Expression of CD24 gene in human malignant pleural mesothelioma and its relationship with prognosis / 中华劳动卫生职业病杂志

Objective: To investigate the expression of CD24 gene in human malignant pleural mesothelioma (MPM) cells and tissues, and evaluate its relationship with clinicopathological characteristics and clinical prognosis of MPM patients. Methods: In February 2021, UALCAN database was used to analyze the correlation between CD24 gene expression and clinicopathological characteristics in 87 cases of MPM patients. The TIMER 2.0 platform was used to explore the relationship between the expression of CD24 in MPM and tumor immune infiltrating cells. cBioportal online tool was used to analyze the correlation between CD24 and MPM tumor marker gene expression. RT-qPCR was used to analyze the expressions of CD24 gene in human normal pleural mesothelial cell lines LP9 and MPM cell lines NCI-H28 (epithelial type), NCI-H2052 (sarcoma type), and NCI-H2452 (biphasic mixed type). RT-qPCR was performed to detect the expressions of CD24 gene in 18 cases of MPM tissues and matched normal pleural tissues. The expression difference of CD24 protein in normal mesothelial tissue and MPM tissue was analyzed by immunohistochemistry. A Kaplan-Meier model was constructed to explore the influence of CD24 gene expression on the prognosis of MPM patients, and Cox regression analysis of prognostic factors in MPM patients was performed. Results: The CD24 gene expression without TP53 mutation MPM patients was significantly higher than that of patients in TP53 mutation (P<0.05). The expression of CD24 gene in MPM was positively correlated with B cells (r(s)=0.37, P<0.001). The expression of CD24 gene had a positive correlation with the expressions of thrombospondin 2 (THBS2) (r(s)=0.26, P<0.05), and had a negative correlation with the expression of epidermal growth factor containing fibulin like extracellular matrix protein 1 (EFEMP1), mesothelin (MSLN) and calbindin 2 (CALB2) (r(s)=-0.31, -0.52, -0.43, P<0.05). RT-qPCR showed that the expression level of CD24 gene in MPM cells (NCI-H28, NCI-H2052 and NCI-H2452) was significantly higher than that in normal pleural mesothelial LP9 cells. The expression level of CD24 gene in MPM tissues was significantly higher than that in matched normal pleural tissues (P<0.05). Immunohistochemistry showed that the expressions of CD24 protein in epithelial and sarcoma MPM tissues were higher than those of matched normal pleural tissues. Compared with low expression of CD24 gene, MPM patients with high expression of CD24 gene had lower overall survival (HR=2.100, 95%CI: 1.336-3.424, P<0.05) and disease-free survival (HR=1.800, 95%CI: 1.026-2.625, P<0.05). Cox multivariate analysis showed that compared with the biphasic mixed type, the epithelial type was a protective factor for the prognosis of MPM patients (HR=0.321, 95%CI: 0.172-0.623, P<0.001). Compared with low expression of CD24 gene, high expression of CD24 gene was an independent risk factor for the prognosis of MPM patients (HR=2.412, 95%CI: 1.291-4.492, P=0.006) . Conclusion: CD24 gene and protein are highly expressed in MPM tissues, and the high expression of CD24 gene suggests poor prognosis in MPM patients.

Biological Functions of CD24 in Malignant Tumor and its Application as a Novel Anti-tumor Target / 中国生物化学与分子生物学报

CD24 is a highly glycosylated protein that is linked to the plasma membrane via a glycosylphosphatidylinositol anchor. As a universally expressed protein on immune cells, CD24 is also overexpressed in nearly 70% of human cancers including hepatocellular carcinoma, lung cancer and bladder cancer et al. Studies revealed that CD24 is involved in regulating cell proliferation, migration and invasion in cancer cells by interacting with P-selectin, activating Wnt and MAPK signaling pathway or other signaling molecules. Therefore, CD24-targeted siRNA or antibody has a great potential to exert anti-tumor effects by blocking the interaction. There are currently several agents or regiments targeting CD24 for the treatment of patients with various kinds of cancers that are undergoing assessment in the preclinical study at present. Recent studies revealed that CD24 was able to interact with the inhibitory receptor sialic-acid-binding Ig-like lectin 10 (Siglec-10), which located on the surface of macrophages, to compose a novel immune checkpoint. The binding of CD24 to Siglec-10 elicits an inhibitory signaling cascade, limits macrophage phagocytosis, evades immune surveillance, and promotes tumor growth, which suggested that CD24 may be a potential target in anti-tumor immunotherapy. In this review, we introduced the structure and function of CD24 and its role in cancer progression and anti-tumor immunity. Moreover, the progression in developing novel anti-cancer drugs or treatment strategies with the target of CD24 was summarized, which aims to provide a new insight in CD24-targeting therapy.

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.

