《超声诊断学》课程教学资源（学术论文）Relationship between microvessel density and cancer stem cells in tumor angiogenesis（a meta-analysis）.pdf_大学文库

Systematic Review Biomarkers For reprint orders,please contact:reprints@futuremedicine.com in Medicine Relationship between microvessel density and cancer stem cells in tumor angiogenesis:a meta-analysis Aim:Several studies have shown that cancer stem cells (CSCs)promote tumor Jiaojiao Lan1,Jun Lit.2 angiogenesis and are involved in tumor growth.A meta-analysis was conducted to Xinxin Ju',Yang Zhou', evaluate the association between cancer stem cell markers expression and microvessel Yan Qi,Yan Ren',Hong Zou', density (MVD).Methods:Relevant literature was identified from diverse databases. Lianghai Wang',Man Li The Stata 12.0 and Review Manager 5.3 software were used to performed meta- Lijuan Pang*.1 Department of Pathology Key analysis.Results:Sixteen studies investigating the correlation between CSCs and Laboratory for Xinjiang Endemic Ethnic tumor angiogenesis were included in a total of 1409 cases.The result showed that Diseases (Ministry of Education),Shihezi positive CSC markers expression were associated with increased MVD count in human University School of Medicine,Shihezi, tumors and CSC-transplanted mouse tumor models(p<0.0001).Conclusion:CSCs may Xinjiang,China be associated with angiogenesis during the growth and development of tumors. 2Department of Ultrasonic Medicine, The First Affiliated Hospital,Shihezi University School of Medicine,Shihezi, First draft submitted:29 January 2016;Accepted for publication:6 June 2016; Xinjiang,China Published online:14 July 2016 *Author for correspondence: Tel.:+8613677565458 Keywords:cancer stem cells.meta-analysis.microvessel density.tumor angiogenesis Fax+869932057136 0cean123456@163.c0m Authors contributed equally Tumors may arise from a small subset of nant role in neovascularization during tumor cancer cells that are capable of self-renewal,growth. unlimited proliferation and tumorigenic- CSCs represent a small subpopulation of ity;such cells are termed cancer stem cells cells withinatumor that express surface mark- (CSCs)[j.CSCs have been identified and ers such as CD133 [o],nestin ABCG2 [2 isolated from tumors and tumor-derived cell and ALDHI [3].A common marker of angio- lines,including melanoma [2l and brain [3],genesis,micro-vessel density(MVD)[41,has breast [4]and lung cancers [51.It is widely been utilized for the quantification of intratu- accepted that angiogenesis is involved in moral angiogenesis in tumor tissue in recent the growth and hematogenous spread of years.Several vascular endothelial cell mark- tumors []Cancer invasion and metastasis ers,such as CD31 [is]and CD34 [6],may be involves multiple complex steps and involves used for the detection of MVD.We speculate a variety of molecules.Angiogenesis,which that CSCs may be involved in angiogenesis enables the supply of blood and nutrients for during tumor growth.Therefore,the present tumor growth,is an important step in can- meta-analysis was performed to investigate cer invasion and metastasis [6.71.According the relationship between CSCs and angio- to the CSC hypothesis,CSCs play a critical genesis by assessing MVD,which may be role in maintaining the capacity for malig- utilized as a clinical parameter for predicting nant proliferation,invasion,metastasis and prognosis during tumor therapy. recurrence of the tumor [8).CSCs may induce cancer metastasis through multiple pathways Materials methods and support tumor progression by promot- Search strategy study selection Future ing angiogenesis [91.Therefore,accumulating We searched MEDLINE using PubMed, evidence indicates that CSCs play a predomi- Medicine paort of CNKI (China National Knowledge Infra- 10.2217/bmm-2016-0026 2016 Future Medicine Ltd Biomark.Med.(Epub ahead of print) 5SN1752-0363

Biomark. Med. (Epub ahead of print) ISSN 1752-0363 part of Systematic Review 10.2217/bmm-2016-0026 © 2016 Future Medicine Ltd Biomark. Med. Systematic Review 2016/07/22 10 8 2016 Aim: Several studies have shown that cancer stem cells (CSCs) promote tumor angiogenesis and are involved in tumor growth. A meta-analysis was conducted to evaluate the association between cancer stem cell markers expression and microvessel density (MVD). Methods: Relevant literature was identified from diverse databases. The Stata 12.0 and Review Manager 5.3 software were used to performed metaanalysis. Results: Sixteen studies investigating the correlation between CSCs and tumor angiogenesis were included in a total of 1409 cases. The result showed that positive CSC markers expression were associated with increased MVD count in human tumors and CSC-transplanted mouse tumor models (p < 0.0001). Conclusion: CSCs may be associated with angiogenesis during the growth and development of tumors. First draft submitted: 29 January 2016; Accepted for publication: 6 June 2016; Published online: 14 July 2016 Keywords: cancer stem cells • meta-analysis • microvessel density • tumor angiogenesis Tumors may arise from a small subset of cancer cells that are capable of self-renewal, unlimited proliferation and tumorigenicity; such cells are termed cancer stem cells (CSCs) [1]. CSCs have been identified and isolated from tumors and tumor-derived cell lines, including melanoma [2] and brain [3], breast [4] and lung cancers [5]. It is widely accepted that angiogenesis is involved in the growth and hematogenous spread of tumors [1]. Cancer invasion and metastasis involves multiple complex steps and involves a variety of molecules. Angiogenesis, which enables the supply of blood and nutrients for tumor growth, is an important step in cancer invasion and metastasis [6,7]. According to the CSC hypothesis, CSCs play a critical role in maintaining the capacity for malignant proliferation, invasion, metastasis and recurrence of the tumor [8]. CSCs may induce cancer metastasis through multiple pathways and support tumor progression by promoting angiogenesis [9]. Therefore, accumulating evidence indicates that CSCs play a predominant role in neovascularization during tumor growth. CSCs represent a small subpopulation of cells within a tumor that express surface markers such as CD133 [10], nestin [11], ABCG2 [12] and ALDH1 [13]. A common marker of angiogenesis, micro-vessel density (MVD) [14], has been utilized for the quantification of intratumoral angiogenesis in tumor tissue in recent years. Several vascular endothelial cell markers, such as CD31 [15] and CD34 [16], may be used for the detection of MVD. We speculate that CSCs may be involved in angiogenesis during tumor growth. Therefore, the present meta-analysis was performed to investigate the relationship between CSCs and angiogenesis by assessing MVD, which may be utilized as a clinical parameter for predicting prognosis during tumor therapy. Materials & methods Search strategy & study selection We searched MEDLINE using PubMed, CNKI (China National Knowledge InfraRelationship between microvessel density and cancer stem cells in tumor angiogenesis: a meta-analysis Jiaojiao Lan‡,1 , Jun Li‡,2, Xinxin Ju1 , Yang Zhou1 , Yan Qi1 , Yan Ren1 , Hong Zou1 , Lianghai Wang1 , Man Li1 & Lijuan Pang*,1 1 Department of Pathology & Key Laboratory for Xinjiang Endemic & Ethnic Diseases (Ministry of Education), Shihezi University School of Medicine, Shihezi, Xinjiang, China 2 Department of Ultrasonic Medicine, The First Affiliated Hospital, Shihezi University School of Medicine, Shihezi, Xinjiang, China *Author for correspondence: Tel.: +86 136 7756 5458 Fax: +86 993 205 7136 ocean123456@163.com ‡ Authors contributed equally For reprint orders, please contact: reprints@futuremedicine.com

