Prognostic Role of Hepatoma-derived Growth Factor in Solid Tumors of Eastern Asia: a Systematic Review and Meta-Analysis

Hepatoma-derived growth factor (HDGF) is a novel jack-of-all-trades in cancer. Here we quantify theprognostic impact of this biomarker and assess how consistent is its expression in solid tumors. A comprehensivesearch strategy was used to search relevant literature updated on October 3, 2014 in PubMed, EMBASE andWEB of Science. Correlations between HDGF expression and clinicopathological features or cancer prognosiswas analyzed. All pooled HRs or ORs were derived from random-effects models. Twenty-six studies, primarilyin Eastern Asia, covering 2,803 patients were included in the analysis, all of them published during the pastdecade. We found that HDGF overexpression was significantly associated with overall survival (OS) (HROS=2.35,95%CI=2.04-2.71, p<0.001) and disease free survival (DFS) (HRDFS=2.25, 95%CI =1.81-2.79, p<0.001) in solidtumors, especially in non-small cell lung cancer, hepatocellular carcinoma and cholangiocarcinoma (CCA).Moreover, multivariate survival analysis showed that HDGF overexpression was an independent predictor ofpoor prognosis (HROS=2.41, 95%CI: 2.02-2.81, p<0.001; HRDFS=2.39, 95%CI: 1.77-3.24, p<0.001). In addition,HDGF overexpression was significantly associated with tumor category (T3-4 versus T1-2, OR=2.12, 95%CI:1.17-3.83, p=0.013) and lymph node status (N+ versus N-, OR=2.37, 95%CI: 1.31-4.29, p=0.03) in CCA. This studyprovides a comprehensive examination of the literature available on the association of HDGF overexpressionwith OS, DFS and some clinicopathological features in solid tumors. Meta-analysis results provide evidence thatHDGF may be a new indicator of poor cancer prognosis. Considering the limitations of the eligible studies, otherlarge-scale prospective trials must be conducted to clarify the prognostic value of HDGF in predicting cancersurvival.     

Basic fibroblast growth factor induces cholangiocarcinoma cell migration via activation of the MEK1/2 pathway

The purpose of this study was to investigate the roles played by basic fibroblast growth factor (bFGF) in the induction of cholangiocarcinoma cell progression and to identify the signal transduction molecules that are activated by bFGF in cholangiocarcinoma cells. FGF receptor-2 (FGFR2) was shown to be expressed in two cholangiocarcinoma cell lines (RMCCA1 and KKU-100). Samples from RMCCA1 and KKU-100 were assayed for the mRNA. Phosphorylation levels were determined by Western blotting. Treatment of the cholangiocarcinoma cells with bFGF enhanced signaling via the phosphorylation of MEK1/2, induced cholangiocarcinoma cell migration and resulted in high levels of actin polymerization. Moreover, treatment with a MEK1/2 inhibitor (U0126) attenuated the effect of bFGF-induced cholangiocarcinoma cell migration. Taken together, these observations indicate that bFGF enhances the migration of cholangiocarcinoma cells and that this enhancement is regulated by the phosphorylation of MEK1/2.     

