CD90^pos Cells Possess Stem Cell And Metastatic Activity In Human Lung Adenocarcinoma

Nowadays lung cancer is the leading cause of cancer incidence and cancer-related deathin CHINA, of which about50percent of lung cancer cases are lungadenocarcinoma(LA).With the heterogeneity and other limitations,the specific mechanism oflung cancer remains unclear.The existence of cancer stem cells（CSCs） reflects the cellularheterogeneity within tumors,which is regarded as a specific subpopulation of cells needed forcancer initiation and progression, metastasis, treatment resistance and so on. Numerous studieshave now provided evidence of the existence and importance of CSCs, and we presume that lungcancer stem cells(LCSCs) play a key role in lung cancer.The epithelial-to-mesenchymal transition (EMT) is a de-differentiation process that has beenimplicated in metastasis and generation in solid tumors. And EMT is also a highly coordinatedprocess observed when epithelial cells lose some or most epithelial characteristics and assumesproperties that are typical of mesenchymal cells.In this study, CD90posCSC were isolated andcharacterized via fluorescence activated cell sorting (FACS) assay from LA cell line A549,andthen we discovered that the specific subtype displayed cancer stem cell andepithelial-mesenchymal transition phenotypes.Main methods, results as follow:1. CD90posCSC were isolated and characterized from LA cell line A549.1) FACS assay was utilized to isolated CD90poscells from LA cell line A549,we foundthat adherent A549cells possess much lower proportion of CD90than A549sphere cells（1.12%±0.23%VS22.43%±0.75%,P＜0.05）,indicating that CD90maybe the potentialCSCs marker.2) Compared with CD90negA549subpopulation, CD90poscells expressed higher stemnessgenes Oct4(0.76±0.045VS0.27±0.022,P＜0.05）and Nanog（0.88±0.052VS0.57±0.048,P ＞0.05）via Western blot analysis.Also the clonal formation（157.33±19.14VS57.33±8.02,P＜0.05）and sphere formation assay(68.89%±6.74%VS30.56%±3.47%,P＜0.05) by CD90posand CD90negcells was performed after seeding wells with the indicated number of cellsalternatively, the results showed that the former highly expressed proliferation capability.3) The tumorigenicity assay in nude mice showed CD90poscells highly expressedtumorigenicity in vivo,which possessed shorter latency stage and formed larger xenograftnodes.2. CD90poscells possessed highly metastasis and invasiveness associated with epithelialmesenchymal transition.1) Migrative and invasive ability of CD90poscells was measured by migration andTranswell invasion assay, and we found that CD90poscells with higher migrative（45.67±12.58VS7.83±2.48，P＜0.05）and invasive（219.12±58.72VS16.43±8.89，P＜0.05）ability in vitro compared with CD90negcells.2) To further confirm upper observation, an experimental tail vein metastasis model wasperformed in vivo. We found that CD90poscells,but not its CD90negcounterparts displayed asuperior ability to metastasize to the lung.3) RT-PCR measurement of gene expression related with EMT showed same tendencycompared with Western blot analysis and immunofluorescence assay.We found that MMP2was higher expression in CD90poscells,which is a key protein with cancer metastasis in matrixmetalloproteinase（MMPs）family,also transcription factors Twist increased significantly,which mould be involved in the EMT phenotype maintaining.4) CD90expression is associated with clinical significance in lung adenocarcinoma.Wedetected the expression of CD90in68primary nodes and26lymphocytic nodes of lungadenocarcinoma,the result showed that CD90expression in metastasis nodes was much higherthan primary node, which was correlated with shorter survival time.To sum up,CD90is anindicator of poor prognosis in lung adenocarcinoma.ConclusionIn summary, CD90is a effective marker for isolation and enrichment of LCSCs.AndCD90poscells highly expressed stem cell related genes,self-renewal capability in vitro,alsoshowed stronger tumorigenicity in vivo.Furthermore, CD90poscells possessed highermetastasis and invasiveness,which was associated with EMT.Our study demonstrated that CD90poscells were endowed with EMT phenotype and showed an decreased expression ofE-cadherin,and increased expression of N-cadherin, vimentin, MMP2,which would beassociated with Twist,an EMT-related transcriptional factor. Expression of CD90is negativelycorrelated with survival time in the metastatic sites instead of primary sites.