molecular chaperones , including heat shock proteins ( hsps ) and glucose - regulated proteins ( grps ) , are some of the most abundant cellular proteins . these proteins can be classified into three major groups by mass ( i.e. , the 90- , 70- , and 50-da to 60-da proteins ) , and can be further subdivided based on localization within the cell . during increased protein production ( e.g. , tumor formation ) and stress , these chaperones bind to exposed hydrophobic sites within nascent polypeptides . the tumor peptide antigens can bind to hsps to form hsp / peptides ( hsp / ps ) , which induce immune responses . the use of hsps as tumor vaccines was first reported by srivastava et al . , who showed that hsps , in particular grp94 , bind peptides that are immunogenic in vitro and in vivo . these hsp / ps induce both specific and innate immune responses , as demonstrated by numerous studies . hsp / ps can present antigenic fingerprints / peptides to major histocompatibility complex antigens and activate dendritic cells , the most efficient antigen - presenting cells , which when used as a vaccine can cause tumor regression . more recently , hsp70 , hsp110 , and grp170 purified from tumors were shown to function as tumor vaccines and cause tumor regression in animal models , which resulted in cd8 t - cell - dependent tumor clearance . these encouraging results in animals have led to cancer vaccine trials of metastatic melanoma , renal cell carcinoma , colorectal cancer , and chronic myelogenous leukemia . the preliminary results from phase ii and iii clinical trials showed that a grp96 ( oncophage ) vaccine has low to no toxicity , although the antitumor effect was not ideal . for example , hsp110 delivers different antigens than does hsp70 . to date , all animal studies and clinical trials have used a single hsp subtype , such as gp96 , hsp70 , or hsp110 . thus , we hypothesized that mixed hsp / ps may deliver more specific tumor antigens and so exhibit greater efficacy . in one animal tumor model , the antitumor activity of the combination of gp96 and hsp70 was superior to that of each protein alone . here , we developed a vaccine comprising a mixture of hsp / ps ( mhsp / ps ) , which included hsp60 and hsp110 in addition to hsp70 and gp96 , and evaluated its antitumor effects in two mouse sarcoma models . female 68-week - old balb / c mice and c57 mice were purchased from the military medical academy ( china ) and bred in the chinese pla general hospital . the institutional animal care and the ascetic mouse s180 sarcoma cell line ( atcc tib-66 ) was obtained from the military medical academy and the mouse mca207 sarcoma cell line was obtained from dr . the s180 line was maintained by several passages through the balb / c mouse peritoneal cavity . anti - hsp60 , -hsp70 , -hsp110 , and -grp96/94 antibodies were purchased from santa cruz biotechnology ( santa cruz , ca , usa ) . sephacryl s-200hr , concanavalin a ( cona ) , and adenosine 5'-diphosphate affinity columns were purchased from pharmacia ( dorval , quebec , canada ) . isolation of mixture of heat shock protein / peptide using molecular chromatography tumor tissue ( s180 or mca207 ) was homogenized at 4c in buffer ( 30 mmol / l nahco3 , ph 7.1 ) , with freshly added protease inhibitor , phenylmethylsulfonyl fluoride ( pmsf , 0.5 mmol / l ) . the homogenate was centrifuged at 10,000  g for 30 min at 4c , after which the supernatant was centrifuged at 100,000  g at 4c for 2 h. the supernatant ( tumor lysate ) was dialyzed against 20 mmol / l tris - hcl and 150 mmol / l nacl , ph 7.2 ; the tumor supernatant was then applied to sephacryl s-200 hr . after equilibration , the protein was eluted with the same buffer and monitored using an ultraviolet monitor . the collected eluted protein fractions were subjected to resolution by sds - page ; fractions 36 contained 40- to 200-kda proteins ; thus , we combined these four fractions as the mhsp / ps . mhsp / ps were isolated from skeletal muscles and liver of wild - type mice using the same methods . purification of grp96 mhsp / ps were applied to a cona - sepharose 4b column ( pharmacia biotech , usa ) equilibrated with binding buffer ( 20 mmol / l tris - hcl , ph 7.5 , 150 mmol / l nacl , 10 mmol / l mgcl2 , 10 mmol / l cacl2 , 15 mmol / l 2-me ) ; the binding proteins were eluted with binding buffer containing 15% -d - methylmannoside ( sigma ) . purification of heat shock protein 70 and heat shock protein 60 the nonbinding fraction of cona was used to purify hsp70 and hsp60 , applied to an adt - sepharose column , and equilibrated with binding buffer ( 20 mmol / l tris - hcl , ph 7.5 , 20 mmol / l nacl , 3 mmol / l mgcl2 , 15 mmol / l 2-me , 0.5 mmol / l pmsf ) . each protein sample was subjected to 10% sds - page and transferred to membranes ( millipore , usa ) . membranes were blocked with 5% nonfat milk in tbst for 