research article expressions of abcg2, cd133, and podoplanin...

Research ArticleExpressions of ABCG2 CD133 and Podoplanin inSalivary Adenoid Cystic Carcinoma

Wuwei Li1 Ryo Tamamura2 Bo Wang3 Qigui Liu4 Han Liu1 Tingjiao Liu1

Naoki Katase5 Jing Xiao1 and Hitoshi Nagatsuka5

1 Medical College of Stomatology Dalian Medical University 9 Western Section Lvshun South Street Dalian 116044 China2Department of Histology Nihon University School of Dentistry at Matsudo 2-870-1 Sakaemachi-nishi Matsudo 271-8587 Japan3 Comprehensive Transplant Center Department of Surgery Feinberg School of Medicine Northwestern University300 E Superior Street Chicago IL 60611 USA

4Department of Health Statistics Dalian Medical University 9 Western Section Lvshun South Street Dalian 116044 China5 Department of Oral Pathology and Medicine Graduate School of Medicine Dentistry and Pharmaceutical SciencesOkayama University 2-5-1 Shikata-cho Okayama 700-8525 Japan

Correspondence should be addressed to Hitoshi Nagatsuka jinmdokayama-uacjp

Received 11 December 2013 Accepted 13 March 2014 Published 6 April 2014

Academic Editor Jun Liao

Copyright copy 2014 Wuwei Li et alThis is an open access article distributed under theCreative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Adenoid cystic carcinoma (ACC) is one of the most common salivary gland malignant tumors with a high risk of recurrence andmetastasis Current studies on cancer stem cells (CSCs) have verified that CSCs are the driving force behind tumor initiation andprogression suggesting that new cancer therapies may be established by effectively targeting and killing the CSCs The primarygoal of this study is to investigate the expression patterns of ABCG2 CD133 and podoplanin in ACC of minor salivary glands byimmunohistochemistry analysisWe found thatABCG2wasweakly expressed in normal looking salivary gland tissues A significantupregulation of ABCG2 expression in ACC was observed with a similar expression pattern of Ki-67 CD133 was detected in apicalmembrane of epithelial cells and podoplanin was expressed positively inmyoepithelial cells of both normal looking tissue andACCHowever no significant difference was found of the expression pattern of CD133 and podoplanin between normal looking tissuesand ACC Our observations suggest that CSCs may exist in quiescent cells with ABCG2 positive staining which are surrounded bycells with positive expression of ABCG2 and Ki-67 in ACC and costaining with ABCG2 and Ki-67 may help predict the locationof CSCs

1 Introduction

Adenoid cystic carcinoma (ACC) is one of the most com-mon malignant neoplasms that affects either the major orminor salivary glands of the oral cavity with high risk ofrecurrence and metastasis [1] About 50 of ACCs occur inthe hard palate and rarely take place in other intraoral sitesincluding the lower lip retromolartonsillar pillar regionsublingual gland and buccal mucosa [2] Currently surgeryand radiotherapy are the most effective treatments but theoutcomes remain unsatisfying [3] Inmany cases ACC showsan indolent clinical course with a considerable risk of localrelapse and late distant metastases to lung or bone whichoccasionally causes fatal outcome after the first episode [3]

The hypothesis of cancer stem cells states that the tumorformation and growth likely depend on a small subset oftumor cells known as cancer stem cells (CSCs) or tumor-initiating cells (TICs) which might be the origin of neoplasia[4] CSCs have been isolated from some types of solidtumors such as breast brain prostate melanoma colonpancreas headneck liver and gastric cancers [5ndash8] andthey are recognized to be more resistant to radiotherapyand chemotherapy and additionally invasive than normaltumor cells If CSCs can be targeted by specific CSC markersand killed efficiently the outcomes of current clinical cancertreatments may be improved [9] Several putative stem cellmarkers such as CD44 CD24 CD166 EpCAM ABCG2CD133 and podoplanin are commonly studied in identifying

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 132349 11 pageshttpdxdoiorg1011552014132349

2 BioMed Research International

and isolating the CSCs from the solid tumors howeversubsequent studies demonstrated that the choice of CSCmarkers differs among cancer originations [10ndash15]

ATP-binding cassette subfamily G member 2 (ABCG2)is a member of the superfamily of ATP-binding cassettetransporter proteins Immunohistochemical analysis showedthatABCG2was expressed ubiquitously in normal and tumortissues [16] Zhou et al [17] found that the expression ofABCG2 gene is an important determinant and a molecularmarker for side population-enriched stem cells (SP cells)and this feature was considered to be related to CSCs [18]Kim et al also reported that ABCG2 is highly expressedin SP cells and its expression is strongly correlated withSP phenotype [10] ABCG2 can be expressed in stem cellsisolated from both normal and tumor tissues which furtherindicates its essential role in stem cell biology [19]

CD133 (prominin-1) a cell-surface glycoprotein carryingfive transmembrane domains was firstly identified as amarker for a subpopulation of CD34-positive haematopoieticstem cells which are derived from human fetal liver fetalbone marrow and peripheral blood [20] CD133 was initiallyregarded as a specific marker of hematopoietic stem celland later found in neural stem cells epidermal stem cellsand endothelial progenitor cells (EPCs) [17 21 22] Previousstudies have identified and isolated a putativeCSCpopulationfrom the brain [23] prostate [24] liver [25] lung [26] andcolon [27] cancers using CD133 antibodies In oral CSC-like cells isolated from primary tumor cells and cell linesscientists found an improved migration and tumorigenicitybehavior of these cells with a higher CD133 expressionlevel [28] CD133-positive cells also showed an enhancedclonogenicity invasiveness and tumorigenicity compared tothose CD133-negative cells [29]

Podoplanin is a mucin-like transmembrane glycoproteinthat is highly and specifically expressed in lymphatic endo-thelial cells of normal tissues and tumors [30] Recent stud-ies have reported that podoplanin-positive cells in tumors actas mediators of metastasis and invasion [31ndash33] Podoplaninis also reported as a potential CSC marker for squamous-cell carcinoma (SCC) Atsumi et al found that A431 cells(a SCC cell line) consisted of both podoplanin-positive andpodoplanin-negative cells in which podoplanin-negativecells only produced podoplanin-negative cells but podo-planin-positive cells generated both podoplanin-positive and-negative cells and podoplanin-positive cells showed highertumorigenicity [34] Rahadiani et al reported that the eso-phageal squamous-cell carcinoma (ESCC) showed defectiveinvasion and tumorigenic activities after the podoplaninexpression were knocked down in esophageal [35]

