Antitumor activity of a homing peptide that targets
tumor lymphatics and tumor cells
Pirjo Laakkonen*†, Maria E. Åkerman*‡, Hector Biliran*§, Meng Yang¶, Fernando Ferrer*, Terhi Karpanen†,
Robert M. Hoffman¶, and Erkki Ruoslahti*?
*The Burnham Institute, La Jolla, CA 92037;†Molecular?Cancer Biology Laboratory, Biomedicum Helsinki, University of Helsinki, P.O.B. 63 (Haartmaninkatu
8), FIN-00014 Helsinki, Finland;‡Department of Bioengineering, University of California at San Diego, La Jolla, CA 92093; and¶AntiCancer, Inc.,
San Diego, CA 92111
Contributed by Erkki Ruoslahti, May 10, 2004
LyP-1 is a peptide selected from a phage-displayed peptide library
that specifically binds to tumor and endothelial cells of tumor
lymphatics in certain tumors. Fluorescein-conjugated LyP-1 and a
related peptide, LyP-1b, strongly accumulated in primary MDA-
MB-435 breast cancer xenografts and their metastases from i.v.
peptide injections, allowing visualization of orthotopic tumors in
intact mice. The LyP peptide accumulation coincided with hypoxic
areas in tumors. LyP-1 induced cell death in cultured human breast
carcinoma cells that bind and internalize the peptide. Melanoma
cells that do not bind LyP-1 were unaffected. Systemic LyP-1
peptide treatment of mice with xenografted tumors induced with
the breast cancer cells inhibited tumor growth. The treated tumors
contained foci of apoptotic cells and were essentially devoid of
lymphatics. These results reveal an unexpected antitumor effect by
the LyP-1 peptide that seems to be dependent on a proapoptotic?
cytotoxic activity of the peptide. As LyP-1 affects the poorly
vascularized tumor compartment, it may complement treatments
directed at tumor blood vessels.
phage display ? tumor targeting ? live imaging ? therapy
vessel markers are related to angiogenesis, but some are selective
for certain tumors (1). Markers that distinguish the vasculature
of tumors at the premalignant stage from the vasculature of fully
malignant tumors in the same tumor system have also been
described (2, 3). Recent data from our laboratory indicate that
lymphatic vessels in tumors are also specialized, because a cyclic
9-amino acid peptide, LyP-1, binds to the lymphatic vessels in
certain tumors, but not to the lymphatics of normal tissues (4).
The lymphatic system is an important route of tumor metas-
Recent discoveries of growth factors and molecular markers for
lymphatic endothelial cells have made possible detailed studies
of the relationship of tumor cells and the lymphatic vasculature
of tumors (5–9). The use of marker proteins such as LYVE-1 (6),
podoplanin (5), and Prox-1 (10) has shown that lymphatic vessels
are abundant in the periphery of tumors and that many tumors
also contain lymphatics within the tumor mass (4, 11). However,
the intratumoral lymphatic vessels are generally not functional in
transporting tissue fluid (12) and are often filled with tumor cells
(4, 13). Recent experimental and clinical data strongly suggest
that the number of lymphatics in a tumor, perhaps their size as
well, and the expression of lymphangiogenic growth factors are
important determinants in the ability of a tumor to metastasize
Thus, it may become possible to reduce metastasis by specif-
ically targeting tumor lymphatics (and the tumor tissue adjacent
to these vessels) for destruction. The LyP-1 peptide, which
specifically binds to tumor lymphatics (4), provides one potential
avenue for developing reagents that can specifically destroy
tumor lymphatics. This peptide also binds to the tumor cells in
umor blood vessels express molecular markers that distin-
guish them from normal blood vessels. Many of these tumor
tumors that contain LyP-1-positive lymphatics, further expand-
ing the potential of this peptide.
