Epithelial Cell Adhesion Molecules (EpCAMs) Promote Drug Resistance in Breast Cancer Cells by Enhancing Tight Junctions / 中国生物化学与分子生物学报

Breast cancer is a malignant tumor with high mortality, and multidrug resistance (MDR) mediated by ABCG2 (ATP-Binding cassette G2) is an important cause of chemotherapy failure. It is an urgent problem to explore the mechanism of ABCG2-mediated drug resistance and its key molecules. Epithelial cell adhesion molecule (EpCAM) is involved in multiple tumor drug resistance and is closely related to breast cancer MDR. However, its role in ABCG2-mediated breast cancer drug resistance has not been clarified. The purpose of this study was to explore the regulation of EpCAM on ABCG2-mediated MDR in breast cancer cells and its mechanism. CCK8 cytotoxicity assays confirmed that the drug resistance of MCF-7/MX cell line to mitoxantrone (MX) was significantly increased compared with MCF-7 drug-sensitive strain of human breast cancer. Western blotting results showed that ABCG2 was highly expressed and EpCAM was up-regulated in MCF-7/MX cells compared with MCF-7. SiRNA knockdown of EpCAM in MCF-7/MX cells down-regulated ABCG2 expression and restored sensitivity to MX. Cell morphology was observed under an inverted microscope, and it was found that knocking down EpCAM reduced cell-cell connections between MCF-7/MX cells. The co-localization of EpCAM and claudin 1 in MCF-7/MX cells was observed by immunofluorescence. Furthermore, Western blotting results showed that EpCAM knockdown reduced claudin 1 expression in MCF-7/MX cells. In conclusion, EpCAM may promote ABCG2-mediated mMDR in breast cancers by enhancing intercellular tight junctions through interaction with claudin 1.

The expression differences and prognostic value of four cancer stem cell markers in hepatocellular carcinoma tissues and circulating tumor cells in peripheral blood / 中华检验医学杂志

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.

Relationship of EpCAM expression and DNA copy number alteration in colorectal carcinoma cells / 临床与实验病理学杂志

Purpose To investigate the clinical pathology significance of epithelial cellular adhesion molecule (EpCAM, CD326) expression in colorectal carcinoma cells. Methods Flow cytometry and immunofluorescence assay were used to detect EpCAM expression in 55 cases of fresh colorectal cancer tissues and adjacent normal mucosa. The percentage of positive cells (PPC) and mean fluorescence index (MFI) were calculated. Correlation of EpCAM expression with DNA ploidy change and its value were investigated in the early diagnosis of colorectal cancer. Results The values of PPC and MFI were significantly higher in colorectal carcinoma tissue than that in the normal colorectal tissue [(PPC: (83.48 ± 7.07)％ vs (43.56±5.29)％, t =39.22, P＜0.001. MFI: 28.90(19.60-45.89) vs4.89(3.79-6.28), Z=-6.45, P＜0.001) ]. There were also significant differences (P＜0.01) in the values of PPC and MFI between invasive type and ulcer types, between well-and moderately-differentiated and poorly-differentiated cancers, between Dukes stages A + B and C + D, (between pTNM stages I+ II and III + IV, between pTl + T2 and pT3 + T4, and between pNO and pNl. DNA content analysis showed that DNA polyploid was detected in 39 of 55 colorectal carcinoma (70.90％ ). The DNA index (DI) and ploid were correlated with differentiation degree and Dukes stages, but uncorrelated with lymph node metastasis. At the same time, in the EpCAM positive cases, DI was increased with increased expression of EpCAM (r = 0.668, P =0.000) and the proliferation index of cells in S phase ratio(Sphase fraction, SPF) (r1 =0.664, P1 =0.000, r2 =0.651, P2 = 0.000 ). Conclusion EpCAM expression is obviously upregulated in colorectal carcinoma, and it is closely correlated with the invasion, metastasis and proliferation of tumor cells. The combination of EpCAM expression and DNA content analysis provides references to the early diagnosis and prognosis evaluation in colorectal carcinoma.

