six patients who were diagnosed as having unilateral highly progressive large retinoblastoma , group 5a in reese - ellsworth classification , were included in this study .
all human retinoblastoma tissue samples were obtained with informed consent and in accordance with the tenets of the declaration of helsinki . for the detection of abcg2 protein ,
mouse monoclonal anti - abcg2 antibody ( 1:100 , santa cruz biotechnology , santa cruz , ca , usa ) was used .
primary goat polyclonal anti - mcm2 antibody ( 1:200 , santa cruz biotechnology ) and primary rabbit polyclonal anti - vegf antibody ( 1:100 , santa cruz biotechnology ) were also used for staining .
sections of archival human specimens were rehydrated with xylene and graded alcohols . after washing in running water for five minutes
, antigen retrieval was performed by placing slides in 10 mm sodium citrate buffer ( ph 6.0 ) , heated at 120 for 10 minutes .
the slides and buffer were then cooled to room temperature for 30 minutes . after washing with
phosphate buffered saline ( pbs ) , the slides were incubated with 0.2% tripton x-100 for 15 minutes . after three rinses in pbs for five minutes each , sections were incubated with blocking solution for 30 minutes , followed by incubation with primary antibodies : five hours for abcg2 and vegf and two hours for mcm2 .
six patients who were diagnosed as having unilateral highly progressive large retinoblastoma , group 5a in reese - ellsworth classification , were included in this study .
all human retinoblastoma tissue samples were obtained with informed consent and in accordance with the tenets of the declaration of helsinki .
for the detection of abcg2 protein , mouse monoclonal anti - abcg2 antibody ( 1:100 , santa cruz biotechnology , santa cruz , ca , usa ) was used .
primary goat polyclonal anti - mcm2 antibody ( 1:200 , santa cruz biotechnology ) and primary rabbit polyclonal anti - vegf antibody ( 1:100 , santa cruz biotechnology ) were also used for staining .
sections of archival human specimens were rehydrated with xylene and graded alcohols . after washing in running water for five minutes
, antigen retrieval was performed by placing slides in 10 mm sodium citrate buffer ( ph 6.0 ) , heated at 120 for 10 minutes .
the slides and buffer were then cooled to room temperature for 30 minutes . after washing with
phosphate buffered saline ( pbs ) , the slides were incubated with 0.2% tripton x-100 for 15 minutes . after three rinses in pbs for five minutes each , sections were incubated with blocking solution for 30 minutes , followed by incubation with primary antibodies : five hours for abcg2 and vegf and two hours for mcm2 .
abcg2 and mcm2 were sporadically expressed in the ganglion cell layer , the inner nuclear layer , and the photoreceptor layer in the non - neoplastic portion of the retina of the tumor - bearing eye .
vegf was sporadically expressed in the inner retina where retinal vessels are abundantly distributed ( fig .
abcg2 was strongly expressed in the wintersteiner rosettes or tumor lobules . in the rosettes or lobules ,
mcm2 was strongly positive in the rosettes , especially in the outer portion of the rosettes ( fig . 2c and 2d ) . within the lobules of the tumor
but unlike mcm2 , vegf was strongly expressed in the inner portion of the rosettes ( fig .
2e and 2f ) . in the lobules of retinoblastoma , vegf was also sporadically expressed , but not directly associated with tumor vessels .
abcg2 and mcm2 were sporadically expressed in the ganglion cell layer , the inner nuclear layer , and the photoreceptor layer in the non - neoplastic portion of the retina of the tumor - bearing eye .
vegf was sporadically expressed in the inner retina where retinal vessels are abundantly distributed ( fig .
abcg2 was strongly expressed in the wintersteiner rosettes or tumor lobules . in the rosettes or lobules ,
mcm2 was strongly positive in the rosettes , especially in the outer portion of the rosettes ( fig . 2c and 2d ) . within the lobules of the tumor , mcm2
but unlike mcm2 , vegf was strongly expressed in the inner portion of the rosettes ( fig .
2e and 2f ) . in the lobules of retinoblastoma , vegf was also sporadically expressed , but not directly associated with tumor vessels .
evidence suggests that cscs exist in human retinoblastoma [ 10 , 11 , 18 ] .
seigel et al .   observed the presence of a small population which express a cancer stem cell marker ( abcg2 ) and a neural stem cell marker ( mcm2 ) in tumors from transgenic mice , human retinoblastoma cell lines , and human retinoblastomas .
abcg2 is a half atp - binding cassette transporter in the g2 subfamily that has been utilized to identify stem cells in several types of cancer .
mcm2 is one of six members of the family of minichromosome maintenance proteins involved in initiating dna replication .
