Supporting Information

Edris et al. 10.1073/pnas.1121629109 SI Materials and Methods with 10 μg/mL mouse isotype control or anti-CD47 clone B6H12 Immunohistochemistry. CD47 protein expression was evaluated by antibodies (eBioscience) for 30 min. Next, 500,000 antibody-coated fluorescent immunohistochemistry (IHC) on OCT-embedded, LMS05 cells were added to each respective chamber and phago- fresh-frozen tissue. In all, 16 LMS, three leiomyoma, and four cytosis was imaged using a BioStation IMQ (Nikon) equilibrated to normal muscletissue samples were evaluated. Tissue wassectioned 37 °C and 5% carbon dioxide. Images were captured at 2-min in- to a thickness of 5 μm and fixed with acetone. NonspecificIg tervals and movies were made using the BioStation IM version 2.12 binding was blocked with 10% goat serum and slides were in- software at a rate of three frames per second. cubated with anti-CD47 antibody (0.2 μg/mL; Abcam ab3283) or IgG-control antibody overnight at 4 °C. Samples were stained with Xenotransplantation Experiments. For primary tumor growth experi- goat anti-mouse Ig secondary antibody conjugated with Alexa-488 ments, LMS04 or LMS05 cells were suspended in RPMI containing (Invitrogen) for 1 h, followed by a nuclear counterstaining with 25% (vol/vol) Matrigel (BD Biosciences) and 100,000 cells Hoechst (Invitrogen). Slides were imaged and analyzed on (LMS04) or 75,000 cells (LMS05) were implanted subcutaneously γnull a fluorescent microscope (Leica). For colorimetric IHC, CD3, on the backs of 4- to 8-wk-old NOD/SCID/ IL-2R (NSG) mice CD4, CD8, CD20, CD68, and CD163, protein expression was (3). After 2 wk (when tumors were small), treatment commenced evaluated on formalin-fixed, paraffin-embedded cell pellets from with humanized anti-CD47 mAb (clone B6H12) or human IgG peripheral blood mononuclear cell (PBMC)-derived human control for 6 wk (treatment details provided in Table S2). Animals fi macrophages. Vimentin protein expression was evaluated on full were killed and tumors were resected, weighed, and xed in for- cross-sections of formalin-fixed, paraffin-embedded mouse or- malin for histologic analysis and IHC; tumors weights were com- t gans, which were dissected after the animals were killed. Slides pared using tests. Concomitantly, the lungs, liver, kidney, spleen, fi from paraffin blocks were cut to a thickness of 4 μm, deparaffi- brain, and heart of each mouse were resected and xed in formalin nized in xylene, and hydrated in a graded series of alcohol. The for histological analysis and Vimentin IHC to check for the pres- deparaffinized slides were then boiled by microwave for 12 min in ence of distant metastases; LMS04-engrafted mice developed lung citrate buffer (pH 6) and stained with primary antibodies. Di- metastasis in almost all animals, but LMS05-engrafted mice lution and manufacturer information for the primary antibodies showed no evidence of metastatic disease. The metastatic burden in fi are as follows: CD3, 1:50 (Dako, clone F7.2.38); CD4, 1:15 (Vi- LMS04 xenografts, which was de ned as the number of cellular foci sion Bio-System, clone IF6); CD8, 1:30 (Dako, clone C8/144B); with Vimentin-positive staining as assessed by eye, was compared t CD20, 1:200 (Dako, clone L26); CD68, 1:50 (Dako, clone KP-1); between anti-CD47 and control-treated mice using tests. To CD163, 1:10 (Novocastra, clone 10D6); Vimentin, 1:200 (Dako). characterize the neoadjuvant model of LMS, 100,000 LMS04 cells The IHC reactions were visualized using mouse versions of the were implanted subcutaneously on the backs of 4- to 8-wk-old NSG EnVision+ system (Dako) using diaminobenzidine. mice, and individual mice were killed each week to check for the appearance of tumor cells in the lung. Lung metastases appeared as Cell Culture. LMS04 and LMS05 cell lines were derived from individual cells after 6 wk and subsequently increased in size and primary clinical specimens [LMS04: retroperitoneal lesion that number. The primary tumors of the eight remaining mice were spread from primary uterine leiomyosarcoma (LMS) tumor; surgically resected and mice were monitored daily and killed upon LMS05: primary thigh LMS tumor]. Both cell lines were main- the outgrowth of axillary lymph-node tumors. Lungs and lymph- tained in RPMI 1640 supplemented with 10% FBS, 100 units/mL node tumors were fixed with formalin and tumor cell presence was fi fi penicillin and streptomycin, and 4 mM L-glutamine (Invitrogen). con rmed by IHC. To test the ef cacy of anti-CD47 treatment in Cell lines were cultured at 37 °C in 5% CO2, and the medium a model of LMS that mimics neoadjuvant therapy, 100,000 LMS04 was replaced every 2–3d. cells were implanted subcutaneously on the backs of NSG mice and mice were randomized based on primary tumor volume and tumor In Vitro Phagocytosis Assays. Human macrophages were prepared growth kinetics on week 6 (Table S3). Treatment with anti-CD47 or from PBMCs as described previously (1). For phagocytosis as- IgG control antibody then commenced, and tumors were resected says, 5 × 104 macrophages were plated per well in a 24-well at week 7. Mice were killed at week 11, or when axillary lymph-node tissue-culture plate. LMS04 and LMS05 cells were labeled with tumor burden necessitated that the animal be killed. All procedures 2.5 μM carboxyfluorescein succinimidyl ester (CFSE) according followed protocols approved by the Stanford Committee on Ani- to the manufacturer’s protocol (Invitrogen). Macrophages were mal Research. incubated in serum-free medium for 2 h before adding 2 × 105 CFSE-labeled LMS cells. Isotype control, nonblocking anti- Morphometrics Analysis. To assess metastatic tumor presence in the CD47 (clone 2D3), or blocking anti-CD47 (clone B6H12) anti- lungs, we performed IHC for human Vimentin, which does not bodies (eBioscience) were added at a concentration of 10 μg/mL react with murine tissue. For the neoadjuvant model, we used and the LMS/macrophage cocultures were incubated for 2 h at morphometric analyses to assess the area of marker stain in the 37 °C. Macrophages were repeatedly washed and subsequently lungs of IgG-treated mice (n = 6), anti-CD47–treated mice (n = imaged with an inverted microscope (Leica DMI6000B). 6), or mice that had not been transplanted with tumor cells (n = 5). Following IHC staining, whole slide images were digitally Live-Cell Imaging of Macrophage Phagocytosis in Vitro. Red fluo- scanned at a magnification of 40×. Vimentin staining area was rescent protein (RFP)-positive mouse macrophages were prepared assessed quantitatively using an image analysis pipeline developed fi fi from C57BL/Ka Rosa26-mRFP1 transgenic mice (2). After 6 d of in De niens Tissue Studio 3.0. The image analysis pipeline rst culture with 10 μg/mL murine M-CSF (Peprotech), macrophages identifies the area of each slide occupied by tissue, and then were replated onto four-well Lab-Tek Permanox chamber slides quantitates the proportion of tissue occupied by positive marker (Thermo Scientific-Nunc) at a density of 50,000 macrophages per staining (Table S4). To assess the statistical significance of stain- chamber. Macrophages were allowed to adhere for 24 h, at which ing differences between the IgG-treated mice and anti-CD47– point LMS05 cells were labeled with 0.5 μM CFSE and incubated treated mice, we performed a Wilcoxon rank-sum test.

