Results Isolation of CSCs from MNNG/HOS cells Célia M. F. Gomes 1,2* , S. R. M. Neves 1 , A. O. G. Lopes 1 , A. do Carmo 3 , A. J. Abrunhosa 2,4 , M. Botelho 1,2 1 Institute of Biophysics/Biomathematics – IBILI – FMUC, Coimbra, PORTUGAL; 2 Center of Investigation in Environment, Genetics and Oncobiology - CIMAGO, Faculty of Medicine, Coimbra, PORTUGAL; 3 Center for Neurosciences and Cell Biology – CNBC, Coimbra, PORTUGAL; 4 Institute for Nuclear Sciences Applied to Health – ICNAS, Coimbra, PORTUGAL Assessing metabolic activity of Cancer Stem Cells during differentiation with [ 18 F]FDG Introduction Figure 1 – Representative diagram of CSCs proliferative and differentiation capacity. [1] Methods • Isolation of a CSCs subpopulation from a human osteosarcoma cell line (MNNG/HOS) using a sphere-culture system Cells were cultured in serum-free medium under anchorage-independent conditions: - DMEM/F12 supplemented with progesterone 20 nM, putrescine 100 μM, insulin-transferrin-selenium-A supplement 1 %, bFGF 10 ng/mL and EGF 10 ng/mL in 1% methylcellulose; - plates coated with poly-HEMA (low adherent conditions). • Differentiation of CSCs The formed spherical clones were transferred to adherent plates and cultured in serum-containing medium for further expansion and differentiation. • Characterisation of CSCs - Analysis of mesenchymal stem cells (MSC) markers by flow cytometry Positive: CD13 + CD90 + CD73 Negative: CD34 + CD45 + CD11b + CD19 + HLA-DR - In vitro differentiation into osteoblasts • Metabolic activity with [ 18 F]FDG ‐ Single-cell suspensions of spherical clones and adherent cells at 1 st ,4 th ,6 th and 12 th passages were incubated with 0.75 MBq/mL of [ 18 F]FDG at 37°C. ‐ Cellular uptake was measured at 15, 30 and 60 minutes. • Cell cycle analysis - Single-cell suspensions were fixed with ice-cold ethanol and incubated with propidium iodide (PI) and RNase solution. - Flow cytometric PI fluorescence analysis References [1] Rebecca G.Bagley, Beverly A.Teicher. Cancer Drug Discovery and Development: Stem Cells and Cancer. 2009. Acknowledgements This project (Ref. 16/09) was funded by CIMAGO – Center of Investigation in Environment, Genetics and Oncobiology, Faculty of Medicine, Coimbra, Portugal. There are no conflicts of interest to declare. Conclusions  The MNNG/HOS cell line contains a sub-population of cells with stem-like properties.  CSCs can be successfully isolated from osteosarcoma using the sphere formation assay.  CSCs have lower energy requirements than the more differentiated ones.  The metabolic activity of CSCs increases progressively during their differentiation status and can be monitored with [ 18 F]FDG.  [ 18 F]FDG uptake is cell cycle-dependent and varies proportionally with the percentage of cells in G 2 /M phase. CSCs possess attributes of MSCs To evaluate the metabolic activity of CSCs during differentiation using [ 18 F]FDG and their correlation with the cell cycle progression. Aim * Corresponding author e-mail: cgomes@ibili.uc.pt 23 rd Annual European Association of Nuclear Medicine Congress, 2010, Vienna, Austria The Cancer Stem Cell theory proposes a hierarchical organisation within the tumour in which a small subset of stem-like cells are responsible for sustaining tumour growth and differentiation. These cells, termed as cancer stem cells (CSC) lying at the apex of the hierarchy, divide asymmetrically, producing an identical progenitor and a more differentiated cell, which in subsequent divisions generates the diverse cells comprising the bulk tumour. The existence of CSCs has important therapeutic implications, since these cells are referred to be more resistant to conventional therapies compared with their more differentiated progeny. 0,00 2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 15 30 60 [ 18 F]FDG uptake (%/10 6 cells) Time (min) CSCs 1st # 4th # 6th # 12th # POSTER: P361 Table 1. Uptake of [ 18 F]FDG during differentiation of CSCs. The stressful growth conditions (serum starvation and anchorage independence) select for primitive clonogenic cells possessing attributes of stem cells (Figure 2) by eliminating the differentiated ones that are unable to survive under such circumstances. MNNG/HOS cells Serum-free medium Low adherent conditions Serum-free medium Low adherent conditions Standard culture conditions Spherical colonies of 1 st generation Spherical colonies of 2 nd generation Spherical colonies adhering to substrate Phenotypic changes of CSCs during differentiation CSCs 4 th passage 16 th passage Spherical colonies adhering to substrate Serum-containing medium Adherent flasks Propagation in serum- containing medium Adherent flasks ... Figure 2. (A) CSCs were positive for MSCs markers (CD13, CD90 and CD73) and (B) deposited a mineralized matrix (calcium deposits), which demonstrates their osteogenic differentiation potential. Figure 3. Uptake of [ 18 F]FDG during differentiation of CSCs. Results are reported as the percentage of cell radioactivity associated with the total radioactivity added, normalised per million of cells. 1 st CSCs 6 th MNNG/HOS 12 th 4 th 0,0 20,0 40,0 60,0 80,0 100,0 CSCs 1st 4th 6th 12th MNNG Cell cycle distribution (%) G2/M S G1 Cell cycle progression during differentiation of CSCs During differentiation of CSCs it was observed a progressive increase in the percentage of cells in G 2 /M phase that was accompanied by a proportional decrease in the percentage of cells in G 1 phase. After 12 passages in differentiation medium, the cell cycle distribution is similar to that of the MNNG/HOS cells. CD90 APC-A CD73 PE-A CD90 APC-A CD13 PE-Cy-7-A A B Metabolic changes occurring during differentiation of CSCs The uptake of [ 18 F]FDG at 60 min in CSCs was 2.94±1.33% and increased progressively with the number of passages under differentiation conditions, reaching the values of parental MNNG/HOS cells (%/10 6 cells=12.52±1.41) after 12 passages. After transferring to adherent growth conditions, CSCs started to expand from the sphere acquiring gradually spindle-shaped morphology similar to the original monolayer. Min CSCs 1 st passage 4 th passage 6 th passage 12 th passage 15 1.86 ± 0.94 2.80 ± 0.64 3.04 ± 1.31 3.86 ± 0.41 4.42 ± 1.47 30 2.01 ± 0.79 4.24 ± 1.00 4.87 ± 2.20 5.70 ± 1.45 6.45 ± 2.19 60 2.94 ± 1.33 7.10 ± 2.03 8.65 ± 2.83 10.77 ± 2.95 11.57 ± 3.55 Figure 4. Cell cycle phase distributions of CSCs during differentiation measured by flow cytometry. The distribution of cells in each cell cycle phase is shown as percentage of the total number of cells.