1 h at room temperature and then incubated with primary antibodies and horseradish peroxidase - conjugated goat anti - rabbit or goat anti - mouse igg . s180 or mca207 mhsp / ps or ns ( 2 , 5 , 10 , 20 , or 30 g ) was injected subcutaneously in all mice 3 times at weekly intervals . seven days after the second injection , balb / c mice were injected subcutaneously with s180 tumor cells ( 4  10 ) in the back hind quarters s.c . , and c57 mice were injected with mca207 tumor cells ( 5  10 ) . the antitumor potency was evaluated based on tumor volume , tumor growth inhibition rates , and survival time . the shortest diameter ( a ) and longest diameter ( b ) were measured using a caliper every 3 days to monitor tumor growth . the tumor volume ( v ) was calculated using the formula v = ( ab/2 ) . the rate was calculated using the formula r = tumor volume of immunized mice / tumor volume of phosphate - buffered saline - treated mice . survival rate or complete regression was considered when the tumor did not appear after more than 3 months . treatment of mice for the analysis of immune responses was the same as for the prophylactic protocol . three days after the third immunization with s180 mhsp / ps , all mice were euthanized and blood and spleen samples collected . control group mice were euthanized at the same time ; each group comprised three mice . assay for t - cell subgroups subgroups of t - lymphocytes in sera from each group of s180-immunized mice were analyzed using a facscan instrument ( becton dickinson , usa ) . cell staining involved the use of a fluorescein isothiocyanate- or phycoerythrin - conjugated goat antibody against mouse cd3 , cd4 , cd8 , or natural killer ( nk ) cells . cytotoxicity assay ( cytotoxic t - lymphocytes ) the in vitro tumor - specific cytotoxic t - lymphocyte ( ctl ) response elicited by immunization with mhsp / p was analyzed by means of lactate dehydrogenase release from damaged cells . splenocytes of mhsp / p - immunized mice were isolated and purified using ficoll - paque density centrifugation and used as effector cells . after re - stimulating with cona and mhsp / p in vitro for 4 days , s180 cells as target cells were first seeded on 96-well plates , after which expanded lymphocytes were serially diluted in 96-well plates in triplicate with effector : target(e : t ) ratios of 40:1 , 20:1 , and 5:1 . wells containing only target cells or lymphocytes with culture medium were spontaneous - release wells ; target cells treated with 0.5% triton x-100 served as maximal release controls . after 24-h incubation at 37c and 5% co2 , 150 l of the supernatant from each well were transferred to a new 96-well plate , scanned using a 96-well plate reader ( bio - rad ) , and optical density read at a wavelength of 490 nm . the percent specific lysis was calculated using the following formula : % specific lysis = 100  ( experimental release  spontaneous release / maximum release  spontaneous release ) . eispot assays for interferon- splenocytes of immunized mice were obtained and expanded as described for the ctl assay . half of the cells were incubated with cona and half were additionally re - stimulated with mhsp / p for 5 days in 96-well elispot plates . the assays were performed according to the manufacturer 's instructions ( utrecht cj , u - cytech b.v . , the netherlands ) . the serum was separated from the blood of immunized mice in each group , diluted , incubated with antibodies against hsp60 and hsp70 , and assayed using elisa . statistical analysis used spss version 13.0 ( spss inc . , a two - tailed paired t - test with welch correction was used for comparison of interferon- ( ifn- ) levels . female 68-week - old balb / c mice and c57 mice were purchased from the military medical academy ( china ) and bred in the chinese pla general hospital . the institutional animal care and the ascetic mouse s180 sarcoma cell line ( atcc tib-66 ) was obtained from the military medical academy and the mouse mca207 sarcoma cell line was obtained from dr . the s180 line was maintained by several passages through the balb / c mouse peritoneal cavity . anti - hsp60 , -hsp70 , -hsp110 , and -grp96/94 antibodies were purchased from santa cruz biotechnology ( santa cruz , ca , usa ) . sephacryl s-200hr , concanavalin a ( cona ) , and adenosine 5'-diphosphate affinity columns were purchased from pharmacia ( dorval , quebec , canada ) . isolation of mixture of heat shock protein / peptide using molecular chromatography tumor tissue ( s180 or mca207 ) was homogenized at 4c in buffer ( 30 mmol / l nahco3 , ph 7.1 ) , with freshly added protease inhibitor , phenylmethylsulfonyl fluoride ( pmsf , 0.5 mmol / l ) . the homogenate was centrifuged at 10,000  g for 30 min at 4c , after which the supernatant was centrifuged at 100,000  g at 4c for 2 h. the supernatant ( tumor lysate ) was dialyzed against 20 mmol / l