Oral squamous cell carcinoma(OSCC)is typified by extensive stromal fibrosis and an immunosuppressive microenvironment,both of which impede effective responses to immune checkpoint blockade.In this study,we identify pr...Oral squamous cell carcinoma(OSCC)is typified by extensive stromal fibrosis and an immunosuppressive microenvironment,both of which impede effective responses to immune checkpoint blockade.In this study,we identify prolyl 3-hydroxylase family member 4(P3H4)as a critical mediator of extracellular matrix(ECM)remodeling,epithelial-mesenchymal transition(EMT),and the exclusion of cytotoxic CD8+T lymphocytes.Elevated P3H4 expression correlates with unfavorable clinical outcomes and resistance to immunotherapy.Genetic ablation of P3H4 significantly attenuates tumor progression and promotes CD8^(+)T cell infiltration.To pharmacologically target P3H4,we engineered a liposomal formulation of 1,4-dihydrophenanthroline-2,5-dicarboxylic acid(1,4-DPCA),a small-molecule prolyl hydroxylase inhibitor.This nanomedicine,designated Lipo-1,4-DPCA,effectively downregulates P3H4 expression,mitigates tumor-associated fibrosis,reprograms the immune microenvironment,and elicits robust anti-tumor responses in vivo.Collectively,our findings establish P3H4 as a promising therapeutic target and highlight Lipo-1,4-DPCA as a dualfunctional nanotherapeutic candidate capable of enhancing the efficacy of immunotherapy in OSCC.展开更多
目的探讨光动力纳米载体联合小干扰-脯氨基3-羟化酶家族成员(si-P3H4)精准治疗膀胱肿瘤的疗效。方法RT-qPCR和Western Blot分别检测转染siRNA后膀胱癌EJ和T24细胞株中的P3H4mRNA和蛋白表达量。CCK8、划痕实验和Transwell小室检测敲低P3H...目的探讨光动力纳米载体联合小干扰-脯氨基3-羟化酶家族成员(si-P3H4)精准治疗膀胱肿瘤的疗效。方法RT-qPCR和Western Blot分别检测转染siRNA后膀胱癌EJ和T24细胞株中的P3H4mRNA和蛋白表达量。CCK8、划痕实验和Transwell小室检测敲低P3H4后对EJ和T24膀胱癌细胞增殖、迁移、侵袭的影响。以氨基树脂为底物合成高分子纳米载体CH3-R9-cRGD,将纳米载体药物包裹si-P3H4、光敏剂Ce6转染至膀胱癌细胞(HCV29细胞),检测不同pH及激光照射条件下药物体外释放情况。同时,探索纳米药物与膀胱癌细胞靶向结合内吞机制,检测细胞内活性氧(ROS)水平。将不同分组纳米复合物转染至膀胱癌细胞,CCK8法检测各组细胞活力,体内试验进一步探索不同分组纳米复合物肿瘤抑制能力。结果RT-qPCR显示EJ组和T24组P3H4mRNA表达量分别降低至对照组的68.4%和57.1%。Western Blot显示EJ组和T24组P3H4蛋白表达较阴性对照组分别下降至20.3%和36.5%。CCK8实验吸光度A值为EJ组相对于对照组96 h:(0.785±0.084) vs (1.358±0.064),t=12.06,P<0.01;T24组相对于对照组96 h:(0.833±0.065) vs (1.346±0.545),t=13.415,P<0.01。划痕实验细胞愈合率:EJ组相对于对照组为(47.8±4.32)% vs (68.60±4.39)%,t=7.545,P<0.01;T24组相对于对照组为(50.40±3.64)% vs (70.61±3.85)%,t=8.521,P<0.01。Transwell细胞穿膜数:EJ组相对于对照组为[(302.71±7.56) vs (130.42±3.95)]个,t=53.40,P<0.01;T24组[(99.56±4.50) vs (35.22±6.28)]个,t=24.974,P<0.01。成像结果显示多肽纳米载体上具有良好的渗透性,可以同时将si-P3H4和Ce6靶向运输至膀胱癌细胞中。CCK8法检测结果显示当纳米复合物CH3-R9-cRGD&Ce6的浓度为50 μg/ml时,激光照射组的细胞活力均低于无激光照射组,且纳米探针+激光+siP3H4组细胞活力最低(F=299.57,P<0.05)。体内试验显示纳米复合物/si-P3H4/激光抑制肿瘤细胞增强效果最为显著。结论纳米载体可以同时将si-P3H4和Ce6靶向运输至膀胱癌细胞中抑制细胞增殖,实现对膀胱癌的综合治疗。光动力和基因治疗在膀胱肿瘤治疗领域具有互补优势,两者联用可达到协同增强肿瘤治疗的效果,可以将其应用于膀胱尿路上皮癌的治疗。展开更多
基金supported by grants from the National Natural Science Foundation of China(Nos.82501207,81700993,52403312,and 82571151)Postdoctoral Fellowship Program of CPSF(No.GZC20251219)+1 种基金the Beijing Nova Program(No.20250484855)the Beijing Natural Science Foundation(No.L252168).