Systematic Review Lan,Li,Ju et al. structure)and the Wan-fang database for relevant animal models of transplanted tumors:author,year, studies as of 4 May 2016,without language restric- model of tumor,type of transplanted animal,the tions.The search strategy used text terms such as 'can-number of transplanted animals,number of injected cer stem cells (CSCs)','stem cell','cancer,'tumor,tumor cells or stem cells,time and mean and standard 'angiogenesis,'vascularization'and 'microvessel den-deviation value of MVD(Table 2).If data from any of sity (MVD)'in PubMed.Eligible articles and relevant the above categories were not reported in the literature, review articles were also manually reviewed.Studies items were regarded as'not applicable.' eligible for inclusion in this meta-analysis satisfied the following criteria:case-control studies assessing the Statistical methods analysis relationship between the expression of CSC markers Statistical analyses of included studies were performed and pathological features of tumors;articles describing by comparing MVD value between the groups that the association between the expression of CSC mark-was positive and negative expression for CSC mark- ers and MVD count;literature reporting data with the ers in human tumor tissues.In addition,we measured mean or standard deviation of MVD as the statisti-the MVD count on animal models of transplanted cal indicator;and studies focusing on the evaluation of tumors between the experimental group and control CSC marker expression in tumor tissue using immu- group.The MVD counts were regarded as continuous nohistochemistry.In addition,with regard to duplicate variables,using mean and standard deviation. studies,only the most recent or most informative was The meta-analysis was performed using STATA included in the present meta-analysis.Two review- 12.0 and Review manager 5.3software.Standard ers (JIL and LIP)independently determined study mean difference (SMD)with a corresponding 95% eligibility.Disagreements were resolved by consensus. confidence interval (CI)was calculated.Meanwhile, odds ratios(ORs)with 95%CI were used to evaluate Data extraction quality assessment the correlation between CSC marker expression and We extracted information regarding the author,year,clinicopathological characteristics.For each analysis, sample number,type of tumor,CSC vascular endo-an OR>1 implied higher levels of expression of CSC thelial markers and the mean and standard deviation markers in the indicated group.The Z test was used value of MVD in human tumor tissues(Table 1).The for evaluating the statistical significance of SMD.The following relevant data were extracted with regard to Cochran's Q-statistic [7]and I2 [s]test were used for Table 1.Main characteristics and results of the eligible studies in human tumor tissues. Study (year) Cancers CSCs Vascular MVD of the high expression/ MVD of the low expression/ Ref. markers endothelial positive of CSC markers negative of CSC markers antibodies N M SD N M SD Feng Li(2011) Lung cancer CD133/ CD34 33 85.4 31.7 112 55.3 24.5 [2o ABCG2+ Jun Dong(2011)Brain tumor ABCG2 CD34 24 31 19.20 68 13 12.09 [21 Huaming Wang Brain tumor CD133 CD34 68 23.57 7.16 4.67 1.53 [22 (2011) Lingjun Kong Brain tumor CD133/ CD34 39 52.5 20.0 11 26.6 13.7 [23) (2012) Nestin Qing Li(2012) Lung cancer CD133/ CD34 14 22.64 5.123 36 19.75 5.872 [24 ALDH1A1 Shiwu Wu(2012)Lung cancer CD133 CD34 149 29.4 9.8 156 15.0 7.2 [25 Liang Pan(2013)Brain tumor CD133 CD34 37 38.76 13.87 9 20.22 5.26 [26 Yungin Huang Breast CD133 CD34 63 22.0 8.3 42 18.0 6.4 [271 (2013) cancer Yiming Song Breast CD133/ CD34 31 28.78 10.55 110 26.92 9.02 [28] (2015) cancer ALDH1 Zhengquan Han Breast CD133 CD34 158 22.1 6.3 167 19.1 6.3 [29 (2015) cancer CSC:Cancer stem cell;M:Mean;MVD:Microvessel density;N:Number;SD:Standard deviation. 10.2217/bmm-2016-0026 Biomark.Med.(Epub ahead of print) future science groupg