We show here that i.v. injected LyP-1 strongly and specifically
accumulates in breast cancer xenografts over time, localizing
preferentially in hypoxic areas. We also report that LyP-1 has a
proapoptotic?cytotoxic effect on tumor cells and that systemic
administration of the LyP-1 peptide inhibits breast cancer xeno-
graft growth in mice. The treated tumors contain foci of
apoptotic cells and reduced numbers of lymphatic vessels. These
findings suggest that LyP-1 may provide a starting point for the
development of new antitumor agents.
Materials and Methods
Cell Lines and Tumors. MDA-MB-435 human breast carcinoma
cells and C8161 human melanoma cells were maintained in
DMEM supplemented with 10% FCS. Nude BALB?c nu?nu
mice were injected s.c. or into the mammary fat pad with 1 ? 106
tumor cells to induce tumors. A vascular endothelial growth
factor (VEGF)-C-transfected MDA-MB-435 cell line was pre-
pared as previously reported for MCF7 cells (13).
Antibodies and Immunohistology. Blood vessels were visualized by
staining tissue sections with monoclonal antibodies against
CD-31, CD-34, or MECA-32 (all rat anti-mouse antibodies from
Pharmingen). A polyclonal rabbit anti-mouse LYVE-1 antibody
(4) and a rat monoclonal anti-mouse podoplanin antibody
(provided by Kari Alitalo, University of Helsinki) were used to
visualize lymphatic vessels. The primary antibodies were de-
tected with goat anti-rabbit or anti-rat Alexa 594 (Molecular
Biodistribution of fluorescein-conjugated peptides was exam-
ined after i.v. injection (100 ?l of 1 mM peptide solution in 200
?l of PBS) into the tail vein of a mouse. The peptide was allowed
to circulate for various periods of time, and the mouse was
perfused through the heart with 4% paraformaldehyde. Tissues
were removed, soaked in 30% sucrose in PBS overnight, and
frozen in OCT embedding medium (Tissue-Tek). Alternatively,
tumor-bearing mice were i.v. injected with 500 ?l of 1 mM
fluorescein-conjugated peptide in PBS, and the peptide was
allowed to circulate for 16–20 h.
imaging system of a fluorescence stereo microscope (model LZ12;
Leica, Deerfield, IL) equipped with a mercury 50-W lamp (19).
Determination of Vessel Density in Tissues. Frozen tumor sections
were stained with antibodies against CD-34 and podoplanin (5)
to visualize the tumor-associated blood and lymphatic vessels.
Using ?200 magnification, each microscopic field in the hori-
Abbreviation: VEGF, vascular endothelial growth factor.
§Present address: Department of Pathology, School of Medicine, Wayne State University,
540 East Canfield Road, Detroit, MI 48201.
?To whom correspondence should be addressed. E-mail: email@example.com.
© 2004 by The National Academy of Sciences of the USA
June 22, 2004 ?
vol. 101 ?
no. 25 ?
zontal and the vertical directions was counted for the presence
of the two types of vessels.
Hypoxia. Hypoxic areas in the tumor were visualized by i.v.
injection of a hypoxia marker 2-nitroimidazole (EF5) (20) into
tumor-bearing mice (10 ?l of 10 mM EF5 per g), followed by
Cy3-conjugated mouse anti-EF5 (provided by Randall S.
Johnson, University of California at San Diego). Cultured
MDA-MB-435 cells were grown on coverslips and incubated
overnight at 37°C to allow for attachment and spreading of the
cells. Half of the cells were transferred to a hypoxia chamber
(0.1% oxygen?5% CO2) and incubated overnight under hy-
poxic conditions. Fluorescein-conjugated peptides (10 ?M)
were added to the cells in 1% BSA in DMEM and incubated
for 3 h, followed by fixation with 4% paraformaldehyde in PBS.
