Supplemental Information Garmy-Susini, et al., PI3Kα activates integrin α4β1 to establish a metastatic niche in lymph nodes

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1 Supplemental Information Garmy-Susini, et al., PI3Kα activates integrin α4β1 to establish a metastatic niche in lymph nodes Supplementary Figure 1: Lymphangiogenesis and metastasis in lymph nodes of tumorbearing animals (A)Lymph nodes from normal mice and mice 7, 14 and 21 days after subcutaneous LLC inoculation were immunostained to detect Lyve-1+ (green) and Prox-1+ (red) lymphatic vessels (arrowheads) or cytokeratin+ metastases (green, arrowheads) and counterstained with DAPI (blue). White dotted lines, edge of lymph node. (B) 30 µm cryosections of lymph nodes from day 21 LLC tumor-bearing mice were immunostained to detect CD31 (red) and Lyve-1 (green) and counterstained with Dapi. CD31+Lyve-1- immunostaining (red) identifies thick walled, high endothelial venules, while CD31+Lyve-1+ staining (yellow) identifies vessels and channels at the periphery of the lymph node (arrowheads). (C) RT-PCR analysis of Lyve-1 expression in lymph nodes of normal and d1-d21 LLC tumor bearing mice indicates that Lyve-1 expression in lymph nodes steadily increases over time after tumor cell inoculation. (D) Lyve-1 (green) and CD31 (red) immunostaining of brachial and mesenteric lymph nodes from normal and LLC bearing mice indicates expansion of lymphatic vessels (green) but not blood vessels (red) in lymph nodes from tumor bearing animals.

2 Supplementary Figure 2: PyMT lymph node lymphangiogenesis and metastasis A) Cryosections of brachial lymph nodes from 6 week old FVB wildtype (WT) at 40X and 6, 9 and 12 week old FVB PyMT+ transgenic mice at 200X immunostained to detect Lyve-1+ or Lyve-1/Prox-1+ lymphatic vessels and cytokeratin+ metastases. Sections were counterstained with DAPI (blue). H&E stained lungs and whole mount lungs indicating metastases (arrows) in 15-week-old PyMT mice. Bar, 50µm. White dotted lines, edge of lymph node. Arrowheads indicate Lyve-1/Prox-1 or cytokeratin positive structures. (B) Mean +/- SEM Lyve-1+ pixels/field in lymph nodes from A (n=10, *P<0.05). (C) Mean +/- SEM Prox-1+ pixels/field in lymph nodes from A (n=10, *P<0.05). (D) Mean number of mice +/- SEM with lymph node (blue and lung (red) metastases from A (n=10, *P<0.05).

3 Supplementary Figure 3: Lyve-1 and myeloid cell markers (A) Immunostaining for integrin α4β1 (red)(arrowheads), Lyve-1 (green) and DAPI (blue) in normal and PyMT tumor draining lymph nodes. White dotted lines, in lymph node edge. (B) Immunostaining to detect macrophages and dendritic cells (CD11b, CD18, F4/80) or B cells (B220) (red) and Lyve-1 (green) in 5 µm thick sections of inguinal lymph nodes from day 21 LLC tumor bearing mice. As Lyve-1 expression does not overlap with these macrophages/b cell markers, Lyve-1 staining does not identify macrophages in lymph nodes. (C) Immunostaining to detect CD11b+ cells (red) and Lyve-1+ vessels (green) in inguinal lymph nodes from day 1 to day 21 LLC tumor bearing mice, indicates that CD11b+ myeloid cells are gradually accumulate in lymph nodes, but remain Lyve-1 negative.

