the microenvironment is a dynamic system that is largely composed of inflammatory cells and cytokines , including tumor cells and stroma ( the latter of which consists of immune cells and the surrounding extracellular matrix ) [ 1 , 2 ] . under specific stimulation conditions , inflammation can result in the production of antiangiogenic cytokines , which suppress tumor growth . despite being antagonistic to the action of tumor suppression , in most tumors , inflammatory cells can promote angiogenesis , growth , tumor cell migration , tissue degradation , and remodeling steps that are characteristic of the chronic inflammation associated with intense tissue disorders typical of acute inflammation [ 1 , 37 ] . the transcription factor nuclear kappa b ( nf-b ) plays a major role in innate immunity and inflammatory responses   and it is considered the major link between inflammation and tumorigenesis . this factor activates antiapoptotic genes [ 911 ] and could promote the transcription of genes associated with growth , invasion , and metastasis , being the key factor that allows precancerous and malignant cells to escape apoptosis . interleukins ( ils ) could stimulate many signaling pathways and thus regulate the transcription of target genes involved in cellular proliferation and survival . besides that , they act as immune mediators   and have been linked to cancer , playing a key role in the recruitment and positioning of leukocytes in carcinogenesis . the recruitment and activation of immune cells and the antitumor effects of il-4 , an anti - inflammatory cytokine , are involved in the inhibition of angiogenesis , inflammation , thrombosis , growth , and invasion in some cancers [ 16 , 17 ] . in addition to these effects on stromal cells , some studies have reported that il-4 has antitumor functions by inducing apoptosis in several types of tumor cells including breast cancer , renal cell carcinoma , and hepatocellular carcinoma . il-10 is a cytokine produced by th1 and th2 cells   as well as by tumor cells and has potent immunosuppressive properties . originally known as an inhibitor factor of the synthesis of cytokines , il-10 suppresses the production of il-1 , il-1 , tumor necrosis factor- ( tnf- )  , il-6 , il-8 , il-12 , il-18 , granulocytes , inflammatory macrophages , and nf-b [ 7 , 21 , 22 ] . this cytokine acts concurrently with il-3 and il-4 to stimulate the proliferation of mast cells , peripheral blood lymphocytes , and macrophages , and their anti - inflammatory action has been suggested in tumor microenvironment . on the other hand , il-6 has strong procarcinogenic activity due to its role in tumor cell proliferation , survival , angiogenesis , inflammation , and metastasis [ 7 , 23 ] . in addition , il-6 is one of the activation signals for nf-b , promoting cell differentiation and subsequent metastasis , thus reinforcing the close link between inflammatory mediators and neoplastic progression . therefore , tnf- induces proinflammatory cytokines such as il-1   and il-6 [ 26 , 27 ] , during the inflammatory response [ 2628 ] . the interaction of this cytokine with its receptor ( tnfr ) induces apoptotic and antiapoptotic effects in different cell systems , and it can promote cell proliferation and inhibits apoptosis in the tumor microenvironment , at least indirectly , through the induction of nf-b . a better understanding of the complexity of these biological factors ' network , together with the characterization of new genes , aids in uncovering the molecular mechanisms of inflammatory diseases and cancer . in this way , the objective of this work was to ascertain the role of anti - inflammatory cytokines , il-4 and il-10 , and proinflammatory cytokines , il-6 and tnf- , in mammary tumors of female dogs and to correlate these results with the gene and protein expression of nf-b . in addition , we sought to verify the correlation of these cytokines with clinicopathological parameters , cancer progression , and overall survival , to determine their prognostic value in mammary tumors . peripheral blood and tumor samples were obtained from 30 female dogs with malignant mammary neoplasia ( test group ) attended to in so jos do rio preto , sp , brazil , veterinary clinics in the years 2011 and 2012 . animals with mammary tumors were included regardless of breed , weight , or age . these animals were followed up for 540 days after tumor excision and blood collection . for control group , peripheral blood samples