Relative studies in identifying CSCs markers in ACCare still in the early stage In this study we examined theexpression patterns of ABCG2 CD133 and podoplanin inACC of minor salivary glands by immunohistochemicalanalysis Meanwhile Ki-67 a nuclear protein associated withcellular proliferation was used to evaluate the proliferation ofthe tumor cells

2 Materials and Methods

21 Tissue Samples Archival paraffin-embedded tissueblocks of 25 ACC cases (9 men 16 women mean age 6072years range 32ndash78 years) were randomly selected Tenof these samples included normal looking tissues Thesesamples were diagnosed at Okayama University HospitalSurgical Pathology Unit (Okayama Japan) from 1980 to2005 All specimens were fixed with 10 neutral bufferedformalin and embedded in paraffin Histological diagnosiswas carried out by routine hematoxylin and eosin (H and E)staining according to World Health Organization (WHO)histological typing of ACC Clinical and histopathologicalcharacteristics of the patients are shown in Table 1 Tennormal looking salivary gland tissues were used as thecontrol group in this study Informed consents were obtainedfrom the patients and the experiment was approved by theResearch Ethics Board of Okayama University

22 Immunohistochemistry Staining Antibodies againstABCG2 (BXP-21) (1 50 mouse monoclonal Abcam Cam-bridge UK) CD133 (1 300 rabbit polyclonal Abcam)podoplanin (1 50 mouse monoclonal DAKO CarpinteriaCA) and Ki-67 (1 50 mouse monoclonal DAKO) were usedin this study

For immunohistochemical staining serial sections of4 120583m thickness were deparaffinized in xylene and rehydratedin decreasing concentrations of ethanol Endogenous per-oxidase activity was blocked by immersing the sections in3 H

2O2with methanol for 30min For antigen retrieval

sections were boiled in 10mmolL citrate buffer (pH 60)for 15min in a pressure cooker After treatment with proteinblock serum at room temperature sections were coveredwith primary antibodies and incubated at 4∘C overnight Forsecondary antibody DAKO Cytomation Envision+System-HRP kit (AEC) was used according to the manufacturerrsquosinstructions Antibody reactions were stained with 331015840-diaminobenzidine and counterstained with hematoxylin Forthe negative control sections were incubated with PBSinstead of the primary antibodies

23 Assessment Standard ABCG2 CD133 and podoplaninwere detected in the cytoplasm andor cell membrane Tenareas were selected randomly in each section under highpower magnification (times400) Also 100 cells were selectedrandomly in each area so that a total of 1000 cells wereanalyzed for counting the positive cells Positive stainingintensity for cancer stem cell markers was classified as nega-tive minus (lt30) weak + (30ndash49) moderate ++ (50ndash74)and strong staining +++ (ge75) For Ki-67 staining thenucleus was stained with brown color and positive stainingintensity was classified as negative minus (lt5) weak + (5ndash9)moderate ++ (10ndash14) and strong staining +++ (ge15)No false positive or negative staining was encountered in thepositive or negative controls respectively

24 Statistical Analysis Statistical analyses were performedwith SPSS version 180 for Windows Wilcoxon test was used

BioMed Research International 3

Table 1 Clinical and histopathological data of 25 patients with ACC

Number Gender Age (years) Primary site Histological pattern1 F 78 Mouth floor Cribriform2 F 62 Mouth floor Cribriform3 F 42 Mouth floor Cribriform4 F 70 Mouth floor Cribriform5 F 78 Mouth floor Cribriform6 F 65 Mouth floor Cribriform7 F 58 Mouth floor Cribriform with tubular8 F 66 Mouth floor Cribriform with tubular9 M 67 Mouth floor Cribriform with solid10 F 46 Mouth floor Cribriform with solid11 M 57 Mouth floor Cribriform with solid12 M 75 Palate Cribriform13 M 77 Palate Cribriform14 M 62 Palate Cribriform15 F 71 Palate Cribriform16 F 66 Palate Cribriform17 F 34 Palate Cribriform18 F 71 Palate Cribriform19 F 32 Palate Cribriform20 M 54 Palate Cribriform with tubular21 M 53 Palate Cribriform with tubular22 F 51 Palate Cribriform with tubular23 M 52 Palate Cribriform with solid24 M 65 Buccal mucosa Cribriform25 F 66 Buccal mucosa Cribriform

to determine the intensity of marker expression and Fisherrsquosexact test was used to determine the positive percentageof tumor cells and clinicopathological characteristics Cor-relations between different markers and Ki-67 expressionintensities in different locations of ACC and normal lookingtissues were assessed by Spearmanrsquos rank correlation analysisAll tests were two-tailed and the differences were consideredstatistically significant when 119875 lt 005

3 Results

31 Expression of ABCG2 CD133 Podoplanin and Ki-67 inNormal Looking Tissues Immunopositivity for ABCG2 wasobserved in the cytoplasm and cell membrane of acinarepithelium cells ductal cells and myoepithelial cells in 6cases and the staining intensities were weak in all 6 samples(Figure 1(a) Table 2) A small number of ABCG2-positivecells were present in the capillary endothelium CD133-positive expressions were localized only in apical membraneof acinar epithelial cells and ductal cells in all 10 samples(Figure 1(b)) in which 5 cases showed moderate stainingintensity and 5 cases showed strong intensity Podoplanin-positive expressions were found in the cytoplasm and cellmembrane of myoepithelial cells in just 1 case with weakstaining intensity (Figure 1(c)) Expression of Ki-67 wasnegative in all 10 cases (Figure 1(d))

32 Expression of ABCG2 CD133 Podoplaninand Ki-67 in ACC

ABCG2 ABCG2-positive stainings could be detected in 23cases including 1 case of weak staining intensity 7 casesof moderate intensity and 15 cases of strong intensity andthe positive expression percentage was 92 (119875 = 0043)(Table 2) The tumor cells showed strong ABCG2 stainingin the invasive front of cancer nests and the intensity inperipheral zone was stronger than the center zone Theintensity of ABCG2 staining was significantly stronger inACC than in the normal looking tissues (119875 = 0000)