4 Supplementary Figure 4: Podoplanin, Lyve-1, CD31 and integrin α4β1 are biomarkers of lymphatic vessels in human ductal breast carcinoma draining lymph nodes. (A-B) Serial 5µm sections of paraffin-embedded axial lymph nodes from patients with ductal breast carcinoma were immunostained for expression of Podoplanin, CD31, or Lyve-1 (brown) and counterstained with hematoxylin (blue). (A-B) Three magnifications (200X, 600X, 1200X) of 2 types of lymphatic channels in lymph nodes are shown. Podoplanin, CD31 and Lyve-1 are each expressed on large, thin-walled lymphatic vessels in lymph nodes, in which leukocytes can be observed. CD31 and Lyve-1 are also strongly expressed on smaller vessels leading to these larger vessels, while podoplanin is only weakly expressed on some of these small vessels. Arrowheads indicate lymphatic vessels staining positively for each marker. (C) Metastases can be observed both within and outside of the lumens of lymphatic vessels (arrowheads). (D) Serial sections of paraffin-embedded axial lymph nodes from patients with ductal breast carcinoma were immunostained for expression of Podoplanin or integrin α4β1 and counterstained with hematoxylin. Arrowheads indicate podoplanin/ integrin α4β1+ lymphatic vessels. (E) Immunofluorescence staining to detect integrin α4β1 (red) and Lyve-1 (green) in cryosections of lymph nodes from normal patients or from patients with ductal breast carcinoma. Arrowheads indicate lymphatic vessels. Yellow color indicates integrin α4β1+podoplanin+ vessels. Bar indicates 50 µm.

5 Supplementary Figure 5: Systemic tumor derived VEGF-C promotes lymph node lymphangiogenesis (A) Relative gene expression levels of GAPDH, VEGF-A and VEGF-C in RNA from normal tissue, primary LLC tumors and inguinal lymph nodes from normal mice and d21 LLC tumorbearing mice. (B) Lyve-1+ lymphatic vessels (green) and DAPI+ nuclei (blue) in lymph nodes from mice stimulated with systemic injections of saline or VEGF-C. White dotted lines, edge of lymph node. Bars, 50µm. (C) Mean +/- SEM Lyve-1+ pixels/field in lymph nodes from animals in B (n=7, *p=0.001). (D) Lyve-1+ lymphatic vessels (green) and DAPI+ nuclei (blue) in inguinal and brachial lymph nodes from mice stimulated with intradermal injections of saline or VEGF-C proximal to the inguinal lymph node. White dotted lines, edge of lymph node. (E) Mean +/- SEM Lyve-1+ pixels/field in inguinal and brachial lymph nodes from D (n=7).

6 Supplementary Figure 6: PI3Kinase control of LEC functions (A) qpcr measurement of mrna levels of PI3K isoforms p110α, β, δ and γ expressed in cultured LEC (red) and HUVEC (blue). While both endothelial cell types express PI3Kalpha and beta, HUVECs express PI3Kgamma and LEC express PI3Kdelta. (B) VEGF-C stimulated LEC migration in the presence and absence of panpi3k inhibitor LY294002, p110α inhibitor PIK2α, p110γ inhibitor TG or an inert chemically matched control compound (control). (C) Western blotting (micrograph) to detect and quantify (graph) p110α and actin in untransfected lymphatic endothelial cells or in cells that had been transfected with control sirna or the p110α sirnas Hs_PIK3CA_5 or Hs_PIK3CA_8.

7 Supplemental Figure 7: Integrin α4β1 promotes lymph node lymphangiogenesis (A) Immunostaining for integrin α4β1 or CD11b (red)(arrowheads), Lyve-1 (green) and DAPI (blue) in normal and VEGF-C stimulated lymph nodes. White dotted lines, lymph node edge. (B) Lyve-1+ lymphatic vessels in saline and VEGF-C stimulated inguinal lymph nodes from Tie2Cre- a4 loxp/loxp, Tie2Cre+ a4 loxp/+ and Tie2Cre+ a4 loxp/loxp mice. (C) Mean +/- SEM Lyve-1+ pixels/field in lymph nodes from B (n=6). (D) Lyve-1+ lymphatic vessels in saline or VEGF-C stimulated lymph nodes from mice that were systemically treated with saline, anti-α4β1 or isotype-matched control antibodies (n=10). (E) Lyve-1 pixels/field +/- SEM from D. (F) Images: Lyve-1 immunofluorescence (red) in saline or ELN treated lymph nodes. Quantification of Lyve-1 immunostaining in VEGF-C stimulated lymph nodes from mice that were treated systemically with the integrin α4β1 inhibitor ELN or saline, *P<0.05.