The coverslips were mounted on glass slides by using Vecta-
Shield mounting media with 4?,6-diamidino-2-phenylindole
Cytotoxicity Assay. Cytotoxic efficacy of the different peptides
was judged by measuring the release of a cytoplasmic enzyme,
lactate dehydrogenase, from damaged cells into the superna-
tant by using a colorimetric assay Cytotoxicity Detection Kit
(LDH assay; Roche Diagnostics). MDA-MB-435 cells were
plated on 96-well plates (6,000 cells per well) and incubated
overnight at 37°C to allow for attachment and spreading of the
cells. Cells were washed once with PBS, and 50 ?l of 2% BSA
in DMEM was added to the cells. Peptides were added in
50 ?l of H2O and incubated for 24–72 h at 37°C. After the
incubation, the cells were spun down (1,000 rpm, 10 min), and
the supernatant was transferred to a new plate. The color
reaction was added to the cells and incubated for 25 min before
the absorbance was read at 492 nm. Cells incubated with 50 ?l
of H2O and 50 ?l of 2% BSA in DMEM served as a background
control, and cells incubated with 1% Nonidet P-40 showed the
maximal cytotoxic value. The cytotoxicity was determined as
a percentage of the maximal value after the subtraction of the
Tumor Treatment Studies. Tumor-bearing mice were treated with
i.v. injections of peptides beginning 4 weeks after tumor cell
inoculation. The injections were administered twice a week for
4–5 weeks. Tumor volumes were measured once a week and
were calculated according to the formula V ? width ?
height ? depth?2, derived from the formula for the volume of
an ellipsoid (21). Student’s t test was used for statistical
analysis of the results. The animal experiments reported here
were approved by The Burnham Institute Animal Research
Synthesis of Fluorescein-Conjugated Peptides. Peptides were syn-
thesized by using Fmoc-protected amino acids (Nova Bio-
chem) and HATU (PE Biosystems, Foster City, CA) as a
coupling reagent in dimethylformamide activated with diisio-
propylethylamine. All peptides were amide-capped at the C
terminus by the use of Fmoc-PAL-PEG-PS resin (PE Biosys-
tems). The peptides were conjugated with fluorescein at the N
terminus by reacting with fluorescein isothiocyanate isomer
(FITC, Aldrich) in dimethylformamide for 20 h in the presence
Intravenously injected LyP-1 peptide was observed to home to
tumor-associated lymphatic vessels and tumor cells in MDA-
MB-435 xenografts and some other tumors (4). In this earlier
work, the peptide was allowed to circulate for ?20 min. To
optimize the accumulation of LyP-1 in these tumors, we
studied the distribution of the peptide for longer periods of
time. We found striking accumulation of fluorescein-
conjugated LyP-1 in tumors several hours after the injection.
At 16–20 h, the tumors of the LyP-1-injected mice were
brightly fluorescent in whole-body fluorescent imaging (19) of
intact mice (Fig. 1A). Tumors from the mice injected with a
control peptide showed no fluorescence (Fig. 1B). Imaging of
dissected tumors and organs from the same animals revealed
strong fluorescence in tumors from mice injected with LyP-1,
whereas no fluorescence was detectable in the tumors from the
control peptide-injected mice. Other tissues showed no spe-
cific fluorescence with either peptide (Fig. 1 C and D).
We then confirmed the tumor-specificity of LyP-1 by quan-
tifying the fluorescence in the dissected tissues. Tumor fluores-
cence from the control peptide injection was too low to be
accurately distinguished from the background, but LyP-1 con-
centration was at least 15- to 40-fold higher in the tumors than
that of the control peptide, whereas fluorescence in other tissues
was not significantly different from the background (Fig. 1E). A
peptide closely related to LyP-1 (CNKRTRGGC; H.B., J. A.
Hoffman, P.L., and E.R., unpublished data) also strongly accu-
mulated in the tumors (LyP-1b in Fig. 1E). These results show
that the LyP-1 peptides accumulate in the MDA-MB-435 tumors
with extraordinary efficiency and that the accumulation is
nuclei (Fig. 1F), whereas the control peptide was essentially
negative in tumor tissue (Fig. 1G). No fluorescence was detected
tissue in Fig. 1H).
