Pancreatic-carcinoma-cell-derived pro-angiogenic factors can induce endothelial-cell ...

15 Pages·2013·2.69 MB·English

Vizioetal.JournalofTranslationalMedicine2013,11:314
http://www.translational-medicine.com/content/11/1/314
RESEARCH Open Access
Pancreatic-carcinoma-cell-derived pro-angiogenic
factors can induce endothelial-cell differentiation
of a subset of circulating CD34+ progenitors
Barbara Vizio1, Fiorella Biasi2, Tiziana Scirelli1, Anna Novarino3, Adriana Prati1, Libero Ciuffreda3,
Giuseppe Montrucchio1, Giuseppe Poli2 and Graziella Bellone1*
Abstract
Background: CD34+ progenitor cells comprise both hematopoietic and endothelial progenitor cells. Recent studies
suggest thatcirculating endothelial progenitor cells are recruited into theangiogenicvascular system ofseveral
cancers, including pancreatic carcinoma, and that theycorrelate withclinical progress. However, whether
endothelial progenitor cell mobilization occurs inresponse to cytokine release by tumor cellsis still unclear.
Methods: The chemotactic- and/or differentiating-activities of thepoorly-differentiated pancreatic carcinoma cell
line PT45,and oftheimmortal H6c7 cell line, a line of near-normal pancreatic duct epithelial cells, on endothelial
progenitor cells were investigated in vitro using circulatingCD34+as model.
Results: The study showed thatVascular EndothelialGrowth Factor produced by PT45 cells and,at lesser extent, by
H6c7cells, predominantly chemoattract peripheral blood CD34+ expressing thetype 2relative receptor. Addition of
PT45-conditionedmediumtoCD34+cells,culturedunder conditions supporting myeloidcelldevelopment, diverted
thedifferentiation of a subset of theseprogenitor cells into cellsexpressing endothelial cell markers, such as CD146,
CD105, VE-cadherinandvonWillebrandFactor-relatedantigen.Moreover,theseendothelial-likecellsformedcapillary
networksinvitro,chieflythroughthereleaseofAngiopoietin-1byPT45cells.
Conclusions:Theresultsdemonstratethatpancreatic-carcinomacellspotentiallyattractcirculatingendothelial
progenitorcellstothetumorsite,byreleasinghighlevelsofpro-angiogenicfactorssuchasVascularEndothelial
GrowthFactorandAngiopoietin-1,andmaydirectthedifferentiationofthesecellsubsetsoftheCD34+cellpopulation
intoendothelialcells;thelattercellsmaybecomeacomponentofthenewly-formedvessels,contributingto
angiogenesis-mediatedtumorgrowthandmetastasis.
Keywords:CD34+cells,Endothelialprogenitorcells,Pancreaticcarcinoma,H6c7cells,Angiogenicgrowthfactors,
Tumorneoangiogenesis
Background Tumor cells may induce angiogenesis directly, by
It is well known that, in order to develop in size and releasing pro-angiogenic factors, and/or indirectly, by
metastatic potential, tumors must make an "angiogenic attracting inflammatory cells that, in turn, release angio-
switch"; they achieve this by perturbing the local genic stimuli [2]. The number of factors known to be
balance of pro-angiogenic and anti-angiogenic factors capable of eliciting an angiogenic response is steadily
[1]. When positive angiogenic regulators overcome the increasing. Vascular Endothelial Growth Factor (VEGF)
effect of negative angiogenic molecules, the tumor is considered a master regulator of the angiogenic
acquires angiogenic capability, leading to the forma- cascade, and is thought to promote endothelial cell
tionofnewbloodvessels. differentiation, migration, proliferation, and survival [3].
The finding that a number of malignant human tumors,
including lung, breast, gastrointestinal tract, ovary, and
*Correspondence:[email protected]
1DepartmentofMedicalSciences,UniversityofTurin,Turin,Italy colon, produce VEGF [4-8], and that the inhibition of
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©2013Vizioetal.;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
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VEGF-induced angiogenesis significantly inhibits tumor CPVE) (Turin, Italy) Ethics Committee has waived the
growth in vivo [9], point to its possessing clinical signifi- need for consent, due to the fact the blood donor mater-
cance intumor growth. ial used was fully anonymized. The study did not involve
Pancreatic carcinoma is a biologically-aggressive humanbeingsdirectlyand,accordingtoarticle2comma
tumor that has an early propensity to spread locally I, letter a) and article 6 of Italian Legislative Decree
and metastasize distally. While not grossly vascular, dated 24. 06. 2003, no. 211, and article 1, comma I of
this tumor often exhibits enhanced foci of endothelial Italian Ministry of Health Decree dated 12. 05. 2006, did
cell proliferation, and over-expresses multiple pro- not requireanopinionfrom theEthical Committee.
angiogenic factors [10,11]. In particular, a positive cor-
relation between blood vessel density, tumor VEGF-A
Celllines
isoform levels, and disease progression has been re-
The pancreatic-cell line PT45 (kindly provided by Dr.
portedinpancreaticcarcinoma[12-14].
