Supplementary Materials. for Garmy-Susini, et al, Integrin 4 1 signaling is required for lymphangiogenesis and tumor metastasis

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1 Supplementary Materials for Garmy-Susini, et al, Integrin 4 1 signaling is required for lymphangiogenesis and tumor metastasis 1

2 Supplementary Figure Legends Supplementary Figure 1: Integrin expression on human lymphatic vessels. Human breast carcinoma or normal breast cryosections were immunostained to detect expression of integrins v 3, 4 1, 5 1 and v 5 (red) and were counterstained to detect Lyve-1 (green), a marker of lymphatic endothelium. Integrin expression on lymphatic vessels is seen as yellow in merged images. Only integrin 4 1 and v 5 were detected on human tumor lymphatic vessels. Of these two, only integrin 4 1 was upregulated on lymphatic vessels. Supplementary Figure 2: Integrin expression on murine lymphatic vessels. Murine PyMT+ breast carcinoma or normal murine mammary gland cryosections were immunostained to detect expression of integrins v 3, 4 1, 5 1 and v 5 (red) and were counterstained to detect Lyve-1 (green), a marker of lymphatic endothelium. Integrin expression on lymphatic vessels is seen as yellow in merged images. Only integrin 4 1 and v 5 were detected on murine tumor lymphatic vessels. Of these, only integrin 4 1 was upregulated on tumor lymphatic vessels. Supplementary Figure 3: Integrin 4 1 and fibronectin are markers of proliferative lymphatic endothelium in human and murine tumor. (A) Integrin 4 1 (red), podoplanin (green) and DAPI (blue) immunostaining of cryosections of human invasive breast ductal carcinoma and normal breast tissue; integrin 2

3 4 1 (red); Prox-1 (green) and DAPI (blue) staining of cryosections of human invasive breast ductal carcinoma; and integrin 4 1; and podoplanin staining (brown) of formalin-fixed, paraffin-embedded serial breast carcinoma sections with hematoxylin counterstaining. (B) FACs analysis of cultured human LECS for integrin 4 1, Lyve-1 (dark lines), isotype matched IgG (gray lines) or VCAM-1 expression (gray filled curve). Images, Fibronectin (green) and Lyve1 (red) immunostaining in PyMT+ spontaneous breast carcinoma tumors. (C) Immunoblots of human LEC and HUVEC lysates for fibronectin and actin expression. Supplementary Figure 4: Role of integrin 4 1 in VEGF-A stimulated lymphangiogenesis. 400 l of ice cold Matrigel containing saline or 400ng of VEGF-A was injected into mice and animals were treated by intraperitoneal injection with saline or 200 g/mouse of function-blocking anti-murine anti- 4 1 antibodies or anti- 5 1 isotype control antibodies every third day. After 7-10 days, Matrigel plugs were removed, and 5 m cryosections were immunostained with anti-lyve-1 antibodies. At least five microscopic fields per tissue section were analyzed for quantification studies. Mean lymphatic vessels/field +/- SEM for each condition is shown (n=10, *p<0.002). Supplementary Figure 5: Integrin 4 1 promotes LEC migration and invasion. (A) TUNEL (green), Lyve-1 (red) and DAPI (blue) immunostained murine LECs in cryopreserved Matrigel plugs from VEGF-C saturated Matrigel 3