Edris et al. www.pnas.org/cgi/content/short/1121629109 1of5 1. Majeti R, et al. (2009) CD47 is an adverse prognostic factor and therapeutic antibody 3. Ito M, et al. (2002) NOD/SCID/gamma(c)(null) mouse: An excellent recipient mouse target on human acute myeloid leukemia stem cells. Cell 138:286–299. model for engraftment of human cells. Blood 100:3175–3182. 2. Ueno H, Weissman IL (2006) Clonal analysis of mouse development reveals a polyclonal origin for yolk sac blood islands. Dev Cell 11:519–533.

Fig. S1. CD47 immunofluorescence staining of fresh-frozen tissue samples. Immunofluorescence staining with anti-CD47 (Upper) or isotype control (Lower) antibodies on three normal muscle (A–C), three leiomyoma (D–F), and 16 LMS samples (G–V) revealed that CD47 protein was more highly expressed on LMS than on benign or normal tissues. Additional information for each tissue sample is available in Table S1. Magnification, 200×.

Edris et al. www.pnas.org/cgi/content/short/1121629109 2of5 100 Anti-CD47 IgG Control

80

60

40

20 HR: 2.81 p=0.22 0 % Mice with Lymph Node Metastases 0 9 18 27 Days Post-Resection

Fig. S2. Time to presence of axillary lymph-node metastasis. Kaplan-Meier curves and log-rank (Mantel-Cox) tests were used to determine whether time to presence of visible axillary lymph-node tumors after primary tumor resection differed in anti-CD47 vs. IgG-treated mice.

Table S1. List of cases examined by IHC for CD47 expression Tissue ID Tissue type Figure CD47 Expression*

STT158 Normal uterus, endometrium Fig. S1A 1 STT216 Normal uterus Fig. S1B 0 STT315 Normal muscle, right calf Fig. S1C 0 STT159 Leiomyoma, uterus Fig. S1D 0 STT1424 Leiomyoma, uterus Fig. S1E 2 STT4662 Leiomyoma, uterus Fig. 1B, Fig. S1F 1 STT1220 Leiomyosarcoma, abdominal Fig. 1B, Fig. S1G 2 STT1979 Leiomyosarcoma, uterus Fig. S1H 1 STT4583 Leiomyosarcoma, uterus Fig. S1I 2 STT4689 Leiomyosarcoma, uterus Fig. S1J 2 STT4657 Leiomyosarcoma, pelvis Fig. S1K 2 STT4608 Leiomyosarcoma, retroperitoneum Fig. S1L 2 STT5114 Leiomyosarcoma, retroperitoneum Fig. S1M 2 STT5116 Leiomyosarcoma, retroperitoneum Fig. S1N 2 STT516 Leiomyosarcoma, retroperitoneum Fig. S1O 2 STT2251 Leiomyosarcoma, thigh Fig. S1P 2 STT5115 Leiomyosarcoma, thigh Fig. S1Q 2 STT5119 Leiomyosarcoma, buttock Fig. S1R 2 STT5327 Leiomyosarcoma, hip Fig. S1S 2 STT4609 Leiomyosarcoma, lung Fig. S1T 1 STT4612 Leiomyosarcoma, lung Fig. S1U 2 STT4730 Leiomyosarcoma, chest wall Fig. S1V 2

