glioblastomas are the most aggressive type of intracranial tumours , highly resistant to combined treatment , in patients displaying a median survival time of 15 months . the molecular mechanisms underlying these clinical features are the existence of specific genetic and molecular profiles of these tumour cells . recent reports show genomic instability ( especially in tumours from short - term survival patients ) , chromosomal alterations , somatic mutations , and polymorphisms . knowing this particular brain tumour cell , one can wonder if , besides the intrinsic cellular features , the inflammatory milieu triggered by the development of such a tumour can not influence the particular clinical development in glioblastomas as well . the relationship between inflammation and cancer has first been suggested in modern time , by virchow in 1863 , who found  lymphoreticular infiltrates  in neoplastic tissues , consequently suggesting that these reflect the origin of cancer of sites of chronic inflammation . massive experimental proofs appeared in the recent years to support virchow 's concept .   in a synthetic formulation , inflammation is defined as  the seventh hallmark of cancer  , by colotta et al . . the body 's response to cancer has many analogies with inflammation and repair ; the inflammatory cells and cytokines present in tumours are more likely to contribute to tumour growth , progression , and immunosuppression , rather than in building an effective antitumour defence . cancer susceptibility and severity are often associated with functional polymorphisms in cytokine genes . as plastically described by balkwill and mantovani , if genetic damage is the  match that lights the fire  of cancer , some types of inflammation may provide the  fuel that feeds the flames  . tumour initiation and progression is a complex process involving genomic mutations , micro environmental factors , and inflammatory mediators . within the tumour environment inflammatory markers are responsible for cell proliferation , tumour invasion , marked angiogenesis , and suppression of certain immune functions . inflammation represents the immune system response to external or internal aggressors , such as injury or infection in certain tissues . typical signs of inflammation include swelling , redness , pain , temperature rise , and subsequently loss of function . numerous studies have shown that the majority of tumour tissues are associated with inflammatory signs . however the most common primary neoplasm of the central nervous system , composing over 40% of all such tumours , with an incidence ranging from 8% to 27% . a broad array of cytokines displays modified expression in cancers , including glioblastoma multiforme [ 7 , 8 ] . the changes arise from the interaction of tumour cells and nontumour cells , like macrophages , lymphocytes , or stromal cells , and provide regulatory support for tumour growth , angiogenesis , invasion , and metastasis [ 810 ] . the vascular system of brain cancers inappropriately expresses membrane proteins , resulting in blood extravasation . the production of inflammatory mediators ( such as cytokines and nitric oxide ) , and tumour hypoxia have been involved in these effects . pain belongs to the  classical  markers of inflammation , described over 2000 years ago by aulus celsus ( calor , rubor , tumour et dolor ) . various molecular actors of inflammation , including mediators of pain have been described in the recent years ; a key role appears to be played by cytokines , which sometimes appear to conduct the orchestra of small molecule mediators , such as nitric oxide and prostaglandins . several cancer risk factors like cigarette smoke , alcohol and growth factors can activate signaling pathways related with inflammation ( such as nfb and stat3 signaling ) . some chronic infections lead to inflammatory conditions and are associated with carcinogenesis ( e.g. , hepatitis b virus ) . chemotherapeutic agents and gamma irradiation can also interfere in the regulation of expression of some genes implicated in inflammation , survival , proliferation , invasion , angiogenesis , and cancer metastasis . inflammation may be involved in carcinogenesis through mutations , genomic instability , and epigenetic modifications . also , inflammation can participate in premalignant cells proliferation , stimulate angiogenesis , and promote metastatic spread ( figure 1 ) .    among the cytokines , often found to be over expressed at tumour level , il1-beta , tnf - alpha , il-6 , il-10 , ifn - gamma , cx3cl1 , to name just a few , have been closely related to pain for a long time [ 1416 ] . in the case of glioblastoma , headache is one of the most frequently claimed signs by the patients , but also , very often the diagnostic is set too late for a successful therapeutical approach .   