, respectively.

                  To assess the ability of the cell lines to matrix invasion, we have assessed their growth characteristics on Matrigel matrix (Figure 4). After 72 hrs of culturing (similarly as in all experiments) on Matrigel CMT-U27, CMT-U309 and P114 cell lines formed colonies, whereas CMT-W1 and CMT-W2 cell lines formed branching structures (Figure 4a) what indicated their invasive phenotype. However, all the cell lines grown as a co-culture with CAFs after 72 hrs were dispersed (Figure 4b) what indicated increase of their matrix invasion ability by CAFs.
                  http://static-content.springer.com/image/art%3A10.1186%2F1746-6148-8-35/MediaObjects/12917_2011_354_Fig4_HTML.jpg
                  Figure 4

                  Canine mammary cancer cell lines growth characteristics in Matrigel matrix. A Growth characteristics of CMT-U27, CMT-U309, P114, CMT-W1 and CMT-W2 cell lines (phase contrast micrographs) grown on Matrigel matrix for 72 hours. B Growth characteristics of CMT-U27, CMT-U309, P114, CMT-W1 and CMT-W2 cell lines (phase contrast micrographs) grown as a co-culture with CAFs on Matrigel matrix for 72 hours. The pictures were taken using phase-contrast microscopy (Olympus) at the magnification of 200×.

                  Gene expression analysis

                  The gene expression analysis showed similar rate of gene expression in each of the dye-swap experiment. This result indicates that all microarray samples were successfully labeled, hybridized, and scanned. The discriminating analysis (with p value cut-off < 0.05; fold change > 2.0) revealed 106 up-regulated (Table 2) and 100 down-regulated (Table 3) genes in cancer cells grown as a co-culture with the CAFs in each of the slides. These up/down-regulated genes were common for each cell line examined individually, compared to the same cell line grown as a mono-culture. Thus, these genes were activated/inactivated in all of the cell lines under co-culture conditions with the CAFs.
                  Table 2

                  Up-regulated genes in cancer cells grown as a co-culture with CAFs

                  No

                  Fold change

                  Gene ID

                  Gene name

                  Molecular function

                  Biological process

                  1

                  4.18

                  TRIM6

                  Tripartite motif-containing protein 6

                  ubiquitin-protein ligase activity; structural constituent of cytoskeleton; RNA binding; cytoskeletal protein binding

                  spermatogenesis; neurotransmitter secretion; intracellular protein transport; exocytosis; cell cycle; signal transduction; synaptic transmission; carbohydrate metabolic process; protein metabolic process; cell-cell signaling; dorsal/ventral axis specification; mesoderm development; mammary gland development

                  2

                  3.76

                  PPP1R12A

                  Protein phosphatase 1 regulatory subunit 12A

                  protein binding; phosphatase regulator activity

                  protein metabolic process

                  3

                  3.73

                  TCHHL1

                  Trichohyalin-like protein 1

                    

                  4

                  3.24

                  QRICH2

                  Glutamine-rich protein 2

                  receptor activity

                  fertilization; cell adhesion

                  5

                  3.14

                  TMEM82

                  Transmembrane protein 82

                    

                  6

                  3.14

                  ZNF212

                  Zinc finger protein 212

                  DNA binding; transcription factor activity

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

                  7

                  3.13

                  PYROXD1

                  Pyridine nucleotide-disulfide oxidoreductase domain-containing protein 1

                  oxidoreductase activity

                  immune system process; respiratory electron transport chain; apoptosis; ferredoxin metabolic process; oxygen and reactive oxygen species metabolic process

                  8

                  3.10

                  PDE5A

                  cGMP-specific 3',5'-cyclic phosphodiesterase

                  hydrolase activity, acting on ester bonds

                  visual perception; sensory perception; signal transduction; nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; signal transduction

                  9

                  3.02

                  CHAD

                  Chondroadherin

                  receptor activity

                  immune system process; cell surface receptor linked signal transduction; cell-cell adhesion; mesoderm development; skeletal system development

                  10

                  3.00

                  QSER1

                  Glutamine and serine-rich protein 1

                    

                  11

                  2.96

                  PRKX

                  Serine/threonine-protein kinase

                  kinase activity

                  muscle contraction; neurological system process; mitosis; intracellular signaling cascade; protein metabolic process; signal transduction;

                  12

                  2.90

                  ZFAND5

                  AN1-type zinc finger protein 5

                  nucleic acid binding

                  sensory perception; respiratory electron transport chain

                  13

                  2.86

                  C2CD3

                  C2 domain-containing protein 3

                    

                  14

                  2.82

                  CEP110

                  Centriolin;Centrosomal protein of 110 kDa

                    

                  15

                  2.80

                  FOXQ1

                  Forkhead box protein Q1

                  DNA binding; transcription factor activity

                  visual perception; sensory perception; cell cycle; cell surface receptor linked signal transduction; carbohydrate metabolic process; nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; cellular component morphogenesis; segment specification; anterior/posterior axis specification; ectoderm development; mesoderm development; embryonic development; nervous system development

                  16

                  2.80

                  NUP210L

                  Nuclear pore membrane glycoprotein 210-like

                   

                  intracellular protein transport; nuclear transport

                  17

                  2.79

                  SLC22A11

                  Solute carrier family 22 member 11

                  ATPase activity, coupled to transmembrane movement of substances; ligase activity; carbohydrate transmembrane transporter activity; cation transmembrane transporter activity

                  cation transport; anion transport; extracellular transport; carbohydrate transport; carbohydrate metabolic process

                  18

                  2.78

                  HERC2

                  Probable E3 ubiquitin-protein ligase HERC2

                  ubiquitin-protein ligase activity

                  protein metabolic process; ectoderm development; mesoderm development; skeletal system development; nervous system development

                  19

                  2.76

                  PCSK6

                  Proprotein convertase subtilisin/kexin type 6

                  peptidase activity

                  cell surface receptor linked signal transduction; cell-matrix adhesion; protein metabolic process; signal transduction; mesoderm development

                  20

                  2.76

                  UCK2

                  Uridine-cytidine kinase 2

                  kinase activity; transferase activity, transferring glycosyl groups

                  carbohydrate metabolic process; nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