10.2217/bmm-2016-0026 Biomark. Med. (Epub ahead of print) future science group Systematic Review Lan, Li, Ju et al. structure) and the Wan-fang database for relevant studies as of 4 May 2016, without language restrictions. The search strategy used text terms such as ‘cancer stem cells (CSCs)’, ‘stem cell’, ‘cancer’, ‘tumor’, ‘angiogenesis’, ‘vascularization’ and ‘microvessel density (MVD)’ in PubMed. Eligible articles and relevant review articles were also manually reviewed. Studies eligible for inclusion in this meta-analysis satisfied the following criteria: case–control studies assessing the relationship between the expression of CSC markers and pathological features of tumors; articles describing the association between the expression of CSC markers and MVD count; literature reporting data with the mean or standard deviation of MVD as the statistical indicator; and studies focusing on the evaluation of CSC marker expression in tumor tissue using immunohistochemistry. In addition, with regard to duplicate studies, only the most recent or most informative was included in the present meta-analysis. Two reviewers (JJL and LJP) independently determined study eligibility. Disagreements were resolved by consensus. Data extraction & quality assessment We extracted information regarding the author, year, sample number, type of tumor, CSC vascular endothelial markers and the mean and standard deviation value of MVD in human tumor tissues (Table 1). The following relevant data were extracted with regard to animal models of transplanted tumors: author, year, model of tumor, type of transplanted animal, the number of transplanted animals, number of injected tumor cells or stem cells, time and mean and standard deviation value of MVD (Table 2). If data from any of the above categories were not reported in the literature, items were regarded as ‘not applicable.’ Statistical methods & analysis Statistical analyses of included studies were performed by comparing MVD value between the groups that was positive and negative expression for CSC markers in human tumor tissues. In addition, we measured the MVD count on animal models of transplanted tumors between the experimental group and control group. The MVD counts were regarded as continuous variables, using mean and standard deviation. The meta-analysis was performed using STATA 12.0 and Review manager 5.3software. Standard mean difference (SMD) with a corresponding 95% confidence interval (CI) was calculated. Meanwhile, odds ratios (ORs) with 95% CI were used to evaluate the correlation between CSC marker expression and clinicopathological characteristics. For each analysis, an OR >1 implied higher levels of expression of CSC markers in the indicated group. The Z test was used for evaluating the statistical significance of SMD. The Cochran’s Q-statistic [17] and I2 [18] test were used for Table 1. Main characteristics and results of the eligible studies in human tumor tissues. Study (year) Cancers CSCs markers Vascular endothelial antibodies MVD of the high expression/ positive of CSC markers MVD of the low expression/ negative of CSC markers Ref. N M SD N M SD Feng Li (2011) Lung cancer CD133+/ ABCG2+ CD34 33 85.4 31.7 112 55.3 24.5 [20] Jun Dong (2011) Brain tumor ABCG2 CD34 24 31 19.20 68 13 12.09 [21] Huaming Wang (2011) Brain tumor CD133 CD34 68 23.57 7.16 3 4.67 1.53 [22] Lingjun Kong (2012) Brain tumor CD133/ Nestin CD34 39 52.5 20.0 11 26.6 13.7 [23] Qing Li (2012) Lung cancer CD133/ ALDH1A1 CD34 14 22.64 5.123 36 19.75 5.872 [24] Shiwu Wu (2012) Lung cancer CD133 CD34 149 29.4 9.8 156 15.0 7.2 [25] Liang Pan (2013) Brain tumor CD133 CD34 37 38.76 13.87 9 20.22 5.26 [26] Yunqin Huang (2013) Breast cancer CD133 CD34 63 22.0 8.3 42 18.0 6.4 [27] Yiming Song (2015) Breast cancer CD133/ ALDH1 CD34 31 28.78 10.55 110 26.92 9.02 [28] Zhengquan Han (2015) Breast cancer CD133 CD34 158 22.1 6.3 167 19.1 6.3 [29] CSC: Cancer stem cell; M: Mean; MVD: Microvessel density; N: Number; SD: Standard deviation

Systematic Review Lan,Li,Ju et al. Study ID SMD(95%CI) Weight(%) Zhengquan Han(2015) 0.48(0.26,0.70) 11.93 Yiming Song (2015) 0.20(-0.20,0.60) 11.06 Yungin Huang(2013) 0.53(0.13,0.92) 11.07 Liang Pan (2013) 1.45(0.67,2.24) 8.47 Shiwu Wu (2012) 1.68(1.42,1.94) 11.76 Qing Li (2012) 0.51(-0.12,1.13) 9.59 Lingjun Kong (2012) 1.37(0.65,2.10) 8.92 Huaming Wang (2011) 2.68(1.44,3.92) 5.79 Jun Dong (2011) 1.26(0.76,1.76) 10.43 Feng Li (2011) 1.15(0.73,1.56) 10.99 Overall (I-squared =88.7%,p=0.000) 1.04(0.63,1.45) 100.00 Note:Weights are from random effects analysis -3.92 0 3.92 Figure 2.Meta-analysis of ten studies evaluating the association between CSC marker expression and MVD in human tumors. CSC:Cancer stem cell;MVD:Microvessel density;SMD:Standardized mean difference. Publication bias was evaluated using the funnel plot Characteristics of the included studies with Begg's bias test 91. The main characteristics of ten eligible studies [2o-291 describing the association between CSCs and MVD in Results human tumor tissues are summarized in Table 1.Addi- A total of 202 studies for CSCs and MVD were iden- tionally,Table 2 shows the characteristics ofsix included tified from a primary literature search of electronic studies [30-35]reporting the relationship between CSCs databases PubMed,CNKI and the Wan-fang database and MVD in tumor transplantation animal models. (Figure 1).After manually screening the titles,abstracts Finally,a total of 1331 cases from ten studies of MVD and key data,151 reviews,commentaries,meta-analy- in human tumor tissues and a total of 78 cases from ses or studies that were not relevant to the present anal- six studies of MVD in tumor transplantation animal ysis were excluded.Of 51 studies selected for detailed models were included.These tem studies in which evaluation:28 studies were excluded;these consisted CSC marker expression was analyzed by immunohis- of 16 studies that did not report MVD values,six stud- tochemistry were included and the Weidner method ies lacking statistical analyses,two studies that lacked was used to calculate MVD. data describing the relationship between MVD and CSC marker expression,four studies were detected by Correlation between the expression of CSCs immunofluorescence staining;seven additional studies markers MVD in human tumor tissues were excluded as they lacked a suitable control group.The characteristics of the ten selected studies that were The final meta-analysis included ten studies for MVD reporting the relationship between CSC markers and and CSC markers in human tumor tissues and six MVD in tumor tissue(Figure 2).The expression of studies for MVD and CSCs in tumor transplantation CSC markers was associated with MVD count;high animal models. levels of expression of CSC markers indicated increased 10.2217/bmm-2016-0026 Biomark.Med.(Epub ahead of print) future science group