In the cribriform pattern of ACC ABCG2 staining wasobserved on both cytoplasm and membrane of the epithelialcells The highest intensity of ABCG2 staining was mainlylocalized in the periphery of cancer nests (Figure 2(a))A similar pattern of ABCG2-positive staining in stromalvascular endothelial cells was also observed in the normallooking salivary gland tissues In tubular pattern ABCG2was expressed in glandular epithelial cells (Figure 3(a)) Insolid pattern the myoepithelial cells were diffusely positivefor ABCG2 and the highest intensity of ABCG2 staining wasfound focally in both periphery and center of cancer nests(Figure 4(a))

CD133 CD133-positive cells were detected in 25 cases (1case with weak intensity 12 cases with moderate intensity


Table2Ex

pressio

nsof

ABC

G2CD

133po

doplaninand

Ki-67in

norm

allook

ingtissues

andAC

C

Group

s119899

ABC

G2intensity

Positivity

()

CD133intensity

Positivity

()

Podo

planin

intensity

Positivity

()

Ki-67intensity

Positivity

()

minus+

++++

+minus

+++

+++

minus+

++++

+minus

+++

+++

Normal

104

60

060

00

55

100

91

00

1010

00

00

ACC

252

17

1592

01

1212

100

194

11

2410

43

860

119875000

0lowast0043lowast

0835

1000

0332

064

40001lowast

0001lowast

Normalnormallook

ingtissuesIntensityminusnegativ

e+weakstaining

++mod

erates

tainingand++

+str

ongstaining

lowastdeno

tessignificantd

ifference

whencomparedwith

then

ormallookingtissue(119875lt005)


ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)

Figure 1 Expression of (a) ABCG2 (b) CD133 (c) podoplanin and (d) Ki-67 in normal looking tissues

ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)

Figure 2 Expression of (a) ABCG2 (b) CD133 (c) podoplanin and (d) Ki-67 in ACC of cribriform pattern


ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)

Figure 3 Expression of (a) ABCG2 (b) CD133 (c) podoplanin and (d) Ki-67 in ACC of tubular pattern

ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)

Figure 4 Expression of (a) ABCG2 (b) CD133 (c) podoplanin and (d) Ki-67 in ACC of solid pattern


Cribriform pattern

ABCG2 Ki-67

(a)

Solid pattern

ABCG2 Ki-67

(b)

ABCG2

Ki-67

(c)

ABCG2

Ki-67

(d)

Figure 5 Positive expressions of ABCG2 and Ki-67 by the tumor cells were shown in the same area The double positive stained cells wereconcentrated mainly in the peripheral zone of the cribriform pattern and also in the periphery and central zones of the solid pattern Only afew strongly positive ABCG2 cells were negative for Ki-67 (scale bar 50 120583m)

and 12 cases with strong intensity) but limited in the apicalmembrane of the luminal cells in cribriform (Figure 2(b))and tubular (Figure 3(b)) patternsThe contents in the ductalstructures and pseudocyst spaces were positive for CD133 Insolid pattern positive expression was mainly diffused amongthe dense myoepithelial cells (Figure 4(b)) There was nosignificant difference of CD133 intensity or positivity betweenACC and normal looking tissues (119875 = 0835 and 119875 = 1000resp)

Podoplanin Podoplanin expressions were detected in 6 casesof ACC including 4 cases with weak staining intensity 1 casewith moderate intensity and 1 case with strong intensity andthe average positive percentage was 24 (119875 = 0644) Theintensity in ACC showed no significant difference comparedwith the normal looking tissues (119875 = 0332) Podoplanincan be only observed in myoepithelial cells and lymphaticendothelial cells Inmost of the samples of cribriformpatternpodoplanin showed negative staining (Figure 2(c)) In tubu-lar pattern of ACC expressions of podoplanin were mainlydetected in myoepithelial cells but not in glandular epithelialcells (Figure 3(c)) Negative podoplanin expressions weredetected in all solid pattern samples (Figure 4(c))

Ki-67 Positive expressions of Ki-67 were found in 15 ACCsamples including 4 cases with weak staining intensity 3cases with moderate intensity and 8 cases with strong

Table 3 The correlation information of ABCG2 CD133 andpodoplanin in normal looking tissues

CD133 Podoplanin119903119904

119875 119903119904

119875

ABCG2 0298 0403 0124 0732CD133 mdash mdash minus0333 0347

intensity The average positive percentage was about 60(119875 = 0001) which was significantly stronger comparedto the normal looking tissues (119875 = 0001) The Ki-67-positive stainings separated broadly in the tumor tissues ofcribriform (Figure 2(d)) and solid pattern (Figure 4(d)) Wealso noticed that most cells overlaid with ABCG2 and Ki-67were mainly found in the same location of the tumor tissueA few cells expressing high level of ABCG2 with Ki-67-negative expression were surrounded by cells expressing bothABCG2 and Ki-67 (Figure 5)

33 Statistical Results The distribution correlation ofABCG2 and Ki-67 in ACC was summarized in Figure 5We also compared the expression correlations of differentmarkers between ACC and normal looking tissues (Table 2)To further identify the expression correlations amongABCG2 CD133 podoplanin and Ki-67 in normal looking


Table 4 The correlation information of ABCG2 CD133 podoplanin and Ki-67 in ACC

CD133 Podoplanin Ki-67119903119904

119875 119903119904

119875 119903119904

119875

ABCG2 minus0320 0119 0155 0459 0620 0001lowast

CD133 mdash mdash 0237 0254 minus0340 0096Podoplanin mdash mdash mdash mdash 0069 0743lowastdenotes significant difference (119875 lt 005)

tissues and ACC we summarized the expression patterns ofeach marker in Tables 3 and 4 In normal looking tissuesthere were no significant correlations between the twomarkers (119875 gt 005) (Table 3) InACC onlyABCG2 andKi-67showed a significant correlation (119903

119904= 0620 and 119875 = 0001)

and no other significant associations were found betweenany two of the four markers (119875 gt 005) (Table 4)

4 Discussion

To date growing evidence has identified that CSC is thedriving force of tumor initiation and progression in severaltumor types [36 37] If CSCs can be identified and isolatedfrom tumors using specific markers new therapies may beestablished by effectively targeting the CSCs and thereforemay improve the clinical outcomes of cancer treatment