肺癌细胞外泌体miR-24-3p调控CD8+T细胞抗肿瘤的功能及机制研究

目的:探究肺癌细胞外泌体miR-24-3p调控CD8+T细胞抗肿瘤的功能及机制。方法:将16HBE exo、A549 exo、H522 exo、H460 exo、miR-NC exo、miR-24-3p exo与CD8+T细胞共孵育（16HBE exo组、A549 exo组、H522 exo组、H460 exo组、miR-NC exo组、miR-24-3p exo组），与PBS共孵育组（PBS组）作为对照，CD8+T与miR-24-3p exo共孵育后转染FGF11质粒（miR-24-3p exo+FGF11组）。CCK8法检测CD8+T细胞的增殖能力。双荧光素酶报告基因实验验证miR-24-3p和FGF11的靶向关系。将各组CD8+T细胞与A549细胞以20∶1的比例共孵育4 h（16HBE exo/CD8+T组、A549 exo/CD8+T组、H522 exo/CD8+T组、H460 exo/CD8+T组、miR-NC exo/CD8+T组、miR-24-3p exo/CD8+T组、PBS/CD8+T组、miR-24-3p exo+FGF11/CD8+T组），Elisa检测共孵育后细胞上清中INF-γ和IL-2的浓度，LDH法检测CD8+T细胞对A549细胞的杀伤能力。结果:相比于PBS组，A549 exo组、H522 exo组、H460 exo组CD8+T细胞增殖能力显著降低（P＜0.05）。A549 exo/CD8+T组、H522 exo/CD8+T组、H460 exo/CD8+T组细胞上清中INF-γ和IL-2的浓度显著下降（P＜0.001），CD8+T细胞杀伤能力亦显著下降（P＜0.001）。双荧光素报告基因检测结果显示FGF11为miR-24-3p的靶基因。miR-24-3p exo组CD8+T细胞增殖能力显著低于miR-NC exo组，miR-24-3p exo+FGF11组CD8+T细胞增殖能力显著高于miR-24-3p exo组（P＜0.05）。miR-24-3p exo/CD8+T组细胞上清中INF-γ和IL-2浓度及CD8+T细胞杀伤能力均显著低于miR-NC exo/CD8+T组（P＜0.001）， miR-24-3p exo+FGF11/CD8+T组细胞上清中INF-γ和IL-2浓度及CD8+T细胞杀伤能力显著高于miR-24-3p exo/CD8+T组（P＜0.001）。结论:肺癌细胞外泌体miR-24-3p可通过靶向抑制FGF11而抑制CD8+T细胞的抗肿瘤功能。     

Identification of CXCL5/ENA-78 as a factor involved in the interaction between cholangiocarcinoma cells and cancer-associated fibroblasts

Knowledge of tumor-stromal interactions is essential for understanding tumor development. We focused on the interaction between cholangiocarcinoma and cancer-associated fibroblasts (CAFs) in intrahepatic cholangiocarcinoma and reported their positive interaction in vitro and in vivo. The aim of this study is to identify the key protein involved in the interaction between cholangiocarcinoma cells and CAFs and its role on cholangiocarcinoma progression. Using the conditioning medium from cholangiocarcinoma cells, hepatic stellate cells and coculture of them, Protein-Chip analysis with SELDI-TOF-MS showed that the peak of an 8,360-Da protein remarkably increased in the coculture medium. This protein was identified as CXCL5/ENA78, epithelial cell-derived neutrophil-activating peptide-78, by q-TOF/MS/MS analysis. Two cholangiocarcinoma cell lines, HuCCT1 and RBE, produced CXCL5 that promoted their invasion and migration in an autocrine fashion. These effects of CXCL5 significantly decreased by inhibition of CXC-receptor 2, which is the receptor for CXCL5. In addition, IL-1β produced by hepatic stellate cells induced the expression of CXCL5 in cholangiocarcinoma cells. In human tissue samples, a significant correlation was observed between CAFs and CXCL5 produced by cholangiocarcinoma cells in intrahepatic cholangiocarcinoma (p = 0.0044). Furthermore, the high-CXCL5-expression group exhibited poor overall survival after curative hepatic resection (p = 0.027). The presence of tumor-infiltrating neutrophils expressing CD66b was associated with CXCL5 expression in tumor cells (p < 0.0001). These data suggest that CXCL5 is important for the interaction between cholangiocarcinoma and CAFs, and inhibition of tumor-stromal interactions may be a useful therapeutic approach for cholangiocarcinoma.     

The upregulation of programmed death 1 on peripheral blood T cells of glioma is correlated with disease progression

Glioma is the most common primary brain tumor. Programmed death 1 (PD-1) is a surface receptor expressed on activated and exhausted T cells, which mediate T cell inhibition upon binding with its ligand. In the current study, we investigated the expression of PD-1 on peripheral CD4+ and CD8+ T cells in glioma patients. Percentage of PD-1+ cells was measured by flow cytometry in 86 glioma cases and 62 healthy controls. Results showed that PD-1 expression was significantly increased in both peripheral CD4+ and CD8+ T cells in glioma (p?<?0.001 and p?<?0.001, respectively). When comparing PD-1 level in glioma patients with different histological types, patients with astrocytomas revealed clearly higher proportion of PD-1 on CD4+ T cells than those with oligodendrogliomas (p?<?0.001), ependymomas (p?<?0.001), or pilocytic astrocytomas (p?<?0.001). Also, patients with the highest tumor grade (IV) demonstrated the most elevated expression of PD-1 on both CD4+ and CD8+ T cells. Interestingly, cases with tumor grade III and IV had downregulated PD-1 level on peripheral CD4+ T cells after surgery, whereas only grade IV patients showed decreased proportion of PD-1 on CD8+ T cells after treatment. In addition, no correlation between PD-1 expression and progression to secondary glioblastoma was observed. These data suggested PD-1 may act as a positive regulator in the pathogenesis and progression of glioma.     