                  21

                  2.76

                  WFDC5

                  WAP four-disulfide core domain protein 5

                  protein binding;peptidase inhibitor activity

                  protein metabolic process

                  22

                  2.75

                  RPS10

                  40S ribosomal protein S10

                  structural constituent of ribosome; nucleic acid binding

                  protein metabolic process

                  23

                  2.75

                  SLC7A14

                  Probable cationic amino acid transporter

                  amino acid transmembrane transporter activity; transmembrane transporter activity

                  amino acid transport; cellular amino acid and derivative metabolic process

                  24

                  2.75

                  UBQLN2

                  Ubiquilin-2;UBQLN2

                   

                  protein metabolic process

                  25

                  2.70

                  CLEC7A

                  C-type lectin domain family 7 member A

                  receptor activity; receptor binding

                  B cell mediated immunity; natural killer cell activation; intracellular protein transport; endocytosis; signal transduction; cell-cell adhesion; signal transduction; cellular defense response

                  26

                  2.70

                  SH3BP1

                  SH3 domain-binding protein 1

                  protein binding; small GTPase regulator activity

                  cell surface receptor linked signal transduction; signal transduction; cellular component morphogenesis

                  27

                  2.68

                  HMGB2

                  High mobility group protein B2

                  DNA binding; chromatin binding; receptor binding; transcription factor activity

                  intracellular signaling cascade; nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; signal transduction; organelle organization; establishment or maintenance of chromatin architecture

                  28

                  2.68

                  TMSB10

                  Thymosin beta-10

                    

                  29

                  2.67

                  NEFM

                  Neurofilament medium polypeptide

                  structural constituent of cytoskeleton

                  cellular component morphogenesis; ectoderm development

                  30

                  2.67

                  ZNF274

                  Zinc finger protein 274

                  DNA binding;transcription factor activity

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

                  31

                  2.66

                  PCDH19

                  Protocadherin-19

                  G-protein coupled receptor activity; calcium ion binding

                  cell surface receptor linked signal transduction; cell-cell adhesion; cell motion; signal transduction; cellular component morphogenesis; ectoderm development; mesoderm development; embryonic development; nervous system development; heart development; muscle organ development

                  32

                  2.61

                  KCMF1

                  E3 ubiquitin-protein ligase KCMF1

                    

                  33

                  2.60

                  PIP4K2B

                  Phosphatidylinositol-5-phosphate 4-kinase type-2 beta

                  kinase activity

                  cell surface receptor linked signal transduction; lipid metabolic process; signal transduction

                  34

                  2.57

                  GNAT3

                  Guanine nucleotide-binding protein G(t) subunit alpha-3

                  GTPase activity; protein binding

                  cell surface receptor linked signal transduction; signal transduction

                  35

                  2.56

                  LPCAT1

                  1-acylglycerophosphocholine O-acyltransferase 1

                  acyltransferase activity; calcium ion binding; calmodulin binding; calcium-dependent phospholipid binding

                  metabolic process

                  36

                  2.56

                  XPR1

                  Xenotropic and polytropic retrovirus receptor 1

                  G-protein coupled receptor activity

                  cell surface receptor linked signal transduction; signal transduction; embryonic development

                  37

                  2.54

                  LPHN2

                  Latrophilin-2

                  G-protein coupled receptor activity

                  immune system process; neurotransmitter secretion; intracellular protein transport; exocytosis; cell surface receptor linked signal transduction; synaptic transmission; cell adhesion; cell-cell signaling; mesoderm development; angiogenesis; heart development

                  38

                  2.54

                  MPHOSPH6

                  M-phase phosphoprotein 6

                   

                  cell cycle

                  39

                  2.52

                  NDUFB10

                  NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10

                  oxidoreductase activity

                  oxidative phosphorylation; respiratory electron transport chain

                  40

                  2.51

                  DSP

                  Desmoplakin

                  structural constituent of cytoskeleton; cytoskeletal protein binding

                  cell adhesion; cellular component morphogenesis; ectoderm development

                  41

                  2.51

                  EXOC3L2

                  Exocyst complex component 3-like protein 2

                   

                  spermatogenesis; immune response; macrophage activation; intracellular protein transport; exocytosis; mesoderm development; angiogenesis; hemopoiesis; response to stimulus

                  42

                  2.50

                  UBXN1

                  UBX domain-containing protein 1

                    

                  43

                  2.49

                  MYOT

                  Myotilin;

                    

                  44

                  2.49

                  SHPRH

                  E3 ubiquitin-protein ligase

                  DNA helicase activity; nucleic acid binding

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; organelle organization; establishment or maintenance of chromatin architecture

                  45

                  2.49

                  SMYD1

                  SET and MYND domain-containing protein 1

                  DNA binding; transcription factor activity; transcription cofactor activity

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

                  46

                  2.48

                  KTN1

                  Kinectin

                  structural constituent of cytoskeleton

                  intracellular protein transport; cellular component morphogenesis

                  47

                  2.48

                  PJA2

                  E3 ubiquitin-protein ligase Praja2

                  DNA binding; transcription factor activity

                   

                  48

                  2.48

                  SRPK1

                  Serine/threonine-protein kinase SRPK1

                  kinase activity

                  immune system process; mitosis; cell surface receptor linked signal transduction; intracellular signaling cascade; carbohydrate metabolic process; protein metabolic process; cell motion; mitosis; signal transduction; segment specification; ectoderm development; mesoderm development; embryonic development; nervous system development; response to stress

                  49

                  2.47

                  ANKS3

                  Ankyrin repeat and SAM domain-containing protein 3

                    

                  50

                  2.47

                  C20orf26

                  Uncharacterized protein C20orf26

                    

                  51

                  2.47

                  TREML2

                  Trem-like transcript 2 protein

                    

                  52

                  2.46

                  TAB2

                  Mitogen-activated protein kinase kinase kinase 7-interacting protein 2

                  DNA binding; transcription factor activity

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

                  53

                  2.45

                  C19orf6

                  Membralin

                  structural molecule activity

                   

                  54

                  2.45

                  EPB41L2

                  Band 4.1-like protein 2

                    

                  55

                  2.44

                  CCDC71

                  Coiled-coil domain-containing protein 71

                    

                  56

                  2.44

                  FGD1

                  FYVE, RhoGEF and PH domain-containing protein 1

                  protein binding; small GTPase regulator activity; guanyl-nucleotide exchange factor activity

                  mesoderm development; skeletal system development

                  57

                  2.44

                  VCAM1

                  Vascular cell adhesion protein 1

                  hydrolase activity, acting on ester bonds; phosphatase activity; receptor activity

                  immune system process; muscle contraction; induction of apoptosis; cell cycle; cell surface receptor linked signal transduction; cell-cell signaling; cell-cell adhesion; protein metabolic process; cell motion; cell cycle; signal transduction; ectoderm development; mesoderm development; angiogenesis; nervous system development; muscle organ development