In this study we characterized the expression patternsof ABCG2 CD133 and podoplanin in ACC of minor sali-vary glands by immunohistochemistry staining We founda significantly higher expression of ABCG2 in ACC thanin normal looking salivary gland tissues We also exploitedKi-67 to detect the proliferative activities of tumor cells inACC and we noticed that ABCG2 was also expressed in thesame location with Ki-67-positive cells These overlaid cellswere mainly located in the peripheral zone of the cancernest in cribriform pattern and in the periphery and centralregions of the cancer nest in solid subtypeMore interestinglywe noticed that a few cells with strong staining of ABCG2but negative staining with Ki-67 were usually surroundedby the cells expressing both ABCG2 and Ki-67 ABCG2 iswidely expressed in many normal vital organs such as liverlung spleen kidney brain nerve skeletal muscle cardiacmuscle testis pancreas small intestine and trachea and alsoin a variety of tumors without tissue specificity [38 39]Meanwhile ABCG2 shows dominantmembrane staining in awide range of stem cells [40] and is recognized as a universalmarker for stem cells [41] In this study we found a diffusionexpression pattern ofABCG2 inACCand the expression levelin ACC is significantly higher than that in normal lookingtissues which suggested that ABCG2might be involved in thedevelopment process of ACC and played a role in enhancingthe proliferation and invasion capabilities of cancer cellsCompared to the expression pattern of Ki67 we also noticedthat most ABCG2-positive cells also had positive stainingwith Ki-67 and mainly located in the periphery of cribriformcancer nests and in both periphery and central zones of solidcancer nests

It is known that Ki-67 as a marker of cell proliferationis absent in quiescent cells but has universal expression

in proliferating cells [42] According to the CSC theoryCSCs are rare and quiescent and have the potential of self-renewing and maintaining tumor growth and heterogeneity[43] Some studies also reported that the Ki-67-positive cellsare less likely to contain cancer stem-like cells due to theirhigh degree of proliferation and differentiation activity [41]Conversely ABCG2 is found to be overexpressed in SP stemcells isolated form bone marrow [44] and a number ofestablished cancer cell lines as well as tumor samples [45ndash47] ABCG2 is also speculated to be critical in maintaininga quiescent state of the stem cells although the mechanismis still unclear [48] From our results a small number ofcells with the ABCG2 strongest staining and Ki-67-negativestaining (ABCG2+++Ki-67minus) could be repetitively foundto be surrounded with cells expressing both ABCG2 andKi-67 We speculate that cancer stem-like cells may existin the cells with ABCG2+++Ki-67minus staining which aresurrounded by cells expressing both ABCG2 and Ki-67 inACC and ABCG2 may play a role in the process of cellularproliferation and development in ACC Our findings suggestthat costaining with ABCG2 and Ki-67 may help predictthe location of CSCs in ACC Besides the statistical analysisbased on the total cases of ACC analyzed in this studyshowed a significant correlation between ABCG2 and Ki-67 which can further support our above findings Howeverour sample size is not big enough for the statistical analysisaccording to the sample varies based on the clinical andhistopathological classifications More samples are needed inthe future studies to identify the above findings based onsample clinical and histopathological varies and additionallydetermine the mechanism of their correlations

We also explored the expression patterns of ABCG2CD133 and podoplanin in normal looking and ACC tissuesWe found that ABCG2 and CD133 had a very similar posi-tivity in ACC (92 and 100 resp) however the positivestaining distributions of ABCG2 and CD133 in ACC weredifferent In the cribriform pattern of ACC ABCG2 stainingswere observed in cytoplasm andmembrane of epithelial cellsIn the tubular pattern ABCG2 was expressed in glandularepithelial cells In the solid pattern the myoepithelial cellswere diffusely positive for ABCG2 CD133 staining couldonly be found in the luminal cells of cribriform and tubularpatterns In the solid pattern CD133-positive expression wasmainly diffused among the dense myoepithelial cells Therewas no significant correlation between CD133 and ABCG2 orKi-67 based on their staining intensity and location CD133has been studied in recent years as a specific surface moleculein detecting CSCs and CD133-positive cancer cells whichare also found possessing many stem cell characteristics


[7 25 49] Its attentiveness as a cancer stem cell markerhas recently grown dramatically in breast cancer [17 50]brain cancer [21] prostate cancer [22] pancreatic cancer [22]kidney cancer [51] and also in hepatocarcinoma [25] andmelanoma [52] In our study we also noticed a similar expres-sion pattern of CD133 in the normal looking tissues of humanminor salivary glands as reported in the mammary glandspancreas and liver [53] However the CD133 expressionpattern and intensity were similar in both normal lookingtissues and in ACC from our study so that no definitiveCD133-positive tumor cells could be distinguished in ourACC samples

Podoplanin has also been investigated for its potential as aCSCmarker in the solid tumors in recent studies Podoplaninis found in various neoplasms such as squamous-cell carci-noma [54] germ cell tumors [55] mesothelioma [56] andsome subtypes of vascular tumors Podoplanin is also con-sidered to be involved in the process of tumor cell migrationinvasion and metastasis From our results podoplanin onlyexhibited weak positive staining in the cytoplasm and cellmembrane of myoepithelial cells of normal looking tissuesand cribriform and tubular patterns of ACC but showednegative staining in solid pattern of ACC Furthermorewe did not find any significant difference of podoplaninexpressions between the normal looking tissues and ACCAll these results are not sufficient enough to support thatCD133 or podoplanin can detect cancer stem cells in ACCWe speculate that a more accurate and specific way to quicklylocate and identify the CSCs with clinical carcinoma samplesis to target the candidate cells by costaining with multipleCSC markers If CSCs can be successfully located anticancerdrugs can be precisely delivered into the cancer nest throughtissue engineering technologies and the efficiency of thecurrent clinical treatments can be significantly improvedTherefore finding out the expression patterns and correla-tions of the CSC surface markers with immunohistologicalstudies is an important and initial step towards this goalIn our study we did not find out any significant associa-tions among the investigated markers (ABCG2 CD133 andpodoplanin) in ACC More efforts will be put in exploringthe expression patterns and correlations of other commonlyused CSC markers such as CD44 CD24 CD29 CD90 andaldehyde dehydrogenase 1 (ALDH1) inACCofminor salivaryglands Still whether CD133 or podoplanin can be selectedas a specific marker of CSCs in ACC and its biologicaland functional contribution to tumorigenesis needs to beanswered in further investigations