Experimental study on ovarian cancer immunotherapy by EpCAM activated dendritic cells induce antigen-specific CD8+ cytotoxic T lymphocytes / 中国免疫学杂志

Objective:To observe the effect on ovarian cancer immunotherapy by dendritic cells (DC) which activated by Epithelial cell adhesion molecule (EpCAM) induce antigen-specific CD8+ cytotoxic T lymphocytes (CTL) and to provide some help to ovarian cancer immunotherapy.Methods: Interleukin (IL)-12,and IL-10 of DC were tested after inducing by EpCAM.Subsequently,EpCAM specific CTL CD8+ was induced by EpCAM-DC.The therapeutic effect and interferon (IFN)-γ of EpCAM-DC-CD8+ CTL on normal ovarian epithelial cells IOSE80 and ovarian cancer cell SKVO3 was detected.After treatment of EpCAM-DC-CD8+ CTL,the volume of ovarian tumor of bearing BALB/c-nu/nu mice was detected.Meanwhile,the morphology changes of tumor tissue were observed by HE staining.Results: Compared with PBS,EpCAM stimulation significantly inceased surface markers DC80,DC83,DC86 and HLA-DR levels,and added up to 4.79,4.85,4.60 and 10.91 times (P0.05).However,the killing rate of EpCAM-DC-CD8+ CTL on SKVO3 cells was 6.82-folds as much as that of DC-CD8+ CTL.Animal experiments showed that ovarian cancer transplantation tumor volume ratio after EpCAM-DC-CD8+ CTL treatment,was significantly lower than PBS group and DC-CD8+ CTL group,which reached 0.27 and 0.28 times,respectively (P<0.05).HE staining showed that EpCAM-DC-CD8+ CTL treatment resulted in significant changes of tumor tissues in pathology.Conclusion: EpCAM protein stimulated the maturation of DC that induced the production of EpCAM specific CD8+ CTL.EpCAM-DC-CD8+ CTL can effectively kill ovarian tumor cells and delay the growth of tumor,which is of great significance for the immunotherapy of ovarian cancer.

Immunohistochemical Expression and Clinical Significance of Suggested Stem Cell Markers in Hepatocellular Carcinoma

BACKGROUND: Increasing evidence has shown that tumor initiation and growth are nourished by a small subpopulation of cancer stem cells (CSCs) within the tumor mass. CSCs are posited to be responsible for tumor maintenance, growth, distant metastasis, and relapse after curative operation. We examined the expression of CSC markers in paraffin-embedded tissue sections of hepatocellular carcinoma (HCC) and correlated the results with clinicopathologic characteristics. METHODS: Immunohistochemical staining for the markers believed to be expressed in the CSCs, including epithelial cell adhesion molecule (EpCAM), keratin 19 (K19), CD133, and CD56, was performed in 82 HCC specimens. RESULTS: EpCAM expression was observed in 56% of the HCCs (46/82) and K19 in 6% (5/82). EpCAM expression in HCC significantly correlated with elevated alpha-fetoprotein level, microvessel invasion of tumor cells, and high histologic grade. In addition, EpCAM expression significantly correlated with K19 expression. The overall survival and relapsefree survival rates in patients with EpCAM-expressing HCC were relatively lower than those in patients with EpCAM-negative HCC. All but two of the 82 HCCs were negative for CD133 and CD56, respectively. CONCLUSIONS: Our results suggest that HCCs expressing EpCAM are associated with unfavorable prognostic factors and have a more aggressive clinical course than those not expressing EpCAM. Further, the expression of either CD133 or CD56 in paraffin-embedded HCC tissues appears to be rare.

Bioinformatics research of CD44 and epithelial cell adhesion molecule related genes and pathways in colorectal cancer / 中华消化杂志