M.F. Di Renzo, Department of Biomedical Sciences and
The formation of new capillaries to provide an oxygen
Human Oncology, University of Turin, Italy) [19] was
supply for tumors was, until recently, believed to be me-
grown in RPMI 1640 medium supplemented with 10%
diated by the sprouting or co-option of pre-existing
fetal calf serum (FCS) (Merck Millipore, Billerica, MA).
neighboring vessels[15].
The cell line was routinely screened for mycoplasma
However, increasing evidence now suggests that circu-
contamination, using the Hoechst dye H33258 (Sigma
lating endothelial progenitor cells (EPC), normally in-
Aldrich, St. Louis, MO, USA). Immortalized human
volved in maintaining vascular integrity, can also home
pancreatic ductal epithelial cells HPDE6-E6E7 (H6c7),
in to the tumor site and contribute to the de novo
established after transduction of the HPV16-E6E7 genes
formationof blood vessels[16].
into primary cultures of normal pancreatic duct epithe-
In previous studies we found that VEGF expression in
lial cells, were generously provided by Dr. Ming-Sound
pancreaticcarcinomacelllinesisbothhighandinversely
Tsao, (Ontario Cancer Institute/Princess Margaret Hos-
correlated with differentiation status [10]. Moreover,
pital, University Health Network,Toronto, Canada) [20].
EPCandVEGF-Aplasmalevelswerefoundtobesignifi-
The cell line demonstrates a near-normal genotype and
cantly elevated in the blood of pancreatic carcinoma
phenotype of pancreatic duct epithelial cells [21]. The
patients, to be positively associated with disease stage, and
H6c7 cells were grown in serum free Keratinocyte Basal
inverselyassociatedwithoverallsurvival[17].Thesefinding
Medium (KBM) fortified with growth factors, cytokines,
suggest that microenvironmental conditions favoring mo- and supplements (SingleQuots™ Kit) (Lonza Group Ltd,
bilization of EPC, which are key contributors to the early
Basel,Switzerland).
steps in neoplastic vascularization [18], might enable the
Inordertoobtainserum-freeconditionedmedium(CM),
tumortogrowandmetastasizefaster.However,thereison-
PT45 and H6c7 cells were trypsinized, extensively washed
going debate about the distribution, contribution, origin, withphosphate-bufferedsaline(PBS),andseededat3×105/
anddifferentiationofEPCintumorvasculogenesis.
ml, in 5 ml of serum-free RPMI 1640 medium containing
The present research aimed to investigate the ability of
0.25% fatty-acid-free bovine serum albumin fraction V
pancreaticcarcinomacellstoattractandskewthedifferen-
(Sigma Aldrich) and KBM, respectively. After 48-hour
tiation of CD34+ progenitor cells toward endothelial cells,
incubationinahumidifiedatmospherecontaining5%CO ,
2
by releasing pro-angiogenic factors. We show that PT45
CM was collected after centrifugation, and stored at -20°C
cells, as normal pancreatic ductal epithelial cells, promote
untiluse.
the recruitment of CD34+ cells. Moreover, when cultured
under conditions that facilitate myeloid-cell development,
CD34+cellsareinsteadredirectedbythetumortodifferen- Humanumbilicalveinendothelialcells(HUVEC)
HUVEC were obtained from Lonza Group Ltd and
tiate into endothelial cells. The resulting cells resemble
propagated inEndoGROmedium(Merck Millipore).
endothelial cellsphenotypically, as well as functionally,
as is shown by the fact they can be stimulated to
reorganize into cord structures. Tumor-derived VEGF IsolationofcirculatingCD34+progenitorcells
contributed significantly to the chemoattractant activity, Low-density peripheral blood mononuclear cells (PBMC)
whereas Angiopoietin (Angio)-1 chiefly provided the in- were isolated from normal buffy coats received from the
structivedifferentiationsignal. CPVE, by centrifugation at 250g over Ficoll-Hypaque.
CD34+ cells were purified using the CD34-isolation mini-
Materials and methods MACS (magnetic cell sorting) kit (Miltenyi, Bergisch
EthicsStatement Gladbach, Germany) or the EasySep human CD34+ selec-
The Hemocomponent Production and Validation Center tion kit (StemCell Technologies, Vancouver, BC, Canada),
(Centro per la Produzione eValidazione di Emoprodotti, following the manufacturers' directions. In both cases, the
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purity of the isolated CD34+ cells was verified by flow cy- the control group (untreated-CD34+ cells) normalized to
tometry,andwasinthe89%-98%range. a reference gene (β-actin). The mRNA expression data
for β-actin showed no significant difference between
RNAextractionandrealtimereversetranscription untreated- and PT45-CM treated- CD34+ cells. The ex-
(RT)-PCR pression ratio (R) is: R=E ΔCt target (mean untreated-mean
target
RNA was extracted from PT45 cells, H6c7 cells, 19-day- treated sample)/E ΔCt reference (mean untreated-mean treated
reference
cultured CD34+ cells in the absence or presence of sample).
