Zhu et al. Journal of Experimental & Clinical Cancer Research 2010, 29:124 http://www.jeccr.com/content/29/1/124 Page 3 of 8 migrated through the filters were fixed, stained with Giemsa (Sigma, St. Louis, MO), photographed, and counted. Laser capture microdissection Fifteen microliters of Matrigel were mounted on ethy- lene vinyl acetate (EVA) membrane (Leica, Wetzlar, Germany) with frame instead of coverslip in 9-cm dishes and treated to establish three-dimensional culture as described above. The density of tumor cells seeded onto gel was adjusted to 1 × 105. After 7 d, samples on EVA membrane were washed with PBS-DEPC and air-dried, channels formed by endothelial-like cells (ELs) were selected by microscopy and microdissected with laser capture microdissection (LCM) system (Leica). About 1,500-2,000 ELs were laser-captured from each EVA membrane. The cells were immersed in digestion buffer for quantitative real-time reverse transcription polymer- ase chain reaction (RT-PCR) and telomerase activity assay. Quantitative real-time RT-PCR Total RNA was extracted from 2 × 104 cells (including HUVEC, SKOV-3, SKOV-3 EL, ES-2, ES-2 EL, or the SKOV-3 or ES-2 cells treated by 50 nM Sirolimus) using TRIzol reagent (Invitrogen, Carlsbad, CA). Ali- quots of RNA were reverse transcribed to cDNA using a Superscribe First-Strand synthesis system (Invitrogen). Real-time PCR analysis was performed to quantify mRNA expression of HIF-1a, VEGF, Flk-1, Cyclin D1, p53, and V-src by a Rotor-Gene3000 PCR system (Cor- bett, Australia) using SYBR-Green PCR Master mix (Qiagen, Hilden, Germany). The PCR reaction consisted of 12.5 μl of SYBR-Green PCR Master mix, 1.0 μl of for- ward and reverse primers (0.4 μM final concentration), and 2.0 μl of 1:10-diluted template cDNA in a total volume of 25 μl. Amplification was initiated at 50°C for 2 min, 95°C for 70 sec, followed by 40 cycles of 95°C for 20 sec, 58°C for 20 sec, and 72°C for 30 sec. To verify only a single product produced, a dissociation protocol was added after thermocycling. The assay included a no-template control, a standard curve of four serial dilu- tion points (in steps by 10-fold) of a cDNA mixture. All data were controlled by Rotor-Gene software (version 6.0) for quantity of RNA input, an endogenous reference gene (b-actin) was performed as control in the same reverse transcription reaction. Data were presented as the means ± S.E from three separate experiments. The primers used in this experiment were shown in Table 1. All primers were designed using Primer3 web software (Whitehead Institute, Cambridge, MA) and were synthe- sized by Sangon Biological Engineering Technology and Service Co., Ltd. (Shanghai, P.R. China). Telomerase activity assay The telomerase activity of all the cells (including HUVEC, SKOV-3, SKOV-3 EL, ES-2, ES-2 EL, or the SKOV-3 or ES-2 cells treated by 50 nM Sirolimus) was tested by telomerase repeat sequence amplification- enzyme linked immunosorbent assay (TRAP-ELISA) using the kit from Huamei Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturer’s instruction. Statistical analysis ANOVA analysis or paired-samples t-test were per- formed to identify differences, using SPSS11.5 statistical software (Lead, US). Statistical significance was assumed at P < 0.05, P-values are presented as two-tailed. Results The morphology of the endothelial-like cells from ovarian cancer shows similarities to HUVEC endothelial cells To investigate the morphology of the endothelial-like cells from ovarian cancer induced by hypoxia, the SKOV-3 and ES-2 cells were cultured in the 3-dimen- sional Matrigel system on EVA membrane under 1% O2 for 7 d before harvested by LCM. The morphology of the endothelial-like cells induced by hypoxia were pic- tured by microscope and shown in Figure 1. As it shown, after incubated under hypoxia, the ovarian can- cer cells extended and reshaped, developed ELs and connected with each other (A and B), eventually form- ing network structures and channels (C and D). The original and microdissected by LCM of the single cell were shown in Fig. 1A and 1B, Fig. 1C and 1D indicated the original and microdissected grouped cells. The biological behaviors such as proliferation, cell cycle, apoptosis and invasion of SKOV-3, ES-2 and HUVEC cells are changed by hypoxia In order to elucidate the biological behaviors changes in SKOV-3, ES-2 and HUVEC cells by hypoxia, the prolif- eration, cell cycle, apoptosis and invasion were detected by MTT, FCM and transwell chamber after induced by hypoxia for 3 or 7 d. As shown in Fig. 2A, the prolifera- tion of SKOV-3 was inhibited significantly on 3rd d while there was no difference after 7d’s incubation. As for the proliferation of ES-2 cells, there has no signifi- cant difference after incubation under hypoxia. The pro- liferation of HUVEC cells were inhibited by incubation under hypoxia for 3 d and further inhibited after 7 d’s incubation. The percent of cells in S-phase and apoptosis after incubation for 3 or 7 d under hypoxia were shown in Fig. 2B and 2C. As they shown, in the case of SKOV-3 and ES-2 cells, the percent in S-phase were decreased