LyP-1 Recognizes Metastatic Lesions. MDA-MB-435 tumor cells
transfected with the lymphangiogenic growth factor VEGF-C
produce tumors with increased number of lymphatic vessels
and enhanced propensity to metastasize into regional lymph
nodes and the lungs (13, 16, 22). In agreement with the ability
of LyP-1 to recognize tumor lymphatics, LyP-1 accumulation
in the VEGF-C-expressing tumors seemed to be stronger than
in the parental-line tumors (data not shown). LyP-1 peptide
also homed to the metastatic lymph nodes of the MDA-MB-
435?VEGF-C tumor mice (Fig. 2 A–E), colocalizing with
lymphatic endothelial markers (arrows in Fig. 2C) and tumor
cells (Fig. 2E) within the lymph nodes. No LyP-1 fluorescence
was detected in the vessels of normal lymph nodes; the nuclei
of a few isolated cells that appeared to be leukocytes were
positive (Fig. 2B Inset). Metastatic foci in lungs were also
positive for Lyp-1 (Fig. 2F). These results show that metastases
can retain the LyP-1 binding of the primary tumor and that the
same tumor can induce the LyP-1-binding epitope in the
lymphatic vessels of more than one tissue.
LyP-1 Peptide Recognizes Hypoxic Areas in Tumors. The tumor cells
that accumulated LyP-1 formed clusters within the tumors, and
these clusters contained few blood vessels (Fig. 3A) but were
positive for lymphatic endothelial markers (Fig. 3B). The LyP-
1-positive tumor cell clusters were strikingly similar to clusters of
tumor cells revealed by uptake of hypoxia markers (23). This
similarity, and the lack of blood vessels, led us to examine a
possible connection between LyP-1 binding and hypoxia. Intra-
venously injected hypoxia reagent EF5 and fluorescein-labeled
LyP-1 accumulated in the same areas in the tumors, but the
staining for the two markers seemed to be mutually exclusive at
the level of individual cells (Fig. 3 C and D). If EF5 was injected
first, the homing of LyP-1 was reduced (Fig. 3C) and vice versa
(Fig. 3D). Moreover, injecting LyP-1 or EF5 alone gave a
stronger tumor signal for both compounds than the coinjections.
In contrast, the accumulation of EF5 in C8161 melanoma
xenografts, which are not recognized by LyP-1 (4), was unaf-
fected by coinjecting LyP-1 (data not shown). These results
www.pnas.org?cgi?doi?10.1073?pnas.0403317101Laakkonen et al.
indicate that LyP-1 preferentially localizes in hypoxic parts of
tumors and that LyP-1 and EF5 specifically affect one another’s
recognition of hypoxic tumor cells.
Serum Starvation Increases Binding of Fluorescein-Conjugated LyP-1
to Cultured MDA-MB-435 Cells. We next sought to reproduce the
effect of hypoxia on tumor cell recognition in vitro. We cultured
MDA-MB-435 cells under hypoxic conditions but detected no
increase in the number of cells that were positive for fluorescein-
conjugated LyP-1 (data not shown). However, we did see an
increase in the number of cells that had taken up LyP-1 when we
maintained the cells in low serum (compare Fig. 3 E and F).
Counting of LyP-1-positive cells showed that the difference was
2.5-fold. These results suggest that LyP-1 homing to tumors may
fluorescein-conjugated LyP-1 (A) or a fluorescein-conjugated control peptide (ARALPSQRSR) (B). The mice were anesthetized 16–20 h later and examined for
fluorescence under blue light. Tumor fluorescence of a LyP-1-injected tumor mouse is shown in A. No fluorescence was detected in tumors of mice injected with
the control peptide (B). After the external examination, the mice were killed, and tumor, kidneys, spleen, and liver were excised and examined for fluorescence.
