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Vascular ligand-receptor mapping by direct
combinatorial selection in cancer patients
Fernanda I. Staquicinia,b,1, Marina Cardó-Vilaa,b,1, Mikhail G. Kolonina,b,1,3, Martin Trepelc, Julianna K. Edwardsa,b,
Diana N. Nunesa,b,4, Anna Sergeevad, Eleni Efstathioua,b, Jessica Suna,b, Nalvo F. Almeidae, Shi-Ming Tub,
Gregory H. Botzf, Michael J. Wallaceg, David J. O’Connellh, Stan Krajewskii, Jeffrey E. Gershenwaldj,
Jeffrey J. Molldremd, Anne L. Flammk,5, Erkki Koivunenl, Rebecca D. Pentzk,6, Emmanuel Dias-Netoa,b,4,
João C. Setubale, Dolores J. Cahillh, Patricia Troncosom, Kim-Ahn Don, Christopher J. Logothetisa,b,
Richard L. Sidmano,2, Renata Pasqualinia,b,p,1,2, and Wadih Arapa,b,p,1,2
aDavid H. Koch Center; bDepartment of Genitourinary Medical Oncology; dDepartment of Stem Cell Transplantation; fDepartment of Critical Care;
gDepartment of Diagnostic Radiology; jDepartment of Surgical Oncology; kDepartment of Clinical Ethics; lDepartment of Leukemia; mDepartment of
Pathology; nDepartment of Biostatistics; pDepartment of Experimental Diagnostic Imaging, University of Texas M. D. Anderson Cancer Center, Houston,
TX 77030; cDepartment of Oncology and Hematology, University Medical Center of Hamburg, 20246 Hamburg, Germany; eVirginia Bioinformatics
Institute and Department of Computer Science, Virginia Polytechnic University, Blacksburg, VA 24060; hConway Institute of Biomedical and Biomolecular
Science, University College Dublin, Belfield, Dublin 4, Ireland; iCancer Center, The Sanford-Burnham Medical Research Institute, La Jolla, CA 92037; and
oDepartment of Neurology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215
Contributed by Richard L. Sidman, September 12, 2011 (sent for review March 28, 2011)
Molecules differentially expressed in blood vessels among organs
or between damaged and normal tissues, are attractive therapy
targets; however, their identification within the human vasculature
is challenging. Here we screened a peptide library in cancer pa-
tients to uncover ligand-receptors common or specific to certain
vascular beds. Surveying ∼2.35 ×106motifs recovered from biop-
sies yielded a nonrandom distribution, indicating that systemic
tissue targeting is feasible. High-throughput analysis by similarity
search, protein arrays, and affinity chromatography revealed four
native ligand-receptors, three of which were previously unrecog-
nized. Two are shared among multiple tissues (integrin α4/annexin
A4 and cathepsin B/apolipoprotein E3) and the other two have a
restricted and specific distribution in normal tissue (prohibitin/
annexin A2 in white adipose tissue) or cancer (RAGE/leukocyte
proteinase-3 in bone metastases). These findings provide vascular
molecular markers for biotechnology and medical applications.
human disease ∣phage display ∣obesity ∣angiogenesis ∣tumor
Despite methodological efforts to systematically map protein
interactions in model organisms (1–4) including human-
derived biological systems (2, 3), transfer of datasets from simple
life forms to patient applications remains challenging. This issue
becomes relevant in the setting of blood vessel formation and
cancer metastasis, because inherent limitations of experimental
models fail to recapitulate the multiple functional human ligand-
receptors involved in processes such as angiogenesis, vasculo-
genesis, or site-specific metastasis (5–7).
To discover or analyze functional ligand-receptor interactions
in blood vessels under disease conditions, we have used combi-
natorial screenings based on phage display, which has enabled
the targeted delivery of agents to specific vascular beds (8, 9).
This approach allows the selection of homing peptides to specific
organs in vivo after systemic administration of random peptide
libraries (10, 11). We have isolated ligand peptides and identified
their tissue-specific receptors in rodents and in a patient, and
have developed a ligand-receptor in prostate cancer (12, 13) that
serves as the basis for an ongoing first-in-man trial.
Over the past few years we have developed a tripartite ap-
proach to enable serial combinatorial selections to humans. First,
we established an ethical framework to ensure respectful research
in patients who were brain-dead or whose families decided to
terminate life support (14, 15). Second, we adapted techniques
that were validated in rodents (16) to enable synchronous selec-
tion of ligands to multiple organs. Third, we integrated genomic
tools, in which recovery of 106peptides is ∼1;000-fold faster and
∼250-fold cheaper (17). Here, these quantitative and qualitative
methods enabled refined combinatorial selections in patients and
identified unique ligand-receptors.
Results
An Integrated Strategy for Combinatorial Selections in Patients. Our
approach (Fig. S1) involved serial rounds of selection in three
cancer patients, which enabled the enrichment of targeting
peptides for identifying ligand-receptors. After systemic delivery
of a peptide library to the first human subject (12), phage were
recovered from organs, pooled, and serially screened in two sub-
sequent patients (Fig. S1, Step #1). Large-scale sequencing was
performed after the third selection round.
To identify targeting peptides with tissue-specificity from this
sequence dataset, we performed Monte Carlo simulation on the
datasets (Fig. S1, Step #2) for each tissue (16). This approach
allowed the predicted selection of targeting peptides over the
random library. The simulation was followed by high-throughput
analysis of peptides divided into tripeptides, which provided suf-
ficient structure for motifs to mediate protein interactions (18).