Objective To investigate genes and involved biological processes closely associated with stem cell markers of colorectal cancer-epithelial cell adhesion molecule (EpCAM) + and CD44+.Methods By the bioinformatics method,with microarray data of colorectal cancer from gene expression omnibus (GEO) database and R2 platform,the genes significantly related with CD44 and EpCAM expression were screened out.The differences in expression of related genes were analyzed on the basis of gender,family history of cancer,alcohol and Dukes stage.The expression of related genes in colorectal cancer was compared with that of other tumors and healthy subjects.At same time,the pathways of the genes and Kyoto encyclopedia of genes and genomes (KEGG) of CD44 and EpCAM significantly related genes were analyzed with gene ontology (GO) and KEGG method.Single factor analysis of variance and Chi-square test of four-fold table with correction for continuity were used for statistical analysis by R2 platform embedded statistical tools.Results The expressions of CD44 and EpCAM were detected in all 315 colorectal cancer samples.A total of 888 and 6 316 genes were screened out which were significantly associated with CD44 and EpCAM expression.CD44 was positively correlated with EpCAM.There was no obvious correlation between the expression of five genes which expressed in all 315 tissues and gender family history of cancer,alcohol and Dukes stage (all P＞0.05).By further compared with the expression in other tumors and tissues,the expressions of two genes solute carrier family 12,member 2 (SLC12A2) and proteome of centriole 1 centriolar protein B (POC1B) in colorectal tumor were significantly higher than that in other tumors (F=289.422、128.456,all P＜0.01),and its expression in colorectal cancer was obviously higher than that in tissues of health subjects (F=349.519、128.456,all P＜0.01).GO analysis indicated there were 15 GO semantics related with both CD44 and EpCAM.The genes related with CD44 and EpCAM were analyzed by KEGG access pathway method,while seven and 10 pathways were found to be statistically significant (all P＜0.01).Conclusions CD44 and FpCAM commonly expressed in colorectal cancer.The genes related with CD44 and EpCAM expression are involved in multiple tumor biological processes.

Cancer stem cells in hepatocellular carcinoma - an immuno- histochemical study with histopathological association.

Background & objectives: Cancer stem cells (CSCs) may be responsible for tumour recurrence and resistance to chemotherapy in hepatocellular carcinoma (HCC). This study was carried out to evaluate the association between histological parameters and liver CSCs (LCSC) in HCC, and to compare distribution of liver CSCs in HCC associated with and without hepatitis B virus (HBV) infection. Methods: Seventy nine tumours (49 surgical resections from 46 patients, and 30 from autopsy) were reviewed. Immunohistochemical staining for the LCSC marker EpCAM (epithelial cell adhesion molecule), liver progenitor cell (LPC) markers CK19 (cytokeratin 19) and neural cell adhesion molecule (NCAM) were performed and were associated with histological features of tumour behaviour. Results: Thirty three tumours (41.8%) showed positive staining for EpCAM. CK19 and NCAM expression were seen in 26 (32.9%) and four (5.1%) tumours, respectively. The expression of EpCAM and CK19 was significantly associated with each other (P<0.001). EpCAM expression was significantly associated with clinical and histological features indicating aggressive tumour behaviour, including younger age of onset, higher serum alpha foetoprotein (AFP) levels, tumour cell dedifferentiation, increased mitotic activity, and vascular invasiveness. There was no significant difference in expression of EpCAM, CK19 and NCAM between HBV positive and negative HCC. Interpretation & conclusions: The LCSC marker EpCAM was expressed in less than half of HCC, was independent of HBV aetiology, and was strongly associated with clinical and histological features of aggressive tumour behaviour. Positive staining for CK19 suggests a possible LPC origin of the EpCAM positive HCCs.

MiR-429 regulates inflammatory infiltration, proliferative ability and differentiation of hepatocellular carcinoma / 第二军医大学学报

