ALDH1 May Act As A Functional Marker Of Lung Cancer Stem Cells

BACKGROUNDTumor stem cell theory holds that tumors derive from tumor stem cells which are the results of cumulative mutations of normal stem cells.Tumor tissues are consist of cells with heterogeneity.There are a very small amount of tumor cells with similar properties to normal stem cells including self-renewal capacity and multilineage differentiation potential, which are the roots of proliferation metastasis and recurrence of tumors.The tumor stem cells are believed to have the following biological characteristics:(1)They are only a very small amount of tumor tissues, which can promote tumor regeneration and tumor heterogeneity. Because the tumor stem cells have self-renewal and differentiation properties, on the one hand they can generate self-renewing cancer stem cells, on the other hand they differentiate into progeny cells. (2) They possess multiple drug resistance and radiation resistance. (3) They have early micro-metastasis property. Tumor stem cells have various mechanisms such as secreting signaling molecules to create micro-enviroments which are appropriate to their differentiation and proliferation.Then the tumor cells can survive in micro-enviroments which are completely different from the in situ tumor microenvironment. (4)They have signaling pathways of the tumor cells and normal stem cells. (5)They express markers of normal stem cells, tumor cells and stem cells-related. Nowadays, people have been successfully separated and cultured tumor stem cells from acute myeloid leukemia, multiple myeloma, breast cancer, brain tumors, non-small cell lung cancer, melanoma, prostate cancer and bladder cancer and other types of cancer patients.Lung cancer is a cancer with the world's highest incidence,accounting for the first of the cancer-related causes of death. Most of the patients with lung cancer finally die from tumor recurrence and metastasis. So overcoming and delaying recurrence and metastasis of lung cancer will be important means to improve survival. The key point of tumor recurrence and metastasis dues to drug resistance. The drug resistance of tumor cells had been extensive studied,and many different theories had been put forward, including:drug transporting or uptaking barriers; drug activation barriers; target enzyme changes in the quantity and quality;metabolic pathways increased; decomposing enzyme increased;repair mechanisms increased;the cell membrane glycoprotein specific in excluding drugs increased; DNA chains cross-linked reduced; hormone receptor reduction or loss of function, and so on. Although these theories explain different aspects of drug resistance of tumor cells, but they are all one-sided and have some limitations, can not fully explain all aspects of drug resistance of tumor cells. In recent years, tumor stem cell theory have been proposed,which can well explain the drug resistance of tumor cells. Tumor stem cells in tumor research has become a hot spot. But the quantities of the tumor stem cells in tumor tissues are very small,so isolation and identification of these cells play a crucial role in the research on tumor stem cells.Usually the isolation and identification of tumor stem cells are based mainly on their characteristic surface markers, their biological characteristics in vitro, SP cell properties, immortal DNA chain characteristics and animal tumorigenicity experiments. However, due to lack of specific surface markers, the separation and purification of lung cancer stem cells have long been one important problems to be solved.Aldehyde dehydrogenase 1 (aldehyde dehydrog enase 1, ALDH 1, ALDH 1A1 or RALDH 1) belong to aldehyde dehydrogenase (ALDH) family. By oxidation of retinol to retinoic acid,ALDH1 takes part in gene expression and tissue differentiation. In human, ALDH 1 is expressed in various tissues, also in immune cells.It plays an important role in the maintenance of normal tissue development and homeostasis. Chute reported that ALDH1 stem cells might play an important role in early differentiation. The human ALDH1 gene is expressed in the cytoplasm, and located in the 9q21 chromosome, the 53×103 base pairs form, contains 13 exons, encoding 501 amino acid sequence, without repeats, N'terminal for the methionine M (Met), has a glutamic acid (Glu) and a cysteine (Cys) active site, the gene promoter region contains a ATA box and a CCAAT box,they located upstream of the transcription start site 32 bp and 74 bp department. Comprehensive in recent years, ALDH1 showed high expression in a variety of tissue types of the tumor stem cells.And tumor cells with high expression of ALDH1 had stem cell characteristics. At present, ALDH1 activity has been successfully used as functional markers for isolation and identification of tumor stem cells in breast cancer, retinoblastoma, head and neck squamous cell carcinoma, prostate cancer, colon cancer and so on.Recent studies in foreign countries found that ALDH1 was a functional marker of the lung cancer stem cells, ALDH 1-positive lung cancer cells had biological functions of stem cells.This experiment on lung cancer A549 cell line mainly studied on the biological functions of the ALDH1-positive cells in vitro and in vivo, as well as the relationship with a number of stem cell surface markers to analyze whether the ALDH 1-positive cells possessed properties of tumor stem cells. The study established a method or platform to isolate and identify lung cancer stem cells,also layed solid foundation for further study of the occurrence, development, metastasis, recurrence, drug resistance of lung cancer, and developing new methods to intervent tumor progression and even cure lung cancer.METHODS1.Cell sortingIn this study,we used the Aldefluor assay and fluorescence-activated cell sorting(FACS) to isolate ALDH1-positive cells and ALDH1-negative cells from A549 lung cancer cell line.2.The biological functions of ALDHl-positive A549 cells2.1Tumor sphere culturation:ALDH1-positive cells, ALDH1-negative cells and unsorted A549 cells were cultured with serum-free medium,then we observed the differences of the tumor sphere formation abilities among them.The serum-free medium was:DMEM-F12+B27+EGF(20ug/ml)+bFGF(20ug/ml)。