This step uses pattern-recognition software (12, 16) that analyzes
the frequencies of ndistinct motifs representing all possible com-
binations of three residue (n3) overlapping sequences in both
directions (n≪n3). Statistical significance was assessed by one-
tailed Fisher’s exact test. Once enriched 3-mers were selected,
full 7-mers containing those tripeptides were identified from the
Author contributions: F.I.S., M.C.V., M.G.K., E.K., D.J.C., R.L.S., W.A., and R.P. designed
research; F.I.S., M.C.V., M.G.K., J.K.E., D.N.N., A.S., E.E., S.-M.T., G.H.B., M.J.W., D.J.O.,
S.K., J.E.G., and E.D.-N. performed research; F.I.S., M.C.V., M.G.K., M.T., J.K.E., D.N.N.,
A.S., E.E., J.S., N.F.A., D.J.O., J.J.M., A.L.F., E.K., R.D.P., E.D.-N., J.C.S., D.J.C., P.T., K.-A.D.,
C.J.L., R.L.S., W.A., and R.P. analyzed data; and F.I.S., M.C.V., M.G.K., R.L.S., W.A., and
R.P. wrote the paper.
Conflict of interest statement: The University of Texas M.D. Anderson Cancer Center and
some of its researchers (W.A. and R.P.) have equity positions in and are paid consultants for
Alvos Therapeutics and Ablaris Therapeutics, which are subjected to certain restrictions
under university policy; the university manages and monitors the terms of these
arrangements in accordance with its conflict-of-interest policy.
Freely available online through the PNAS open access option.
1F.I.S., M.C.V., M.G.K., R.P., and W.A. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: rpasqual@mdanderson.org, or
warap@mdanderson.org, or richard_sidman@hms.harvard.edu.
3Present address: Institute of Molecular Medicine, University of Texas, Houston, TX 77030.
4Present address: A.C. Camargo Hospital, São Paulo, Brazil 01509-010.
5Present address: Cleveland Clinic, Cleveland, OH 44195.
6Present address: Winship Cancer Institute, Emory University, Atlanta, GA 30322.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1114503108/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1114503108 PNAS ∣November 15, 2011 ∣vol. 108 ∣no. 46 ∣18637–18642
APPLIED BIOLOGICAL
SCIENCES
original dataset, which served to isolate tissue-specific and shared
ligand-receptors (Fig. S1, Step #3).
Next, three approaches were used for isolation of functional
ligand-receptors (Fig. S1, Step #4): (i) protein purification and
identification of candidate receptors were performed by affinity
chromatography and mass spectrometry, (ii) identification of
native ligands was achieved from protein arrays with antipeptide
antibodies or BLAST analysis, and (iii) supervised online data-
base searches to evaluate known ligand-receptor interactions.
Biochemical validation of ligand-receptors was ultimately carried
out (Fig. S1, Step #5).
Large-Scale Analysis and Combinatorial Selection. A total of three
rounds of synchronous combinatorial screening in patients
produced 2,348,940 tripeptides, allowing assessment of virtually
every peptide displayed in the selected tissues. For each round,
we analyzed frequencies and tissue distribution of all tripeptides
in each direction within the recovered full peptide sequences. The
same procedure was applied to the unselected phage display
library. Peptide sequences were evaluated by one-sided Fisher’s
exact tests, which identified a pool of tripeptide motifs (n¼23)
enriched (P<0.05) in targeted tissues in round three relative to
the unselected library (Table S1).
Analysis of large-scale derived sequences established a non-
random distribution of tripeptides among tissues (Fig. 1A) with
specific saturation curves (Fig. 1B), and revealed ∼78% agree-
ment between the DNA sequencing methods (Table S1) used
(17). Monte Carlo simulations confirmed a progressive accumu-
lation of enriched motifs from the first to the third round
(Fig. 1A), in a tissue-specific manner (Fig. 1C); such simulations
also revealed that the analytical design used had a >95% prob-
ability of detecting enriched motifs (P<0.05). Saturation plots
of high-throughput DNA pyrosequencing performed in the third
round confirmed the identification of most peptides present with-
in the selected tissues (Fig. 1B). Moreover, we observed that each
tissue had a reproducible ligand saturation curve, a result sugges-
tive of intrinsic tissue diversity among vascular receptor pools. In
contrast, we could not reach ligand saturation with the unselected
library, an indication of true randomness (Fig. 1B).
By comparing tripeptide frequencies in targeted vs. nontar-
geted tissues, we showed that these motifs were not evenly dis-
tributed and some were enriched with tissue-specificity (Table S1,
underlined motifs). Of note, 9 of 23 motifs (39%) recovered
from white adipose tissue (WAT) were found only in WAT, 14
of 17 bone marrow-homing motifs (82%) were found only in bone
marrow, and all tripeptides selected in skin and muscle were un-
ique to the tissue-of-origin. Full peptide sequences with apparent
tissue-specificity (for tumor-containing bone marrow, skin, WAT,
and muscle) were obtained from selected tripeptides. We also
observed progressive enrichment of ligand peptides common to
all tissues examined, a result consistent with selection of an ubi-
quitous ligand or one shared in the several targeted vasculatures;
peptide-based affinity chromatography and mass spectrometry
served to uncover candidate receptors for each selected peptide
in the human vasculature (Table S2). Additionally, protein array
screenings with polyclonal antibodies against the targeting pep-
tides and similarity data mining guided the identification of native
ligands. We reasoned that some ligand peptides likely mimic
circulating proteins that bind to receptors exposed on vascular
endothelial cells. Thus, we refined our similarity searches accord-
ing to protein structure, glycosylation state, subcellular location,
and membrane orientation (www.hprd.org). As a proof-of-con-
cept (i.e., specific binding), we present examples of ligand-recep-
tors in shared (n¼2) and tissue-specific (n¼2; WAT and bone
marrow) settings selected from human blood vessels of normal
and tumor-containing tissues. The validation of these four candi-
date ligand-receptors suggests a reasonable success rate for this
approach; the remaining eleven candidate ligand-receptors were
not pursued here.