miR-497-5p靶向调控CCNE1基因抑制胰腺癌细胞增殖

目的 明确miR-497-5p在胰腺癌（PaCa）中的表达及临床意义,并探究其对PaCa细胞增殖的影响及机制。方法 实时荧光定量PCR实验检测miR-497-5p的表达,卡方检验和Kaplan-Meier生存法分析miR-497-5p的表达与临床病理特征及预后的关系;CCK-8实验和流式细胞术检测过表达miR-497-5p对Capan-2和PANC-1细胞增殖和周期的影响,Spearman相关性检验分析miR-497-5p表达与G₁/S特异性细胞周期蛋白E1（cyclin E1, CCNE1）mRNA表达的关系;双荧光素酶报告基因实验和蛋白质印迹法验证miR-497-5p对CCNE1表达的调控作用。结果 miR-497-5p在癌组织中的表达显著低于癌旁正常组织（P<0.001）,T3+T4期患者癌组织中miR-497-5p的表达显著低于T1+T2期癌组织（P<0.001）;低表达miR-497-5p 与较高的T分期相关（P=0.003）;低表达miR-497-5p的患者5年总体生存率显著低于高表达者（P=0.036）。与对照组相比,miR-497-5p过表达组细胞增殖显著降低,G₀/G₁期比例增加,S期比例减少。CCNE1 mRNA在癌组织的表达显著高于癌旁正常组织（P<0.001）,且与miR-497-5p表达呈负相关（P<0.001）。miR-497-5p可直接与CCNE1 mRNA 3’-UTR互补结合从而抑制CCNE1蛋白的表达。结论 miR-497-5p在PaCa中低表达,且与较高的T分期及不良预后相关;过表达miR-497-5p通过靶向调控CCNE1基因诱导细胞周期阻滞进而抑制PaCa细胞增殖。     

Impact of CD44+CD24- cells on non-sentinel axillary lymph node metastases in sentinel node-positive breast cancer

Although complete axillary lymph node dissection (ALND) is the standard for evaluating axillary status after the identification of a positive sentinel lymph node (SLN) in breast cancer; approximately 40-60% of SLN-positive patients have negative non-SLN. In this study, to explore putative breast cancer stem cells with CD44+CD24- in the SLN, we retrospectively analyzed the expression of CD44+CD24- on metastatic tumor cells within SLNs as a predictive factor for positive non-SLNs (NSLNs). We tested 271 patients for SLNs using serial sectioning with cytokeratin immunohistochemistry (IHC) and hematoxylin-eosin staining and identified 67 patients who had a positive SLN biopsy and complete ALND. CD44 and CD24 expression was detected using double-staining IHC. Twenty-eight (41.8%) out of 67 patients had positive NSLN metastases. Seven positive SLNs with micrometastases were not available for the evaluation of CD24 and CD44 expression. Out of the remaining 60?patients, 19?(31.7%), 44?(73.83%) and 37?(61.7%) patients had CD24+, CD44+ and CD44+CD24- metastatic tumor cells in SLNs, respectively. Positive NSLN metastasis was significantly associated with the primary tumor size (P=0.004), CD24- expression (P=0.04), CD44+ expression (P=0.01) and CD44+CD24- expression (P=0.02). This report provides the first evidence of the existence of a putative stem-like phenotype within the SLN, which is significantly associated with positive NSLN in early breast cancer patients.     

CD44 expression and its relationship with MMP-9, clinicopathological factors and survival in oral squamous cell carcinoma

We investigated the expression of CD44 and MMP-9 in primary oral squamous cell carcinoma (OSCC) and evaluated their association with each other and clinicopathological factors as well as their prognostic value during long term follow up. Histological samples from 138 OSCC patients were immunohistochemically stained for the expression of CD44 and MMP-9. The staining results were compared with conventional prognostic factors and their impacts to patients' prognosis were also studied with survival analyses. Irregular staining of CD44 in tumour cells was associated with poor tumour differentiation (p=0.003), higher clinical stage (III-IV) (p=0.049), and the presence of T3-4 tumour stage (p=0.03). Strong stromal MMP-9 staining intensity was correlated with poor tumour differentiation (p=0.03). In univariate survival analysis irregular staining of CD44 in tumour cells was related to poor disease free and overall survival (p=0.001 and p<0.001, respectively). In multivariate analysis CD44 staining was a significant independent predictor for overall (p=0.03) and disease free survival (p=0.003). MMP-9 expression showed no statistical significance in survival analyses. Strong stromal staining intensity of MMP-9 correlated with irregular staining of CD44 in tumour cells, but had no prognostic significance in the present cohort. However, irregular staining of CD44 predicted more advanced disease and shortened survival of the patients.     