PT45-CM, and from appropriate positive controls, com-
prising the colon carcinoma cell line DLD-1, the hepato- EvaluationofVEGF-A,CXCL12,Angio-1,andAngio-2
cellular carcinoma cell line HepG2, bone marrow (BM) SecretedlevelsofVEGF-A,CXCL12,Angio-1,andAngio-2
stroma, and HUVEC, using TRIzol Reagent (Invitrogen, were determined in cell-free CM from the pancreatic-
Carlsbad, CA) and following the manufacturer’s instruc- carcinoma cell lines PT45 and H6c7, using commercially-
tions. To remove traces of genomic DNA, total RNA availableELISAkits(R&DSystems,Minneapolis,MN,and
(3 μg) was treated with DNase I (Invitrogen) and
reverse-transcribed to cDNA, using SuperScript II Re- Table1Primersequencesforhousekeeping,cytokine
verse Transcriptase (Invitrogen). Lastly, cDNA speci- andendothelialmarkerquantificationbyrealtime
menswere diluted with 130μlofH O. RT-PCR
2
Real Time RT-PCR analysis was run on the iCycler iQ Primerset GenBank Primersequence RT-PCR
system (Bio-Rad, Hercules, CA) by the SYBR green I dye Accession# E(%)
detection method. Amplification of β-actin, VEGF-A, β-actin NM_001101 FW-GCGAGAAGA
Angio-1, Angio-2, SDF-1A (CXCL12), VE-cadherin, von TGACCCAGATC-
Willebrand factor (vWF),VEGF receptor (R)-1 (flt-1) and RW-GGATAGCAC 100
VEGFR-2 (flk-1/KDR) transcripts was run in duplicate, AGCCTGGATAG-
in a PCR optical 96-well reaction plate (Bio-Rad); the VEGF-A NM_001025366 FW-ATGAGGACA
PCR mixture in each well contained cDNA correspond- CCGGCTCTGACCA-
ing to 100 ng of total RNA, the relevant sequence- RW-AGGCTCCTG 94.5
specific primers (150 nM for β-actin and 300 nM for AATCTTCCAGGCA-
the other cytokines) and 1X iQ SYBR Green Supermix Angio-1 NM_001146 FW-GAGGCACGG
AAGGAGTGTGCTG-
(Bio-Rad). Table 1listsprimersequences,designed using
Beacon Designer 5 Software (Bio-Rad) to be cDNA spe- RW-CGGCGCTGA 92.0
TTGCTGCACCCTA-
cific, and to work under equivalent reaction conditions.
PrimersweresynthesizedbyInvitrogenandreconstituted Angio-2 NM_001147 FW-AAAAGCTGA
CACAGCCCTCCCA-
in nuclease-free water before use. Negative PCR controls
RW-ACTGCTGTGTTC 86.0
without cDNA template, and a positive control sample
TCTCCAGGCA-
with a known cycle threshold value (Ct), were included
CXCL12 NM_000609 FW-ATGCCCATG
ineachassay.Optimizedthermalcyclingconditionswere
CCGATTCTTCG-
as follow: 5 min at 95°C followed by 40 cycles of 15 s at
RW-GCCGGGCTA 100
95°C and 1 min at 60°C (two-step PCR). Specificity of
CAATCTGAAGG-
the PCR products was confirmed by the melting curve
VE-cadherin NM_001795 FW-GGTCCCTGA
program at the end of the reaction (55°C to 95°C with ACGCCCTGGTAA-
a heating rate of 0.5°C/10 sec and continuous fluores-
RW-GGAGTGGAGTAT 95.5
cence measurements). PCR efficiency (E) was deter- GGAGTTGGAGCA-
mined by the method described by Ramakers et al. [22]
vonWillebrand NM_000552 FW-GCCTGCTTC
using the iCycler iQ software. For each sample, the Ct Factor(vWF) TGCGACACCATTG-
was acquired using the Fit Point Method [23]. Mean RW-GCCACTCACACT 96.2
normalized gene expression (MNE) was calculated from CATACCCGTTCT-
the following formula: NE=Ereference Ct reference/Etarget Flt1(VEGF-R1) NM_002019 FW-CACGCAGGA
Ct target. The statistical significance of the difference in CCAGTTTGATTGAG-
mRNA expression of the receptors examined, in RW-GCTAGAGGACTC 96.7
PT45-CM treated- and untreated-CD34+ cells, was ana- CCGAGATGTTG-
lyzed using the Relative Expression Software Tool (REST) Flk-1/KDR(VEGF- NM_002253 FW-GCAGGGGAC
R2) AGAGGGACTTG-
for group-wise comparison, and statistical analysis of
relative expression results in real-time PCR [24]. This RW-GAGGCCATC 95.3
GCTGCACTCA
software package calculates an expression ratio relative to
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RayBiotech,Norcross,GA,respectively)followingtheman- wells of 6-well plates coated with fibronectin, in the ab-
ufacturers’directions.Theminimumdetectabledoseswere senceorpresenceof30%PT45-CM.Cellswereallowedto
below 9 pg/ml for VEGF-A, below 18 pg/ml for CXCL12, incubatefor15-19daysat37°Cina5%CO atmosphere.
