dendritic cells ( dcs ) have been identified as a key component in manipulating and stimulating the immune system . activated dcs are potent antigen presenting cells that express both major histocompatibility complex ( mhc ) class i and ii molecules ( signal 1 ) and costimulatory molecules ( signal 2 ) and secrete immune modulating cytokines ( signal 3 ) resulting in activation of t lymphocytes . depending on the cytokine environment , dcs may elicit either a th ( t helper ) 1 or th2 cd4 t - cell response . for tumor immunotherapy , induction of a th1 t - cell response is pivotal , and secretion of il-12 ( interleukin 12 ) by dcs is of critical importance for differentiation of naive t cells into th1 cells . furthermore , il-12 stimulates the production of interferon - gamma ( ifn- ) and tumor necrosis factor - alpha ( tnf- ) from t cells and natural killer cells . in contrast , th2 responses , associated with cytokines il-4 , il-5 , il-6 , and il-10 , suppress th1 activity and may anergize effector t cells to tumor antigens . one of these strategies involves fusing dcs with tumor cells using electrical currents in a method called electrofusion , hence combining the antigen presenting properties of dcs with the full repertoire of antigens present within a tumor cell in order to stimulate effector t cells [ 6 , 7 ] . while dc - tumor hybrids alone are insufficient to elicit significant immune responses in vivo and are critically dependent upon exogenously administered 3rd signal adjuvants , murine studies using dc - tumor hybrids for vaccination given concomitantly with an adjuvant third signal , such as il-12 , ox-40- , 4 - 1bb - monoclonal antibody , or toll - like receptor agonists , showed regression of tumor metastases after a single vaccination in several tumor types including melanoma , breast , sarcoma , and squamous cell carcinoma [ 811 ] . however , systemic delivery of 3rd signal along with a dc - tumor fusion vaccine is clinically problematic due to 3rd signal toxicity and/or availability . therefore , a better understanding of the mechanisms affecting the dependence of dc - tumor fusions on 3rd signal adjuvants is of paramount importance for optimizing this immunotherapeutic approach . in this study , we show that production of the th1 skewing cytokine il-12 was dramatically downregulated in dc - tumor fusion cells . in addition , gene products that are involved in signaling pathways including nfb ( nuclear factor kappa - light - chain - enhancer of activated b - cells ) , pi3k / akt / mtor ( phosphatidylinositol 3-kinase / akt , protein kinase b / mammalian target of rapamycin ) , wnt ( wingless - related integration site ) , and mapk ( mitogen - activated protein kinase ) were differentially expressed in fusion cells . inhibitor studies revealed that interruption of the canonical wnt pathway did not affect il-12 production by dc - tumor fusion cells and that inhibition of mek ( mitogen extracellular signal - regulated kinase ) only increased il-12 production marginally . in contrast , il-12 production could significantly be enhanced by treatment of dc - tumor hybrids with inhibitors of the pi3k and mtor . given the critical role of the pi3k / akt / mtor signaling pathway in cancer biology and the immunostimulatory effect of pi3k / akt / mtor inhibitors on dc - tumor hybrids , combination therapy may represent a promising and novel cancer vaccine with enhanced clinical impact . animals were housed in a specific pathogen - free environment at the animal facility of the durham veteran affairs medical center . all mice used in this study were cared for in accordance with the guide for humane care and use of laboratory animals published by the national institutes of health . all the animal experimental protocols were approved by the duke university medical center institutional animal care and use committee . d5lacz is a -galactosidase expressing derivative of the b16 f10.9 melanoma cell line and has been shown to be poorly immunogenic . cells were cultured in complete media ( cm ) composed of rpmi 1640 media supplemented with 10% fetal bovine serum , 2  mm l - glutamine , 0.1  mm nonessential amino acids , 1  mm sodium pyruvate , 100  u / ml penicillin , 100  g / ml streptomycin , 0.5  g / ml fungizone , 50  g / ml gentamicin , and 5  10  m 2-mercaptoethanol ( invitrogen , carlsbad , ca ) . these cells were maintained at 37c with 5% co2 , harvested following a short incubation