Biological and Genetic Characteristics of Tumor-Initiating Cells in Colon Cancer

Background Human prominin-1 (PROM1, CD133) was used as a marker to detect stem cells (progenitor cells) and cancer stem cells (tumor-initiating cells) in various tissues. The purpose of this study was to investigate the biological and genetic characteristics of tumor-initiating cells in colon cancer with both in vitro and in vivo analyses. Methods The CD133 expression of 12 colon cancer cell lines was evaluated. CD133⁺ cells were isolated by flow cytometry and examined for in vivo tumor formation, in vitro proliferation, colony formation, and invasion ability. Additionally, we used microarray analysis to compare gene expression profiles between CD133⁺ and CD133^– isolated cells. Results CD133⁺ cells were found in 5 of 12 colon cancer cell lines. Isolated CD133⁺ cells from the HT29 colon cancer cell line exhibited a higher tumorigenic potential than CD133^– cells in the in vivo tumor formation assay. Furthermore, it was shown that CD133⁺ cells are more proliferative and have higher colony-forming and invasive abilities than CD133^– cells in vitro. Microarray analysis found differential gene expression correlating with CD133 expression. Conclusions It was confirmed that CD133⁺ cells in colon cancer are useful markers for the detection of tumor-initiating cells. Intimate biological and genetic features of CD133⁺ cells in colon cancer cell lines were also revealed. The biological characteristics of CD133⁺ cells and differentially expressed genes in these cells will help elucidate more details of tumor-initiating cells in colon cancer. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users     

The percentage of CD31<Superscript>+</Superscript> T cells decreases after open but not laparoscopic surgery

Background: Efficient killing of tumor cells depends on T cells that migrate from the circulation to the peripheral tissues; these cells express CD31. This study was undertaken to determine the impact of open (OS) and laparoscopic (LS) colorectal surgery on the percentage of circulating CD3⁺CD31⁺ cells. Methods: Peripheral blood was collected from 27 OS and 24 LS colon cancer patients preoperatively (preOP) and on postoperative days 1 (POD1) and 3 (POD3). CD31⁺ T cells were assessed by flow cytometry using monoclonal antibodies. Results: In the OS group, the percentage of CD3⁺CD31⁺ cells was significantly lower in POD1 and POD3 samples compared to the preOP results. LS surgery did not result in a significant change in the percentage of these T cells. A significant correlation was found between the decrease in the percentage of CD3⁺CD31⁺ cells and the length of incision in OS patients. Conclusions: The percentage of CD3⁺CD31⁺ cells decreases following OS but not LS and may be related to incision length. This may compromise T cell function in the peripheral tissues in the postoperative period.     

Different prognostic significance of CD24 and CD44 expression in breast cancer according to hormone receptor status

Objective

CD44⁺CD24^−/low-expressing tumor cells have been studied as tumorigenic stem cells in vitro study. This study was designed to determine the clinical implication of the CD44 and CD24 expression in breast cancer.

Methods

Tissue microarray blocks containing 643 consecutive cases of invasive breast carcinomas from 1993 to 1998 were immunostained for CD44 and CD24. The median follow-up period was 127 months.

Results

CD44⁻CD24⁺ phenotype was associated with frequent hormone receptor positivity and Her2/neu positivity (P = 0.000; Both). The CD44⁺CD24⁻ phenotype was inversely associated with lymph node metastasis (P = 0.002), and it showed positive associations with prolonged disease-free survival (DFS; P = 0.003) and overall survival (OS; P = 0.002). 10-year DFS and OS were 68.9% and 74.6% for CD24 negative group, 55.6% and 60.9% for CD24 positive group (P = 0.001; Both). 10-year DFS and OS were 62.2% and 68.1% for CD44 negative group, 73% and 77.7% for CD44 positive group (P = 0.012, P = 0.013, respectively). In a multivariate analysis, CD24 expression was negatively related to OS only in the receptor positive group (Hazard ratio = 2.03; P = 0.003; 95% CI: 1.27-3.24) and CD44 expression was positively related to OS only in the hormone receptor negative group (hazard ratio = 0.58; P = 0.022; 95% CI: 0.36-0.92).