Expression and clinical significance of CD24 in preoperative peripheral blood and cancerous tissues of hepatocellular carcinoma patients undergoing liver transplantation / 中华器官移植杂志

Objective:To explore the relationship between CD24 expression in preoperative peripheral blood as well as cancer tissue and clinical parameters and prognosis in patients with hepatocellular carcinoma (HCC) after liver transplantation (LT).Methods:From November 2018 to November 2019, clinical data were collected for 65 HCC patients and 41 patients with benign liver disease.The preoperative peripheral blood level of CD24 was detected by enzyme-linked immunosorbent assay (ELISA) and the expression of CD24 in cancerous foci and adjacent tissues examined by immunohistochemistry.Kaplan-Meier survival curves of differential CD24 expression were plotted and survival differences compared by Log-rank method.One-way ANOVA was utilized for examining the relationship between the expression level of CD24 and various clinicopathological parameters and multivariate Cox analysis for screening independent risk factors affecting patient prognosis.Results:The concentration of CD24 in preoperative peripheral blood (p-CD24) of HCC patients (6.51±2.33 μg/L) was significantly higher than that of patients with benign liver disease (4.10±0.91) μg/L, P<0.05.The positive rate of CD24 was obviously higher in cancerous tissues than that in adjacent tissues (87.7% vs. 4.6%, P<0.05). The peripheral blood level of CD24 was positively correlated with the expression intensity of CD24 in tumor tissues (t-CD24, r=0.570, P<0.001). The expression of CD24 (both in blood and cancer foci) was significantly correlated with preoperative level of gamma-glutamyl transferase (GGT), maximal tumor diameter, microvascular invasion, portal vein tumor thrombus, vessel carcinoma embolus and satellite focus ( P<0.05). The expression of CD24 in patients exceeding the Milan/UCSF criteria was higher than those fulfilling the criteria ( P<0.005). Patients with a higher expression of CD24 had worse overall survival and recurrence-free survival rates as compared to those a lower expression of CD24 ( P<0.05). Multivariate Cox analysis indicated that t-CD24 [OS: HR=3.661(1.005-13.333)], P=0.049; recurrence-free survival (RFS): [HR=4.331(1.887-9.942), P=0.001] and preoperative level of alpha fetoprotein (AFP) [OS: HR=4.900(1.590-15.097), P=0.006]; RFS: [HR=3.414(1.614-7.221), P=0.001] were independent risk factors for overall survival and recurrence-free survival in HCC patients undergoing LT. Conclusions:The preoperative peripheral blood level of CD24 in HCC patients undergoing LT indirectly reflects the expression of CD24 in cancerous tissues to a certain extent.And the expression of CD24 in cancerous tissue is one of the independent risk factors affecting OS and RFS of LT patients.

Specific inhibition of Notch1 signaling suppresses properties of lung cancer stem cells

Objective: Lung cancer is the leading cause of cancer-related death worldwide with a relatively low 5-year relative survival rate of 16%. Novel and efficient therapeutic approach for lung cancer is desperately needed. Materials and Methods: Targeting cancer stem cells (CSCs) provides a promising strategy to eradicate malignancies. The Notch signaling pathway plays an important role in the control of cell fates and developmental processes including CSCs. The function of Notch1 in the regulation of CSCs and whether targeting Notch1 could be a potential therapy for lung cancer were explored in this study. Lung CSCs (LCSCs) were isolated from A549 cells and identified as CD44+/CD24– cells by magnetic-assisted cell sorting, then the putative LCSCs were treated with Notch1 inhibitor and Notch1 small interfering RNAs (siRNAs); the growth and proliferation of LCSCs were investigated to test the effect of Notch1 blocking on the growth and viability of LCSCs. Results: CD44+/CD24– cells isolated from A549 cells possessed stem cell-like properties with high expression of Notch1. Blocking Notch1 by inhibitor DAPT or siRNA both inhibited the growth capacity of LCSCs. Conclusion: Our discovery demonstrated a depression of growth in CD44+/CD24– and A549 cells caused by blockade of Notch signaling pathway. Further studies are needed to demonstrate the detailed effects of Notch1 blocking on the LCSCs. Nevertheless, targeting the Notch pathway has exhibited great potential to be an improved lung cancer treatment

Expression of CD24 in prostate cancer tissues and its clinical significance / 中国肿瘤生物治疗杂志

@#Objective: To investigate the expression of CD24 in prostate cancer (PC) tissues, and explore its relationship with clinicopathological features of PC patients. Methods:Atotal of 40 cases of PC tissues and 36 cases of corresponding para-cacerous tissues resected during surgery at the Department of Urology Surgery, Quanzhou First HospitalAffiliated to Fujian Medical University from February 2016 to March 2019 were collected for this study; in addition, 46 cases of benign prostatic hyperplasia tissues were collected from patients underwent TURP surgery. Flow cytometry was used to detect the expression of CD24 in above mentioned tissues; One-way analysis of variance was used to analyze the relationship between the expression of CD24 and the age, tumor distribution, preoperative serum PSA, postoperative Gleason score, clinical stage and distant metastasis of PC patients. Results: The positive expression rate and MFI (mean fluorensece intensity) value of CD24 in prostate cancer tissues were significantly higher than those in para-cancerous prostate tissues and benign prostatic hyperplasia tissues (all P<0.05); CD24 positive expression rate and MFI value in PC tissues of patients with preoperative serum PSA≥10 ng/ml, postoperative Gleason score≥8 (low differentiation), clinical stage of T4 and distant metastasis were significantly higher than corresponding control group (all P<0.05); The expression of CD24 gradually increased with the progression of postoperative Gleason score and clinical stage ( P <0.05). Conclusions: The expression of CD24 is increased in prostate cancer tissues. The detection of CD24 expression level can help to determine the occurrence, development, invasion and metastasis of prostate cancer, and has potential clinical application value.