LyP-1 produced intense fluorescence in the tumor, whereas no fluorescence was detectable in other organs (C). Even when imaged directly, no fluorescence was
observed in the control peptide-injected tumor (D). The gallbladder is autofluorescent and appears as a green spot in C and D. E shows quantification of the
(F–H) Mice injected with peptides as in A and B were perfused through the heart, and their tumors were examined microscopically. Strong LyP-1 fluorescence
tissue (G), and all normal tissues tested were negative for all peptides (the result for LyP-1 in the brain is shown in H). T, tumor; B, brain; H, heart; Lu, lungs; Li,
liver; S, spleen; K, kidneys. Magnification: F and G, ?100; H, ?200.
Specific accumulation of lymphatic homing peptides in tumors. Mice bearing orthotopic MDA-MB-435 xenograft tumors were i.v. injected with
Laakkonen et al.
June 22, 2004 ?
vol. 101 ?
no. 25 ?
not be directly related to hypoxia but may result from the
attendant nutrient starvation.
LyP-1 Binding and Internalization Induce Cell Death. Studying the
internalization of the fluorescein-conjugated LyP-1 peptide in
cultured cells, we noticed that the LyP-1 positive cells tended to
round up, and the morphology of their nuclei frequently sug-
we incubated MDA-MB-435 cells with unlabeled LyP-1 and
monitored cell lysis. Incubation with LyP-1 resulted in a con-
centration-dependent increase in cell lysis with an IC50of ?66
?M (Fig. 4A). C8161 human melanoma cells, which do not bind
LyP-1 (4), were not affected by the peptide. Control peptides
that resemble LyP-1 in their amino acid composition and?or
cyclic structure (CRVRTRSGC, Fig. 4A) and two other peptides
[CGEKRTRGC, a variant of LyP-1, which has no cell-binding
activity (4); and KECQSRLLSCP, (data not shown)] had no
effect on the viability of either cell line. Thus, LyP-1 specifically
kills cells that bind this peptide.
Systemic Treatment with LyP-1 Inhibits Tumor Growth and Reduces
the Number of Tumor Lymphatics. Given that LyP-1 had an in vitro
cytotoxic effect on the MDA-MB-435 tumor cells, we examined
the effect of LyP-1 on tumor growth in vivo. We gave MDA-
MB-435 or MDA-MB-435?VEGF-C tumor mice biweekly i.v.
injections of the LyP-1 peptide, starting after the mice had
established palpable tumors. Fig. 4B shows one of three similar
treatment experiments. The LyP-1 peptide inhibited tumor
significant (P ? 0.005). Increasing the dose of the LyP-1 peptide
did not improve the efficacy of the compound (data not shown).
The tumors of the LyP-1-treated animals contained numerous
TUNEL-positive cells, indicating apoptosis, whereas little apo-
ptosis was detected in the tumors of the control-treated mice
(Fig. 4 C and D). The increased apoptosis in the LyP-1 group was
specific for the tumor tissue; other tissues did not contain
significant numbers of TUNEL-positive cells (data not shown).
LyP-1-treatment selectively reduced the number of lymphatic
vessels in the tumors, while having a less prominent effect on the
in agreement with the in vivo homing pattern of the fluorescein-
conjugated LyP-1 peptide to the lymphatics in MDA-MB-435
tumor-bearing mice (4). It seems that the lymphatic endothelial
cells in the tumor are also susceptible to LyP-1.
mice bearing orthotopic VEGF-C-expressing MDA-MB-435 tumors and al-
lowed to circulate for 15 min. The tumor, lymph nodes, lungs, kidneys, and
liver were removed and prepared for immunohistology. Lymphatic vessels in
lymph node metastases (A–D) were visualized by staining with anti-LYVE-1
antibodies followed by goat anti-rabbit Alexa 594 (red, A and C). Nuclei were
visualized by 4?,6-diamidino-2-phenylindole staining (blue, B and D). A and B
and C and D show the same microscopic fields with different staining. LyP-1
peptide (green) is present in the nuclei of cells in and around enlarged
lymphatic vessels in lymph node metastases. These cells are tumor cells as
judged by their intense staining with anti-VEGF-C antibody (red, E). The
peptide is also seen in the nuclei of lymphatic endothelial cells (arrows in C).
tumor also accumulates LyP-1 (F; LyP-1, green; nuclei, blue). Magnification,
?200; Inset, ?50.