from 30 female dogs and five samples of mammary tissue adjacent to mammary cancer were obtained from animals that underwent elective hysterectomy as a preventive measure , after completion of the consent form by their owners . the criteria for the control group were rigorously followed and included animals outside the estrus period , animals with no tumor history , and animals with no detectable disease / inflammation in the period or surgery in the next study period . in addition , animals that died of causes other than tumor recurrence or metastasis were excluded from the study . peripheral blood ( 3  ml ) was collected in a corvac serum separator tube ( labor import , so paulo , sp ) , stored at 4c , processed by centrifugation ( 1000  g , 25  min ) , and cryopreserved at 80c for posterior enzyme linked immunosorbent assay ( elisa ) and xmap protein analysis . the first one was fixed in 10% formaldehyde buffer for 24 hours and paraffin embedded for histopathology classification after hematoxylin - and - eosin ( he ) stain . tumor grading was ascertained according to misdorp et al .   by the armed forces institute of pathology ( afip ) . the second piece was collected in a falcon tube containing the rna - stabilizing solution , rnalater ( life technologies ) , stored at room temperature for 24 hours , and posteriorly used for rna extraction and qpcr analysis . female dogs from the test group were evaluated with respect to physical ( age ) , pathological ( time course ( interval between tumor diagnosis and surgical removal ) , tumor nodules location , lymph node involvement , tumor mass size , clinical staging , ulceration , and vascularization ) , and clinical ( metastasis , local recurrence , and death ) characteristics ( table 1 ) . the parameters employed for the classification of clinical tumor grading were in accordance with the tnm ( size , lymph node involvement , and metastasis ) system established by the world health organization ( who ) for canine mammary gland tumors . the clinical grade was assigned as i , ii , iii , or iv according to the extent of the tumor and the prognosis . the age of the animals varied from 7 to 14 years ( mean = 10 years ) and there was predominance of poodle ( 19% ) and dachshund ( 24% ) races . in terms of the clinicopathological characteristics , most animals had a tumor for more than six months ( 54% ) and were of clinical stage iii or iv ( 54% ) . the proportions of animals with lymph node involvement , animals with metastasis / local recurrence , or animals that died during the study were 16% , 27% , and 27% , respectively ( table 1 ) . for total rna extraction , the tumor samples were manually processed into smaller pieces ( 100  mg / piece ) , and the pieces were then immersed in trizol reagent ( invitrogen life technologies ) and macerated with a politron ( dremel , mexico ) . previously , tumor samples were treated with dnase and 10x buffer ( 10  mm tris - hcl , ph 7.5 ; 50  mm cacl2 ; and 10  mm mgcl2 ) ( rneasy mini kit , qiagen ) followed by the addition of ethylenediaminetetraacetic acid ( edta ) to deactivate or eliminate proteins binding to template dna . the qpcr was performed in triplicate using the stepone plus real time pcr system ( applied biosystems ) and taqman universal master mix ( applied biosystems ) . the following specific primers were used : nf-b , cf02622551_m1 ( applied biosystems ) ; il-4 , cf02623112_m1 ( applied biosystems ) ; il-6 , cf02624153_m1 ( applied biosystems ) ; il-10 , cf02624265_m1 ( applied biosystems ) ; and tnf- , cf02628236_m1 ( applied biosystems ) . the following qpcr conditions were used : an initial step of 2 minutes at 50c ; denaturation for 10 minutes at 95c ; 40 cycles at 95c for 15 seconds and 60c for one minute to anneal primers and extend the chains , respectively ; and 35 seconds at 65c to collect the signal . the dissociation curve was generated after amplification and included a step of 15 seconds at 95c followed by 1  min at 60c . each transcript level was normalized to the expression of rps19 ( sense ( 5-gcc ttc ctc aaa aag tct ggg-3 ) , antisense ( 5-gct tgc tcc cta cga tga gaa c-3 ) , and probe ( 5-ccc tga atg ggt gga c-3 ) ( applied biosystems ) ) and rpl8 , cf02663820_m1 ( applied biosystems ) , which were used as endogenous control genes . the relative quantification ( rq ) value of the expression of interest genes was determined with dataassist v3.0 , by the quantification method related to the average of the