Stem cell analysis with intraductally treated mammary epithelial cells

PURPOSE: The activity of mammary stem cells (MaSCs) is essential to mammary growth, differentiation and regeneration in cycles of pregnancy, lactation, and involution. The capability to recruit the mammary gland through the cycles is attributed to stem cells. It was shown that the intraductal (i.duc) injection of pegylated liposomal doxorubicin (PLD) to multiparous FVB/N mice was associated with a significantly reduced outgrowth potential of mammary gland cells. We have explored i.duc PLD's effect on stem cell number and function in mouse mammary gland and aldehyde dehydrogenase (ALDH)'s availability as a mouse MaSC marker.METHODS: The total mammary epithelium was purified from 6 to 8-month-old FVB/N control and i.duc PLD-administered mice treated twice and analyzed by flow cytometry and limiting dilution cleared mammary fat pad transplants.RESULTS: There was no significant difference in the proportions of stem cell-enriched population (CD49(fhigh)CD24(med)) between control and i.duc PLD-treated groups. However, we found a significant reduction in the outgrowth potential of CD49(fhigh)CD24(med) and CD49(fhigh)CD24(med)ALDH(+) cells from i.duc PLD-treated mammary glands. We discovered that adding ALDH to CD49(fhigh)CD24(med) had the possibility of better marker selection for MaSC of mice.CONCLUSION: We present i.duc administration of PLD to reduce MaSC function, but not the number; and ALDH activity may add further selection of MaSCs to CD49f CD24 in mouse mammary glands. Screening of chemotherapeutic drugs or other natural products by this method of stem cell analysis may provide safe i.duc treatment in breast cancer.

CD24 regulates Ly6Chi macrophages and BDL-induced liver fibrosis / 中华微生物学和免疫学杂志

Objective To investigate the regulatory effects of CD24 on Ly6Chi macrophages in liv-er and its influences on bile duct ligation ( BDL)-induced hepatic fibrosis in mice. Methods Liver fibrosis was induced in wild-type ( WT) and CD24-/- mice by surgical ligation of the biliary duct. Levels of alanine amino transferase ( ALT) in serum were detected and liver sections were stained with haematoxylin and eosin ( H&E) staining to assess the severity of liver injury. Sirius Red staining was used to observe the degree of liver fibrosis. Real-time PCR was performed for detecting the expression of hepatic fibrosis-related markers and TGF-β1 at mRNA level. The percentage of macrophages and the number of TGF-β1-producing macro-phages were measured by flow cytometry. Results BDL-induced liver fibrosis was exacerbated in CD24-/-mice than in WT mice as demonstrated by more serious hyperplasia in bile duct, more inflammatory infiltra-tion at the portal area and higher levels of ALT in serum. Results of Sirius red staining also showed that the liver fibrosis was more severe in CD24-/- mice than in WT mice. Moreover, α-SMA and collagen typeⅠalpha 1 (Col1a1) were significantly upregulated in CD24-/- mice. Flow cytometry analysis revealed that CD24 was highly expressed by hepatic macrophages in BDL-induced WT mice, and the percentages of hepat-ic macrophages were significantly elevated in CD24-/-mice compared with those in WT mice. Further analy-sis revealed that the percentages of Ly6Chi hepatic macrophages in CD24-/- mice were higher than those in WT mice, but there was no significant difference in the percentages of Ly6Clo macrophages. The expression of TGF-β1 at mRNA level was increased in CD24-/-mice as compared with that in WT mice after BDL. Mo-reover, intracellular staining showed that Ly6Chi hepatic macrophages in CD24-/- mice secreted more TGF-β1 than the macrophages in WT mice. Conclusions CD24 might attenuate the BDL-induced liver fibrosis in mice via regulating the percentage of hepatic Ly6Chi macrophages and the secretion of TGF-β1.