Objective To observe the effects of miR-429 on inflammatory infiltration, differentiation and proliferation of hepatocelluar carcinoma (HCC). Methods HCCLM3 cells were sorted into positive and -negative cells using magnetic activated cell sorting (MACS) according to EpCAM marker. The EpCAM-positive and negative cells were transfected with AntagomiR- 429 and miR-429mimic to construct over-expressed and down-regulated cells of miR-429, respectively. To validate the effect of transfection, control cells and constructed cells were tested by flow cytometry (FC) and real-time polymerase chain reaction (PCR). The proliferative curves of transfected cells were plotted using cell counting kit-8 (CCK8) assay. Furthermore, tumor- bearing mice model was established by subcutaneous inoculation with transfected cells and related control cells into non-obese diabetic, severe combined immunodeficient mice (NOD-SCID). Mice were sacrificed after 6 weeks, and protein expressions of Ki67, AFP and F4/80 in tumors were determined via immunohistochemistry (IHC) to examine the status of proliferation, differentiation and inflammatory infiltration, respectively. Results The proportion of EpCAM-positive cells was decreased by down-regulating miR-429, and EpCAM-negative cells could partially transfer to EpCAM-positive cells by miR-429 ove- expression. In vivo study showed that over-expressing miR-429 in EpC AM-negative cells cound enhance the tumor forming ability; whereas EpCAM-positive cells had an inhibited tumor-formation when down-regulating miR-429. Interestingly, F4/80, a marker of macrophage, was strongly stained in tumor tissue with miR-429 over-expression. It was also found that in tumor tissue, Ki67 and AFP staining was suppressed with miR-429 down-regulation. Meanwhile, compared to the control, miR-429 significantly accelerated the proliferation of HCCLM3 in vitro compared with the control (P$0. 05). Conclusion miR-429 can regulate the proportion of EpCAM-positive cells of HCC in vitro and in vivo, and greatly increase the inflammatory infiltration and proliferative ability, accompanied by decreased differentiation.

Enteropatia com formação de tufos epiteliais e mutação do gene EpCAM: relato de caso / Tufting enteropathy with EpCAM mutation: case report

Tufting enteropathy (TE), also known as intestinal epithelial dysplasia (IED), is a rare congenital enteropathy related to an earlyonset of severe intractable diarrhea due to specific abnormalities of the intestinal epithelium and mutations of the EpCAM gene. TE is characterized by clinical and histological heterogeneity, such as with low or without mononuclear cell infiltration of the lamina propria, and abnormalities of basement membrane. TE can be associated with malformations, other epithelial diseases, or to abnormal enterocytes development and/or differentiation. The authors report a case of a Brazilian child with TE associated with c.556-14A>G mutation in the EpCAM gene (NM_002354.2)...

Enteropatia com formação de tufos epiteliais (ETE), também conhecida como displasia epitelial intestinal (DEI), é uma rara enteropatia congênita relacionada com um início precoce de diarreia intratável grave devido a anormalidades específicas do epitélio intestinal e mutações do gene EpCAM. ETE caracteriza-se por uma heterogeneidade clínica e histológica, como ausência ou leve infiltrado de células mononucleares na lâmina própria e anormalidades de membrana basal. Pode ser associada a malformações, outras doenças epiteliais ou anormalidades no desenvolvimento/na diferenciação dos enterócitos. Os autores relatam um caso de ETE, em uma criança brasileira, associada à mutação c.556-14A> g do gene EPCAM (NM_002354.2)...

Comparison of putative circulating cancer stem cell detection between the hepatic portal system and peripheral blood in colorectal cancer patients

PURPOSE: The present pilot study was conducted to detect putative cancer stem cell (CSC) from the hepatic portal system and peripheral blood in the colorectal cancer patients and to compare them to healthy donor and diverticulitis patients. METHODS: Laboratory study was performed to identify the expression of cell surface markers, epithelial cell adhesion molecule (EpCAM), cytokeratin (CK) 18, CK20, CD44, and CD133, on several colon cancer cell lines. Clinical pilot study was conducted to detect putative circulating CSC as EpCAM+CD133+ cell in colorectal cancer (n = 10), diverticulitis (n = 5), and four healthy donors, by using flow cytometry. Blood was drawn from the hepatic portal system and peripheral vein. RESULTS: On laboratory study, EpCAM was expressed in whole colon cancer cell lines, and CD44 and CD133 were simultaneously expressed in 50% of the cell lines with stemness phenotype, but CK18 and CK20 were not expressed in most of the cell lines. On clinical study, the mean EpCAM+CD133+ cell counts of 11.6/105 in the hepatic portal system were somewhat lower than 15.4/105 in peripheral vein (P = 0.241). As for diverticulitis patients, EpCAM+CD133+ cells were also detected to have steeper dropped to near zero, after the surgery. CONCLUSION: The numbers of putative CSC were not statistically different between the detection sites of the portal vein and peripheral vein in the colon cancer patients. Therefore, we may not have benefitted by getting the cells from the hepatic portal system. In addition, the CD133+EpCAM+ cells in the colon cancer patients might contain normal stem cells from cancer inflammation similar to diverticulitis.

