Stem cell marker 인 Aldehyde dehydrogenase1 을 이용한 뇌종양 유발세포의 분리 및 특성 분석

Abstract

Aldehyde dehydrogenase 1 (ALDH1) has been identified in stem cells from both normal and cancerous tissue. This study aimed to evaluate the potential of ALDH1 as an universal brain tumor initiating cell (BTIC) marker applicable to primary brain tumors and their biological role in maintaining stem cell status. Cells from various primary brain tumors (medulloblastoma, ependymoma, atypical teratoid/rhabdoid tumor (ATRT), pilocytic astrocytoma, subependymal giant cell astrocytoma (SEGA), pineoblastoma, choroid plexus papilloma, choroid plexus carcinoma, glioblastoma and anaplastic astrocytoma) were stained with Aldefluor and sorted by flow cytometry. We analyzed CD133 and ALDH1 co-expressing cells by dual staining. For further studies, we sorted ALDH1+ and ALDH1− cells from four different brain tumors (medulloblastoma, ependymoma, ATRT and glioblastoma) using a fluorescence activated cell sorting (FACS). We performed sphere forming assay and BrdU assay for comparing ALDH1+ and ALDH1− cells. The characterization and differentiation potential of ALDH1+ cells were investigated by immunostaining. To observe the differences in the molecular profiles of ALDH1+ and ALDH1- cells, we evaluated gene expression levels of reprogamming-related factors. For functional study, we inhibited ALDH1 using ALDH1 inhibitors or short hairpin RNA (shRNA) in vitro. We confirmed the tumorigenic potential and histological characteristics in vivo. Primary cultured brain tumor cells showed universal expression of ALDH1 with 0.3 to 28.9% of the cells in various tumors identified as ALDH1+ (average 12.7 ± 8.3 %). ALDH1+ cells generated neurospheres (or tumor-spheres) with high proliferative potential, expressed neural stem cell markers (nestin and mushashi) and differentiated into multiple nervous system lineages. ALDH1+ cells were phenotypically distinct from CD133+ cells. They showed high expression of induced pluripotent stem cell related genes. Notably, ALDH1 inhibitors significantly reduced sphere forming capability. Moreover, targeted knockdown of ALDH1 by shRNA interference in BTICs potently disrupted their self-renewing ability. At the three months end point, ALDH1+ cells gave rise to tumors in 93% of mice, whereas ALDH1- cells did not. Xenograft tumor showed the same pathology as the parental human brain tumors. Tumor xenografts derived from ALDH1+ cells showed high proliferative potential and displayed a mixture of both ALDH1+ cell and ALDH1− cells. ALDH1/CD133 double staining showed few co-expressed cells in pediatric tumors. The majority of ALDH1+ cells exhibited co-expression of CD133 in glioblastoma. Our data suggest that primary brain tumors contain distinct subpopulation of cells that have high expression levels of ALDH1 and BTIC characteristics. ALDH1 might be a potential therapeutic target applicable to primary brain tumors.