Defective mitogen-induced cellular cytotoxicity in myelodysplastic syndromes. Recovery after alpha-interferon administration

Peripheral blood mitogen - induced cellular cytotoxicity (MICC) and natural killer- cell cytotoxicity (NKCC) were assessed in 25 patients with myelodysplastic syndromes (MDS). Both MICC and NKCC were examined under the same experimental conditions using the 18 hr chromium release assay, except that cultures for MICC were stimulated in vitro by the addition of phytohemagglutinin (PHA). Patients' MICC was found significantly reduced, in relation to controls (p less than 0.001), but significantly higher than patients' NKCC (p less than 0.001). Furthermore, patients CD3+ cells and CD4+ cells, as well as the CD4+/CD8+ ratio, were significantly decreased (p less than 0.01, p less than 0.001 and p less than 0.001, respectively), while CD8+ cells and CD16+ cells were within normal limits. No relationship was noted between patients' MICC and total lymphocyte count or any lymphocyte subpopulation. In eleven patients who were subsequently subjected to a-interferon (a-IFN) administration, MICC values were found within normal range one month after the cessation of alpha-IFN, while NKCC values were significantly increased (p less than 0.01), but they still remained below the lower limit of the control (p less than 0.001). Percentages of CD3+, CD4+ and CD8+ cells, as well as the CD4+/CD8+ ratio, did not change after alpha-IFN, but the absolute numbers of CD3+ cells and CD8+ cells were significantly reduced. A statistically significant rise was noted in CD16+ cells. Post- IFN rises in MICC did not correlate with lymphocyte subpopulations. The findings indicate that MDS patients display very low MICC, which can be restored by alpha-IFN administration. The cause of this disturbance and the mechanism of its restoration by alpha-IFN remain unclear.     

A CD44?/CD24+ phenotype is a poor prognostic marker in early invasive breast cancer

A CD44(-)/CD24(+) phenotype is a poor prognostic marker in early invasive breast cancer. Breast cancer cells with high CD44 and low or absent CD24 (i.e. CD44(+)CD24(-)/low phenotype) are reported to have stem cell features. However, the clinical impact of CD24 and CD44 expression in tumours remains unclear. To explore the immunohistochemical expression of CD44 and CD24 (individually and combined) and their clinical value as prognostic and predictive markers. Immunohistochemical expression of CD24 and CD44 was studied in a large series of early primary invasive breast cancer tumours (n = 1036) prepared as a tissue microarray. Associations between the expression of each marker individually and in combination and clinico-pathological, molecular variables and patients' outcome were investigated. CD24 cytoplasmic expression was significantly associated with poor prognostic variables including high tumour grade, ER-, PR-, HER2(+), p53+ and triple negative (TN) phenotype; P < 0.05. However, CD24 expression was not significantly associated with patients' outcome. Conversely, CD44 expression was associated with favourable prognostic criteria including lower Nottingham prognostic index, ER+, HER2- and luminal phenotype; P < 0.05. Moreover, CD44 expression was found to be an independent predictor of good prognosis. In combination, the CD44(+)/CD24(-) phenotype was associated with the most favourable outcome (84 and 80% 10 year breast cancer survival [BCSS] and metastasis free survival [MFS], respectively). Contrasting this, the CD44(-)/CD24(+) phenotype was associated with the most dismal outcome (62 and 60% 10 years BCSS and MFS, respectively). CD24 and CD44 expression can individually yield prognostic data in breast cancer, but importantly, when both markers are considered; the CD44(+)/CD24(-) phenotype had the best prognosis, while the CD44(-)/CD24(+) phenotype had the worst prognosis. This shows that the relationship between basic cell biology and clinical behaviour is not always straightforward and warrants further investigations of the true clinical impact of breast cancer stem cells.     