acknowledging the worldwide research effort in the field of glioblastoma , we have embarked in the study of circulatory cytokines to pinpoint the serum inflammatory pattern that can characterize the glioblastoma patient 's evolution . therefore , our study investigated the serum levels of several pro- and anti - inflammatory cytokines and of angiogenic factors in brain tumour patients diagnosed in stages iii and iv ( glioblastoma ) , in order to establish their roles and behaviour in tumour progression . samples ( serum ) were collected from 55 patients with glioblastoma ( 28 men and 27 women ; mean ages : 58 and 62 years , resp . , range : 3779 years ) from neurology and neurovascular diseases national institute , elias hospital neurosurgery department and 20 controls ( healthy individuals with no known history of inflammatory or neoplastic diseases , 12 men and 8 women ; mean age : 57 years , range : 2570 years ) . written informed consent has been obtained upon sample prelevation according to helsinki ii declaration and ethics committee of victor babes national institute of pathology that has approved the study . the collection of total peripheral blood from patients and controls has been achieved in vacutainers ( systems , becton dickinson ) without anticoagulant . the xmap assay was performed according to the manufacturers ' protocols , and the plates were analysed using luminex 200 system . cytokines levels and angiogenic factors were determined using the human cytokine 12-plex kit , with 12 analyte - specific bead sets ( simultaneous quantification)pro - inflammatory il-1 , il-2 , il-6 , il-8 , tnf , gm - csf , and inf , anti - inflammatory il-4 , il-10 , and il-12 , and angiogenic factors vegf and fgf-2 . values for individual proteins measured by this multiplexed protein array technology have been shown to correlate with single elisa measurements . all samples were assayed in triplicate , and the mean values of cytokines were taken into account . optical density was measured at 450  nm on an anthos zenith 3100 multimode micro plate reader . minimum detectable concentrations were found to be less than 9.0  pg / ml for vegf and less than 3.0  pg / ml for bfgf . data were collected and expressed as the mean  standard error of three independent repeats . differences between groups were analysed by one way anova ; p values less than 0.05 were considered statistically significant ; pearson correlation ( r , p ) was used to explore the association between cytokine expressions . from multiplex assay ( luminex 200 ) a strong overexpression was detected for il-6 , il-1 , tnf- , and il-10 ( over 3-fold stimulation in glioblastoma patients ) . significant up - regulation ( up to 2-fold ) was found for vegf , fgf-2 , il-8 , il-2 , and gm - csf . cytokines expression was significantly higher and strongly correlated with tumour grade , proliferation markers , and clinical aggressiveness in glioblastomas . comparing the patient groups and control for growth factors , the obtained values by xmap array were comparable to the outline obtained by the elisa analysis . based on xmap analysis , the changes in average serum levels ( compared to the controls ) are presented in figure 2 . several molecules display a modification in plasma levels of more than 2-fold , which is , as a general practice , a criterion of acceptance as potential marker . however , it also appears evident that , for several cytokines , the intervals of variations in patients were broad . further details on expression are provided in figures 3 , 4 , and 5 , where the distributions can be better examined and also cover the behaviour of controls . the enhanced expression of il-1 appears to directly correlate with il-6 and il-8 levels and inversely correlate with il-4 . in brain , il-1 regulates survival and invasiveness of glioblastoma cells , and anti - il-1 antibodies inhibit both the growth and invasion of glioblastoma cells . wang et al . showed that in ln-229 glioma cell line , il-1 and tgf- can induce glioma stem cells phenotype and contribute to carcinogenesis . enhanced secretion of il-1 , il-6 , and il-8 by glioma cells was reported by yeung et al . , and these cytokines are related with the expansion of gbm ( glioblastoma multiforme ) . in other types of cancers ( gastric and oesophageal ) , il-1 was involved in carcinogenesis and proliferation and played a crucial role in the development of chemical carcinogen - induced tumours [ 20 , 21 ] . the determined serum levels are consistent with the ability of tumour cell to secrete pro - inflammatory cytokines , as well as with il-6 role in stimulation of angiogenesis . according to our data , il-6 expression correlates