HER2-expressing breast tumors are associated with breast cancer stem-cell phenotype CD44+/CD24- / Tumores de mama que superexpressam HER2 estão associados ao fenótipo de células-tronco tumorais CD44+/CD24-

ABSTRACT INTRODUCTION: According to the cancer stem-cell theory, tumors originate from a small population of cancer stem cells, which lose the mechanism of self-regulation and begin to differentiate and proliferate indefinitely. The CD44+/CD24- phenotype may be considered a stem-cell marker in breast cancer. OBJECTIVE: To evaluate the correlation between CD44+/CD24- phenotype and different molecular subtypes of breast cancer in invasive ductal carcinoma samples. METHODS: The expression of CD44, CD44v6, and CD24 markers was investigated in 133 cases of invasive mammary carcinoma with immunohistochemistry. CD44+/CD24- phenotype was identified and correlated with the molecular subtypes and classical prognostic factors such as age, histological grade, tumor size, and lymph node status. RESULTS: Eighteen (14%) cases were positive for CD44+/CD24- (CD44+/CD24- or CD44v6+/CD24-) phenotype; among these, 11.1%, 27.8%, 38.9%, and 22.2% were luminal, luminal B-human epidermal growth factor receptor 2 (HER2), HER2, and triple-negative subtype, respectively. CD44+/ CD24- phenotype was more common in HER2 subgroup (p = 0.0197). CONCLUSION: CD44+/CD24- phenotype was correlated with molecular subtypes of breast cancer. The highest expression of CD44+/CD24- phenotype was reported in patients with HER2+ disease, a molecular subtype associated with more aggressive behavior and worse prognosis.

RESUMO INTRODUÇÃO: De acordo com a teoria das células-tronco tumorais, os tumores são originários de uma pequena população de células-tronco que perdem o mecanismo de autorregulação e começam a se diferenciar e proliferar indefinidamente. O fenótipo CD44+/CD24- pode ser considerado um marcador de células-tronco tumorais no câncer de mama. OBJETIVO: Avaliar a correlação entre o fenótipo CD44+/CD24- e os diferentes subtipos moleculares do câncer de mama em amostras de carcinoma ductal invasor. MÉTODOS: A expressão dos marcadores CD44, CD44v6 e CD24 foi investigada em 133 casos de carcinoma mamário invasor por meio de imuno-histoquímica. O fenótipo CD44+/CD24- foi identificado e correlacionado com os subtipos moleculares e os fatores prognósticos clássicos, como idade, grau histológico, tamanho do tumor e status do linfonodo. RESULTADOS: Dezoito (14%) casos foram positivos para o fenótipo CD44+/CD24- (CD44+/CD24- ou CD44v6+/CD24-), sendo 11, 1%, 27, 8%, 38, 9% e 22, 2% dos subtipos luminal, luminal B-human epidermal growth factor receptor 2 (HER2), HER2 e triplo negativo, respectivamente. O fenótipo CD44+/CD24- foi mais comum no subgrupo HER2 (p = 0, 0197). CONCLUSÃO: O fenótipo CD44+/CD24- foi correlacionado com os subtipos moleculares do câncer de mama. A maior expressão do fenótipo CD44+/CD24- foi encontrada em pacientes com doença HER2+, subtipo molecular associado a um comportamento mais agressivo e a um pior prognóstico.

High Mobility Group Box 1/ CD 24 Receptor/β-EP Signaling in "Zusanli" (ST 36) Region Contributes to Electroacupuncture Analgesia in Rats with Neuropathic Pain / 针刺研究

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of high mobility group box 1 (HMGB 1) and its receptor CD 24 proteins and β-endorphin (β-EP) content in "Zusanli" (ST 36) region in rats with chronic constriction injury (CCI), so as to explore its mechanisms underlying pain relief. METHODS: Thirty male Wistar rats were rando-mized into control, CCI model and EA groups (n＝ 10 rats in each). The neuropathic pain model was established by ligature of the left sciatic nerve to induce CCI in the model and EA groups, and sham operation was performed in rats of the control group. Paw with drawal latency (PWL, thermal pain threshold) of the bilateral hind-limbs was detected by using an algesia-detector. Eight days after CCI operation, EA was applied to bilateral "Zusanli" (ST 36) and "Yanglingquan" (GB 34) for 30 min, once daily for 5 days. The acetylated-HMGB 1 expression was determined by immunoprecipitation, and the expression of HMGB 1 and toll like receptor 4 (TLR 4) proteins and CD 24 mRNA were detected using Western blot and fluorescent quantitative real time-PCR, respectively, and the content of β-EP in the acupoint region was assayed by enzyme linked immunosorbent assay (ELISA). Anti-CD 24 neutralizing antibody (200 µL, 100 µg/mL) was injected into ST 36 region once daily for 3 days for verifying the involvement of HMGB 1/CD 24 signaling in EA analgesia. RESULTS: Compared with the control group, the bilateral PWL difference values in the other two groups were significantly increased (P0.05). After local injection of anti-CD 24 antibody, EA-induced increases of β-EP content and reduction of thermal pain threshold were significantly suppressed (P<0.05). CONCLUSION: EA of ST 36 and GB 34 can alleviate neuropathic pain in CCI rats, which is associated with its effects in up-regulating β-EP content, and HMGB 1 protein and CD 24 mRNA expression levels in ST 36 region. The activated HMGB 1/CD 24/β-EP signaling contributes to EA-ST 36 induced analgesia.

