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Endothelial CD276 (B7-H3) expression
is increased in human malignancies
and distinguishes between normal and
tumour-derived circulating endothelial cells
J Kraan*
,1
, P van den Broek
1
, C Verhoef
2
, D J Grunhagen
2
, W Taal
3
, J W Gratama
1
and S Sleijfer
1
1
Department of Medical Oncology, Erasmus MC Cancer Institute, box 2040, 3000 CA Rotterdam, The Netherlands;
2
Department
of Surgical Oncology, Erasmus MC Cancer Institute, box 2040, 3000 CA Rotterdam, The Netherlands and
3
Department of
Neuro-oncology, Erasmus MC Cancer Institute, box 2040, 3000 CA Rotterdam, The Netherlands
Background: Mature circulating endothelial cells (CEC) are surrogate markers of endothelial damage. CEC measured in patients
with advanced cancer are thought not only to derive from damaged normal vasculature (n-CEC), but also from damaged (t-CEC).
Therefore, assays that allow the discrimination between these two putative types of CEC are thought to improve the specificity of
the enumeration of CEC in cancer.
Methods: Identification of tumour-associated endothelial markers (TEM) by comparing antigen expression on normal vs t-CEC
and assess the presence of t-CEC in peripheral blood of cancer patients by incorporating TEM in our novel flow cytometry-based
CEC detection assay.
Results: No difference in antigen expression between normal and malignant endothelial cells (ECs) was found for CD54, CD109,
CD137, CD141, CD144 and CXCR7. In contrast, overexpression for CD105, CD146, CD276 and CD309 was observed in tumour ECs
compared with normal ECs. CD276 was most differentially expressed and chosen as a marker for further investigation. CD276-
expressing CEC were significantly higher in 15 patients with advanced colorectal cancer (median 9 (range 1–293 cell per 4 ml);
Po0.005), in 83 patients with a glioblastoma multiforme (median 10 (range 0–804); Po0.0001) and in 14 patients with advanced
breast cancer (median 14 (range 0–390) Po0.05) as compared with 24 healthy individuals (median 3 (range 0–11)). Of all patients
with malignancies, 58% had CD276
þ
CEC counts above the ULN (8 cell per 4 ml).
Conclusions: The present study shows that CD276 can be used to discriminate ECs from malignant tissue from ECs from normal
tissue. In addition, CD276
þ
CEC do occur in higher frequencies in patients with advanced cancer.
Malignant tumours are critically dependent on the formation of
new blood vessels for their growth and dissemination (Naumov
et al, 2006; Strijbos et al, 2008). Unlike normal tissues, which use
sprouting angiogenesis, vasculogenesis and intussusception to form
new vessels, tumours also use additional modes of vessel formation.
These include the co-optation of pre-existing vessels, the imitation
of tumour vasculature (t-CEC) by tumour cells (also known as
vascular mimicry), and the differentiation of putative cancer
stem-like cells into tumour endothelium. (Carmeliet and Jain,
2011) As a result, t-CEC differs morphologically from normal
vasculature (n-CEC), as it features an apparently chaotic mixture of
abnormal and hierarchically disorganised vessels with an increased
permeability (Nagy et al, 2010).
Next to morphologic differences, in recent years many tumour-
associated endothelial markers (TEM) have been identified by
comparing gene and protein expression levels between normal and
*Correspondence: J Kraan; E-mail: j.kraan@erasmusmc.nl
Received 17 February 2014; revised 10 April 2014; accepted 25 April 2014; published online 3 June 2014
&2014 Cancer Research UK. All rights reserved 0007 – 0920/14
FULL PAPER
Keywords: circulating endothelial cells (CEC); tumour endothelial markers (TEM); CD276; pericytes; endothelial cells;
flowytometry; rare events
British Journal of Cancer (2014) 111, 149–156 | doi: 10.1038/bjc.2014.286
www.bjcancer.com | DOI:10.1038/bjc.2014.286 149
tumour endothelial cells (ECs), demonstrating distinctive gene and
protein profiles (Griffioen et al, 1996; Lijnen et al, 2000; Bertolini
et al, 2007; Seaman et al, 2007; Nagy et al, 2010; Wurth et al, 2011;
Maishi et al, 2012).