tris - hcl and 150 mmol / l nacl , ph 7.2 ; the tumor supernatant was then applied to sephacryl s-200 hr . after equilibration , the protein was eluted with the same buffer and monitored using an ultraviolet monitor . the collected eluted protein fractions were subjected to resolution by sds - page ; fractions 36 contained 40- to 200-kda proteins ; thus , we combined these four fractions as the mhsp / ps . mhsp / ps were isolated from skeletal muscles and liver of wild - type mice using the same methods . purification of grp96 mhsp / ps were applied to a cona - sepharose 4b column ( pharmacia biotech , usa ) equilibrated with binding buffer ( 20 mmol / l tris - hcl , ph 7.5 , 150 mmol / l nacl , 10 mmol / l mgcl2 , 10 mmol / l cacl2 , 15 mmol / l 2-me ) ; the binding proteins were eluted with binding buffer containing 15% -d - methylmannoside ( sigma ) . purification of heat shock protein 70 and heat shock protein 60 the nonbinding fraction of cona was used to purify hsp70 and hsp60 , applied to an adt - sepharose column , and equilibrated with binding buffer ( 20 mmol / l tris - hcl , ph 7.5 , 20 mmol / l nacl , 3 mmol / l mgcl2 , 15 mmol / l 2-me , 0.5 mmol / l pmsf ) . after 30 min , each protein sample was subjected to 10% sds - page and transferred to membranes ( millipore , usa ) . membranes were blocked with 5% nonfat milk in tbst for 1 h at room temperature and then incubated with primary antibodies and horseradish peroxidase - conjugated goat anti - rabbit or goat anti - mouse igg . s180 or mca207 mhsp / ps or ns ( 2 , 5 , 10 , 20 , or 30 g ) was injected subcutaneously in all mice 3 times at weekly intervals . seven days after the second injection , balb / c mice were injected subcutaneously with s180 tumor cells ( 4  10 ) in the back hind quarters s.c . , and c57 mice were injected with mca207 tumor cells ( 5  10 ) . the antitumor potency was evaluated based on tumor volume , tumor growth inhibition rates , and survival time . the shortest diameter ( a ) and longest diameter ( b ) were measured using a caliper every 3 days to monitor tumor growth . the tumor volume ( v ) was calculated using the formula v = ( ab/2 ) . the rate was calculated using the formula r = tumor volume of immunized mice / tumor volume of phosphate - buffered saline - treated mice . survival rate or complete regression was considered when the tumor did not appear after more than 3 months . treatment of mice for the analysis of immune responses was the same as for the prophylactic protocol . three days after the third immunization with s180 mhsp / ps , all mice were euthanized and blood and spleen samples collected . control group mice were euthanized at the same time ; each group comprised three mice . assay for t - cell subgroups subgroups of t - lymphocytes in sera from each group of s180-immunized mice were analyzed using a facscan instrument ( becton dickinson , usa ) . cell staining involved the use of a fluorescein isothiocyanate- or phycoerythrin - conjugated goat antibody against mouse cd3 , cd4 , cd8 , or natural killer ( nk ) cells . cytotoxicity assay ( cytotoxic t - lymphocytes ) the in vitro tumor - specific cytotoxic t - lymphocyte ( ctl ) response elicited by immunization with mhsp / p was analyzed by means of lactate dehydrogenase release from damaged cells . splenocytes of mhsp / p - immunized mice were isolated and purified using ficoll - paque density centrifugation and used as effector cells . after re - stimulating with cona and mhsp / p in vitro for 4 days , s180 cells as target cells were first seeded on 96-well plates , after which expanded lymphocytes were serially diluted in 96-well plates in triplicate with effector : target(e : t ) ratios of 40:1 , 20:1 , and 5:1 . wells containing only target cells or lymphocytes with culture medium were spontaneous - release wells ; target cells treated with 0.5% triton x-100 served as maximal release controls . after 24-h incubation at 37c and 5% co2 , 150 l of the supernatant from each well were transferred to a new 96-well plate , scanned using a 96-well plate reader ( bio - rad ) , and optical density read at a wavelength of 490 nm . the percent specific lysis was calculated using the following formula : % specific lysis = 100  ( experimental release  spontaneous release / maximum release  spontaneous release ) . eispot assays for interferon- splenocytes of immunized mice were obtained and expanded as described for the ctl assay . half of the cells were incubated with cona and half were additionally re - stimulated with mhsp / p for 5 days in 96-well elispot plates . the assays were performed according to the manufacturer 's instructions ( utrecht cj , u - cytech b.v . , the serum was separated from the blood of immunized mice in each group , diluted , incubated with antibodies against hsp60 and hsp70 , and assayed using elisa . statistical analysis used spss version 