Evaluation of expression of CD15 and sCD15 in non-small cell lung cancer

Changes in cell membrane carbohydrate antigens play an important role in metastatic potential associated with carcinogenesis and in prognostic factors. We investigated immunohistochemically the expression of CD15 and sialyl CD15 (sCD15) in lung cancer tissue by using Leu-M1 antibody and MXKM-93 antibody, respectively, and then assessed the relationship between their expression and the patient outcome. Lung cancer tissue expression of CD15 was significantly higher in adenocarcinoma (55.9%) and squamous cell carcinoma (44.7%) than in small cell carcinoma (10%) (p=0.01, p=0.006). Expression of sCD15 was significantly higher in adenocarcinoma (52.9%) than in squamous cell carcinoma (10.5%) or small cell carcinoma (10%) (p<0. 0001, p=0.016). No association was found between CD15 expression and clinical stage, but sCD15 expression increased with clinical stage (stage I+II vs. III+IV: 16.7% vs. 39.6%; p=0.049). Expression of CD15 (1.5%) was significantly lower than expression of sCD15 (12.3%) in normal surrounding tissue. Examination of associations with outcome in NSCLC revealed that expression of sCD15 in resected cases, and expression of CD15 in non-resected cases were significantly correlated with shortening of median survival time (p<0.05). When associations with prognostic factors were assessed by univariate analysis, expression of sCD15 was found to be correlated with distant metastasis, and expression of CD15 with decrease in performance status (PS). In the multivariate analysis by the Cox proportional hazard model, sCD15 and CD15 negativity contributed to longer survival time after PS and clinical stage. The results of a combination assay of CD15 and sCD15 showed that expression of both carbohydrate antigens significantly shortened survival time in both the resected and non-resected group (log-rank test, p<0.05). This combination assay also appeared to be extremely useful in predicting the outcome in all clinical stages of NSCLC.     

miR-502-3p通过靶向GTPBP2基因调控结直肠癌干细胞增殖与凋亡

目的：探讨miR-502-3p通过靶向GTP结合蛋白2(GTPBP2)调控结直肠癌干细胞（CCSC）增殖、细胞周期和凋亡的分子机制。方法：利用免疫磁珠分选技术从结直肠癌细胞HCT116中分选CCSC(CD133+CD44+双阳性细胞和CD133-CD44-双阴性细胞),用qPCR法检测细胞中miR-502-3p表达水平。利用脂质体转染法分别将miR-NC、miR-502-3p、si-miR-NC、si-miR-502-3p、miR-502-3p+vector和miR-502-3p+GTPBP2转染至CD133+CD44+细胞中,记作miR-NC、miR-502-3p、si-miR-NC、si-miR-502-3p、miR-502-3p+vector和miR-502-3p+GTPBP2组。用qPCR法检测细胞中miR-502-3p、GTPBP2 mRNA表达水平,MTT法、流式细胞术分别检测细胞增殖率、细胞周期和凋亡率,WB法检测细胞中Ki67、CDK1、Bcl2、BAX和GTPBP2蛋白的表达水平。双荧光素酶报告基因实验验证miR-502-3p与GTPBP2基因的靶向关系。结果：CD1...     

Tumor infiltration by FcγRIII (CD16)+ myeloid cells is associated with improved survival in patients with colorectal carcinoma

The prognostic significance of macrophage and natural killer (NK) cell infiltration in colorectal carcinoma (CRC) microenvironment is unclear. We investigated the CRC innate inflammatory infiltrate in over 1,600 CRC using two independent tissue microarrays and immunohistochemistry. Survival time was assessed using the Kaplan–Meier method and Cox proportional hazards regression analysis in a multivariable setting. Spearman's rank correlation tested the association between macrophage and lymphocyte infiltration. The Basel study included over 1,400 CRCs. The level of CD16+ cell infiltration correlated with that of CD3+ and CD8+ lymphocytes but not with NK cell infiltration. Patients with high CD16+ cell infiltration (score 2) survived longer than patients with low (score 1) infiltration (p = 0.008), while no survival difference between patients with score 1 or 2 for CD56+ (p = 0.264) or CD57+ cell (p = 0.583) infiltration was detected. CD16+ infiltrate was associated with improved survival even after adjusting for known prognostic factors including pT, pN, grade, vascular invasion, tumor growth and age [(p = 0.001: HR (95% CI) = 0.71 (0.6–0.9)]. These effects were independent from CD8+ lymphocyte infiltration [(p = 0.036: HR (95% CI) = 0.81 (0.7–0.9)] and presence of metastases [(p = 0.002: HR (95% CI) = 0.43 (0.3–0.7)]. Phenotypic studies identified CD16+ as CD45+CD33+CD11b+CD11c+ but CD64? HLA‐DR‐myeloid cells. Beneficial effects of CD16+ cell infiltration were independently validated by a study carried out at the University of Athens confirming that patients with CD16 score 2 survived longer than patients with score 1 CRCs (p = 0.011). Thus, CD16+ cell infiltration represents a novel favorable prognostic factor in CRC.     

