| Increasing evidence indicates that cancer stem cells (CSCs) are initiators of the occurrence, development and recurrence of malignant tumors. Due to the great efforts of many researchers, more and more specific features of CSCs have been revealed, such as proliferation/division, cytotoxic resistance, invasiveness, immunogenicity, hypoxic tolerance and the capacity of neovessels induction. These findings aid in understanding the underlying mechanisms of carcinogenesis, recurrence after therapy and invasion/metastasis of malignant tumors. However, the mitochondrial and energy/metabolism-related phenotypes of CSCs, the stem cell-like subpopulation in cancer cells, are still unkown.Mitochondria are important organelles in eukaryotic cells, not only responsible for converting nutrients into the energy-yielding molecule adenosine triphosphate (ATP) to fuel the cell's activities, but also play essential roles in processes such as steroid metabolism, calcium homeostasis, apoptosis and cellular proliferation. At the same time, mitochondrial-related abnormalities have also been considered to have an important role in the origin and development of tumors. Interestingly, in recent years, some mitochondrial features, such as the mitochondrial morphous and spatial arrangements, the mass of mitochondria and the energy/metabolism-related parameter, have been studied by an increasing number of researchers. It has been suggested that these features may influence the self-renewal, multipotency, proliferative and differentiation potential of normal stem cells. Therefore, the investigation of the mitochondrial and energy/metabolism-related phenotypes of CSCs would help to understand the biologic characteristics of CSCs deeply. Meanwhile, considering the central role performed by CSCs in the tumorigenesis, progression and recurrence of cancer, the results would also advance our understanding of cancer biology.In this study, after the isolation and identification of lung CSCs (LCSCs) from the A549 lung cancer cell line, some of mitochondrial and energy/metabolism-related features, such as the mass of mitochondria, the mitochondrial arrangement, the quantity of mitochondrial DNA (mtDNA), the mitochondrial membrane potential (??m), oxygen/glucose consumption and the intracellular concentration of reactive oxygen species (ROS) and ATP, were examined. In addition, the heterogeneity of??m of cance cells and its role as a novel tool to isolate and enrich LCSCs from A549 lung cancer cell line, were investigated. The results help to provide new experiment data for understanding the biological characteristics of LCSCs.OBJECTIVE1. Isolatation and identification LCSCs from A549 lung cancer cell line.2. To investigate the mitochondrial and energy/metabolism-related features of LCSCs.3. To explore the possibility that heterogeneity of??m as a novel tool to isolate and enrich LCSCs from A549 lung cancer cell line.METHODS1. Isolation and identification of LCSCsA549 cells were seeded in 6-well plates at 1×103 cells/well with Dulbecco's modification of eagle's medium (DMEM) containing 10% bovine serum bovine serum (FBS) for 2 weeks. Thereafter, holoclones were isolated and cultured in innovated serum-free stem cell medium for another 2-3 weeks until primary tumorspheres appear. Then, the self-renewal capability of these primary tumorspheres was assessed by serial sphere forming assay. After immunofluorescence staining, the expression of different makers (stem cell and differentiation markers) under serum-free and 10% FBS containing culture medium was detected by laser confocal microscopy (LCM) respectively. The mRNA expression level of more stem cell molecules was assessed by real time PCR. Finally, we observed the tumorigenicity in vivo, and examined the histology of tumors drived from tumorspheres, monolayer cells and A549 cell line by HE and immunofluorescence staining.2. Investigation of mitochondrial and energy/metabolism-related properties of lung CSCs2.1 The spatial arrangement of mitochondria of A549 cell line was analyzed by LCM after MitoTracker Green and 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) staining. 2.2 The distribution and amount of mitochondria of SP cells and no-SP cells in A549 cell line were analyzed by LCM after MitoTracker Green and Hoechst 33342 staining. Meanwile, the amount of mitochondria of different groups (A549 spheres, monolayer cells, A549 cell line and differentiated descendants derived from A549 spheres) were analyzed by flow cytometry after MitoTracker Green staining.2.3 The quantity of mtDNA represented by the Cox I/?