normalizing genes used as endogenous controls ( ct )   and showed by log10 . the gene expression of each sample was separately analyzed and classified as underexpressed ( samples giving < 0 log10 measurement ) and overexpressed ( samples with quantification > 0 log10 ) . nf-b ( dog nuclear factor ( nf ) kappa b p105 subunit ( nf-b1 ) , cusabio biotech co. , hubei , china ) and il-4 ( uscn life science inc . , houston , tx , usa ) protein expression analysis was performed according to the manufacturers recommendations . first of all , the plate was precoated with a specific antibody for nf-b and il-4 . then , the standards and samples were added in each well and incubated for 2 hours at 37c . a biotin - conjugated antibody and a horseradish peroxidase- ( hrp- ) avidin conjugate were added to the plate to amplify the signal and to increase the detectability of the target molecule . a chromogenic substrate ( 3 - 3 - 5 - 5-tetramethylbenzidine , tmb ) finally , sulfuric acid ( stop solution ) was added to stabilize the color development to enable accurate measurement of the intensity at 450  nm . the analysis of il-6 , il-10 , and tnf- proteins was performed using the milliplex map kit ( ccytomag-90k ; millipore corporation , usa ) , and the protein levels were analyzed using the luminex xmap magpix ( millipore corporation , usa ) . the standard , control , and samples were added to the appropriate wells as were magnetic beads coated with specific antibodies for il-6 , il-10 , and tnf-. the plate was sealed and incubated overnight at 4c with agitation on a plate shaker . measurements were performed at 635  nm to excite the magnetic beads and 525  nm to detect phycoerythrin . the calculation of od was determined through the adjustment curve four - parameter logistic ( 4-pl ) using the skanit software for the multiskan fc 2.5.1 . clinicopathological characteristics , gene expression , and protein quantification were analyzed in the test and control groups , and the differences between these groups were determined by student 's t - test or anova followed by the bonferroni test . the cutoff points for the protein analyses were established by the receiver operating characteristic ( roc ) curve . for the roc curve , the protein expression levels from female dogs that died were compared with those that survived until the end of the followup period . survival curves were plotted by the kaplan - meier method and the differences between the curves were evaluated by a log - rank test and hazard function . spearman 's rank correlation analysis was performed to describe the correlation between proinflammatory and anti - inflammatory cytokines protein and gene expression with nf-b . in addition , the mutual relationship between proinflammatory and anti - inflammatory cytokines was analyzed . a multivariate logistic regression analysis was employed to evaluate the simultaneous influence of the prognostic factors on animal death . the nf-b gene was significantly overexpressed in animals with tumor time course greater than six months ( p < 0.0001 ) ; multiple tumor locations ( p = 0.005 ) ; abundant tumor vascularization ( p = 0.009 ) ; tumor mass size greater than three centimeters ( cm ) ( p = 0.005 ) ; lymph node involvement ( p = 0.008 ) ; metastasis ( p = 0.02 ) ; recurrence ( p = 0.01 ) ; and clinical stage iii or iv ( p < 0.0001 ) ( figure 1(a ) ) . in the same way , the nf-b protein expression was significantly higher in animals older than 10 years ( p = 0.0001 ) , as well as in animals with a tumor time course of less than six months ( p = 0.001 ) , multiple tumor locations ( p = 0.01 ) , abundant tumor vascularization ( p = 0.03 ) , and metastasis ( p = 0.0005 ) ( figure 1(b ) ) . the best cutoff point for nf-b established by the roc curve to discriminate the high risk of death was 219.5  pg / ml ( sensitivity ( 95% ci ) = 71% , specificity ( 95% ci ) = 41% ) . no correlation was observed between nf-b serum levels and survival time ( or 2.003 ; 95% ci = 0.5553 to 8.983 ; p = 0.29 ) ( data not shown ) . il-4 was significantly overexpressed in animals aged  10 years ( p = 0.02 ) and animals with tumor time course of less than six months ( p = 0.01 ) ; single tumor location ( p = 0.0001 ) ; moderate tumor vascularization ( p = 0.0002 ) ; tumor mass size less than three centimeters ( p = 0.0006 ) ; no lymph node involvement ( p = 0.001 ) ; metastasis ( p < 0.0001 ) ; recurrence ( p = 0.0003 ) ; and clinical stage i or ii ( p < 0.0001 ) and