Jung Shin, Kim Hyun-Woo, Lee Je-Hyuk, Kang Sam-Suk, Rhu Hyang-Hwa, Jeong Young-Il, Yang Soo-Yeon, Chung Hee-Young, Bae Choon-Sang, Choi Chan, Shin Boo-Ahn, Kim Kyung-Keun, Ahn Kyu-Youn
Chonnam National University Research Institute of Medical Sciences, Chonnam National University, Medical School, Dong-Ku, Kwangju, South Korea.
J Cancer Res Clin Oncol. 2002 Sep;128(9):469-76. doi: 10.1007/s00432-002-0366-x. Epub 2002 Aug 27.
The primary cause of local recurrence and therapeutic failure in the treatment of malignant gliomas is the invasion of tumor cells into the surrounding normal brain. While it is known that malignant gliomas infiltrate diffusely into regions of normal brain, it is frequently very difficult to unequivocally identify the solitary invading glioma cell in histopathological preparations, or in experimental glioma models. We have developed an experimental invasion assay system, which allows us to track the solitary invasive glioma cell, using human brain tissue obtained from routine craniotomies for seizures or trauma.
This tissue is cut into 1-mm thick slices and cultured in the upper chamber of Transwell culture dishes on top of a 0.4- micro m pore size polyester membrane, which is fed on medium provided in the lower chamber. Glioma cells are stably transfected with vectors containing a green fluorescent protein (GFP) cDNA. Stable, high-level expression GFP transfectants were selected by direct visualization under fluorescence microscope. In addition, various tumor spheroids are stained with vital dye, DiI, to track the invading cells. GFP-expressing glioma cells or stained spheroids were then implanted on the center of the brain slice, and the degree of brain tumor invasion into the brain tissue was evaluated at different time points by optical sectioning using a confocal microscope.
We observed that GFP-expressing glioma cells or stained spheroids could be readily tracked and followed with this model system. Individual tumor cells that exhibited green or red fluorescence could be identified and their migration path through the brain slices unequivocally followed.
This experimental invasion system may be of considerable utility in studying the process of brain tumor invasion and in evaluating its invasiveness in individual brain tumor because it not only provides a better representation of extracellular matrix molecules normally encountered by invading glioma cells, but also provides the fluorescent tag applied to the tumor cells.
恶性胶质瘤治疗中局部复发和治疗失败的主要原因是肿瘤细胞侵入周围正常脑组织。虽然已知恶性胶质瘤会弥漫性浸润到正常脑区，但在组织病理学标本或实验性胶质瘤模型中，通常很难明确识别单个侵入的胶质瘤细胞。我们开发了一种实验性侵袭检测系统，利用因癫痫发作或创伤而进行常规开颅手术获取的人脑组织，来追踪单个侵袭性胶质瘤细胞。
将该组织切成1毫米厚的切片，培养在Transwell培养皿的上室，置于孔径为0.4微米的聚酯膜上，下室提供培养基。用含有绿色荧光蛋白（GFP）cDNA的载体稳定转染胶质瘤细胞。通过荧光显微镜直接观察筛选出稳定、高水平表达GFP的转染子。此外，用活性染料DiI对各种肿瘤球体进行染色，以追踪侵袭细胞。然后将表达GFP的胶质瘤细胞或染色的球体植入脑片中心，在不同时间点使用共聚焦显微镜通过光学切片评估脑肿瘤向脑组织的侵袭程度。
我们观察到，利用该模型系统可以很容易地追踪表达GFP的胶质瘤细胞或染色的球体。可以识别出发出绿色或红色荧光的单个肿瘤细胞，并明确其在脑片中的迁移路径。
这个实验性侵袭系统在研究脑肿瘤侵袭过程以及评估个体脑肿瘤的侵袭性方面可能具有相当大的实用价值，因为它不仅能更好地呈现侵袭性胶质瘤细胞通常遇到的细胞外基质分子，还能为肿瘤细胞提供荧光标记。
