Analysis of the invasive capacity at the invasive front of colon cancer, using crypt isolation technique and microarray

为了分析癌细胞的侵袭能力，我们筛选了在侵袭前沿表达特定变化的基因。我们从20个手术切除的样本中提取了位于肿瘤侵袭前沿和肿瘤中心的癌细胞。虽然我们最初尝试了隐窝分离技术，但要从每个部位正确地分离和采集癌细胞是非常困难的。因此，我们通过激光显微切割技术获取癌细胞，进行微阵列分析，并鉴定出在侵袭前沿表达发生特异性变化的基因。通过学生t检验，我们获得了5个信号在侵袭前线上调的基因和1个信号下调的基因。通过聚类分析，24个基因表达上调，29个基因表达下调。此外，最近的一些报道表明，侵袭性前沿低分化簇的存在与预后不良相关，当检查具有这种组织学特征的11例病例时，我们可以通过学生t检验来阐明4个上调基因和6个下调基因。通过聚类分析，共鉴定出13个表达上调的基因和18个表达下调的基因。我们的结果表明，许多与蛋白分解或细胞外基质无关的分子可能与癌细胞的侵袭或低分化相关。我们现在正在通过RT-PCR来证实所拾取的基因的表达。

To analyze the invasive capacity of cancer cells, we screened the genes whose expression specifically changes at the invasive front. We harvested the cancer cells at the invasive front and the center of the tumor from 20 surgically resected samples. Though we attempted the crypt isolation technique at first, it was very difficult to isolate and harvest cancer cells from each part correctly. So we harvested cancer cells by laser micro-dissection technique, conducted microarray analysis, and identified the genes whose expression changed specifically at the invasive front. By Student-t test, we picked up five genes whose signals were up-regulated at the invasive front, and one gene whose signal was down-regulated. By cluster analysis, twenty-four genes were up-regulated, and twenty-nine genes were down-regulated. Moreover, some recent reports revealed that the presence of poorly differentiated clusters at the invasive front was correlated with poor prognosis.When examined the eleven cases with this histological feature, we could elucidate four up-regulated and six down-regulated genes by Student-t test. By cluster analysis, thirteen up-regulated and eighteen down-regulated genes were identified.Our results suggested that many molecules not related with proteolysis or extra-cellular matrix might correlate with cancer cell invasion or poor differentiation. We are now confirming expression of the picked-up genes by RT-PCR.