Differential Features of Microsatellite-Unstable Colorectal Carcinomas Depending on EPCAM Expression Status

BACKGROUND: Recent studies have revealed that a small subset of Lynch syndrome-associated colorectal carcinomas (CRCs) is caused by a germline EPCAM deletion-induced MSH2 epimutation. Based on the finding of this genetic alteration, we investigated the implications of EPCAM expression changes in microsatellite instability-high (MSI-H) CRCs. METHODS: Expression of EPCAM and DNA mismatch repair proteins was assessed by immunohistochemistry in 168 MSI-H CRCs. Using DNA samples of these tumors, MLH1 promoter methylation status was also determined by methylation-specific real-time polymerase chain reaction method (MethyLight). RESULTS: Among 168 MSI-H CRCs, complete loss (CL) and focal loss (FL) of EPCAM expression was observed in two (1.2%) and 22 (13.1%) cases, respectively. Both of the EPCAM-CL cases were found in MSH2-negative tumors without MLH1 promoter methylation. However, only nine of the 22 EPCAM-FL tumors had MSH2 deficiency. Of the 22 EPCAM-FL tumors, 13 showed MLH1 loss, and among them, nine cases were determined to have MLH1 methylation. EPCAM-FL was significantly associated with advanced stage (p=.043), distant metastasis (p=.003), poor differentiation (p=.001), and signet ring cell component (p=.004). CONCLUSIONS: Loss of EPCAM expression is differentially associated with clinicopathological and molecular features, depending on the completeness of the loss, in MSI-H CRCs.

Diagnostic value of chromoendoscopy combined with EpCAM detection in Barrett′s esophagus / 重庆医学

Objective To investigate the diagnostic value of chromoendoscopy combined with EpCAM detection in Barrett′s e-sophagus .Methods 95 patients diagnosed BE under routine endoscopy were recruited in the study ,and in which 49 patients were stained by 2% Lugol′s solution and undergone biopsy as group A ;46 patients were undergone biopsy by routine endoscopy as group B .The expressions of EpCAM were also detected by immunohistochemical SP methods in biopsies of group A .Results 39 patients were diagnosed BE by routine histology in group A ,in which 15 Colonic type metaplasia of BE were found ,an EpCam expression was observed in 28 Barrett′s esophagus patients conformed by histology with HE staining .And 23 patients were diagnosed BE by routine histology in group B ,in which 8 Colonic type metaplasia of BE were found .Both diagnostic rate of BE and colonic type meta-plasia of BE were significantly different between group A and B .Conclusion Targeted biopsies directed by Lugol′s solution staining can improve the diagnostic rate of Barrett′s esophagus ,and diagnostic rate of colonic type metaplasia of BE can be improved fatherly by detecting the expression of EpCAM .

EpCam and Wnt / β-catenin pathway expression in colon cancer / 中国医师杂志

Objective EpCam and Wnt/ β-catenin pathway in colon carcinoma and its clinic-pathological significance of the distribution,studying the relationship between EpCam and Wnt / β-catenin.Methods Retrospective analysis detected by immunohistochemistry 60 cases of colon cancer,20 cases of adjacent atypical hyperplasia,60 normal colon tissue EpCam and Wnt / β-catenin protein expression.Results (1)normal colon tissue,cancer tissue and cancer tissue showed positive expression EpCam clear upward trend,were 23.5％,62.3％,96.5％ ; with normal colonic mucosa to cancer transformation,β-catenin in the membrane expression of the positive rate decreased,while the cytoplasm is followed by increased expression rate in poorly differentiated carcinoma or even nuclear expression,EpCam strong positive expression of Wnt / β-catenin cytoplasmic-positive rate of histological type,depth of invasion,lymph node metastasis; (2)the EpCam with the Wnt / β-catenin expression showed a positive correlation (r =0.653,P ＜0.05) ;(3)high expression EpCam and Wnt / β-catenin in patients with colon significant increase in cancer recurrence rate and 5-year survival rate was significantly reduced.Conclusion EpCam and Wnt / β-catenin pathway in colon cancer positively is correlated,EpCam and Wnt / β-catenin is connected with high expression and tumor invasion,metastasis and prognosis.