Native Ligands and Shared Receptors in Human Endothelial Cells. To
identify shared molecular targets in human blood vessels, we ana-
lyzed peptides recovered from biopsies of multiple tissues. Statis-
tical analysis revealed that the selected peptides CMRGFRAAC
and CMGGHGWGC (Table S2) contained multiple tripeptides
showing the highest frequencies of recovery in all sampled tissues
by round three and were therefore chosen as representative
ligands for further analysis.
We used peptide-based affinity chromatography to purify
interacting molecules for CMRGFRAAC. Total protein extract
from human tissue was loaded onto a peptide column, and inter-
acting proteins were eluted with a solution of the synthetic
peptide. Phage binding assays in vitro were used to identify eluted
fractions containing the highest concentration of receptors. Pro-
teins in fractions of interest were separated by SDS-polyacryla-
mide gel electrophoresis (PAGE), and protein bands were
identified by mass spectrometry and database search. As a result,
human annexin A4 (ANXA4) was isolated as a candidate recep-
Fig. 1. Combinatorial selection in patients. (A) Monte Carlo simulations with
peptides analyzed in serial rounds of selection show nonrandom distribution
of tripeptides. Thick black lines represent the Fisher’s exact test; thin blue
lines represent the corresponding random permutated dataset. (B) A satura-
tion plot (modified from ref. 17) shows the number of distinct peptides as a
function of the total number of peptide sequences. All tissues reached satura-
tion, as indicated by flattening of the slope; in contrast, unselected library
showed no evidence of saturation. (C) Isolated peptides were grouped
according to tissue-of-origin and subjected to Monte Carlo simulation. For
every simulation, the pool of peptides was randomly distributed into groups
corresponding to the number of sequences analyzed for each targeted tissue.
Frequencies were calculated for each simulated organ, and Fisher’s exact test
applied on the permutated dataset. A nonrandom selection of tripeptides
was observed in all organs tested.
18638 ∣www.pnas.org/cgi/doi/10.1073/pnas.1114503108 Staquicini et al.
tor for CMRGFRAAC (Fig. S2A). Annexins are a highly con-
served family of membrane-binding proteins with intracellular
and extracellular functions (19, 20); ANXA4 function in angio-
genesis is currently unknown.
A series of in vitro phage- and protein-based assays was de-
signed to determine whether ANXA4 is a receptor for CMRGF-
RAAC (Fig. 2). We assessed specificity by evaluating the binding
of CMRGFRAAC-displaying phage or insertless phage to immo-
bilized ANXA4 or controls. We observed binding of CMRGFR-
AAC-displaying phage to ANXA4, compared to the family re-
lated control proteins ANXA1 and ANXA5. The unrelated
proteins vascular endothelial growth factor receptor (VEGFR)
and bovine serum albumin (BSA) were additional controls, bind-
ing at background levels (Fig. 2A). Competition assays in the pre-
sence of the targeted synthetic peptide or controls confirmed
binding specificity (Fig. 2B).
Having isolated ANXA4 as a potential shared human vascular
marker, we next screened a representative panel of normal
human tissues (n¼38) in tissue microarray (TMA) format as
a broader survey of the expression and location of ANXA4
(Table S3). We found ANXA4-positive staining in the vasculature
of 50% of tissues examined (Fig. 2G,Table S3), including the
more intensively analyzed WAT, skin, muscle, and bone marrow.
Other ANXA4-positive sites included the vascular endothelium
of human thymus, lymph nodes, spleen, heart, stomach, colon,
and various areas of the brain. Overall, expression of ANXA4
did not correlate with the embryologic origin of tissues/organs
and in most cases was not restricted to endothelial cells.
Next, we sought native ligands for this vascular receptor. Simi-
larity searches and data mining suggested that CMRGFRAAC is
a mimic of the integrin α4 subunit (Table S2, residues 147–152), a
cell adhesion molecule with a well established role in lymphocyte
and monocyte transendothelial migration (21). Enzyme-linked
immunosorbent assays (ELISA) in the presence or absence of the
synthetic peptide (Fig. 2 Cand D), and immunoblotting (Fig. 2E)
with an antibody against a keyhole limpet hemocyanin (KLH)-
conjugated CMRGFRAAC, confirmed integrin α4 subunit as a
native ligand. Negative controls included integrins αvβ5, α5β1,
and αvβ3. Protein interaction experiments showed that binding
of soluble ANXA4 to immobilized α4 integrin is concentration-
dependent (Fig. 2F). Evaluation of integrin α4 subunit expression
in a representative panel of normal tissue samples (Fig. S2B)
showed expression mostly restricted to the surface of leukocytes,
suggesting a role for the ANXA4/integrin α4 in adhesion of
leukocytes to the vascular endothelium.