mcm2 is a proven marker of neural stem cells and is known to be related to the aggressiveness of neuroectodermal tumors and retinoblastoma [ 11 , 21 ] . in this study ,
the expression of abcg2 and mcm2 in human archival retinoblastoma tumors and the non - neoplastic retina in the same eye was observed by immunofluorescence staining .
concurrent with previous studies , mcm2 positivity was observed surrounding blood vessels in both well- and poorly - differentiated tumor tissues .
in normal retinas , mcm2 positivity was observed in the ganglion cell layer , inner nuclear layer , and photoreceptor layer .
the abcg2 staining pattern was similar in the normal retina . in retinoblastoma tumor tissues , on the other hand ,
this result differed from previous reports . with our data , abcg2 and mcm2 stained different portions of the retinoblastoma tumor tissue . according to the cancer stem cell hypothesis
, the cancer stem cell - vascular niche is an important aspect in understanding cscs and in differentiating them from normal stem cells .
stem cells reside in " niches " , defined as physical spaces in the tissue used to maintain homeostasis .
cscs might escape from normal niche control , and the dysregulated stem cell division may give rise to turmor- igenesis .
cscs secrete angiogenic factors , such as vegf , that promote the recruitment and formation of tumor vasculature [ 5 , 6 ] .
vegf had been studied as an important regulator of physiologic and pathologic angiogenesis before the concept of cscs emerged .
vegf expression in retinoblastoma has also been studied because retinoblastomas are highly vascularized tumors that are dependent on angiogenesis [ 13 , 14 , 22 ] .
recent studies have revealed that vegf - targeted treatments suppress tumor angiogenesis and growth significantly in retinoblastomas . in these studies ,
the anti- angiogenic effect by blocking vegf was considered the most important mode of action [ 15 - 17 ] .
until now , the mechanism of action with anti - vegf agents has not been fully understood . in this study , we speculate that vegf is related to cscs or the cscs - perivascular niche complex .
vegf is known to be secreted from neoplastic cells and influences endothelial cells as paracrine mediators , but the relationship between vegf and cscs in retinoblastomas is unknown .
we stained the vegf protein in retinoblastoma tissues to locate the expression and to compare it to that of abcg2 and mcm2 .
consistent with previous studies , vegf immunoreactivity was observed sporadically at the inner retina where there is abundant retinal vasculature in the normal retina .
however , this staining was weak and not directly related to retinal vasculature . in retinoblastoma tumor tissues , vegf expression was observed in the rosettes or tumor lobules , similar to mcm2 expression . unlike mcm2 , strong vegf expressed was observed in the inner portion of the rosettes .
one possible reason that vegf expression is observed at the inner portion of the rosettes is that it is a secretary protein .
the lobules of the retinoblastoma also showed sporadic vegf positivity but that was not directly associated with tumor vessels .
if cscs or the cscs - perivascular niche have pivotal roles in vegf secretion , vegf immunopositivity must be observed in similar locations as csc markers .
similarly , if vegf directly contributes to the cscs - perivascular niche complex , then vegf must be located around the tumor vasculature .
however , our data show that vegf expression is not related to abcg2 expression or tumor vasculature .
instead , vegf expression was similar to that of mcm2 except with specific topographical distribution .
our study suggests that , although vegf might not directly contribute to the csc - perivascular niche complex , it could play some role in the differentiation of tumor cells and in rosette formation .
 purposeto investigate the relationship between vascular endothelial growth factor ( vegf ) and the cancer stem cell - vascular niche complex in human retinoblastoma tissue.methodssix human retinoblastoma specimens primarily enucleated for reese - ellsworth classification stage 5a were stained to detect cancer stem cell markers , including abcg2 for the stem cell marker and mcm2 for the neural stem cell marker , as well as to detect vegf for the angiogenic cytokine . 
 using immunofluorescence , the expression of these proteins was analyzed , and their relative locations noted.resultsin non - neoplastic retina of tumor - bearing eyes , abcg2 and mcm2 were sporadically expressed in the ganglion cell layer and the inner nuclear layer , whereas vegf was sporadically expressed in inner retina where retinal vessels are abundantly distributed . in the tumor , 
 abcg2 was strongly expressed out of wintersteiner rosettes , whereas mcm2 and vegf were strongly stained in the rosettes . 
 interestingly , the outer portion of the rosettes was positive for mcm2 , and the inner portion of the rosettes was positive for vegf.conclusionsour data demonstrated that mcm2 and vegf are strongly expressed in the rosettes of the tumor , which were far from the area of abcg2-positive cells . 
 although vegf might not directly contribute to the cancer stem cell - vascular niche complex , it could play some role in the differentiation of tumor cells to build up the rosettes . 
Materials and Methods
Patients
Antibodies and chemicals
Immunofluorescence
Results
ABCG2, MCM2 and VEGF expression in the non-neoplastic retina
ABCG2, MCM2 and VEGF expression in retinoblastoma tumors
Discussion