2
below 30 pg/ml for Angio-1, and below 10 pg/ml for
Angio-2. Evaluationofacetylatedlow-densitylipoproteinuptake
andofbindingtoUlexeuropaeuslectin,bylaserscanning
Cellmigrationassay confocalmicroscopy(LSCM)
Cell migration assays were run in transwell plates (8 μm To measure the uptake of acetylated low-density lipo-
pore size, Costar, Cambridge, MA). Either CD34+ or protein, and adherence to Ulex europaeus lectin, CD34+
HUVEC cell suspension (3×104 cells/ml) was added to cells were grown in thick-glass-base dishes (Asahi
the upper compartment of the transwell chamber. 30% Techno Glass Co., Singapore). After 15-19 days’ culture
of PT45- or of H6c7-CM, as such or pre-treated with in the presence of IL-3, SCF, and GM-CSF, with or with-
neutralizing polyclonal goat antibodies against VEGF-A out 30% of PT45-CM, cells were incubated for 1 h at
(Chemicon International, Temecula, CA) and Angio-1 37°CinRPMI+10%FCScontaining10μg/ml1,1-dioctade-
(Sigma Aldrich), or with appropriate irrelevant antibody cyl-3,3,3,3-tetramethylindocarbocyanine-labeledacetylated
as control, was added to the lower well of the transwell low-density lipoprotein (Dil-AcLDL; Molecular Probes,
chamber as chemoattractant. In selected experiments, Eugene, USA) and fixed with 2% paraformaldehyde for
CD34+ cells were also pretreated with an anti-human 10 min. After washing, fluorescein isothiocyanate (FITC)-
VEGFR-1/Flt-1polyclonalgoatantibodyblockingreceptor- conjugated Ulex europaeus lectin (Sigma Aldrich) was
ligand interaction (25 μg/ml) (R&D Systems). The cham- added at a concentration of 10 μg/ml in PBS, and cells
bers were incubated for 24 h at 37°C, 5% CO . After wereincubatedfor1hatroomtemperatureinthedark.
2
removing the cells from the upper side of the mem- HUVEC were used as positive control. Direct fluores-
brane,withacottonswab,migratedcellswerefixedand cence staining was analyzed by LSCM (Zeiss LSM 510;
stained with crystal violet dye. Stained cells were visual- Carl Zeiss SpA, Arese, Milan, Italy) equipped with a
ized and quantified by counting cells in three high- Zeiss inverted microscope with a “plan neofluar” lens.
powerfields(100X).Dataareshownasmeans±SEfrom Exciting light intensity, black level, and photomultiplier
three/sixseparateseriesofassays. gain were adjusted on control specimens; the same set-
tings were used to scan experimental slides. The laser
Proliferationassay band of excitation was 488/543 nm; the emission was
CD34+ cells were seeded in triplicate at a concentration set with a 560 nm long-pass filter for Dil-AcLDL (red)
of 5×104 cells/well in 96-well plates, in RPMI 1640 and a 505-530 nm band pass for FITC-conjugated Ulex
medium containing 10%FCS, inthe absence orpresence europaeus lectin (green). The fluorescent images were
of 30% of either PT45-CM or H6c7-CM. 50 ng/ml of processed using LSCM 510 Image Examiner software
human recombinant Stem Cell Factor (SCF) and Fms- fromZeiss.
related tyrosine kinase (FLT)-3 Ligand (PeproTech,
Rocky Hill, NJ) was used as control, to activate stem-cell Flowcytometryanalysis
and immature-progenitor-cell cycling. To determine Expression of endothelial cell markers CD146 and
DNA synthesis, after 20 hr incubation, cells were pulsed CD105 was analyzed on untreated and on PT45-CM-
with triziated-Thymidine(3H)-TdR (Perkin Elmer,1μCi/ treated CD34+ cells, after 19 days’ culture, in the pres-
well) for a further 4 hr. Cellular DNA was collected ence of GM-CSF, IL-3 and SCF. The cells were stained
on glass fiber filters, and 3H-TdR incorporation was with FITC-conjugated CD146 (Chemicon International)
measured in a β-counter. Results are expressed as me- and CD105 (Millipore) mouse monoclonal antibodies
dian cpm (count per minute) (range) of two separate for 30 min at 4°C. After washing, cells were analyzed
experiments. by flow cytometry in a Coulter Epics IV Cytometer
(BeckmanCoulter,Inc.,Fullerton,CA).Resultsareexpressed
DifferentiationofCD34+cells as percentages of positive cells. Statistical analyses were
To determine the effect of tumor-derived soluble factors based on at least 30,000 events gated on the population
on CD34+ cell differentiation, normal peripheral blood of interest.