                  58

                  2.42

                  ANKRA2

                  Ankyrin repeat family A protein 2

                  DNA binding; transcription factor activity

                  antigen processing and presentation of peptide or polysaccharide antigen via MHC class II; cellular defense response

                  59

                  2.42

                  KCNK17

                  Potassium channel subfamily K member 17

                  cation transmembrane transporter activity; voltage-gated potassium channel activity; cation channel activity

                  neurological system process; cation transport

                  60

                  2.42

                  TXNDC15

                  Thioredoxin domain-containing protein 15

                    

                  61

                  2.41

                  TBPL2

                  TATA box-binding protein-like protein 2

                  DNA binding; transcription factor activity

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

                  62

                  2.40

                  ALDH3A2

                  Fatty aldehyde dehydrogenase

                  oxidoreductase activity

                  carbohydrate metabolic process; nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; cellular amino acid and derivative metabolic process

                  63

                  2.40

                  LAMP3

                  Lysosome-associated membrane glycoprotein 3

                   

                  lysosomal transport; intracellular protein transport; protein metabolic process

                  64

                  2.40

                  PNLIP

                  Pancreatic triacylglycerol lipase

                  hydrolase activity, acting on ester bonds

                  lipid metabolic process

                  65

                  2.37

                  C17orf28

                  UPF0663 transmembrane protein C17orf28

                    

                  66

                  2.36

                  GPR137

                  Integral membrane protein GPR137

                    

                  67

                  2.35

                  WARS

                  Tryptophanyl-tRNA synthetase, cytoplasmic

                  aminoacyl-tRNA ligase activity

                  protein metabolic process

                  68

                  2.34

                  C11orf35

                  Uncharacterized protein C11orf35

                    

                  69

                  2.34

                  SCML2

                  Sex comb on midleg-like protein 2

                  DNA binding; chromatin binding; transcription factor activity

                  cell cycle; nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; cell cycle; organelle organization; establishment or maintenance of chromatin architecture; ectoderm development; mesoderm development; nervous system development

                  70

                  2.33

                  GNL1

                  Guanine nucleotide-binding protein-like 1

                  GTPase activity; nucleic acid binding; receptor binding

                  intracellular protein transport; cell surface receptor linked signal transduction; intracellular signaling cascade; nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; signal transduction

                  71

                  2.33

                  MORN1

                  MORN repeat-containing protein 1

                  kinase activity

                  cell surface receptor linked signal transduction; signal transduction

                  72

                  2.33

                  TMEM149

                  Transmembrane protein 149

                    

                  73

                  2.32

                  AK4

                  Adenylate kinase isoenzyme 4, mitochondrial

                  kinase activity

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

                  74

                  2.32

                  TOMM34

                  Mitochondrial import receptor subunit TOM34

                   

                  immune system process; protein metabolic process; response to stress

                  75

                  2.32

                  ZNHIT6

                  Zinc finger HIT domain-containing protein 6

                    

                  76

                  2.31

                  BTG1

                  Protein BTG1

                   

                  cell cycle; intracellular signaling cascade; signal transduction

                  77

                  2.30

                  GRAMD1A

                  GRAM domain-containing protein 1A

                    

                  78

                  2.30

                  GRIK5

                  Glutamate receptor, ionotropic kainate 5

                  glutamate receptor activity; ligand-gated ion channel activity

                  neurological system process; cation transport; cell surface receptor linked signal transduction; synaptic transmission; signal transduction; cell-cell signaling

                  79

                  2.30

                  TDRKH

                  Tudor and KH domain-containing protein

                  hydrolase activity, acting on ester bonds;nucleic acid binding

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

                  80

                  2.30

                  TGIF1

                  Homeobox protein TGIF1

                  DNA-directed RNA polymerase activity; DNA binding; transcription factor activity

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; ectoderm development; nervous system development

                  81

                  2.29

                  FAM81B

                  Protein FAM81B

                    

                  82

                  2.29

                  MRPL9

                  39S ribosomal protein L9, mitochondrial

                  structural constituent of ribosome; nucleic acid binding

                  protein metabolic process

                  83

                  2.28

                  ANKRD46

                  Ankyrin repeat domain-containing protein 46

                    

                  84

                  2.27

                  INSM1

                  Insulinoma-associated protein 1

                   

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

                  85

                  2.27

                  PBX1

                  Pre-B-cell leukemia transcription factor 1

                  DNA-directed RNA polymerase activity; DNA binding; transcription factor activity

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; ectoderm development; mesoderm development; nervous system development; hemopoiesis

                  86

                  2.27

                  TPRG1

                  Tumor protein p63-regulated gene 1 protein

                    

                  87

                  2.26

                  SEPT4

                  Septin-4

                  GTPase activity;structural constituent of cytoskeleton; protein binding

                  mitosis; cytokinesis

                  88

                  2.25

                  FAM159B

                  UPF0514 membrane protein FAM159B

                    

                  89

                  2.24

                  AKAP8

                  A-kinase anchor protein 8

                   

                  mitosis; chromosome segregation

                  90

                  2.24

                  FAM83A

                  Protein FAM83A

                    

                  91

                  2.24

                  MAG

                  Myelin-associated glycoprotein

                  receptor activity; structural constituent of myelin sheath; receptor binding

                  B cell mediated immunity; cell surface receptor linked signal transduction; cell-cell adhesion; signal transduction; ectoderm development; nervous system development; response to stimulus

                  92

                  2.24

                  SCN9A

                  Sodium channel protein type 9 subunit alpha

                  cation transmembrane transporter activity; voltage-gated sodium channel activity; cation channel activity

                  neuronal action potential propagation; cation transport

                  93

                  2.23

                  ASTN1

                  Astrotactin-1

                    

                  94

                  2.23

                  RAB7L1

                  Ras-related protein Rab-7 L1

                  GTPase activity; protein binding

                  neurotransmitter secretion; intracellular protein transport; exocytosis; endocytosis; cell cycle; cell surface receptor linked signal transduction; intracellular signaling cascade; signal transduction

                  95

                  2.22

                  RASA3

                  Ras GTPase-activating protein 3

                  protein binding; small GTPase regulator activity

                  signal transduction

                  96

                  2.22

                  TMEM59L

                  Transmembrane protein 59-like

                    