13.0 ( spss inc . , chicago , il , usa ) . a two - tailed paired t - test with welch correction was used for comparison of interferon- ( ifn- ) levels . mhsp / ps were obtained from s180 and mca207 tumors and liver or muscles of wild - type mice . hsp60 , hsp70 , gp96 , and hsp110 isolated from s180 tumors were subjected to sds - page [ figure 1a ] and identification by western blotting analysis [ figure 2 ] . in total , 22.5-mg mhsp / p was obtained from each gram ( wet weight ) of the tumor . this yield was higher than that of pure hsp70 ( 100150 g ) and pure hsp60 ( 400500 g ) . mhsp / p obtained from wild - type mice liver is shown in figure 1b . ( a ) sds - page of mixed heat shock proteins / peptides from ( a ) s180 sarcoma cells and ( b ) tissue of wild - type mice . ( a ) s180 cells : lane 1 , standard ; lanes 2 and 3 , bands for hsp110 , grp96 , hsp70 , and hsp60 . ( b ) wild - type mice : lane 1 , standard ; lanes 2 and 3 , bands for hsp110 , grp96 , hsp70 , and hsp60 in muscle . lanes 4 and 5 , bands for hsp110 , grp96 , hsp70 , and hsp60 in liver ( hsp : heat shock protein ) . sds - page and western blotting analysis of heat shock protein / peptides of s180 cells after concanavalin a affinity chromatography . western blot analysis : lane 3 , proteins that did not bind the atp column ; lane 4 , protein that bound to atp ; lane 5 , anti - grp96 antibody staining ; lane 6 , anti - heat shock protein 110 antibody staining . balb / c mice were administered s180 mhsp / ps at 2 , 5 , 10 , 20 , and 30 g . after vaccination , tumors transiently developed in some vaccinated mice , while no measurable tumor mass developed in others . tumor growth volume was significantly smaller in vaccinated than unvaccinated mice at 30 days ( p < 0.01 ) . the most effective dose was 20 g : the growth inhibition rate was 82.3% and complete regression occurred in 7 of 17 mice ( 41.2% ) [ table 1 ] . prophylactic antitumor effects of varying doses of mhsp / ps in s180 model mhsp / ps : mixed heat shock protein / peptides . c57 mice were administered mca207 mhsp / ps at 5 , 10 , 20 , and 30 g . all doses produced antitumor effects ; the mean tumor regression was 50% , and the most effective dose was 30 g ( 70% tumor regression ) . tumor growth volume at 30 days was significantly smaller than that in the controls ( p < 0.01 ) [ table 2 and figure 3 ] . in this model , prophylactic antitumor effects of varying doses of mhsp / ps in mca207 model mhsp / ps : mixed heat shock protein / peptides . effect of immunization with mca207 tumor - cell derived mixture of heat shock protein / peptides on tumor growth . three mixture of heat shock protein / peptide treatments in mca207 tumor - bearing mice compared with the saline - treated group ( c ) ; the tumor growth volume in the mixture of heat shock protein / peptides group was significantly lower than that in the control group ( p < 0.01 ) . in these two mouse models , the antitumor effects were not in parallel with the vaccinated doses , which showed that a large dose range of antigen could stimulate an immune reaction . to determine whether this antitumor activity induces long - term immunity against tumors , we challenged mice that survived with 10 s180 cells 12 months after the first challenge , but no mouse developed tumors . to explore the specificity of mhsp / p vaccine , three mice were vaccinated with the mca207 mhsp / p , and the mca207 tumor was rejected . we next compared the antitumor effects of mhsp / ps and single hsp / p ( hsp60 or hsp70 ) vaccines in the s180 model . all mice were subcutaneously vaccinated with 20-g hsp / ps ( as this amount exerted the greater antitumor effects ) 3 times at weekly intervals . the antitumor effects of mhsp / ps were superior to those of hsp60 , hsp70 , and tumor lysates [ table 3 and figure 4 ] , with no significant differences between mhsp / ps and single hsps or tumor lysates ( p > 0.05 ) . comparison of antitumor effects of various hsps of s180 hsps : heat shock proteins ; mhsp / p : mixed heat shock protein / peptide . effect of immunization with various tumor - derived mixtures of heat shock protein / peptides , hsp70 , and hsp60 on the survival of s180 tumor - bearing mice . comparison of immunization with mixture of heat shock protein / peptide , hsp70 , hsp60 , and tumor lysates in s180 tumor - bearing mice ; survival following vaccination with mixture of heat shock protein / peptides ( 40% ) was higher than that with hsp70 ( 33.3% ) , hsp60 ( 20% ) , and tumor lysates ( 20% ) when compared to mixture of heat shock protein / peptide and phosphate - buffered saline control treatment , p < 0.01 . no significant difference between mixture of heat shock protein / peptide and hsp70 , hsp60 , and tumor lysates ( p > 0.01 ) , ( hsp : heat shock protein ) . numbers of cd8 cells and nk cells were elevated following s180 mhsp / p treatment in