Conclusions

The CD44⁺CD24⁻ group is considered a favorable prognostic subgroup in breast cancer. CD24 expression was a poor prognosis marker in hormone receptor positive breast cancer, and CD44 expression was a good prognostic marker in the receptor negative group.     

CD 133 and CXCR4 colon cancer cells as a marker for lymph node metastasis

Introduction

Colorectal cancer (CRC) stem cells or tumor-initiating cells (Co-TIC) are implicated in both cancer recurrence and extranodal metastasis. CD133 and CXCR4 are specific cell surface markers that are indicators of Co-TIC. The presence of lymph node (LN) metastases is one of the strongest negative prognostic factors for CRC patients. We examined the relationship between the Co-TIC markers CD133 and CXCR4 and LN involvement in CRC.

Methods

CRC cells were isolated via enzymatic digestion. CD133⁺, CXCR4⁺, and double-positive CRC cells were detected by fluorescence-activated cell sorting analysis. The percentages of CD133⁺, CXCR4⁺, and double-positive cells were identified and correlated to the number and percentage of positive LN on staging.

Results

Twenty-seven samples underwent fluorescence-activated cell sorting analysis. The mean percentage of CD133⁺ cells was 3.94% (range 0.15%–19.06%). The mean percentage of CXCR4⁺ cells was 6.15% (range 0%–27.11%). The mean percentage of CD133⁺CXCR4⁺ cells was 0.45% (range 0%–2.08%). Thirteen patients had LN metastasis: 8 N1 disease and 5 N2 disease. The correlation coefficients between the percentage of Co-TIC marker–positive cells and percentage of positive LN were r = 0.58 (P = 0.0016) for CD133⁺ cells, r = 0.36 (P = 0.5868) for CXCR4⁺ cells, and r = 0.56 (P = 0.0022) for double-positive cells.

Discussion

Our results show CD133⁺ and CD133⁺CXCR4⁺ cancer cells correlate with the presence of LN metastasis in CRC. Further studies will examine whether these markers can give consistent prognostic information and may help to develop novel diagnostic and therapeutic options.     

Overexpression of B7-H3 in CD133 colorectal cancer cells is associated with cancer progression and survival in human patients

Cancer stem–like cells are enriched in CD133-positive (CD133⁺) colorectal cancer (CRC) cells. To date, the biological significance of CD133 expression in cancer stem–like cells is still unknown. B7-H3, a costimulatory molecule, plays a pivotal role in tumor immune escape by inhibiting the functions of T cells. To identify a new marker to predict the tumor grade of CRC, we analyzed the expression of B7-H3 and CD133 in colorectal tumor samples, and their clinical significance was determined. By using a series of techniques including pathologic tissue microarray technology, immunohistochemistry, and immunofluorescent staining, we found B7-H3 was expressed in 56.73% of the CRC cases (59/104) sampled; CD133 was detected in 26.92% of the CRC cases (28/104) sampled. Further analysis indicated that 22 of these CD133⁺ samples expressed B7-H3. We also found coexpression of CD133 and B7-H3 in tumor tissue samples (r = 0.321, P < 0.01). Moreover, in contrast to individual CD133 or B7-H3 expression, the coexpression of B7-H3 and CD133 was evidently associated with the depth of tumor invasion, lymphatic metastasis, distant metastasis, and Dukes' stage, suggesting it is a valuable biomarker for the progression of CRC. Indeed, the patients with coexpression of B7-H3 and CD133 had a poorer survival than the other patients (P < 0.05). In summary, our results reveal that B7-H3 was aberrantly expressed in CD133⁺ CRC cells, and the expression level was closely associated with tumor progression.     