Lymphatic homing peptide recognizes metastases of VEGF-C-
contain lymphatics. Fluorescein-conjugated LyP-1 peptide was i.v. injected
into MDA-MB-435 tumor-bearing mice and allowed to circulate for 15 min.
vessel endothelial marker, MECA-32 (red, A) but were often positive for the
lymphatic endothelial markers, LYVE-1 (red, B) and podoplanin (not shown).
The hypoxia marker EF5 (red), injected 8 h before LyP-1 (green), localized in
the LyP-1-positive patches within the tumors (C). Reversing the order of the
injections reduced the amount of EF5 in the LyP-1-positive patches (D). The
presence of the two compounds at the cellular level seemed to be mutually
exclusive. (E and F) LyP-1 binding to cultured cells is increased by serum
starvation. Fluorescein-conjugated LyP-1 peptide was added to MDA-MB-435
cells cultured either in 10% (E) or 0.1% (F) serum, and the binding and uptake
of the peptide by the cells was determined 3 h later. Serum starvation
increased the number of LyP-1-positive cells (LyP-1, green; nuclei, blue).
Magnification: A–D, ?200; E and F, ?100.
LyP-1 peptide recognizes cell clusters that lack blood vessels but
www.pnas.org?cgi?doi?10.1073?pnas.0403317101Laakkonen et al.
We report here that LyP-1, a peptide that specifically binds to
tumor lymphatics and tumor cells, strongly accumulates in breast
cancer xenografts after an i.v. injection. The peptide and a
closely related variant of it preferentially localize in hypoxic
areas within the tumors. We also show that systemically admin-
istered LyP-1 causes tumor cell apoptosis, reduces the number of
tumor lymphatics, and inhibits tumor growth in mice bearing
breast cancer xenografts. These results suggest that it may be
possible to develop LyP-1-based cancer therapies.
The LyP-1 peptide shows strong accumulation in the MDA-
MB-435 tumors, including metastases from these tumors. The
efficacy and specificity of this peptide was sufficient to allow us
to visualize orthotopic tumors in intact mice based on fluores-
cence. Although fluorescence-based imaging of tumors formed
by GFP-producing cells in intact animals is possible (19), achiev-
ing it with an i.v. injected material may be unique. The remark-
able tumor-homing efficiency of the LyP-1 peptide may be
because of the propensity of this peptide to become internalized
by cells. Cells that bind the LyP-1 peptide transport it across the
cell membrane, into the cytoplasm and the nucleus. In this
regard, LyP-1 is similar to the Tat peptide and other cell-
penetrating peptides, which are also taken up by cells (24). An
important difference is that our LyP-1 peptides are cell type-
specific and deliver a payload to specific target cells: the
lymphatic endothelial and tumor cells in tumors that display the
‘‘receptor’’ for these peptides. The internalization is likely to
contribute to the effectiveness of these peptides in becoming
concentrated in the targeted tumors. If this efficacy can be
reproduced in clinical settings, LyP-1-directed targeting of con-
trast agents may become useful in tumor detection.
dehydrogenase from the cultured MDA-MB-435 cells, whereas a control peptide (CRVRTRSGC, E) has no effect. LyP-1 does not release lactate dehydrogenase
from human C8161 melanoma cells (F, dotted line). (B) Mice bearing MDA-MB-435 tumors were injected twice a week with 60 ?g of LyP-1 or its inactive variant
(CGEKRTRGC), or with PBS. There were five mice?group; the treatment was started 4 weeks after the inoculation of the tumor cells (1–2 weeks after the tumors
staining (red) reveals clusters of apoptotic cells in the LyP-1-treated (C), but not control-treated (D), tumors. Blue, 4?,6-diamidino-2-phenylindole staining of
nuclei. Magnification, ?200. The error bars in B show SEM.