10.2217/bmm-2016-0026 Biomark. Med. (Epub ahead of print) future science group Systematic Review Lan, Li, Ju et al. Publication bias was evaluated using the funnel plot with Begg’s bias test [19]. Results A total of 202 studies for CSCs and MVD were identified from a primary literature search of electronic databases PubMed, CNKI and the Wan-fang database (Figure 1). After manually screening the titles, abstracts and key data, 151 reviews, commentaries, meta-analyses or studies that were not relevant to the present analysis were excluded. Of 51 studies selected for detailed evaluation: 28 studies were excluded; these consisted of 16 studies that did not report MVD values, six studies lacking statistical analyses, two studies that lacked data describing the relationship between MVD and CSC marker expression, four studies were detected by immunofluorescence staining; seven additional studies were excluded as they lacked a suitable control group. The final meta-analysis included ten studies for MVD and CSC markers in human tumor tissues and six studies for MVD and CSCs in tumor transplantation animal models. Characteristics of the included studies The main characteristics of ten eligible studies [20–29] describing the association between CSCs and MVD in human tumor tissues are summarized in Table 1. Additionally, Table 2 shows the characteristics of six included studies [30–35] reporting the relationship between CSCs and MVD in tumor transplantation animal models. Finally, a total of 1331 cases from ten studies of MVD in human tumor tissues and a total of 78 cases from six studies of MVD in tumor transplantation animal models were included. These tem studies in which CSC marker expression was analyzed by immunohistochemistry were included and the Weidner method was used to calculate MVD. Correlation between the expression of CSCs markers & MVD in human tumor tissues The characteristics of the ten selected studies that were reporting the relationship between CSC markers and MVD in tumor tissue (Figure 2). The expression of CSC markers was associated with MVD count; high levels of expression of CSC markers indicated increased Figure 2. Meta-analysis of ten studies evaluating the association between CSC marker expression and MVD in human tumors. CSC: Cancer stem cell; MVD: Microvessel density; SMD: Standardized mean difference. Zhengquan Han (2015) Yiming Song (2015) Yunqin Huang (2013) Liang Pan (2013) Shiwu Wu (2012) Qing Li (2012) Lingjun Kong (2012) Huaming Wang (2011) Jun Dong (2011) Feng Li (2011) Overall (I-squared = 88.7%, p = 0.000) Note: Weights are from random effects analysis Study ID SMD (95% CI) Weight (%) 0.48 (0.26, 0.70) 0.20 (-0.20, 0.60) 0.53 (0.13, 0.92) 1.45 (0.67, 2.24) 1.68 (1.42, 1.94) 0.51 (-0.12, 1.13) 1.37 (0.65, 2.10) 2.68 (1.44, 3.92) 1.26 (0.76, 1.76) 1.15 (0.73, 1.56) 1.04 (0.63, 1.45) 11.93 11.06 11.07 8.47 11.76 9.59 8.92 5.79 10.43 10.99 100.00 -3.92 0 3.92

Relationship between microvessel density cancer stem cells in tumor angiogenesis Systematic Review MVD count.Pooled meta-analysis showed that,com-effect)indicated that the MVD count in the CSC pared with low expression of CSC markers,high groups was higher than in the tumor cell groups.The CSCs marker expression correlates with high MVD difference between the groups was statistically signifi- count(SMD=1.04,95%CI=0.63-1.45,Z=4.99,cant(SMD=3.69,95%CI-2.195.19,Z-4.83, p50%;tissues tumor type random effect). In order to investigate the relationship between angio- genesis and tumor type,we determined the MVD count Correlation of expression of CSC markers with of tumor tissues according to tumor type.The CSC MVD in tumor transplantation animal models markers and MVD of tumor tissues varied according The characteristics of the six selected studies describing to the specific type of tumor (Figure 4).The subgroup the relationship between CSCs and MVD in tumor analysis was performed on four studies investigating transplantation animal models are shown (Figure 3).In the association between CSC marker expression and order to elucidate the association between CSCs and MVD count in brain tumor tissue.High CSC marker angiogenesis,data for in vivo models were required.In expression was significantly associated with high MVD the present meta-analysis,studies of five tumor types count(SMD-1.50,95%CI-1.05-1.94,Z=6.55, were included:brain tumor,hepatoma and cancers of p0.05, It is considered that CSCs possess stronger angio-I2=30.8%).Three studies analyzing the association genic capability than well-differentiated tumor between CSC marker expression and MVD count in cells.Meta-analysis using the random effect model breast cancer were included.The difference between (X2=17.31,p =0.004,I2=71.1%>50%;random high and low CSC marker expression was statisti- Study ID SMD (95%CI) Weight(%) Lin Lin (2015) 7.27(3.50,11.04) 9.76 Hao Song(2012) 2.04(0.94,3.15) 22.53 Feng Jin (2011) 1.97(0.40,3.54) 20.01 XuHe(2010) 5.65(3.34,7.96) 15.92 Weigi Nian (2009) 2.74(1.00,4.47） 19.09 Dawei Kong (2008) 5.55(2.56.8.53) 12.69 Overall (I-squared 71.1%,p =0.004) 3.69(2.19,5.19) 100.00 Note:Weights are from random effects analysis -11 0 11 Figure 3.Meta-analysis of six studies evaluating the association between CSCs and MVD in tumor transplantation animal models. CSC:Cancer stem cell;MVD:Microvessel density;SMD:Standardized mean difference. future science group www.futuremedicine.com 10.2217/bmm-2016-0026

www.futuremedicine.com 10.2217/bmm-2016-0026 Figure 3. Meta-analysis of six studies evaluating the association between CSCs and MVD in tumor transplantation animal models. CSC: Cancer stem cell; MVD: Microvessel density; SMD: Standardized mean difference. Lin Lin (2015) Hao Song (2012) Feng Jin (2011) Xu He (2010) Weiqi Nian (2009) Dawei Kong (2008) Overall (I-squared = 71.1%, p = 0.004) Note: Weights are from random effects analysis 7.27 (3.50, 11.04) 2.04 (0.94, 3.15) 1.97 (0.40, 3.54) 5.65 (3.34, 7.96) 2.74 (1.00, 4.47) 5.55 (2.56, 8.53) 3.69 (2.19, 5.19) 9.76 22.53 20.01 15.92 19.09 12.69 100.00 Study ID SMD (95% CI) -11 0 11 Weight (%) future science group Relationship between microvessel density & cancer stem cells in tumor angiogenesis Systematic Review MVD count. Pooled meta-analysis showed that, compared with low expression of CSC markers, high CSCs marker expression correlates with high MVD count (SMD = 1.04, 95% CI = 0.63∼1.45, Z = 4.99, p 50%; random effect). Correlation of expression of CSC markers with MVD in tumor transplantation animal models The characteristics of the six selected studies describing the relationship between CSCs and MVD in tumor transplantation animal models are shown (Figure 3). In order to elucidate the association between CSCs and angiogenesis, data for in vivo models were required. In the present meta-analysis, studies of five tumor types were included: brain tumor, hepatoma and cancers of the lung, breast and cervix. It is considered that CSCs possess stronger angiogenic capability than well-differentiated tumor cells. Meta-analysis using the random effect model (X2 = 17.31, p = 0.004, I2 = 71.1% > 50%; random effect) indicated that the MVD count in the CSC groups was higher than in the tumor cell groups. The difference between the groups was statistically significant (SMD = 3.69, 95% CI = 2.19∼5.19, Z = 4.83, p 0.05, I2 = 30.8%). Three studies analyzing the association between CSC marker expression and MVD count in breast cancer were included. The difference between high and low CSC marker expression was statisti-