animals that were still alive at the end of the followup period ( p < 0.0001 ) ( figure 2(a ) ) . therefore , il-4 protein expression was significantly higher in animals aged  10 years ( p < 0.0001 ) and animals with tumor time course of less than six months ( p = 0.04 ) ; moderate tumor vascularization ( p = 0.03 ) ; tumor mass size of less than three centimeters ( p = 0.001 ) ; no metastasis ( p = 0.01 ) ; no recurrence ( p = 0.02 ) ; and clinical stage i or ii ( p = 0.008 ) and animals that were still alive at the end of the followup period ( p = 0.005 ) ( figure 2(b ) ) . using the roc curve , the best cutoff point for il-4 to discriminate the high risk of death was 368.4  pg / ml ( sensitivity ( 95% ci ) = 88% , specificity ( 95% ci ) = 95% ) . the kaplan - meier test demonstrated a positive correlation between il-4 level and survival time ( or 0.068184 ; 95% ci = 0.783029 to 1 ; p = 0.007 ) ( figure 3 ) . for il-10 , significant gene overexpression was also correlated with tumor time course of less than six months ( p = 0.003 ) ; moderate tumor vascularization ( p = 0.02 ) ; no lymph node involvement ( p = 0.0005 ) ; no metastasis ( p = 0.01 ) ; no recurrence ( p = 0.01 ) ; clinical stage i or ii ( p = 0.02 ) ; and animals that were still alive at the end of the followup period ( p = 0.009 ) ( figure 4(a ) ) . likewise , il-10 protein expression was significantly higher in animals with tumor time course of less than six months ( p = 0.003 ) ; single tumor location ( p = 0.0009 ) ; moderate vascularization ( p = 0.04 ) ; no recurrence ( p = 0.04 ) , and clinical stage i or ii ( p = 0.03 ) and animals that were still alive during the followup ( p = 0.04 ) ( figure 4(b ) ) . for the roc curve for il-10 , the best cutoff point to discriminate the high risk of death was 243  pg / ml ( sensitivity ( 95% ci ) = 100% , specificity ( 95% ci ) = 53% ) . no correlation was observed between il-10 protein expression and survival time ( or 0.3267 ; 95% ci = 0.06811 to 1.564 ; p = 0.16 ) ( data not shown ) . in contrast , il-6 was significantly overexpressed in animals aged > 10 years ( p = 0.03 ) and animals with multiple tumor locations ( p = 0.0005 ) ; abundant tumor vascularization ( p = 0.003 ) ; tumor mass size greater than three centimeters ( p = 0.01 ) ; lymph node involvement ( p = 0.04 ) ; metastasis ( p = 0.003 ) ; recurrence ( p = 0.03 ) ; and clinical stage iii or iv ( p = 0.02 ) and animals that died during followup ( p = 0.008 ) ( figure 5(a ) ) . similarly , il-6 protein expression was significantly higher in animals with tumor time course greater than six months ( p = 0.001 ) ; multiple tumor locations ( p = 0.01 ) ; abundant vascularization ( p = 0.02 ) ; tumor mass size greater than three centimeters ( p = 0.001 ) ; lymph node involvement ( p < 0.0001 ) ; metastasis ( p < 0.0001 ) ; recurrence ( p = 0.007 ) ; and clinical stage iii or iv ( p = 0.001 ) and animals that died during followup ( p = 0.04 ) ( figure 5(b ) ) . the best cutoff point for il-6 established by the roc curve to discriminate the high risk of death was 76.95  pg / ml ( sensitivity ( 95% ci ) = 75% , specificity ( 95% ci ) = 86% ) . no correlation was observed between il-6 protein expression and survival time ( or 1.719 ; 95% ci = 0.4045 to 9.580 ; p = 0.43 ) ( data not shown ) . tnf- was also overexpressed in animals with tumor time course greater than six months ( p = 0.01 ) ; multiple tumor locations ( p = 0.005 ) ; abundant tumor vascularization ( p = 0.007 ) ; tumor mass size greater than three centimeters ( p = 0.02 ) ; metastasis ( p = 0.001 ) ; recurrence ( p = 0.003 ) ; and clinical stage iii or iv ( p = 0.0002 ) and animals that died during followup ( p = 0.005 ) ( figure 6(a ) ) . the protein expression of tnf- was significantly higher in animals aged > 10 years ( p = 0.04 ) and animals with tumor time course greater than six months ( p = 0.004 ) ; abundant vascularization ( p = 0.01 ) ; lymph node involvement ( p = 0.0007 ) ; metastasis ( p = 0.02 ) ; and recurrence ( p = 0.01 ) and animals that died during followup ( p = 0.04 ) ( figure 6(b ) ) . the best cutoff point to discriminate the high risk of death established by the roc curve was 16.3  pg / ml ( sensitivity ( 95% ci ) = 87% , specificity ( 95% ci ) = 62% ) . this analysis demonstrated a negative correlation between tnf- protein expression and survival time ( or 5.906 ; 95% ci = 1.387 to 25.6 ; p = 0.01 ) ( figure 7 ) . spearman 's rank correlation demonstrated that proinflammatory