Supporting Information
DeLisser et al. 10.1073/pnas.1004654107
SI Materials and Methods
FACS Analysis. H5V [a murine CD31-positive vascular endothelial
cell (VEC) line], B16-F10, LOX, or 4T1 tumor cells were treated
with rat IgG or mAb 390 for 1 h at 4 °C. The primary Ab then was
removed, the cells were washed with PBS, and a 1/200 dilution of
FITC-labeled goat anti-mouse or anti-rat secondary Ab (Cappel)
was added for 30 min at 4 °C. After cells were washed in PBS, flow
cytometry was performed using an Ortho Cytofluorograph 50H
cell sorter equipped with a 2150 data-handling system (Ortho In-
struments).
Tumor–Endothelial Cell Adhesion Assay. Confluent monolayers of the
murine H5V endothelial cell line were cultured in 24-well plates. The
cells then were incubated for 30 min with 500 μL of DMEM medium
with 1% serum containing 100 μg/mL IgG or antibody. Then 1 × 104
tritium-labeled B16-F10 tumor cells in 500 μL of DMEM medium
were added and incubated at 37 °C for an additional 30 min. The
wells were washed gently with PBS, the cells were lysed with a de-
tergent buffer, and the beta-emitting activity of the cell lysates was
counted using a liquid scintillation counter (Beckman Coulter).
Tumor Transendothelial Migration Assay. H5V endothelial cells (2 ×
105) were plated on fibronectin-coated 8-μm pore Transwell inserts
(BD Biosciences) and cultured for 24–48 h to permit the formation
of tight monolayers. Then 1 × 105 tritium-labeled B16 tumor cells in
500 μL of DMEM medium with 1% serum and IgG or antibody at
a concentration of 100 μg/mL were added to the Transwell inserts
and were incubated at 37 °C for 8 h. DMEM with 10% serum was
added to each outer well. (The B16 tumor cells migrate through the
H5V endothelial cells and the pores of the filter to the undersurface
of the filter.) The upper surfaces of the Transwell inserts were washed
and wiped with a cotton swab. The filters were cut out, and their beta-
emitting activity was counted using a liquid scintillation counter.
Tumor–Platelet Adhesion Assay. Blood was obtained via cardiac
puncture from mice injected i.p. with 100 U of heparin and anes-
thetizedwithketamine/xylazine.Onemilliliterofbloodwascombined
with 7 mL of PBS and was centrifuged at 100 × g for 10 minat 4 °C. The
resulting supernatant was centrifuged at 3,000 × g for 20 min at 4 °C.
The resulting platelet pellet was resuspended in PBS. Then 1 × 108
platelets in 200 μL PBS were added to the wells of a 96-well flat-
bottomed plate and incubated for 24 h at 4 °C to allow the platelets to
settle to bottom of the wells. Then 150 μL of the PBS was removed
1. Malorny U, Michels E, Sorg C (1986) A monoclonal antibody against an antigen present
on mouse macrophages and absent from monocytes. Cell Tissue Res 243:421–428.
2. Vailhé B, Vittet D, Feige JJ (2001) In vitro models of vasculogenesis and angiogenesis.
Lab Invest 81:439–452.
DeLisser et al. www.pnas.org/cgi/content/short/1004654107
and replaced with an equal amount of PBS with 1% BSA for 1 h at
37 °C. The PBS/BSA was removed, and 150 μL of IgG or antibody
at a concentration of 100 μg/mL was added for 1 h at 37 °C. The
antibodies were removed, and 5 × 103 B16 tumor cells in PBS with
0.9 mM CaCl2 were added for an additional 1 h. The wells were
washed gently with PBS to remove nonadherent tumor cells. For-
malin was added to each well to fix the cells, the cells were stained
with toluidine blue, and the adherent tumor cells were counted.
Immunohistochemistry to Detect F4/80. A rat anti-mouse mAb
(Invitrogen) was used to detect F4/80 (1). Paraffin-embedded
sections were processed in a fully integrated Leica BOND IHC
autoprocessor incorporating a peroxidase-based detection system.
3D Coculture Assay. Each well of a six-well polystyrene plate (Corning)
was coated with 200–300 μL of Matrigel (BD Biosciences). One hour
later, 2 × 105 cells of either of two different murine CD31-positive
VEC lines (CD3 or H5V) were plated on the Matrigel. Once plated
on Matrigel, VEC form tubes but do not divide (2). Seven to 8 h later,
after VEC capillary-like structures had stably formed, 2 × 105 B16-
F10 or 4T1 cells were added, together with 200 μg/mL of either anti-
platelet endothelial cell adhesion molecule 1 (PECAM-1) or isotype
control mAb. mAbs were added again at 24 and 48 h, and all cells, as
well as culture supernatants for conditioned media, were harvested
at 72 h. All wells were trypsinized and cells counted using a hemo-
cytometer by an individual blinded to the identity of each group.
Conditioned media were frozen at −80 °C until further use. Apo-
ptosis counts did not differ between tumor cells treated with anti–
PECAM-1 and those treated with control mAb.
Time-Lapse Photography of Cultured Tumor Cells. At the start of each
experiment, cell cultures were transferred from the incubator to
a time-lapse microscope equipped with a heated stage and incubation
chamber (Axiovert 200; Zeiss). The incubation chamber maintained
optimum environmental conditions (37 °C, 5% CO2) by independent
digital control units (Zeiss). Four sets of phase-contrast images from
each of three wells were acquired over a 44-h observation period
using a Cohu 2600 Series compact monochrome interline transfer
CCD camera and were taken at 300-s intervals in accordance with
previously published methods (3). Velocity software automation
(Improvision) operated the camera and stage movements and
compiled the acquired phase images. Images then were processed as
Quicktime movies using the above software.
3. Yount G, Yang Y, Wong B, Wang HJ, Yang LX (2007) A novel camptothecin analog
with enhanced antitumor activity. Anticancer Res 27(5A):3173–3178.
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 A Murine VEC B16 4T1 LOX CT26

