Supplementary Materials for

Development of a prosaposin-derived therapeutic cyclic peptide that

targets ovarian cancer via the tumor microenvironment

Suming Wang, Anna Blois, Tina El Rayes, Joyce F. Liu, Michelle S. Hirsch,

Karsten Gravdal, Sangeetha Palakurthi, Diane R. Bielenberg, Lars A. Akslen,

Ronny Drapkin, Vivek Mittal, Randolph S. Watnick*

*Corresponding author. E-mail: randy.watnick@childrens.harvard.edu

Published 9 March 2016, Sci. Transl. Med. 8, 329ra34 (2016)

DOI: 10.1126/scitranslmed.aad5653

This PDF file includes:

Materials and Methods

Fig. S1. Expression of CD36 in patient-derived ovarian cancer cells.

Fig. S2. FACS analysis of rhTSP-1 effects on ovarian cancer cell apoptosis.

Fig. S3. Effects of CD36 blocking antibody on rhTSP-1 induction of ovarian

cancer cell apoptosis.

Fig. S4. Body weight tracking of mice bearing patient-derived ovarian cancer

metastases.

Fig. S5. Bioluminescent imaging of peptide-treated patient-derived ovarian cancer

metastases.

Fig. S6. Bioluminescent imaging of control-treated patient-derived ovarian cancer

metastases.

Fig. S7. CD36 staining of ovarian cancer patient TMA.

Table S1. Characterization of platinum sensitivity of patient-derived ovarian

cancer cells.

References (38, 39)

Other Supplementary Material for this manuscript includes the following:

(available at www.sciencetranslationalmedicine.org/cgi/content/full/8/329/329ra34/DC1)

Table S2. Original data (provided as an Excel file).

www.sciencetranslationalmedicine.org/cgi/content/full/8/329/329ra34/DC1

Materials and Methods

Mice and cell lines

All animal work was conducted in accordance with a protocol approved by the

Institutional Animal Care and Use Committee of Boston Children’s Hospital. C57BL6/J

mice were obtained from The Jackson Laboratory. The C.B-17 severe combined

immunodeficiency (SCID) mice were obtained from Massachusetts General Hospital.

Human WI-38 lung fibroblast cells were purchased from American Type Culture

Collection. The 1D8 cell line was a generous gift from Jack Lawler (Beth Israel

Deaconess Medical Center and Harvard Medical School) and is previously described

(38). The human primary ovarian cancer cells (DF lines) were isolated directly from

peritoneal paracentesis of patients with advanced-stage ovarian cancer at the time of

initial cytoreductive surgery or recurrence. Red blood cells were lysed and tumor cells

were enriched either by using immunomagnetic beads coupled to EpCAM antibodies

(Dynal, Invitrogen) or by filtration with a 40-m nylon cell strainer (BD Falcon) to isolate

tumor cell spheres. In all cases, the epithelial nature of the cells was confirmed by

EpCAM and Pax8 immunostaining, as well as RT-PCR detection of cytokeratin 7 as

previously described (24). All cells were collected with approval of the institutional

review board. DF14 and 1D8 cells were retrovirally transduced with pLucNeo (a

generous gift of Andrew Kung, Dana Farber Cancer Institute and Harvard Medical

School) to express firefly luciferase as previously described (6).

WI-38 cells were cultured in Minimum Essential Medium (GIBCO) and

supplemented with 10% fetal bovine serum (GIBCO). Both PC3M-LN4 and DF lines

were cultured in RPMI 1640 Medium (Life Technologies) supplemented with 10% FBS.

DF lines were cultured in ultra low adherent cell culture dishes coated with 1% Poly(2-

hydroxyethyl methacrylate) (Sigma-Aldrich) in a mixture of methanol and ethanol (1:4).

Western blotting

Cells were lysed in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% sodium

deoxycholate, 1% NP-40, 0.1% SDS, 2 nM DTT, and Complete Mini protease inhibitor

cocktail (Roche). Western blots were performed as previously described with the

following antibodies: TSP-1 (mouse mAb Ab-11, Thermo Scientific), CD36 (rabbit pAb,

Abcam), and -actin (mouse mAb AC-15, Abcam).

In vitro peptide activity assay

WI-38 lung fibroblasts were serum-starved for 3 hours. Cells were then treated

with psap peptide for 8 hours at a concentration of 10 g/mL. Cells were harvested and

lysed for western blot analysis as described above. All experiments were repeated a

minimum of 5 independent times with 2-3 replicates per experiment.

