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Objective:To investigate the expression of four cancer stem cell (CSC) markers (EpCAM, CD133, CD90 and CD24) in hepatocellular carcinoma tissues and peripheral blood circulating tumor cells (CTC),their value in the prognosis of patients with hepatocellular carcinoma.Methods:A total of 50 hepatocellular carcinoma tissues and 29 peripheral blood sample from 50 patients with hepatocellular cancer treated in Zhongshan Hospital Fudan University from October 2013 to September 2014 were collected and analyzed by flow cytometry or qRT-PCR to examine the expression of EpCAM, CD133, CD90 and CD24. The clinical data of patients were collected, including tumor size, tumor number, satellite lesions, vascular invasion, Edmondson stage, BCLC stage and liver cirrhosis, etc. The correlation between the expression of four markers in hepatocellular carcinoma tissues and CTC with the clinical data and survival time of patients were compared.Results:The positive expression rates of EpCAM, CD133, CD90 and CD24 in hepatocellular carcinoma tissues were 66% (33/50), 18% (9/50), 60% (30/50) and 56% (28/50); the positive expression rates in CTC were 55% (16/29), 38% (11/29), 31% (9/29) and 59% (17/29). CD90 expression in hepatocellular carcinoma tissue was positively correlated with the occurrence HCC liver cirrhosis ( P<0.05), while CD133 expression was negatively correlated with the 5-year survival rate of patients ( P<0.05). The expression of EpCAM and CD24 in peripheral blood CTC were closely related to the patient′s Edmondson stage ( P<0.05). The survival time of patients with CD133 positive expression in hepatocellular carcinoma tissue was lower than those without CD133 expression ( P<0.05); the survival rate of patients with EpCAM expressed in either tissue or peripheral blood CTC was lower than that of patients with EpCAM double negative expression ( P<0.05). The survival rate of patients with CD90 negative in HCC tissue and positive in peripheral blood was lower than that in patients with double negative/double positive in tissue and peripheral blood or patients positive in hepatocellular carcinoma tissue and negative in peripheral blood ( P<0.01). Conclusion:Different expression characteristics of four markers in cancer tissues and peripheral blood CTC might provide useful information about predicting prognosis of hepatocellular carcinoma. The expression of CD133 in tissues can be used as an important survival predictor of hepatocellular carcinoma patients. The differential expression of cancer markers in tissue samples and blood samples can provide more clinical prognostic information.
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Objective:To investigate the expression and distribution of human dermal papillary fibroblasts (Fp) , reticular fibroblasts (Fr) , and myofibroblasts (MFB) in keloid tissues.Methods:Keloid tissues were collected from 15 outpatients (including 8 males and 7 females) aged 20-50 years, who were diagnosed in the Department of Dermatology, Renmin Hospital of Wuhan University from May to December 2019. Normal skin tissues were taken from 15 age-matched women who underwent mammoplasty, and served as controls. The distribution of fibroblast activation protein (FAP) , CD90 and alpha-smooth muscle actin (α-SMA) was observed in the keloid tissues and normal skin tissues by dual immunofluorescence staining. Furthermore, fibroblasts were isolated from 3 normal skin and 3 keloid tissue samples, and subjected to primary culture. Subsequently, the fibroblasts were treated with 10 ng/ml transforming growth factor-β1 (TGF-β1) for 48 hours in vitro, during which, changes in fibroblast phenotypes were observed in the 2 groups. Fluorescence-based quantitative RT-PCR and Western blot analysis were performed to determine the mRNA and protein expression of FAP, CD90 and α-SMA. Measurement data were compared between 2 groups by using t test. Results:Immunofluorescence staining of the normal skin tissues revealed that FAP +/CD90 - fibroblasts were predominantly distributed in the superficial dermis, FAP -/CD90 + fibroblasts in the deep dermis, and CD90 + cells hardly expressed α-SMA; however, a large number of FAP + fibroblasts and CD90 + fibroblasts were observed in the deep keloid tissues, and many CD90 + fibroblasts also expressed α-SMA. Dual immunofluorescence staining showed that normal tissue-derived fibroblasts hardly expressed α-SMA, and keloid-derived fibroblasts expressed α-SMA. The fluorescence intensity of α-SMA + cells significantly increased in the normal tissue-and keloid-derived fibroblasts after 24-hour treatment with TGF-β1 (21.058 ± 0.709, 27.112 ± 0.097, respectively) compared with that in the corresponding untreated fibroblasts (11.312 ± 0.636, 21.306 ± 0.464, t=22.430, 13.370, respectively, both P < 0.05) . RT-PCR and Western blot analysis showed that the mRNA and protein expression of FAP, CD90 and α-SMA significantly increased in the keloid-derived fibroblasts after 48-hour treatment with TGF-β1 (mRNA: 92.610 ± 3.667, 1.366 ± 0.105, 3.240 ± 0.141; protein: 0.652 ± 0.073, 1.046 ± 0.119, 0.946 ± 0.117, respectively) compared with the untreated keloid-derived fibroblasts (all P < 0.05) . Conclusion:CD90 + Fr aberrantly proliferated in the deep dermis of keloid tissues, suggesting that directional intervention in aberrantly proliferating FAP -/CD90 + Fr in the deep dermis may promote the efficacy for keloids.