LEA-135 expression: its association with a lower risk of recurrence and increased overall survival of patients with lymph node-positive primary invasive breast cancer

A retrospective study was undertaken to determine and compare the prognostic significance of LEA-135 protein expression by immunohistochemistry with other prognostic pathological parameters, with respect to recurrence and overall survival. This study was conducted in freshly-frozen tissue sections from a cohort of 367 patients having primary invasive breast cancer, with axillary lymph node metastasis. The association of LEA-135 expression was compared with estrogen and progesterone receptor status, segmentectomy or radical mastectomy and hormonal therapy or chemotherapy in terms of recurrence or disease-free survival. Pathologic parameters including tumor size, histological tumor type and histological grade, as well as age of patients at the time of initial diagnosis, and the treatments, together with a median follow-up of 8.8 years were contemplated for the study. Among these parameters, tumor size and histological grade were individually and significantly associated with an increased probability of recurrence (log rank p<0.001 in both cases) and short survival (log ranks p<0.001 and p=0.002, respectively), whereas age was only significantly associated with an increased probability of recurrence (log rank p=0.002) by univariate analysis. By multivariate analysis, both tumor size and histological grade remained statistically significant for recurrence (log rank p<0.001 and p=0.013, respectively) and overall survival (log ranks p<0.001 and p=0.016, respectively). Among the prognostic biomarkers, both ER and PR expression were associated with a decreased rate of recurrence (log ranks p<0.001 and p=0.008, respectively) and overall survival (log ranks p<0.001 and p=0.002, respectively) by univariate analysis. By multivariate analysis, only the ER expression remained significantly associated with a decreased recurrence and increased overall survival (log ranks p=0.023 and p=0.002, respectively). Patients with high (>50% positive cells) or moderate (5-50% positive cells) number of LEA-135-positive cells had a lower probability (46%) of recurrence at 10 years after surgery compared to 76% in LEA-135-negative patients (log rank p<0.001) by univariate analysis. Moreover, the probability of overall survival was higher in patients with high or moderate expression of LEA-135 (46% and 47%, respectively) compared to LEA-135-negative patients (24%) by univariate analysis (log rank p=0.009). By multivariate analysis, the association remained statistically significant for recurrence (log rank p<0.001) and survival (log rank p=0.002). However, there was no significant association between LEA-135 and any of the pathological parameters, age, hormone receptor status, the mode of surgery or the form of therapy (chemo- and/or hormonal) received by this cohort of patients. The results show that an improved prognosis was directly associated with the density of LEA-135-positive cancer cells, while loss of LEA-135 expression was associated with an aggressive phenotype of cancer cells during breast cancer progression. Thus, LEA-135 expression can be implicated as a significant and independent biomarker to identify and distinguish high- from low-risk patients with lymph node-positive invasive breast cancer for an aggressive treatment. Moreover, according to the present results, LEA-135 expression appears to be associated with the tumor cells that have retained certain normal biological characteristics, leading to their lack of aggressiveness and hence a better prognosis.     