with il-1 , il-8 , and ifn-. ancrile et al . showed il-6 is involved in carcinogenesis by angiogenesis and tumour growth and may be a potential anti - invasion target . in u251 , t98 g and u87 mg glioblastoma cell lines , il-6 promotes vascular endothelial cell migration and facilitates tumour angiogenesis and invasion [ 23 , 24 ] . amplification of the il-6 gene in patients with glioblastoma multiforme is correlated with decreased survival . serum levels of tnf appeared significantly enhanced ( p = 2.5e  8) , suggesting a strong correlation with the disease ; however , the correlation with other molecules is not so strong , suggesting its implication in distinct / complementary regulatory cascades . hagemann et al . suggested that tnf is involved in tumour cell invasion through upregulation of migration - inhibitory factor ( mif ) and through enhanced mmps production in tumour cells via nf-- and jnk - signalling . in ovarian cancer , tnf stimulated other cytokines ( il-6 ) , angiogenic factors ( vegf ) , and chemokines ( ccl2 and cxcl12 ) that promoted tumour growth and metastases . other studies have showed that tnf over - expression enhances migration and metastasis through induction of cxcr4 , mcp-1 , and il-8 and matrix metalloproteinase [ 28 , 29 ] . recent studies on u373 mg and c6 human glioma cell lines showed that tnf- induces il-6 synthesis through the jak / stat3 pathway and tnf inhibitors can reduce tumour cell invasion [ 30 , 31 ] . il-8 has been found to be up - regulated ( fold stimulation 1.9 ) in patient sera , compared to controls . many studies showed that il8 is upregulated in gliomas and is involved in the promotion of angiogenesis . in pten - deficient glioblastoma cells , studies on murine models showed that transplanted glioblastoma tumour cells , which express high levels of il-2 , il-4 , or gm - csf show enhanced tumour survival . u87-mg glioblastoma cell line expressed high levels of gm - csf , and gm - csf over - expression is found exclusively in cultures derived from astrocytomas .  
 in glioblastoma patients , il-2 overexpression averaged 2-fold , but the patient group has been distributed in one subgroup of patients ( 55% ) who displayed strongly enhanced levels of expression ( up to 5-fold ) while 45% of the patients displayed a moderate enhancement of expression  on average 1.4-fold increase . statistical analysis ( t - test ) suggests that the two subgroups represent distinct subpopulation of glioblastoma patients , and so further investigations and integration of more data is required to consolidate and explain this segregation . il-2 is reported in use for cancer treatment , as a stimulator of t - cell mediated anti - tumour activity .   anti - inflammatory cytokines il-4 and il-12 appear at lower level in patient 's sera at 5780% compared to controls group ( figure 3 ) . il-4 is involved in inhibition of cell proliferation , regulation of adhesion molecules , and induction jak / stat signalling ; il-4 receptor is overexpressed in malignant glioma cell lines and tumour specimens from patients with glioblastoma , but his mechanism is still unclear . many studies on murine models showed that il-12 is a powerful anticancer factor which can inhibit growth of implanted glioblastoma and the increase survival time . il-10 levels are significantly increased ( p < 0.001 ) . at a first glance , the strong increase in il-10 looks paradoxical in the general balance of pro- and anti - inflammatory cytokines ; however , the finding confirms previous reports of kumar et al . , who reported significant increase of il-10 serum levels in patients with anaplastic astrocytoma and glioblastoma . the increase in serum levels of il-10 may also be correlated with glioma induced immunosuppression . the same study also detected significant decrease in serum levels of glioblastoma and anaplastic astrocytoma , suggesting a systemic impact of brain tumours on the immune system . rt - pcr and immunoassay studies on glioblastoma showed that il-10 expression is significantly higher in stem - cell - derived tumour sphere cells than in primary cultured glioma cells from the same tumour . il-10 is significantly overexpressed in high grade tumours and can contribute to progression of astrocytomas . serum levels of angiogenic factors were considerably elevated in glioblastoma patients , as measured by xmap analysis and confirmed by elisa ; both vegf and bfgf were significantly overexpressed ( bfgf , 3.05-fold modification , p = 0.002 , vegf , 3.2-fold modification , p = 0.005 ) , see figure 5 . the expression and distribution profiles of angiogenic factors were similar in both detection methods , with an increase for bfgf of 2.99-fold in xmap analysis and 3.22 in elisa and for vegf of 3.12 and 3.08 , respectively . in gbm , vegf - vegfr2 signalling is maintained by continuous secretion of vegf ligand and promotes tumour growth , invasiveness and enhanced resistance to some treatments . anti - vegf therapy and vegfr inhibitors can delay progression of glioblastoma , but this mechanism is not well understood . in vitro and in vivo studies showed that stem - cell - like glioma cells secrete elevated levels of vegf induced by hypoxia , and anti - vegf therapy cancel proangiogenic effects of glioma [ 4143 ] . fgf-2 is involved in neoplastic transformation of glioma cells by activating ras / raf / erk signalling and can stimulate angiogenesis in glioblastoma [ 44 , 45 ] . our study was primarily aimed on the estimation of serum level of several cytokines and angiogenic factors in glioblastoma patients , in order to assess their suitability as diagnostic , prognostic and monitoring biomarkers . nevertheless , many components of the investigated panel are pleiotropic molecules , and , besides their primary role as regulators of cell behaviour , they also play major roles in inflammation and/or tumour related pain . the serum levels of il-1 , tnf , ifn- , and gm - csf were significantly increased in glioblastoma patients . a previous study of makimura et al . investigated the plasma levels of 26 cytokines in cancer patients in correlation with responses to morphine treatment ; they could not correlate the levels with pain levels , but were able to correlate some of the investigated molecules with responsiveness to morphine treatment . according to kawasaki et al . tnf and il-1 cause an increase in the activity of ampa ( 2-amino-3-(3-hydroxy-5-methyl - isoxazol-4-yl ) propanoic acid ) or nmda ( n - methyl - d - aspartate ) receptors , while il-1 and il-6 inhibit gamma - amino - butyric acid ( gaba ) and glycine - induced ion currents in rexed lamina ii nociceptors , demonstrating that these pro - inflammatory cytokines favour the increase in neuronal excitability . several studies showed that pro - inflammatory and anti - inflammatory cytokines dysregulation is associated with carcinogenesis and tumour progression of solid cancers , like pancreatic and colorectal [ 4850 ] . our findings demonstrate that cytokines and angiogenic factors levels are closely linked to the brain tumour behaviour . out of all potential biomarkers for glioblastoma staging and prognostic , a panel of inflammatory cytokines and angiogenic factors is more relevant than single molecules , as proven by our study . moreover , further investigation could generate a multimolecular panel for better patient stratification and more adequate therapeutical approaches . the involvement of cytokines in inflammation and pain , as well as the relevance of pain in glioblastoma makes them reliable targets for investigation , with potential diagnostics and therapeutic applications . the advantages of xmap technology could be less invasive techniques , screening for molecular markers , and validation of putative therapeutic targets . further analysis on protein expression and signalling , protein interaction networks , associated with the implementation of a clinical panel for pain scoring , may lead to establish more clearly the connection between mediators of inflammation , signaling pathways and targets for tumoural progression , cancer therapy , and cancer pain therapy . pain is a frequent symptom accused by glioblastoma patients and one of the primary signs conducting to investigation and diagnostics , but , unfortunately , most often the diagnostics appear late or too late ; the set of cytokines could add in speed and accuracy , making thus possible earlier diagnostics and onset of therapy .
inflammation represents the immune system response to external or internal aggressors such as injury or infection in certain tissues . 
 the body 's response to cancer has many parallels with inflammation and repair ; the inflammatory cells and cytokines present in tumours are more likely to contribute to tumour growth , progression , and immunosuppression , rather than in building an effective antitumour defence . using new proteomic technology , we have investigated serum profile of pro- ( il-1 , il-6 , il-8 , il-12 , gm - csf , and tnf- ) and anti - inflammatory cytokines ( il-4 , il-10 ) , along with angiogenic factors ( vegf , bfgf ) in order to assess tumoural aggressiveness . 
 our results indicate significant dysregulation in serum levels of cytokines and angiogenic factors , with over threefold upregulation of il-6 , il-1 , tnf- , and il-10 and up to twofold upregulation of vegf , fgf-2 , il-8 , il-2 , and gm - csf . 
 these molecules are involved in tumour progression and aggressiveness , and are also involved in a generation of disease associated pain .