文摘Oral squamous cell carcinoma(OSCC)is typified by extensive stromal fibrosis and an immunosuppressive microenvironment,both of which impede effective responses to immune checkpoint blockade.In this study,we identify prolyl 3-hydroxylase family member 4(P3H4)as a critical mediator of extracellular matrix(ECM)remodeling,epithelial-mesenchymal transition(EMT),and the exclusion of cytotoxic CD8+T lymphocytes.Elevated P3H4 expression correlates with unfavorable clinical outcomes and resistance to immunotherapy.Genetic ablation of P3H4 significantly attenuates tumor progression and promotes CD8^(+)T cell infiltration.To pharmacologically target P3H4,we engineered a liposomal formulation of 1,4-dihydrophenanthroline-2,5-dicarboxylic acid(1,4-DPCA),a small-molecule prolyl hydroxylase inhibitor.This nanomedicine,designated Lipo-1,4-DPCA,effectively downregulates P3H4 expression,mitigates tumor-associated fibrosis,reprograms the immune microenvironment,and elicits robust anti-tumor responses in vivo.Collectively,our findings establish P3H4 as a promising therapeutic target and highlight Lipo-1,4-DPCA as a dualfunctional nanotherapeutic candidate capable of enhancing the efficacy of immunotherapy in OSCC.
文摘目的探讨光动力纳米载体联合小干扰-脯氨基3-羟化酶家族成员(si-P3H4)精准治疗膀胱肿瘤的疗效。方法RT-qPCR和Western Blot分别检测转染siRNA后膀胱癌EJ和T24细胞株中的P3H4mRNA和蛋白表达量。CCK8、划痕实验和Transwell小室检测敲低P3H4后对EJ和T24膀胱癌细胞增殖、迁移、侵袭的影响。以氨基树脂为底物合成高分子纳米载体CH3-R9-cRGD,将纳米载体药物包裹si-P3H4、光敏剂Ce6转染至膀胱癌细胞(HCV29细胞),检测不同pH及激光照射条件下药物体外释放情况。同时,探索纳米药物与膀胱癌细胞靶向结合内吞机制,检测细胞内活性氧(ROS)水平。将不同分组纳米复合物转染至膀胱癌细胞,CCK8法检测各组细胞活力,体内试验进一步探索不同分组纳米复合物肿瘤抑制能力。结果RT-qPCR显示EJ组和T24组P3H4mRNA表达量分别降低至对照组的68.4%和57.1%。Western Blot显示EJ组和T24组P3H4蛋白表达较阴性对照组分别下降至20.3%和36.5%。CCK8实验吸光度A值为EJ组相对于对照组96 h:(0.785±0.084) vs (1.358±0.064),t=12.06,P<0.01;T24组相对于对照组96 h:(0.833±0.065) vs (1.346±0.545),t=13.415,P<0.01。划痕实验细胞愈合率:EJ组相对于对照组为(47.8±4.32)% vs (68.60±4.39)%,t=7.545,P<0.01;T24组相对于对照组为(50.40±3.64)% vs (70.61±3.85)%,t=8.521,P<0.01。Transwell细胞穿膜数:EJ组相对于对照组为[(302.71±7.56) vs (130.42±3.95)]个,t=53.40,P<0.01;T24组[(99.56±4.50) vs (35.22±6.28)]个,t=24.974,P<0.01。成像结果显示多肽纳米载体上具有良好的渗透性,可以同时将si-P3H4和Ce6靶向运输至膀胱癌细胞中。CCK8法检测结果显示当纳米复合物CH3-R9-cRGD&Ce6的浓度为50 μg/ml时,激光照射组的细胞活力均低于无激光照射组,且纳米探针+激光+siP3H4组细胞活力最低(F=299.57,P<0.05)。体内试验显示纳米复合物/si-P3H4/激光抑制肿瘤细胞增强效果最为显著。结论纳米载体可以同时将si-P3H4和Ce6靶向运输至膀胱癌细胞中抑制细胞增殖,实现对膀胱癌的综合治疗。光动力和基因治疗在膀胱肿瘤治疗领域具有互补优势,两者联用可达到协同增强肿瘤治疗的效果,可以将其应用于膀胱尿路上皮癌的治疗。