乳腺癌干细胞富集与鉴定的初步研究

目的:通过从MCF-7、ZR-75-1、MDA-MB-231乳腺癌细胞系中培养富集及鉴定乳腺癌干细胞(breast cancer stem cell,BCSC),寻找培养与富集乳腺癌干细胞的方法。方法:贴壁培养MCF-7、ZR-75-1、MDA-MB-231细胞系,倒置显微镜观察各细胞形态;流式细胞仪分别分选收集CD44-CD24-、CD44-CD24+、CD44+CD24-及 CD44+CD24+ 细胞,其中CD44+CD24-为乳腺癌干细胞,其余三类为对照组;MTT法计数细胞,绘制MCF-7、ZR-75-1、MDA-MB-231细胞系生长曲线;MCF-7细胞系进行无血清悬浮培养1个周期,流式细胞仪检测分子表面标记物CD44+CD24-含量,贴壁培养的CD44+CD24-乳腺癌干细胞为对照组;将分选的MCF-7（CD44+CD24-）和分选的其余MCF-7细胞（非CD44+CD24-）进行干性成球实验,鉴定CD44+CD24-干性表达。结果:MCF-7、MDA-MB-231细胞系富含表面标志物CD44-CD24-的乳腺癌细胞;ZR-75-1细胞系富含分子表面标志物CD44+CD24+的乳腺癌细胞;生长曲线显示MCF-7、ZR-75-1、MDA-MB-231均呈持续增长,MDA-MB-231细胞生长较MCF-7、ZR-75-1细胞快;通过无血清悬浮培养CD44+CD24-乳腺癌干细胞由19.4%富集到88.9%;成球实验中CD44+CD24-表型细胞成球数量较分选的其余MCF-7细胞（非CD44+CD24-表型）明显增多,成球率分别为（36.5±1.7）%,（1.1±0.5）%。结论:流式细胞仪可成功分选出分子表面标志物为CD44+CD24-的乳腺癌干细胞;CD44+CD24-可能不是乳腺癌干细胞唯一的表面标志物;MDA-MB-231细胞系较MCF-7、ZR-75-1细胞系生长快;无血清悬浮培养法可简便、高效地富集乳腺癌干细胞;CD44+CD24-乳腺癌干细胞干性表达较强。     

肿瘤干细胞在食管癌放射线抵抗中的作用及分子机制研究

目的：探究肿瘤干细胞在食管癌放射线抵抗中的作用及其分子机理,为食管癌的放疗提供理论参考。方法采用8 GyX线照射食管癌细胞系TE1,建立并筛选出放射线抵抗的食管癌细胞系TE1－res。利用细胞计数法比较增殖情况,采用流式细胞术比较CD44（ high） CD24（－） CD133（＋）分子表达及其凋亡情况,克隆形成实验比较克隆形成率及细胞存活曲线,BSP方法比较抑癌基因甲基化程度。成组t 检验或方差分析差异。结果 TE1－res 细胞比 TE1细胞增殖能力增强（平均值20．84×105∶4．46×105／d,P＝0．008）,CD44（high） CD24（－）CD133（＋）细胞比例升高[（38．0±2．9）％∶（10．1±1．3）％,P＝0．001],抗凋亡能力增强（平均值33．23％∶10．50％,P＝0．003）,8 Gy照射后克隆形成率升高[（14．3±2．6）％∶（0．9±0．3）％,P＝0．011],D0值升高（3．28 Gy ∶2．19 Gy,P＝0．125）, SPINT2、CDKN1B、DKK1、TP53、PPP2R1B抑癌基因启动子区甲基化水平升高[（89．7±4．9）％∶（5．0±0．5）％（P＝0．001）、（92．3±4．7）％∶（10．4±0．7）％（P＝0．001）、（90．7±3．7）％∶（7．9±0．4）％（P＝0．001）、（83．4±5．7）％∶（17．2±1．2）％（P＝0．002）、（90．2±6．7）％∶（4．4±1．2）％（P＝0．002）]。结论肿瘤干细胞在食管癌放射线抵抗中具有重要作用,放射线抵抗与SPINT2、CDKN1B、DKK1、TP53及PPP2R1B等抑癌基因启动子区的高甲基化水平密切相关。     

Identification of Homer1 as a Potential Prognostic Marker for Intrahepatic Cholangiocarcinoma

Background: The aim of the present study was to analyze whether Homer1 is a potential prognostic markerfor intrahepatic cholangiocarcinoma (ICC). Materials and Methods: The expression of Homer1 in ICC tissuewas detected with immunohistochemistry and levels of protein in ICC and paratumor tissues were evaluated byWestern blotting. Survival analysis by the Kaplan-Meier method was performed to assess prognostic significance.Results: Homer1 expression was high in 67.4% (58/86) of ICC samples, and there was significant differencebetween ICC and adjacent noncancerous tissues (p<0.001); high expression was associated with poor histologicdifferentiation (p=0.019), TNM stage (p=0.014), lymph node metastasis (p=0.040), and lymphatic invasion(p=0.025). On Kaplan-Meier analysis, a comparison of survival curves of low versus high expressors of Homer1revealed a highly significant difference in OS (p=0.001) and DFS (p=0.006), indicating that high expressionof Homer1 was linked with a worse prognosis. Multivariate analyses showed that Homer1 expression was anindependent risk factor predicting overall survival[Hazard ratio(HR), 7.52; 95% confidence interval (CI), 2.63-21.47; p=0.002] and disease-free survival (HR, 11.56; 95%CI, 5.17-25.96; p<0.001) in ICC. Conclusions: Homer1promotes lymphatic invasion and associates with lymph node metastasis and poor prognosis of ICC. The currentstudy shows that Homer1 may be an independent prognostic factor for ICC patients after curative resection,and it provides an important basis for screening/treating high-risk patients.     

