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

为了阐明结直肠癌侵袭边缘肿瘤腺体、基底膜和纤维间质之间的三维关系，我们尝试了几种利用免疫扫描电子显微镜(免疫扫描电子显微镜)识别胶原纤维类型的技术。在项目期间，对标本进行免疫金标记法和免疫扫描电子显微镜背向散射和二次电子成像观察，对类似制备的标本进行德克萨斯红标记和激光共聚焦扫描显微镜(CLSM)观察并进行三维图像重建，其中激光共聚焦扫描显微镜(CLSM)用于估计免疫扫描显微镜上金标记的位置。Stromel胶原纤维为I型，倾向于形成厚束并平行于腺泡外表面运行。癌腺泡(或无基膜的腺泡)的基底膜与间质胶原纤维之间几乎没有纤维联系。形成癌腺泡基底膜的IV型胶原纤维呈波状排列，纤维束排列密集，但网状不宽，偶见不连续或缺失。结论：1.与正常隐窝相比，肿瘤腺泡的基底膜既不能为癌细胞提供牢固的锚定，也不利于物质的穿透。目前的结果可能支持这一理论，即它是一种新沉积的结构，而不是被肿瘤细胞破坏的先前存在的基底膜。2.免疫扫描电子显微镜在组织(而非细胞学)水平上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，许多来自不同肿瘤学领域的研究人员对此进行了研究。

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.