CD24 attenuates carbon tetrachloride-induced murine liver fibrosis / 中华微生物学和免疫学杂志

Objective To investigate the effects of CD24 on CCl4-induced murine liver fibrosis and to analyze the possible molecular mechanism.Methods Wild type (WT) and CD24 knockout (CD24-/-) C57BL/6 mice were treated with CCl4 through intraperitoneal injection.Levels of ALT in serum samples were detected and liver tissues were stained with hematoxylin and eosin (HE) to assess liver tissue injury.Sirius Red staining was used to observe liver fibrosis.Real-time PCR was performed to detect the expression of α-SMA (α-smooth muscle actin), Col1a1 (Collagen, typeⅠ, alpha 1), TGF-β1 (transforming growth factor-β1) and CD24 at mRNA level in liver tissues.Western blot was performed to analyze the expression of α-SMA and Col1a1 at protein level.Flow cytometry analysis was used to detect the macrophages in liver tissues.ELISA was used to detect the expression of TGF-β1 in the culture supernatants of M1 and M2 macrophages.Results The expression of CD24 at both mRNA and protein levels were up-regulated in mice with CCl4-induced liver fibrosis.HE staining showed that liver inflammation in CD24-/-mice was more severe than that in WT mice after treated with CCl4.Sirius Red staining of paraffin-embadded liver sections revealed that compared with WT mice, CD24-/-mice presented with more severe liver fibrosis.Moreover, α-SMA and Col1a1, indicators of liver fibrosis, in CD24-/-mice were significantly higher than those in WT mice.Flow cytometry analysis showed that murine hepatic macrophages significantly increased in CD24-/-mice than in WT mice following CCl4 treatment.Real-time PCR analysis also confirmed that significantly enhanced expression of TGF-β1 at mRNA level in liver tissues was observed in CD24-/-mice than in WT mice.TGF-β1 secreted in the culture supernatant of M2 macrophages derived from CD24-/-mice group was more than that of the WT mice group.No significant difference in TGF-β1 secretion in culture supernatant of M1 macrophages was observed between the two groups.Conclusion Taken together, these data suggest that CD24 plays an important role in attenuating CCl4-induced chronic inflammation and hepatic fibrosis in mice.The mechanism of CD24 in alleviating liver fibrosis might be through regulating intrahepatic macrophages, inhibiting the secretion of TGF-β1 by M2 macrophages and suppressing the activation of hepatic stellate cells.

The biological characteristics of adriamycin-resistant breast cancer cell line / 肿瘤

Objective: To investigate biological characteristics of adriamycin (ADR)-resistant breast cancer cell line. Methods: Using MCF-7 cells as parent cell line, an ADR-resistant cell line named MCF-7/ADR was established. The half maximal inhibitory concentration (IC50) values of MCF-7/ADR and MCF-7 cells were detected by resazurin test. The ability of colony formation of MCF-7/ADR and MCF-7 cells was measured by colony formation assay. The percentages of CD44+/CD24- and acetaldehyde dehydrogenase 1 (ALDH1)+ subtypes of cancer stem cells in MCF-7/ADR and MCF-7 cells were detected by FCM and immunofluorescent assay, respectively. The expressions of ALDH1 and smoothened (Smo) and Gli1 proteins in Hedgehog signaling pathway related to modulation of stem cells in MCF-7/ADR and MCF-7 cells were detected by Western blotting. Results: The IC50 value of ADR in MCF-7/ADR cells was higher than that in MCF-7 cells (P < 0.01), the resistant fold was 385. The colony formation rate of MCF-7/ADR was higher than that of MCF-7 cells (P < 0.01). The percentages of CD44+/CD24- and ALDH1+ subtypes of cancer stem cells in MCF-7/ADR cells were higher than those in MCF-7 cells (both P<0.01). The expression levels of ALDH1 protein and the Smo and Gli1 proteins in Hedgehog pathway related to modulation of stem cells in MCF-7/ADR cells were higher than those in MCF-7 cells (P < 0.01, P<0.01 and P<0.05, respectively). Conclusion: The percentages of CD44+/CD24- and ALDH1+ subtypes of cancer stem cells in MCF-7/ADR cells are higher, and the expressions of ALDH1 protein and the Smo and Gli1 proteins in Hedgehog pathway related to modulation of stem cells in MCF-7/ADR cells are higher. This mechanism may be responsible for ADR resistance in breast cancer cells.