Monoclonal antibodies in myeloid diseases: prognostic use in acute myeloid leukaemia

Bone marrow cells from 109 patients (median age 60) with newly diagnosed acute myeloid leukaemia (AML) were prospectively immunophenotyped (IP) and the prognostic value of monoclonal antibody (MAB) reactivities was analysed to detect differences in complete remission rates and survival, not only between groups of MAB + and - bone marrow cells, but also in cases with or without prominent MAB reactivity as compared to normal BM reactivity of the respective MABs. This approach was based on the assumption that the qualitative expression of antigens is not an all or none phenomenon, but that different degrees of expression of antigens exist. Patients with significantly elevated CD13 (MY7+) cells in bone marrows (CD13 greater than reference value + one standard deviation) (S.D.) showed decreased probability of entering CR (p less than 0.05) and a significantly shorter survival (p less than 0.05). Superior CR rates (p less than 0.05) without difference in long-term survival were seen in patients with low CD33 (MY9) or low HLA-DR expression, while high CD14 (MY4) expression showed a trend towards an adverse factor (p = 0.12). No other antibody reactivities showed differences in CR rates (CD3, CD20, CDw65 (VIM-2) and NAT-9). The more prominent bone marrow expression of CD33 antigen than CD13 (CD33/CD13 greater than 1) correlated to a better chance of entering CR (p = 0.01) and to improved survival (p = 0.002), while the expression of high numbers of VIM-2+ cells was a favourable prognostic factor regarding length of survival (p = 0.002). The importance of a high CD33/CD13 ratio as a positive prognostic factor was evaluated using stratified analysis according to age or leucocyte counts at presentation. In both cases, CD33/CD13 was associated with longer survival (age: p = 0.05, leucocyte counts: p = 0.03). A Cox multiparameter analysis revealed that the CD33/CD13 ratio was a favourable prognostic factor (p = 0.03) together with age (p = 0.001) and leucocyte counts in peripheral blood (p less than 0.01). We conclude that establishing the immunologic phenotype can be of prognostic value in cases of AML, especially with regard to the relationship between the CD33 and CD13 antigens.     

Tim-3 Expression by Peripheral Natural Killer Cells and Natural Killer T Cells Increases in Patients with Lung Cancer - Reduction after Surgical Resection

Background: The purpose of this study was to investigate Tim-3 expression on peripheral CD3-CD56+natural killer (NK) cells and CD3+CD56+ natural killer T (NKT) cells in lung cancer patients. Materials andMethods: We analyzed Tim-3+CD3-CD56+ cells, Tim-3+CD3-CD56dim cells, Tim-3+CD3-CD56bright cells, and Tim-3+CD3+CD56+ cells in fresh peripheral blood from 79 lung cancer cases preoperatively and 53 healthy controlsby flow cytometry. Postoperative blood samples were also analyzed from 21 members of the lung cancer patientcohort. Results: It was showed that expression of Tim-3 was significantly increased on CD3-CD56+ cells, CD3-CD56dim cells and CD3+CD56+ cells in lung cancer patients as compared to healthy controls (p=0.03, p=0.03 andp=0.04, respectively). When analyzing Tim-3 expression with cancer progression, results revealed more elevatedTim-3 expression in CD3-CD56+ cells, CD3-CD56dim cells and CD3+CD56+ cells in cases with advanced stages(III/IV) than those with stage I and II (p=0.02, p=0.04 and p=0.01, respectively). In addition, Tim-3 expression wassignificantly reduced on after surgical resection of the primary tumor (p<0.01). Conclusions: Tim-3 expressionin natural killer cells from fresh peripheral blood may provide a useful indicator of disease progression of lungcancer. Furthermore, it was indicated that Tim-3 might be as a therapeutic target.