5 Conclusion

In conclusion the expression of ABCG2 showed more sig-nificant upregulation in ACC than in normal looking tissuesand it showed a similar expression pattern with Ki-67 inACC We speculate that the cancer stem-like cells may existwith ABCG2+++Ki-67minus staining which are surrounded bycells with positive expression of ABCG2 and Ki-67 in ACCCostaining with ABCG2 and Ki-67 may help predict thelocation of CSCs in ACC

In future studies we will study more clinical samplesbased on clinical and histopathological classification to findout the relationships between the expression level of ABCG2and other CSC markers and the prognosis and clinicalimplications of ACC patients We will explore the expressionpatterns and correlations of other surface markers that arecommonly used in identifying CSCs such as CD44 CD24CD29 CD90 and aldehyde dehydrogenase 1 (ALDH1) inACC of minor salivary glands

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by JSPS KAKENHI Grant nos24592766 and 25463084The authors thank Japanese Interna-tional Cooperation Agency (JICA) for the financial supportand also all the faculty members in Department of OralPathology andMedicine Graduate School of Medicine Den-tistry and Pharmaceutical Sciences of Okayama Universityfor their support in experimental samples and facilities

References

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[2] P J Giannini K V Shetty S L Horan W D Reid and L LLitchmore ldquoAdenoid cystic carcinoma of the buccal vestibule acase report and review of the literaturerdquo Oral Oncology vol 42no 10 pp 1029ndash1032 2006

[3] J D Casler and J J Conley ldquoSurgical management of adenoidcystic carcinoma in the parotid glandrdquo OtolaryngologymdashHeadand Neck Surgery vol 106 no 4 pp 332ndash338 1992

[4] I L Weissman ldquoStem cells units of development units ofregeneration and units in evolutionrdquo Cell vol 100 no 1 pp157ndash168 2000

[5] J Lessard andG Sauvageau ldquoBmi-1 determines the proliferativecapacity of normal and leukaemic stem cellsrdquo Nature vol 423no 6937 pp 255ndash260 2003

[6] M Al-Hajj M S Wicha A Benito-Hernandez S J Morrisonand M F Clarke ldquoProspective identification of tumorigenicbreast cancer cellsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 7 pp 3983ndash3988 2003

[7] S K Singh I D Clarke M Terasaki et al ldquoIdentification of acancer stem cell in human brain tumorsrdquo Cancer Research vol63 no 18 pp 5821ndash5828 2003

[8] S Takaishi T Okumura S Tu et al ldquoIdentification of gastriccancer stem cells using the cell surface marker CD44rdquo StemCells vol 27 no 5 pp 1006ndash1020 2009

[9] J Burkert N A Wright and M R Alison ldquoStem cells andcancer an intimate relationshiprdquo Journal of Pathology vol 209no 3 pp 287ndash297 2006

[10] M Kim H Turnquist J Jackson et al ldquoThe multidrug resis-tance transporter ABCG2 (breast cancer resistance protein 1)


effluxes Hoechst 33342 and is overexpressed in hematopoieticstem cellsrdquo Clinical Cancer Research vol 8 no 1 pp 22ndash282002

[11] I L Botchkina R A Rowehl D E Rivadeneira et al ldquoPhe-notypic subpopulations of metastatic colon cancer stem cellsgenomic analysisrdquo Cancer Genomics and Proteomics vol 6 no1 pp 19ndash30 2009

[12] E CAndersonCHessman TG LevinMMMonroe andMHWong ldquoThe role of colorectal cancer stem cells in metastaticdisease and therapeutic responserdquoCancers vol 3 no 1 pp 319ndash339 2011

[13] L Du H Wang and L He ldquoCD44 is of functional importancefor colorectal cancer stem cellsrdquo Clinical Cancer Research vol14 no 21 pp 6751ndash6760 2008

[14] E L-H Leung R R Fiscus J W Tung et al ldquoNon-small celllung cancer cells expressing CD44 are enriched for stem cell-like propertiesrdquoPLoSONE vol 5 no 11 Article ID e14062 2010

[15] H J Lee G Choe S Jheon S-W Sung C-T Lee and J-HChung ldquoCD24 a novel cancer biomarker predicting disease-free survival of non-small cell lung carcinomas a retrospectivestudy of prognostic factor analysis from the viewpoint offorthcoming (Seventh) New TNM classificationrdquo Journal ofThoracic Oncology vol 5 no 5 pp 649ndash657 2010

[16] P A Fetsch A Abati T Litman et al ldquoLocalization of theABCG2 mitoxantrone resistance-associated protein in normaltissuesrdquo Cancer Letters vol 235 no 1 pp 84ndash92 2006

[17] S Zhou JD Schuetz KD Bunting et al ldquoTheABC transporterBcrp1ABCG2 is expressed in a wide variety of stem cells andis a molecular determinant of the side-population phenotyperdquoNature Medicine vol 7 no 9 pp 1028ndash1034 2001

[18] A-M Bleau D Hambardzumyan T Ozawa et al ldquoPTENPI3KAkt pathway regulates the side population phenotype andABCG2 activity in glioma tumor stem-like cellsrdquo Cell Stem Cellvol 4 no 3 pp 226ndash235 2009

[19] X-W Ding J-H Wu and C-P Jiang ldquoABCG2 a potentialmarker of stem cells and novel target in stem cell and cancertherapyrdquo Life Sciences vol 86 no 17-18 pp 631ndash637 2010

[20] S Miraglia W Godfrey A H Yin et al ldquoA novel five-transmembrane hematopoietic stem cell antigen isolationcharacterization and molecular cloningrdquo Blood vol 90 no 12pp 5013ndash5021 1997

[21] T Chiba K Kita Y-W Zheng et al ldquoSide population purifiedfrom hepatocellular carcinoma cells harbors cancer stem cell-like propertiesrdquo Hepatology vol 44 no 1 pp 240ndash251 2006

[22] M D Brown P E Gilmore C A Hart et al ldquoCharacterizationof benign and malignant prostate epithelial Hoechst 33342 sidepopulationsrdquo Prostate vol 67 no 13 pp 1384ndash1396 2007

[23] S K Singh C Hawkins I D Clarke et al ldquoIdentification ofhuman brain tumour initiating cellsrdquo Nature vol 432 no 7015pp 396ndash401 2004

[24] A T Collins P A Berry C Hyde M J Stower and N JMaitland ldquoProspective identification of tumorigenic prostatecancer stem cellsrdquo Cancer Research vol 65 no 23 pp 10946ndash10951 2005