Given the central role of vasculature for the growth and
development of tumours, various anti-angiogenic agents have been
developed and are now under study with the aim to interfere with
the vascularisation of tumours. Several clinical trials have
demonstrated a benefit in terms of prolonged survival of cancer
patients treated with such anti-angiogenic therapies. Further
development of this field requires the identification of clinically
effective biomarkers that assist the determination of the optimal
dose of such agents, the monitoring of their biologic activity, and
the selection of patients who are most likely to benefit from this
treatment (Bertolini et al, 2007).
Circulating endothelial cells (CEC) are a relative new candidate to
monitor vascular effects of anti-angiogenic treatments. CEC are
mature ECs, which have been shed from the vascular cell lining as a
result of vascular damage. As numerous diseases, including cancer,
are associated with vascular damage, enumeration of CEC is being
considered a promising tool to monitor disease activity with a
potential to assess prognosis and response to treatment (Kraan et al,
2012a). Importantly, CEC measured in patients with advanced
cancer are thought not only to derive from damaged n-CEC, but also
from damaged t-CEC. Therefore, assays that allow the discrimina-
tion between these two putative types of CEC are thought to
improve the specificity of the enumeration of CEC in cancer.
Following dissociation, vascular ECs from tumour and normal
tissues can be analysed by multiparameter flow cytometry using a
combination of exclusion and inclusion markers (Griffioen et al,
1996; Zimmerlin et al, 2010). The aim of the current study is to
identify TEM, in order to enable the distinction between t-CEC
and n-CEC and to assess the possible detection of t-CEC in
peripheral blood (PB) of cancer patients by incorporating TEM
enumeration in our novel flow cytometry-based CEC detection
assay (Kraan et al, 2012b).
MATERIALS AND METHODS
Blood and tissue collection. Tumour and macroscopically normal
tissues were obtained from patients (n¼17) undergoing standard
surgical procedures for liver metastasis resection of metastatic
colorectal cancer (CRC; n¼11) or for resection of a retroperitoneal
liposarcoma (n¼6). Peripheral blood was collected using EDTA-
containing or CellSave tubes (Veridex, Raritan, NJ, USA) from
healthy donors (n¼18) or patients with advanced CRC (n¼13),
advanced breast cancer (n¼9) or patients with a glioblastoma
multiforme (GBM; n¼73) who relapsed after prior chemoradia-
tion. Samples were stored at room temperature and examined
within 8 h (EDTA-containing tubes) or within 96 h (CellSave
tubes) after venipuncture. All patients provided written informed
consent and the study protocols were approved by the local
research and ethics committee (protocols METC: 2011-260, 2006-
248, 2009-405 and P05.182).
Isolation of ECs from tissues. For endothelial cell isolation the
resected specimens containing normal or malignant tissue were
dissociated using a standardised, semi-automated protocol based
on a combination of mechanical tissue disruption and collagenase
IV digestion, using a gentleMACS dissociator (Miltenyi Biotec,
Bergisch Gladbach, Germany). Briefly, tissues were cut in small
pieces (2–4 mm) in a petri dish prior to collection in a dedicated
gentleMACS tube, to which 10 ml of RPMI 1640 (Lonza,
BioWhittaker, Walkersville, MD, USA) with 1% L-Glutamin, 1%
Penicillin/Streptomycin, 10% heat-inactivated FBS (Sigma Aldrich,
St Louis, MO, USA), as well as collagenase IV (Sigma Aldrich),
1mgml
1
, and DNAse I (Sigma Aldrich), 10 mgml
1
, had been
added. The tissues were mechanically minced on the gentleMACS
after incubation for 30 min at 37 1C in a shaking water-bath. This
procedure was repeated once. After the second dissociation step at
the gentleMACS, the cell suspension was passed through a 70 mm
Falcon cell strainer (BD Biosciences, San Jose, CA, USA). The cell
suspension was then collected in a 50 ml conical tube, and
centrifuged at 1000 gfor 5 min. The resulting pellet was washed
with 50 ml phosphate buffered saline (PBS) and concentrated at
210
6
cells ml
1
in PBS for flow cytometry to detect and
characterise the tissue-derived ECs.