period with 0.05% trypsin with edta , and irradiated at 100  gy prior to use . b- and t - lymphocytes were depleted using antibody - coated magnetic beads ( dynal biotech , carlsbad , ca ) . the dc - enriched cell fraction was then cultured in cm supplemented with 10  ng / ml gm - csf and 10  ng / ml il-4 ( peprotech , rocky hill , nj ) at a concentration of 0.5  10  cells / ml at 37c with 5% co2 . on day 6 , cells were harvested , resuspended in fresh cm + gm - csf / il-4 media at 1  10  cells / ml , and incubated at 37c with 5% co2 for 24 hours . then , lps ( lipopolysaccharide , 100  ng / ml , sigma - aldrich , saint louis , mo ) was added to stimulate dc maturation . after 24 hours after 24 hours , dcs were stained intracellularly with cfse prior to use ( molecular probes , eugene , or ) . irradiated tumor cells and cfse stained dc were mixed in a 1  :  1 ratio and washed in prefusion media , followed by resuspension in fusion media at a concentration of 20  10  cells / ml . for electrofusion , the pulse generator ( model ecm 2001 generator , btx instruments , san diego , ca ) was used . cells were exposed to two consecutive , independent electrical currents : ( 1 ) a low voltage alternating current of 120  v / cm for 10 seconds to achieve alignment and chain formation , and ( 2 ) a high voltage direct current of 1100  v / cm for 25 microseconds to cause a reversible breakdown of cell membranes . the multinucleated hybrid cells were allowed to stand for at least 5 minutes before incubation in culture media overnight at 37c with 5% co2 . to separate unfused tumor cells ( t ) from t - t hybrids and unfused dcs from dc - dc hybrids facs sorting by size on forward scatter ( fsc ) and side scatter ( ssc ) dc - t hybrids were purified using a combination of mechanical and facs sorting techniques , based on their plastic adherence characteristics as well as cfse staining . therefore , after electrofusion and overnight culture , the nonadherent cell population representing unfused dcs and dc - dc hybrids was discarded . facs was then performed only on the adherent cell population containing unfused tumor cells , t - t hybrids , and dc - t hybrids . since only dcs were stained with cfse , facs sorting was used to separate cfse positive cells from the cfse negative populations ( unfused tumor cells and t - t hybrids ) . all cell samples were analyzed using the facs aria ii ( bd biosciences , san jose , ca ) . 24 hours after electrofusion , total rna was isolated using the rneasy plus mini kit protocol ( qiagen , valencia , ca ) . the cdna template was synthesized from 0.51.0  ug of total rna using the rt first strand kit protocol ( sabiosciences , frederick , md ) . each template was then combined with rt sybr green qpcr master mix ( sa biosciences ) and aliquoted into a 96-well mouse common cytokine plate array ( sa biosciences ) . the pcr cycling program was 95c for 10 minutes , followed by 40 cycles of 95c for 15 seconds , and then 60c for 1 minute on a stratagene mx3005p qpcr machine . briefly , all threshold values ( ct ) reported as greater than 35 indicated no detectable gene expression . genomic dna ( gdna ) contamination was detected if the gdna control ct value was below 35 . a reverse transcription control ( rtc ) detected impurities in the rna sample that affect the reverse transcription of the template and was considered positive if the ct was greater than 5 . gene expression associated with th1 ( ifn- , il-2 , il-12p40 , il-15 , il-18 , and tnf- ) and th2 ( il-4 , il-10 , il-13 , and il-25 ) immune responses was analyzed . total rna was isolated from tumor cells , dcs , and dc - t fusion cells using the rneasy plus mini kit protocol ( qiagen , valencia , ca ) . rna isolation for tumor and dendritic cells was done in triplicate . for the dc - t fusion cells , triplicate samples of d5lacz tumor cells , dcs , and dc - t fusion cells were each run through a microarray chip ( affymetrix ) by the duke dna microarray core facility . multiway anova was performed and fold change was determined to select target genes that were differentially expressed between fusion cells and dcs , or fusions cells and tumors cells , respectively . top differentially expressed genes were selected with p value cutoff of 0.01 based on anova test and fold change cutoff of > 5 . hierarchical clustering was performed on differentially expressed genes based on average linkage with pearson 's dissimilarity . the