[25] S Yin J Li C Hu et al ldquoCD133 positive hepatocellularcarcinoma cells possess high capacity for tumorigenicityrdquo Inter-national Journal of Cancer vol 120 no 7 pp 1444ndash1450 2007

[26] A Eramo F Lotti G Sette et al ldquoIdentification and expansionof the tumorigenic lung cancer stem cell populationrdquoCell Deathand Differentiation vol 15 no 3 pp 504ndash514 2008

[27] M TodaroM P Alea A BDi Stefano et al ldquoColon cancer stemcells dictate tumor growth and resist cell death by production ofinterleukin-4rdquo Cell Stem Cell vol 1 no 4 pp 389ndash402 2007

[28] S-H Chiou C-C Yu C-Y Huang et al ldquoPositive correlationsof Oct-4 and Nanog in oral cancer stem-like cells and high-grade oral squamous cell carcinomardquo Clinical Cancer Researchvol 14 no 13 pp 4085ndash4095 2008

[29] Q Zhang S Shi Y Yen J Brown J Q Ta and A D Le ldquoAsubpopulation of CD133+ cancer stem-like cells characterizedin human oral squamous cell carcinoma confer resistance tochemotherapyrdquo Cancer Letters vol 289 no 2 pp 151ndash160 2010

[30] M Kreppel M Scheer U Drebber L Ritter and J E ZollerldquoImpact of podoplanin expression in oral squamous cell car-cinoma clinical and histopathologic correlationsrdquo VirchowsArchiv vol 456 no 5 pp 473ndash482 2010

[31] E Martın-Villar F G Scholl C Gamallo et al ldquoCharacteri-zation of human PA226 antigen (T1120572-2 podoplanin) a smallmembrane mucin induced in oral squamous cell carcinomasrdquoInternational Journal of Cancer vol 113 no 6 pp 899ndash910 2005

[32] E Martın-Villar D Megıas S Castel M M Yurrita S Vilaroand M Quintanilla ldquoPodoplanin binds ERM proteins to acti-vate RhoA and promote epithelial-mesenchymal transitionrdquoJournal of Cell Science vol 119 no 21 pp 4541ndash4553 2006

[33] K Mishima Y Kato M K Kaneko R Nishikawa T Hiroseand M Matsutani ldquoIncreased expression of podoplanin inmalignant astrocytic tumors as a novel molecular marker ofmalignant progressionrdquo Acta Neuropathologica vol 111 no 5pp 483ndash488 2006

[34] N Atsumi G Ishii M Kojima M Sanada S Fujii and AOchiai ldquoPodoplanin a novel marker of tumor-initiating cellsin human squamous cell carcinoma A431rdquo Biochemical andBiophysical Research Communications vol 373 no 1 pp 36ndash412008

[35] N Rahadiani J-I Ikeda T Makino et al ldquoTumorigenic role ofpodoplanin in esophageal squamous-cell carcinomardquo Annals ofSurgical Oncology vol 17 no 5 pp 1311ndash1323 2010

[36] T Reya S J Morrison M F Clarke and I L Weissman ldquoStemcells cancer and cancer stem cellsrdquo Nature vol 414 no 6859pp 105ndash111 2001

[37] H Kitamura K Okudela T Yazawa H Sato and H Shimoya-mada ldquoCancer stem cell Implications in cancer biology andtherapy with special reference to lung cancerrdquo Lung Cancer vol66 no 3 pp 275ndash281 2009

[38] M Maliepaard G L Scheffer I F Faneyte et al ldquoSubcellularlocalization and distribution of the Breast Resistance ProteinTransporter in normal human tissuesrdquo Cancer Research vol 61no 8 pp 3458ndash3464 2001

[39] M Huls C D A Brown A S Windass et al ldquoThe breastcancer resistance protein transporter ABCG2 is expressed inthe human kidney proximal tubule apical membranerdquo KidneyInternational vol 73 no 2 pp 220ndash225 2008

[40] J E Trosko and R J Ruch ldquoCell-cell communication incarcinogenesisrdquo Frontiers in Bioscience vol 3 pp D208ndashD2361998

[41] G A Sathi R Tamamura H Tsujigiwa et al ldquoAnalysis ofimmunoexpression of common cancer stem cell markers inameloblastomardquo Experimental andTherapeutic Medicine vol 3no 3 pp 397ndash402 2012

[42] J H Van Dierendonck R Keijzer C J H Van de Velde and CJ Cornelisse ldquoNuclear distribution of the Ki-67 antigen duringthe cell cycle comparisonwith growth fraction in human breast


cancer cellsrdquo Cancer Research vol 49 no 11 pp 2999ndash30061989

[43] V Tirino V Desiderio F Paino et al ldquoCancer stem cells in solidtumors an overview and new approaches for their isolation andcharacterizationrdquo The FASEB Journal vol 27 no 1 pp 13ndash242013

[44] S Zhou J J Morris Y Barnes L Lan J D Schuetz and BP Sorrentino ldquoBcrp1 gene expression is required for normalnumbers of side population stem cells in mice and confersrelative protection to mitoxantrone in hematopoietic cells invivordquo Proceedings of the National Academy of Sciences of theUnited States of America vol 99 no 19 pp 12339ndash12344 2002

[45] C Hirschmann-Jax A E Foster G G Wulf et al ldquoA distinctldquoside populationrdquo of cells with high drug efflux capacity inhuman tumor cellsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 101 no 39 pp14228ndash14233 2004

[46] N Haraguchi H Ishii K Mimori et al ldquoCD13 is a therapeutictarget in human liver cancer stem cellsrdquo Journal of ClinicalInvestigation vol 120 no 9 pp 3326ndash3339 2010

[47] M Olempska P A Eisenach O Ammerpohl H UngefrorenF Fandrich and H Kalthoff ldquoDetection of tumor stem cellmarkers in pancreatic carcinoma cell linesrdquo Hepatobiliary andPancreatic Diseases International vol 6 no 1 pp 92ndash97 2007

[48] O Tsinkalovsky A O Vik-Mo S Ferreira O D Laerumand A Fjose ldquoZebrafish kidney marrow contains ABCG2-dependent side population cells exhibiting hematopoietic stemcell propertiesrdquo Differentiation vol 75 no 3 pp 175ndash183 2007

