4NQO was painted on tongue of Swiss mice every third day at a dose of 25µl of 5mg/ml stock solution. After five consecutive treatment with 4NQO (starting Day7), one group of mice was treated with NLGP (s.c., 25µg/mice/week), keeping a group as PBS control. Mice were sacrificed in different time-intervals to harvest tongues and studied using histology, immunohistochemistry, flow-cytometry and RT-PCR on different immune cells and EMT markers (e-cadherin, vimentin) to elucidate their phenotypic and secretory status.
Local administration of 4NQO for consecutive 300 days promotes significant alteration in tongue mucosa including erosion in papillae and migration of malignant epithelial cells to the underlying connective tissue stroma with the formation of cell nests (exophytic-hyperkeratosis with mild dysplasia). Therapeutic NLGP treatment delayed pre-neoplastic changes promoting normalization of mucosa by maintaining normal structure. Flow-cytometric evidences suggest that NLGP treatment upregulated CD8+, IFNγ+, granzyme B+, CD11c+ cells in comparison to 4NQO treated mice with a decrease in Ki67+ and CD4+FoxP3+ cells in NLGP treated cohort. RT-PCR demonstrated a marked reduction of MMP9, IL-6, IL-2, CD31 and an upregulation in CCR5 in tongues from 4NQO+NLGP treated mice in comparison to 4NQO treated group. Moreover, 4NQO mediated changes were associated with reduction of e-cadherin and simultaneous up-regulation of vimentin expression in epithelium that was partially reversed by NLGP.
Efficacy of NLGP was tested first time in sequential carcinogenesis model and proved effective in delaying the initial progression. NLGP normalizes type 1 immunity including activation of the CD8+T effector functions, reduction of regulatory T cell functions, along with changes in EMT to make the host systemically alert to combat the carcinogenic threat.
■每三天将4NQO以25μl的5mg/ml储备溶液的剂量涂在瑞士小鼠的舌头上。在连续5次4NQO治疗后(第7天开始),一组小鼠用NLGP治疗(s.c.,25µg/小鼠/周),保持一组作为PBS对照。在不同的时间间隔处死小鼠以收获舌头,并使用组织学进行研究。免疫组织化学,不同免疫细胞和EMT标志物的流式细胞术和RT-PCR(e-cadherin,波形蛋白)以阐明其表型和分泌状态。
■连续300天局部施用4NQO促进舌粘膜的显著改变,包括乳头糜烂和恶性上皮细胞迁移到下面的结缔组织基质,形成细胞巢(外生性角化过度伴轻度发育不良)。治疗性NLGP治疗延迟了肿瘤前的变化,通过维持正常结构来促进粘膜正常化。流式细胞术证据表明NLGP治疗上调CD8+,IFNγ+,颗粒酶B+,与4NQO处理的小鼠相比,CD11c+细胞在NLGP处理的组群中Ki67+和CD4+FoxP3+细胞减少。RT-PCR显示与4NQO治疗组相比,4NQO+NLGP治疗小鼠的舌中MMP9、IL-6、IL-2、CD31的显著减少和CCR5的上调。此外,4NQO介导的变化与上皮中e-cadherin的减少和波形蛋白表达的同时上调有关,NLGP部分逆转了这一变化。
■首次在序贯癌变模型中测试了NLGP的功效,并证明其在延缓初始进展方面是有效的。NLGP使1型免疫正常化,包括激活CD8+T效应子功能,减少调节性T细胞功能,随着EMT的变化,使宿主系统警惕以对抗致癌威胁。