                  97

                  2.21

                  CACNB3

                  Voltage-dependent L-type calcium channel subunit beta-3

                  cation transmembrane transporter activity; voltage-gated calcium channel activity; cation channel activity

                  muscle contraction; neurotransmitter secretion; synaptic transmission; cell-cell signaling

                  98

                  2.21

                  PLCD3

                  1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase delta-3

                  hydrolase activity, acting on ester bonds; calcium ion binding

                  cell surface receptor linked signal transduction; lipid metabolic process; signal transduction

                  99

                  2.21

                  PLCH1

                  1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase eta-1

                  hydrolase activity, acting on ester bonds; calcium ion binding; receptor binding; small GTPase regulator activity; guanyl-nucleotide exchange factor activity

                  cell surface receptor linked signal transduction; intracellular signaling cascade; lipid metabolic process; signal transduction

                  100

                  2.21

                  TULP1

                  Tubby-related protein 1

                   

                  visual perception; sensory perception; ectoderm development; nervous system development

                  101

                  2.20

                  EFCAB6

                  EF-hand calcium-binding domain-containing protein 6;EFCAB6

                  calcium ion binding; receptor binding; calmodulin binding; enzyme regulator activity

                  cation transport; cell cycle;signal transduction; cell cycle; signal transduction

                  102

                  2.19

                  PTPN6

                  Tyrosine-protein phosphatase non-receptor type 6

                  hydrolase activity, acting on ester bonds; phosphatase activity; receptor activity

                  immune system process; intracellular protein transport; mitosis; cell surface receptor linked signal transduction; intracellular signaling cascade; cell-matrix adhesion; cell-cell adhesion; protein metabolic process; cytokinesis; cell motion; signal transduction; nervous system development; cellular glucose homeostasis

                  103

                  2.18

                  GPR155

                  Integral membrane protein GPR155

                  receptor activity

                   

                  104

                  2.18

                  PSPN

                  Persephin

                  receptor binding

                  neurological system process; cell surface receptor linked signal transduction; cell-cell signaling; signal transduction; ectoderm development; nervous system development

                  105

                  2.18

                  STAM2

                  Signal transducing adapter molecule 2

                  transmembrane transporter activity; protein binding; kinase activator activity; kinase regulator activity

                  lysosomal transport; intracellular protein transport; endocytosis; intracellular signaling cascade; signal transduction

                  106

                  2.17

                  AGXT2L2

                  Alanine--glyoxylate aminotransferase 2-like 2

                  transaminase activity

                  visual perception; sensory perception; vitamin biosynthetic process; cellular amino acid and derivative metabolic process

                  The list of up-regulated genes in cancer cells grown as a co-culture with carcinoma-associated fibroblasts (CAFs). Gene ID, name, molecular function and biological process are listed according to the Panther Database classification www.​pantherdb.​org. The analyzed genes were significantly up-regulated at the level of p < 0.05, fold change > 2.0.

                  Table 3

                  Down-regulated genes in cancer cells grown as a co-culture with CAFs

                  No

                  Fold change

                  Gene ID

                  Gene name

                  Molecular function

                  Biological process

                  1

                  2.17

                  EPS8L1

                  Epidermal growth factor receptor kinase substrate 8-like protein 1

                   

                  intracellular signaling cascade; cell motion; signal transduction

                  2

                  2.17

                  OR4X1

                  Olfactory receptor 4X1

                    

                  3

                  2.18

                  C2orf61

                  Uncharacterized protein C2orf61

                    

                  4

                  2.18

                  DSN1

                  Kinetochore-associated protein DSN1 homolog

                    

                  5

                  2.18

                  PFAS

                  Phosphoribosylformylglycinamidine synthase

                  ligase activity

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

                  6

                  2.18

                  SLC5A2

                  Sodium/glucose cotransporter 2

                  carbohydrate transmembrane transporter activity; cation transmembrane transporter activity

                  cation transport; extracellular transport; amino acid transport; carbohydrate metabolic process; cellular amino acid and derivative metabolic process

                  7

                  2.18

                  TMEM138

                  Transmembrane protein 138

                    

                  8

                  2.19

                  FOLR1

                  Folate receptor alpha

                  receptor activity;transmembrane transporter activity

                  vitamin transport

                  9

                  2.19

                  MFN2

                  Mitofusin-2

                  hydrolase activity, acting on ester bonds; phosphatase activity

                  intracellular protein transport; organelle organization; mitochondrion organization

                  10

                  2.19

                  NDRG3

                  Protein NDRG3

                    

                  11

                  2.19

                  UNC13D

                  Protein unc-13 homolog D

                   

                  intracellular protein transport; exocytosis

                  12

                  2.20

                  GOLPH3

                  Golgi phosphoprotein 3

                    

                  13

                  2.20

                  SLC22A13

                  Solute carrier family 22 member 13

                  ATPase activity, coupled to transmembrane movement of substances; ligase activity; carbohydrate transmembrane transporter activity; cation transmembrane transporter activity

                  cation transport; anion transport; extracellular transport; carbohydrate transport; carbohydrate metabolic process

                  14

                  2.20

                  USP54

                  Inactive ubiquitin carboxyl-terminal hydrolase 54

                  ubiquitin-protein ligase activity

                  protein metabolic process

                  15

                  2.22

                  NMI

                  N-myc-interactor

                  DNA binding; transcription factor activity; transcription cofactor activity

                  response to interferon-gamma; intracellular signaling cascade; signal transduction; cellular defense response

                  16

                  2.22

                  PDCD1

                  Programmed cell death protein 1

                    

                  17

                  2.22

                  SNRPN

                  Small nuclear ribonucleoprotein-associated protein N

                  RNA splicing factor activity, transesterification mechanism; RNA binding

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

                  18

                  2.23

                  SPSB1

                  SPRY domain-containing SOCS box protein 1

                    

                  19

                  2.24

                  ADAMTS15

                  A disintegrin and metalloproteinase with thrombospondin motifs 15

                  peptidase activity; protein binding; peptidase inhibitor activity

                  fertilization; signal transduction; cell-matrix adhesion; cell-cell adhesion; protein metabolic process; signal transduction; ectoderm development; mesoderm development; skeletal system development; angiogenesis; nervous system development; muscle organ development

                  20

                  2.24

                  MTRF1L

                  Peptide chain release factor 1-like, mitochondrial

                  translation factor activity, nucleic acid binding; translation release factor activity

                  protein metabolic process

                  21

                  2.24

                  TOMM7

                  Mitochondrial import receptor subunit TOM7 homolog

                  transmembrane transporter activity

                  intracellular protein transport

                  22

                  2.25

                  ATG7

                  Autophagy-related protein 7

                  ligase activity

                  intracellular signaling cascade; coenzyme metabolic process; protein metabolic process; signal transduction