CD34+ progenitor cells, isolated immunomagnetically as
described above, were resuspended in Iscove medium Immunohistochemistry
(Merck Millipore) containing 20% FCS, 10 ng/ml re- PT45 cells, and 19-day cultured CD34+ cells in the pres-
combinant Interleukin (IL)-3, 10 ng/ml SCF, and 50 ence of IL-3, SCF and GM-CSF, with or without 30% of
ng/mlGranulocyte-Macrophage ColonyStimulatingFac- PT45-CM, were harvested, cytospinned and fixed in
tor (GM-CSF) (PeproTech); they were then seeded in acetone. After 5 min exposure to 0.3% solution of H O
2 2,
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to inhibit endogenous peroxidase activity, cytospinswere Statisticalanalysis
treated for 1 h with a blocking buffer (10% normal To assess statistically-significant differences between data-
mouse/rabbit/goat serum in PBS) to prevent nonspecific sets,Student'sttestsforpairedsamples,orMann-Whitney
staining, incubated overnight at 4°C with the primary Rank Sum tests, were performed using SigmaStat software
antibodies listed in Table 2, and then washed twice (JandelScientific,SanRafael,CA).
with PBS for 5 min each time. For negative controls,
the primary antibodies were replaced with appropriate
Results
non-immune sera. The slides were then treated with ap-
ExpressionofangiogenicfactormRNAinPT45andin
propriate biotinylated secondary antibodies, followed by
H6c7cellsbyrealtimeRT-PCR
incubationwithavidin-biotinperoxidasecomplexsolution,
Steady-state angiogenic factormRNA levels werequanti-
for 30 min, and diaminobenzidine tetrahydrochloride as
tatively assessed in PT45 and H6c7 cell lines, using real-
chromogen (Dako LSAB® kit, Dako, Glostrup, Denmark).
time RT-PCR, and normalized gene expressions were
The slides were finally counterstained with Mayer's
compared (Table 3). Both cell lines expressed similar
hematoxylin for 5 seconds, dehydrated, and mounted in
levels of VEGF mRNA. Conversely, the Angio-1 tran-
Clarion(Biomeda,FosterCity,California,USA).
script was expressed at a markedly lower level in H6c7
cells than in PT45 cells (fourteen times lower). Both cell
Invitroangiogenesisassays
lines were found to be negative for Angio-2 mRNA. The
Matrigelbasementmembranematrix(BectonDickinson,
CXCL12 message was expressed at a very low level in
Franklin Lakes, NJ USA) was added (100 μl) to a 96-well
PT45 cells andnotatallinH6c7cells.
plate and allowed to polymerize. HUVEC or CD34+
cells, cultured for 19 days in IL-3, SCF and GM-CSF, in
the presence or absence of 30% of PT45-CM, were re- ExpressionofVEGF-A,Angio-1,Angio-2andCXCL12
suspended in Medium 199 (Millipore) and 0.5% FCS proteinsbyPT45andbyH6c7cells
(2×104 cells/1 ml) in the presence of 30% of PT45-CM, The release of VEGF-A, Angio-1, Angio-2, and CXCL12
pre-treated or not with neutralizing antibodies against proteins was evaluated in PT45-CM and in H6c7-CMby
VEGF or Angio-1, or with the appropriate irrelevant ELISA. As shown in Figure 1 (upper panel), significantly
antibody; they were then plated (100 μl) on the top of higher levels of VEGF-A and Angio-1 were detected
the solidified matrix solution and incubated at 37 C for in PT45-CM than in H6c7-CM (mean±SE: 1413.8±
24 hr. Tubule formation was visualized using an 311.6 pg/ml vs. 971.3±186.5 pg/ml, p=0,042; mean±
inverted-light microscope with a 20X objective (Zeiss), SE: 362.9±37.9 pg/ml vs. 224.4±33.14 pg/ml, p=
and images of ten random fields were captured using a 0,0267, respectively). Consistently with mRNA expres-
digital camera. Each cell within the optical field was sion, Angio-2 and CXCL12 levels were undetectable by
counted, and this number referred to as the "total num- ELISA in either cell supernatants. Immunohistochemical
ber of cells per field". The angiogenic potential was analysis of these molecules, run on PT45 cell cytospins
quantifiedfrom thefollowing formula: using specific antibodies, detected intense staining for
Angiogenic score=[(number of sprouting cells x 1]+ VEGF-A and Angio-1 in the cytoplasm of the majority
number of connected cells x 2+number of polygons x of PT45 cells, while Angio-2 and CXCL12 expression
3)]/ Total number of cells per field. The final individual was negligible (Figure 1, lower panel), consistently with
score derived from the formula was then taken as one the ELISA results. The combined expression of VEGF-A
value in a total of ten, with median [range] represented and Angio-1 suggests that the poorly-differentiated
againstothertreatment conditions. PT45 cells possess higher vascular remodeling capacity
than doH6c7cells.
Table2Antibodiesandimmunohistochemicalprocedures
Reactivity Antibody Source Antibody
Table3LevelsofangiogenicfactormRNAinPT45and
Species Dilution
H6c7cells
VEGF-A Rabbit SantaCruzBiotech.,Santa 1:20 Gene mRNAlevels(relativetoβ-actin)
Cruz,CA
PT45cellsH6c7cells
Angio-1/4 Goat SantaCruzBiotech 1:200
VEGF-A 0,016821a 0,016270
Angio-2 Goat SantaCruzBiotech 1:200
Angio-1 0,000083 0,000006
CXCL12 Rabbit SantaCruzBiotech 1:30
Angio-2 ndb nd
VE-cadherin Mouse SantaCruzBiotech 1:100
CXCL12 0,000001 nd
vWFrelated Rabbit ShandonImmunon, 1:20
antigen Pittsburgh,PA aMeannormalizedgeneexpression(MNE).
bnotdetectable.