balb / c mice , as determined using flow cytometry analysis . the mean proportion of cd8 cells among total mononuclear cells was 5.89%  0.36% in wild - type mice [ figure 5 ] . after mhsp / p immunization , the mean proportion of cd8 cells among total mononuclear cells increased to 9.21%  1.45% . the mean proportion of nk cells among total mononuclear cells in wild - type mice was 1.69%  0.32% . after mhsp / p immunization , the mean proportion of nk cells among total mononuclear cells increased to 5.29%  0.25% . ( a and b ) analysis of natural killer cells ( a ) and cd8 cells ( b ) in s180 mixture of heat shock protein / peptide - immunized mice using flow cytometry . inf- level increased in s180 mixture of heat shock protein / peptide - treated mice the level of ifn- from splenocytes expanded with cona and stimulated with mhsp / p in vitro was higher than that of cells expanded with cona alone in vitro . upon stimulation with cona and mhsp / ps , the mean ifn- level in mhsp / p - immunized mice was 246.6  7.45/10 cells , higher than that of wild - type ( 180  3.082/10 cells , p < 0.05 ) and tumor - bearing ( 121.3  1.00/10 , p < 0.01 ) mice [ figure 6 ] . the level of ifn- from splenocytes stimulated with cona alone was significantly higher in mhsp / p - immunized than in untreated tumor - bearing mice and wild - type mice . interferon- release in s180 mixture of heat shock protein / peptide - immunized mice evaluated by elispot . interferon- released from splenocytes stimulated with both concanavalin a and mixture of heat shock protein / peptide in vitro in mixture of heat shock protein / peptide immunized s180-bearing mice ( 246.6  7.45/10 cells ) was higher than that in untreated tumor - bearing ( 121.3  1.00/10 , p < 0.01 ) or wild - type ( 180  3.082/10 cells , p < 0.05 ) mice . cytotoxic t - lymphocyte generated by mixture of heat shock protein / peptide were capable of killing target cells to assess the functional effector properties of ctls generated by mhsp / p , we performed in vitro cytotoxicity assays of t - lymphocytes isolated from mice vaccinated with s180 mhsp / ps . target cells ( s180 ) pulsed with effector t - cells were killed in an e / t ratio - dependent manner : 1:5 = 21% , 1:20 = 27% , 1:40 = 44% , which is significantly higher than that of tumor - bearing mice that did not receive mhsp / ps ( p < 0.01 ) and wild - type mice ( p < 0.05 ) [ figure 7a ] . t - lymphocytes isolated from mice vaccinated with s180 mhsp / ps did not kill mca207 cells at e / t ratios of 5:1 , 20:1 , and 40:1 . the cytolytic ratios were 6.1% , 8.3% , and 10.0% [ figure 7b ] . in addition , t - lymphocytes isolated from liver or muscle of wild - type mice vaccinated with mhsp / ps did not kill the s180 sarcoma cells ; all cytolytic ratios were lower than 1% . cytotoxicity of t - lymphocytes from s180 mixture of heat shock protein / peptide - immunized mice based on lactate dehydrogenase release . ( a ) comparison of mixture of heat shock protein / peptide - immunized mice with tumor - bearing mice and wild - type mice . the cytotoxicity of mixed lymphocytes against s180 tumor cells in mice immunized with mixture of heat shock protein / peptide was higher than that in untreated tumor - bearing ( e / t = 5:1 , 20:1 and 40:1 , * p < 0.01 ) and wild - type ( e / t = 5:1 , 20:1 and 40:1 , p < 0.05 ) mice . ( b ) cytotoxicity of lymphocytes from mice vaccinated with s180 mixture of heat shock protein / peptides against mca207 . antibody detected in immunized mice all mice in the control and mhsp / p groups were positive for anti - hsp70 and -hsp60 antibodies using serum dilutions of 1:5001:2000 . moreover , there were no differences in levels of antibodies against hsp60 and hsp70 [ figure 8 ] . ( a and b ) cytotoxicity of lymphocytes from mice vaccinated with s180 mixture of heat shock protein / peptides . mhsp / ps were obtained from s180 and mca207 tumors and liver or muscles of wild - type mice . hsp60 , hsp70 , gp96 , and hsp110 isolated from s180 tumors were subjected to sds - page [ figure 1a ] and identification by western blotting analysis [ figure 2 ] . in total , 22.5-mg mhsp / p was obtained from each gram ( wet weight ) of the tumor . this yield was higher than that of pure hsp70 ( 100150 g ) and pure hsp60 ( 400500 g ) . mhsp / p obtained from wild - type mice liver is shown in figure 1b . ( a ) sds - page of mixed heat shock proteins / peptides from ( a ) s180 sarcoma cells and ( b ) tissue of wild - type mice . ( a ) s180 cells : lane 1 , standard ; lanes 2 and 3 , bands for hsp110 , grp96 , hsp70 , and hsp60 . ( b ) wild - type mice : lane 1 , standard ; lanes 2 and 3 , bands for hsp110 , grp96 , hsp70 , and hsp60 in muscle . lanes 4 and 5 , bands for hsp110 , grp96 , hsp70 , and hsp60 in liver ( hsp : heat shock protein ) . sds - page and western blotting analysis of heat shock protein / peptides of s180 cells