Systematic Review Lan,Li,Ju et al. Study ID SMD(95%CI) Weight(%) Breast cancer Zhengquan Han(2015) 0.48(0.26,0.70) 11.93 Yiming Song (2015) 0.20(-0.20,0.60) 11.06 Yunqin Huang(2013) 0.53(0.13,0.92) 11.07 Subtotal (I-squared =0.0%,p=0.431) 0.43(0.26,0.61) 34.05 Brain tumor Liang Pan(2013) 1.45(0.67,2.24) 8.47 Lingjun Kong (2012) 1.37(0.65,2.10 8.92 Huaming Wang(2011) 2.68(1.44,3.92) 5.79 Jun Dong(2011)】 1.26(0.76,1.76) 10.43 Subtotal (I-squared 30.8%,p =0.227) 1.49(1.05,1.94) 33.60 Lung cancer Shiwu Wu(2012) 1.68(1.42,1.94) 11.76 Qing Li (2012) 0.51(-0.12,1.13) 9.59 Feng Li (2011) 1.15(0.73,1.56) 10.99 Subtotal (I-squared =85.4%.p =0.001) 1.16(0.54,1.79) 32.34 Overall (I-squared 88.7%,p=0.000) 1.04(0.63.1.45) 100.00 Note:Weights are from random effects analysis -3.92 3.92 Figure 4.Forest plots of SMD showing the association between CSC marker expression and MVD in human tumors,based on tumor type. CSC:Cancer stem cell;MVD:Microvessel density:SMD:Standardized mean difference. cally significant (SMD =0.43,95%CI=0.26~0.61,shown (Figure 5A).A total of three studies reported Z=4.89,p0.05,P=0.0%).Three studies assessing the and the non-adenocarcinoma group.Meta-analysis relationship between CSC marker expression and MVD of the fixed effect model shows that the difference count in lung cancer were included.The pooled SMD between the two groups was not statistically significant was1.16(95%CI-0.54-1.79,Z=3.66,p50%)was observed due sues (well-moderate differentiation group vs poor dif to lack of sufficient statistical data for evaluation of the ferentiation group)is shown in Figure 5B.Three stud- relationship between CSCs markers and angiogenesis in ies reported the overexpression of three CSC markers the subgroups. (ABCG2,CD133 and ALDH1Al)in the well-moderate differentiation group and poor differentiation group. Correlation between CSC marker expression Meta-analysis of the fixed effect model showed that clinicopathological features in lung cancer over-expression of CSC markers in the poor differentia- The histological types of lung cancer tissues(adeno- tion group was higher than in the well-moderate differ- carcinoma group vs nonadenocarcinoma group)are entiation group.The difference between the groups was 10.2217/bmm-2016-0026 Biomark.Med.(Epub ahead of print) future science group

10.2217/bmm-2016-0026 Biomark. Med. (Epub ahead of print) Figure 4. Forest plots of SMD showing the association between CSC marker expression and MVD in human tumors, based on tumor type. CSC: Cancer stem cell; MVD: Microvessel density; SMD: Standardized mean difference. Breast cancer Zhengquan Han (2015) Yiming Song (2015) Yunqin Huang (2013) Subtotal (I-squared = 0.0%, p = 0.431) Brain tumor Liang Pan (2013) Lingjun Kong (2012) Huaming Wang (2011) Jun Dong (2011) Subtotal (I-squared = 30.8%, p = 0.227) Lung cancer Shiwu Wu (2012) Qing Li (2012) Feng Li (2011) Subtotal (I-squared = 85.4%, p = 0.001) Overall (I-squared = 88.7%, p = 0.000) Note: Weights are from random effects analysis 0.48 (0.26, 0.70) 0.20 (-0.20, 0.60) 0.53 (0.13, 0.92) 0.43 (0.26, 0.61) 1.45 (0.67, 2.24) 1.37 (0.65, 2.10) 2.68 (1.44, 3.92) 1.26 (0.76, 1.76) 1.49 (1.05, 1.94) 1.68 (1.42, 1.94) 0.51 (-0.12, 1.13) 1.15 (0.73, 1.56) 1.16 (0.54, 1.79) 1.04 (0.63, 1.45) 11.93 11.06 11.07 34.05 8.47 8.92 5.79 10.43 33.60 11.76 9.59 10.99 32.34 100.00 Study ID SMD (95% CI) Weight (%) -3.92 0 3.92 future science group Systematic Review Lan, Li, Ju et al. cally significant (SMD = 0.43, 95% CI = 0.26∼0.61, Z = 4.89, p 0.05, I2 = 0.0%). Three studies assessing the relationship between CSC marker expression and MVD count in lung cancer were included. The pooled SMD was 1.16 (95% CI = 0.54∼1.79, Z = 3.66, p 50%) was observed due to lack of sufficient statistical data for evaluation of the relationship between CSCs markers and angiogenesis in the subgroups. Correlation between CSC marker expression & clinicopathological features in lung cancer The histological types of lung cancer tissues (adenocarcinoma group vs nonadenocarcinoma group) are shown (Figure 5A). A total of three studies reported the overexpression of three CSC markers (ABCG2, CD133 and ALDH1A1) in the adenocarcinoma group and the non-adenocarcinoma group. Meta-analysis of the fixed effect model shows that the difference between the two groups was not statistically significant (OR = 0.99, 95% CI: 0.72–1.36, p = 0.94). Significant heterogeneity was not observed between the studies (I2 = 0%, p = 0.70). The histological differentiation of lung cancer tissues (well-moderate differentiation group vs poor differentiation group) is shown in Figure 5B. Three studies reported the overexpression of three CSC markers (ABCG2, CD133 and ALDH1A1) in the well-moderate differentiation group and poor differentiation group. Meta-analysis of the fixed effect model showed that over-expression of CSC markers in the poor differentiation group was higher than in the well-moderate differentiation group. The difference between the groups was