                  23

                  2.25

                  C19orf52

                  Uncharacterized protein C19orf52

                    

                  24

                  2.25

                  USF2

                  Upstream stimulatory factor 2

                  DNA binding; transcription factor activity

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; lipid metabolic process

                  25

                  2.26

                  POGK

                  Pogo transposable element with KRAB domain

                  DNA binding

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; ectoderm development; nervous system development

                  26

                  2.26

                  ZNF804B

                  Zinc finger protein 804B

                    

                  27

                  2.27

                  CD274

                  Programmed cell death 1 ligand 1

                  ubiquitin-protein ligase activity; receptor activity; DNA binding; receptor binding; transcription factor activity; transcription cofactor activity

                  immune system process; neurotransmitter secretion; intracellular protein transport; exocytosis; signal transduction; synaptic transmission; nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; protein metabolic process; cell-cell signaling; organelle organization; establishment or maintenance of chromatin architecture; mesoderm development; mammary gland development; response to stress; cellular defense response

                  28

                  2.27

                  FAM132B

                  Protein FAM132B

                    

                  29

                  2.27

                  STK32C

                  Serine/threonine-protein kinase 32 C

                  kinase activity

                  cell cycle; intracellular signaling cascade; protein metabolic process; cell cycle; signal transduction

                  30

                  2.28

                  CADM4

                  Cell adhesion molecule 4

                  receptor activity

                   

                  31

                  2.28

                  GLIPR2

                  Golgi-associated plant pathogenesis-related protein 1

                   

                  immune system process

                  32

                  2.28

                  GPR160

                  Probable G-protein coupled receptor 160

                    

                  33

                  2.29

                  GALNT2

                  Polypeptide N-acetylgalactosaminyltransferase 2 soluble form

                  transferase activity, transferring glycosyl groups

                  carbohydrate metabolic process; protein metabolic process

                  34

                  2.29

                  KRT20

                  Keratin, type I cytoskeletal 20

                  structural constituent of cytoskeleton

                  cellular component morphogenesis; cellular component morphogenesis; ectoderm development; cellular component morphogenesis

                  35

                  2.29

                  PTPN6

                  Tyrosine-protein phosphatase non-receptor type 6

                  hydrolase activity, acting on ester bonds; phosphatase activity; receptor activity

                  immune system process; intracellular protein transport; mitosis; cell surface receptor linked signal transduction; intracellular signaling cascade; cell-matrix adhesion; cell-cell adhesion; protein metabolic process; cytokinesis; cell motion; mitosis; signal transduction; nervous system development; cellular glucose homeostasis

                  36

                  2.29

                  SOHLH1

                  Spermatogenesis- and oogenesis-specific basic helix-loop-helix-containing protein 1

                    

                  37

                  2.31

                  CENPM

                  Centromere protein M

                    

                  38

                  2.31

                  HPCAL1

                  Hippocalcin-like protein 1

                  calcium ion binding; calmodulin binding; small GTPase regulator activity

                  visual perception; sensory perception; cell surface receptor linked signal transduction; signal transduction

                  39

                  2.31

                  TMEM81

                  Transmembrane protein 81

                    

                  40

                  2.32

                  HOXA1

                  Homeobox protein Hox-A1

                  DNA binding; transcription factor activity

                  female gamete generation; nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; segment specification; ectoderm development; gut mesoderm development; embryonic development; skeletal system development; angiogenesis; nervous system development; muscle organ development

                  41

                  2.32

                  SMO

                  Smoothened homolog

                  G-protein coupled receptor activity; receptor binding

                  cell surface receptor linked signal transduction; cell-cell signaling

                  42

                  2.33

                  LAMP2

                  Lysosome-associated membrane glycoprotein 2

                   

                  lysosomal transport; intracellular protein transport; protein metabolic process

                  43

                  2.33

                  RNF121

                  RING finger protein 121

                  ubiquitin-protein ligase activity

                  protein metabolic process

                  44

                  2.34

                  PLA2G2E

                  Group IIE secretory phospholipase A2

                  hydrolase activity, acting on ester bonds

                  signal transduction; lipid metabolic process; signal transduction

                  45

                  2.34

                  POU6F2

                  POU domain, class 6, transcription factor 2

                  DNA binding; transcription factor activity

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

                  46

                  2.34

                  PPP2R4

                  Serine/threonine-protein phosphatase 2A regulatory subunit B'

                  protein binding;phosphatase activator activity; phosphatase regulator activity

                  protein metabolic process

                  47

                  2.34

                  STX5

                  Syntaxin-5

                  SNAP receptor activity

                  neurotransmitter secretion; intracellular protein transport; exocytosis; endocytosis; synaptic transmission; cell-cell signaling

                  48

                  2.35

                  DMC1

                  Meiotic recombination protein DMC1/LIM15 homolog

                  hydrolase activity; DNA binding

                  immune system process; meiosis; nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; meiosis; response to stress

                  49

                  2.35

                  ERRFI1

                  ERBB receptor feedback inhibitor 1

                   

                  signal transduction;signal transduction

                  50

                  2.36

                  TSPYL4

                  Testis-specific Y-encoded-like protein 4

                  protein binding; phosphatase inhibitor activity; phosphatase regulator activity

                  apoptosis; cell cycle; nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; protein metabolic process; cell cycle; organelle organization; establishment or maintenance of chromatin architecture

                  51

                  2.37

                  USF1

                  Upstream stimulatory factor 1

                  DNA binding; transcription factor activity

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; lipid metabolic process

                  52

                  2.40

                  ARFGAP3

                  ADP-ribosylation factor GTPase-activating protein 3

                  nucleic acid binding; protein binding; small GTPase regulator activity

                  cell surface receptor linked signal transduction; cell adhesion

                  53

                  2.40

                  CNBP

                  Cellular nucleic acid-binding protein

                  nucleic acid binding

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; lipid metabolic process

                  54

                  2.40

                  NKD1

                  Protein naked cuticle homolog 1

                    

                  55

                  2.41

                  MRPL51

                  39S ribosomal protein L51, mitochondrial;

                  structural constituent of ribosome; nucleic acid binding

                  protein metabolic process

                  56

                  2.41

                  OPRK1

                  Kappa-type opioid receptor

                  G-protein coupled receptor activity

                  sensory perception; cell surface receptor linked signal transduction; synaptic transmission; cell motion; signal transduction; cell-cell signaling