8 Supplemental Figure 8: Integrin α4β1 mediated lymph node lymphangiogenesis promotes experimental metastasis. (A) Model of experimental lymph node metastasis: mice were injected for 5 days adjacent to the left inguinal LN with intradermal injections of VEGF-C or saline to stimulate lymph node lymphangiogenesis and then were inoculated with red fluorescent tumor cells in the left rear footpad. (B) Lyve1+ lymphatic vessels (green) and LLC tumor cells (red) in brachial and mesenteric lymph nodes from animals stimulated with VEGF-C proximal to the inguinal lymph node. (C) Mean +/- SEM Lyve-1 positive pixels/field in lymph nodes (n=10). (D) Tumor cells (red) in VEGF-C or saline stimulated inguinal lymph nodes that were treated with or without intradermal injections of anti-vegf-r3, anti-α4β1 or cigg to block lymphangiogenesis. (E) Mean +/- SEM tumor cells (as red fluorescent pixels/field) in treated lymph nodes from B (n=8). Significance testing was performed by one way Anova. Bar, 50 µm.

9 Supplemental Figure 9: Integrin α4β1 knockdown or antibody mediated inhibition blocks LEC-tumor cell adhesion and lymph node lymphangiogenesis. (A) Western blot analysis of integrin α4 and β-actin expression in sirna transfected LEC and relative integrin α4 expressed in transfected cells normalized to actin expression. (B) Mean +/- Lyve-1 positive pixels in anti-integrin α4β1 antibody treated lymph nodes.

10 Supplemental Figure 10: VCAM promotes human tumor cell adhesion to LECs (A) Merged brightfield, red (LLC) and blue (DAPI) fluorescence images of in vitro adhesion of CMTMR labeled-llc cells to LEC in the absence or presence of function-blocking anti-murine VCAM-1, isotype matched control (cigg), or no (Medium) antibodies. (B) Western blot and histogram of VCAM-1 and beta-actin expression in transfected LLC cells. (C) Human R40P pancreatic carcinoma cells were sorted into high (R40P hi) or low (R40P lo) VCAM expressing cell populations by flow cytometry. Graph represents adhesion of fluorescently labelled R40P subpopulations to monolayers of LECs. (D) Mice were stimulated intradermally with VEGF-C proximal to inguinal lymph nodes and injected in the footpad with red fluorescent LLC cells that were pre-incubated with anti-vcam-1 or cigg antibodies (n=10). Mean +/- SEM Lyve-1 positive pixels in treated lymph nodes.

11 Supplementary Figure 11: Lymphatic endothelial cell integrin α4β1 promotes spontaneous lymph node metastasis (A) Lyve-1+ vessels and cytokeratin or H&E positive metastases in lymph nodes from WT or α4y991a mice with LLC (21 day), B16 (28 day), or Panc02 (28 day) tumors. Arrowheads indicate metastases. (B) Mean +/- SEM Lyve-1+ pixels/field and (C) mean lymph node metastases in tumor-draining inguinal (LLC, B16) and hilar (Panc02) lymph nodes from WT or α4y991a mice with LLC (21 day), B16 (28 day) or Panc02 (28 day) tumors (n=10).

12 Supplementary Figure 12: Lymphatic endothelial cell integrin α4β1 promotes spontaneous lymph node metastasis (A) Animals with LLC or B16 tumors were systemically treated with saline, anti-α4β1 and isotype-matched control antibodies. Representative images of Lyve-1+ vessels (green) in lymph nodes. (B) Mean +/- SEM Lyve-1+ pixels per field in lymph nodes from A (n=10). (C) H&E stained lymph nodes (LN) and lungs from mice with B16 tumors. (D) Percent mice with lymph node (LN) metastases..