after concanavalin a affinity chromatography . western blot analysis : lane 3 , proteins that did not bind the atp column ; lane 4 , protein that bound to atp ; lane 5 , anti - grp96 antibody staining ; lane 6 , anti - heat shock protein 110 antibody staining . balb / c mice were administered s180 mhsp / ps at 2 , 5 , 10 , 20 , and 30 g . after vaccination , tumors transiently developed in some vaccinated mice , while no measurable tumor mass developed in others . all doses produced antitumor effects . tumor growth volume was significantly smaller in vaccinated than unvaccinated mice at 30 days ( p < 0.01 ) . the most effective dose was 20 g : the growth inhibition rate was 82.3% and complete regression occurred in 7 of 17 mice ( 41.2% ) [ table 1 ] . prophylactic antitumor effects of varying doses of mhsp / ps in s180 model mhsp / ps : mixed heat shock protein / peptides . c57 mice were administered mca207 mhsp / ps at 5 , 10 , 20 , and 30 g . all doses produced antitumor effects ; the mean tumor regression was 50% , and the most effective dose was 30 g ( 70% tumor regression ) . tumor growth volume at 30 days was significantly smaller than that in the controls ( p < 0.01 ) [ table 2 and figure 3 ] . prophylactic antitumor effects of varying doses of mhsp / ps in mca207 model mhsp / ps : mixed heat shock protein / peptides . effect of immunization with mca207 tumor - cell derived mixture of heat shock protein / peptides on tumor growth . three mixture of heat shock protein / peptide treatments in mca207 tumor - bearing mice compared with the saline - treated group ( c ) ; the tumor growth volume in the mixture of heat shock protein / peptides group was significantly lower than that in the control group ( p < 0.01 ) . in these two mouse models , the antitumor effects were not in parallel with the vaccinated doses , which showed that a large dose range of antigen could stimulate an immune reaction . to determine whether this antitumor activity induces long - term immunity against tumors , we challenged mice that survived with 10 s180 cells 12 months after the first challenge , but no mouse developed tumors . to explore the specificity of mhsp / p vaccine , three mice were vaccinated with the mca207 mhsp / p , and the mca207 tumor was rejected . we next compared the antitumor effects of mhsp / ps and single hsp / p ( hsp60 or hsp70 ) vaccines in the s180 model . all mice were subcutaneously vaccinated with 20-g hsp / ps ( as this amount exerted the greater antitumor effects ) 3 times at weekly intervals . the antitumor effects of mhsp / ps were superior to those of hsp60 , hsp70 , and tumor lysates [ table 3 and figure 4 ] , with no significant differences between mhsp / ps and single hsps or tumor lysates ( p > 0.05 ) . comparison of antitumor effects of various hsps of s180 hsps : heat shock proteins ; mhsp / p : mixed heat shock protein / peptide . effect of immunization with various tumor - derived mixtures of heat shock protein / peptides , hsp70 , and hsp60 on the survival of s180 tumor - bearing mice . comparison of immunization with mixture of heat shock protein / peptide , hsp70 , hsp60 , and tumor lysates in s180 tumor - bearing mice ; survival following vaccination with mixture of heat shock protein / peptides ( 40% ) was higher than that with hsp70 ( 33.3% ) , hsp60 ( 20% ) , and tumor lysates ( 20% ) when compared to mixture of heat shock protein / peptide and phosphate - buffered saline control treatment , p < 0.01 . no significant difference between mixture of heat shock protein / peptide and hsp70 , hsp60 , and tumor lysates ( p > 0.01 ) , ( hsp : heat shock protein ) . numbers of cd8 cells and nk cells were elevated following s180 mhsp / p treatment in balb / c mice , as determined using flow cytometry analysis . the mean proportion of cd8 cells among total mononuclear cells was 5.89%  0.36% in wild - type mice [ figure 5 ] . after mhsp / p immunization , the mean proportion of cd8 cells among total mononuclear cells increased to 9.21%  1.45% . the mean proportion of nk cells among total mononuclear cells in wild - type mice was 1.69%  0.32% . after mhsp / p immunization , the mean proportion of nk cells among total mononuclear cells increased to 5.29%  0.25% . ( a and b ) analysis of natural killer cells ( a ) and cd8 cells ( b ) in s180 mixture of heat shock protein / peptide - immunized mice using flow cytometry . inf- level increased in s180 mixture of heat shock protein / peptide - treated mice the level of ifn- from splenocytes expanded with cona and stimulated with mhsp / p in vitro was higher than that of cells expanded with cona alone in vitro . upon stimulation with cona and mhsp / ps , the mean ifn- level in mhsp / p - immunized mice was 246.6  7.45/10 cells , higher than that of wild - type ( 180  3.082/10 cells , p < 0.05 ) and tumor - bearing ( 121.3  1.00/10 , p the level of ifn- from