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Objective The objectives of this study were to screen and identify monoclonal antibodies against hepatoma stem cells by screening for hepatoma spheroid cells,and to provide candidate therapeutic monoclonal antibodies for targeting cancer stem cells to treat hepatic cancer. Methods Hepatic cancer stem cells were enriched by serum-free suspension culture. Immunofluores-cence,cisplatin resistance assay, Real -time qPCR, subcutaneous tumor formation in nude mice, and other methods were used to screen and identify anti-hepatocarcinoma stem cell monoclonal antibodies. Immunohistochemistry was used to identify the expression of antigen recognized by monoclonal antibody in liver cancer tissues. The antigen was identified by mass spectrometry. Results MH-CC97L cells were able to form cell spheres in serum -free suspension culture and were labeled with PKH26 dye. Flow cytometry showed that the expression of CD90 + in MHCC97L spheroid cells was 3. 4 times higher than that in the parental cells. In the inhibition experiment of serum-free spheroid,6 monoclonal antibodies significantly inhibited MHCC97L cells in serum-free medium,and in-hibitory rates were 54. 67% ,50. 33% ,45. 73% ,42. 26% ,39. 11% ,and 37. 63% ,respectively. The results of immunofluorescence showed that monoclonal antibodies 28C10 and CD90 were colocalized in MHCC97L cells. The results of real-time qPCR showed that the expression of Sox-2 and Oct-4 in MHCC97L 28C10 + cells was significantly higher than those of MHCC97L 28C10 - cells. Flow cytometry showed that the ratio of 28C10 + in MHCC97L cells and its sphere cells were 7. 98% and 10. 7% ,respectively. The ratio of 28C10 + cells was increased by 1. 34 times. The in vitro globing ability and invasive ability of 28C10 + cells obtained by flow cytometry were significantly higher than those of 28C10 - cells. The results of CCK-8 assay showed that 28C10 + cells were resistance to cispla-tin in 28C10 - cells,which are 1. 96 g/ml and 1. 16 g/ml,respectively. Tumorigenic assay showed that 28C10 + cells were inoculated subcutaneously with 2×104 cells into the nude mice,and tumors were formed in 2 months,with 40% of tumor formation rate. Another nude mouse that did not form a tumor had formed a lung metastasis(1/5). Immunohistochemistry showed that the target antigen posi-tive rate of monoclonal antibody 28C10 in hepatic cancer tissues was about 72. 0% (77/107),while it was lowly expressed in adjacent tissues,and the difference was significant. Mass spectrometry showed that the antigen recognized by 28C10 was HSP90α. Conclusion The MHCC97L spheroid cell model is successfully used to identify a monoclonal antibody that specifically recognizes hepatoma stem cells,which provides a foundation for antibody therapy targeting hepatic cancer stem cells.