化疗对CD133+结肠癌细胞的作用效果

目的 观察化疗前后结肠癌细胞株HT29、SW620中CD133+结肠癌细胞的数量变化,探讨化疗对CD133+结肠癌细胞的作用效果.方法 取对数生长的结肠癌细胞株HT29、SW620,采用80 mg/L 5-氟尿嘧啶(5-Fu)和25 mg/L奥沙利铂(Oxaliplatin)分别作用8 h,并设对照组,利用CD133抗体进行标记后再用流式细胞仪进行检测,观察化疗前后CD133+细胞的比例.结果 流式细胞仪检测结肠癌细胞株HT29,80 mg/L 5-Fu处理8 h组中CD133+肿瘤细胞为45.00%,与未处理组(32.80%)比较结果差异有统计学意义(P＜0.01);25 mg/L奥沙利铂处理8 h组中CD133+肿瘤细胞为55.30%,与未处理组和80 mg/L 5-Fu处理8 h组比较结果差异有统计学意义(P＜0.01).流式细胞仪检测结肠癌细胞株SW620,80 mg/L 5-Fu处理8 h组中CD133+肿瘤细胞为47.10%,与未处理组(33.90%)比较结果差异有统计学意义(P＜0.01);25 mg/L奥沙利铂处理8 h组中CD133+肿瘤细胞为56.50%,与未处理组和80 mg/L 5-Fu处理8 h组比较结果差异有统计学意义(P＜0.01).结论 CD133+结肠癌细胞能明显抵抗化疗.

Abstract：

Objective To observe the changes of quantity of CD133 + cells in HT29 and SW620 cell lines before and after chemotherapy, and detect the effect of chemotherapy on CD133 + cells. Methods HT29 and SW620 cells in logarithmic growth phase were separately treated with 80 mg/L 5-fluorouracil(5-Fu) and 25 mg/L oxaliplatin for 8 h, and analyzed by flow cytometry after being marked by CD133 antibody. The proportion of CD133 + cells before and after chemotherapy was measured. Results For HT29 cells, the proportion of CD133 + cancer cells in the group treated with 80 mg/L 5-Fu for 8 h was 45.00% with the difference being remarkable ( P ＜ 0. 01 ) compared to the non-treatment group ( 32. 80% ). The proportion of CD133 + cancer cells in the group treated with 25 mg/L oxaliplatin for 8 h was 55.30% with the difference being remarkable ( P ＜ 0. 01 ) compared with both the non-treatment group and the group treated with 80 mg/L 5-Fu for 8 h. For SW620 cells, the proportion of CD133 + cancer cells in the group treated with 80 mg/L 5-Fu for 8 h was 47. 10% with the difference being remarkable ( P ＜0. 01 ) compared with the non-treatment group (33.90%). The proportion of CD133 + cancer cells in the group treated with 25 mg/L oxaliplatin for 8 h was 56. 50% with the difference being remarkable ( P ＜ 0. 01 ) compared with both the non-treatment group and the group treated with 80 mg/L 5-Fu for 8 h. Conclusion CD133 positive colon cancer cells have anti-chemotherapy activity obviously.     

Apoptosis is not correlated with the presence of CD57<Superscript>+</Superscript> NK-cells in brain metastases

Summary ¶Background. Cell apoptosis in solid tumours has been related to immunological attack by NK-cells. The purpose of the present study is to verify in the tissue of brain metastases a possible relationship among the degree of NK-cell infiltration and the number of apoptotic tumour cells. Methods. Twenty brain metastases whose tumour cells expressed CD95 (Fas/APO1) have been studied. NK-cells were identified by using the monoclonal antibody to CD57, and apoptotic tumour cells by means of the immunostain with the anti-ssDNA monoclonal antibody F7-26. The Spearman rank correlation test was used to study the relationship between the degree of CD57-NK-cell infiltration and the apoptosis labelling-index. Findings. Positivity to F7-26 was present in all tumour samples, but the number of immunostained cells showed a wide variability, with a mean apoptosis labelling-index of 11.48%. All the studied tumours showed CD57 immunostained cells, with a number that ranged between 4 and 20 per microscopical field at 200× (mean±standard deviation: 8.4±3.7). Statistical studies showed that there was no correlation between the number of CD57 immunostained NK-cells and the apoptosis labelling-index (p>0.05). Interpretation. These findings suggest that in brain metastases, apoptosis related to immune r esponse is mainly mediated by activated tumour-infiltrating mononuclear cells other than CD57⁺ NK-cells.Published online August 8, 2003     

Protective Effects of Early CD4<Superscript>+</Superscript> T Cell Reduction in Hepatic Ischemia/Reperfusion Injury

Aim  

CD4⁺ T cells contribute to disturbances of liver microcirculation after warm ischemia/reperfusion (I/R). The aim of this study was to investigate a possible protective role of FTY720 (Sphingosine-1 phosphate receptor agonist) in this setting.     