Expression,roles and therapy target values of CD24 in oral squamous cell carcinoma / 北京大学学报(医学版)

Objective:To determine the expression profile and potential roles of CD24 in oral squamous cell carcinoma and explore the values of CD24 function as a potential target of clinical therapy.Me-thods:Semi-quantitative immunohistochemistry was used to construct the expression profile of CD24 in 78 human oral tissues and 59 Hamster buccal pouch tissues.Real-time RT-PCR and Western blot were used to analyze the CD24 expression levels in oral DOK4 cells,oral cancer CAL-27 and WSU-HN6 cells. Then these two cancer cell lines were selected to evaluate the effect of all-trans retinoic acid (ATRA)and CD24 antibody on CD24 expression,and the proliferation and tumorsphere formation capacity of these two cell lines.Results:CD24 expression was found significantly elevated in both human and animal tissues compared with normal and benign tissues (P<0.05),as well as in oral cancer CAL-27 and WSU-HN6 cells compared with DOK cells (P<0.05).CAL-27 and WSU-HN6 cells possess increased proliferative and specific tumorsphere formation capability compared with DOK cells (P<0.05 ).Both ATRA and CD24 antibody were able to effectively inhibit the proliferation and tumorsphere formation of CAL-27 and WSU-HN6 cells (P<0.05).Among them ATRA at least involved partially in the proliferation by down-regulating the CD24 expression (P<0.05 ),while CD24 antibody blocking had no effect on the CD24 expression.Conclusion:CD24 was upregulated in oral cancer and functioned as a potential factor that promoted the proliferation and tumorsphere formation of CAL-27 and WSU-HN6 cells.Both ATRA and CD24 antibody might effectively inhibit the proliferation and tumorsphere formation of CAL-27 and WSU-HN6 cells and function as a potential therapy target.

Expression and significance of c-myc and CD24 in colorectal cancer / 国际肿瘤学杂志

Objective To determine the expression of c-myc and CD24 in colorectal carcinoma,colo-rectal polyp and normal mucosa,and to explore the role and correlation of them in the carcinogenesis of colorec-tal carcinoma. Methods The expression of c-myc and CD24 in colorectal carcinoma(n = 60),colorectal ade-nomatous polyp(n = 45),colorectal hyperplastic polyp(n = 15)and the adjacent non-cancerous tissue(n =30)was observed by immunohistochemical assay. Results The positive rate of c-myc in colorectal carcinoma were 73. 3% ,significantly higher than that in colorectal adenomatous polyp 44. 4%( χ2 = 9. 016 8,P <0. 01),colorectal hyperplastic polyp 13. 3%(χ2 = 18. 215 9,P < 0. 01)and adjacent non-cancerous tissue 6. 7%(χ2 = 25. 133 0,P < 0. 01);the positive rate of CD24 in colorectal carcinoma was 76. 7% ,significantly higher than that in colorectal hyperplastic polyp 6. 7%(χ2 = 25. 133 0,P < 0. 01)and adjacent non-cancerous tissue 3. 3%(χ2 = 43. 107 4,P < 0. 01). c-myc expression in colon cancer was significantly correlated with cancer site(χ2 = 8. 352 3,P < 0. 01),lymph node metastasis(χ2 = 4. 275 1,P < 0. 05),differentiation (χ2 = 4. 115 3,P < 0. 05)and TNM stage(χ2 = 5. 739 9,P < 0. 05). CD24 expression in colon cancer was significantly correlated with cancer size(χ2 = 9. 333 6,P < 0. 01),lymph node metastasis(χ2 = 7. 693 0,P <0. 01),differentiation(χ2 = 5. 870 0,P < 0. 05)and TNM stage(χ2 = 4. 498 7,P < 0. 05). There was a pos-itive correlation relationship between CD24 and c-myc in colorectal carcinoma tissue(χ2 = 10. 824 9,r = 0. 39, P < 0. 05). Conclusion The expression of c-myc and CD24 are high in colorectal cancer,having a significant correlation with some of the clinicaopathological features. c-myc is likely to act as a downstream target gene of CD24 signaling pathway,whose expression is probably regulated by CD24 in colorectal carcinoma tissue.

Expression and significance of CD24 in colorectal cancer / 国际肿瘤学杂志

Objective To determine the expression of CD24 in colorectal carcinoma, and to explore the relationship between CD24 and the clinicopathological features of colorectal carcinoma.Methods The expression of CD24 in 62 cases of colorectal carcinoma, 47 cases of adenomas, 15 cases of colorectal polyps and 30 cases of the adjacent non-cancerous tissues were observed by immunohistochemical assay.The relationship between CD24 and the clinicopathological features was analyzed.Results The positive rates of CD24 in colorectal carcinoma 72.6％ and adenomas 63.8％ were significantly higher than those in colorectal hyperplastic polyps 13.3％ (x2 =17.83, P =0.00;x2 =11.61, P =0.00) and adjacent non-cancerous tissues 6.7％ (x2 =35.15, P =0.00;x2 =24.64, P =0.00).The expression of CD24 in colorectal carcinoma had a significant correlation with the tumor diameter (x2 =5.48, P =0.02), tumor differentiation (x2 =8.86, P =0.00), Duke staging (x2 =11.47, P =0.00) and lymph node metastasis (x2 =8.92, P =0.00).Conclusion The expression of CD24 is high in colorectal carcinoma, having a significant correlation with the size of tumor, degree of differentiation, Duke stage and lymph node metastasis.