For plasma stability/activity studies, 100 g of d1,3 or cyclic prosaposin peptide

was incubated in 1 mL of pooled human plasma (Innovative Research) at 37oC for 2, 4,

8, and 24 hours. After the incubation, 500 L of the peptide plasma mixture was added

to a 10 cm plate of WI-38 fibroblasts and incubated overnight at 37oC. After this

incubation, the conditioned medium was centrifuged at 2,000 rpm at room temperature

for 5 minutes in a Beckman GS-6KR centrifuge. The concentration of TSP-1 in the cell-

free conditioned medium was then measured by ELISA (R&D systems) using the

manufacturer’s protocol. TSP-1 concentration determined by ELISA was then

normalized to the total protein concentration in the cells from which the conditioned

medium was obtained.

In vivo peptide activity assay

For the in vivo peptide activity assay, 8-week-old C57BL6/J mice (n=3 per group)

were treated with 300 L of the conditioned medium from PC3M-LN4 cells alone or in

combination with psap peptides, diluted in PBS, at a dose of 30 mg/kg/day via

intraperitoneal injection for 3 days (6). After 3 days, the mice were euthanized by

cervical dislocation under isoflurane anesthetic. The lungs were harvested, lysed, and

analyzed for TSP-1 expression by western blot as described above.

Cell proliferation assays

For the cell proliferation assay, all cells were grown in the same complete

medium described above. 4,000 cells/well were plated in 96-well plates pre-coated as

described above, and incubated with 1 g/mL rhTSP-1 (R&D Systems). Fresh rhTSP-1

was added every 24 hours. The number of cells in culture was calculated at 0, 8, 24, 48,

and 72 hours using WST-1 cell proliferation reagent (Roche Diagnostics Corporation).

Each proliferation assay was repeated 3 times, and 3 replicates were performed for

each time point and condition. Statistical significance was determined by ANOVA.

FACS analyses

For the apoptosis assay, cells were cultured in 6-well plates pre-coated as

described above and treated with 1 g/mL rhTSP-1. Fresh rhTSP-1 was added every 24

hours. For blocking antibody experiments, cells were treated with 3 g/mL of CD36

antibody FA6-152 (Abcam) alone or in combination with 1 g/mL rhTSP-1 (n=3/group).

Cells were collected after centrifugation (1,200 rpm for 2 min at 4°C) and incubated with

Dead Cell Apoptosis Kit with Annexin V Alexa Fluor-488 & Propidium Iodide (Life

Technologies), according to the manufacturer’s protocol. Cells were analyzed with a BD

FACSCalibur machine (BD Biosciences).

Analysis of bone marrow cells in ascites fluid was performed by collecting cells

after centrifugation (1,200 rpm for 2 min at 4°C) and washing them with FACS buffer

(PBS plus 2% FBS). Cells were blocked with PBS plus 5% BSA for 15 min at 4°C. After

blocking, cells were washed once (1,200 rpm for 2 min at 4°C) with FACS buffer and

then incubated with antibodies against CD11b (Clone M1/70, BD Pharmingen) and GR1

(Clone RB6-8C5, BD Pharmingen) for 30 min (antibodies diluted 1:20 in FACS buffer) at

4°C with light protection. Antibodies were directly conjugated to Alexa Fluor dyes (Alexa

488-CD11b and Alexa 647-GR1) using antibody-labeling kits (Invitrogen) as per the

manufacturer’s instructions and purified over BioSpin P30 Gel (Bio-Rad). The cells were

then washed three times. Finally, cells were resuspended in FACS buffer and analyzed

with a BD FACSCalibur machine. All FACS experiments were repeated a minimum of 3

times with 3 replicates per experiment.

Animal Tumor Models

Orthotopic injection into the ovary

Female, C57BL6/J mice (n=8/group) (8 weeks old) were shaved 1 day before surgery.

Mice were anesthetized with isoflurane, sterilized with betadine and alcohol, and placed

on their right side. A small incision was made on the left side, and the ovary was

exposed and placed on a sterile cotton swab. Syngeneic ovarian carcinoma cells, 1D8-

luciferase (1 x106 cells/10 l), diluted in HBSS and mixed 1:1 with Matrigel, were

injected into the bursa (20 l total volume). The ovary was returned and the incision was

closed with wound clips. Mice received analgesic (buprenorphine) for 72 hours after

surgery, and wound clips were removed after one week. Mice were treated daily with

the d1,3 psap peptide at a dose of 40 mg/kg/day. Treatment was initiated 31 days after

tumor cell injection. Treatment was terminated 20 days later, and mice were left

untreated for 32 days, at which point treatment with the psap peptide was reinitiated for

33 days. After that, all mice were euthanized with CO2 and necropsied.