Flow cytometry of ECs from tissues. Multiparameter flow
cytometry was performed on 100 ml of cells stained with 10 mlof
each of the following monoclonal antibodies (mAb): fluorescein
isothiocyanate-conjugated CD34 (clone 8G12; BD Biosciences);
peridinin chlorophyll protein-conjugated CD45 (clone 2D1; BD
Biosciences); allophycocyanin-conjugated CD146 (clone 541-10B2;
Miltenyi Biotec), and 10 ml of the DNA dye 5-bis[2-(di-methyla-
mino) ethyl]amino-4, 8-dihydroxyanthracene-9,10-dione (DRAQ5;
Biostatus, Shepshed, UK) and 5 ml of the nuclear stain 40,6-
diamidino-2-phenylindole (DAPI). Coexpression of ECs was
studied using the following phycoerythrin (PE)-labelled mAb:
CD54 (clone LB2), CD109 (clone TEA 2/16), CD137 (clone 4B4-1)
(BD Biosciences), CD105 (clone 1G2), CD144 (clone TEA 1/31)
(Beckman Coulter, Miami, FL, USA), CD276 (B7-H3, clone
185504), CD309 (clone 89106) (R&D systems, Minneapolis, MN,
USA), CD141 (clone AD5-14H12, Miltenyi Biotec) and CXCR7
(clone 8F11-M16, Biolegend, San Diego, CA, USA). All reagents
were diluted in PBS supplemented with 1% bovine serum albumin
(BSA) based on titration experiments (i.e., absence of non-specific
staining on negative populations and optimal discriminatory
power between negative and positive populations). After 15 min
of incubation in darkness at room temperature, 2 ml of PBS were
added, and the suspension was centrifuged for 5 min at 500g. After
removal of supernatant, the cells were resuspended in 250 ml of PBS
for acquisition on a BD LSR Fortessa flow cytometer equipped with
BD FACSDiva software.
CEC enumeration. CEC enumeration and characterisation was
performed as described before (Kraan et al, 2012b). The assay is
based on absolute CD34 counts after analysis of all CD34-positive
events in a total blood volume of 4 ml using a ‘live gate’ on CD34
þ
events to exclude most of the cells that are not of interest from this
analysis. Following this acquisition, CEC are defined as CD34
þþ
,
CD45
neg
, CD146
þ
and DNA
þ
events and combined with PE-
labelled markers for their further characterisation. The true
endothelial origin of this population was previously confirmed by
sorting this specific population and characterising it further using
morphology, immunohistochemistry, multi-colour FCM and gene
expression profiling (Kraan et al, 2012b). In that study, we
demonstrated for this population a specific EC morphology,
coexpression of vWF, CD31, CD105 and CD144, which are all
considered endothelial cell markers, and additionally found that
this population exhibits an EC specific gene profile.
Statistical analysis. Statistical analysis was performed using Prism
software (GraphPad Software, La Jolla, CA, USA). The Mann–
Whitney U-test was used to evaluate differences between ECs
populations in tissue and PB. P-values o0.05 are considered
significant.
RESULTS
Immunophenotype of tissue-derived ECs. Based on the previous
work of ourselves (Kraan et al, 2012b) and others (Goon et al,
2006; Dignat-George et al, 2007), viable ECs were identified by
BRITISH JOURNAL OF CANCER CD276 expression identifies tumour-derived CEC
150 www.bjcancer.com | DOI:10.1038/bjc.2014.286
multiparameter flow cytometry using a combination of exclusion
and inclusion markers combined with a permeant DNA-specific
dye (DRAQ5) to detect nucleated cells, and a non-permeant dye
(DAPI) to exclude dead cells. EC were identified by staining with
two pan-endothelial antibodies (CD34 and CD146), whereas CD45
served to exclude leukocytes and non-specific binding of mAb on
tissue cells. Using these criteria we were able to identify a small
population of vital EC from tissue specimens containing normal or
malignant tissue defined as CD34
þþ
, CD146
þ
, CD45
neg
, DNA
þ
and DAPI
neg
(Figure 1). In addition, we also identified a
population of viable cells with a phenotype reported to be specific
for pericytes (Table 1): CD34
neg
, CD146
þþ
, CD45
neg
, DNA
þ
and
DAPI
neg
. In order to verify the nature of ECs and pericytes, we
performed additional stainings with a panel of antibodies contain-
ing endothelial and pericyte-associated markers that were not used
in the flow cytometric marker definition for the detection of these
events. These results confirmed that the events assigned as EC and
pericytes by this assay indeed represent these cells (Table 1 and
Figure 2).