By a similar approach, we identified apolipoprotein E3
(ApoE3) as a potential native receptor for the ligand CMGGHG-
WGC (Fig. S2C). ApoE is a lipoprotein constituent and functions
in lipid metabolism; it has a major role in cholesterol efflux and
has been linked to atherosclerosis (22). However, no direct link
has been described between ApoE3 and blood vessels formation
in normal or pathological conditions. The relationship between
CMGGHGWGC and ApoE3 was validated by in vitro assays,
and binding specificity confirmed via phage binding to ApoE3 or
controls (ApoE4 and ApoC; Fig. 3A), plus inhibition with the
cognate synthetic peptide (Fig. 3B). Moreover, screening of nor-
mal human TMA revealed ApoE3-positive staining in ∼40% of
examined organs, including muscle, liver, WAT, and distinct areas
of the brain (Fig. 3F,Table S3). Similar to ANXA4, expression of
ApoE3 in normal organs did not correlate with the embryologic
origin of each tissue or organ, and it was not restricted to en-
dothelial cells. Protein similarity search identified CMGGHGW-
GC as a mimic of cathepsin B. This finding is of interest as
cathepsin B participates in the release of atherosclerotic plaque
in animal models (23), supporting the hypothesis that ApoE3
and cathepsin B interact in vivo. Notably, structural analysis
located the CMGGHGWGC peptide within the active catalytic
site of cathepsin B (Fig. S3A). ELISA (Fig. 3D), inhibition assay
(Fig. 3C), and immunoblotting with a polyclonal antibody against
KLH-conjugated CMGGHGWGC (Fig. 3E), support the candi-
dacy of cathepsin B as a ligand mimicked by CMGGHGWGC.
Finally, evaluation of cathepsin B expression in a panel of normal
tissue samples (Fig. S3B) showed widespread expression of the
protein in different cellular compartments, including the blood
vessels of WAT and muscle.
Selection and Validation of a Ligand-Receptor System in Human WAT.
In previous work, we have found that the peptide CKGGRAKDC
was localized to and internalized by cells of mouse WAT vascu-
lature, and that its native vascular target was prohibitin, a multi-
functional protein expressed selectively on WATendothelium (8).
Given that mouse and human prohibitin differ by only a single
residue, we asked whether this protein might also serve as a target
for WAT-homing peptides in patients, and evaluated this possibi-
lity here by analysis of the binding of a large pool of peptide-
targeted phage clones (n¼851) isolated after three rounds of
selection in human WAT to bind to immobilized prohibitin. A
subset of nonredundant peptides (n¼66) that showed specific
binding to prohibitin was subjected to individual database
searches for similarity to human proteins, leading to identifica-
tion of a smaller peptide subset (n¼14) similar to the prohibitin-
binding sequence CKGGRAKDC that matched a region of
Fig. 2. Discovery of integrin α4 subunit/ANXA4 as a shared ligand-receptor
in the vasculature of multiple human tissues. (A) Binding of phage clones
to the receptor ANXA4. Phage displaying the peptide CMRGFRAAC bound
preferentially to its receptor, relative to negative controls. Experiments were
performed three times in triplicate with similar results. Bars represent
mean standard error of the mean (SEM). (B) Competition with the synthetic
peptide shows that binding of selected phage to the purified receptor is
specific. Binding of unrelated control phage, insertless phage, binding to
BSA and inhibition with an unrelated peptide served as controls. (C) ELISA
with preimmune and postimmune rabbit polyclonal antibodies against
CMRGFRAAC and performed on recombinant integrin α4 or a control (shown
is α5β1 integrin). (D) Binding of postimmune antibodies to recombinant
integrin α4 is inhibited by CMRGFRAAC but not by an unrelated control
peptide. (E) Binding specificity was confirmed by immunoblotting. Integrins
αvβ5, α5β1, and αvβ3 were used as negative controls. Arrow points to integrin
α4. (F) Direct interaction between ANXA4 and integrin α4. The binding is
concentration-dependent, indicating specificity. (G) Immunostaining of
sections of normal human tissues with an anti-ANXA4 polyclonal antibody.
Arrows point to ANXA4-positive blood vessels. Insets show negative control
staining. (Scale bar, 100 μm).
Staquicini et al. PNAS ∣November 15, 2011 ∣vol. 108 ∣no. 46 ∣18639
APPLIED BIOLOGICAL
SCIENCES
human ANXA2 (Fig. S3C). Reciprocal analysis revealed a wide
range of sequences among all human WAT-selected peptides that
mimicked prohibitin and that were clustered within the prohibitin
N-terminal segment (Fig. S3C), a region involved in lipid raft
location and interaction with other proteins such as ANXA2
(24). To map the ANXA2 domains involved in the binding to
prohibitin, we performed automated alignment of human WAT-
homing peptides against ANXA2 and identified two similarity
hotspots in the N-terminal domain of the protein (Fig. S3D).
Strikingly, these segments correspond to the connector loops
between ANXA2 repeats 1∕2and 2∕3, which interact with mem-
brane-bound proteins (24).
To confirm that CKGGRAKDC mimics the candidate ligand
ANXA2 and binds to prohibitin, we showed that antibodies
against KLH-conjugated CKGGRAKDC recognize recombinant
ANXA2 (Fig. 4A). Phage-CKGGRAKDC also bound to prohibitin
in vitro, an interaction specifically inhibited by the synthetic pep-
tide (Fig. 4B). Next, membrane fractions extracted from WAT
(Fig. 4C) showed that ANXA2 and prohibitin are located in
noncaveolar lipid rafts (Fig. 4D). Lastly, we used recombinant glu-
tathione S-transferase (GST)-conjugated protein to show that pro-
hibitin binds to ANXA2, but not to the control ANXA5 (Fig. 4E).