Enrichment of breast cancer stem cells by serum-free culture / 临床与实验病理学杂志

Purpose To identify whether serum-free culture can enrich breast cancer stem cells from MCF-7 human breast cancer cell line. Methods MCF-7 human breast cancer cell line by serum-free culture and serum culture technology were cultured, its cell mor-phology and growth pattern were observed by the inverted microscope. The expression of stem cell surface molecular makers CD24, CD44 was observed by the inverted fluorescence microscope and the flow cytometry, the proportion of different subpopulation cells was detected by the flow cytometry. At the same time, difference of the cell cycle was detected by the flow cytometry. Results The cell line cultured by serum-free culture grew in the form of suspended microspheres of different sizes in the medium, but the cell line cul-tured by serum culture grew in the form of monolayer adherent growth. There was no obvious difference in the expression of stem cell surface molecular makers CD44 between the suspended microsphere cells and the adherent cells, but the expression of CD24 in the sus-pended microsphere cells decreased compared to the adherent cells. The proportion of CD44 + /CD24 -/low phenotype cells in the suspen-ded microsphere cells and the adherent cells was (86. 93 ± 0. 53)% and (19. 98 ± 0. 62)%, respectively (P<0. 05), the proportion of CD44 + /CD24 + phenotype cells was (12. 68 ± 0. 59)% and (79. 90 ± 0. 57)%, respectively (P<0. 05). The proportion of mitotic cells in the suspended microsphere cells and the adherent cells was ( 18. 85 ± 2. 26 )% and ( 43. 91 ± 1. 81 )%, respectively ( P<0. 05), the proportion of quiescent cells was (64. 92 ± 2. 07)% and (39. 82 ± 1. 77)%, respectively (P<0. 05). Conclusion CD44 + /CD24 -/low breast cancer stem cells can be effectively enriched by the serum-free culture technology.

Study on the chemoresistance of CD44+/CD24+ Siha cells to cisplatin and its mechanisms / 中国癌症杂志

Background and purpose:One of the reasons why cancer cells are resistant to chemotherapy is the existence of cancer stem cells. The purpose of this study was to investigate the chemoresistance of CD44+/CD24+ Siha cells to cisplatin and its mechanisms.Methods:Siha cells were cultivatedin vitro. The CD44+/CD24+ Siha cells were sorted out by fluorescence activated cell sorter (FACS) andin vitro proliferation was detected by MTT assay after treatment with the different concentrations of cisplatin. The cell apoptosis rate was detected by flow cytometry after 10 μg/mL cisplatin acted on CD44+/CD24+ Siha cells for 24, 48 and 72 h. The relative mRNA and protein expressions of Bcl-2, Oct-4 and ABCG2 were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. Results:The survival rates of CD44+/CD24+ Siha cells treated with different concentrations of cisplatin (0.1, 1, 5, 10, 15 and 20 μg/mL) were higher than those of their parental Siha cells [(88.42±1.51)%vs (92.87±1.5)%, (79.94±1.05)%vs (84.72±1.09)%, (69.78±0.81)%vs (75.13±2.86)%, (58.97±0.70)%vs (65.79±2.71)%, (49.60±0.88)%vs (52.10±0.52)%, (45.13±0.69)%vs (48.84±1.02)%,P<0.05]. Compared with their parental Siha cells, the apoptosis rates of CD44+/CD24+ Siha cells were lower after 10 μg/mL of cisplatin acting on them for 24, 48 and 72 h, respectively [(3.05±0.16)%vs (5.17±0.27)%, (17.94±2.02)%vs (32.60±4.28)% and (40.14±3.01)%vs (56.62±5.32)%,P<0.05]. The results from both qRT-PCR and Western blot indicated that Oct-4, ABCG2 and Bcl-2 were highly expressed on CD44+/CD24+ Siha cells. A significant difference was found in Oct-4, ABCG2 and Bcl-2 expression between CD44+/CD24+Siha cells and their parental cells (P=0.015<0.05).Conclusion:CD44+/CD24+ Siha cells could be resistant to apoptosis induced by cisplatin and expressed high levels of cancer stem cell markers such as Oct-4 and ABCG2. This study lays the basis for useful isolation and further targeted therapy of cervical cancer stem cells.

Avaliação da imunomarcação de células-tronco tumorais em carcinossarcomas mamários e carcinomas em tumores mistos em cadelas / Evaluation of immunostaining of tumor stem cells in mammary carcinosarcomas and carcinomas in mixed tumors in bitches

As células-tronco tumorais (CTTs) pertencem a uma pequena população de células dentro do tumor com propriedades de autorrenovação e diferenciação em outros tipos celulares. Neste estudo avaliou-se o comportamento tanto das porções mesenquimais quanto das epiteliais de seis carcinossarcomas (CSs), 11 carcinomas em tumores mistos (CTMs) grau I, 11 grau II e 10 grau III. Nas porções epiteliais dos CS e CTM foram observadas imunomarcações para os anticorpos CD44, CD24, Oct-4 e ALDH-1. Nas porções mesenquimais dos CS, nas porções epiteliais dos CTMs graus II e III não houve imunomarcação para o ALDH-1. Concluiu-se que as CTTs são expressas em proporções iguais tanto nas porções mesenquimais quanto nas epiteliais dos CSs e ausentes nas porções mesenquimais bem diferenciadas de CTMs.