Increased Expression of Fractalkine is Correlated with a Better Prognosis and an Increased Number of Both CD8<Superscript>+</Superscript> T Cells and Natural Killer Cells in Gastric Adenocarcinoma

Background Fractalkine (CX3CL1) is the only CX3C chemokine that can chemoattract natural killer (NK) cells, CD8⁺ T cells, monocytes, and dendritic cells. Although experimental studies have demonstrated that Fractalkine expression by tumor cells is related to the infiltrating lymphocytes and initiates antitumor immunity, the clinical significance of Fractalkine remains to be elucidated in gastric adenocarcinoma. Methods Tissue sections from 158 patients with curatively resected T2 or T3 gastric adenocarcinoma were immunohistochemically stained for Fractalkine. Furthermore, to evaluate CD8⁺ T cells and NK cells infiltration, antibodies to CD8 and CD57 protein were respectively used for immunohistochemistry. Results A significant direct correlation was observed between the Fractalkine scores and the number of CD8⁺ T cells and NK cells using the Spearman rank correlation coefficient test (P = .0080, .0031, respectively). Furthermore, the high Fractalkine expression group (n = 67) showed a significantly better prognosis than the low Fractalkine expression group (n = 91) regarding the disease-free survival (P = .0016). In a multivariate analysis, the Fractalkine expression was identified as one of the independent prognosticators for disease-free survival (risk ratio, 2.5; P = .0147). Conclusions These data suggest that the expression of Fractalkine by tumor cells enhances the recruitment of CD8⁺ T cells and NK cells and induces both innate and adaptive immunity, thereby yielding a better prognosis in gastric adenocarcinoma. Fractalkine is a new independent predictor of the prognosis and can be a novel candidate for development of a more effective therapeutic strategy for gastric adenocarcinomas.     

Expression and prognostic significance of cancer stem cell markers CD24 and CD44 in urothelial bladder cancer xenografts and patients undergoing radical cystectomy

ObjectivesTo evaluate CD24/CD44/CD47 cancer stem cell marker expressions in bladder cancer (BCa) and provide data on their prognostic significance for clinical outcome in patients undergoing radical cystectomy (RC).Material and methodsPrimary BCa tissue was used for xenograft studies. A tissue microarray was prepared using specimens from a cohort of 132 patients. All patients underwent RC for urothelial BCa between 2001 and 2010. Expression of CD24, CD44, and CD47 was examined in primary samples and xenografts by fluorescence-activated cell sorting. Populations of CD24^low- and CD24^high- expressing cells were sorted and evaluated for tumorigenicity in vivo. Tissue microarray was analyzed for CD24/CD44 staining intensity and tumor-specific vs. stromal cell staining. Associations with BCa survival, BCa stage, and lymph node status were evaluated by univariate and multivariate analyses.ResultsCD24 and CD44/CD47 expressions mark distinct cell populations within the normal urothelium as well as in BCa. CD24^high/low expression was not sufficient to characterize CD24 as a BCa-initiating marker in in vivo primary xenotransplants. CD24 and CD44 expressions correlated with lower cancer-specific survival in patients. However, multivariate analyses of CD24 or CD44 did not demonstrate significantly increased hazards for cancer-specific death if analyzed together with stage, grade, and nodal status of patients.ConclusionsCancer stem cell markers CD24/CD44/CD47 are differentially expressed in cells of urothelial BCa in patients undergoing RC and influence cancer-specific survival of patients. Further evaluation of CD24/CD44/CD47 protein expression could be of high therapeutic value in BCa. However, both CD24 and CD44 expressions cannot be regarded as independent prognostic parameters for patients undergoing RC.     