murine il-4 elisa kit ( ebioscience , san diego , ca ) and the murine il-12p70 elisa kit ( bd biosciences , san jose , ca ) were used according to the manual provided by the manufacturer . to determine cytokine secretion by dcs or dt - tumor fusion cells , 2  10 cells in 1  ml of aimv media ( invitrogen , carlsbad , ca ) were incubated in the presence of 100  ng / ml of lps for 24 hours at 37c , 5% co2 . where indicated , lps stimulation was performed in the presence of the following inhibitors ( purchased from sigma - aldrich , saint louis , mo ) : u0126 ( 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio ] butadiene ) is a highly selective inhibitor of both mek1 and mek2 and was used at a concentration of 100  nm , jw 74 ( 4-[4-(4-methoxyphenyl)-5-[[[3-(4-methylphenyl)-1,2,4-oxadiazol-5-yl]methyl]thio]-4h-1,2,4-triazol-3-yl]-pyridine ) an inhibitor of the canonical wnt pathway was used at a concentration of 10  m , rapamycin ( 23,27-epoxy-3h - pyrido[2,1-c]oxaazacyclohentriacontine ) forms a complex with fkbp12 ( fk506 binding protein 12 ) that binds to and inhibits mtor which was used at 0.5  m , and wortmannin which inhibits the pi3k / akt signal transduction cascade was used at 100  nm . experiments were performed in duplicate and error bars represent the sem ( standard error of the mean ) . in a first set of experiments , d5lacz tumor - tumor ( t - t ) cell hybrids , dc - dc hybrids , and dc - t hybrids were generated by electrofusion . fusion cells were purified by facs and rna isolated from hybrid cells was analyzed by quantitative real - time pcr ( qpcr ) for expression levels of mrnas encoding the th1 cytokines ifn- , tnf- , il-2 , il-12p40 ( the -subunit of bioactive il-12p70 ) , il-15 , and il-18 or the th2 cytokines il-4 , il-10 , il-13 , and il-25 .   figure 1 shows the results of our qpcr analyses . comparison of dc - t hybrids with dc - dc fusion cells ( white bars ) reveals that cytokines associated with a th1 response including il-12p40 and il-15 were downregulated by more than 100- and 15-fold , respectively . in contrast , the th2 cytokine il-4 was dramatically upregulated by 115-fold . among all cytokines analyzed , only tnf- and il-12p40 exhibited higher expression levels in dc - t fusion cells when compared to t - t fusions ( figure 1 , black bars ) . in another series of experiments , the th1 and th2 cytokine expression profile of cells exposed to electrofusion was compared to unexposed cells . however , no significant changes in cytokine gene expression between tumor cells and t - t fusion cells or dcs and dc - dc fusion cells were observed ( data not shown ) . for this reason , we focused on the comparison of gene expression levels between dc - t hybrid cells and dcs in the subsequent analyses presented in this study . we next sought to determine changes in the expression of genes that may negatively impact the immunologic properties of dc - t fusion cells . in order to do so , rnas were isolated from facs - isolated dc - t hybrids cells , dcs , or d5lacz tumor cells , and microarray assays were performed . consistent with our qpcr data , expression of il12p40 and il-15 by dc - t fusion cells was markedly downregulated ( 13.2- and 8-fold ) when compared to dcs , albeit to a lesser degree than observed in pcr analyses ( figure 2(a ) ) . also , il-4 was upregulated 59.4-fold in dc - t fusions . the proinflammatory cytokines il-1 and il-1 were downregulated 5.5- and 8.2-fold , respectively , while tgf3 was upregulated 8.8-fold . furthermore , we observed a downregulation of receptors for colony - stimulating factor ( csfr1 ) , tnf- ( tnfr2 ) , and il-7 ( il-7r ) . in contrast the receptors for tweak ( tnf - like weak inducer of apoptosis , tweakr ) and for il-17 ( il-17rc ) were upregulated 9.5- and 6.5-fold . while overexpression of il-17rc has been implicated in bcl-2- and bcl - xl - independent protection of cancer cell lines from tnf-induced apoptosis , tweakr signaling has been shown to enhance the expression of nfb ( nuclear factor kappa - light - chain - enhancer of activated b - cells)-regulated genes including il-6 , il-8 , rantes , and icam-1 ( cd54 ) . however , upregulation of none of these gene products was observed in our study ( figures 2(a ) , 2(c ) , and 3(a ) ) . in addition to cytokine gene and cytokine receptor expression , there were also significant changes in the expression level of gene products that are involved in cytokine signaling ( figure 2(b ) ) . expression of tgfi ( transforming growth factor beta - induced ) , a protein that is induced by tgf and that acts to inhibit cell adhesion , was downregulated 14.1-fold . downregulation of this gene product was unexpected given that tgf3 was upregulated in dc - t hybrids ( figure 2(a ) ) and implies that the tgf-signaling pathway may not be hyperactive in dc - t hybrid cells . there were no differences in expression levels of tgf-receptors between dcs and dc - t hybrid cells . however , nedd4l ( neural precursor cell expressed developmentally downregulated gene 4-like ) was upregulated 13.8-fold in dc - t hybrid cells . nedd4l negatively regulates tgf signaling by ubiquitination - mediated degradation of tgf- receptor 1 and receptor - regulated smad2 ( mothers against decapentaplegic homolog 2 ) . as such , it is reasonable to assume that neddl4 overexpression suppressed transcriptional activity induced by tgf. expression of il-1ra , the interleukin-1 receptor antagonist , which modulates a variety of il-1 related immune and inflammatory responses , was downregulated 7.5-fold . moreover , expression of irfs ( interferon regulatory factors ) 4 , 7 , and 9 , which are involved in transcriptional regulation of type i interferon genes , interferon signaling , and hence the janus kinase- ( jak- ) signal transducer and activator of transcription ( stat ) pathway , was downregulated 7- , 6- , and 10.5-fold , respectively . also , jak-2 and stat-4 , known to be involved in il-12 receptor signaling , are downregulated 6.5- and 11.3-fold . last , three gene products that are associated with nf-b signaling were found to be downregulated in dc - t hybrid cells , namely , relb ( reticuloendotheliosis viral oncogene homolog b , 5.3-fold ) , traf-1 ( tnf receptor associated factor-1 , 11.3-fold ) , and the nf-b inhibitor ib ( nuclear factor of kappa light polypeptide gene enhancer in b - cells inhibitor alpha , 7.3-fold ) . relb is known to form heterodimers with nf-b p50 or p52 , and traf-1 forms a heterodimeric complex with traf2 , which is required for tnf--mediated activation of mapk8/jnk ( jun kinase ) and nf-b . on the other hand , these results are somewhat contradictory and argue that nf-b activity in dc - tumor fusion cells is not regulated at the transcriptional level . as shown in figure 2(c ) , expression of chemokines or their receptors which are involved in chemotaxis of neutrophils , monocytes , dcs , t cells , and nk cells were generally downregulated in dc - tumor fusions , with the exception of cxcl-10 ( ip-10 , interferon - gamma - induced protein 10 ) . surprisingly , even chemokines involved in chemotaxis of th2 cells and regulatory t cells ( ccl-17 and ccl-22 ) were downregulated while ip-10 which is implicated in the induction of th1 responses and chemotaxis of th1 cells was significantly upregulated [ 23 , 24 ] . we therefore hypothesize that the chemokine expression profile of dc - tumor hybrids does not have a major impact on the th - polarizing capacity of dc - tumor hybrid cells . it has been demonstrated that the expression of matrix metalloproteinases mt-1 ( mmp-14 ) and mmp-9 is a major contributing factor to the migratory capacity of dcs to lymph nodes through the degradation of extracellular matrix components . in this context , mmp-9 activity is of particular importance since it cleaves collagen iv , a major component of basement membranes . furthermore , it has been shown that the balance of mmp-9 and timp ( tissue inhibitor of mmps ) expression is crucial for dc migration in vivo . our data reveal that timp-2 was upregulated 11.8-fold in dc - t fusion cells , while mmp-9 is downregulated 7-fold ( figure 2(d ) ) . as such , these results suggest that the migratory capacity of dc - t hybrids toward lymph - node derived chemokines , namely , ccl-19 and ccl-21 , may be impaired . as shown in figure 3(a ) , expression of genes involved in antigen presentation in the context of mhc classes i and ii or cd1d was downregulated 5.7- , 16.5- , and 6-fold in fusion cells . furthermore , the expression of all well - established costimulatory molecules , including cd40 , cd54 , cd80 , cd83 , cd86 , 4 - 1bb , gitr ( glucocorticoid - induced tnfr - related protein ) , ox40l , and slam ( signaling lymphocytic activation molecule ) , was downregulated in dc - tumor fusion cells . these data explain to some degree why targeting of costimulatory molecules with agonistic