Isolation And Identification Of Lateral Population Of Stem Cells In Undifferentiated Thyroid

Object:Recently the identification of cancer stem cells (CSCs) in leukemia, as well as in brain and breast cancer has renewed the interest in the hypothesis that cancer may arise from adult stem/progenitor cells. In thyroid gland,it has also been confirmed that the pluripotent stem cell subsets originating from the endoderm exist in human thyroid cells. In2007, cells with stem-cell properties as a side population been isolated from hunman thyroid cancer cell lines by Fluorescent Activated Cell Sortin(FACS), which is the further confirmation of the CSCs in thyroid tumors. Based on the findings above, the aim of this research is to further identify the existence of cancer stem-like cells (side population,SP) in anaplastic thyroid cancer cell lines by FACS, compare the growth pattern, clonogenity and invasive potential between cancer SP and non-SP cells, and clarify the aberrant activation of epithelial-mesenchymal transition(EMT) in cancer SP cells.Method:(1) Three different human anaplastic thyroid cancer cell lines (including HTh74, SW1736, C643) were stained with Hoechst33342dye and sorted for a small fraction of side population by Fluorescence-activated Cell Sorting (FACS).(2)The gene expression of stem cell marker—Oct4(octamer-binding transcription factor4), genes related to cancer resistance and relapse—ABCG2(ATP-binding cassette superfamily G member2) and MDRl(multidrug resistance1), some epithelial-mesenchymal transition (EMT) associated genes and genes of the matrix metalloproteinases (MMPs) and members of the urokinase plasminogen activator system, which involved in extracellular matrix (ECM) degradation for tumour invasion and metastasis, in SP and non-SP cells were compared by performing RT-PCR and Immunofluorescent staining.(3) Cancer SP and non-SP cells were maintained individually in culture for2weeks, then stained with Hoechst33342, and resorted into SP and non-SP fractions to examine whether they have the capacity to self-renew and give rise to diverse progenies.(4)The clonal formation assay was performed to evaluate the self-renewal potential of SP cells, and cell invasive potential was measured by using Matrigel invasion assay.(5) Morphology of HTH74 cells cultured in defferent conditions for10-14days was observed, then FACS and BRDU were applied to evaluate the proliferation of HTH74-SP cells.Results:(1) The established human anaplastic thyroid cancer cell lines, including C643, HTh74and SW1736contained a small percentage of side populations.(2) They displayed high gene expression of stem cell marker Oct4, as well as ABC transporter genes ABCG2and MDR1, which endow SP cells with the ability to pump out diverse dyes and drugs, and therefore probably let such SP cells be highly resistant to chemotherapeutic agents and to escape cancer therapy.(3) In vitro, these cancer SP cells exhibited the capacity to self-renew and generate heterologous descendents of non-SP cells, whereas the non-side population (non-SP) cells only produced other non-SP progenies.(4) clonal formation assay revealed that thyroid cancer SP cells displayed markedly higher clonogenic potential than non-SP cells.(5) Matrigel invasion assay revealed that thyroid cancer SP cells displayed markedly higher invasive potential than non-SP cells.(6) SP cells show preferential expression of β-catenin, Snail, Slug, LAMC2and L1CAM, but decreased expression of E-cadherin, which indicates the activation of EMT in these cells. Furthermore, the SP cells also displayed high levels of metastasis-associated proteinase genes such as MMP-2, uPA and uPAR, which participate in the process of extracellular matrix degradation for tumor invasion and metastases.(7) In the presence of EGF and bFGF, HTh74cells can propagate as spheres which possess increased percentage of SP cells. Furthermore, after sorting, the SP cells could proliferate and re-form three-dimensional spheres in the presence of these two growth factors, whereas the non-SP cells could not.Conclusion:These findings suggest that the side population in thyroid cancer cell lines is enriched with stem-like cancer cells. These cancer stem-like cells, which may be closely related to tumor recurrence and metastasis, exhibited some typical sternness phenotypes, including preferential expression of stem cell marker and ABC transporter genes, the capacity to generate both SP and non-SP cells, and high invasive potential.Consequently, these cancer stem-like cells may have significant impact on future treatment strategies for thyroid cancer.