Analysis of micro and molecular mechanism of lymphatic metastasis, and development of new strategy targeting for tumor lymphatic vessel

我们研究了CD 44及其配体透明质酸之间的相互作用对结直肠癌淋巴结转移的影响。在90%的裸鼠中，通过表达高水平CD 44的LS 174 T和LS 174 T-NEO的同种异体移植建立了淋巴结转移。而转染反义CD 44基因的Colo 320和LS 174 T-AS 2细胞在裸鼠同种异体移植中均无CD 44表达，也未发生淋巴结转移。在淋巴管的免疫组织化学中，LS 174 T-NEO显示出显著的淋巴浸润，而LS 174 T-AS 2和Colo 320中的淋巴浸润最小。LS 174 T和LS 174 T-NEO广泛附着于正常淋巴结，并被抗透明质酸处理抑制。另一方面，LS 174 T-AS 2细胞对淋巴结的粘附能力显著降低。我们评价了大肠癌细胞系对淋巴结转移的能力，并通过cDNA微阵列筛选了转移（或抗转移）基因。本文研究了23株人结直肠癌细胞系在异基因移植模型中的淋巴结转移情况，其转移率差异很大，分别为：KM 12 c 100%（7/7），CloneA 100%（9/9），HT 29 94.4%（17/18），. SW 1116 25%（2/8），DLD 1 0%（0/13）。根据淋巴结转移率将细胞系分为高、中、低三组。基于cDNA微阵列分析和体内实验，共筛选出107个转移相关基因。其中一些基因的表达水平也被实时RT-PCR证实升高或降低。此外，候选基因的表达水平与结直肠癌的临床淋巴结转移有关。

We investigated the interaction between CD44 and its ligand, hyaluronic acid, on lymph node metastasis of colorectal cancers. In 90% of nude mice, lymph node metastasis was established by alloic transplantation of LS174T and LS174T-NEO, which expressed high levels of CD44. In contrast, Colo320 and LS174T-AS2, which were transfected with antisense CD44 gene, had no expressions of CD44 and never metastasized to lymph nodes by alloic transplantation in nude mice. In immunohistochemistry of lymphatic vessels, LS174T-NEO showed marked lymphatic invasion, in contrast to minimal lymphatic invasion in LS174T-AS2 and Colo320. LS174T and LS174T-NEO widely attached to normal lymph nodes, and it was inhibited by anti-hyaluronic acid processing. On the other hand, the attachment to lymph nodes was significantly reduced in LS174T-AS2.We evaluated the ability of colorectal cancer cell lines on metastasis to lymph nodes, and metastatic (or anti-metastatic) genes were screened by cDNA microarray. Twenty three of human colorectal cancer cell lines were investigated on the lymph node metastasis in alloic transplantation model and the incidence of metastasis was varied widely as follows : KM12c 100%(7/7), CloneA 100%(9/9), HT29 94.4%(17/18),..., SW1116 25%(2/8), DLD1 0%(0/13). Cell lines were divided into three groups (high, middle and low) according to the incidence of lymph node metastasis. Based on cDNA microarray analysis together with in vivo experiments, 107 candidates of metastasis-related genes were extracted. The expression levels of some of those genes were confirmed elevated or depressed with real-time RT-PCR as well. Further, there was a relationship between expression levels of candidate genes and clinical lymph node metastasis in colorectal cancers.