New Culturing System For Liver Cancer Stem Cells And Therapeutic Target Screening

It is commonly suggested tumors exist cancer stem cells, leading to its formation,persistence, recurrence and metastasis. But still, the isolated CSCs are too rare to supportthe subsequent experiment, emphasizing the culture and expansion CSCs in vitro isimportant. Using culture conditions that support the growth of undifferentiated CSCsmay help to devise novel diagnostic and therapeutic procedures. Recent studies havedocumented that serum-free medium with EGF and b FGF can expansion adult stem cellsand maintain pluripotency. This reminds us to find new culture medium for liver cancerstem cells. As far as we know, it is already available to separate and cultureCSC-like-cells from breast cancer, glioma, colon cancer and other malignancies. And it ispossible to maintain the potential of proliferation and differentiation in the serum-freeculture medium with EGF and b FGF. What we are striving for is optimal culturemedium for liver cancer stem cells.1. Optimized the culture medium for CSCs and expansion of human liver cancerstem cell-like cells from different sourcesIn order to successfully enrich liver CSCs, we optimized our culture system firstly.After long time exploration and screening,7effective culture systems were selected.These systems contain ingredients such as DMEM/F12, Neurobasal-A medium, KO-SR,N2, B27, B27without VA, BSA, EGF, bFGF and2cytokines as FGF-10and IGF-1. Weconfirmed C3culture formula as the ultimate culture system, which contains B27withoutVA, EGF and the cytokines FGF-10and IGF-1.By using C3culture formula, human liver cancer stem cell-like cells from differentsources were cultured and expansion.We first cultured hepatoma cell lines includingHep3B and Huh7in our optimized culture medium. The results showed that Hep3B-Cand Huh7-C displayed clone-like forms and maintained the same state after3months cellpassage. Next we cultured CSC-like cells derived from cells lines with different invasionability. HCCLM3and MHCC97-H were high-invasive cell lines and MHCC97-L waslow-invasive one. All of these cell lines were able to form clone and be stable for cellpassage. We called them as HCCLM3-C, MHCC97-H-C and MHCC97-L-C.We also got cells from tumor tissue in patients. After surgical resection, liver tumortissues were dissociated into single cells. EHBH-HCSC-1and EHBH-HCSC-2cells grewwell for more than2months in vitro as undifferentiated tumor spheres in serum-freemedium. Another way to gain cells was to engraft patient's tumor tissues and gave rise to subcutaneous tumors in severe combined immuno-deficient mice. We took thesubcutaneous tumor for digestion and culture. These tumor cells were cultured in2different culture mediums—one was in DMEM with10%FBS and the other one was inour optimized culture system. EHBH-3display epithelial-like growth tumor cells whilethe EHBH-HCSC-3show clone-like growth.2. Biological characteristics assayWe assayed biological characteristics of these cells when we got the CSC-like cells.(1) Fluorescent quantitative PCR was used to detect the expression of Wnt-1,Wnt-1,CD90,Ep-CAM, NOTCH1, NOTCH2, NOTCH3in hepatocellularcarcinoma cells, such as: Hep3B,Huh7,MHCC97-L,HCCLM3and CSC-likecells, such as Hep3B-C,Huh7-C,MHCC97-L-C,HCCLM3-C; CD90expressedhigher in clonal cells than the corresponding adherent hepatocellular carcinomacells. Among them, CD90expression in Huh7-C was10times higher than Huh7cells. NOTCH mRNA expression level were decreased in clonal cells comparedto corresponding cell lines, but Wnt-1and Ep-CAM mRNA expression levelappeared inconsistent phenomenon.(2) We evaluated the cell source of these CSC-like cells using glycogen staining andICG uptake experiments, the results showed that these cells are indeed fromhepatocellular carcinoma cells,but not due to other cell contamination.(3) Furthermore, we performed in vitro Matrigel invasion assay to detect invasionability of Huh7-C and corresponding Huh7cells, the results showed that both ofthem had the strong invasiveness. Through the quantitative analysis we can seethat Huh7-C like cells invasion extent were nearly3times higher than Huh7.(4) Through the cell cycle analysis, the CSC-like cells Hep3B-C and Huh7-C weremainly in phase G0-G1, with percentage at74.09%and59.20%respectively,which were significantly different from cancer cell lines Hep3B and Huh7.(5) Based on both the RT-PCR and FCM analysis, expression of CD90in CSC-likecells were upregulated in different degrees. Consistent with previous study onCSC phenotype, CD90expression level in Hep3B-C was10times higher thanHep3B. Simultaneously, we detected the expression of cell surface antigenEp-CAM, and inconsistent with previous research, both two CSC-like cellsweakened their expression.