In vivo bioluminescent imaging

For in vivo determination of tumor burden, mice were anesthetized and injected

intraperitoneally with 75 mg/kg of D-luciferin (100 μL of 30 mg/mL in PBS). Tumor

growth was monitored in real time by bioluminescence imaging performed 5 minutes

after D-luciferin injection with a Xenogen IVIS system coupled to Living Image

acquisition and analysis software (Xenogen). For bioluminescent imaging (BLI) plots,

photon flux was calculated for each mouse by using the same circular region of interest

encompassing the abdomen of the mouse.

Immunofluorescence

Thrombospondin-1 and GR1

Samples were fixed in 10% paraformaldehyde and subsequently paraffin-embedded for

sectioning. Four-micrometer sections were deparaffinized with xylene and rehydrated in

decreasing concentrations of ethanol to water. Antigen retrieval was performed using

proteinase K (Roche Diagnostics) at a final concentration of 20 µg/ml in 0.2 M Tris PH

7.2 at 37°C for 25 min. Slides were then incubated for 30 min in blocking buffer

consisting of PBS with 3% goat serum. Slides were incubated overnight at 4°C with

primary antibodies rat anti-rabbit TSP-1 (ab85762, Abcam) 1:50 in blocking buffer. The

next day, slides were washed and incubated with secondary antibodies Alexa anti rabbit

-488 1:500 for 1 hour, washed, re-blocked, and incubated with mouse anti-rat Gr-1,

(clone RB6-8C5, BD-Pharmingen), 1:50 in blocking buffer at room temperature for 2

hours. Sections were washed and incubated with secondary antibodies Alexa anti rat -

555 for 1 hour at room temperature. Samples were then washed and mounted using

VECTASHIELD mounting medium with DAPI (Vector Laboratories). Stained slides were

immediately evaluated with a fluorescence microscope. Statistical significance of

differences in the number of TSP-1 and GR1 -positive cells was determined by ANOVA.

TUNEL

Tumor cell apoptosis was analyzed with the DeadEnd Fluorometric TUNEL

System according to the manufacturer’s protocol (Promega). Samples were fixed in

10% paraformaldehyde and subsequently paraffin-embedded for sectioning. Four-

micrometer sections were deparaffinized with xylene and rehydrated in decreasing

concentrations of ethanol to water. Slides were fixed in 4% methanol-free formaldehyde

in PBS and then permeabilized with Proteinase K (20 g/ml in PBS). Samples were

then incubated with Nucleotide Mix and rTdT Enzyme at 37°C for 1 hour. The reaction

was stopped with 2X sodium citrate. Slides were mounted using VECTASHIELD

mounting medium with DAPI (VECTOR laboratories). Apoptotic cells (fluorescein-12-

dUTP) and blue cell nuclei were detected by fluorescence microscopy.

Immunohistochemistry

Thrombospondin-1, CD31, and MAC3

Samples were fixed in 10% paraformaldehyde and subsequently paraffin-

embedded for sectioning. Four-micrometer sections were deparaffinized with xylene

and rehydrated in decreasing concentrations of ethanol to water. Antigen retrieval was

performed with proteinase K (Roche Diagnostics) at a final concentration of 20 µg/ml in

0.2 M Tris pH 7.2 at 37°C for 25 min. Slides were then incubated for 30 min with

blocking buffer consisting of PBS with 3% goat serum.

Slides were incubated overnight at 4°C with the following primary antibodies: rat

anti-rabbit TSP-1 (ab85762, Abcam), CD31 (MEC13.3, BD Biosciences Pharmingen), or

MAC3 (M3/84, BD Biosciences Pharmingen) 1:200 in Tris-NaCl blocking (TNB) buffer.

The next day, slides were washed, re-blocked, and incubated with biotinylated anti-

rabbit IgG secondary antibody for TSP-1 and anti-rat IgG secondary antibody for CD31

and MAC3 (Vector Laboratories) 1:200 in TNB at room temperature for 30 min. Sections

were incubated with streptavidin-HRP (provided in the Tyramide Signal Amplification kit,

Perkin Elmer Life & Analytical Science) once before and once after biotin-labeled

tyramide 1:100 in TNB for 30 min at room temperature before using peroxidase

substrate kit Vector Novared (VECTOR laboratories) for 10 to 15 min. Sections were

counterstained with hematoxylin (Sigma-Aldrich).

After staining, the number and percent area of blood vessels, as defined by

CD31 positivity, as well as the number of macrophages, as defined by Mac-3 positivity,

were counted in both control- and peptide-treated tumors. For this analysis, control

treated tumors were subdivided into 17 high-power (400x) fields, and peptide-treated

tumors were subdivided into 7 high-power fields. The percent area and number of

blood vessels were then counted for each field.