Relationship between microvessel density cancer stem cells in tumor angiogenesis Systematic Review Q AD N-AD Odds ratio Odds ratio Study or subgroup Events Total Events Total Weight M-H,fixed,95%CI M-H,fixed,95%Cl Feng Li(ABCG2+) 21 60 3 85 23.7% 0.810.41,1.60j Feng Li(CD133+) 18 60 28 85 21.1% 0.87[0.43,1.78 Qing Li (ALDH1A1+) 10 29 10 21 9.9% 0.58[0.18,1.83 Qing Li(CD133+) 16 29 11 21 7.4% 1.12[0.36,3.45 Shiwu Wu (CD133+) 50 95 99 210 37.9% 1.25[0.77,2.02 Total (95%CI) 273 422100.0% 0.99[0.72,1.36] Total events 115 182 Heterogeneity:Chi2=2.20,df =4(P=0.70);12=0% 0.1 Test for overall effect:Z=0.07(P =0.94) 0.01 10 100 AD N-AD ⑧ W+M Poor Odds ratio Odds ratio Study or subgroup Events Total Events Total Weight M-H,fixed,95%Cl M-H.fixed,95%CI Feng Li(ABCG2+) 38 101 17 44 16.9% 0.96[0.46,1.98] Feng Li(CD133+) 31 101 15 44 16.6% 0.86[0.40.1.82] Qing Li(ALDH1A1+) 10 29 10 21 8.7% 0.58[0.18,1.83] Qing Li (CD133+) 12 29 15 21 11.7% 0.28[0.08,0.94] Shiwu Wu(CD133+) 103 237 46 68 46.2% 0.37[0.21,0.65] Total (95%CI) 497 198 100.0% 0.56[0.40,0.78)] Total events 194 103 Heterogeneity:ChiP=6.66.df=4(P=0.15);12=40% 0.1 10 100 Test for overall effect:Z=3.36(P=0.0008) 0.01 W+M Poor © TNM1+I TNM IlI+IV Odds ratio Odds ratio Study or subgroup Events Total Events Total Weight M-H,random,95%CI M-H,random,95%CI Qing Li(ALDH1A1+) 9 33 11 17 28.1% 0.200.06,0.72 Qing Li(CD133+) 16 33 11 17 29.0% 0.51[0.15,1.72 Shiwu Wu (CD133+) 29140 120165 42.9% 0.10[0.06,0.17刀 Total (95%CI) 206 199100.0% 0.19[0.07,0.541 Total events 54 142 Heterogeneity:Tau2=0.56;Chi2=6.52,df=2(P=0.04);12=69% 0.01 0.1 10 100 Test for overall effect:Z=3.14 (P =0.002) TNMI+I TNM II+IV ⊙ LNM Non-LNM Odds ratio Odds ratio Study or subgroup Events Total Events Total Weight M-H,fixed,95%Cl M-H.fixed,95%Cl Qing Li (ALDH1A1+) 11 25 9 2519.3% 1.40[0.45,4.35] Qing Li (CD133+) 15 25 12 25 18.4% 1.63[0.53,4.981 Shiwu Wu(CD133+) 114 185 35 120 62.3% 3.90[2.38,6.38 Total (95%CI) 235 170100.0% 3.00[1.98,4.54] Total events 140 % Heterogeneity:Chi2=3.98,df=2(P=0.14);12=50% 0.01 0.1 10 100 Test for overall effect:Z=5.19 (P <0.00001) LNM Non-LNM Figure 5.Forest plots of CSC marker expression in lung cancer.(A)the result was not significant for association between CSC marker expression and histological type (p=0.94).However,there was a significant association between CSC marker expression and histological differentiation(B),clinical TNM stage (C)and lymph node metastasis(D)(p<0.05). AD:Adenocarcinoma;CSC:Cancer stem cell;LNM:Lymph node metastasis;W+M:Well-moderate. ifuture science group www.futuremedicine.com 10.2217/bmm-2016-0026