antibodies can enhance the potency of dc - tumor fusion - based vaccines , as has been described previously . last , expression of pd - l2 ( programmed death ligand 2 ) , an inhibitory immune checkpoint molecule , was suppressed 7.8-fold in dc - fusion cells . no differences in pd - l1 expression between dcs and dc - t hybrid cells were observed . the development of melanocytes is highly dependent on the action of the microphthalmia - associated transcription factor ( mitf ) which has been shown to regulate a broad variety of genes , whose functions range from pigment production to cell - cycle regulation , migration , and survival . mitf was upregulated in dc - tumor fusion cells ( figure 3(b ) ) . concomitantly , also mitf - regulated mrnas encoding melanoma antigens , including tyr ( tyrosinase ) , trp-1 and trp-2 ( tyrosinase - related protein ) , gp100 ( silver ) , melan , melanophilin , m - cam ( melanoma cell adhesion molecule ) , and matp ( membrane - associated transporter protein also known as solute carrier family 45 member 2 ( slc45a2 ) or melanoma antigen aim1 ) , were also highly upregulated . moreover , expression of mitf - regulated mcr1 ( melanocortin 1 receptor ) , trpm1 ( transient receptor potential cation channel subfamily m member 1 ) , gpr143 ( g protein - coupled receptor 143 ) , and mbp ( myelin basic protein ) was highly upregulated in fusion cells . expression of mbp by melanoma cells is somewhat surprising , but it has been shown that b16f10 cells undergo differentiation to a myelinating glial phenotype characterized by induction of the transcriptional activity of the mbp promoter . last , osteonectin ( secreted protein acidic and rich in cysteine ( sparc ) ) , which has been implicated in metastasis of melanoma to the lungs , and plagl1 ( pleomorphic adenoma gene - like 1 ) , a potential tumor suppressor gene , were also overexpressed in dc - tumor fusions . these results suggest that the entire antigenic repertoire of melanoma cells is indeed strongly expressed in dc - tumor hybrid cells , as has been hypothesized . next , we analyzed expression levels of genes that are involved in signaling pathways known to be aberrantly regulated in cancer cells . the transcription factor tcf7l1 ( transcription factor 7-like 1 ) which is activated by -catenin and thus mediated wnt signaling was upregulated 6.2-fold ( figure 4(a ) ) . also , expression of frzb ( frisbee ) , a wnt - binding protein and competitor for the cell - surface receptor frizzled , and expression of wntless ( g protein - coupled receptor 177 ) , another receptor for wnt proteins , were increased 12.2- and 61.3-fold . furthermore , target genes of the canonical wnt pathway , wisp-1 ( wnt1-inducible - signaling pathway protein 1 )   and nrcam ( neuronal cell adhesion molecule ) , were upregulated 13.7- and 148.7-fold , indicating activation of the wnt pathway in dc - tumor fusion cells . expression of the fk506 binding proteins fkbp4 , fkbp6 , and fkbp9 , immunophilins known to interact with mtor , was upregulated in dc - tumor fusions 5.3- , 6.7- , and 28-fold ( figure 4(b ) ) . furthermore , expression of pik3r1 ( phosphatidylinositol 3-kinase regulatory subunit alpha , p85 ) was downregulated 6.5-fold , which may indicate aberrant activity of pi3k in dc - tumor fusion cells . nedd4 directly binds to and poly - ubiquitinates pten ( phosphatase and tensin homolog ) , targeting it for proteasomal degradation . therefore , posttranslational suppression of its expression level may lead to hyperactivation of the pi3k / akt signaling pathway . the lps - inducible mitogen - activated protein kinase 12 ( mapk12 ) , also known as extracellular signal - regulated kinase 6 ( erk6 ) or p38- , was upregulated 11.7-fold in dc - tumor fusions ( figure 4(c ) ) . in contrast , the lps - inducible gadd45 ( growth arrest and dna damage - inducible 45 ) was downregulated 10.8-fold . gadd45 is an nf-b target gene which , in combination with mekk4 , activates p38mapk . furthermore , mitogen - activated protein kinase kinase kinase 14 ( map3k14 ) also known as nf - kappa - b - inducing kinase was downregulated 7.25-fold in fusion cells . lastly , expression of c - jun amino - terminal kinase interacting protein 1 ( mapk8ip1 ) , a negative regulator of mapk8 ( c - jun amino - terminal kinase ) , was upregulated 6.9-fold in dc - tumor hybrid cells . lipid mediators such as prostaglandins have been implicated in tumor - mediated immunosuppression [ 37 , 38 ] . as presented in figure 4(d ) , several genes involved in eicosanoid biosynthesis and signaling were differentially expressed in dc - tumor fusion cells . additionally , cyclooxygenase-2 was downregulated 16.2-fold and the cysteinyl - leukotriene c4 synthase ( ltc4s ) was downregulated 12.1-fold . in contrast , phospholipase a2 ( pla2 ) and prostaglandin d2 synthase ( pgds ) were upregulated 47.2- and 61.3-fold , respectively . we next sought to determine whether inhibition of signaling pathways , for which inhibitors are available clinically , could restore secretion of bioactive il-12p70 by dc - tumor fusion cells . we chose wortmannin as an inhibitor of pi3k upstream of akt ( pkb ) , u0126 as an inhibitor of mek1 and mek2 , rapamycin as an inhibitor of mtor , and jw74 as an inhibitor of the canonical wnt pathway . admittedly , our data provide several lines of evidence that nf-b - signaling is impaired in dc - tumor fusion cells , but , even though nf-b - inhibitors are starting to emerge in the clinic , it would obviously not make sense to administer nf-b - agonists to cancer patients . dcs and dc - tumor fusion cells were stimulated with lps in the presence or absence of inhibitors as indicated in figure 5(a ) and supernatants were analyzed for il-12p70 secretion by elisa . as expected , dc - tumor fusions did not produce il-12p70 in response , while dcs responded to lps stimulation . u0126 led to a modest increase of il-12p70 by both dc - tumor fusions and dcs . inhibition of pi3k with wortmannin and inhibition of mtor with rapamycin increased secretion of il12-p70 significantly ( 11 - 13-fold ) . inhibition of the canonical wnt pathway with jw74 did not have any impact on il-12p70 production by dcs or dc - tumor fusion cells . we next asked whether combined inhibition of pi3k and of mtor could further enhance il-12p70 secretion by dc - tumor fusion cells . as shown in figure 5(b ) , combining wortmannin and rapamycin to inhibit pi3k and mtor did not significantly enhance il-12p70 secretion by dc - tumor hybrid cells , hence excluding a synergistic or additive effect of these inhibitors . this study is the first to investigate the mechanisms responsible for the dependence of dc - tumor hybrid vaccines on exogenously provided 3rd signal adjuvants . these include the induction of apoptosis of immune cells via expression of fas ligand , trail ( tnf - related apoptosis - inducing ligand ) [ 39 , 40 ] , or pd - l1 and pd - l2 ( programmed death ligand ) . furthermore , induction of tolerance through cytokines such as tgf- , il-6 , and il-10   or lipid mediators [ 37 , 38 ] has been described . lastly , activation of the mapk pathway by melanoma cells has been described as a mechanism to inhibit il-12 production by dcs in a paracrine manner . our results do not provide evidence for overexpression of apoptosis - inducing ligands by dc - tumor cell hybrids , nor did we observe an enhanced production of tolerance - inducing cytokine il-6 or il-10 by these cells . tgf-3 was upregulated 8.8-fold in dc - tumor fusions , but the observed downregulation of the tgf-induced protein in combination with upregulation of nedd4l argues against a major impact of this cytokine on fusion cells . surprisingly , despite a profound upregulation of mrna encoding il-4 in dc - tumor hybrids , there was no evidence of il-4 signaling in these cells . we did not observe any upregulation of target genes of the il-4 receptor i or ii , including socs-1 , il-4 receptor  , ccl11 ( eotaxin 1 ) , or fc receptor ii . in addition , there were no changes in expression of gene products that are components of the il-4 receptors , namely , il-4 receptor  , il13 receptor  , and cd25 . even though the expression levels of phospholipases a2 , which release arachidonic acid from phospholipids , and of prostaglandin d2 synthase were upregulated in dc - tumor fusions , expression of cyclooxygenase , which catalyzes the downstream conversion of arachidonic acid into eicosanoids , was downregulated . furthermore , while pgd-2 has been shown to upregulate cd80 and to downregulate ip-10 in lps - matured dcs , the exact opposite was observed in our experiments ( figures 2(c ) and 3(a ) ) . accordingly , we conclude that pgd-2 may not be the main culprit for the dramatic downregulation of il-12 production in dc - tumor hybrids . however , it has been described that , in the spontaneous b16f10 melanoma