Isolation And Identification Of Cancer Stem Cells From Rats Metastatic Hepatocellular Carcinoma In The Lung

It has been verified by pathological research that there is close relation between liver cancer tumorigenesis and liver stem cells and liver tumor stem cells could be found in liver tumor tissue. Specific markers of liverstem/progenitor cells are reported to be present on liver cancer cell lines. As localization and specific markers of liver stem cells is not clarified yet, liver cancer stem cells has not been isolated so far. Since Goodel etc. enriched hemopoietic stem cells from bone marrow by Hoechst 33342 staining to segregate side population (SP) cells, tumor stem cells have been isolated by SP segregation from several types of solid tumor. It is confirmed that liver cancer stem cells can be enriched by SP segregation from liver cancer cell lines and primary heptocellular cancer tissue. Liver tumor stem cell is estimated to be associated with either tumorigenesis or invasion and metastasis. Stemcell like liver cancer cells have never been separated from liver cancer distant metastasis so far. We intended to isolate stemcell like liver cancer cells by Hoechst 33342 stained SP cell segregation from pulmonary metastasis of diethylnitrosamine induced rat primary heptocellular cancer and investigate the stemcell like feature of SP cells. Methods1 Rat model construction of hepatocellular carcinoma pulmonary metastasis. Rats were administered P.O. for 4 weeks with 0.1% DEN, 3 times per week, and killed 20 weeks later for observation of lung and liver.2 Primary culture of HPC pulmonary metastasis. Pulmonary metastasis tissue was cut into pieces of 1 mm3, digested with 0.25% collagenase IV, 37 oC, 10 min, and washed 3 times by Hank's buffer. Tissue pieces were planted into bottles and cultured with William's E medium and 10% fetal calf serum, 37 oC, 5% CO2.3 SP cell segregation of PHC pulmonary metastasis. Tissue pieces were digested into single cells and stained with Hoechst 33342 as described by Goodel etc. Cells were incubated with PE-labeled c-kit and sca-1 antibody respectively, then detected with FACS. When UV wavelength of 355nm was cast onto cells, scattering light signal was acquired at FITC and Percp-cy5.5 channel. Data were analyzed to render scatter plot by FACS Diva software of BD, with Percp-cy5.5 channel fluorescence intensity as Hoechst red staining intensity projected on X axis, and FITC channel fluorescence intensity as Hoechst blue staining intensity projected on Y axis. Side population cells were count by cytometer and isolated from total cells after the gate were set on scatter plot.4 Detection of stemcell marker on SP cells. Expression rate of c-kit and sca-1 on SP and non-SP cells were determined by FACS at PE channel when metastasis cells were segregated by Hoechst/FACS.5 In-vitro culture in serum-free medium of SP and non-SP metastasis cells. SP and non-SP cells isolated were planted into dishes pre-incubated respectively, then cultured with serum-free DMEM-F12 medium containing HGF. Medium were changed every two days. 6 Soft agar clone formation assay. SP and non-SP cells were mixed with 0.3% top medium respectively, then planted on 0.6% bottom agar medium. Clones were counted 3 weeks later. Averages of SP and non-SP cells were calculated and analyzed with Student's t test.7 Self proliferation assay. Two groups of SP and non-SP cells were cultured in serum-free medium respectively for 2 weeks followed by Hoechst33342 staining. Ratios of SP and non-SP cells in each group were determined by Hoechst33342/FACS to evaluate self proliferation potential of SP and non-SP cells.8 Invasion assay by transwell. SP, non-SP and unsegregated cells were planted on top chamber of transwell dishes which were pre-incubated with Matrigel and chemotaxised by medium in which NIH3T3 cells were cultured previously. 12 hours later, cells penetrating into bottom chamber were counted by microscope. Averages of each group were calculated and analyzed by ANOVA with SPSS 10.09 In vivo tumorigenesis assay. 2×10~4 SP cells and non-SP cells were injected subcutaneously into nude mice respectively. Growth of xenograft tumor was observed every 3 days. Rats were killed 30 days later with tumorigenesis rate,volume and weight of xenograft tumors measured.Results1 Pulmonary metastasis in later stage of rat HCC. Rats of control group were in well condition and organs were not found abnormal after anatomy. Livers of rats which were administrated with DEN were covered by tumor nodules. Metastasis tumor nodules can be found in lungs of two DEN-treated rats. It was determined by H.E. staining that tumor nodules in lungs of these two rats were pulmonary metastasis HCC. 2 Pulmonary metastasis HCC cells were primary cultured from rat tumor nodules. Cells migrated from tumor frags 24 hours after tumor frags were planted into culture bottles. Epithelial-like cell isles can be found at the original site of tumor frags when the frags were removed in 7 days.3 Segregation of SP cells from pulmonary metastasis HCC. On the 2-D plot figure of cells stained with Hoechst33342, side population cells can be found on the down-left corner with low Hoechst blue and Hoechst Red density, which was far different from most HCC cells.4 Stem-cell markers were highly expressed on SP cells from pulmonary metastasis HCC. C-kit and Sca-1 expression ratio on SP cells were 85% and 75% respectively while expression ratio were 8% and 3% on non-SP cells respectively.5 SP and non-SP cells from pulmonary metastasis HCC cultured in serum-free medium. SP cells proliferated in serum-free medium whereas non-SP cells were stalled. Isle-like cell clone can be found in SP cells after being cultured in serum-free medium for 10 days in contrast to stalled non-SP cells.6 Higher soft agar clone formation ability of SP cells compared with non-SP cells. Clone formation ratio of SP cells were determined to be 69.90% in contrast with that of non-SP cells to be 15.25%. t=20.46, n=5, P<0.017 Self proliferation ability of SP cells were higher than that of non-SP cells. SP cells can be detected in group of SP cells after cultured in serum-free medium for 2 weeks. However, SP cells can hardly be detected in group of non-SP cells after serum starvation.8 Invasion potential of SP cells were higher than that of non-SP cells. Average number of SP cells penetrating matrigel member were much more than that of non-SP cells and unsegregated cells. F=9.519481, P=0.002142 9 Tumorigenesis potential of SP cells were stronger than that of non-SP cells. Tumorigenesis rate, volume and weight of xenograft tumors in SP cell injected nude mice were significantly more than that of non-SP cell injected nude mice.ConclusionStem-like cells can be enriched in side population cells isolated from rat pulmonary hepatocellular carcinoma metastasis. Liver tissue can be formed and reconstructed by SP cells which are featured with potent invasion ,migration and self renewal ability. Rat pulmonary metastasis of HCC cells are heterogeneous containing stem cells and non-stem cells. HCC stem-cell featured SP cells can be segregated by Hoechst33342/FACS stragedy from Rat pulmonary metastasis of HCC. SP cells are determined to display higher ability to self-proliferate, invade, migrate, and form tumor.