Detection, Isolation And Cultivation Of Cancer Stem Cells From Human Gliomas

Recent studies indicated that tumor is a kind of stem cell disease. Neoplasm is composed of abnormal tissues derived from a small portion of special tumor cells capable of self-renewal and tumorogenicity. These cells were named cancer stem cells. Their characteristics include unlimited proliferation, clonogenity, tumorogenicity, symmetrical and asymmetrical division. Cancer stem cells are vital to the formation, maintenance and progression of tumor. The non-stem cells possess none or very little capacity of proliferation, and will die in short term.Cancer stem cells were firstly identified in leukemia. Later studies demonstrated that solid tumors including glioma involve cancer stem cells too. In the traditional theory, the heterogeneity of tumor cells was owing to the instability of inherited and acquired factors. However, the recent studies supposed that in the process of proliferation and differentiation of cancer stem cells, a population of heterogenic tumor cells formed, mainly because of the abnormal differentiation.Singh and his colleagues isolated CD133⁺ and CD133^- cells from fresh samples of astrocytoma, ependymocytoma and oligodendroglioma. These cells were implanted into the brains of NOD/SCID mice. Merely 100 CD133⁺ cells were sufficient to generate a glioma in vivo, whice resembled the original tumors immunocytochemically. However, 10⁵ CD133^- tumor cells could not give rise to a tumor. Thus CD133⁺ cells stand for the cancer stem cells of the gliomas.Another characteristic of brain tumor stem cell lies in the negative staining of Hoechst33342, a nuclear dye. In the flow cytometry based on the Hoechst33342 staining, the cancer stem cells were enriched in side population (SP), because the stem cells expressed ABCG2, or named breast cancer-resistant protein 1(BCRP1), whichbelongs to the ATP-Binding Cassette superfamily. Therefore, ABCG2 was also a special marker for brain tumor stem cells.In order to investigate the location of cancer stem cells in human glioma, we detected stem cells in the tumor samples of both primary and recurrent gliomas in different WHO grade, with the markers of CD133 and ABCG2. Meanwhile, cancer stem cells were isolated from fresh glioma samples and expanded in the serum-free medium.Materials and Methods1. EthicsFor each tumor sample, written consent was assigned by the patient or his/her authorized surrogate. The whole process and the result of this study were completely harmless to the patient. This study was in accordance to the guideline of the Ethics committee of the Medical School, Zhejiang University.2. AgentsDMEM, DMEM/F12 media (GIBCO). R-h EGF, r-h bFGF (Sigma), B27 serum-free supplement(50×) liquid (Gibco), 0.25%Trypsin (Gibco), BSA, EDTA, DMSO (BBI). Goat anti-CD133 lgG, goat anti-ABCG2 lgG (Santa Cruz). Other agents were purchased in the local market.3. Detection of cancer stem cellsGlioma samples were fixed in 4% formaldehyde. Paraffin slices of these samples were prepared in routine procedures. Immunocytochemical staining with CD133 and ABCG2 IgG and HE staining were performed. Count the positive cells and total cells in 10 random visual fields of each slice. The labeling index equals to positive cell number divided by total cell numbers.4. Isolation and cultivation of cancer stem cells1) Primary culture: Glioma samples were put into a sterile Eppendoff tube with DMEM medium and sent from the surgical room to the laboratory in less than 5min in 4°C. They were cut into small pieces (1mm³) and then incubated in Trypsin-EDTA solution for 5 min. When the tumor tissue was digested into single-cell suspension, cells were collected by centrifugation and resuspended in DMEM/F12 serum-free medium at a density of (2—5)×10⁵/ml, and then culturedin the Corning flasks. Meanwhile, 2%B27, 20ng/ml bFGF and 20ng/ml EGF were supplemented into the culture. The cells were cultured in 37°C with 5%CO₂. Fresh medium was added every other day. Cells were passaged every one week approximately.2) Passage: Cell clusters (tumorospheres) were collected by centrifugation and then digested into single-cell suspension in Trypsin-EDTA solution. The cells were collected, resuspended and seeded in new flasks with fresh serum-free medium.3) Freezing and resuscitation: The stem cells were collected and adjusted to the dencity of 2×10⁶ cells/ml with freezing solution (serum-free medium+10%DMSO+0.1%BSA). They were transferred to the freezing tube with 1 ml per tube. The tubes were store in 4°C for 30min, -20°C for 2h, -80°C overnight and in the liquid nitrogen for ever. During resuscitation, the freezing tube was transferred from liquid nitrogen quickly to the 37°C water. Ten minutes later, when thawed completely, the cells were transferred to a centrifuge tube containing 9ml fresh medium, and then centrifuged to collect the cells. These cells were resuspended and cultured in the same way as above.4) Identification: Immunocytochemical staining was performed. The CD133(+) cells were identified as cancer stem cells.5. StatisticsData were expressed as means±SD. X² test was used to compare the difference. P≤0.05 was accepted as significant.Results1. Clinic dataThe study includes 36 patients of glioma, all of which were confirmedpathologically. Primary glioma 28 cases, recurrent glioma 8 cases. Astrocytoma 21cases, oligodendroglioma 5 cases, mixed glioma 8 cases and glioblastoma 2 cases. WHO grades:I-II grade 13 cases, III—IV grade 23 cases. All patients were reported as glioma in the MRIdiagnosis.2. Glioma of different pathological types includes cancer stem cells.In all slices of these 36 gliomas, CD133(+) and ABCG2(+) cells were found, in a form of "nest" or diversely in the midst of massive negative tumor cells. This resultwas in accordance with the opinion that cancer stem cell was the general source of gliomas of different pathological types. According to the ICC staining of adjacent slices, CD133(+) and ABCG2(+) cells were located overlapping. Therefore, CD133 and ABCG2 coexpressed on the cancer stem cells, and are both special markers for human gliomas.3. Differentiation was not related to cancer stem cell ratio directly.The cancer stem cell ratio of high grade glioma (I - II, 0. 37%) was not significantly different from that of the low grade (III-IV, 0. 38%). In fact, we often noticed that low grade glioma grows much quicker. We deduced that the cancer stem cells are quiet cell type and did not contribute to the expansion of tumor. The difference of growth rate between high grade and low grade gliomas did not lie in the cancer stem cell ratio, but in the proliferating velocity.4. The cancer stem cell ratio of recurrent glioma was higher than primary glioma.The cancer stem cell ratio of recurrent glioma was higher than primary glioma (0.35% vs 0.46%) . Because of the quiescent nature and the drug-resistance protein ABCG2, cancer stem cells were superior to the non-stem cells, in the selective pressure of tumor immunity, radiotherapy and chemotherapy. Thus we deduced that in the course of clinic treatment and recurrence, more cancer stem cells were selected and reserved.5. Cancer stem cells were successfully cultured in vitro.Cancer stem cells from 2 cases of glioma were successfully cultured in vitro, in a form of floating sphere, named tumorosphere. Though the culture medium and conditions were identical, the tumorosphere from the glioblastoma was expanded much quickly.After passage, the single cells would aggregated to form new spheres the other day. It is the same case as for resuscitation from liquid nitrogen.Immunohistochemistry revealed that about 46.6% cells in the tumorospheres were CD133 positive. Thus it is clear that cancer stem cells were successfully expanded in serum-free medium.Conclusions1. Glioma of different pathological types includes cancer stem cells.2. Differentiation was not related to cancer stem cell ratio directly.3. The cancer stem cell ratio of recurrent glioma was higher than primary glioma.4. Cancer stem cells were successfully cultured in vitro.