cytokines il-6 and tnf- are mediated by nf-b . there was a positive correlation between the proinflammatory tnf- protein and gene expression when compared to nf-b protein and gene expression ( r = 0.5233 , p = 0.003 ; r = 0.6645 , p < 0.0001 ; figures 8(a ) and 8(b ) , resp . ) . in the same way , there was a positive correlation between il-6 and nf-b gene expression ( r = 0.4012 ; p = 0.02 ) ( figure 8(b ) ) . furthermore , the mutual balance between proinflammatory cytokines and anti - inflammatory ones was analyzed by spearman 's rank correlation and showed no correlation ( p > 0.05 ; data not shown ) . the multivariate analysis of high levels of the proinflammatory cytokines , low levels of the anti - inflammatory cytokines , and clinicopathological features of poor prognosis with regard to an increased risk of death showed no correlation ( p > 0.05 ) ( table 2 ) . nf-b is a crucial factor between chronic inflammation and cancer development   and it regulates both anti- and proinflammatory cytokines at various stages of tumorigenesis and inflammation [ 35 , 36 ] . we observed high gene and protein expression levels of nf-b in animals with tumor progression for greater than six months , multiple tumor locations , abundant vascularization , and metastasis , indicating a role of this factor in angiogenesis support and inhibition of apoptosis in tumor cells . in accordance with our prediction , kiliccioglu et al .   observed that nf-b inhibition , through proteasome inhibition , induces apoptosis in human prostate cancer cell line ( pc3 ) , resulting in significantly increased protein levels of caspase-3 , which plays a central role in the execution phase of cell apoptosis . similarly , chien et al .   reported that pancreatic cancer cell growth is suppressed by inhibiting the nf-b pathway . in the same way , high protein levels of nf-b were observed in female dogs above 10 years old which could indicate the correlation between this nuclear factor and cell oxidative stress , leading to tissue damage and chronic inflammation . corroborating with this , a review of oxidative stress and breast cancer , recently published by nourazarian et al . , suggests that the increase of free radical levels in tumor cells occurs under the influence of the increased expression of cellular enzymes and enhanced activity of some dependent tumor cells , such as cancer - associated fibroblasts ( cafs ) and tumor - associated macrophages ( tams ) [ 39 , 40 ] . once nf-b has been activated through one of many pathways , crucial properties of the malignant phenotype are activated , promoting tumor cell adaptation to the tumor environment ; then , we also reported nf-b overexpressed in female dogs with tumor masses greater than three centimeters , lymph node involvement , recurrence , and clinical stage iii or iv , leading to a poor survival rate of the animals . it is known that nf-b is required for the expression of many cytokine genes that encode proteins associated with inflammatory mediators . then , through spearman 's rank correlation , we showed that high expression of nf-b modulates positively the il-6 and tnf- expression levels in mammary tumors . the proinflammatory cytokines il-1 , il-6 , and tnf- could be modulated by lipopolysaccharides ( lps ) administration as it occurs in inflammatory diseases . according to cheon et al . , some drugs , as saxifragin , inhibited the lps - induced nuclear translocation of p65 and the activation of caspase-1 in raw 264 cells , suggesting that the nf-b - regulated gene transcription is responsible for anti - inflammatory response of macrophages . in addition , osnes et al .   affirmed that the inhibitory synthesis of tnf- by drugs as acetylsalicylic acid and sodium salicylate in human monocytes is due to interfering of nuclear translocation of nf-b / c - rel proteins . besides that , corroborating with our study , al - halabi et al .   