                  57

                  2.41

                  PTX3

                  Pentraxin-related protein PTX3

                   

                  immune response; response to stress; defense response to bacterium

                  58

                  2.42

                  GPRC5B

                  G-protein coupled receptor family C group 5 member B

                  G-protein coupled receptor activity

                  cell surface receptor linked signal transduction; signal transduction

                  59

                  2.42

                  NGLY1

                  Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase

                  hydrolase activity

                  protein metabolic process

                  60

                  2.43

                  CAPN12

                  Calpain-12

                  peptidase activity; calcium ion binding; calmodulin binding; calcium-dependent phospholipid binding

                  induction of apoptosis; intracellular signaling cascade; protein metabolic process; signal transduction

                  61

                  2.43

                  POLD1

                  DNA polymerase delta catalytic subunit

                  DNA-directed DNA polymerase activity; hydrolase activity, acting on ester bonds; nucleic acid binding

                  cell cycle; nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; cell cycle

                  62

                  2.44

                  TBC1D8

                  TBC1 domain family member 8

                  hydrolase activity; protein binding; small GTPase regulator activity

                  intracellular protein transport; exocytosis; cellular component morphogenesis

                  63

                  2.45

                  DHRS11

                  Dehydrogenase/reductase SDR family member 11

                  oxidoreductase activity

                  visual perception; sensory perception; lipid metabolic process

                  64

                  2.46

                  LZTR1

                  Leucine-zipper-like transcriptional regulator 1

                  structural constituent of cytoskeleton; DNA binding; chromatin binding; protein binding; small GTPase regulator activity; transcription factor activity

                  spermatogenesis; immune system process; intracellular protein transport; vesicle-mediated transport; cell cycle; nitrogen compound metabolic process; nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; protein metabolic process

                  65

                  2.46

                  OR6V1

                  Olfactory receptor 6 V1

                    

                  66

                  2.47

                  C12orf65

                  Uncharacterized protein C12orf65

                  translation factor activity, nucleic acid binding; translation release factor activity

                  protein metabolic process

                  67

                  2.47

                  C2orf56

                  Protein midA homolog, mitochondrial

                    

                  68

                  2.47

                  RBM42

                  RNA-binding protein 42

                  RNA splicing factor activity, transesterification mechanism; DNA binding; RNA binding

                  spermatogenesis; neurological system process; cell cycle; nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; protein metabolic process; ectoderm development; nervous system development

                  69

                  2.48

                  DPM2

                  Dolichol phosphate-mannose biosynthesis regulatory protein

                    

                  70

                  2.48

                  TDRD12

                  Tudor domain-containing protein 12

                  RNA helicase activity; translation factor activity, nucleic acid binding; translation initiation factor activity

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; protein metabolic process

                  71

                  2.53

                  CCDC85B

                  Coiled-coil domain-containing protein 85B

                    

                  72

                  2.54

                  MATN1

                  Cartilage matrix protein

                  extracellular matrix structural constituent

                  immune system process; sensory perception of sound;sensory perception; signal transduction; cell-cell adhesion; cellular component morphogenesis; mesoderm development; skeletal system development; blood coagulation

                  73

                  2.55

                  FLYWCH1

                  FLYWCH-type zinc finger-containing protein 1

                    

                  74

                  2.55

                  PLA2G2D

                  Group IID secretory phospholipase A2

                  hydrolase activity, acting on ester bonds

                  signal transduction; lipid metabolic process; signal transduction

                  75

                  2.56

                  TMEM38A

                  Trimeric intracellular cation channel type A

                    

                  76

                  2.58

                  RGS11

                  Regulator of G-protein signaling 11

                  protein binding;small GTPase regulator activity

                  cell surface receptor linked signal transduction; signal transduction; dorsal/ventral axis specification

                  77

                  2.61

                  BAHD1

                  Bromo adjacent homology domain-containing 1 protein

                  DNA binding

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

                  78

                  2.67

                  CHD5

                  Chromodomain-helicase-DNA-binding protein 5

                  DNA helicase activity; nucleic acid binding

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; organelle organization; establishment or maintenance of chromatin architecture

                  79

                  2.67

                  CHD5

                  Tryptophan-rich protein;

                  structural constituent of ribosome; nucleic acid binding

                  protein metabolic process

                  80

                  2.67

                  CYP39A1

                  Cytochrome P450 39A1

                  oxidoreductase activity

                  respiratory electron transport chain; lipid metabolic process

                  81

                  2.68

                  SUDS3

                  Sin3 histone deacetylase corepressor complex component SDS3

                   

                  cell cycle

                  82

                  2.69

                  C16orf62

                  UPF0505 protein C16orf62

                    

                  83

                  2.69

                  FLOT2

                  Flotillin-2

                   

                  intracellular protein transport; vesicle-mediated transport

                  84

                  2.69

                  NINJ1

                  Ninjurin-1

                   

                  neurological system process; cell adhesion; cell adhesion; ectoderm development; nervous system development

                  85

                  2.70

                  FAM84A

                  Protein FAM84A

                    

                  86

                  2.71

                  PAF1

                  RNA polymerase II-associated factor 1 homolog

                    

                  87

                  2.71

                  PAF1

                  Peroxisome assembly factor 1

                   

                  protein metabolic process

                  88

                  2.77

                  POLR2F

                  DNA-directed RNA polymerases I, II, and III subunit

                  DNA-directed RNA polymerase activity; nucleic acid binding

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

                  89

                  2.78

                  C12orf34

                  Uncharacterized protein C12orf34

                    

                  90

                  2.80

                  GGT6

                  Gamma-glutamyltransferase 6 light chain

                  acyltransferase activity; peptidase activity

                  cellular amino acid and derivative metabolic process; protein metabolic process

                  91

                  2.83

                  TSC22D4

                  TSC22 domain family protein 4

                  DNA binding; transcription factor activity

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

                  92

                  2.84

                  CLRN2

                  Clarin-2

                    

                  93

                  2.92

                  KCNS1

                  Potassium voltage-gated channel subfamily S member 1

                  cation transmembrane transporter activity; voltage-gated potassium channel activity; cation channel activity

                  muscle contraction; blood circulation; neuronal action potential propagation; cation transport; signal transduction; synaptic transmission; signal transduction; cell-cell signaling

                  94

                  2.94

                  OR51T1

                  Olfactory receptor 51 T1

                    