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DoPT45-CMandH6c7-CMchemoattractcirculating treated CD34+ cells [median cpm (range): control 438
CD34+? (334-527); PT45-CM 395 (296-478); H6c7-CM 380
To test this hypothesis, since production of endothelial (297-502)]. In the presence of the mitogenic factors SCF
chemotactic factors, such as VEGF-A and Angio-1 [25], and FLT-3, the DNA-synthetic activity of CD34+ cells
was detected in PT45-CM and in H6c7-CM, the super- slightly increased over control values, but not to a
natants were assessed for in vitro chemotaxis of circulat- statistically-significant extent (median cpm (range): 531
ing CD34+ cells, which are known to express the relative (344-727) vs. 438 (334-527), p=0,400). To determine
receptors [26]. HUVEC were used as positive controls. the factor responsible for chemoattraction of CD34+
As shown in Figure 2, both HUVEC and CD34+ cells cells, the supernatants were pre-treated with isotype-
demonstrated a significant chemotactic response, both control or with neutralizing VEGF-A and Angio-1 anti-
to PT45-CM and to HPDE-CM, although in the latter bodies. Treatment with antibodies neutralizing Angio-1
case to a lesser extent (CD34+ cells migrated/HPF: activity only partially reduced the CD34+ migration
PT45-CM, 16.333±3.082 vs. H6c7-CM, 9.667±3.41, induced by either CM (% CD34+ cells migration inhib-
p=0.003).Since factors secreted byPT45andH6c7cells ition: PT45-CM, 24.167±8.503%; H6c7-CM, 29.134±
are also likely to have an effect on cell proliferation, to 13.429%), whereas when the neutralizing VEGF-A
ensure that the results are a true measure of cell antibody was added, migration was almost entirely
migration, CD34+ cells were cultured for 24 hr in the inhibited (% CD34+ cells migration inhibition: PT45-
presence or absence of PT45-CM or H6c7-CM, and CM, 75.167±25.87%; H6c7-CM, 59.476±17.359%).
DNA-synthetic activity was assessed in terms of 3H-TdR By contrast, exposure to a neutralizing VEGF-R1 poly-
uptake. No significant difference in DNA synthesis clonal antibody did not significantly decrease CD34+
(p >0.05) was observed between untreated and CM- recruitment (% CD34+ cells migration inhibition: PT45-
Figure1ConcentrationsofVEGF-A,Angio-1,Angio-2andCXCL12inCMderivedfromPT45andH6c7celllinesmeasuredbyELISA.
Valuesaremeans±SEofangiogenicfactorsdetectedinduplicateCMsamples,infiveseparateexperimentsforPT45-CM,andinthreeforH6c7-CM
(upper panel). Detection of VEGF-A, Angio-1, Angio-2 and CXCL12 in pancreatic carcinoma cells PT45, determined by immunohistochemistry.
A representative negative control is also shown (original magnification 250X) (lower panel).
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Figure2Chemo-attractionofCD34+cellsbyPT45-CMandH6c7-CM.PBCD34+cellsandHUVECwereseededintotheuppercompartment
ofachemotaxischamber.PT45-orH6c7-CM(30%)wasplacedinthelowercompartment,assuchorpre-treatedwithneutralizinganti-VEGF-A
andanti-Angio-1antibodies.Inselectedexperiments,CD34+cellswerepre-treatedwithaneutralizinganti-VEGF-R1antibodybeforeexposureto
PT45-ortoH6c7-CM.After24hrincubation,cellsintheuppercompartmentwereremoved,andthenumberofCD34+orHUVECcellsthathad
migratedfromtheuppercompartmentthroughthefilterintothelowercompartmentwasdetermined,bycountingthecellsinthreerandom
high-powerfields.(A)RepresentativeimagesofHUVECandCD34+cellsmigratinginresponsetomediumalone(control),toPT45-CMorto
H6c7-CM,inatranswellmigrationassay.(B)Numberofcellsmigratinginatranswellmigrationassay,intheabsenceorpresenceofPT45-CMor
H6c7-CM.Resultsaremeanvalues±SEofsixseparateseriesofstudiesforPT45-CMandofthreeseriesforH6c7-CM.(C)Inhibitoryeffectofa
neutralizinganti-VEGF-R1antibodyonCD34+cellchemotaxis;valuesareexpressedaspercentageinhibitionofCD34+cellmigrationinthe
presenceofPT45-CM/H6c7-CMversusmediumalone(control).Datashownaremeans±SEfromthreeseparateseries.