splenocytes stimulated with cona alone was significantly higher in mhsp / p - immunized than in untreated tumor - bearing mice and wild - type mice . interferon- release in s180 mixture of heat shock protein / peptide - immunized mice evaluated by elispot . interferon- released from splenocytes stimulated with both concanavalin a and mixture of heat shock protein / peptide in vitro in mixture of heat shock protein / peptide immunized s180-bearing mice ( 246.6  7.45/10 cells ) was higher than that in untreated tumor - bearing ( 121.3  1.00/10 , p < 0.01 ) or wild - type ( 180  3.082/10 cells , p < 0.05 ) mice . cytotoxic t - lymphocyte generated by mixture of heat shock protein / peptide were capable of killing target cells to assess the functional effector properties of ctls generated by mhsp / p , we performed in vitro cytotoxicity assays of t - lymphocytes isolated from mice vaccinated with s180 mhsp / ps . target cells ( s180 ) pulsed with effector t - cells were killed in an e / t ratio - dependent manner : 1:5 = 21% , 1:20 = 27% , 1:40 = 44% , which is significantly higher than that of tumor - bearing mice that did not receive mhsp / ps ( p < 0.01 ) and wild - type mice ( p < 0.05 ) [ figure 7a ] . t - lymphocytes isolated from mice vaccinated with s180 mhsp / ps did not kill mca207 cells at e / t ratios of 5:1 , 20:1 , and 40:1 . in addition , t - lymphocytes isolated from liver or muscle of wild - type mice vaccinated with mhsp / ps did not kill the s180 sarcoma cells ; all cytolytic ratios were lower than 1% . cytotoxicity of t - lymphocytes from s180 mixture of heat shock protein / peptide - immunized mice based on lactate dehydrogenase release . ( a ) comparison of mixture of heat shock protein / peptide - immunized mice with tumor - bearing mice and wild - type mice . the cytotoxicity of mixed lymphocytes against s180 tumor cells in mice immunized with mixture of heat shock protein / peptide was higher than that in untreated tumor - bearing ( e / t = 5:1 , 20:1 and 40:1 , * p < 0.01 ) and wild - type ( e / t = 5:1 , 20:1 and 40:1 , p < 0.05 ) mice . ( b ) cytotoxicity of lymphocytes from mice vaccinated with s180 mixture of heat shock protein / peptides against mca207 . antibody detected in immunized mice all mice in the control and mhsp / p groups were positive for anti - hsp70 and -hsp60 antibodies using serum dilutions of 1:5001:2000 . moreover , there were no differences in levels of antibodies against hsp60 and hsp70 [ figure 8 ] . ( a and b ) cytotoxicity of lymphocytes from mice vaccinated with s180 mixture of heat shock protein / peptides . the efficacy of current standard therapies for the treatment of sarcoma remains limited . to facilitate the development of vaccine - based immunotherapy for sarcoma , we evaluated mhsp / ps as a vaccine treatment for sarcoma in mice . vaccination with gp96 ( oncophage ) and hsp70 as immunotherapy has been tested in clinical trials . however , use of a mixture of hsp60 , hsp70 , hsp110 , and gp96 ( mhsp / p ) as a vaccine for immunotherapy of sarcoma has not been reported . here , we examined the capacity of mhsp / p , a mixture of hsp60 , hsp70 , hsp110 , and gp96 , as a vaccine for immunotherapy of sarcoma in mouse models . mhsp / p vaccination induced an antitumor immune response and significantly inhibited s180 and mca207 tumor growth in mice , and significantly prolonged their survival . the antitumor effects of mhsp / ps may improve the antitumor effects of vaccination with hsp70 , hsp60 , or tumor lysate . the hsp / p profile expressed in sarcoma we examined 110 cases of human orthopedic tumors , including osteosarcoma , chondrosarcoma , fibrosarcoma , liposarcoma , giant cell sarcoma , and synovial sarcoma . hsp60 was 100% highly expressed in those tumors based on immunohistochemistry , and was abundant in s180 and mca207 cell lines , as shown by sds - page and western blot analysis . hsp110 was 90% expressed , besides hsp70 , and gp96 , which are highly expressed in cancer cells . thus , we isolated all subtypes of hsp / ps from mouse sarcoma . in our experiments , the antitumor effect of mhsp / ps was greater than that of any single hsp ; hsp60 is a potent adjuvant that induces innate immune responses and has been used in vaccines as an effective adjuvant . another major component , hsp110 , was more effective than hsp70 in binding the peptide chain and can bind various peptides originating from hsp70 or gp96 . our mixture of hsp / ps included hsp60 and hsp110 , in addition to hsp70 and gp96 , and could bind more antigen peptides and potentially induce multifarious immune responses . this may explain why use of multiple mhsp / ps results in greater antitumor effects compared to use of single hsp70 , hsp60 alone , although the differences between mhsp / p and hsp70 or hsp60 were not significant . in addition , mhsp / ps isolated from mca207 cells were more