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Objective To study the expression and clinical significance of stem cell markers CD44 and CD90 in ovarian cancer tissue.Methods A total of 45 ovarian cancer patients received surgery resection were col-lected into the study group,45 patients received hysteromyomectomy were collected into the control group. Immunohistochemical method was used to detect the expression of stem cell markers CD44 and CD90 in the o-varian tissue of two groups,then the relationships between the expression of CD44,CD90 and 1 year survival rate were analyzed.Results The positive rates of CD44 and CD90 in the study group were 64.44% and 68.89%,which were significant higher than those of 0.00% and 0.00% in the control group.The positive rates of CD44 in tissues of clinical staging Ⅲ - Ⅳ stage,histological grade G2+G3 stage and with lymph node metastasis were 86.36%,88.46%,88.24% respectively,which were significant higher than 43.48%,31.58%, 50.00% inⅠ - Ⅱ period clinical stage,histological grade G1 phase and without lymph node metastasis.The positive rates of CD90 in tissues of clinical staging Ⅲ - Ⅳ stage,histological grade G2+ G3 stage and with lymph node metastasis were 90.91%,92.31%,88.24% respectively,which were significant higher than 47.83%,36.84%,57.14% inⅠ - Ⅱ period clinical stage,histological grade G1 phase and without lymph node metastasis,the differences had statistical significance(P<0.05).Follow-up was conducted for 45 cases of pa-tients with ovarian cancer,the survival rate of CD44 positive patients was 62.07%,less than patients with CD44 negative expression 87.50%,the survival rate of CD90 positive patients was 64.52%,less than patients with CD90 negative expression 85.71%,but the differences had no statistical significance(P>0.05).Conclu-sion T he expression of stem cell markers CD44 and CD90 might be involved in the occurrence and develop-ment of ovarian cancer,metastasis and infiltration process,and the expression level of the two markers could be used as clinical assessment of biological indicators of prognosis.
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Purpose To detect the expression of CD90 in invasive ductal breast carcinoma with different molecular subtypes and to explore its clinical significance.Methods The expression of CD90,ER,PR,Ki-67 and HER-2 were detected in 80 cases of invasive ductal breast carcinoma tissue and 20 cases of breast benign lesion with immunohistochemical method.The relationship between CD90 and clinicopathological parameters were analyzed.Results The positive expression rate of CD90 in invasive ductal breast carcinoma and breast benign lesion tissues were 62.5％ and 20.0％,respectively (P ＜0.001).The expression of CD90 in invasive ductal breast carcinoma was correlated with lymph node metastasis (P ＜ 0.05),but not with age,tumor size,TNM staging and histological grade (P ＞ 0.05).Among different molecular subtypes,CD90 expression level in Luminal A type was the lowest (40.0％),and the level in triple negative type was the highest (82.4％) (P ＜0.05).CD90 expression level was negatively correlated with ER (r=-0.342,P＜0.05) orPR (r=-0.374,P＜0.05) expression level,but not with Ki-67 (r =0.084,P ＞ 0.05).Condusion The over-expression of CD90 is related with molecular subtypes of breast carcinoma,and its high expression suggests a poor prognosis.
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Glioblastoma multiforme (GBM) is the most malignant World Health Organization grade IV brain tumor. GBM patients have a poor prognosis because of its resistance to standard therapies, such as chemotherapy and radiation. Since stem-like cells have been associated with the treatment resistance of GBM, novel therapies targeting the cancer stem cell (CSC) population is critically required. However, GBM CSCs share molecular and functional characteristics with normal neural stem cells (NSCs). To elucidate differential therapeutic targets of GBM CSCs, we compared surface markers of GBM CSCs with adult human NSCs and found that GD2 and CD90 were specifically overexpressed in GBM CSCs. We further tested whether the GBM CSC specific markers are associated with the cancer stemness using primarily cultured patient-derived GBM cells. However, results consistently indicated that GBM cells with or without GD2 and CD90 had similar in vitro sphere formation capacity, a functional characteristics of CSCs. Therefore, GD2 and CD90, GBM specific surface markers, might not be used as specific therapeutic targets for GBM CSCs, although they could have other clinical utilities.
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Adult , Humans , Brain Neoplasms , Drug Therapy , Glioblastoma , Neoplastic Stem Cells , Neural Stem Cells , Prognosis , World Health OrganizationABSTRACT
Objective To investigate the expression of stem cell associated protein CD90 in primary hepatocellular carcinoma (HCC) and its relationship with clinical pathological characters ;to analyze the relation between CD90 and epithelial mesenchymal transition (EMT) markers E‐cadherin (E‐cad) and N‐cadherin (N‐cad) .Methods The expressions of CD90 ,E‐cad and N‐cad in 53 tissues of HCC were detected by immunohistochemistry streptavidin‐perosidase (SP ) method , and 10 surrounding of liver benign lesions were studied as control .The expression of E‐cad and N‐cad in CD90 positive cells of two HCC cell lines (M HCC97‐L and HCCLM‐3 ) was measured by flow cytometry .Chi square test and Spearman rank correlation analysis were performed for count data .Independent sample t test was used for measurement data comparison .Results There was no CD90 expression in control liver tissue . The positive rate of CD90 expression in HCC tissue was (34% ,18/53) ,and the difference between two groups was statistically significant (χ2 = 4 .755 , P< 0 .05) .The expression of CD90 in HCC was positively correlated with portal vein cancer embolus (r= 0 .378 , P< 0 .05) and was negatively correlated with histological differentiation degree and capsular infiltration (r= -0 .398 and -0 .519 ,both P<0 .05) .The expression of CD90 was negatively correlated with the expression of E‐cad (r= -0 .341 , P<0 .05) ,however was positively correlated with the expression of N‐cad (r=0 .441 ,P<0 .05) .The positive expression rate of E‐cad in CD90 positive HCCLM‐3 cells was lower than that of MHCC97‐L cells ((2 .56 ± 0 .29)% and (4 .31 ± 0 .18)% ,t= 8 .757 ,P< 0 .05) ,while the positive expression rate of N‐cad was higher than that of MHCC97‐L cells ((8 .10 ± 1 .45)% and (5 .51 ± 0 .44)% ,t= -9 .667 ,P<0 .05) .Conclusions The expression of stem cell associated protein CD90 is correlated with the EMT of HCC .Their interaction promote tumor infiltration metastasis w hich may be a new target of HCC treatment .