Expression of ANXA2 on the surface of endothelial cells
has been reported (25), but without organ comparisons. The ex-
pression pattern of prohibitin and ANXA2 in human tissue sam-
ples appeared coincident in human WAT but not in non-WAT
(Fig. 4 Fand G,Fig. S3E). We conclude that the vascular coex-
pression and interaction between ANXA2 and prohibitin are
likely restricted to WAT.
Discovery of a Specific Ligand-Receptor in Tumor-Containing Human
Bone Marrow. Human bone marrow is often affected by primary
hematologic tumors or metastatic solid tumors. In all three
patients selected in this study, bone marrows were replete with
tumor cells. Within the bone marrow microenvironment, we as-
sumed that the molecular crosstalk between nonmalignant cells
of the vascular endothelium and cancer cells might have at least
some common elements, independent of the tumor type.
Statistical analysis revealed tripeptides enriched in bone
marrow after three rounds of selection (Table S1). In particular,
selected peptides containing the motif Gly-Gly-Gly-Pro were
identified within RAGE, the receptor for advanced glycation end-
products (Table S2). A computer-assisted modeling of RAGE and
three 7-mer peptides containing an embedded Gly-Gly-Gly-Pro
motif (CWELGGGPC, CHVLGGGPC, and CVQGGGGPC)
showed high similarity to an exposed surface of the ligand-binding
extracellular domain of the protein (Fig. S4A).
To query whether CWELGGGPC behaves as a molecular
mimic of RAGE, we developed a polyclonal antibody against
KLH-conjugated CWELGGGPC and used ELISA to evaluate
binding to immobilized RAGE. We showed that the anti-CWEL-
GGGPC antibody recognizes the segment of RAGE containing
Fig. 3. Discovery of cathepsin B/ApoE3 as a shared ligand-receptor in the
vasculature of multiple human tissues. (A) Binding of CMGGHGWGC-phage
to ApoE3. CMGGHGWGC- phage bound preferentially to its receptor relative
to negative controls. Experiments were performed three times in triplicate
with similar results. Bars represent mean SEM (B) Competition assay with
the cognate synthetic peptide shows that binding of CMGGHGWGC-phage
to the purified ApoE3 is specific. Binding of unrelated control phage, insert-
less phage, binding to BSA and inhibition with an unrelated peptide served
as controls. (Cand D) ELISA with preimmune and postimmune polyclonal
antibodies against CMGGHGWGC (C) and performed on purified cathepsin
B or control protein (D). (E) Binding specificity was confirmed by immunoblot-
ting. (F) Immunostaining of human sections with an anti-ApoE3 antibody
confirms that the candidate receptor ApoE3 is expressed in the normal vas-
culature of several human tissues (arrows). (Scale bar, 100 μm).
Fig. 4. Discovery of ANXA2/prohibitin as a tissue-specific ligand-receptor
targeting normal human tissue. (A) Immunoblotting of His6-ANXA2 or con-
trol proteins with antiserum against CKGGRAKDC or control preimmune ser-
um, as indicated. Arrow: His6-ANXA2. (B) Binding of CKGGRAKDC-displaying
phage is specifically inhibited by the synthetic peptide. Binding of unrelated
control phage, insertless phage, binding to BSA and inhibition with an
unrelated peptide served as controls. (Cand D) Association of prohibitin
and ANXA2 with membrane lipid rafts. Membrane proteins extracted from
human WATwere subjected to immunoblotting or to fractionation enriching
for noncaveolar or caveolar lipid rafts. Proteins recognized by anti-ANXA2
(C), antiprohibitin (D, upper box), and anticaveolin 1 antibodies (D, lower
box) are indicated by arrows. (E) Binding of prohibitin and ANXA2 in vitro.
Increasing concentrations of GST-prohibitin or GST control were captured
with His6-ANXA2 or control His6-ANXA5. Specific binding was assessed with
anti-GST antibodies. Arrow indicates GST-prohibitin (migrating as several
bands). (Fand G) Vascular expression of ANXA2 in human WAT. Immunohis-
tochemistry with anti-ANXA2 and antiprohibitin antibodies on human WAT
demonstrated colocalization of ANXA2 and prohibitin in the vasculature.
Blood vessels identity was confirmed by staining with anti-VE-cadherin anti-
body (G, inset). Arrows point to blood vessels. Red insets show lower mag-
nification of the corresponding area. (Scale bar, 100 μm).
18640 ∣www.pnas.org/cgi/doi/10.1073/pnas.1114503108 Staquicini et al.
CWKLGGGPC (Fig. 5A), whereas preimmune serum produces
only a background signal. Immunoblotting with the anti-CWKL-
GGGPC antibody confirmed reactivity with the native protein
extracted from human prostate cancer cells (Fig. 5B, arrow).