13 Supplemental Figure 13: Lymphangiogenesis in lymph nodes promotes tumor metastasis independently of the primary tumor. Mice were inoculated with LLC cells and treated systemically (A-C) or locally proximal to the inguinal lymph node (D-F) with saline or anti-α4β1, anti-vegfr3 or isotype-matched control antibodies (cigg) for 14 days from day (A, D) Mean +/- SEM Lyve-1+ pixels/field in lymph nodes (n=10). (B, E) Percent mice with cytokeratin+ lymph node metastases. (C, F) Mean +/- SEM primary tumor volume.

14 Supplemental Figure 14: Schematic of the role of integrin α4β1 in lymph node lymphangiogenesis VEGF-C/PI3Kα mediated integrin α4β1 activation promotes lymph node lymphangiogenesis, resulting in lymph node metastasis, which can contribute to distant metastasis.

15 Supplemental Experimental Procedures Reagents Purified recombinant human VEGF-A and VEGF-C were purchased from R&D Systems (Minneapolis, MN). Rabbit anti-human and rabbit-anti-mouse Lyve-1 antibodies (RDI- 102PA50 and RDI-103PA50) were purchased from Research Diagnostics Incorporated (Concord, MA). Rat anti-mouse CD31 (MEC 13.3) was from BD Bioscience (San Diego, CA). Hamster anti-murine podoplanin (103-M40) was from Research Diagnostics Incorporated (Concord, MA). Rabbit anti-human podoplanin (D2-40) was from Biocare Medical LLC (Concord, CA). Murine anti-pan-species Prox-1 (MAB5652, clone 5G10) was from Millipore. Murine anti-human fibronectin (TEV-1) was from Chemicon International. Goat anti integrin alpha 4 (sc-6590) was from Santa Cruz Biotechnology. Murine anti-human α4β1 (HP1/2) and rat anti-murine α4β1 (PS2) were gifts from Biogen-Idec (Cambridge, MA and San Diego, CA). Isotype matched control antibody (IgG2b) was a gift from Biogen-Idec. Alexa488-conjugated murine anti-pan-cytokeratin was from Cell Signaling Technology. Anti-murine VEGFR3 (AFL4) was from ebioscience. Cell tracker orange CMTMR (C2927) was obtained from Invitrogen. Donkey anti-goat, rabbit and mouse IgG conjugated with Alexa Fluors 488, 568 or 647 were from Invitrogen. Primer sets to detect murine and human VEGF-A and VEGF-C were purchased from Qiagen. Growth factor depleted Matrigel was from Becton- Dickinson. ELN was a gift from Elan Pharmaceuticals. Immunohistochemistry Five µm thickness cryosections were prepared using a Leica cryostat CM1900. Slides were fixed in ice cold acetone or 3.7% paraformaldehyde for 5 min, permeabilized in 0.1% Triton X-100 in phosphate buffered saline (PBS), blocked in 8% normal donkey serum diluted in PBS for 1.5 h at RT and incubated with primary antibodies diluted in 8% normal donkey serum overnight at 4ºC. After extensive washing, slides were incubated with 1-2 µg/ml cross-absorbed donkey anti-goat, antirabbit or anti-mouse IgG (H+L) conjugated with Alexa Fluor 488, 568 or 647 (Invitrogen). Slides were counterstained with DAPI or TOPRO-3 (Invitrogen). Coverslips were mounted with Dako Cytomation fluorescent mounting medium. Lymph node cryosections were immunostained to detect lymphatic vessels with 2 µg/ml anti-human Lyve-1 (RDI-102PA50) or anti-human podoplanin (D240). Blood and lymphatic vessels were detected by anti-cd31 immunostaining with 5 µg/ml MEC13.1. Integrin α4β1 was detected in human tissues with antibody 6590 from Santa Cruz Biotechnology. For quantification, number of lymphatic or blood vessels or pixels in 5-10 microscopic fields per cryosection (per animal) was quantified and the mean number of vessels +/- SEM for the entire treatment group was determined. Quantitative, automated pixel density determinations were performed using Metamorph (Version 6.3r5, Molecular Devices). Murine lymph node cryosections were immunostained to detect lymphatic vessels with 2 µg/ml anti-murine Lyve-1 (RDI-103PA50), anti-prox-1 (MAB5652, clone 5G10), or anti-murine podoplanin (103M40). Blood and lymphatic vessels were detected by anti-cd31 immunostaining with 5 µg/ml MEC13.1. Integrin α4β1 was detected in murine tissues with antibody 6590 from Santa Cruz Biotechnology. For anti-prox-1