2.2The self-renewal and self-proliferation ability in vitro among ALDHl-positive cells, ALDH1-negative cells and unsorted A549 cells were detected by the plate colony formation test and MTT growth curve.2.3 To examine the migration ability of ALDH1-positive cells, ALDH1-negative cells and unsorted A549 cells, we performed in vitro transwell migration test.2.4 Tumorigenesis of ALDH1-positive cells, ALDH1-negative cells and unsorted A549 cells in mice by subcutaneous injection.To address the issue of whether tumorigenic activity differs among ALDH1-positive cells, ALDH1-negative cells and unsorted A549 cells,1000,5000, 1×104, 1×105 of ALDH1-positive cells,ALDH1-negative cells and unsorted A549 cells were separately injected into nude mice.3. Real-time-PCR was used to measure the mRNA expressions of CD133 and CD44 in ALDH1-positive cells and ALDH1-negative cells.RESULTS1. The proportion of ALDH1-positive cells in lung cancer cell line A549The result of sorting ALDH1-positive cells by flow cytometry showed that ALDH1-positive cells in A549 lung cancer cell lines accounted for a very low percentage of only 2.000±0.265%.2. The biological functions of ALDHl-positive A549 cells2.1 The tumor sphere formation ability56.00±8.000 tumor spheres were formed with 100 ALDH1-positive cells, while 16.67±1.528 with 100 unsorted A549 cells and 6.33±1.528 with 100 ALDH1-negative cells. It demonstrated statistical significance (F=90.015 P±0.001). The LSD test showed that compared to ALDH1-positive cells,the numbers of tumor sphere formed by unsorted A549 cells and ALDH1-negative were significantly reduced (both P＜0.001). While A549 cells and unsorted cells formed tumors ALDH1-ball the number of statistically significant differences (P=0.038). The ALDH1-positive cells had the strongest tumor sphere formation abilities among ALDHl-positive cells and ALDH1-negative cells and unsorted A549 cells.2.2 The self-renewal and self-proliferation abilityThe MTT test showed that the self-renewal and self-proliferation ability of ALDH1-positive cells,unsorted A549 cells andALDHl-negative cells was different.The ODs demonstrated statistical significance (F= 22.079 P＜0.001). The numbers of the colony formation were assessed by counting under microscope. Good views were photographed. The ratio of clony formation of ALDH1-positive cells and ALDH1-negative cells and unsorted A549 cells were (39.833±2.363)%,(12.5±0.1000) % and (22.833±3.253)%. It demonstrated statistical significance (F=99.864 P＜0.001). The LSD test showed that compared to ALDH1-positive cells,the self-renewal and self-proliferation of unsorted A549 cells and ALDH1-negative cells were significantly reduced (both P<0.001). While A549 cells and unsorted cells were statistically significant differences, too (P=0.002). The plate colony formation test showed that the self-renewal and self-proliferation ability of ALDH1-positive cells was the best among ALDH1-positive cells and ALDH1-negative cells and unsorted A549 cells.2.3 The migration ability in vitroThe transwell test showed that the cells that penetrated artificial basement membrane was more per high power field for ALDH1-positive cells than ALDHl-negative cells and unsorted A549 cells. The difference was significant (F=301.199 P<0.001). The Dunnett T3 test showed that compared to ALDH1-positive cells,the migration ability of unsorted A549 cells and ALDH1-negative were significantly reduced (both P<0.001). While A549 cells and unsorted cells formed tumors ALDH1-ball the number of statistically significant differences (P=0.001).2.4 Tumorigenesis of ALDH1-positive cells and ALDH1-negative cells and unsorted A549 cells in mice by subcutaneous injection.As low as 5000 ALDH1-positive cell injection could initiate tumors. However, 1×105 unsorted A549 cells could initiate tumors,and the ALDHl-negative cells injection consistently failed to form tumors. The result suggested that the ALDH1-positive had the strongest tumorigenic capacity.3. Real-time-PCR was used to measure the mRNA expressions of CD133 and CD44 in ALDH1-positive cells and ALDH1-negative cells.The results of Real-time PCR show that the expression of the stem cell surface marker CD133 on ALDH1-positive cells was higher than ALDH1-negative cells, the difference was statistically significant (t=18.055 P<0.001); CD44 expression on ALDH1-positive cells was higher than ALDH1-negative cells, The same difference was statistically significant (t=32.362 P＜0.001).Conclusion1. ALDH1-positive A549 cells display features of tumor stem cells. In vitro experiments showed that the ALDH1-positive cells were capable of proliferation, self-renew,differentiation and strong invasion and metastasis propertiesln vivo experiment showed that the ALDH1-positive cells possessed high tumorigenic capacity. ALDH1 may be used as a sorting marker of lung cancer stem cells.2. ALDH1-positive A549 cells were with high expression of stem cell surface markers CD 133 and CD44,further confirmed that ALDH1-positive cells may be tumor stem cells of lung cancer.