effective than those isolated from s180 cells , possibly because s180 is more aggressive than mca207 . in the s180 model , metastasis of the tumor to lung occurred in 90% of the mice , compared to 0% in the mca207 model . the mean survival of s180 tumor - bearing mice was 35 days , and that of mca207 tumor - bearing mice was 54 days . we investigated the specificity of the mhsp / p vaccine in our experiments because the mhsp / p of the s180 model did not induce cytolysis of mca207 ( < 10% cytolysis ) , and mice vaccinated with the mca207 mhsp / ps showed protection against challenge only with mca207 , not s180 , in vivo . in addition , mhsp / ps isolated from liver and muscle of wild - type mice had no cytolytic effect against s180 sarcoma in vitro . thus , the mhsp / p induced an immune reaction was against only an autologous tumor - specific antigen , as with individual vaccines . in addition to tumor antigenic peptides , a vaccine may contain an array of self - peptide complexes , thus raising the possibility of eliciting auto - immunity ; in particular , hsp60 may be associated with the development of arthritis , systemic lupus erythematosus , atherosclerosis , autoimmune thyroid disease , and diabetes . however , in such cases the autoantibody level was considerably higher than that in normal individuals . in our experiment , the levels of antibody against hsp60 and hsp70 in immunized mice were similar to those in wild - type mice . the levels of antibodies against hsp60 and hsp70 were elevated in wild - type mice , perhaps because of natural bacterial infection , which resulted in the production of anti - hsp antibodies against xenogeneic organisms . antibody against hsp60 can be detected in normal humans ; however , our results indicated that immunization with mhsp / p did not increase the level of antibodies against hsp60 or hsp70 , which suggested that mhsp / ps may not induce autoimmune diseases . we found that the number of nk cells and the inf- level were elevated in mhsp / p - treated mice , which increased the frequency of ifn--producing cells and ifn--secreting nk cells . thus , mhsp - mediated nk cell activation could play a role in eliciting antitumor responses . this finding is consistent with the use of hsp70 as a vaccine in a clinical trial . vaccination with whole tumor cells or tumor cell lysates have been investigated in phase iii clinical trials of colon cancer and melanoma . in our mouse model , the mhsp / ps contained mainly high - molecular - weight proteins ( about 40200 kda ) , as determined by chromatography . small molecules  such as transforming growth factor  , interleukin 10 , prostaglandin , or immunosuppressive cytokines  were excluded . in summary , this preliminary study of a mhsp / p tumor vaccine showed induction of effective immune responses in immunized mice . the use of mhsp / ps as a vaccine can provide a broad antigen repertoire of tumor cell peptides , and sufficient quantities of mhsp / p can be prepared from a small amount of tumor tissue . overall , mhsp / p functions as a potent antitumor vaccine and will facilitate future clinical studies of autologous , patient - specific hsp - based vaccines for immunotherapy of human sarcoma .
background : to develop a vaccine - based immunotherapy for sarcoma , we evaluated a mixture of heat shock proteins ( mhsps ) as a vaccine for sarcoma treatment in a mouse model . 
 heat shock protein / peptides ( hsp / ps ) are autoimmune factors that can induce both adaptive and innate immune responses ; hsp / ps isolated from tumors can induce antitumor immune activity when used as vaccines.methods:in this study , we evaluated the effects of mhsp / ps on prophylactic antitumor immunity . 
 we extracted mhsp / ps , including hsp60 , hsp70 , gp96 , and hsp110 , from the mouse sarcoma cell lines s180 and mca207 using chromatography . the immunity induced by mhsp / ps 
 was assessed using flow cytometry , elispot , lactate dehydrogenase release , and enzyme - linked immunosorbent assay.results:of s180 sarcoma - bearing mice immunized with mhsp / ps isolated from s180 cells , 41.2% showed tumor regression and long - term survival , with a tumor growth inhibition rate of 82.3% at 30 days . of mca207 sarcoma - bearing mice immunized with mhsp / ps isolated from mca207 cells , 50% showed tumor regression and long - term survival with a tumor growth inhibition rate of 79.3% . 
 all control mice died within 40 days . 
 the proportions of natural killer cells , cd8 + , and interferon--secreting cells and tumor - specific cytotoxic t - lymphocyte activity were increased in the immunized group.conclusions:vaccination with a polyvalent mhsp / p cancer vaccine can induce an immunological response and a marked antitumor response to autologous tumors . 
 this mhsp / p vaccine exerted greater antitumor effects than did hsp70 , hsp60 , or tumor lysates alone .