The role of CD133 in the identification and characterisation of tumour-initiating cells in non-small-cell lung cancer

Objective: Emerging evidence suggests that specific sub-populations of cancer cells with stem cell characteristics within the bulk of tumours are implicated in the pathogenesis of heterogeneous malignant tumours. The cells that drive tumour growth have been denoted cancer-initiating cells or cancer stem cells (hereafter CSCs). CSCs have been isolated initially from leukaemias and subsequently from several solid tumours including brain, breast, prostate, colon and lung cancer. This study aimed at isolating and characterising the population of tumour-initiating cells in non-small-cell lung cancer (NSCLC). Methods: Specimens of NSCLC obtained from 89 patients undergoing tumour resection at the Cancer National Institute of Naples were analysed. Three methods to isolate the tumour-initiating cells were used: (1) flow cytometry analysis for identification of positive cells for surface markers such as CD24, CD29, CD31, CD34, CD44, CD133 and CD326; (2) Hoechst 33342 dye exclusion test for the identification of a side-population characteristic for the presence of stem cells; (3) non-adherent culture condition able to form spheres with stem cell-like characteristics. Definition of the tumourigenic potential of the cells through soft agar assay and injection into NOD/SCID mice were used to functionally define (in vitro and in vivo) putative CSCs isolated from NSCLC samples. Results: Upon flow cytometry analysis of NSCLC samples, CD133-positive cells were found in 72% of 89 fresh specimens analysed and, on average, represented 6% of the total cells. Moreover, the number of CD133-positive cells increased markedly when the cells, isolated from NSCLC specimens, were grown as spheres in non-adherent culture conditions. Cells from NSCLC, grown as spheres, when assayed in soft agar, give rise to a 3.8-fold larger number of colonies in culture and are more tumourigenic in non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice compared with the corresponding adherent cells. Conclusions: We have isolated and characterised a population of CD133-positive cells from NSCLC that is able to give rise to spheres and can act as tumour-initiating cells.     

CD44+-ESA+在结肠癌干细胞分选中的应用

目的 分选结肠癌细胞株SW480细胞中的CD133+-CD44+-ESA+亚群细胞,并观察其致瘤性.方法 用流式细胞仪分选SW480细胞中CD133+-CD44+-ESA+、CD133--CD44+-ESA+及CD133--CD44--ESA-亚群细胞.将这3组细胞分别接种于NOD/SCID小鼠,每组5只,观察肿瘤生长...     

PKI-587 and sorafenib alone and in combination on inhibition of liver cancer stem cell proliferation

Background

Deregulated Ras/Raf/mitogen-activated protein kinase and PI3 K/AKT/mTOR signaling pathways are significant in hepatocellular carcinoma proliferation (HCC). In this study we evaluated differences in the antiproliferative effect of dual PI3 K/Akt/mTOR and Ras/Raf/mitogen-activated protein kinase inhibition of non liver cancer stem cell lines (PLC and HuH7) and liver cancer stem cell (LCSC) lines (CD133, CD44, CD24, and aldehyde dehydrogenase 1-positive cells).

Materials and methods

Flow cytometry was performed on the resulting tumors to identify the LCSC markers CD133, CD44, CD24, and aldehyde dehydrogenase 1. Methylthiazol tetrazolium assay was used to assess cellular proliferation. Finally, a Western blot assay was used to evaluate for inhibition of specific enzymes in these two signaling pathways.

Results

Using flow cytometry, we found that LCSC contain 64.4% CD133 + cells, 83.2% CD44 + cells, and 96.4% CD24 + cells. PKI-587 and sorafenib caused inhibiton of LCSC and HCC cell proliferation. PLC cells were more sensitive to PKI-587 than LCSC or Huh7 (P < 0.001). Interestingly, HuH7 cells were more sensitive to sorafenib than LCSC or PLC cells. Additionally, combination therapy with PKI-587 and sorafenib caused significantly more inhibition than monotherapy in HuH7, PLC, and LCSC. Using the methylthiazol tetrazolium assay, we found that the LCSC proliferation was inhibited with sorafenib monotherapy 39% at 5 μM (P < 0.001; n = 12) and 67% by PKI-587 at 0.1 μM (P = 0.002, n = 12) compared with control. The combination of PKI-587 and sorafenib, however, synergistically inhibited LCSC proliferation by 86% (P = 0.002; n = 12).