Comparison of antigen expression on normal vs tumour
vasculature. We have studied the expression of endothelial
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0 256
AB
CD
512
FSC-A
Nucleated
CD45 PerCP-A
CD146+, 45–
EC
Pericytes
CD146 APC-A
CD34 FITC-A
CD146 APC-A
Viable
DRAQ5
DAPI
FSC-H
CD9 +
CD31 +
CD54 +
CD104b –
CD105 +
CD144 +
CD146 +
CD9 +
CD31+
CD54 –
CD104b +
CD105(+)
CD144 –
CD146 ++
768 1024 0 256 512 768 1024
Figure 1. Flow cytometric analysis of endothelial cells and pericytes from dissected normal liver tissue. Viable nucleated cells were selected
on DNA content as evidenced by the permeant dye DRAQ5 expression (A) and the absence of reactivity of the non-permeant dye DAPI (B).
Endothelial cells were identified by their expression of CD146 and CD34
bright
, and absence of CD45 expression. Finally, pericytes were identified
by their strong expression of CD146 and dim expression of CD34 (Cand D). Residual lymphocytes(green dots) in the cell isolate were used as an
internal (negative) control. The seven additional markers listed on the right were evaluated using separate PE-labelled antibodies
as shown in Figure 2.
Table 1. Reported expression of different endothelial and pericyte-associated antigens on isolated endothelial and pericyte populations from healthy
donors by flow cytometry
Marker Endothelial Cells
a
Pericytes
a
References
CD9 (TSPAN29) þþ þþ (Lijnen et al, 2000)
CD31 (PECAM-1) þþ (Crisan et al, 2008; Kraan et al, 2012a)
CD34 (Stem cell marker) þþ (Crisan et al, 2008; Zimmerlin et al, 2010; Kraan et al, 2012a)
CD45 (pan-leucocyte) (Crisan et al, 2008; Kraan et al, 2012a)
CD54 (ICAM-1) þ(Kraan et al, 2012a)
CD105 (Endoglin) þ±(Rowand et al, 2007; Kraan et al, 2012a)
CD133 (Stem cell marker) (Kraan et al, 2012a)
CD140b (PDGF-R)þ(Crisan et al, 2008)
CD141 (Thrombomodulin) þþ(Rowand et al, 2007; Kraan et al, 2012a)
CD144 (VE-Cadherin) þ(Crisan et al, 2008; Kraan et al, 2012a)
CD146 (MelCAM) þþþ(Crisan et al, 2008; Zimmerlin et al, 2010; Kraan et al, 2012a)
a
Expression levels: þþ¼strong, þ¼ moderate, ¼negative.
CD276 expression identifies tumour-derived CEC BRITISH JOURNAL OF CANCER
www.bjcancer.com | DOI:10.1038/bjc.2014.286 151
cell-specific and associated antigens on paired cell suspensions
prepared from 17 tumours and normal tissues from the same
patients.
Macroscopically normal tissues were obtained from (partially)
resected liver (N¼10), vein (N¼4), peritoneum (n¼2) and
kidney (N¼1) tissue. These markers included thrombomodulin
(CD141), VE-cadherin (CD144), MelCAM (CD146) and
VEGFR-2 (CD309). In this study, we also addressed the
following putative malignancy-associated antigens, based on
published data (Table 2): ICAM-1 (CD54), endoglin (CD105),
CD109 (a GPI-linked member of the complement superfamily),
CD137 (tumour necrosis superfamily factor 90), CXCR7 (C-X-C
chemokine receptor 7) and CD276 (a member of the B7 family of
costimulatory molecules), all cell surface expressed proteins.