Next, we identified the human leukocyte proteinase-3 (PR-3)
by peptide affinity chromatography and mass spectrometry as
a candidate receptor for CWKLGGGPC (Fig. S4B). This result
was confirmed through a second affinity purification with human
bone marrow cell membrane extracts as the protein source
(Fig. S4C). While PR-3 is a serine protease abundant within the
bone marrow of patients with chronic myelogenous leukemia
(26), it has not yet been implicated in bone metastases. In support
of the hypothesis that PR-3 interacts with RAGE, protein analysis
demonstrated that PR-3 does share epitopes with other estab-
lished RAGE ligands (27), such as the advanced glycosylation
end-products (AGE), high mobility group protein B1 (HMGB1),
and S100 calcium-binding protein A12 (EN-RAGE) (Fig. S4D).
Indeed, 7 of 15 HMGB1 residues (47%) and 13 of 21 EN-RAGE
residues (60%) critical for RAGE binding (28) were identical or
conserved within PR-3. Moreover, the C-terminal α-helix of
PR-3 is highly similar to the corresponding part of EN-RAGE
(Fig. S4D), revealing a previously unrecognized structural rela-
tionship between PR-3 and RAGE partners.
For functional characterization of this ligand-receptor, we per-
formed binding experiments in vitro between the extracellular
portion of human RAGE and endogenous PR-3. RAGE—but
not control proteins—bound to immobilized PR-3 (Fig. 5C).
We showed that binding between PR-3 and RAGE is concen-
tration-dependent (Fig. 5D) and that competition assays with
targeted phage and the synthetic peptide (Fig. 5E) support the
specificity of this interaction. These results indicate that the
selected human bone marrow-targeting motif mimics a functional
site within the extracellular domain of RAGE and that RAGE
binds to PR-3 through its WKLGGGP-spanning region. Notably,
elevation of RAGE mRNA transcripts has been reported in
human prostate cancer (29), which can metastasize to the bone
marrow, and the expression of RAGE in prostate cancer patients
has been documented at the protein level (www.proteinatlas.org).
We assessed RAGE expression in a large annotated panel of
human tumor samples (n¼164) from prostate cancer patients
(Fig. 5F), including low-grade (n¼76) and high-grade locally
advanced primary tumors (n¼76), and prostate cancer-infil-
trated bone marrow biopsy samples (n¼12). We applied a linear
regression model to assess biomarker expression and distribution
among the groups. Significant differences in RAGE expression
were observed between low-grade (Fig. 5F, left box) and high-
grade (Fig. 5F, middle box) tumors (t-test, P<0.0001). More-
over, expression of RAGE was significantly higher in bone
marrow-infiltrated metastases compared to low-grade tumors
(Fig. 5F, right and left boxes; t-test, P¼0.0002 for the black bars).
We did not observe statistically significant differences between
high-grade primary tumors and bone marrow metastases (t-test,
P¼0.61). Finally, we detailed RAGE expression immunohisto-
chemically in human prostate cancer, in a representative patient
sample set (n¼12) including primary tumors (Fig. 5G), lymph
node metastases (Fig. 5H), and bone marrow metastases (Fig. 5I).
While RAGE expression was barely detectable in normal pros-
tate glands (Fig. 5G), it was strongly expressed and widespread
in tumor cells within the marrow cavity of all cases of prostate
cancer patients with bone metastatic disease (Fig. 5I), but not
within lymph node metastases (Fig. 5H).
These results are consistent with the hypothesis that RAGE-
expressing tumor cells can arise focally in primary tumors and
might provide at least part of an advantageous setting for bone
marrow metastases.
Discussion
We have initiated the creation of a ligand/receptor-based mole-
cular map of human blood vessels towards a targeted vascular
pharmacology. Our first patient screening (12, 13) served as a
foundation for an ongoing clinical trial of a ligand-directed drug,
and we have improved quantitative and qualitative methodology
(16, 17) in an ethics framework that includes cancer center-
specific guidelines (14) and national recommendations (15) to har-
monize this line of patient-oriented research with current practices
of transplantation medicine. Our efforts may lead to an improved
understanding of vascular proteomics with clinical implications.
Four ligand-receptors were validated functionally in shared or
tissue-specific settings. Two shared human vascular addresses
were found by our selection. First, ANXA4, a membrane protein
identified in Caþ2-dependent membrane trafficking (30), was
characterized as a marker of the human vasculature that interacts
with α4 integrins. Among the α4 integrin family, α4β1 (31) and/or
α4β7 (32) are membrane constituents of leukocytes that mediate
cell adhesion to the vascular endothelium and affect inflamma-
tion, immune response, and tumor dissemination (31). Vascular
cell adhesion molecule-1 and fibronectin are among the mole-
cules that mediate the binding of α4β1-expressing lymphocytes
Fig. 5. Discovery of RAGE/PR-3 as a ligand-binding targeting human bone
marrow containing cancer cells. (A) Anti-CWELGGGPC antibodies recognize
human recombinant RAGE. ELISA with post- and preimmune polyclonal anti-
bodies against CWELGGGPC was performed on immobilized CWELGGGPC,
a control peptide, recombinant Fc-tagged proteins, and a control protein.
(B) Anti-CWELGGGPC antibodies recognize native human RAGE. Protein ex-
tracts from human prostate cancer cell lines PC3 and DU145, or from human
bone marrow mononuclear control cells, along with recombinant RAGE
protein, were immunoblotted with post- and preimmune polyclonal antibo-
dies against CWELGGGPC. Arrow points to RAGE. (C) Validation of RAGE
binding to PR-3 in vitro. Either immobilized PR-3 or control protein (BSA)
was subjected to RAGE, BMPRIA, BSA, and anti-PR-3 antibody. Bars represent
mean SEM (D) RAGE binding to active PR-3 is concentration-dependent.