[49] H You W Ding and C B Rountree ldquoEpigenetic regulation ofcancer stem cell marker CD133 by transforming growth factor-120573rdquo Hepatology vol 51 no 5 pp 1635ndash1644 2010

[50] T Kondo T Setoguchi and T Taga ldquoPersistence of a smallsubpopulation of cancer stem-like cells in the C6 glioma celllinerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 101 no 3 pp 781ndash786 2004

[51] N H Bander C L Finstad C Cordon-Cardo et al ldquoAnalysisof a mouse monoclonal antibody that reacts with a specificregion of the human proximal tubule and subsets renal cellcarcinomasrdquo Cancer Research vol 49 no 23 pp 6774ndash67801989

[52] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[53] T Welsch S Keleg F Bergmann L Degrate S Bauer and JSchmidt ldquoComparative analysis of tumorbiology and CD133positivity in primary and recurrent pancreatic ductal adenocar-cinomardquo Clinical and Experimental Metastasis vol 26 no 7 pp701ndash711 2009

[54] Y Kato M Kaneko M Sata N Fujita T Tsuruo and MOsawa ldquoEnhanced expression of aggrus (T1alphapodoplanin)a platelet-aggregation-inducing factor in lung squamous cellcarcinomardquo Tumor Biology vol 26 no 4 pp 195ndash200 2005

[55] K Mishima Y Kato M K Kaneko et al ldquoPodoplanin expres-sion in primary central nervous system germ cell tumors auseful histological marker for the diagnosis of germinomardquoActa Neuropathologica vol 111 no 6 pp 563ndash568 2006

[56] NKimura and I Kimura ldquoPodoplanin as amarker formesothe-liomardquo Pathology International vol 55 no 2 pp 83ndash86 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


and isolating the CSCs from the solid tumors howeversubsequent studies demonstrated that the choice of CSCmarkers differs among cancer originations [10ndash15]

ATP-binding cassette subfamily G member 2 (ABCG2)is a member of the superfamily of ATP-binding cassettetransporter proteins Immunohistochemical analysis showedthatABCG2was expressed ubiquitously in normal and tumortissues [16] Zhou et al [17] found that the expression ofABCG2 gene is an important determinant and a molecularmarker for side population-enriched stem cells (SP cells)and this feature was considered to be related to CSCs [18]Kim et al also reported that ABCG2 is highly expressedin SP cells and its expression is strongly correlated withSP phenotype [10] ABCG2 can be expressed in stem cellsisolated from both normal and tumor tissues which furtherindicates its essential role in stem cell biology [19]

CD133 (prominin-1) a cell-surface glycoprotein carryingfive transmembrane domains was firstly identified as amarker for a subpopulation of CD34-positive haematopoieticstem cells which are derived from human fetal liver fetalbone marrow and peripheral blood [20] CD133 was initiallyregarded as a specific marker of hematopoietic stem celland later found in neural stem cells epidermal stem cellsand endothelial progenitor cells (EPCs) [17 21 22] Previousstudies have identified and isolated a putativeCSCpopulationfrom the brain [23] prostate [24] liver [25] lung [26] andcolon [27] cancers using CD133 antibodies In oral CSC-like cells isolated from primary tumor cells and cell linesscientists found an improved migration and tumorigenicitybehavior of these cells with a higher CD133 expressionlevel [28] CD133-positive cells also showed an enhancedclonogenicity invasiveness and tumorigenicity compared tothose CD133-negative cells [29]

Podoplanin is a mucin-like transmembrane glycoproteinthat is highly and specifically expressed in lymphatic endo-thelial cells of normal tissues and tumors [30] Recent stud-ies have reported that podoplanin-positive cells in tumors actas mediators of metastasis and invasion [31ndash33] Podoplaninis also reported as a potential CSC marker for squamous-cell carcinoma (SCC) Atsumi et al found that A431 cells(a SCC cell line) consisted of both podoplanin-positive andpodoplanin-negative cells in which podoplanin-negativecells only produced podoplanin-negative cells but podo-planin-positive cells generated both podoplanin-positive and-negative cells and podoplanin-positive cells showed highertumorigenicity [34] Rahadiani et al reported that the eso-phageal squamous-cell carcinoma (ESCC) showed defectiveinvasion and tumorigenic activities after the podoplaninexpression were knocked down in esophageal [35]

Relative studies in identifying CSCs markers in ACCare still in the early stage In this study we examined theexpression patterns of ABCG2 CD133 and podoplanin inACC of minor salivary glands by immunohistochemicalanalysis Meanwhile Ki-67 a nuclear protein associated withcellular proliferation was used to evaluate the proliferation ofthe tumor cells

2 Materials and Methods

21 Tissue Samples Archival paraffin-embedded tissueblocks of 25 ACC cases (9 men 16 women mean age 6072years range 32ndash78 years) were randomly selected Tenof these samples included normal looking tissues Thesesamples were diagnosed at Okayama University HospitalSurgical Pathology Unit (Okayama Japan) from 1980 to2005 All specimens were fixed with 10 neutral bufferedformalin and embedded in paraffin Histological diagnosiswas carried out by routine hematoxylin and eosin (H and E)staining according to World Health Organization (WHO)histological typing of ACC Clinical and histopathologicalcharacteristics of the patients are shown in Table 1 Tennormal looking salivary gland tissues were used as thecontrol group in this study Informed consents were obtainedfrom the patients and the experiment was approved by theResearch Ethics Board of Okayama University

22 Immunohistochemistry Staining Antibodies againstABCG2 (BXP-21) (1 50 mouse monoclonal Abcam Cam-bridge UK) CD133 (1 300 rabbit polyclonal Abcam)podoplanin (1 50 mouse monoclonal DAKO CarpinteriaCA) and Ki-67 (1 50 mouse monoclonal DAKO) were usedin this study

For immunohistochemical staining serial sections of4 120583m thickness were deparaffinized in xylene and rehydratedin decreasing concentrations of ethanol Endogenous per-oxidase activity was blocked by immersing the sections in3 H

2O2with methanol for 30min For antigen retrieval

sections were boiled in 10mmolL citrate buffer (pH 60)for 15min in a pressure cooker After treatment with proteinblock serum at room temperature sections were coveredwith primary antibodies and incubated at 4∘C overnight Forsecondary antibody DAKO Cytomation Envision+System-HRP kit (AEC) was used according to the manufacturerrsquosinstructions Antibody reactions were stained with 331015840-diaminobenzidine and counterstained with hematoxylin Forthe negative control sections were incubated with PBSinstead of the primary antibodies

