An immuno-scanning electron microscope study of the tumor-stromal border in the invasive edge of colorectal cancers

为了阐明结直肠癌浸润边缘的肿瘤腺体、基底膜和纤维间质之间的三维关系，我们尝试了几种技术，通过免疫扫描电子显微镜（immune-SEM）来识别胶原纤维的类型。在项目期间，对标本进行免疫金标记，并通过免疫扫描电镜进行背散射和二次电子成像观察，对同样制备的标本进行德克萨斯红标记，并通过共聚焦激光扫描显微镜（CLSM）进行三维图像重建观察，其中进行CLSM以估计免疫扫描电镜上的金标记位点。倾向于形成厚束并平行于腺泡外表面延伸。癌腺泡基底膜（或无基底膜的腺泡）与间质胶原纤维之间几乎没有纤维连接。形成癌腺泡基底膜的IV型胶原纤维呈波浪状、排列密集的纤维束，但不呈宽网状，偶尔不连续或缺失。而正常腺窝基底膜呈网状连续排列，并与间质纤维有大量纤维连接。肿瘤腺泡的基底膜与正常腺泡的基底膜不同，既不能为癌细胞提供牢固的锚定，也不适合物质的渗透。它是一种新沉积的结构，而不是预先存在的基底膜被肿瘤细胞破坏的理论，可能会得到目前的结果的支持。2.免疫扫描电镜在组织水平（而非细胞水平）的应用可能为研究肿瘤-间质界面提供一种新的途径，近年来，来自肿瘤学不同领域的许多研究者都在研究肿瘤-间质界面。

To elucidate three-dimensional relationships among the tumor gland, basement membrane, and fibrous stroma in the invasive edge of colorectal cancers, we have attempted several techniques to identify types of collagen fibers by using immuno-scanning electron microscopy (immune-SEM) . During the term of project, immuno-gold labeling of the specimens and observation by immune-SEM with both backscattered and secondary electron imaging, and Texas-red labeling of the similarly prepared specimens and observation by confocal laser-scanning microscopy (CLSM) with three-dimensional image reconstruction were performed, where CLSM being performed to estimate the gold-labeled site on immune-SEM.Stromel collagen fibers, identified as type I,tended to form thick bundles and run parallel to the acinar outer surface. Few fibrous connections were noted between the basement membrane of the cancer acinus (or acinus without basement membranes) and the stromal collagen fibers. Type IV collagen fibers, forming the basement membrane of cancer acini, were observed as waving and densely-arranged bundles of fibers but not wide-meshed, being occasionally discontinuous or absent. In contrast, the basement membrane of the normal crypt formed a wide-meshed and continuous lattice of fibers, and numerous fibrous connections with the interstitial cellagen fibers were noted.Conclusions : 1. The basement membrane of the tumor acini may neither provide a firm anchorage to cancer cells nor be suitable for the penetration of substances, in contrast to that of the normal crypt. The theory that it is a newly-deposited structure, rather than the preexisting basement membrane destroyed by tumor cells, might be supported by the present results. 2. Application of immune-SEM in tissue (not cytologic) level may provide a new way for studying the tumor-stromal interface, which, recently, has been studied by so many investigators from different fields of onocology.