(E) Binding of CWELGGGPC-phage is specifically inhibited by the synthetic
peptide. Binding of unrelated control phage, insertless phage, binding to
BSA and inhibition with an unrelated peptide served as controls. (F) Relative
quantification of RAGE expression on prostate cancer patient samples.
Expression of RAGE is represented as low, moderate and high expression
according to a standardized pathology score. (G–I) Immunohistochemistry
with RAGE-specific antibodies performed on human tissue sections derived
from a panel of prostate cancer patients. (G) Organ-confined prostate cancer;
(H), lymph node metastasis; and (I), bone marrow metastasis. Asterisks repre-
sent lymphoid (H) and bone (I) tissues. (Scale bar, 100 μm).
Staquicini et al. PNAS ∣November 15, 2011 ∣vol. 108 ∣no. 46 ∣18641
APPLIED BIOLOGICAL
SCIENCES
to endothelial and dendritic cells (31). Unlike α4β1, the integrin
α4β7 is active mainly in intestinal immune response, mediated
through interaction with mucosal cell adhesion molecule-1 (32).
Whether the association between ANXA4 in human vascular
endothelium and heterodimers of α4βx integrin on the circulating
cells occurs in widespread (α4β1) or localized (α4β7) patterns
remains to be determined.
Cathepsin B and ApoE3 were also validated as an unrecog-
nized ligand-receptor shared in several tissues. ApoE is a secreted
protein that acts through the low-density lipoprotein receptor to
mediate lipoprotein binding and catabolism (33). Although ApoE
has not been generally considered a conventional cell surface re-
ceptor, our studies revealed a presence of ApoE3 on the luminal
surface of blood vessels in normal human tissues. As documented
for other secreted proteins, binding of ApoE3 to its conventional
receptors or to the extracellular matrix surrounding the vascular
endothelium plausibly explains the isolation of ApoE3 as a recep-
tor for CMGGHGWGC.
We also uncovered two tissue-specific vascular targeting sys-
tems. ANXA2 and prohibitin were found as a ligand-receptor
in human WAT vasculature. Studies with yeast two-hybrid meth-
odology confirmed ANXA2 and prohibitin as interacting compo-
nents in lipid rafts (24, 34). Given the weight loss in obese rodents
by the targeting of prohibitin in the vasculature with an apoptotic
agent (8, 35), we predict that the selective mapping of this protein
in human WAT will have translational value.
Lastly, we report RAGE and PR-3 as molecular partners in
human tumor-containing bone marrow resulting from tumorigen-
esis or metastasis. RAGE and PR-3 appeared unexpectedly as a
molecular complex, mediating the homing of human metastatic
prostate cancer cells to the bone marrow. These proteins have,
until now, been considered to be active in unrelated pathways
and therefore, functionally distinct. PR-3 is secreted by activated
bone marrow-derived polymorphonuclear leukocytes (26, 27) and
deposited on the surface of endothelial cells in inflammation
(36). Given our additional data, PR-3 appears functionally rele-
vant to bone marrow-specific tumorigenesis and metastasis.
Finally, one should note that established vascular markers such
as aminopeptidase N and cadherin-5 have also been affinity-
purified (Table S2) further supporting this screening strategy.
In summary, it is clear that a large-scale analysis of protein
interactions in blood vessels of healthy and diseased organs can
uncover many as yet unidentified or unique molecular networks
in the human vasculature. As such, generation and annotation
of a comprehensive molecular map based on accessible ligand-
receptors in blood vessels may be used as a starting point for
functional discovery and elucidation of protein networks in the
human vasculature.
Materials and Methods
The series of primary and secondary antibody reagents are detailed in the
SI Text. Selection of the three patients and their study adhered strictly to
institutional guidelines specified in the SI Text, where administration of the
phage display library, sample collection, postbiopsy processing of human
tissue samples, statistical methods, chemical analytical methods, and immu-
nostaining methods are also fully described.
ACKNOWLEDGMENTS. We thank Drs. Ricardo Brentani, Webster Cavenee,
Roy Lobb, and Helene Sage for manuscript reading and David Bier, Pauline
Dieringer, Cherie Perez, and Dallas Williams for infrastructure. This work was
supported by grants from the National Institutes of Health, National Cancer
Institute, Department of Defense, and by awards from AngelWorks, the
Gillson-Longenbaugh Foundation, and the Marcus Foundation.
1. Barrios-Rodiles M, et al. (2005) High-throughput mapping of a dynamic signaling
network in mammalian cells. Science 307:1621–1625.
2. Stelzl U, et al. (2005) A human protein-protein interaction network: a resource for
annotating the proteome. Cell 122:957–968.
3. Rual JF, et al. (2005) Towards a proteome-scale map of the human protein-protein
interaction network. Nature 437:1173–1178.
4. Breitkreutz A, et al. (2010) A global protein kinase and phosphatase interaction
network in yeast. Science 328:1043–1046.
5. Zetter BR (1990) The cellular basis of site-specific tumor metastasis. N Engl J Med
322:605–612.
6. Risau W, Flamme I (1995) Vasculogenesis. Annu Rev Cell Dev Biol 11:73–91.
7. Folkman J (2007) Angiogenesis: an organizing principle for drug discovery? Nat Rev
Drug Discov 6:273–286.
8. Kolonin MG, Saha PK, Chan L, Pasqualini R, Arap W (2004) Reversal of obesity by
targeted ablation of adipose tissue. Nat Med 10:625–632.