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O câncer de mama é a doença maligna que mais acomete as mulheres no mundo. Apesar dos inúmeros tratamentos, o óbito se deve principalmente à doença metastática que pode se desenvolver a partir do tumor primário. Esta progressão tumoral decorre da dificuldade de se estabelecer um prognóstico mais preciso. Atualmente, a teoria de células iniciadoras de tumor vem sendo estudada para tentar explicar a biologia do câncer e descrever novos alvos para prognósticos e terapias. O carcinoma mamário foi o primeiro tumor sólido para o qual foi identificada uma subpopulação celular, definida como CD44+/CD24-, apresentando as características de células iniciadoras tumorais. Embora este fenótipo venha sendo muito utilizado para descrever as células iniciadoras tumorais de mama, muitos trabalhos tem questionado a relevância clínica desses marcadores, enfatizando que outros marcadores devem ser identificados. Assim, o objetivo deste trabalho é analisar e caracterizar marcadores de células-tronco que possam estar relacionados com o grau de malignidade no modelo de câncer de mama. Inicialmente, analisou-se a expressão de 10 marcadores de células-tronco em diferentes linhagens de câncer de mama que apresentam graus crescentes de malignidade. O CD90 foi selecionado devido à alta expressão desse marcador na linhagem mais agressiva Hs578T. Para a caracterização deste marcador, realizou-se ensaios funcionais, através do silenciamento do CD90 na linhagem tumorigênica Hs579T e sua superexpressão na linhagem não-tumorigênica MCF10A. As linhagens celulares geradas foram caracterizadas quanto ao crescimento celular, potencial invasivo e metastático. Foi possível observar que houve uma alteração da morfologia nas linhagens transformadas com o CD90 e, também, um maior tempo de dobramento na linhagem Hs578T-CD90- e um menor na MCF10A-CD90+. Além disso, a linhagem MCF10-CD90+ foi capaz de crescer independentemente de EGF. Através da análise da via EGF, foi possível observar que houve um aumento da expressão da forma fosforilada do receptor e dos fatores Erk, c-Jun, e Jnk na linhagem MCF10A-CD90+ e uma diminuição dos mesmos na linhagem Hs578T-CD90-. A análise da atividade do elemento responsivo do fator de transcrição AP1 comprovou que a via de EGF é funcional na linhagem MCF10-CD90+. Também foram analisados os marcadores de transição epitélio-mesenquimal, verificando-se aumento da expressão dos marcadores mesenquimais na linhagem MCF10A-CD90+ e diminuição na linhagem Hs578T-CD90-. Os ensaios in vitro de invasão mostraram que as células MCF10-CD90+ são capazes de migrar e invadir e as células Hs578T-CD90- apresentam diminuição significativa da habilidade de migração e invasão. Além disso, os ensaios de metástase in vitro e in vivo, mostraram que a superexpressão de CD90 levou à malignização das células MCF10A. Por outro lado, a linhagem Hs578T-CD90- apresentou menor potencial metastático in vitro. Portanto, neste trabalho, pela primeira vez, o CD 90 foi caracterizado funcionalmente como um marcador envolvido na transformação maligna do carcinoma mamário, contribuindo, assim, para melhor entendimento da biologia do câncer de mama e para que se possa desenvolver novas ferramentas de diagnóstico/prognóstico e novos protocolos clínicos e terapêuticos
Breast cancer is the malignant disease which affects the highest number of women in the world. In spite of the numerous treatments available, death is primarily due to the metastatic disease that may develop from the primary tumor. This tumor progression occurs because of the difficulty in establishing an accurate diagnosis/prognosis. Currently, the tumor initiating cells theory is being applied in an attempt to explain cancer biology and to unveil new diagnostic and therapeutic targets. Mammary carcinoma was the first solid tumor in which a cellular subpopulation, defined as CD44+/CD24-, was associated with tumor initiating cells. Although this phenotype has been widely used to describe breast tumor initiating cells, several studies have questioned the clinical relevance of these markers, emphasizing that additional