Conclusions

LCSC (CD133+, CD44+, CD24+) were able to develop very aggressive tumors with low cell concentrations at 4 to 6 wk. Cells CD133+, CD44+, CD24+, which demonstrated at least moderate resistance to therapy in vitro. The combination of PKI-587 and sorafenib was better than either drug alone at inhibiting of LCSC and on HCC cell proliferation.     

CD154 Deficiency Uncouples Allograft CD8+ T‐Cell Effector Function from Proliferation and Inhibits Murine Airway Obliteration

Obliterative bronchiolitis (OB) limits the long‐term success of lung transplantation, while T‐cell effector mechanisms in this process remain incompletely understood. Using the murine heterotopic tracheal transplant model of obliterative airway disease (OAD) to characterize airway allograft rejection, we previously reported an important role for CD8⁺ T cells in OAD. Herein, we studied the role of CD154/CD40 costimulation in the regulation of allospecific CD8⁺ T cells, as airway rejection has been reported to be CD154‐dependent. Airway allografts from CD154^−/− recipients had significantly lower day 28 OAD scores compared to wild‐type (WT) recipients, and adoptive transfer of CD8⁺ T cells from WT recipients, but not CD154^−/− recipients, were capable of airway rejection in fresh CD154^−/− allograft recipients. Intragraft CD8⁺ T cells from CD154^−/− mice showed similar expression of the surface markers CD69, CD62L^low CD44^high and PD‐1, but markedly impaired IFN‐γ and TNF‐α secretion and granzyme B expression versus WT controls. Unexpectedly, intragraft and systemic CD8⁺ T cells from CD154^−/− recipients demonstrated robust in vivo expansion similar to WT recipients, consistent with an uncoupling of proliferation from effector function. Together, these data suggest that a lack of CD154/CD40 costimulation results in ineffective allospecific priming of CD8⁺ T cells required for murine OAD.     

Effective enrichment of prostate cancer stem cells from spheres in a suspension culture system

BackgroundStem-like prostate cancer cells are also called prostate cancer stem cells (PrCSCs). These rare cells are supposed to be highly tumorigenic and to be involved in maintenance of tumor homeostasis and mediation of tumor metastasis. Methods for sorting PrCSCs are mainly based on sorting cells with the marker (CD133⁺/CD44⁺) or side population cells. However, CD133⁺/CD44⁺ cells or side population cells are very rare or even undetectable. The scarcity of approaches for isolation and purification of PrCSCs is the main obstacle to studying PrCSCs.MethodsIn the present study, suspension culture was used for enrichment of PrCSCs. And PrCSCs were verified by side population technology, drug sensitivity assays, and the molecular marker analysis of prostate cancer stem cell.ResultsPC3 cells survived and formed spheres in nonadherent suspension culture. The percentage of CD44⁺/CD133⁺ cells was 18-fold higher in the nonadherent sphere-forming cell population than in the adherent PC3 cell population (13.94% vs. 0.77%, respectively). This side population was increased to 3.1% in the nonadherent population but undetectable in adherent population. Resistance to cisplatin was higher in the nonadherent cells than adherent cells.ConclusionSuspension culture can be used to enrich for PrCSCs. This approach will aid prostate stem cell biology research and facilitate identification of novel therapeutic agents for prostate cancer.     

无血清培养人结肠癌球囊样细胞生物学特性研究

目的 采用无血清培养方法从人结肠癌细胞株中分选出富含肿瘤干细胞的球囊样细胞,并分析其增殖和迁移特性.方法 人结肠癌细胞株HCT116、HT29细胞在特殊配制的无血清培养基(SFM)中悬浮培养,观察球囊样细胞的生成.流式细胞仪检测结肠癌干细胞分子标记CD133表达,利用CCK-8比色法以及Transwell小室法研究结肠癌球囊样细胞的增殖情况和迁移能力.采用成组设计资料的t检验分析检测数据.结果 HCT116、HT29细胞在SFM培养条件下可以获得可稳定传代的球囊样细胞,SFM培养下HCT116球囊样细胞CD133阳性细胞占75.44％±11.41％,HT29球囊样细胞CD133阳性细胞占76.22％±14.23％.与常规含血清培养基(SSM)培养的同系细胞比较,HCT116及HT29球囊样细胞中结肠癌干细胞分子标记CD133阳性细胞数明显增多(t=11.43,9.17,P＜0.05),其持续增殖能力和运动迁移能力均增强.结论 无血清培养获得的结肠癌球囊样细胞高表达结肠癌干细胞分子标记CD133,且具有更强的增殖和迁移能力.它可作为进一步研究结肠癌干细胞的良好模型.     

