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Transtumoral targeting enabled by a novel neuropilin-binding peptide
Lise Roth, Ph.D.1,2, Lilach Agemy, Ph.D.1,2, Venkata R. Kotamraju, Ph.D1,2, Gary Braun,
Ph.D3, Tambet Teesalu, Ph.D.1,2, Kazuki N. Sugahara, M.D., Ph.D.2, Juliana Hamzah,
Ph.D.1,2 and Erkki Ruoslahti, M.D., Ph.D.1,2*
1Vascular
Mapping Center, Sanford-Burnham Medical Research Institute, UCSB
campus, Biology II building, University of California, Santa Barbara, CA 93106-9610,
USA 2Cancer Research Center, Sanford-Burnham Medical Research Institute, 10901
North Torrey Pines Road, La Jolla, CA 92037, USA 3Institute for Collaborative
Biotechnologies, University of California, Santa Barbara, California 93106, USA
*Corresponding author. Mailing address:
Sanford-Burnham Medical Research Institute at UCSB, Bio II, Rm. #3119, University
of California, Santa Barbara, Santa Barbara, CA 93106-9610. Phone: (805) 893-5327.
Fax: (805) 893-5805. E-mail: [email protected].
Author contributions: E.R. and L.R. designed research; L.R., L.A., T.T., K.N.S. and J.H.
performed research; L.A., G.B. and V.R.K. contributed reagents; L.R. and E.R.
analyzed data and wrote the paper. All authors discussed the results and
commented on the manuscript.
Running title: Tumor-penetrating peptide
Grant support: This work was supported by grant number W81XWH-09-1-0698
and W81XWH-08-1-0727 from the USAMRAA for the Department of Defense (ER).
L.R. was supported by Susan G. Komen for the Cure post-doctoral fellowship
(KG091411) and G.B. by a fellowship from the Santa Barbara Cancer Center. E.R. is
supported in part by CA30199 the Cancer Center Support Grant from the NCI.
The authors declare no conflict of interest.
Word count: 4300 (Figure count: 7 figures, 6 supplemental figures)
Supplemental figure S1: Neuropilin expression in cancer cell lines. A. Fluorescenceactivated cell sorting (FACS) analysis was done on live human tumor cells to detect
cell-surface expression of NRP1 (red line) and NRP2 (blue line). Approximately 106
cells were stained with mouse anti-NRP1 (monoclonal, Miltenyi), mouse anti-NRP2
(monoclonal, R&D), or mouse IgG (BD PharMingen) at 4 °C. DU145 prostate
carcinoma cells expressed exclusively NRP1, whereas MDA-MB-435 breast tumor
cells expressed only NRP2. PPC1 prostate tumor cells, while expressing very high
levels of NRP1, also expressed low levels of NRP2. Representative of 3 experiments.
B. Confocal microscope images of mouse 4T1 tumor cells stained with anti-NRP1
(polyclonal, Chemicon) and anti-NRP2 (polyclonal, Invitrogen) antibodies. Note that
4T1 cells express both NRPs. (Original magnification, 40×; scale bars: 50 m).
Supplemental figure S2: Phage binding on DU145 and MDA-MB-435 cells. A
suspension of 2x105 cells was incubated with 7x108 pfu/mL of T7 phage in
DMEM/BSA 1% for 1 h at 4 °C. The cells were washed four times with DMEM/BSA
1%, and lysed with lysogeny broth/1% Nonidet P-40 (LB/NP40). The lysate was
subjected to phage titration. Results are expressed as a binding ratio over insertless
phage control. (mean  SEM; n=4/group) A. Binding to DU145 cells. B. Binding to
MDA-MB-435 cells.
Supplemental figure S3: tLyP-1 binding to LyP-1 receptor p32. Binding of FAMtLyP-1 and FAM-LyP-1 to p32 was measured by an ELISA-based assay. Microtiter
wells coated with 3 g/mL of purified p32 (Fogal et al 2008) were incubated with
increasing concentrations of FAM-peptides at 37 °C for 1 h. After washing with cold
PBS added with 0.01% Tween 20 and 0.2 M NaCl, fluorescence was read at 495 nm.
Note that tLyP-1 bound weakly to p32 compared to LyP-1.
Supplemental figure S4: tLyP-1 and LyP-1 phage binding to 4T1 cells in vitro.
Confocal microscope images of murine 4T1 cells incubated in the presence of 109
pfu of LyP-1 or tLyP-1 phage at 37 °C. Phage were detected by staining with anti-T7
phage polyclonal antibody (red). Nuclei were stained with DAPI (blue). Note that
both tLyP-1 and LyP-1 phage bound and internalized into 4T1 cells, which express
NRP1, NRP2, and the LyP-1 primary receptor, p32 (Supplemental Fig. S1 and (Fogal
et al 2008)). (Original magnification, 40×; scale bars, 50 m).
Supplemental figure S5: tLyP-1-NW homing in 4T1 tumors over time. NWs (5 mg
iron/kg mouse) conjugated to FAM-tLyP-1 peptide were intravenously injected into
4T1 tumor bearing mice and allowed to circulate for 30 min (upper panels), 6 h
(middle panels) or 16 h (bottom panels). Note that the NWs were still localized and
spread in the tumor after extended circulation, whereas their presence decreased
over time in other organs involved in non-specific uptake. tLyP-1-NW: green, nuclei:
blue. (Original magnification, 20×; scale bars: 100 m).
Supplemental figure S6: tLyP-1 NWs home specifically to MDA-MB-231 tumors. To
produce MDA-MB-231 breast tumors, BALB/C athymic nude mice were
orthotopically injected into the mammary fat pad with 5x106 cells suspended in 100
L of PBS/matrigel (BD Biosciences) (50/50). tLyP-1-NWs (5 mg iron/kg mouse)
were intravenously injected into tumor bearing mice. Tumors and organs were
collected and processed for immunofluorescence. A. The NWs were allowed to
circulate for 4 h. Note the strong fluorescence in the tumor compared to the other
organs. tLyP-1-NW: green, nuclei: blue. (Original magnification, 20×; scale bars: 100
m). B. The NWs (green) were allowed to circulate for 30 min (left panel) or 4 h
(right panel). Blood vessels were stained with an anti-CD31 antibody (red). Note the
extravasation in the tumor parenchyma over time. (Original magnification, 40×;
scale bars: 50 m).