markers should be identified. The objective of the present study is to analyze and characterize stem cell markers that may be related to malignancy stages in the breast cancer model. Initially, the expression of 10 stem cell markers was analyzed in different breast cancer cell lines displaying different malignancy grades. CD90 was selected due to its high expression levels in the most aggressive cell line, namely: Hs578T. In order to further characterize this marker, a functional study was performed in which CD90 was silenced in the Hs578T tumorigenic cell line and overexpressed in the non-tumorigenic MCF10A cell line. The resulting cell lines were characterized relative to growth rate and invasive and metastatic potential. A change in morphology readily was observed in the cell lines overexpressing CD90. In addition, the Hs578T-CD90-cell line presented an increased doubling time (DT), while the MCF10A-CD90+ cell line displayed a lower DT.. Furthermore, MC10-CD90+ cells were able to grow in the absence of EGF. Analysis of components of the EGF pathwayrevealed increased expression levels of the phosphorylated form of Erk, c-Jun and Jnk receptors in the MCF10-CD90+ cell line, while Hs578T-CD90- cells presented decreased expression of the same factors and receptors. Analysis of the activity of the AP1 responsive element allowed confirmation that the EGF pathway is functional in the MCF10-CD90+. . Epithelial-mesenquimal transition markers presented increased expression levels in the MCF10A-CD90+ cell line, accompanied by decreased expression levels in Hs578T-CD90- cells. In vitro invasion assays showed that MCF10A-CD90+ cells are capable of migrating and invading, while Hs578T-CD90- cells presented a significant decrease in their ability to migrate and invade. Additionally, in vitro and in vivo metastasis assays showed that malignization ensued upon overexpression of CD90 in MCF10A cells and a lower tendency to form metastasis in vitro was observed for the Hs578T-CD90- cell line. Therefore, the present study presents, for the first time in the literature, the functional characterization of CD90 as a genetic marker involved in the malignant transformation of mammary carcinoma, leading to a better understanding of the breast cancer biology, which may, in turn, lead to the development of new clinical and therapeutic protocols
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Biomarkers, Tumor , Stem Cells/metabolism , Thy-1 Antigens/analysis , Breast Neoplasms/physiopathology , Clinical Protocols/classification , Gene Silencing , Plasmids/administration & dosage , Therapeutics/methodsABSTRACT
Objective: To observe the relationship between stem cells marker CD90 and CDDP (cisplatin) resistance in lung adenocarcinoma A549 cells, and to explore whether CD90 is a marker of lung cancer stem cells. Methods: Self-renewal abilities of A549 and A549/CDDP cells were detected by sphere-forming assay, CCK-8 (cell counting kit-8) and plate colony-formation assay, respectively. The proportions of CD90+ in A549 cells, A549/CDDP adherent cells and the spheres cells were measured by FCM (flow cytometry). The CDDP resistance in CD90+A549 cells and CD90-A549 cells was detected by cytotoxicity experiment. The CDDP resistance in CD90+A549 cells in vivo was detected by using transplanted tumor in nude mice. Results: As compared with A549 cells, the abilities of sphere formation, proliferation and colony-formation efficiency of A549/CDDP cells were increased (P < 0.01). The proportions of CD90+ in A549/CDDP adherent cells and the sphere cells were higher than those in the corresponding A549 adherent cells and the sphere cells (P < 0.05). IC50 (half inhibitory concentration) values of CDDP in CD90+A549 and CD90-A549 cells were 8.50 and 1.50 μmol/L, respectively. The tumor xenograft volume of CD90 +A549 cells was larger than that of CD90-A549 cells after treatment with CDDP in nude mice (P < 0.05). Conclusion: A549/CDDP cells show high self-renewal ability and CD90+ proportion. CD90+A549 cells have high ability of CDDP resistance. CD90 may be a marker of lung cancer stem cells. Copyright © 2013 by TUMOR.