showed that the inhibition of nf-b significantly lowers microvessel density ( cd31 ) and mrna expression levels of il-6 , tnf- , and il-1 , as well as protein levels of proliferation marker ( ki-67 ) and vascular endothelial growth factor- ( vegf- ) a in human colon cancer , confirming the primordial role of nf-b in angiogenesis and , therefore , tumor progression . regarding anti - inflammatory interleukins , our study sustains a protector action of the anti - inflammatory cytokines il-4 and il-10 in female dogs ' mammary tumors . il-4 was highly expressed in gene and protein levels in female dogs with a better prognostic . this cytokine acts on endothelial cells , inhibiting tumor - induced vascularization and starving tumor cells , and also induces secretion of il-10 , the th2 cytokine with a strong suppressive effect on tumors [ 46 , 47 ] , which could explain high levels of this cytokine in animals that are still alive after the followup period and in overall survival . furthermore , according to okada and kuwashima , the sustained expression of il-4 may provide effective means for therapy of a variety of diseases , including cancer . in addition , high gene and protein expression of il-10 were correlated with characteristics of better prognosis . il-10 downregulates proinflammatory cytokine expression [ 48 , 49 ] and inhibits the expression of cd31 . lin and karin   reported that il-10 could modulate apoptosis and suppress angiogenesis during tumor regression , downregulating vegf , tnf- , and il-6 production by tams . jindal and borges   showed that il-10 inhibits tumor growth by preventing chemokine expression and angiogenesis when administered at higher doses in cancer animal models . correlated high il-10 expression in female dogs with no metastasis or recurrence and high overall survival . contrarily , gene overexpression of il-10 in animals that had metastasis could suggest posttranscriptional regulation of this cytokine , once the protein levels do not show this correlation . il-6 mediates a plethora of physiological functions , including the developmental differentiation of lymphocytes , cell proliferation , and cell survival during tumorigenesis [ 48 , 53 ] . this cytokine had high gene and protein expression correlated with poor prognosis and a negative correlation was found in animals that died during the followup . in the same way , a study by madeddu et al . demonstrated high expression of il-6 through activation of akt / pi3k / mtor cascade due to elevated oxidative stress , promoting oncogenesis and tumor progression . owing to its association with poor prognosis , researchers have proposed il-6 as a therapeutic target in cancer . several phase i / ii clinical trials are currently evaluating antibodies against il-6 or il-6r as therapeutic alternatives for prostate cancer [ 53 , 55 ] and renal cancer [ 56 , 57 ] . these results suggest that further studies are needed to determine the appropriate use of anti - interleukin monoclonal antibodies as a therapeutic treatment , and studies similar to ours , evaluating interleukin expression , may be useful as a foundation for further clinical trials of therapy using cytokines . increasing lines of evidence suggest that tnf- regulates many of the critical processes of tumor promotion and progression [ 48 , 58 ] . in our study , high protein levels of tnf- were found in animals that died during the followup and correlated with worse prognostic features . its cytokine has been implicated yet as a tumor promoter in different tumor types like ovarian cancer , gallbladder cancer , and oral squamous cell carcinoma . tnf- binds to two receptors , namely , the ubiquitously expressed tnf receptor 1 ( tnfr1 ) and hematopoietically restricted tnfr2 , and modulates a signaling cascade that induces mediators of transcriptional regulation that are key to cell survival , invasion , angiogenesis , and impairment of immune surveillance in tumor biology [ 49 , 58 , 59 , 62 ] . altogether , our data showed the effect of cytokines on tumor development and progression , as well as positive modulation of nf-b and proinflammatory interleukins in mammary tumors . analysis of the active genes and proteins in cancer can be used to target treatment more specifically , and our results suggest the use of these biological factors as prognostic biomarkers in cancer . our results are of utmost importance for a better understanding of the dynamic network of cytokines and the influence of nf-b on tumorigenesis . these cytokines could be employed as noninvasive prognostic markers in female dogs with mammary tumors and could be useful for predicting disease progression and tumor recurrence . there is lack of studies devoted to diagnostic , therapeutic , and prognostic markers in canine mammary tumors ; so , this study defines potential prognostic and predictive markers for routine use in the clinic , allowing the detection of recurrence and metastasis , which could allow the early adoption of more precise conduct to improve the overall survival of animals .