-actin ratio were analyzed by real time PCR.2.4 After the incubation with Rhodamine-123(Rh123), the??m of different groups (A549 spheres, monolayer cells, A549 cell line and differentiated descendants derived from A549 spheres) were analyzed by flow cytometry.2.5 Using an ATP assay kit (luciferin-luciferase), the intracellular ATP concentration of different groups (A549 spheres, monolayer cells, A549 cell line and differentiated descendants derived from A549 spheres) were analyzed by luminometer.2.6 The intracellular ROS concentration of different groups (A549 spheres, monolayer cells, A549 cell line and differentiated descendants derived from A549 spheres) were analyzed by flow cytometry after DCF-DA staining.2.7 After the incubation in serum-free stem cell medium or DMEM containing 10% FBS, the glucose consumption rates of different groups(A549 spheres, monolayer cells, A549 cell line) were calculated as glucose concentration pre-incubation minus the glucose concentration at 24 or 48 hour respectively.2.8 After the isolation of mitochondria form different groups (A549 spheres, monolayer cells, A549 cell line and differentiated descendants derived from A549 spheres), the mitochondrial respiratory functional status was measured by a Clark-type oxygen electrode (The respiration control of rate, RCR).3. Application of??m heterogeneity in isolation and enrichment of LCSCs3.1 Detection of??m heterogeneity between LCSCs and non-LCSCsThe??m Heterogeneity of SP cells and no-SP cells in A549 cell line were analyzed by LCM after Rh123 or JC-1 staining respectively.Then, the??m of different groups (A549 spheres, monolayer cells, A549 cell line) were detected by flow cytometry after JC-1 staining.3.2 Enrichment of LCSCs based on the heterogeneity of??m After the incubation with Rh123, A549 cells were sorted by flow cytometry into two populations with the lowest 5% (L??m) or highest 5% (H??m)??m. The expression of CD133 in the two groups (L??m and H??m) was analyzed under LCM after immunofluorescence staining. Then, the self-renewal capability of the two groups was assessed by serial clone forming assay in serum-free stem cell medium or DMEM containing 10% FBS, respectively. At last, the tumorigenicity of the two groups was compared in vivo.RESULTS1. Isolated and identified LCSCs from A549 lung cancer cell line1.1 Obtained tumorspheres with capability of self-renewalAfter the incubation of A549 holoclone in serum-free stem cell medium, primary tumorspheres were obtained after two weeks. Meanwile, some monolayer non-sphere-forming cells could be seen in the bottom of the wells. The sprimary pheres were dissociated into single cell suspension, and cultured in fresh serum-free stem cell culture medium. Secondary spheres were obtained again after two or three weeks.1.2 Tumorspheres overexpress stem cell markersFluorescent immunostaining revealed that some stem cell-related markers, such as Sca-1, CD133, CD44s, Oct4, and Nanog were positive in these secondary tumorspheres. The mRNA transcript level of more stem cell molecules, including CD34, CD133, c-kit, Twist1, Sox2, Oct4, Nanog, and Bmi-1, were overexpressed in secondary spheres compared to monolayer cells.1.3 Tumorspheres possess the capability of multi-directional differentiationWhen secondary tumorspheres were cultured in DMEM containing 10% FBS, they adhered to the plastic and then proliferated and differentiated. Two weeks post seeding, the expression of Sca-1, Clara cell secretory protein (CCSP) and prosurfactant protein C (SP-C) were detected by LCM. The results reveal that the the positivity rate of these three markers in the differentiated descendants were similar to that of the A549 cell line.1.4 Tumorspheres possess the capability of tumorigenicity in vivoSecondary sphere cells, monolayer nonsphere-forming cells and the A549 cell line were transplanted subcutaneously (s.c.) into nude mice. Tumors form secondary sphere cells displayed most rapid growth than monolayer cells. In contrast, there are no tumors were observed after three weeks in mice implanted with monolayer cells. Histology of subcutaneous tumors revealed that tumors formed by subspheres grew more rapidly with large amounts of necrosis in the center of the tumor tissue. The tumor cells were small and had a fusiform to polygonal shape, little cytoplasm, hypersecreted mucous, numerous hyperchromatic nuclei with pleomorphisms and were arranged in a crypt form. A high density of microvessels was found in the stroma of the tumor tissue. Tumors formed by monolayer A549 cells contained less necrotic regions, crypt-like structures and microvessels, although they shared a similar size, appearance and differentiation status with the tumor cells originating from spheres. Tumors formed by the A549 cell line consisted of large and round tumor cells with abundant cytoplasm.The density of microvessels was fairly low, and no crypt-like structures were detected in the tumor tissue. These results indicate that the tumors