23 Assessment Standard ABCG2 CD133 and podoplaninwere detected in the cytoplasm andor cell membrane Tenareas were selected randomly in each section under highpower magnification (times400) Also 100 cells were selectedrandomly in each area so that a total of 1000 cells wereanalyzed for counting the positive cells Positive stainingintensity for cancer stem cell markers was classified as nega-tive minus (lt30) weak + (30ndash49) moderate ++ (50ndash74)and strong staining +++ (ge75) For Ki-67 staining thenucleus was stained with brown color and positive stainingintensity was classified as negative minus (lt5) weak + (5ndash9)moderate ++ (10ndash14) and strong staining +++ (ge15)No false positive or negative staining was encountered in thepositive or negative controls respectively

24 Statistical Analysis Statistical analyses were performedwith SPSS version 180 for Windows Wilcoxon test was used





3 Results







Table2Ex

pressio

nsof

ABC

G2CD

133po

doplaninand

Ki-67in

norm

allook

ingtissues

andAC

C

Group

s119899

ABC

G2intensity

Positivity

()

CD133intensity

Positivity

()

Podo

planin

intensity

Positivity

()

Ki-67intensity

Positivity

()

minus+

++++

+minus

+++

+++

minus+

++++

+minus

+++

+++

Normal

104

60

060

00

55

100

91

00

1010

00

00

ACC

252

17

1592

01

1212

100

194

11

2410

43

860

119875000

0lowast0043lowast

0835

1000

0332

064

40001lowast

0001lowast

Normalnormallook


e+weakstaining

++mod

erates

tainingand++

+str

ongstaining

lowastdeno

tessignificantd

ifference

whencomparedwith

then



ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)


ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)



ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)


ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)



Cribriform pattern

ABCG2 Ki-67

(a)

Solid pattern

ABCG2 Ki-67

(b)

ABCG2

Ki-67

(c)

ABCG2

Ki-67

(d)







119875 119903119904

119875







119875 119903119904

119875 119903119904

119875




119904= 0620 and 119875 = 0001)


4 Discussion









5 Conclusion





Acknowledgments


References


































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3 Results







Table2Ex

pressio

nsof

ABC

G2CD

133po

doplaninand

Ki-67in

norm

allook

ingtissues

andAC

C

Group

s119899

ABC

G2intensity

Positivity

()

CD133intensity

Positivity

()

Podo

planin

intensity

Positivity

()

Ki-67intensity

Positivity

()

minus+

++++

+minus

+++

+++

minus+

++++

+minus

+++

+++

Normal

104

60

060

00

55

100

91

00

1010

00

00

ACC

252

17

1592

01

1212

100

194

11

2410

43

860

119875000

0lowast0043lowast

0835

1000

0332

064

40001lowast

0001lowast

Normalnormallook


e+weakstaining

++mod

erates

tainingand++

+str

ongstaining

lowastdeno

tessignificantd

ifference

whencomparedwith

then



ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)


ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)



ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)


ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)



Cribriform pattern

ABCG2 Ki-67

(a)

Solid pattern

ABCG2 Ki-67

(b)

ABCG2

Ki-67

(c)

ABCG2

Ki-67

(d)







119875 119903119904

119875







119875 119903119904

119875 119903119904

119875




119904= 0620 and 119875 = 0001)


4 Discussion









5 Conclusion





Acknowledgments


References


































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table2Ex

pressio

nsof

ABC

G2CD

133po

doplaninand

Ki-67in

norm

allook

ingtissues

andAC

C

Group

s119899

ABC

G2intensity

Positivity

()

CD133intensity

Positivity

()

Podo

planin

intensity

Positivity

()

Ki-67intensity

Positivity

()

minus+

++++

+minus

+++

+++

minus+

++++

+minus

+++

+++

Normal

104

60

060

00

55

100

91

00

1010

00

00

ACC

252

17

1592

01

1212

100

194

11

2410

43

860

119875000

0lowast0043lowast

0835

1000

0332

064

40001lowast

0001lowast

Normalnormallook


e+weakstaining

++mod

erates

tainingand++

+str

ongstaining

lowastdeno

tessignificantd

ifference

whencomparedwith

then



ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)


ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)



ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)


ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)



Cribriform pattern

ABCG2 Ki-67

(a)

Solid pattern

ABCG2 Ki-67

(b)

ABCG2

Ki-67

(c)

ABCG2

Ki-67

(d)







119875 119903119904

119875







119875 119903119904

119875 119903119904

119875




119904= 0620 and 119875 = 0001)


4 Discussion









5 Conclusion





Acknowledgments


References


































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)


ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)



ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)


ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)



Cribriform pattern

ABCG2 Ki-67

(a)

Solid pattern

ABCG2 Ki-67

(b)

ABCG2

Ki-67

(c)

ABCG2

Ki-67

(d)







119875 119903119904

119875







119875 119903119904

119875 119903119904

119875




119904= 0620 and 119875 = 0001)


4 Discussion









5 Conclusion





Acknowledgments


References


































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)


ABCG2

50120583m

(a)

CD133

50120583m

(b)

Podoplanin

50120583m

(c)

50120583m

Ki-67

(d)



Cribriform pattern

ABCG2 Ki-67

(a)

Solid pattern

ABCG2 Ki-67

(b)

ABCG2

Ki-67

(c)

ABCG2

Ki-67

(d)







119875 119903119904

119875







119875 119903119904

119875 119903119904

119875




119904= 0620 and 119875 = 0001)


4 Discussion









5 Conclusion





Acknowledgments


References


































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cribriform pattern

ABCG2 Ki-67

(a)

Solid pattern

ABCG2 Ki-67

(b)

ABCG2

Ki-67

(c)

ABCG2

Ki-67

(d)







119875 119903119904

119875







119875 119903119904

119875 119903119904

119875




119904= 0620 and 119875 = 0001)


4 Discussion









5 Conclusion





Acknowledgments


References


































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















119875 119903119904

119875 119903119904

119875




119904= 0620 and 119875 = 0001)


4 Discussion









5 Conclusion





Acknowledgments


References


































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















5 Conclusion





Acknowledgments


References


































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of







































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article expressions of abcg2, cd133, and podoplanin...

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