4 from saline, anti- 4 1, anti- 5 1 or recombinant soluble VCAM-treated mice. Arrows indicate TUNEL positive LECs. (B) LEC adhesion to CS-1 fibronectin in the absence (medium) or presence of anti- 4 1 and anri- v 3 antibodies, *p< (C) Brightfield images of VEGF-C stimulated LEC migration in the presence of medium, anti- 4 1 or anti- 5 1 antibodies (cigg). (D) LEC transwell migration on CS-1 fibronectin in the presence of 25 g/ml of isotype matched control (anti- anti- v 3) or function-blocking anti- 4 1 antibodies (*p < 0.01). (E) Brightfield images of LEC in vitro vessel formation in the presence of anti- 4 1 or isotype matched control (anti- 5 1) antibodies. Supplementary Figure 6: VEGF-C stimulation of integrin 4 1 activation. (A) LECs immunostained to detect integrin 4 (red) and paxillin (green). Arrowheads indicate overlap. (B) Immunoblotting for presence of integrin 4 1 (Santa Cruz-6590), paxillin (Cell Signaling-2542) and IgG (donkey anti-goat- HRP) in integrin 4 1 immunoprecipitates [immunoprecipitated with anti-integrin 4 1 (PS2)] from mild detergent lysates of murine LECs in the presence or absence of VEGF-C. (C) FACs analysis of integrin 4 1 cell surface expression on WT and 4Y991A endothelial cells. (C). Microvessel outgrowth from WT thoracic duct explants immunostained to detect Lyve-1 (red) or integrin 4 1 (green). Overlap indicated by yellow color. (E) Lyve-1 (green), integrin 4 1 (red), paxillin (cyan) and DAPI (Blue) immunostaining of cryosections from VEGF-C Matrigel implanted in WT and 4Y991A mice. 4

5 Supplementary Figure 7: VEGFR3 and integrin 4 1 function together in promoting lymphangiogenesis. (A-B) C57BL/6 mice were inoculated subcutaneously in the dorsum with 400 l of Matrigel containing 1 g/ml VEGF-C. (A) Mice were systemically treated by intravenous injection every third day for 14 days with saline or 200 g/mouse of sterile, endotoxin-free rat anti-mouse 4 integrin (PS2), anti-vegf-r3 (AFL4) or isotype control antibodies (n=8). (B) Mice were systemically treated by intravenous injection every third day for 14 days with saline or 200 g/mouse of sterile, endotoxin-free rat anti-mouse 4 integrin (PS2), anti-vegf-r3 (AFL4), both antibodies in combination or isotype control antibodies (n=8). Matrigel plugs were sectioned and immunostained for Lyve-1. Pixel density was determined using Metamorph imaging software. Asterisk indicates statistical significance, P<0.05. Supplementary Figure 8. Inhibition of tumor lymphangiogenesis and metastasis in integrin 4 mutant animals. (A) Mice with LLC or B16 melanoma tumors were treated with saline, anti- 4 1 and isotype-matched control antibodies and LLC tumor cryosections were immunostained to detect Lyve-1. Arrows indicate lymphatic vessels. (B) Images of lymph node metastases at 200X or 10X from mice in A. (C) Images of Lyve-1 staining (green) in 5 or 50 micron thick cryosections of tumors from LLC or Panc02 tumors from WT and 4Y991A mice. (D) Cytokeratin+ metastases in LLC inguinal lymph node and Panc02 hilar lymph node in WT and 4Y991A mice. Scale bars, 50 m. 5

6 Supplementary Figure 9: Suppression of distant tumor metastasis in animals with an integrin 4Y991A mutation. Panc02 orthotopic pancreatic carcinoma tumor cells were implanted into syngeneic mice. After thirty days, animals were sacrificed and primary tumors as well as metastases were retrieved. Metastases were macroscopically visible and confirmed by hematoxylin and eosin staining of cryosections. The percent of mice with colon metastases was determined. Results were averaged for each study and presented as mean percent metastasis positive mice +/- SEM. Asterisk indicate statistical significance, P<0.05. Supplementary Figure 10: Host contributions of 4 1 integrin to tumor angiogenesis inflammation and tumor growth. LLC tumors were implanted in integrin 4Y991A mice transplanted with WT or 4Y991A bone marrow and in WT mice transplanted with 4Y991A or WT bone marrow. (A) Quantification of mean +/-SEM tumor weight after 21 days of tumor growth, (B) mean +/- SEM percent CD11b+ monocytes in 21 day tumors and (C) CD31+ blood vessel density expressed as mean +/-SEM pixels/field in tumors from BM transplanted animals, *p<

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