derived from secondary spheres displayed lung adenocarcinoma features more similar to the human source of the original tumor.2. The mitochondrial and energy/metabolism-related properties of LCSCs were different from that of differentiated tumor cells2.1 LCSCs displayed more perinuclear mitochondrial distributionSimilar to normal adult stem cell lines, A549 cells also displayed three distinct spatial arrangements of mitochondria: perinuclear (P), homogeneous (H) and aggregated (A). More A549 SP cells displayed perinuclear mitochondrial distribution (P, 0.33; H, 0.37; A, 0.30). However, a large quantity of cells with an aggregated mitochondrial arrangement was observed in the non-SP subpopulation (P, 0.07; H, 0.33; A, 0.60).2.2 There was no difference in mitochondrial mass between LCSCs and non-LCSCsAs revealed by LCM, the average intensities of MitoTracker Green in SP cells and non-SP cells were no significant difference (SP, 54.14±15.5; non-SP, 45.08±11.2; P>0.05). These results were further confirmed using?ow cytometry, no difference in the intensity of MitoTracker Green was found between spheres and monolayer cells.2.3 The quantity of mtDNA was low in LCSCsThe copies of mtDNA (calculated by Cox I/?-actin) were lower in secondary spheres than in monolayer cells (sphere, 0.20±0.03; monolayer, 0.11±0.02; P<0.01). The amount of mtDNA per cell of spheres increased significantly from day 1 to day 8 after differentiating.2.4 Higher??m was observed in LCSCs The intensity of Rh123 was higher in the secondary sphere group than monlayer cells (sphere, 135.3; monlayer, 73.4; n=2×104). When these spheres were differentiated, the intracellular intensity of Rh123 increased significantly within the first 12 hour and decreased rapidly after 2 day.2.5 LCSCs shown low intracellular ATP concentrationThe intracellular ATP concentration was lower in the sphere subpopulation than in the monlayer subpopulation (sphere, 1.99±0.15?mol/L/mg; monolayer, 3.06±0.92?mol/L/ mg; cell line, 3.86±1.73?mol/L/mg; P<0.05). When these spheres were differentiated, the ATP content also rapidly increased 12 hour postinduction, then decreased unevenly and reached the level of the A549 cell line 4 weeks after differentiation.2.6 The consumption of glucose and oxygen, and the concentration of ROS in LCSCs were lower than in non-LCSCs.Results showed that the sphere population contained considerably lower levels of ROS than the non-LCSC population (sphere, 23; monolayer, 13.9; n=3×104). During differentiation, the intracellular ROS concentration quickly increased in the first 12 hour and then dropped slightly. Meanwhile, the spheres subpopulation consumed less glucose than their differentiated descendants in serum-free stem cell medium (sphere, 48 hour, 6.11mmol/L; monolayer, 48 hour, 10.76mmol/L; cell line, 48 hour, 9.70mmol/L). In addition, these spheres also utilized less oxygen (sphere, 1.46±0.16; monolayer, 2.52±0.72; P<0.05). When these spheres were induced to differentiate in DMEM containing 10% FBS, their oxygen consumption was slightly enhanced.3. Heterogeneity of??m may provide a new tool to isolate and enrich LCSCs3.1??m heterogeneity exists among different subpopulations of a tumor massThe??m in SP and non-SP A549 cells was determined by LCM. A higher 590/520 ratio of JC-1 (P<0.001) and intensity of Rh123 (P<0.05) were detected in the subpopulation of SP cells compared with their non-SP counterparts. In addition, the??m was analyzed by flow cytometry, the results also shown that the 590/520 ratio of JC-1 higher in the secondary sphere group than in monolayer cells (P<0.001).3.1 Isolated and enriched LCSCs by??m HeterogeneityAfter incubation with Rh123, the A549 cells were sorted by flow cytometry into populations with the lowest 5% (L??m) or highest 5% (H??m)??m successfully. In the H??m group, obvious CD133 expression could be detected. However, the expression of this stem cell marker was nearly absent in the L??m subpopulation. Moreover, H??m group has higher clone forming rate in serum-free stem cell medium (P<0.05) or DMEM supplemented with 10% FBS (P<0.01) than L??m group. These all indicted that H??m group possess more stem-like properties.CONCLUSION1. We obtained a subset from A549 cell line successfully. These cells, which express mult-stem cell markers and possess the capability of self-renewal, multipotency and tumorigenicity, were identificated as LCSCs.2. Compared with the differentiated cancer cells, LCSCs displayed different mitochondrial-related properties, such as perinuclear arrangement, low copies of mtDNA, high??m, low intracellular ATP and ROS concentration, less glucose and oxygen consumption.3. Heterogeneity of??m could be used as a novel tool to isolate and enrich LCSCs... |
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