| Backgroud:Cryoablation is becoming the primary theray for local prostate cancer,while the exploration of cryoimmunology response will provide the combination oflocal therapy with immunnol therapy,and would provide rationale and experimentalfoundation for synthetic therapy of advanced cancer.Objective:To make the modelsof hormone refractory prostate cancer (HRPC) with lung metastasis.To assess theanti-tumor immune response after cryoablation,and to assess the possibility andavailability of making in-situ tumor vaccine by combined cryoablation and GM-CSFtreatment.Material and Methods:Mouse models of HRPC were made bysubcutaneouly injection of RM-1 prostate cancer cells,and the tumor-bearing micewere divided into different groups:control group,surgery group,GM-CSF group,cryoablation group,cryoablation+GM-CSF group.Cryoablation was applied bycryosurgery needle with 1.7mm diameter from Endocare Argon-Helium cryosurgerysystem.The rate of lung or lymponode metastasis were assessed by pathology,serumcytokine (IFN-γ,IL-12,IL-4,IL-10) levels were analyzed by enzyme-linkedimmunosorbent assay (ELISA),and the Th1/Th2 ratio was estimated from theIFN-γ/IL-4 ratio.The number and maturation changes of dendritic cells in tumor,draining lymph nodes (DLN),spleens were assessed by immunohistochemistry andflow cytometry.Lymphocytes of DLN and spleen were isolated,the numbers oftumor-specific IFN-γ~+CD4+cells after treatment were measured by enzyme linkedimmunospot assay (ELISPOT),and tumor-specific cytolytic activity of cytotoxic Tlymphocyte (CTL) was measured by LDH assay.Result:(1) The Th1/Th2 ratioswere 6.24±0.58,9.30±0.73,13.71±0.57,10.31±0.30 on the day before cryoablation,seventh days,14th day,21st day after cryoablation;(2) The number of IFN-γ~+cellsevery 10~6 CD4+ T cells stimulated by prostate cancer cells in DLN was 23.2±1.48,243.4±46.21,95.8±6.83,25.2±1.64 on the day before cryoablation,seventh day after,14th day after,21 st day after cryoablation respectively,there were no changes inIFN-γ~+cells after stimulated by renal cancer cells,and there were no changes in IFN-γ~+cells in spleen after stimulated by renal cancer cells or prostate cancer cells;(3)Cytolytic activities of CTL in DLN against prostate cancer cells were (14.8±0.84)%, (62.6±2.30)%,(49.8±3.70)%,(15.6±1.14)% on the day before cryoablation,sevenday after,14th day after,21 st day after cryoablation respectively,There were nochanges in cytolytic activity of CTL in DLN against renal cancer cells,and Therewere no changes in cytolytic activity of CTL in spleen against renal cancer cells orprostate cancer cells;(4) On the day before cryoablation,seventh day after,14th dayafter,21st day after cryoablation,the DC numbers per high power field in tumor were1.6±0.55,8.6±1.14,4.4±1.14,1.8±0.84 respectively,and the ratios of DC in D LNwere (3.84±0.50)%,(8.56±0.50)%,(5.32±0.39)%,(3.66±0.51)% respectively,thematuration ratios of DC in DLN were (11.2±0.84)%,(25.8±0.84)%,(18.6±1.52)%,(11.6±1.14)% respectively,but there were no changes in the number or maturationratios of DC in spleen;(5) Relation analysis proved that there was direct correlationbetween the tumor-specific immune response and DC ratio or DC maturation in DLN;(6) On the day before,seventh day after,14th day after,21 st day aftercryoablation+GM-CSF treatment,the DC numbers per high power field in tumorwere 1.6±0.55,18.4±1.14,15.8±0.84,8.2±1.30 respectively,and The ratios of DC inDLN were (3.88±0.31)%,(13.38±1.18)%,(11.08±0.84)%,(9.14±0.34)% respectively,the maturation ratio of DC in DLN were (11.2±0.84)%,(31.4±1.82)%,(24.8±0.84)%,(19.0±1.58)% respectively,the ratios of DC in spleen were (4.80±0.20)%,(13.2±1.30)%,(9.16±0.47)%,(6.94±0.32)% respectively,the maturation ratio of DCin spleen were (12.8±1.3)%,(32.8±2.39)%,(26.4±1.14)%,(21.0±1.58)% respectively;(7) On the day before,seventh day after,14th day after,21st day aftercryoablation+GM-CSF treatment,the numbers of IFN-γ~+cells every 10~6 CD4+ T cellsstimulated by prostate cancer cells in DLN were 23.2±1.48,360.4±4.28,239.8±4.66,109.8±6.94 respectively,the numbers of IFN-γ~+cells every 10~6 CD4+ T cellsstimulated by prostate cancer cells in spleen were 23.6±1.14,129.8±3.49,91.0±2.92,46.6±3.29 respectively,there were no changes in IFN-γ~+ cells in DLN or spleen afterstimulated by renal cancer cells;(8) On the day before,seventh day after,14th dayafter,21st day after cryoablation+GM-CSF treatment,cytolytic activities of CTL inDLN against prostate cancer cells were (14.8±0.84)%,(75.6±1.14)%,(64.6±1.52)%,(37.6±2.07)% respectively,cytolytic activities of CTL in spleen against prostatecancer cells were (12.8±0.84)%,(47.4±1.14)%,(40.4±1.82)%,(32.2±1.48)% respectively,there were no changes in cytolytic activity of CTL in DLN or spleenagainst renal cancer cells;(9) The Th1/Th2 ratios were 6.24±0.58,13.16±0.38,14.91±0.63,15.13±0.50 on the day before,seventh days after,14th day after,21st dayafter cryoablation+GM-CSF treatment;(10) On The 21st day after cryoablation orcryoablation+GM-CSF treatment,the rates of DLN metastasis were 80% vs 40%,Therates of lung metastasis were 80% vs 20%,the median survival times were (55.0±1.38)days vs (72.4±1.84) days respectively.Conclusion:Cryoablation could increase theratio of Th1/Th2,inducing the Th1 advantage transformation of anti-tumorimmunology response,although gradually lowered by time.Cryoablation couldinduce tumor-specific immune response in DLN not in spleen,while thetumor-specific immune response was related with the number and activation ratio ofDC.Combined cryoablation with GM-CSF treatment could increase the number ofDC in tumor microenvironment,DLN and spleen,increase the activation of DC inDLN and spleen,then increase The tumor-specific immune response in DLN andspleen,improve the systemic anti-tumor immune response,increase the ratio ofTh1/Th2,decrease the rate of lung metastasis,prolong the median survival times,achieve the therapeutic effect of an in-situ tumor vaccine.
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