16 immunostaining of cryosections of lymph nodes, sections were blocked with 10µg/ml rat anti-mouse CD16/CD32 (murine Fc block from BD Bioscience) for 30 min at room temperature, incubated in anti-prox1 antibody (5G10, Millipore) for 2h at RT, followed by secondary antibodies. For quantification, number of lymphatic or blood vessels or pixels in 5-10 microscopic fields per cryosection (per animal) was quantified and the mean number of vessels +/- SEM for the entire treatment group was determined. Quantitative, automated pixel density determinations were performed using Metamorph (Version 6.3r5, Molecular Devices). Metastases of PyMT+ breast carcinoma, Lewis lung carcinoma and Panc02 pancreatic adenocarcinoma were detected by immunostaining triplicate cryosections of bilateral lymph nodes, lungs and other tissues with 5 µg/ml Alexa 488 conjugated antimurine cytokeratin (C11); metastases were quantified at in lymph nodes from all animals. The number of mice with metastases in lymph nodes was quantified, and the average incidence of metastasis positive lymph nodes in three studies was determined. Standard error of the mean for metastases was determined from the results of three or more replicate experiments. B16 melanoma metastases were detected by hematoxylin and eosin staining of lymph node and lung tissue sections. Haematoxylin and eosin staining was performed by the Moores UCSD Cancer Center Histology Shared Resource. Human tissue immunohistochemistry Patients at the Moores UCSD Cancer Center in La Jolla, CA, underwent planned procedures for breast surgical treatment. All surgeries were performed at the University of California, San Diego, and standard techniques were used for resection of breast tissue. Normal tissue was also obtained from patients undergoing breast reduction or prophylactic mastectomy. Specimens were removed, sent to the UCSD Medical Center pathology laboratory for analysis, and reviewed by a pathologist to assess the surgical margin tissue. Tissues not needed for diagnosis were embedded in paraffin. Tissues were evaluated for the presence of integrin α4+ lymphatic vessels by immunostaining of fixed or frozen sections. Formalin-fixed, paraffin-embedded human lymph nodes were sectioned at 4µm thickness and de-waxed according to standard protocols. Antigen retrieval was performed in Dako Target Retrieval Solution in a steamer for 20 min or by Proteinase K digestion for 10 min (for integrin α4). Slides were blocked for 2 h in 8% normal goat serum in a humidified chamber. Slides were incubated in primary antibodies overnight at 4 C in a humidified chamber as follows: 4µg/ml anti-vcam-1 (E-10) from Santa Cruz Biotechnology, 2 µg/ml PECAM-1 (sc-1505-r) from Santa Cruz Biotechnology, 2 µg/ml anti-podoplanin (D2-40) from Covance, 2 µg/ml anti-lyve-1 (70R-LR006) from Fitzgerald Industries International, and 2 µg/ml integrin α4 (SC-14008) from Santa Cruz. Slides were then incubated in biotinylated cross-absorbed donkey anti-rabbit or mouse immunoglobulin from Jackson ImmunoResearch and developed using Vectastain ABC kit (Vector Laboratories) with DAB as a color substrate. Cell Culture