(6) After surveying uptake of doxorubicin, Hep3B-C and Huh7-C took in lessdoxorubicin compared with corresponding normal HCC cell lines, and this was thought to be a critical condition. Further detection of cell survival in differentdrug concentrations verified Huh7-C wass more drug resistant than Huh7cellline. CSC-like cells show resistant difference even to sorafenib, the firstclinically confirmed liver cancer targeted drug. We applied Huh7-C cellscultured in serum-free medium for in vitro drug resistant experiment, finding46%cell survival after incubation in40μmol/L sorafinib for48hours, while thisconcentration makes adherent Huh7cells no survival. Taken together, theCSC-like cells cultured in serum-free medium showed better drug resistancethan corresponding adherent HCC cell lines.(7) ELISA quantitative detection results in both the adherent HCC cells andCSC-like cells showed great expression of VEGF, and the expression level wasinconsistent in different CSC-like cells from different HCC cell lines, whichmay be related to many factors.(8) Next we detected the differentiation capacity of clone-like cells in vitro. Wechoose CK8, CK18keratins whose presence is essentially restricted to Liver,while CK19expressed in biliary ducts and vascular cell marker Tie2. We foundthat Huh7expressed CK8and CK18but no CK19and Tie2, while Huh7-Cshowed weak or no expression of these4markers. Interestingly, differentiatedcells express all of these4markers. The result reminded us the clone cell had thepluripotency. If we can find the optimal condition to induced, we can learn morefrom the clone cells.(9) Tumorigentic potential of liver CSC-like cells were evaluated in vivo. Theinjection of1,000Huh7-C cells generated visible tumors after4-5weeks fromthe transplant while100,000live cancer cells Huh7did not induce tumorformation; We injected100MHCC97-L-C cells into NOD/SCID mices,the ratioof tumor formation is100%; For HCCLM3-C cell line, we injected5NOD/SCID mices,1,000cells per mice,the tumor formation rate is100%,whilethe tumor formation rate of corresponding adherent cancer cells is very low. Wefound100MHCC97-H-C cells could generate tumors, and when we injected10,000cells per mice, all of them could generate tumors, while the samequantity adherent MHCC97-H cells could not induce tumor formation. All thedata indicating that the CSC-like cells showed significant higher tumorogenicitythan corresponding adherent cancer cell lines.From the results above we concluded that we got a subset of cells which have the ability of CSCs, we can use them to do more research about the liver cancer stem cells.3. Deep sequencing-based expression profiling analysis of liver CSC-like cellsDeep sequencing were carried out an in-depth analysis of the expression of mRNAsin three hepatoma cells(Hep3B,Huh7and MHCC97-H) and three liver cancer stem celllines(Hep3B-C,Huh7-C and MHCC97-H-C).We identified1012differentially expressedgenes overlapped among Huh7-C vs Huh7, MHCC97-H-C vs MHCC97-H and Hep3B-Cvs Hep3B groups via foldchange method. The406of1012genes whose fold change>2were up or down regulated consistently among these samples.90genes wereup-regulated and316genes were down-regulated. In order to identify the functionalclassification of these406genes, we performed functional enrichment analysis inGeneOntology database. In addition, to identify differential genes enriched in which kindof biological pathways, pathway enrichment analysis was performed in GeneGOdatabase via MetaCore software. We showed top10significant pathways whosecorrected p value was less than0.05. We chose hypergeometric distribution to calculatethe significance values (p values) and the FDR method to correct the p value.In addition,we applied HiSeq deep sequencing to carry out an in-depth analysis ofthe expression of sRNAs in two hepatoma cells(Hep3B,Huh7) and two liver cancer stemcell lines(Hep3B-C,Huh7-C).We first mapped the small RNA tags to genome by SOAPto analyze their expression and distribution on the genome.The small RNA tags wasannotated with known miRNA, repeat, piRNA, exons, introns, rRNA, scRNA, snoRNA,snRNA and tRNA from Genbank, Rfam and miRBase. Then we used Foldchange methodto find out the differentially expressed sRNAs between hepatoma and hepatoma stemcells. Poisson distribution was used to calculate the p value, and Benjamini multiplehypothesis testing was used to correct the p value. Results revealed4repeats,37highexpressed and9low expressed piRNAs,9high expressed and9low expressed miRNAsthat are significantly diffenrentially expressed between hepatoma and liver cancer stemcells with p-value<0.01and q-value<0.01.Through bioinformatics analysis we get a lot of differences betweengene-expression. We need to verify the different function of these genes in liver cancerstem cells and tumor cells by further experiments. Also we need to find the uniquemarker and characteristic of liver cancer stem cells. In the end to find specific targetedliver cancer stem cells drugs and cured liver cancer completely.