                  95

                  2.96

                  OR51I1

                  Olfactory receptor 51I1

                    

                  96

                  3.01

                  ZNF135

                  Zinc finger protein 135

                  DNA binding;transcription factor activity

                  nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

                  97

                  3.03

                  QDPR

                  Dihydropteridine reductase

                  oxidoreductase activity

                  cellular amino acid and derivative metabolic process

                  98

                  3.11

                  C9

                  Complement component C9b

                  peptidase activity; receptor activity

                  complement activation; signal transduction; cell-cell adhesion; protein metabolic process; signal transduction; response to stimulus

                  99

                  3.16

                  VDAC1

                  Voltage-dependent anion-selective channel protein 1

                  voltage-gated ion channel activity; anion channel activity

                  anion transport

                  100

                  3.85

                  FDX1L

                  Adrenodoxin-like protein, mitochondrial

                  oxidoreductase activity

                  respiratory electron transport chain; ferredoxin metabolic process; vitamin metabolic process; lipid metabolic process; protein metabolic process

                  The list of down-regulated genes in cancer cells grown as a co-culture with carcinoma-associated fibroblasts (CAFs). Gene ID, name, molecular function and biological process are listed according to the Panther Database classification www.​pantherdb.​org. The analyzed genes were significantly down-regulated at the level of p < 0.05, fold change > 2.0.

                  The PANTHER binomial statistics tool to compare classifications of multiple clusters of lists to a reference list of Canis familiaris genes allowed us to statistically determine over-manifestation of PANTHER biological process and pathways classification categories. The PANTHER biological process analysis revealed that most of the up-regulated genes in cancer cells grown as a co-culture with the CAFs were involved in: cell surface receptor linked signal transduction (p = 9.05E-03), lysosomal transport (p = 9.2E-03), developmental process (p = 3.64E-02), antigen processing and presentation (p = 4.22E-02), signal transduction (p = 4.35E-02), cell communication (p = 4.55E-02), nervous system development (p = 4.97E-02).

                  Because our concern involved interactions between cancer cells and carcinoma associated fibroblasts which could predispose cancer to metastasis, we specifically focused on the up-regulated genes involved in cell adhesion and cellular morphogenesis: QRICH2, CHAD, FOXQ1, PCSK6, CLEC7A, SH3BP1, NEFM, PCDH19, LPHN2, DSP, KTN1, VCAM1, MAG, and PTPN6 (Table 2). Interestingly, the gene expression analysis also revealed an up-regulation of 24 genes involved in developmental processes such as: a mammary gland development, a mesoderm development, an ectoderm development, a skeletal system development, a nervous system development, an embryonic development, a heart development, and a muscle-organ development. The pathway analysis revealed that significantly over-manifested were: the salvage pyrimidine ribonucleotides (p = 02.87E-03), the oxytocin receptor mediated signaling pathway (p = 4.76E-03), the thyrotropin-releasing hormone receptor signaling (p = 5.21E-03), the 5HT2 type receptor mediated signaling pathway (p = 6.99E-03), the metabotropic glutamate receptor III (p = 8.14E-03), the metabotropic glutamate receptor group II (p = 3.33E-02), the metabotropic glutamate receptor I (p = 1.75E-02), the Beta 2 and Beta1 adrenergic receptor signaling (p = 2.54E-02) and the histamine H1 receptor mediated signaling (p = 2.87E-02).

                  Among the down-regulated genes no significant pathways or biological processes and their over-manifestation were found (Table 3) comparing to a reference list of Canis familiaris genes.

                  The results were confirmed at mRNA level using real-time qPCR analysis

                  For the purposes of the microarray data validation, we have randomly selected 3 of all the genes that may play the most important role in the cancer cells-CAFs interactions: PCDH19, DSP and MAG. Real-time qPCR results showed similar trends in gene expression changes as were observed in microarray studies (Figure 5).
                  http://static-content.springer.com/image/art%3A10.1186%2F1746-6148-8-35/MediaObjects/12917_2011_354_Fig5_HTML.jpg
                  Figure 5

                  Selected genes expression assessed using real-time qPCR. Expression of randomly selected genes in canine mammary cancer lines growing as a monoculture and co-culture with CAFs. The changes in gene expression differed highly significant (p < 0.001, N = 3).

                  MUC1 expression detection in cancer cell lines

                  Because the MAG gene up-regulation was found in cancer cells grown as a co-culture with the CAFs, the expression of MUC1 (which binds MAG) was examined immunohistochemically (Figure 6). The MUC1 expression was confirmed in all of the examined cell lines.
                  http://static-content.springer.com/image/art%3A10.1186%2F1746-6148-8-35/MediaObjects/12917_2011_354_Fig6_HTML.jpg
                  Figure 6

                  MUC1 expression in canine mammary cancer cell lines. MUC1 expression (brown color) in canine mammary cancer cell lines: A. CMT-U27, B. CMT-U309, C. P114, D. CMT-W1 and E. CMT-W2. The pictures were obtained using Olympus BX60 microscope (at the magnification of 200×).

                  Discussion

                  Canine mammary cancers in bulk arise from epithelial cells. Several genetic alterations have been detected, that may predispose these cells to the malignant transformation [1012]. However, researches over the last few years suggested that concomitant changes also occur in stromal cells that form the tumour microenvironment [1, 2]. The hypothesis of stromal cells involvement in tumorigenesis is based on a study of embryological development where interactions between various cells are necessary for programming and maintaining epithelial structure and function. The embryonic epithelial and stromal cells of mesenchymal origin engage in a molecular dialogue that ensures the proper organ development and function [3].

                  The study showed in cancer cells after a co-culture with CAFs an up-regulation of 23 genes (Table 2) involved in developmental processes (a nervous system development, an embryonic development, a mesoderm and ectoderm development).

                  The involvement of fibroblasts in the malignant transformation of epithelial cells has previously been documented [3, 2628]. Moreover, the histology and growth characteristics of CAFs were found different from those of the fibroblasts associated with normal epithelial cells [3]. Mishra et al. [29] have proposed bidirectional cross-talk between the CAFs and the cancer cells which release proteins that increase the fibroblasts ability to secrete a variety of tumour-promoting factors, which then act back on the malignant cells to change their gene expression and promote their proliferative, migratory, and invasive properties. On the other hand, other studies showed that only direct contact of fibroblasts with cancer cells is able to cause changes in their gene expression and biology [3032].