Residual lymphocytes in the cell isolate were used as an internal
(negative) control. No difference in antigen expression between
normal and malignant ECs was found for CD54, CD109, CD137,
CD141, CD144 and CXCR7. In contrast, overexpression for
CD105, CD146, CD276 and CD309 was observed in tumour ECs
compared with normal ECs. CD276 was most differentially
expressed and chosen as a marker for further investigation
(Figure 3).
CD276 expression on CEC in carcinoma patients. As CD276
was the marker with the largest difference in expression between
tumour and normal ECs, it was explored whether or not a
subpopulation of CD276
þ
CEC could be identified in the PB of
solid cancer patients with metastatic disease.
Figure 4 shows the expression of CD276 in two representative
patients, one with partial expression and a low number of CD276
þ
CEC (panel H) and one with a high number of CD276
þ
CEC
(panel I). CD276
þ
CEC were undetectable or below 12 cells per
4 ml in healthy donors (Figures 4G and 5).
Although the investigation of CD276
þ
CEC levels in different
types of cancer other than for validation purposes is beyond the
scope of this paper, Figure 5 shows that CD276
þ
CEC levels in
healthy individuals ranged from 0 to 11 cells per 4 ml (n¼24,
median 3) and that the levels of these cells were significantly higher
in patients with advanced CRC (ranging from 1 to 293 (n¼15,
median 9, Po0.005)), in patients with a GBM (range: 0 to 804
(n¼83, median 10, Po0.0001)) and patients with advanced breast
cancer (range: 0 to 390 (n¼14, median 13.5, Po0.05)). Taking all
patients with malignancies together, 58% of them had more
CD276
þ
CEC than the upper limit of the normal range (ULN) for
healthy individuals, defined as: mean þ196 s.d., which is 8
CD276
þ
CEC per 4 ml. When stratified for the different types of
cancer, the percentage of patients with CD276
þ
CEC above ULN
were 53%, 58% and 64%, respectively for patients with advanced
CRC, GBM and advanced breast cancer, respectively.
DISCUSSION
Recent studies suggest that tumour-derived EC possess different
characteristics from normal EC, including morphologic
(McDonald and Choyke, 2003), cytogenetic (Hida et al, 2004)
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Unstained PE-ACD105 PE-A
CD9 PE-A
CD31 PE-ACD141 PE-A
CD144 PE-A CD54 PE-A
CD140b PE-A
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EF GH
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CD146 APC-A
Figure 2. (A–H) Immunophenotypic characteristics of endothelial cells (blue dots) and pericytes (red dots) isolated from tissue dissections.
Lymphocytes (green dots) are serving as an internal (negative) control.
Table 2. Reported tumour-associated endothelial markers (TEM)
Marker Cell surface expression on tumour endothelium References
CD54 (ICAM-1) Downregulated in renal cell carcinoma (Griffioen et al, 1996)
CD105 (endoglin) Upregulated in breast, colon, cervix, lung, prostate and brain tumours (Duff et al, 2003; Minhajat et al, 2006)
CD109 Upregulated in breast, colon and lung carcinoma (Seaman et al, 2007)
CD137 (4-1BB) Upregulated in colon and lung carcinoma (Broll et al, 2001; Seaman et al, 2007)
CXCR7 Upregulated in renal cell carcinoma and meningioma (Wurth et al, 2011; Maishi et al, 2012)
CD276 (B7-H3) Upregulated in breast, colon, lung, renal carcinoma and Glioblastoma (Seaman et al, 2007; Lemke et al, 2012; Qin et al, 2013)
BRITISH JOURNAL OF CANCER CD276 expression identifies tumour-derived CEC
152 www.bjcancer.com | DOI:10.1038/bjc.2014.286
and pathophysiologic (Hagendoorn et al, 2006) features as well as a
different gene expression profile (St Croix et al, 2000).