CM, 3.594±5.843%; H6c7-CM, 2.891±2.3%). Inclu- vascularization process, prompted us to investigate
sion of the isotype control antibodies had no effect whether tumor cells might cause differentiation of these
(data not shown). These results demonstrate that, both CD34+ progenitor cells along the myeloid lineage to be
in normal and in neoplastic conditions, the chemo- switched toward the endothelial cell lineage. This was
attractantactivityexertedbyH6c7cellsonCD34+cells investigatedbyculturingpurified normal PB CD34+cells
is mainly due to VEGF, and that the responding cells for 19 days, under experimental conditions recognized
are included in the CD34+/VEGF-R2+ progenitor cell as supporting their differentiation into myeloid cells
subset. (IL-3, SCFandGM-CSF) [27],but inthepresenceorab-
sence of PT45-CM. The hypothesis that tumor-CM was
MorphologicalchangesofCD34+cellsculturedinthe actively altering the differentiation of CD34+ cells was
presenceofPT45-CM initially supported by morphological evaluation of the
The finding that the CD34+ cell subset is recruited by resulting cells: as shown in Figure 3, in control cultures,
PT45-CM, along with the higher tumor-derived levels of the cells that developed from the CD34+ cells tended to
VEGF-A and Angio-1 that play prominent roles in the be rounded. By contrast, in cultures with tumor-CM, a
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cells [28,29]. We assayed these capacities in the 19-day
cultures, by both double immunofluorescence staining
and LSCM. We identified and quantified a small pro-
portion (9-15%) of cells showingdouble-positive staining
for Dil-AcLDL uptake and binding of Ulex europeus
lectin (Figure 4). These results indicate that this
endothelial-like fraction originates in the hematopoietic
stem-cell-containing CD34+ cell population only in the
presenceoftumor-CM.
QuantitativeanalysisofmRNAexpressionofVE-cadherin,
vWF,VEGF-R1andVEGF-R2
Using real timeRT-PCR, the expression of genes corre-
lated with endothelial cell lineage differentiation was
then evaluated, in CD34+ cells cultured for 19 days in
the presence of IL-3, GM-CSF and SCF, with or without
30% PT45-CM. The results, shown in Figure 5, de-
monstrate that VE-cadherin, vWF and in particular
the VEGF-R1 (flt-1) transcripts were more strongly
expressed in CD34+ cells cultivated in the presence of
PT45-CM, versus controls (p=0.03, p=0.03 and p=
0.02, respectively). Conversely, the expression of
VEGF-R2 [KDR or flk-1] was not significantly modu-
lated in the presence of PT45-CM.
Expressionofendothelial-cellmarkersonCD34+cells
culturedinthepresenceofPT45-CM
Cells derived from CD34+ cells cultured for 19 days with
or without PT45-CM, under conditions that support
myeloid differentiation, were analyzed for endothelial-
cell-marker expression, by flow cytometry (CD146 and
CD105) and by immunohistochemistry (vWF and VE-
cadherin). As shown in Table 4, in the absence of
tumor-CM, a small proportion of cells were positive for
CD146 and CD105. However, when PT45-CM was also
added to the CD34+ cell cultures, a significantly greater
Figure3AlteredmorphologyduringdifferentiationofCD34+ number of cells expressed CD146 and CD105 (p=0.003
cellsinthepresenceofPT45-CM.CirculatingCD34+cellswere and p=0.006, respectively,). As shown in Figure 6, ex-
culturedfor19daysinthepresenceofGM-CSF,IL-3andSCF,either posure to tumor-derived angiogenic factors promoted
intheabsenceorinthepresenceofPT45-CM.Shownarerepresen- the expression of VE-cadherin and vWF, both of which
tativeexamplesofcellsculturedinGM-CSF,IL-3andSCFalone(A);
are endothelial-specific markers, in a subset of cultured
aspanelA,butincludingPT45-CM(B).
CD34+cells(24±8%and23±10%, respectively).
number of cells (median 7%, range 4-18) appeared Invitroangiogenesisassays
more elongated, spindle-shaped, with areas within the Using an in vitro angiogenesis assay on Matrigel, the an-
culture containing cells organized in long head-to-tail giogenic potential of HUVEC was compared to that of
configurations. untreated or PT45-CM-treated CD34+ cells, cultured for
19 days in the presence of IL-3, GM-CSF and SCF.
CharacterizationofCD34+culturedinthepresenceof Figure 7 A-E shows a representative tubulogenesis assay.
PT45-CM On Matrigel, HUVEC formed a well-organized network
Tests were then run to determine whether or not endo- of cordlike structures, with a median (range) angiogenic
thelial cells develop in cultures containing PT45-CM, by score of 2.19 (1.29-3.46) (E); conversely, in CD34+ cell
means of uptake of Dil-AcLDL and Ulex europeus lectin control cultures, no endothelial organization was ob-
binding; these activities are characteristic of endothelial served [median (range) angiogenic score: 0 (0-0.02)] (A).
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Figure4FluorescencestainingofculturedCD34+cellswithDil-AcLDLandUlexeuropeuslectinunderanLSCM.CirculatingCD34+
cellswereculturedfor19daysinthepresenceofGM-CSF,IL-3andSCF,eitherwithoutorwithPT45-CM.Thelaserbandofexcitationwas488/543nm;
theemissionwassetwitha560nmlong-passfilterforDil-AcLDL(red)anda505–530nmbandpassforFITCconjugatedUlexeuropeuslectin(green).