TMA analysis of CD36 and psap expression

A high-density tissue microarray (TMA) of human ovarian tumors was constructed as

previously described (31, 38, 39). Briefly, the TMA was constructed from 134 samples

of high-grade late-stage ovarian serous carcinoma, from patients who underwent

primary cytoreductive surgery at the Brigham and Women’s Hospital between 1999 and

2005. Patients with known BRCA mutations or with a history of another malignancy in

the previous 5 years were excluded. Four 0.8 mm cores were taken for each case. All

pathology specimens and clinical data were collected under the approval of the

institutional review board.

Sections were deparaffinized with xylene and rehydrated in decreasing

concentrations of ethanol to water. Antigen retrieval was performed with heat

pretreatment in 0.01 M citric acid, pH 6.0 after 8 min incubation with 3% hydrogen

peroxide (Sigma-Aldrich) in methanol. Slides were then blocked for 1 hour at room

temperature and incubated overnight at 4°C with rabbit polyclonal CD36 antibody

(ab78054, Abcam) diluted 1:250. The next day, slides were washed and incubated with

anti-rabbit IgG secondary antibody (VECTOR laboratories) 1:200 for 1 hour at room

temperature followed by DAB peroxidase (HRP) substrate kit (VECTOR laboratories) for

10–15 min. Sections were counterstained with hematoxylin (Sigma-Aldrich). Psap

expression was analyzed immunohistochemically as previously described (6).

Statistical significance of differences in staining indices was determined by Mann-

Whitney U-test.

Supplementary Figures

Figure S1. Expression of CD36 in patient-derived ovarian cancer cells

Western blot analysis of CD36 and -actin expression in patient-derived ovarian cancer

cells.

Figure S2. FACS analysis of rhTSP-1 effects on ovarian cancer cell apoptosis

(A) FACS analysis of propidium iodide uptake and Annexin V staining in DF216 patient-

derived ovarian cancer cells treated with saline, cisplatin (10 g/mL), or recombinant

human TSP-1 at a dose of 1 g/mL (rhTSP-1);

(B) FACS analysis of propidium iodide uptake and Annexin V staining in DF118 patient-

derived ovarian cancer cells treated with saline or recombinant human TSP-1 at a dose

of 1 g/mL (rhTSP-1).

Figure S3. Effects of CD36 blocking antibody on rhTSP-1 induction of ovarian

cancer cell apoptosis

FACS analysis of propidium iodide uptake and Annexin V staining in DF14 patient-

derived ovarian cancer cells treated with saline (control), CD36 blocking antibody at a

dose of 3 g/mL (CD36 Ab), recombinant human TSP-1 at a dose of 1 g/mL (rhTSP-

1), or recombinant human TSP-1 at a dose of 1 g/mL in combination with CD36

blocking antibody 3 g/mL (rhTSP-1+CD36 Ab).

Figure S4. Body weight tracking of mice bearing patient-derived ovarian cancer

metastases

Plot of the body weights of mice treated with saline (control), cisplatin (4 mg/kg every

other day), or linear d1,3 prosaposin peptide (40 mg/kg/day).

Figure S5. Bioluminescent imaging of peptide-treated patient-derived ovarian

cancer metastases

Images acquired by Xenogen In Vivo Imaging System, showing mice bearing patient-

derived ovarian cancer metastases before treatment (Day 17) and after 34 days of

treatment with the linear d1,3 prosaposin peptide (Day 51).

Figure S6. Bioluminescent imaging of control-treated patient-derived ovarian

cancer metastases

Images acquired by Xenogen In Vivo Imaging System, showing mice bearing patient-

derived ovarian cancer metastases before treatment (Day 17) and after 34 days of

treatment with saline (Day 51).

Figure S7. CD36 staining of ovarian cancer patient TMA

Photograph of ovarian cancer patient tissue microarray immunohistochemically stained

for CD36. Photograph was taken with a Nikon D2X with a Nikkor 60mm F2.8D lens.

Supplemental Table 1. Characterization of platinum sensitivity of patient-derived ovarian cancer cells.

Cell line Platinum status

DF09 unknown (discarded patient sample)

DF14 unknown (discarded patient sample)

DF37 unknown (discarded patient sample)

DF43 unknown (discarded patient sample)

DF59 unknown (discarded patient sample)

DF83 platinum-sensitive

DF106 platinum-resistant

DF118 platinum-resistant

DF149 platinum-resistant

DF164 platinum-resistant

DF181 platinum-resistant

DF216 platinum-resistant