In a previous study, we have described a flow cytometric
method to enumerate ECs circulating in the PB and in the same
study, we extensively validated the true endothelial origin of this
population assigned as CEC (Kraan et al, 2012b). This method
enabled us to identify markers that can be used to differentiate
between CECs from n-CEC vs CEC derived from damaged t-CEC
as measured in the PB of cancer patients. Using this flow
cytometric approach, we were successful to isolate a pure
endothelial cell population from normal and malignant tissues.
To the best of our knowledge, this is the first time that such a
method is described, which enables the isolation of EC from
dissociated tissue by multiparameter flow cytometry without pre-
enrichment and the possibility to distinguish EC from non-EC
such as pericytes. We feel that this method can be further used to
investigate the characteristics of ECs in more detail.
Subsequently, we studied ECs from normal and malignant tissue
for their expression of cell surface markers that have been
suggested in the literature as being differentially expressed.
Potential TEM such as CD54, CD109, CD137 and CXCR7 did
not differ in expression between ECs derived from normal and
malignant tissue in a series of colorectal carcinomas and
liposarcomas. In contrast, higher expression of CD105, CD146
10 000
1000
100
10
1
10 000
1000
100
10
1
10 000
1000
100
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1
10 000
1000
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10
1
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1000
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1
10 000
1000
100
10
1
CD54
CD141
CD146
CD309 CXCR7
CD276
CD144
CD105
MFIMFI
MFIMFI
MFIMFI MFI MFI
ns **
**
**
ns
ns
ns
ns
ns
*
***
***
*** ***
**
***
**
**
ns
ns
ns
ns
ns
ns
Normal
Lymphocytes EC - Tumour EC -
Tumour Normal All Sarc CRC Normal
Lymphocytes EC - Tumour EC -
Tumour Normal All Sarc CRC
Normal
Lymphocytes EC - Tumour EC -
Tumour Normal All Sarc CRC Normal
Lymphocytes EC - Tumour EC -
Tumour Normal All Sarc CRC
Normal
Lymphocytes EC - Tumour EC -
Tumour Normal All Sarc CRC Normal
Lymphocytes EC - Tumour EC -
Tumour Normal All Sarc CRC
Normal
Lymphocytes EC - Tumour EC -
Tumour Normal All Sarc CRC Normal
Lymphocytes EC - Tumour EC -
Tumour Normal All Sarc CRC
Figure 3. Antigen expression (mean fluorescence intensity) on lymphocytes (left two bars) and endothelial cells (right four bars) isolated from
dissected normal and tumour tissue. Sarcoma (N¼6), colorectal cancer (N¼11) and all tumours (N¼17; sarcoma and colorectal taken together)
are separately displayed. Solid lines indicate the median value for each group. P-values: *Po0.05; **Po0.01; ***Po0.001.
CD276 expression identifies tumour-derived CEC BRITISH JOURNAL OF CANCER
www.bjcancer.com | DOI:10.1038/bjc.2014.286 153
and CD309 was found on tumour EC compared with EC from
normal tissue. These observations are in line with other reports.
Overexpression of CD105 on tumour-associated EC has been
reported for hepatocellular carcinoma (Xiong et al, 2009), breast,
colon, lung, prostate and brain tumours (Duff et al, 2003), whereas
upregulation of CD146 and CD309 has been described in many
cancers including breast, prostate, melanoma and glioma (Plate
et al, 1993; Ouhtit et al, 2009). In our series, the largest difference
was found for CD276 with higher expression on tumour EC
compared with normal tissue EC. CD276 is a member of the B7
family of immunoregulatory molecules that can be induced on
T cells, B cells and dendritic cells by a variety of inflammatory
cytokines (Chapoval et al, 2001; Steinberger et al, 2004). The ligand
for CD276 has not been identified yet as well as its immuno-
regulatory role. CD276 has been suggested to have a role in the
interaction between tumours and the immune system. Lemke et al,
2012 showed that natural killer cell-mediated cell lysis was
proportionally suppressed as a function of CD276 expression on
tumour target cells. In addition, several others have shown that
expression of CD276 was associated with a relatively aggressive
tumour behaviour in a range of various carcinomas (Sun et al,
2006; Roth et al, 2007; Crispen et al, 2008; Yamato et al, 2009; Zang
et al, 2010; Qin et al, 2013) and GBM (Lemke et al, 2012). In line
with our data, also others have shown that CD276 is differentially
expressed between normal and malignant ECs. St Croix and
colleagues used SAGE technology in a transcriptomic comparison
of gene expression patterns of EC derived from normal resting
tissue, from tumours and from regenerating liver in order to reveal
differences between physiological and pathological angiogenesis
(Seaman et al, 2007). Twenty-five transcripts were identified to be
overexpressed in tumour vs normal endothelium, including 13 that
were undetectable in angiogenic endothelium of regenerating liver.