                  So far several papers have been published about gene expression in tumour microenvironment. Most of them describe gene expression in fibroblasts, but not in cancer cells, however there are some papers available about the changes in gene expression in cancer cells [3336]. These reports indicated up-regulation of genes involved in angiogenesis, EMT and migration in cancer cells grown with fibroblasts. Surprisingly, some of the genes identified, even though functionally identical turned out to be of different names. The studies have been conducted using various cancer models (various species) and various cell lines, so the differences are possible.

                  The results of the study hereby revealed increased expression of 13 genes involved in cell adhesion (Table 2) among cancer cells co-cultured with the CAFs. As much as 10 of them are involved in developmental processes as well. These genes seem to be particularly significant because the cell adhesion is responsible for tumour progression and metastasis, detachment from the primary tumour and spreading to the circulatory system. Moreover, the up-regulated genes responsible for adhesion are by rule involved in angiogenesis and lymphangiogenesis. For example, the vascular cell adhesion molecule-1 (VCAM-1) up-regulated in cancer cells grown as a co-culture with CAFs may be involved in tumor progression and metastasis particularly via lymphangiogenesis promotion [37, 38]. It also has previously been demonstrated that the VCAM-1 plays a crucial role in the endothelial-carcinoma cell adhesion [38].

                  The study also revealed an up-regulation of desmoplakin (DSP), which is a key component of cellular adhesion junctions known as desmosomes. These junctions are found at contact sites between endothelial cells that form capillaries, thus DSP play a role in de novo capillary formation and branching during tumourigenesis, embryonic development and cardiovascular development [39]. Moreover, desmoplakin isoform 2 was only detected in tumours associated with a poor clinical outcome. It may suggest its potentially specific function in the regulation of cancer cells proliferation, differentiation, invasion and metastasis [40, 41].

                  Moreover, desmosomes may also be important in the epithelium-mesenchymal transition (EMT). The epithelium-mesenchymal transition is an indispensable mechanism for morphogenesis during embryonic development, and is implicated in conversion of early-stage tumours into invasive cancers. During EMT, epithelial cells undergo changes in morphology and acquire the migratory and invasive characteristics of mesenchymal cells [30]. EMT is also promoted by the FOXQ1, another up-regulated gene in cancer cells grown under co-culture conditions with the CAFs [42]. It also increases expression of several junction proteins promoting cancer cells to gain the stem-cell-like properties and ensuring resistance to apoptosis [4244]. Moreover, the down regulation of keratin 20 (Table 3) in cancer cells following the co-culture with the CAFs may indicate the EMT induction [45, 46].

                  Interestingly, another up-regulated gene in cancer cells grown with the CAFs, which contributes to cancer invasion is myelin-associated glycoprotein (MAG) that binds to the oncogenic glycoprotein MUC1 [47]. Swanson et al. [47] described an interaction between the MUC1 and the MAG in cancers that invade perineurally, including prostate, salivary, and breast carcinomas. Furthermore, breast cancers may metastasize to the brain where the MAG is abundantly expressed. Interactions between the MUC1 and the MAG have not fully been defined yet. We confirmed the MUC1 expression in all of the examined cell lines (Figure 6). Thus, based on our own observations and those of Swanson et al. [47], we suppose that the MAG up-regulation in cancer cells grown with the CAFs and its binding to the MUC1 may contribute to the adhesion between tumour cells and Schwann cells promoting metastasis to the nervous system.

                  We also found a down-regulation of 5 key genes associated with adhesion. Subject literature suggests 3 of them play a role in cancer development. The down-regulation of these genes is associated with poor prognosis and cancer metastases. One of these genes is the ADAMTS15 (a disintegrin and metalloproteinase with thrombospondin motif 15) which is an anti-angiogenic factor [48]. Our study also revealed a down-regulation of the CADM4. Nagata et al. [49] found decrease in the CADM4 expression in most of renal cell carcinomas and the cancer cell lines. Moreover, the CADM4 expression was decreased in carcinomas with vascular infiltration, suggesting that loss of the CADM4 is involved in tumour angiogenesis and invasion. We have also found a down-regulation of the MATN1 gene which has been defined an angiogenesis inhibitor [50].

                  In the current study we showed a significant over-manifestation of genes involved in the oxytocin receptor mediated signaling pathway, the thyrotropin-releasing hormone receptor signaling, the Beta 2 and Beta1 adrenergic receptor signaling, and the histamine H1 receptor mediated signaling in cancer cells grown with the CAFs. Entschladen et al. [51] described the role of neurotransmitters in cancer progression and metastasis. They found that similarly to chemokines, neurotransmitters are regulators of cell migration. Sadly though, we noticed that only a few results are available on the expression of neurotransmitter receptors in tumour tissues. Among them the best understood is the role of catecholamines in carcinogenesis and tumour progression. These are the stress hormones, whereas stress in turn is a major risk factor for the development of cancer. Norepinephrine has been shown to strongly induce the migration of tumor cells [52, 53], whereas epinephrine was found a modulator for the carcinogenesis in the lung [54].

                  An interesting gene in cancer cells grown as a co-culture with the CAFs is the up-regulated protocadherin 19 (PCDH19) (Table 2). Up-to-date there is no information available on the involvement of this gene in tumor progression or metastasis. However, a PCDH19 mutation was found to be responsible for epilepsy and mental retardation confined to females (EFMR) [55]. There has been an on-going debate about the relationship between epilepsy and cancer. It has been hypothesized that the incidence of cancer is increased in people with epilepsy owing to the cancer promotion by antiepileptic drugs [56]. Perhaps the increased risk of cancer in epileptic patients is caused by the PCDH19 mutation and over-expression, not however related to drugs toxicity. This hypothesis requires further studies.

                  Conclusions

                  The results of the current study showed that the co-culture of cancer cells and the CAFs caused significant changes in expression of genes involved in adhesion, angiogenesis and the EMT that take part in developmental processes.

                  Declarations

                  Acknowledgements

                  This work was supported by grant no N N308012939 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. Małgorzata Gajewska for the confocal analysis and Dr. Alicja Majewska for Bioanalyzer analysis.

                  Authors’ Affiliations

                  (1)
                  Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences - WULS
                  (2)
                  Department of Animal Environment Biology, Faculty of Animal Sciences, Warsaw University of Life Sciences - WULS
                  (3)
                  Institute of Computer Engineering, Control and Robotics I-6, Wroclaw University of Technology
                  (4)
                  Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences - WULS
                  (5)
                  Area Diagnostica Integrata Istologia e Microscopia Elettronica Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche

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                  © Król et al; licensee BioMed Central Ltd. 2012

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