Clinical significance of tumor-infiltrating lymphocytes in neoplastic progression and lymph node metastasis of human breast cancer

To investigate the clinical significance of tumor-infiltrating lymphocytes (TILs) within the tumor milieu, we quantitatively measured and compared the subpopulations of TILs in 24 patients with stage I–III breast carcinoma. Peripheral blood mononuclear cells (PBMCs), normal breast parenchyma-infiltrating lymphocytes (NILs), and TILs were isolated from tissue specimens and quantified by flow cytometry. The results showed that increased proportion of CD8⁺ T cells, with decreased proportion of CD4⁺ T cells, was significant in gated CD3⁺ TILs as compared to autologous NILs or PBMCs (P < 0.001). The tumor-infiltrating CD8⁺ T cells significantly increased with stage progression, reflected in a more strongly decreased CD4/CD8 percentage (P = 0.003). The CD4/CD8 percentage of TILs was strongly correlated with lymphovascular permeation and subsequent lymph node metastasis (P < 0.001). Increased percentages of tumor-infiltrating CD8⁺ T cells with decreased CD4/CD8 percentages are of prognostic importance for cancer progression in human breast cancer.     

RNA干扰抑制CD44和CD133的表达对肺腺癌A549细胞增殖能力的影响

目的 观察CD44和CD133的表达对人肺腺癌A549细胞株增殖能力的影响.方法 筛选可持续增殖的A549细胞并培养,按CD44和133表达的不同将筛选出的细胞分成5组,非转染组:CD44+CD133+细胞(A组)、CD44-/CD133-细胞(B组)、未分选A549细胞(C组);转染组:CD44-和CD133-细胞(D组)及阴性对照组(E组).筛选得到沉默效果最佳的小干扰RNA(siRNA)片段,并用其转染各组细胞,采用MTS法检测并比较干扰前后各组细胞的增殖能力.结果　沉默效果最佳的siRNA片段及浓度为siCD44-h_002 25 nmol/L和siCD133-h_002 25 nmol/L,沉默效率分别为62%和82%.用其转染各组样本后,CD44和CD133的表达明显被抑制,比较转染前后各组样本的灰度值,差异有统计学意义(P＜0.01).通过分析MTS图显示,转染后细胞的增殖能力显著下降.结论　CD44和CD133的阳性表达,与人肺腺癌A549细胞株较强的增殖能力有关,CD44和CD133可能是肺腺癌干细胞的表面标志物.

Abstract：

Objective To study the effect of expression of CD44 and CD133 on the tumorigenesis of human lung adenocarcinoma cell line A549. Methods A549 cells having continuous self-renewal ability were cultured, and classified into 5 subpopulations based on differential expression patterns for CD133and CD44. The small interfering RNA (siRNA) with optimal sequences was screened out, and transfected the A549 cells. The methods of MTS and cell counting were used to investigate the proliferation of A549cells before and after the expression of CD133 and CD44 was inhibited by RNAi. Results For the optimal sequences and concentrations for siRNA transfection efficiency being 25 nmol/L for both siCD44-h_002 25and siCD133-h_002 25, the transfection efficiency was 62% and 82% respectively. Stably transfected cells were screened, and the expression of CD44 and CD133 was inhibited significantly. The gray values before transfection were significantly different from those after transfection ( P ＜ 0. 01 ). The MTS curve showed that the cell viability was significantly decreased after transfection. Conclusion The proliferation ability of A549 cells was related to the positive expression of CD44 and CD133. CD133 and CD44 may be important surface markers of lung cancer stem cells.