Seven of this transcripts encoded known cell surface proteins, of
which CD276(B7-H3) was the most differentially expressed TEM.
As CD276 was the most differentially expressed TEM, we
subsequently determined the presence of CD276
þ
CEC in healthy
controls and in three groups of patients with advanced tumours
(colorectal cancer, GBM and breast cancer).
1024
ABC
DE F
GHI
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512256
FSC-A
CD45 PerCP-A
CD45 PerCP-A
CD34 FITC-A
CD34 FITC-A CD34 FITC-A
CD276 PE-A CD276 PE-A CD276 PE-A
6% 94%
68%
32%
97% 3%
Gate 1
Gate 1
Gate 2
Gate 3
Gate 4
SSC-HSSC-H
CD146 APC-ACD146 APC-A
CD146 APC-A
SSC-H
CD146 APC-ACD146 APC-A DRAQ5 APC-Cy7-A
0
256
0
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Figure 4. CD276 expression on circulating endothelial cells (CEC, purple dots). CEC are distinguished as a small population within the CD34
þ
progenitor cells (red dots: CD34
þ
, CD45
neg/dim
, DNA/DRAQ5
þ
;A–C). For optimal detection of the rare CEC population, all CD34
þ
cells in a total
of 4 ml blood were analysed using a threshold on CD34 (D). CEC are identified as positive for CD146 and negative for CD45 (E). Eand Fare
overlay histograms combining the lymphocytes (internal control, including a small subset of CD146
þ
T lymphocytes; green dots) from the first
50 000 events acquired without a CD34 threshold, and all CD34-positive events using a threshold on the remaining cells (red and violet dots).
(G–I) shows the absence of CD276 expression on CEC from a healthy individual (G)vs partial (H) and high (I) expression on CEC from two
representative carcinoma patients.
BRITISH JOURNAL OF CANCER CD276 expression identifies tumour-derived CEC
154 www.bjcancer.com | DOI:10.1038/bjc.2014.286
The levels of CD276
þ
CEC were significantly increased in all
patient groups and 58% of all patients had CD276
þ
CEC numbers
above the ULN (53, 58 and 64%, respectively). Although no clear
differences in incidence or numbers of CD276
þ
CECs between
these three tumour types could be identified, this study was not
powered to detect such differences.
The relatively small series of this study is one of its limitations.
Another limitation is that the CD276
þ
CEC numbers were not
correlated to clinical outcome as a consequence of which the
clinical relevance of this cell population remains to be defined.
In conclusion, the present study shows that CD276 can be used
to discriminate ECs from malignant tissue from ECs from normal
tissue. In addition, CD276
þ
CEC do occur in higher frequencies in
patients with advanced cancer. In addition to defining potential
differences between various tumour types and tumour stages, it is
important to further investigate the potential clinical relevance of
these cells in oncology.
To do so, further clinical studies are needed to investigate the
association of CD276
þ
CEC levels at baseline with outcome, to
examine the use of CD276
þ
CEC as a marker for response
assessment by serially determining CD276
þ
CEC levels during
treatment and to establish whether or not changes are associated
with outcome to treatment as determined by conventional means,
and to determine whether targets for treatment can be revealed on
these cells. This will require large studies in homogenous groups of
patients.
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***
**
*
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