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ObjectiveTo study the expressions of CD90 and hTERT in hepatocellular carcinoma (HCC),and their relationships to progression of tumor.MethodsThe expressions of CD90 and hTERT in hepatocellular carcinoma were detected by S-P immunohistochemical staining.Twenty patients with hemangiomas of liver were used as control.ResultsCompared with the control group,the positive rates of CD90 and hTERT in HCC were significantly higher (63.9％ and 47.2％ vs 0％ and 0％).The positive rates of CD90 and hTERT were significantly higher in patients with tumors at UICC Ⅲ-Ⅳ stage than at UICC stage Ⅰ -Ⅱ (79.1％ and 62.5％ vs 33.3％ and 16.6％).The CD90 expression correlated with hTERT positively.There were significant differences in survival between patients with CD90+ and CD90- or hTERT+ and hTERT-.The median postoperative survivals for patients with CD90+ and hTERT+,CD90- and hTERT- were 85 d and 76 d,505 d and 463 d,respectively.ConclusionsCD90 expression correlated positively with progression of HCC.It has the potential to serve as a prognostic marker for HCC.
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Objective To investigate the expressions of hepatorna stem cell surface marker CD133 CD90 in tissues of hepatocellular carcinoma (HCC) and evaluate their related clinical significances. Method The expressions of CD133 CD90 were detected by immunohistochemical method in HCC tissues of 93 patients, and normal liver tissues of 10 cases. Results Among 93 cases with HCC, the positive expression of CD133 were found in 71 cases (76.3%), and CD90 positive expression in 64 cases (68.8%), and the percentage of positive cells were (6.4±3.3)% and (4.3±3.9)% respectively. No positive expression of CD133 and CD90 was found in normal liver tissues (P<0.01). CD133, CD90 expressions in the HCC tissues of TNM stage Ⅲ-Ⅳ [(8.1±3.7)%,(5.7±4.2)%] were higher than those of TNM stage Ⅰ - Ⅱ [(4.1±2.3)%,(2.3±1.9)%] (P<0.01). Spearman correlation analysis indicated that the expressions of CD133 and CD90 were up-regulated as the pathohistology grades increased (P<0.05). Positive correlation was observed between CD133 and CD90 expression (r=0.402, P<0.01). Conclusions CD133, CD90 positive cells exist in HCC tissues, their expressions positively relate to the TNM stage and the pathohistology grades for HCC patients.
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Background and purpose:Small cell lung cancer(SCLC) is a highly aggressive malignancy with a 5-year survival rate of less than 10%.These features suggest the enrichment in cancer stem cells.Our study was aimed to establish a small cell lung cancer cell line from primary cultured from lung cancer tissue,and to identify and isolate cancer stem cells from the early generations of the established cell line.Methods:By using a serum-free medium,lung cancer primary cells were cultured from a small fresh lung cancer cell sample,and a small cell lung cancer cell line was established after passaging the cultured cells.Lung cancer stem cell markers were searched by using flow cytometry analysis to sort through the third or forth generation cells of the established cell line.Biological characteristics of lung cancer stem cells were studied by using the single cell clone formation test,plat colony formation test and cell sphere formation test.Results:A small cell lung cancer cell line was established by primarily culturing a fresh lung cancer sample.The cell line could easily passaged more than 25 generations.When the third or fourth generation cells of the established cell line were checked by flow cytometry,there was a small population of cells that were obviously with CD44 stronger positive(CD44++ cells,5.1%) than the main population cells,which were CD44 weak positive(CD44+ cells).CD44++ cells showed stronger colony formation ability than the CD44+ cells.Furthermore,only the CD44++ cells could form stem cells when a single cell was seeded in a well of 96 well plates.Most importantly,only the CD44++ cells could form cell spheres in ultra low attachment 96 well plates.These results indicated that the CD44++ cells enriched more cancer stem cells in the small cell lung cancer cell line.When CD44 and CD90 antibodies were co-stained,the cells of the established cell line could be separated into 4 populations,i.e.CD44+CD90-,CD44+ CD90+,CD44++ CD90+ and CD44++ CD90-cells.CD44+ CD90+ cells were the smallest population cells in the cell line,with a ratio of about 1.9%.When cultured in ultra low attachment 96 well plates,CD44++ CD90+ cells had the strongest cell sphere formation ability when compared with other population cells,indicating that CD90 might also be a small cell lung cancer stem cell marker.Conclusion:There were cancer stem-like cells in primary cultured small cell lung cancer cell lines,CD44 and CD90,which might mean that they could be lung cancer stem cell markers.