LyP-1 peptide causes cell death in vitro and inhibits tumor growth in vivo. (A) LyP-1 (F, solid line) causes a dose-dependent release of lactate
sections were stained with antibodies against CD-34 and podoplanin to
visualize and count tumor-associated blood vessels and lymphatics. LyP-1
reduced the number of lymphatic vessels by an average of 85% (three exper-
iments). Blood vessel density in the same tumors was affected less (average
reduction, 39%). The error bars show SD.
LyP-1 peptide reduces the number of tumor lymphatics. Tumor
Laakkonen et al.
June 22, 2004 ?
vol. 101 ?
no. 25 ?
Our results show that treatment of tumor cells with the LyP-1 Download full-text
peptide causes cell death. This effect is specific because cells that
do not bind LyP-1 were not affected. The tumor cell apoptosis
we observed in vivo indicates that the LyP-1-binding cells die by
Whereas the mechanism whereby LyP-1 kills cells remains to
be elucidated, the proapoptotic effect seems to be directed
against tumor cells that are under stress, as LyP-1 colocalized
with a tissue hypoxia marker in vivo, and serum starvation
enhanced LyP-1 binding and internalization by cultured tumor
cells in vitro. It will be important to identify the molecule
(receptor) to which LyP-1 binds at the cell surface (and that may
mediate the proapoptotic effect of LyP-1). Our efforts to isolate
a LyP-1 receptor by affinity chromatography and various cloning
methods have not yet been successful.
Treatment of tumor-bearing mice with the LyP-1 peptide
suppressed tumor growth. It also drastically reduced the expres-
latter result suggests that LyP-1 is also cytotoxic?proapoptotic
for lymphatic endothelial cells in tumors. As tumor lymphatics
have not been shown to be important for tumor growth (25), it
is likely that the antitumor activity of LyP-1 is related to its effect
on tumor cells rather than tumor lymphatics. However, given the
demonstrated role of tumor lymphatics in metastasis (15, 16, 22),
destroying tumor lymphatics with LyP-1 may be particularly
effective in curtailing lymphatic spread of tumors. As lymphatics
appear to be the first target of LyP-1 in tumors (4), the antitumor
effect of LyP-1 may be particularly pronounced on tumor cells
within and close to the lymphatics, which are likely to be the cells
most probable to spread through the lymphatic system.
Hypoxia enhances metastasis (23, 26), and LyP-1 selectively
targets tumor cells in the hypoxic areas of tumors. This may be
another pathway through which LyP-1 could suppress metasta-
sis. MDA-MB-435 tumors are highly metastatic, and the VEGF-
study, we evaluated the effects of LyP-1 on established primary
tumors. As metastasis had already occurred at the time the
metastatic spread. Studies to determine the effects of LyP-1 on
metastasis are underway. Nonetheless, the data already at hand
define this peptide as a potentially unique tool for tumor
diagnosis and treatment.
We thank Dr. Randall Johnson for reagents, Drs. Kari Alitalo and Eva
Engvall for comments on the manuscript, and Roslind Varghese for
editing. This work was supported by National Cancer Institute Grant
Cancer Center Support Grant CA30199, and National Cancer Institute
Grant CA099258-01 (to AntiCancer, Inc.). P.L. received support from
the Academy of Finland and Biocentrum Helsinki. M.E.A. was sup-
ported by Department of Defense Fellowship DAMD17-02-1-0308.
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www.pnas.org?cgi?doi?10.1073?pnas.0403317101Laakkonen et al.