前列腺癌冷冻免疫反应与原位肿瘤疫苗的实验研究
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摘要
背景：冷冻消融技术逐渐发展成为局限性前列腺癌的一线治疗手段，冷冻免疫反应研究将冷冻消融局部治疗与全身免疫治疗有机结合起来，为肿瘤综合治疗提供理论和实验依据。目的：评价冷冻消融治疗前列腺癌移植瘤后对机体抗肿瘤免疫状态的影响，探讨冷冻消融联合GM-CSF制作体内原位肿瘤疫苗的可行性与有效性。材料与方法：复制RM-1细胞激素非依赖性前列腺癌肺转移模型，将动物随机分为对照组、手术切除组、冷冻治疗组、GM-CSF治疗组、冷冻+GM-CSF治疗组。利用Endocare公司氩氦冷冻系统直径2mm冷冻针行皮下移植瘤冷冻消融治疗。HE染色观察肿瘤引流淋巴结和肺转移发生率；免疫组织化学、流式细胞术评价肿瘤局部、引流淋巴结、脾脏DC的数量及活化比例；ELISPOT法检测引流淋巴结、脾脏CD4+T细胞肿瘤特异性IFN-γ分泌情况；LDH法检测引流淋巴结、脾脏CD8+CTL细胞肿瘤杀伤活性；ELISA法检测外周血IFN-γ、IL-12、IL-4、IL-10水平，以IFN-γ/IL-4计算Th1/Th2。结果：(1)冷冻消融术前、术后7天、术后14天、术后21天外周血Th1/Th2比值分别为6．24±0．58、9．30±0．73、13．71±0．57、10．31±0．30(P＜0．05)；(2)冷冻消融术前、术后7天、术后14天、术后21天，肿瘤引流淋巴结CD4+T细胞由前列腺癌细胞刺激后IFN-γ分泌阳性细胞数每10~6T细胞分别为23．2±1．48、243．4±46．21、95．8±6．83、25．2±1．64(P＜0．05)，而肾癌细胞刺激后IFN-γ分泌无变化。脾脏CD4+T细胞由肾癌细胞、前列腺癌细胞刺激后IFN-γ分泌均没有明显变化；(3)冷冻消融术前、术后7天、术后14天、术后21天，肿瘤引流淋巴结CD8+CTL对前列腺癌细胞杀伤活性分别为(14．8±0．84)%、(62．6±2．30)%、(49．8±3．70)%、(15．6±1．14)%，对肾癌细胞杀伤活性无明显变化，而脾脏CD8+CTL对前列腺癌细胞、肾癌细胞杀伤活性均没有明显变化；(4)冷冻消融治疗术前、术后7天、术后14天、术后21天肿瘤局部DC数量分别为每高倍视野下(1．6±0．55)个、(8．6±1．14)个、(4．4±1．14)个、(1．8±0．84)个(P＜0．05)，引流淋巴结DC比例分别为(3．84±0．50)%、(8．56±0．50)%、(5．32±0．39)%、(3．66±0．51)%(P＜0．05)，DC活化比例分别为(11．2±0．84)%、(25．8±0．84)%、(18．6±1．52)%、(11．6±1．14)% (P＜0．05)，而脾脏DC数量及活化比例无明显变化；(5)相关性分析表明引流淋巴结CD4+T细胞、CD8+CTL细胞的肿瘤特异性免疫反应与DC比例及活化直接相关(P＜0．05)；(6)冷冻+GM-CSF治疗术前、术后7天、术后14天、术后21天肿瘤局部DC数量分别为每高倍视野下(1．6±0．55)个、(18．4±1．14)、(15．8±0．84)、(8．2±1．30)个(P＜0．05)，引流淋巴结DC数量比例分别为(3．88±0．31)%、(13．38±1．18)%、(11．08±0．84)%、(9．14±0．34)%(P＜0．05)，DC活化比例分别为(11．2±0．84)%、(31．4±1．82)%、(24．8±0．84)%、(19．0±1．58)%(P＜0．05)；而脾脏DC数量比例分别为(4．80±0．20)%、(13．2±1．30)%、(9．16±0．47)%、(6．94±0．32)%(P＜0．05)，DC活化比例分别为(12．8±1．3)%、(32．8±2．39)%、(26．4±1．14)%、(21．0±1．58)%(P＜0．05)；(7)冷冻+GM-CSF治疗术前、术后7天、术后14天、术后21天，肿瘤引流淋巴结CD4+T细胞由前列腺癌细胞刺激后IFN-γ分泌阳性细胞数每10~6T细胞分别为23．2±1．48、360．4±4．28、239．8±4．66、109．8±6．94(P＜0．05)，而肾癌细胞刺激后IFN-γ分泌无变化。脾脏CD4+T细胞由前列腺癌细胞刺激后IFN-γ分泌阳性细胞数每10~6T细胞分别为23．6±1．14、129．8±3．49、91．0±2．92、46．6±3．29(P＜0．05)，而肾癌细胞刺激后IFN-γ分泌无变化；(8)冷冻+GM-CSF治疗术前、术后7天、术后14天、术后21天，肿瘤引流淋巴结CTL对前列腺癌细胞杀伤活性分别为(14．8±0．84)%、(75．6±1．14)%、(64．6±1．52)%、(37．6±2．07)%(P＜0．05)，对肾癌细胞杀伤活性治疗前后无明显变化。脾脏CTL对前列腺癌细胞杀伤活性分别为(12．8±0．84)%、(47．4±1．14)%、(40．4±1．82)%、(32．2±1．48)%(P＜0．05)，对肾癌细胞杀伤活性治疗前后无明显变化；(9)冷冻+GM-CSF治疗术前、术后7天、术后14天、术后21天外周血Th1/Th2比值分别为6．24±0．58、13．16±0．38、14．91±0．63、15．13±0．50(P＜0．05)；(10)冷冻消融组、冷冻+GM-CSF治疗组术后21天淋巴结转移率分别为80%、40%，肺转移率分别为80%、20%，中位生存期分别为(55．0±1．38)天、(72．4±1．84)天。结论：冷冻消融治疗可升高Th1/Th2比值，诱导机体抗肿瘤免疫向Th1优势转化，但随时间延长有逐渐降低趋势。冷冻消融可诱导局部引流淋巴结T细胞的特异性抗肿瘤免疫反应，但不能诱导脾脏T细胞的特异性抗肿瘤免疫反应。冷冻消融诱导的肿瘤特异性免疫反应与DC数量及活化比例有关。冷冻联合GM-CSF治疗可增加肿瘤组织局部DC浸润、增加局部淋巴结、脾脏DC数量及活化比例，明显增强局部引流淋巴结以及脾脏T细胞的特异性抗肿瘤免疫反应，提高Th1/Th2比值，有效降低肺转移率，延长生存期，达到原位DC肿瘤疫苗的疗效。
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.
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.
引文
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