cell line , expression of p16ink4a ( inhibitor of cdk4a ) , which inhibits cell - cycle progression by inactivating cyclin - dependent kinases , and of p19arf ( alternate reading frame tumor suppressor ) , which causes mdm2 ( mouse double minute 2 homologue ) induced translational silencing and p53 degradation , is lost and that there is no evidence of activation of the mapk - signaling pathway in this cell line . it is therefore highly unlikely that the mapk - signaling pathway would be a major contributor to the loss of immunostimulatory capacity of dc - tumor hybrid cells . nevertheless , our data reveal that treatment of dc - tumor hybrid cells with mek inhibitor u0126 led to a modest increase in il-12 secretion . this however might be a result of the previously published observation that treatment with u0126 can result in a slight but significant inhibition of p70 ( s6 ribosomal protein kinase ) activation , a downstream target of akt . our results further indicate that molecules that are involved in wnt signaling , including tcf7l1 , frzb , and wntless , were upregulated in dc - tumor fusions . additionally , wisp-1 and nrcam , targets of the canonical wnt pathway , were upregulated 12- and 148.7-fold , respectively . in the canonical wnt pathway , activation of wnt receptors leads to stabilization and import of -catenin into the nucleus where -catenin associates with t - cell factor / lymphoid enhancer factor ( tcf / lef ) and activates target genes . however , treatment of dc - tumor fusions with jw74 , a specific inhibitor of the canonical wnt pathway , had no impact on il-12 secretion by these cells . on the other hand , it is conceivable that the mtor pathway was activated through the noncanonical wnt / ca pathway , wnt - dependent activation of pka ( protein kinase a ) and creb ( camp response element - binding protein ) , or mtor activation via wnt - mediated inhibition of glycogen synthase kinase 3 . alternatively , we can not exclude that the pi3k / akt / mtor pathway was activated independent of wnt signaling . the pi3k / akt / mtor pathway has been shown to play a critical role in cell proliferation , survival , and metastasis of cancer cells , and we observed that inhibition of the pi3k / akt and inhibition of the mtor pathway enhanced the immune - stimulatory capacity of dc - tumor fusions through induction of bioactive il-12p70 secretion . the fact that combined inhibition of pi3k and mtor signaling did not further improve il-12p70 secretion by dc - tumor fusions may indicate that inhibition acted on the same signaling pathway , likely to involve p70 as has been described previously . in sum , we conclude that combining pi3k / akt / mtor inhibition with dc - melanoma fusion cell - based cancer vaccination appears to be a promising strategy and warrants further studies in vitro and in animal models . ultimately , this research may lead to the development of improved dc - fusion - based cancer vaccines with enhanced clinical impact .
background . 
 dendritic cell- ( dc- ) tumor fusion cells stimulate effective in vivo antitumor responses . however , therapeutic approaches are dependent upon the coadministration of exogenous 3rd signals . 
 the purpose of this study was to determine the mechanisms for inadequate 3rd signaling by electrofused dc - tumor cell hybrids . 
 methods . 
 murine melanoma cells were fused with dcs derived from c57bl/6 mice . 
 quantitative real - time pcr ( qpcr ) was used to determine relative changes in th ( t helper ) 1 and th2 cytokine gene expression . 
 in addition , changes in gene expression of fusion cells were determined by microarray . 
 last , cytokine secretion by fusion cells upon inhibition of signaling pathways was analyzed by elisa . 
 results . 
 qpcr analyses revealed that fusion cells exhibited a downregulation of th1 associated cytokines il-12 and il-15 and an upregulation of the th2 cytokine il-4 . 
 microarray studies further showed that the expression of chemokines , costimulatory molecules , and matrix - metalloproteinases was deregulated in fusion cells . 
 lastly , inhibitor studies demonstrate that inhibition of the pi3k / akt / mtor signaling pathway could restore the secretion of bioactive il-12p70 by fusion cells . conclusion . 
 our results suggest that combining fusion cell - based vaccination with administration of inhibitors of the pi3k / akt / mtor signaling pathway may enhance antitumor responses in patients .