Epithelial cell adhesion molecule and tumor / 国际肿瘤学杂志

Epithelial cell adhesion molecules (EpCAMs) play an important role in the process of gene expression and regulation by combining with nuclear receptor.Aberrant expression of EpCAMs has been detected in different types of cancers,such as breast cancer,lung cancer,gastric cancer,and etc.The research of EpCAMs in circulating tumor cells enrichment,tumor pathogenesis and prognosis will be conducive to achieve new breakthroughs in cancer targeted therapy.

Relationship between ganglioside expression and anti-cancer effects of the monoclonal antibody against epithelial cell adhesion molecule in colon cancer

The human colorectal carcinoma-associated GA733 antigen epithelial cell adhesion molecule (EpCAM) was initially described as a cell surface protein selectively expressed in some myeloid cancers. Gangliosides are sialic acid-containing glycosphingolipids involved in inflammation and oncogenesis. We have demonstrated that treatment with anti-EpCAM mAb and RAW264.7 cells significant inhibited the cell growth in SW620 cancer cells, but neither anti-EpCAM mAb nor RAW264.7 cells alone induced cytotoxicity. The relationship between ganglioside expression and the anti-cancer effects of anti-EpCAM mAb and RAW264.7 was investigated by high-performance thin-layer chromatography. The results demonstrated that expression of GM1 and GD1a significantly increased in the ability of anti-EpCAM to inhibit cell growth in SW620 cells. Anti-EpCAM mAb treatment increased the expression of anti-apoptotic proteins such as Bcl-2, but the expression of pro-apoptotic proteins Bax, TNF-alpha, caspase-3, cleaved caspase-3, and cleaved caspase-8 were unaltered. We observed that anti-EpCAM mAb significantly inhibited the growth of colon tumors, as determined by a decrease in tumor volume and weight. The expression of anti-apoptotic protein was inhibited by treatment with anti-EpCAM mAb, whereas the expression of pro-apoptotic proteins was increased. These results suggest that GD1a and GM1 were closely related to anticancer effects of anti-EpCAM mAb. In light of these results, further clinical investigation should be conducted on anti-EpCAM mAb to determine its possible chemopreventive and/or therapeutic efficacy against human colon cancer.

Expression of AC133 and EpCAM in human pulmonary adenocarcinoma and isolation of their double positive cells / 第二军医大学学报

Objective: To determine the expression of AC133 and EpCAM in human pulmonary adenocarcinoma by dual immunofluorescent labeling technique and to isolate AC133+ EpCAM+ cells by flow cytometry, so as to provide a basis for further investigation of human pulmonary adenocarcinoma stem cells. Methods: The human lung adenocarcinoma tissues were obtained and subjected to cryosection and dual immunofluorescent staining. AC133+ EpCAM+ cells in human pulmonary adenocarcinoma were identified by using laser confocal microscopy. The fresh adenocarcinoma tissues were prepared into single cell suspension with the collagen and red blood cell removed. AC133 and EpCAM were used to label cells and the AC133+ EpCAM+ cells were isolated by flow cytometry. ResuIts:AC133+ EpCAM + cells were found in human pulmonary adenocarcinoma and they could be isolated by flow cytometry. Conclusion:The existence of AC133+ EpCAM+ cells has been confirmed in the lung adenocarcinoma tissues; the double positive cells can be isolated by flow cytometry, which provides a basis for further investigation of lung cancer stem cells.

Biological Characteristics of Human Colon Cancer Cell Line CW-2 Stem Cells / 中国医科大学学报

Objective To isolate cancer stem cells from human colon cancer cell line CW-2,and observe the biological characteristics.Methods The percentage of CD44＋EpCAM＋ cancer stem cells which were isolated by multi-combination method was evaluated by flow-cytometry.The biological characteristics of the stem cells were analyzed by cell cycle analysis,in vitro invasion assay and in vivo tumorigenicity assay.Results The percentage of CD44＋EpCAM＋ cancer stem cells was 89.57%,and their capabilities of generation,invasion,tumorigenicity were higher than non-cancer stem cells（P 0.05）.Conclusion CD44＋EpCAM＋ cancer stem cells showed low generation,high invasiveness,high tumorigenesis,and they could be isolated by multi-combination.