9. Staquicini FI, Pasqualini R, Arap W (2009) Ligand-directed profiling: applications to
target drug discovery in cancer. Expert Opin Drug Dis 4:51–59.
10. Pasqualini R, Ruoslahti E (1996) Organ targeting in vivo using phage display peptide
libraries. Nature 380:364–366.
11. Smith GP, Scott JK (1993) Libraries of peptides and proteins displayed on filamentous
phage. Methods Enzymol 217:228–257.
12. Arap W, et al. (2002) Steps toward mapping the human vasculature by phage display.
Nat Med 8:121–127.
13. Zurita AJ, et al. (2004) Combinatorial screenings in patients: the interleukin-11 recep-
tor alpha as a candidate target in the progression of human prostate cancer. Cancer
Res 64:435–439.
14. Pentz RD, Flamm AL, Pasqualini R, Logothetis CJ, Arap W (2003) Revisiting technical
guidelines for research with terminal wean and brain-dead patients. Hastings Cent
Rep 33:20–26.
15. Pentz RD, et al. (2005) Ethics guidelines for research with the recently dead. Nat Med
11:1145–1149.
16. Kolonin MG, et al. (2006) Synchronous selection of homing peptides for multiple
tissues by in vivo phage display. FASEB J 20:979–981.
17. Dias-Neto E, et al. (2009) Next-generation phage display: integrating and comparing
available molecular tools to enable cost-effective high-throughput analysis. PLoS One
4:1–11.
18. Vendruscolo M, Paci E, Dobson CM, Karplus M (2001) Three key residues form a critical
contact network in a protein folding transition state. Nature 409:641–645.
19. Swairjo MA, Seaton BA (1994) Annexin structure and membrane interactions: a
molecular perspective. Annu Rev Biophys Biomol Struct 23:193–213.
20. Burgoyne RD, Geisow MJ (1989) The annexin family of calcium-binding proteins.
Review article. Cell Calcium 10:1–10.
21. Rose DM, Alon R, Ginsberg MH (2007) Integrin modulation and signaling in leukocyte
adhesion and migration. Immunol Rev 218:126–134.
22. van Vlijmen BJ, et al. (1994) Diet-induced hyperlipoproteinemia and atherosclerosis
in apolipoprotein E3-Leiden transgenic mice. J Clin Invest 93:1403–1410.
23. Lutgens SPM, Cleutjens KB, Daemen MJ, Heeneman S (2007) Cathepsin cysteine
proteases in cardiovascular disease. FASEB J 21:3029–3041.
24. Liu J, Deyoung SM, Zhang M, Dold LH, Saltiel AR (2005) The stomatin/prohibitin/
flotillin/HflK/C domain of flotillin-1 contains distinct sequences that direct plasma
membrane localization and protein interactions in 3T3-L1 adipocytes. J Biol Chem
280:16125–16134.
25. Zhang J, McCrae KR (2005) Annexin A2 mediates endothelial cell activation by
antiphospholipid/anti-beta2 glycoprotein I antibodies. Blood 105:1964–1969.
26. Molldrem JJ, et al. (2000) Evidence that specific T lymphocytes may participate in the
elimination of chronic myelogenous leukemia. Nat Med 6:1018–1023.
27. Campanelli D, et al. (1990) Cloning of cDNA for proteinase 3: a serine protease,
antibiotic, and autoantigen from human neutrophils. J Exp Med 172:1709–1715.
28. Huttunen HJ, Fages C, Kuja-Panula J, Ridley AJ, Rauvala H (2002) Receptor for
advanced glycation end products-binding COOH-terminal motif of amphoterin inhi-
bits invasive migration and metastasis. Cancer Res 62:4805–4811.
29. Ishiguro H, et al. (2005) Receptor for advanced glycation end products (RAGE) and
its ligand, amphoterin are overexpressed and associated with prostate cancer devel-
opment. Prostate 64:92–100.
30. Jeon YJ, et al. (2010) Annexin A4 interacts with NF-kappaB p50 subunit and modulates
NF-kappaB transcriptional activity in a Caþ-dependent manner. Cell Mol Life Sci
67:2271–2281.
31. Lobb RR, Hemler ME (1994) The pathophysiologic role of α4 integrins in vivo. J Clin
Invest 94:1722–1728.
32. Gorfu G, Rivera-Nieves J, Ley K (2009) Role of β7 integrins in intestinal lymphocyte
homing and retention. Curr Mol Med 9:836–850.
33. Hatters DM, Peters-Libeu CA, Weisgraber KH (2006) Apolipoprotein E structure:
insights into function. Trends Biochem Sci 31:445–454.
34. Bacher S, Achatz G, Schmitz ML, Lamers MC (2002) Prohibitin and prohibitone are
contained in high-molecular weight complexes and interact with alpha-actinin and
annexin A2. Biochimie 84:1207–1220.
35. Kim DH, Woods SC, Seeley RJ (2010) Peptide designed to elicit apoptosis in adipose
tissue endothelium reduces food intake and body weight. Diabetes 59:907–915.
36. Uehara A, Sugawara Y, Sasano T, Takada H, Sugawara S (2004) Proinflammatory
cytokines induce proteinase 3 as membrane-bound and secretory forms in human oral
epithelial cells and antibodies to proteinase 3 activate the cells through protease-
activated receptor-2. J Immunol 173:4179–4189.
18642 ∣www.pnas.org/cgi/doi/10.1073/pnas.1114503108 Staquicini et al.
