| Although the existence of cancer stem cells (CSCs) has been confirmed in some primary tumors and cancer cell lines, it has not been well documented whether cancer stem cells have the potential of differentiating into different types of cancer cells. Therefore, to investigate the multi-potential of cancer stem cells will have great significance for developing a novel anti-tumor method by targeting cancer stem cells as well as for elucidating the initiation, progression and metastasis of cancer.Objectives: To investigate and confirm the potential that cancer stem cells could differentiate into different types of cancer cells.Methods: A population of small cells (T3A) with rapidly proliferation capacity were obtained during isolation and culture of primary human liver cancer microvascular endothelial cells. Undergo subcutanously inoculation with NOD/SCID mice and monoclonal screening, a single cell clone (T3A-A3) with rapid proliferation and high tumorigenicity was selected by MTT assay and immune deficienct mice tumorigenesis. Expression of stem cell related genes was analyzed by RT-PCR in T3A-A3. The stem cell-related markers were detected by flow cytometry in T3A-A3. Self-renewal potential of T3A-A3 was evaluated by methylcellulose secondary colony fomation assay and secondary tumorigenesis assay. Chromosome karyotype assay was made with T3A-A3 tumorigenicity and metastasis ability was evaluated by subcutaneous inoculation and intrahepatic inoculation respectively.In order to investigate the multiple differentiation potential of T3A-A3, human melanoma cell line SK-MEL-1 and human lymphoma cell line Daudi were selected to prepare the cancer inducing conditioned mediums (including cell conditioned mediums and tissue conditioned mediums):the cell conditioned mediums were made from culture supernatant of melanoma/lymphoma after incubated under hypoxia/reoxygenation, while the tissue conditioned medium were made from tissue homogenate of tumors formed by both cell transplanted in NOD/SCID mice. T3A-A3 was cultured with melanoma/lymphoma cell conditioned mediums or with melanoma/lymphoma tissue conditioned mediums for 21 days respectively, then the expression of melanoma specific molecule (gp100) and lymphoma correlative molecule (CD10) were detected by RT-PCR, immunofluorescence staining, western blot and immunohistochemistry technology.In addition, the monoclonal antibody against T3A was developed and applied to immunohistochemistry staining on 6 types of human cancer tissues and 4 types of human normal tissues.Results: By RT-PCR, mRNA of such genes related to stem cell proliferation and self-renewal as Notch, Bmi-1,β-catenin, Oct-4 and SMO were fund to express in T3A-A3. mRNA of several transcription factors related to induced pluripotent stem cell, Oct-4, Nanog, Lin28, K1f4, Sox2 and C-myc were expressed in T3A-A3 as well as stem cell classic markers CD133, CD34, ABCG2, Nestin, C-kit and SCF. By flow cytometry, expressions of the classic stem cell markers CD133,Nestin,CD34,CD44, ABCG2 and Oct-4 were confirmed on T3A-A3 cells. Chromosome karyotype analysis showed that T3A-A3 is polyploidy. The ability of secondary colony formation and secondary tumorigenicity ability were exhibited with T3A-A3 cells. Tumorigenesis was found when just 1000 T3A-A3 cells were subcutaneously inoculated into NOD/SCID mice. The metastatic focuses were found in stomach, lung and colon two months later 1×106 T3A-A3 cells were inoculated in NOD/SCID mice. The morphological changes were found in T3A-A3 cells after induced with melanoma/lymphoma cell conditioned mediums or melanoma/lymphoma tissue conditioned mediums. The specific marker of melanoma (gp100) was found in T3A-A3 after cultured with melanoma conditioned mediums or melanoma tissue mediums by RT-PCR, immunofluorescence staining, western blot and immunohistochemical staining. It was indicated that T3A-A3 was induced to differentiate toward melanoma cell. The fact specific marker of lymphoma (CD 10) was detectable in T3A-A3 after cultured with lymphoma cell conditioned mediums or tissue conditioned mediums by RT-PCR, immunofluorescence staining, western blot and immunohistochemical staining indicated that T3A-A3 was induced to differentiate toward to lymphoma cell. In addition, by immunohistochemical staining with mAb against T3A, positive cell subsets were detected in several different types of cancers including hepatoma, giant cell glioblastoma, breast cancer, gliosarcoma, esophageal cancer and gastric signet ring cell carcinoma.At the same time, no positive cell was detected in normal liver, normal brain, normal esophagus, and normal stomach tissues.Conclusions:Human cancer stem cells have the potential of differentiating into different types of cancer cells, and the different types of cancer cells may come from one homogeneous cancer stem cells. |
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