Photographsare fromonerepresentative experiment. Thelens usedwas 20×/0.5 (0.849×0.849 mm2 image dimension).Thefluorescentimages
were processedusing an LSCM 510 Image ExaminerProgram from Zeiss: theyellowmergedimagerepresents theresult ofdouble-fluorescence
labeling.
PT45-CMexposedCD34+cellsexhibitedextendedprotru- (range) angiogenic score of 0.45 (0-1), p<0.001 vs. PT45-
sions,connections,andalignments,buttubeformationwas CM-exposed CD34+ cells] (C), but not when PT45-CM
less pronounced [median (range) angiogenic score of 1.47 was pre-treated with a neutralizing anti-VEGF-A antibody
(0.98-1.70), p=0,002 vs. HUVEC and p<0.001 vs. CD34+ [median (range) angiogenic score of 1.39 (0.20-1.72),
controlcells](B).Theformationofcapillary-likestructures p=0.345 vs. PT45-CM exposed CD34+ cells] (D). Non-
byPT45-CMtreatedCD34+cellswassignificantlyreduced immune goat IgG did not affect cell organization (not
bytheuseofaneutralizinganti-Angio-1antibody[median shown).
Discussion
Expression of the CD34 surface antigen characterizes a
heterogeneous population of BM-derived cells, including
hematopoietic progenitor cells (HPC) and EPC [30],
which can differentiate into mature endothelial cells
when recruited to angiogenic sites. This in situ develop-
ment of vessels, known as vasculogenesis, is prominent
during embryonic development. However, although tis-
sue remodeling and regeneration in postnatal life are
mainlydue toangiogenesis,involvingpreexisting vessels,
it has recently been found that also vasculogenesis can
occur in the adult, particularly during wound healing
andtumor vascularization[31,32].Incancerpatients,in-
Figure5LevelsofVE-cadherin,vWF,VEGF-R1andVEGF-R2
cluding those with advanced pancreatic carcinoma, EPC
mRNAinCD34+culturedwithorwithoutPT45-CM.Cellswere
are numerous compared to healthy controls; EPC num-
culturedfor19daysunderconditionssupportingtheir
differentiationtowardmyeloidlineagecells,inthepresenceor bers also correlate positively with circulating VEGF-A
absenceofPT45-CM.ResultswereassessedbyrealtimeRT-PCR,and levels, and inversely with disease stage and prognosis
normalizedtoβ-actinmRNAlevels.Normalizedgeneexpression [17,33,34].Thesefindingscombinetosuggestthattumor
valuesrelativetothatofcontrolCD34+cells(culturedintheabsence
microenvironmental conditions may foster the recruit-
ofPT45-CM,arbitrarilysetto1)areshown.
ment, mobilization, and differentiationofEPC [35].Both
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Table4Flowcytofluorimetricanalysisofendothelial CXCL12, PT45 cells also express Angio-1 mRNA and
markerCD146andCD105 protein. However, these vasculogenic cytokines are not
Coltureconditions %positivecells(mean±SE) tumor-exclusive products, but are thought to be part of
ofCD34+cells CD146paCD105p the complex milieu involved in maintaining the vascula-
ture in normal tissues: they are also expressed, albeit to
GM-CSF,IL-3,SCF 12.14±5.9 18.14±3
a lesser extent, in the near-normal HPDE H6c7 cells.
GM-CSF,IL-3,SCF+ 21.84±7.7 0.003 28.1±2.1 0.006
30%PT45-CM VEGF-A, released by both malignant (PT45) and normal
avsCD34+culturedinthepresenceofIL-3,GM-CSFandSCF. (H6c7) cells, appears to play a major role, with regard to
cell targets (CD34+ VEGF-R2+) and functional chemo-
tumor cells themselves, and tumor-educated immune tactic activity.
cells, locally secrete a broad spectrum of biologically- Unlike the physiological restoration of vascular perfu-
active factorsthatfacilitate cancergrowth. sion to damaged tissues, blood vessel development in
In a previous study that examined an array of tumor- solid tumors is not closely controlled, but continues re-
derived cytokines affecting the immune response, we lentlessly. Tumors exploit a number of strategies to
found that the poorly-differentiated pancreatic carcin- achieve vascularization (alteration of local normal equi-
oma cell line PT45 produced high levels of VEGF-A librium of anti and pro-angiogenic stimuli, co-optation
[10]. The present study shows that, among other rele- of pre-existing vessels, stimulation of intussusceptive
vant angiogenic factors including the angiopoietins and microvascular growth, post-natal vasculogenesis, and/or
Figure6DetectionbyimmunohistochemistryofVE-cadherinandvWFinCD34+cellsculturedwithorwithoutPT45-CM.Cellswere
culturedfor19daysunderconditionssupportingtheirdifferentiationtowardmyeloidlineagecells,inthepresenceorabsenceofPT45-CM.
Specimensarerepresentativeexamplesofninedifferentexperiments.Representativenegativecontrolsarealsoshown(originalmagnification250X).