肿瘤病变中CD8(+)T细胞(CTL)的局部浸润预测了癌症患者来自免疫检查点阻断的总体临床结果和临床益处。在目前的研究中,我们评估了前列腺癌病变中不同类别趋化因子的局部产生,以及它们调节以促进CTL选择性进入前列腺肿瘤的可行性。
通过基于TaqMan的定量PCR分析前列腺癌病灶中趋化因子的表达,共聚焦荧光显微镜和ELISA。对于离体趋化因子调制分析,从接受原发性前列腺癌切除术的患者的前列腺肿瘤外植体培养24小时,在不存在或存在聚-I:C的组合的情况下,IFNα,和塞来昔布(PAC)。通过共聚焦显微镜分析组织中产生确定的趋化因子的细胞数量。在标准的体外迁移测定中评估了效应CD8()T细胞对未处理和PAC处理的肿瘤外植体上清液的趋化性,使用24孔反式孔板。通过流式细胞术计数迁移的效应细胞的数量。采用Pearson(r)相关性分析趋化因子与免疫滤液的相关性,而配对双尾学生t检验用于治疗组之间的比较。
前列腺肿瘤显示均匀低水平的CTL/NK/Th1募集趋化因子(CCL5,CXCL9,CXCL10),但表达高水平的趋化因子,涉及髓源性抑制细胞(MDSC)和调节性T细胞(Treg)的吸引:CCL2,CCL22和CXCL12。在CXCL9和CXCL10与局部CD8表达之间观察到强正相关。CCL2、CCL22和CXCL12的肿瘤表达水平与肿瘤内MDSC/Treg标志物FOXP3、CD33和NCF2的表达相关。用PAC处理抑制了肿瘤内Treg引诱剂CCL22和Treg/MDSC引诱剂的产生,CXCL12,在增加CTL引诱剂产量的同时,CXCL10.局部趋化因子产生的这些变化伴随着离体治疗的肿瘤吸引CD4(+)FOXP3(+)Treg细胞的能力降低,和强烈增强的CD8(+)颗粒酶B(+)CTL的吸引力。
我们的数据表明,前列腺癌中的趋化因子环境可以重新编程,以选择性地增强1型效应免疫细胞的吸引力并减少MDSC和Tregs的局部吸引力。前列腺76:1095-1105,2016。©2016威利期刊,Inc.
Local infiltration of CD8(+) T cells (CTLs) in tumor lesions predicts overall clinical outcomes and the clinical benefit of cancer patients from immune checkpoint blockade. In the current study, we evaluated local production of different classes of chemokines in prostate cancer lesions, and the feasibility of their modulation to promote selective entry of CTLs into prostate tumors.
Chemokine expression in prostate cancer lesion was analyzed by TaqMan-based quantitative PCR, confocal fluorescence microscopy and ELISA. For ex vivo chemokine modulation analysis, prostate tumor explants from patients undergoing primary prostate cancer resections were cultured for 24 hr, in the absence or presence of the combination of poly-I:C, IFNα, and celecoxib (PAC). The numbers of cells producing defined chemokines in the tissues were analyzed by confocal microscopy. Chemotaxis of effector CD8(+) T cells towards the untreated and PAC-treated tumor explant supernatants were evaluated in a standard in vitro migration assays, using 24 well trans-well plates. The number of effector cells that migrated was enumerated by flow cytometry. Pearson (r) correlation was used for analyzing correlations between chemokines and immune filtrate, while paired two tailed students t-test was used for comparison between treatment groups.
Prostate tumors showed uniformly low levels of CTL/NK/Th1-recruiting chemokines (CCL5, CXCL9, CXCL10) but expressed high levels of chemokines implicated in the attraction of myeloid derived suppressor cells (MDSC) and regulatory T cells (Treg ): CCL2, CCL22, and CXCL12. Strong positive correlations were observed between CXCL9 and CXCL10 and local CD8 expression. Tumor expression levels of CCL2, CCL22, and CXCL12 were correlated with intratumoral expression of MDSC/Treg markers: FOXP3, CD33, and NCF2. Treatment with PAC suppressed intratumoral production of the Treg -attractant CCL22 and Treg /MDSC-attractant, CXCL12, while increasing the production of the CTL attractant, CXCL10. These changes in local chemokine production were accompanied by the reduced ability of the ex vivo-treated tumors to attract CD4(+) FOXP3(+) Treg cells, and strongly enhanced attraction of the CD8(+) Granzyme B(+) CTLs.
Our data demonstrate that the chemokine environment in prostate cancer can be reprogrammed to selectively enhance the attraction of type-1 effector immune cells and reduce local attraction of MDSCs and Tregs . Prostate 76:1095-1105, 2016. © 2016 Wiley Periodicals, Inc.