www.futuremedicine.com 10.2217/bmm-2016-0026 Figure 5. Forest plots of CSC marker expression in lung cancer. (A) the result was not significant for association between CSC marker expression and histological type (p = 0.94). However, there was a significant association between CSC marker expression and histological differentiation (B), clinical TNM stage (C) and lymph node metastasis (D) (p < 0.05). AD: Adenocarcinoma; CSC: Cancer stem cell; LNM: Lymph node metastasis; W + M: Well-moderate. 38 31 10 12 103 194 101 101 29 29 237 497 17 15 10 15 46 103 44 44 21 21 68 198 16.9% 16.6% 8.7% 11.7% 46.2% 100.0% 0.96 [0.46, 1.98] 0.86 [0.40, 1.82] 0.58 [0.18, 1.83] 0.28 [0.08, 0.94] 0.37 [0.21, 0.65] 0.56 [0.40, 0.78] 0.01 0.1 1 10 100 W + M Poor Feng Li (ABCG2+) Feng Li (CD133+) Qing Li (ALDH1A1+) Qing Li (CD133+) Shiwu Wu (CD133+) Total (95% CI) Total events Heterogeneity: Chi2 = 6.66, df = 4 (P = 0.15); I 2 = 40% Test for overall effect: Z = 3.36 (P = 0.0008) Study or subgroup Events W + M Poor Total Events Total Weight Odds ratio Odds ratio M-H, fixed, 95% CI M-H, fixed, 95% CI 9 16 29 54 33 33 140 206 11 11 120 142 17 17 165 199 28.1% 29.0% 42.9% 100.0% 0.20 [0.06, 0.72] 0.51 [0.15, 1.72] 0.10 [0.06, 0.17] 0.19 [0.07, 0.54] 0.01 0.1 1 10 100 TNM I + II TNM III + IV Qing Li (ALDH1A1+) Qing Li (CD133+) Shiwu Wu (CD133+) Total (95% CI) Total events Heterogeneity: Tau2 = 0.56; Chi2 = 6.52, df = 2 (P = 0.04); I 2 = 69% Test for overall effect: Z = 3.14 (P = 0.002) Study or subgroup Events TNM I + II TNM III + IV Total Events Total Weight Odds ratio Odds ratio M-H, random, 95% CI M-H, random, 95% CI 11 15 114 140 25 25 185 235 9 12 35 56 25 25 120 170 19.3% 18.4% 62.3% 100.0% 1.40 [0.45, 4.35] 1.63 [0.53, 4.98] 3.90 [2.38, 6.38] 3.00 [1.98, 4.54] 0.01 0.1 1 10 100 LNM Non-LNM Qing Li (ALDH1A1+) Qing Li (CD133+) Shiwu Wu (CD133+) Total (95% CI) Total events Heterogeneity: Chi2 = 3.98, df = 2 (P = 0.14); I 2 = 50% Test for overall effect: Z = 5.19 (P < 0.00001) Study or subgroup Events LNM Non-LNM Total Events Total Weight Odds ratio Odds ratio M-H, fixed, 95% CI M-H, fixed, 95% CI Feng Li (ABCG2+) Feng Li (CD133+) Qing Li (ALDH1A1+) Qing Li (CD133+) Shiwu Wu (CD133+) Total (95% CI) Total events Heterogeneity: Chi2 = 2.20, df = 4 (P = 0.70); I 2 = 0% Test for overall effect: Z = 0.07 (P = 0.94) 21 18 10 16 50 115 60 60 29 29 95 273 Study or subgroup Events AD N-AD Total Events Total Weight Odds ratio Odds ratio 0.01 0.1 1 10 100 AD N-AD M-H, fixed, 95% CI M-H, fixed, 95% CI 34 28 10 11 99 182 85 85 21 21 210 422 23.7% 21.1% 9.9% 7.4% 37.9% 100.0% 0.81 [0.41, 1.60] 0.87 [0.43, 1.78] 0.58 [0.18, 1.83] 1.12 [0.36, 3.45] 1.25 [0.77, 2.02] 0.99 [0.72, 1.36] future science group Relationship between microvessel density & cancer stem cells in tumor angiogenesis Systematic Review

Relationship between microvessel density cancer stem cells in tumor angiogenesis Systematic Review with regard to the six studies evaluating the relation-as resistance to current therapeutic regimens.Specific ship between CSCs and MVD in tumor transplanta- cell surface markers have been used for the identifi- tion animal models(Begg's p-value =0.06>0.05);the cation and isolation of CSCs in tumors.CD133 [431 funnel plot is shown in Figure 9. is a common stem cell surface marker expressed in CSCs.Additionally,CD133 expression levels are cor- Discussion related with the tumor-promoting ability of CSCs [441. The role of CSCs in tumor initiation,progression and CSCs have been found to express the marker ALDHI, metastasis [6]has been extensively validated in numer- and the CSC-enriched ALDHI'fraction of human ous tumor types,such as leukemia [37]and cancers of breast cancer cells plays a pivotal role in tumor angio- the colon [38],prostate [39]and pancreas [401.Recent genesis [31.Notably,cancer cells with stem cell-like studies [41.421 have shown that CSCs are responsible properties,that express markers of vascular endothe- for tumorigenicity,invasion and recurrence,as well lium [45]and form tumors,have been described.CSCs ④ W+M Poor Odds ratio Odds ratio Study or subgroup Events Total Events Total Weight M-H,fixed,95%Cl M-H,fixed.95%CI Yiming Song (ALDH1+/CD133+) 24 99 7 14 8.5%0.32[0.10,1.001 Yunqin Huang(CD133+) 42 75 21 30 12.1% 0.55[0.22,1.35] Zhengquan Han(CD133+) 125 235 42 90 26.0% 1.300.80.2.111 Zhengquan Han(CD44+) 116 235 34 90 22.8% 1.61[0.98.2.64 Zhengquan Han(CD82+) 134 235 54 90 30.7% 0.88[0.54,1.45] Total (95%CI) 879 314100.0%1.07[0.82,1.39] Total events 441 158 Heterogeneity:Chi2=10.15,df=4 (P=0.04);12=61% Test for overall effect:Z=0.49 (P =0.62) 0.01 0.1 10 100 W+M Poor ⑧ TNMI+II TNM III+IV Odds ratio Odds ratio Study or subgroup Events Total Events Total Weight M-H,random,95%Cl M-H,random,95%CI Yungin Huang(CD133+) 21 59 42 4620.3% 0.050.02,0.17可 Zhengquan Han(CD133+) 104 177 63 14826.6% 1.921.23.2.991 Zhengquan Han(CD44+) 91 177 59 14826.6% 1.60[1.03.2.48] Zhengquan Han(CD82+) 93 177 95 14826.5% 0.62[0.39,0.97 Total (95%CI) 590 490100.0% 0.65[0.24,1.76] Total events 309 259 Heterogeneity:Tau2=0.92;Chi2=42.28,df =3(P 0.00001):12=93% Test for overall effect:Z=0.84 (P=0.40) 0.01 0.1 10 100 TNMI+II TNM III+IV © LNM Non-LNM Odds ratio Odds ratio Study or subgroup Events Total Events Total Weight M-H,random,95%CI M-H,random,95%CI Yiming Song(ALDH1+/CD133+) 16 60 15. 6018.9% 1.09[0.48.2.47J Yunqin Huang (CD133+) 49 59 14 4618.2c% 11.20「4.44.28.271 Zhengquan Han(CD133+) 102 155 6517021.0% 3.11[1.98.4.89] Zhengquan Han(CD44+) 83 155 6717021.0% 1.77[1.14.2.75 Zhengquan Han(CD82+) 74 155 11417021.0% 0.45[0.29,0.70] Total (95%CI) 584 616100.0% 1.91[0.74,4.88] Total events 324 275 Heterogeneity:Tau2=1.04;Chi2=56.89,df=4(P0.05). CSC:Cancer stem cell;LNM:lymph node metastasis;W+M:Well-moderate. ifuture science group www.futuremedicine.com 10.2217/bmm-2016-0026