Rat IgG

mAb 390

B C

D E

Fig. S1. Functional activities of anti–PECAM-1 mAb 390. (A) FACS analysis was used to assess the binding of rat IgG and mAb 390, an anti-murine PECAM-1
antibody, to H5V murine VEC (CD31-positive) and to B16-F10, 4T1, or LOX tumor cells. Anti–PECAM-1 mAb bound to murine VEC but not to any of the four tumor
lines (hatched lines represent mean fluorescent intensity of control mAb in each cell line tested). (B) mAb 390 (60 mg/mL) did not affect tumor cell proliferation
(over 24 h) as assessed by a colorimetric assay and measurement of the reaction mixture at 490 nm. (C–E) The effects of anti–PECAM-1 mAb on the interactions of
tumor cells with murine endothelial cells and platelets were studied as described in SI Materials and Methods. Compared with control (no antibody) or with anti–
ICAM-1, anti–PECAM-1 mAb did not inhibit the adhesion of B16-F10 tumor cells to platelets in 96-well plates (n = 6) (C), the adhesion of various tumor cell lines
(B16-F10, 4T1, or LOX) to confluent monolayers of murine endothelial cells in 96-well plates (n = 3) (D), or the migration of B16-F10 tumor cells across endothelial
cell monolayers plated on Transwell filters (n = 4) (E). Error bars indicate SEM. The potential statistical significance of differences was assessed using an unpaired
two-tailed Student’s t test. ICAM, intercellular adhesion molecule.

Fig. S2. A 3D coculture assay recapitulates the antiproliferative effect anti–PECAM-1 mAb produces against advanced metastatic growth. (A) Each well of
a six-well polystyrene plate was coated with 200–300 μL of Matrigel, and 2 × 105 CD3 or H5V cells were plated 1 h later. Seven to 8 h later, after VEC capillary-
like structures formed, 2 × 105 B16-F10 or 4T1 cells were added, together with 200 μg/mL of anti–PECAM-1 or isotype control mAb. mAbs were added again at
24 and 48 h, and all cells were harvested at 72 h and counted (n = 3). Values are mean ± SD. The potential statistical significance of differences was assessed
using an unpaired two-tailed Student’s t test. (B and C) B16-F10 cells were photographed at 40× magnification after being grown over VEC tubes on Matrigel
in the presence of isotype control mAb (B) or anti–PECAM-1 mAb (C) for 48 h.

DeLisser et al. www.pnas.org/cgi/content/short/1004654107 2 of 4

Fig. S3. Metastatic lung tumor nodules from mice treated for 6 consecutive days with anti–PECAM-1 or isotype control mAb were surveyed for the presence of
F4/80 antigen-positive macrophages using immunohistochemistry. (F4/80 is a cell-surface glycoprotein present on mature, tissue-fixed macrophages including
some dendritic cells.) The presence of macrophages within individual tumor nodules varied widely, from nonexistent to moderate; however, this pattern was
not influenced by treatment. Fig. 3 shows a representative distribution of F4/80-positive brown-staining macrophages within tumors from mice treated with six
doses of anti–PECAM-1 (A) or isotype control (B) mAb.

1. Yount G, Yang Y, Wong B, Wang HJ, Yang LX; S3 (2007) A novel camptothecin analog with enhanced antitumor activity. Anticancer Res 27(5A):3173–3178.

Table S1. Quantitation of tumor cell lesions 4 d after tumor injection

Mouse strain Density of metastatic lesions (lesions/mm2) No. cells per metastatic lesion

WT 0.75 ± 0.7 2.1 ± 0.05

PECAM-1–null 0.73 ± 1.0 2.2 ± 1.0

Lung tissues harvested from WT and PECAM-1–null mice 4 d after injection of B16-F10 tumor cells were fixed
and stained with H&E. The density (lesions/mm2) and size (cells per lesion) of subclinical tumor cell lesions were
determined by computer-assisted image analysis (n = 200 nodules from four mice for each strain). Even small
numbers of cells were identified as tumor cells, based on characteristic morphological features of the cell and
nucleus, often confirmed by the presence of melanin. There were no significant differences between the two
strains of mice (P > 0.5). Data are mean ± SEM.

DeLisser et al. www.pnas.org/cgi/content/short/1004654107 3 of 4

Movie S1. B16-F10 cells grown in conditioned media from anti–PECAM-1 mAb-treated cocultures. Conditioned media (CM) from cocultures maintained in the
presence of anti–PECAM-1 mAb suppresses tumor cell proliferation. Conditioned media (CM) harvested from CD3 CLS-B16-F10-Matrigel cocultures after ex-
posure to either anti–PECAM-1 or isotype control mAb was added to B16-F10 cells. At the start of each experiment, cell cultures were transferred from the
incubator to a time-lapse microscope equipped with a heated stage and incubation chamber. Four sets of phase-contrast images from each of three wells were
acquired over a 44-h observation period. Images then were processed as Quicktime movies as described in SI Materials and Methods.

Movie S1

Movie S2. B16-F10 cells grown in conditioned media from isotype control mAb-treated cocultures. Conditioned media (CM) harvested from CD3 CLS-B16-F10-
Matrigel cocultures after exposure to isotype control mAb was added to B16-F10 cells. The cells were subsequently processed and Quicktime movies recorded
exactly as described in the legend to Movie S1.

Movie S2

DeLisser et al. www.pnas.org/cgi/content/short/1004654107 4 of 4