Targeting Carcinoma-Associated Fibroblasts Within the Tumor Stroma With a Fibroblast Activation Protein-Activated Prodrug
Targeting Carcinoma-Associated Fibroblasts Within the Tumor Stroma With a Fibroblast Activation Protein-Activated Prodrug
复制标题
DOI：
10.1093/jnci/djs336
复制
发表时间：
2012-09-01
影响因子：
10.3
通讯作者：
Denmeade, Samuel R.
中科院分区：
文献类型：
--
作者：
Brennen, W. Nathaniel;Rosen, D. Marc;Denmeade, Samuel R.
在肿瘤微环境中，成纤维细胞会发生形态转变为反应性表型，其特征是表达诸如成纤维细胞活化蛋白（FAP）等蛋白质，FAP是一种脯氨酰后内切肽酶，其表达主要局限于癌相关成纤维细胞。毒胡萝卜素（TG）是一种毒性很强的天然植物产物，可引发细胞内钙水平升高和细胞凋亡。因此，FAP是激活由与TG的一种强效细胞毒性类似物偶联的FAP特异性肽组成的前药的一个有吸引力的靶点。在人癌细胞系中通过细胞增殖试验以及对细胞内钙的影响在体外测试了FAP激活的肽基 - TG前药的功效。在Balb - C裸鼠MCF - 7和LNCaP异种移植小鼠（每组n = 9 - 11）中测试了FAP激活的前药对肿瘤生长和宿主毒性的影响。使用基于50000次排列的排列检验计算P值。使用混合效应模型来解释重复测量之间的相关性。所有统计检验都是双侧的。FAP激活的前药在低纳摩尔浓度下杀死人癌细胞（MCF - 7细胞：半数抑制浓度IC50 = 3.5 nM）。来自FAP切割的前药的氨基酸 - 12ADT类似物，而非未切割的前药，在暴露几分钟内使细胞内钙迅速升高。对暴露于FAP前药的异种移植物进行的免疫组织化学分析记录了成纤维细胞、周细胞和内皮细胞的基质选择性细胞死亡，其程度足以抑制MCF - 7和LNCaP异种移植物的生长，且全身毒性极小，而不可被FAP切割的前药则无活性。用FAP激活的前药治疗的MCF - 7和LNCaP异种移植物在治疗后第21天的治疗组与对照组肿瘤体积比最大分别为0.36（治疗组：平均值 = 0.206 mm³，95%置信区间 = 0.068至0.344 mm³；对照组：平均值 = 0.580 mm³，95%置信区间 = 0.267至0.893 mm³）和0.24（治疗组：平均值 = 0.131 mm³，95%置信区间 = 0.09至0.180 mm³；对照组：平均值 = 0.543 mm³，95%置信区间 = 0.173至0.913 mm³）。这项研究验证了FAP的蛋白水解活性作为激活全身给药的细胞毒性前药的一个靶点，并证明了对肿瘤微环境的基质区室中的细胞进行靶向杀伤可产生治疗反应。
Fibroblasts undergo a morphological transformation to a reactive phenotype in the tumor microenvironment characterized by the expression of proteins such as fibroblast activation protein (FAP), a post-prolyl endopeptidase with expression largely restricted to carcinoma-associated fibroblasts. Thapsigargin (TG) is a highly toxic natural plant product that triggers a rise in intracellular calcium levels and apoptosis. FAP is therefore a provocative target for the activation of prodrugs consisting of a FAP-specific peptide coupled to a potent cytotoxic analog of TG.The efficacy of FAP-activated peptidyl-TG prodrugs was tested in vitro in cell proliferation assays and effects on intracellular calcium in human cancer cell lines. The effects of FAP-activated prodrugs on tumor growth and host toxicity were tested in Balb-C nude MCF-7 and LNCaP xenograft mice (n = 911 per group). P values were calculated using permutation tests based on 50 000 permutations. Mixed effects models were used to account for correlations among replicate measures. All statistical tests were two-sided.FAP-activated prodrugs killed human cancer cells at low nanomolar concentrations (MCF-7 cells: IC50 = 3.5nM). Amino acid-12ADT analogs from FAP-cleaved prodrugs, but not uncleaved prodrugs, produced a rapid rise in intracellular calcium within minutes of exposure.Immunohistochemical analysis of xenografts exposed to FAP-prodrugs documented stromal-selective cell death of fibroblasts, pericytes, and endothelial cells of sufficient magnitude to inhibit growth of MCF-7 and LNCaP xenografts with minimal systemic toxicity, whereas non-FAP cleavable prodrugs were inactive. MCF-7 and LNCaP xenografts treated with a FAP-activated prodrug had maximal treated-to-control tumor volume ratios of 0.36 (treated: mean = 0.206mm(3), 95% CI = 0.068 to 0.344mm(3); control: mean = 0.580mm(3), 95% CI = 0.267 to 0.893mm(3)) and 0.24 (treated: mean = 0.131mm(3), 95% CI = 0.09 to 0.180mm(3); control: mean = 0.543mm(3), 95% CI = 0.173 to 0.913mm(3)), respectively, on day 21 after therapy.This study validates the proteolytic activity of FAP as a target for the activation of a systemically delivered cytotoxic prodrug and demonstrates that targeted killing of cells within the stromal compartment of the tumor microenvironment can produce a therapeutic response.