*CD47 immunofluorescence protein expression on fresh-frozen tissue sections was given a score of 0 (no ex- pression), 1 (low expression), or 2 (high expression). Images from the tissue sections whose scores are recorded in above are shown in Fig. 1 and Fig. S1.

Table S2. Parameters of primary LMS xenograft tumor-growth experiments † ‡ Cell line No. of cells injected Engraftment period* Group size Anti-CD47 Ab Control Ab Treatment frequency Ab dose

LMS04 100,000 14 d 5 Humanized B6H12 Human IgG Twice weekly 500 μg LMS05 75,000 14 d 5 Humanized B6H12 Human IgG Twice weekly 500 μg

*Duration between injection of tumor cells and initiation of antibody treatments. †Number of mice per treatment group (anti-CD47 or control). ‡ Per mouse. Antibody administered by intraperitoneal injections in PBS.

Edris et al. www.pnas.org/cgi/content/short/1121629109 3of5 Table S3. Week 6 tumor sizes and treatment group assignments for mice in neoadjuvant therapy experiment Mouse ID Tumor length (mm)* Tumor width (mm)* Tumor volume† Group assignment

2798 7.89 5.87 77.19 IgG 550 8.04 7.19 96.35 Anti-CD47 648 9.63 7.89 126.63 IgG 650 10.77 7.25 130.14 Anti-CD47 649 9.94 8.45 139.99 IgG 2794 9.81 8.87 145.02 Anti-CD47 527 10.66 8.63 153.33 IgG 540 14.52 7.39 178.84 Anti-CD47 534 10.97 9.99 182.65 IgG 546 12.24 9.86 201.14 Anti-CD47 528 13.28 9.38 207.61 IgG 548 11.98 11.06 220.83 Anti-CD47

*As measured in millimeters using digital calipers. † Tumor volume calculated using the following equation: (length × width)/(0.6).

Table S4. Histological and morphometric analysis of LMS04 mouse lungs in neoadjuvant therapy experiment Mouse ID Treatment group* % Vimentin positive† Histological observations of vimentin IHC

648 IgG 8.97 Innumerable large foci + single cells 528 IgG 0.26 36 foci of > 4 cells + single cells 534 IgG 0.12 35 foci of > 4 cells + single cells 649 IgG 0.09 38 foci of > 4 cells + single cells 527 IgG 0.04 7 foci of > 4 cells + single cells 2798 IgG 0.01 4 foci of > 4 cells + single cells 548 Anti-CD47 0.0045 Approximately 20 single cells 550 Anti-CD47 0.0025 Approximately 17 single cells + 1 focus of > 4 cells 650 Anti-CD47 0.0023 Approximately 15 single cells + 4 foci of > 4 cells 540 Anti-CD47 0.0023 None (one area of artifact) 546 Anti-CD47 0.0016 Approximately 23 single cells + 2 foci of 2 cells 2794 Anti-CD47 0.0001 5 single cells + 1 focus of 2 cells — Negative control 0.0001 None — Negative control 0.0001 None — Negative control 0 None — Negative control 0 None

*Negative Control mice were those that were killed without ever having been exposed to tumor cells. † Calculated as the area of Vimentin positivity divided by the total surface area of the lungs (see SI Materials and Methods for additional details).

Movie S1. Fluorescently labeled LMS05 cells (green) were incubated with control IgG antibodies and then cocultured with RFP-positive mouse macrophages (red) and visualized using video microscopy. IgG treatment resulted in no phagocytosis.

Movie S1

Edris et al. www.pnas.org/cgi/content/short/1121629109 4of5 Movie S2. Fluorescently labeled LMS05 cells (green) were incubated with anti-CD47 antibodies and then cocultured with RFP-positive mouse macrophages (red) and visualized using video microscopy. Anti-CD47 treatment resulted in robust phagocytosis of the LMS cells by the macrophages.

Movie S2

Movie S3. Fluorescently labeled LMS05 cells (green) were incubated with anti-CD47 antibodies and then cocultured with RFP-positive mouse macrophages (red) and visualized using video microscopy. Anti-CD47 treatment resulted in robust phagocytosis of the LMS cells by the macrophages.

Movie S3

Edris et al. www.pnas.org/cgi/content/short/1121629109 5of5