In vivo and in vitro characterization of bone-marrow derived fibroblast recruited into cancer induced stroma.

成纤维细胞是癌症诱导间质的主要组成部分，可对邻近恶性上皮的进展产生重大影响。为了描述成纤维细胞被募集到癌症诱导的基质中，我们检测了9种人类成纤维细胞系在免疫缺陷小鼠中产生的实验性肿瘤中的募集效率。分别在不同部位注射绿色荧光蛋白（GFP）标记的成纤维细胞株和人胰腺癌细胞株Capan-1；然后分析异种移植物中gfp标记的细胞。KM104GFP（骨髓）和VA-13GFP（肺）比其他细胞系更有效地选择性募集到癌间质中。KM104GFP细胞不影响肿瘤体积；然而，VA-13GFP细胞使肿瘤体积增加约2倍。在Capan-1中对KM104GFP进行5次体内循环传代后，我们选择了一个招募效率比亲本KM104GFP高8.4倍的亚群（KM104GFP- 5g）。与KM104GFP相比，KM104GFP- 5g在对癌症释放的化学引诱剂的反应中也表现出更高的趋化性和化学侵袭活性。寡核苷酸芯片分析鉴定出KM104GFP-5G中>3倍上调基因8个，>3倍下调基因6个。免疫组织化学证实，募集到胰腺癌基质中的成纤维细胞强烈表达碳酸酐酶IX和角蛋白8，其转录本在KM104GFP-5G中上调，而在非癌性胰腺基质中的成纤维细胞中表达低于检测水平。我们的研究结果表明，就器官来源而言，成纤维细胞募集不是选择性的，具有特定表型特征的特定成纤维细胞亚群可以有效地募集到癌症诱导的基质中。

Fibroblasts, which are a major component of cancer-induced stroma, can have a significant impact on the progression of adjacent malignant epithelia. To characterize fibroblasts recruited into cancer-induced stroma, we examined the recruitment efficiency of 9 human fibroblast cell lines into experimental tumors generated in immunodeficient mice. Green fluorescence protein (GFP)-labeled fibroblast cell lines and human pancreatic cancer cell line Capan-1 were injected i.p. at different sites ; the GFP-labeled cells within xenografts were then analyzed. KM104GFP (bone marrow) and VA-13GFP (lung) were selectively recruited into cancer stroma more efficiently than the other cell lines. KM104GFP cells did not affect tumor volume ; however, VA-13GFP cells increased tumor volume by about 2-fold. After 5 cyclic in vivo passages of KM104GFP in Capan-1, we selected a subpopulation with an 8.4-fold higher recruitment efficiency (KM104GFP-5G) compared to parental KM104GFP. KM104GFP-5G also exhibited higher chemotaxis and chemoinvasion activity compared to KM104GFP in response to cancer-released chemoattractant(s). Oligonucleotide microarray analysis identified 8 genes with >3-fold upregulation and 6 genes with >3-fold downregulation in KM104GFP-5G. Immunohistochemistry confirmed that fibroblasts recruited into pancreatic cancer stroma strongly expressed carbonic anhydrase IX and keratin-8, whose transcripts were upregulated in KM104GFP-5G by oligonucleotide microarray analysis, whereas their expression in fibroblasts within noncancerous pancreatic stroma were under the detection level. Our results indicate that fibroblast recruitment is not selective with respect to organ origin and that particular fibroblast subpopulations with specific phenotypic characteristics could be recruited efficiently into cancer-induced stroma.