Cancer stem cells belong to a small population of cells within the tumor with properties of self-renewal and differentiation into other cell types. In this study, the behavior of both portions, mesenchymal and epithelial, was evaluated. Six carcinosarcomas (CSs), 11 carcinomas within mixed tumors (CWMTs) grade I, 11 grade II, and 10 grade III were evaluated. In the epithelial portions of the CS and CWMTs was observed immunostaining for antibodies CD44, CD24, Oct-4 and ALDH-1. In the mesenchymal portions of the CS, in the epithelial portions of CMTs grades II and III no immunostaining for ALDH-1 was found. It was concluded that the tumor stem cells are expressed in equal proportions in the epithelial and mesenchymal portions of the CS. No immunostaining in the mesenchymal portions of well-differentiated CWMTs was seen.

The expression and clinical significance among CD44 +/CD24 -,E-cadherin and ivm entin in non-sma ll cell lung canc er / 实用肿瘤学杂志

Objective To detect the expression of CD 44 +/CD24 -and E-cadherin and vimentin in non-small cell lung cancer and its clinical prognostic relevance .Methods By immunohistochemistry , detecting , 500 cases of non-small cell lung cancer E -cadherin ,Vimentin and CD44 +/CD24 -expressions′correlation ,we analyzed the relationship with non -small cell lung cancer .Results In non-small cell lung cancer , vimentin was related to tissue differentiation,TNM staging(P<0.05);there was relationship between CD44 +/CD24 -and EMT by E-cadherin expression(P <0.05);CD44 +/CD24 -expression was related to lymph node metastasis , differentiation(P<0.05);their expressions were not obviously related to age ,sex,smoking history,clinical patho-logical type .The survival time of CD44 +/CD24 -high expression in NSCLC was significantly shorter than lower expression in NSCLC(P<0.05).Conclusion EMT malignant phenomenon raises cancer stem cells ,which can lead to the development of malignant cells poorly differentiated trends ,they are not an isolated process;important role in the development of EMT and CD 44 +/CD24 -union occurs in non -small cell lung cancer and prognosis with patients ,which provides a potential breakthrough in the treatment of cancer prevention and treatment targets .

Identification and function of a monoclonal antibody against pancreatic cancer stem cell / 肿瘤研究与临床

Objective To identify-and study a monoclonal antibody (McAb) against pancreatic cancer stem cell in vitro,as well as to provide candidate antibody-drug for cancer stem cell-targeted therapy of pancreatic cancer.Methods Cell culture in serum-free medium and PKH26 staining were used to determine the existence of cancer stem cell in PANC-1 cell line.Flow cytometry was used to detect the expression of CD24 and CD44 in PANC-1 cells and sphere cells,Immunofluorescence was used to detect the expression of CD24 and antigen recognized by 15D2.The effects of 15D2 on self-renewal,proliferation and chemosensitivity to gemcitabine of PANC-1 parent or sphere cells were identified by serum-free suspension culture and CCK-8 assay,Immunohistochemistry was applied to detect the level of antigen recognized by 15D2 in cancer and adjacent tissues.Results PANC-1 cells could survive,proliferate and form sphere cells in serum-free medium.The sphere-forming rate was (2.5±0.5) ％.The percentage of CD44+ CD24+ cells population in sphere cells increased by 11.4 folds compared to PANC-1 cells,in which single nearly 97 ％ CD24+ cells was CD44+ CD24+ cells.Therefore,CD24+ was selected for cancer stem cell marker in PANC-1 in this study.The two-color immunofluorescence assay showed that 15D2 could recognize cells which was also stained by CD24.In vitro functional experiments demonstrated that 15D2 significantly suppressed the sphere formation of PANC-1 cells,with the inhibitory rate being 30.4 ％.Meanwhile,the combination of 15D2 and gemcitabine can significantly attenuate the growth of PANC-1 sphere cells.The IC50 was 0.10 μmol/L in 15D2+gemeitabine group,and 0.39 μmol/L in mlgM+gemcitabine group,Immunohistochemical results showed that the antigen recognized by 15D2 was greatly expressed in about 76.9 ％ (11/13) human pancreatic cancer tissues and hardly detected in adjacent normal tissues (10.0 ％,1/10).Conclusion McAb 15D2 can inhibit self-renewal and drug-resistance of pancreatic cancer stem cell in PANC-1 cell line,and it might become a candidate drug for target pancreatic cancer stem cell treatment.