Systematic Review Lan,Li,Ju et al. 3 Begg's funnel plot with pseudo 95%confidence limits 2 1 0 0.0 0.2 0.4 0.6 s.e.of:SMD Figure 8.The Begg's test(with pseudo 95%Cl)showed no statistically significant evidence of publication bias for studies of human tumor tissues(Begg's p-value =0.107>0.05). SMD:Standardized mean difference. may contribute to tumor angiogenesis.Bao et al.[46] studies report the evaluation of MVD by immunohisto- have found that CSCs produce much higher levels of chemistry.Markers of endothelial cells,such as CD31 VEGF than non-CSCs,and that this CSC-mediated and CD34,are used for immunohistochemical stain- VEGF production leads to amplified endothelial cell ing and counting.A high MVD count is indicative of migration and tube formation in vitro.Fantozzi et al. tumors that are more prone to metastasis,and predic- [47]confirmed that CSCs produce VEGF and other tive of a poor prognosis [501.In addition,MVD count factors to induce angiogenesis.The identification and is indicative of the relationship between tumor angio- characterization of CSCs is critical for understanding genesis and CSCs.CSCs have been predicted to act as their function in angiogenesis during tumor growth critical drivers of tumor angiogenesis due to their self- and development.Therefore,elucidation of the rela- renewal and limitless proliferative potential,which indi- tionship between CSCs and angiogenesis is significant cate that these cells possess stronger angiogenic capacity for exploring the role of CSCs in tumor development.than tumor cells.Compared with the low expression Increasing evidence shows that neovasculariza-of CSCs markers,high expression of these markers is tion 9 plays an important role in tumor development correlated with increased MVD count.Recently,stud- and progression,and that angiogenesis is essential for ies (45]have shown that CD133'tumor cell population, further growth and expansion of tumors [71.In addi-derived from the human glioblastoma cell line,exhibits tion,CSCs that express markers of vascular endo-a significantly stronger capacity for promoting angio- thelium and form tumor blood vessels have been genesis than CD133 tumor cells.Evidence from these described.Recent studies demonstrated that CSCs studies indicates that CSCs play a critical role in tumor with the capacity of self-renewal and differentiation neovascularization. may promote tumorignesis.Interestingly,some studies The present meta-analysis systematically evaluates have suggested that CSCs initiate tumor neovascular-the association between angiogenesis and CSCs in ization.Kaur et al [48]have demonstrated that CSCs tumors by assessing MVD count.The significant cor- are capable of transdifferentiation into endothelial cells relation observed between CSCs and MVD in tumors, involved in tumor angiogenesis. in both human tumor tissue and tumor transplanta- MVD evaluation,the most commonly used tech-tion animal models,suggests that CSCs contribute to nique to assess the formation of new blood vessels tumor angiogenesis.As tumor angiogenesis may vary in tumor tissue,was developed by Weidner et al.in according to the specific tumor type,further subgroup 1991 1491;this technique utilizes immunohistochemical analyses were conducted and stratified based on tumor staining for detection of blood microvessels.Numerous type.Data from these analyses showed that CSCs occur 10.2217/bmm-2016-0026 Biomark.Med.(Epub ahead of print) future science group

10.2217/bmm-2016-0026 Biomark. Med. (Epub ahead of print) Figure 8. The Begg’s test (with pseudo 95% CI) showed no statistically significant evidence of publication bias for studies of human tumor tissues (Begg’s p-value = 0.107 > 0.05). SMD: Standardized mean difference. 1 2 0 -1 3 0.0 0.2 0.4 0.6 s.e. of: SMD SMD Begg’s funnel plot with pseudo 95% confidence limits future science group Systematic Review Lan, Li, Ju et al. may contribute to tumor angiogenesis. Bao et al. [46] have found that CSCs produce much higher levels of VEGF than non-CSCs, and that this CSC-mediated VEGF production leads to amplified endothelial cell migration and tube formation in vitro. Fantozzi et al. [47] confirmed that CSCs produce VEGF and other factors to induce angiogenesis. The identification and characterization of CSCs is critical for understanding their function in angiogenesis during tumor growth and development. Therefore, elucidation of the relationship between CSCs and angiogenesis is significant for exploring the role of CSCs in tumor development. Increasing evidence shows that neovascularization [9] plays an important role in tumor development and progression, and that angiogenesis is essential for further growth and expansion of tumors [7]. In addition, CSCs that express markers of vascular endothelium and form tumor blood vessels have been described. Recent studies demonstrated that CSCs with the capacity of self-renewal and differentiation may promote tumorignesis. Interestingly, some studies have suggested that CSCs initiate tumor neovascularization. Kaur et al. [48] have demonstrated that CSCs are capable of transdifferentiation into endothelial cells involved in tumor angiogenesis. MVD evaluation, the most commonly used technique to assess the formation of new blood vessels in tumor tissue, was developed by Weidner et al. in 1991 [49]; this technique utilizes immunohistochemical staining for detection of blood microvessels. Numerous studies report the evaluation of MVD by immunohistochemistry. Markers of endothelial cells, such as CD31 and CD34, are used for immunohistochemical staining and counting. A high MVD count is indicative of tumors that are more prone to metastasis, and predictive of a poor prognosis [50]. In addition, MVD count is indicative of the relationship between tumor angiogenesis and CSCs. CSCs have been predicted to act as critical drivers of tumor angiogenesis due to their selfrenewal and limitless proliferative potential, which indicate that these cells possess stronger angiogenic capacity than tumor cells. Compared with the low expression of CSCs markers, high expression of these markers is correlated with increased MVD count. Recently, studies [45] have shown that CD133+ tumor cell population, derived from the human glioblastoma cell line, exhibits a significantly stronger capacity for promoting angiogenesis than CD133- tumor cells. Evidence from these studies indicates that CSCs play a critical role in tumor neovascularization. The present meta-analysis systematically evaluates the association between angiogenesis and CSCs in tumors by assessing MVD count. The significant correlation observed between CSCs and MVD in tumors, in both human tumor tissue and tumor transplantation animal models, suggests that CSCs contribute to tumor angiogenesis. As tumor angiogenesis may vary according to the specific tumor type, further subgroup analyses were conducted and stratified based on tumor type. Data from these analyses showed that CSCs occur