17 Lymphatic endothelial cells (HMVEC-dLyNeo, Lonza) were cultured in endothelial growth medium (EGM-2) containing 10% fetal bovine serum (Lonza). Integrin α4 and VCAM-1 expression on lymphatic endothelial cells (LEC) or LLC cells was examined by immunostaining cultured cells with µg/ml of monoclonal antibodies. Evaluation of positive cells was carried out using flow cytometry, and more than 100,000 events were collected for each sample tested. To exclude dead cells, 2.5µg/ml propidium iodide (PI) was added before data acquisition by FACs Calibur (BD Bioscience). Lewis lung carcinoma (LLC) and B16 melanoma cells were obtained from the American Type Culture Collection (ATCC). Panc02 pancreatic ductal carcinoma cells were obtained from the NCI DCTDC Tumour Repository. LLC, B16 and Panc02 cells were cultured in DMEM medium containing 10% FBS and antibiotics. Gene and protein expression Total RNA was isolated from lymph nodes, cultured lymphatic endothelial cells or tumor cells using ISOGEN (Nippon Gene). cdna was prepared from 1µg RNA from each sample and qpcr was performed using primers for lyve-1, vegf-a and vegf-c from Qiagen (QuantiTect Primer Assay). Transcript levels were normalized to gapdh expression. Expression of p110 PI3K isoforms in HUVEC and LEC was performed using anti-bodies directed against p110α, p110β, p110γ and p110δ. Expression of actin served as a negative control. PI3K activation in response to VEGF-C stimulation was determined by Western blotting to detect phosphoakt and total Akt. Integrin activation and cell migration studies Lymphatic endothelial cells were plated on rsvcam-coated, non-tissue culture treated 48 well plates in the presence or absence of 200 ng/ml VEGF-C and were allowed to adhere for 30 minutes at 37 C before washing 3 times with PBS. Adherent cells were quantified in quadruplicate replicates. Additionally, adhesion assays were performed in the presence of 1µM PI3K2α or upon transfection with p110α or control sirna. Cultured human lymphatic endothelial cells were transfected using an AMAXA Nucleofection kit with 100 nm of sirna for p110α (Hs_PIK3CA_5 or Hs_PIK3CA_8) or non-silencing sirna (Ctrl_AllStars_1) purchased from Qiagen. After transfection, cells were cultured for h in media containing 20% serum. Cell migration assays were performed in the presence of 1µM PI3K isoform inhibitors, including p110α inhibitor PI3K2α, p110γ inhibitor TG , pan-pi3k inhibitor LY and an inert, chemically similar control (1) as previously described (2). Transgenic and other animals Male PyMT mice on an FVB background were randomly bred with FVB females lacking the PyMT transgene to obtain female mice heterozygous for the PyMT transgene. Female mice heterozygous for the PyMT transgene were compared to wild type FVB female mice. All PyMT+ females exhibit hyperplasias by 6 weeks of age, the majority exhibit adenomas/ early carcinomas by 9 weeks of age and lymph node and lung

18 metastases by weeks of age. C57Bl6 mice were from Charles River, and Tie2Cre mice were from Jackson Laboratories. Integrin α4y991a mice in the C57Bl6 background were derived as previously described (3). Male Tie2Cre+ mice were crossed with female integrin α4 loxp/loxp mice (4) to generate Tie2Cre+ α4 loxp/+ mice, which were then crossed to with α4 loxp/loxp mice to obtain sibling Tie2Cre- α4 loxp/loxp, Tie2Cre+ α4 loxp/+ and Tie2Cre+ α4 loxp/loxp mice for studies. Tumor studies For time courses, 5X10 5 LLC cells were injected subcutaneously into anesthetized C57Bl6 mice and tumours and lymph nodes were excised at 3, 7, 10, 14 or 21 days, weighed, cryopreserved in O.C.T., or lysed for RNA purification (n=10). Additionally, normal tissues from age-matched mice, including lymph nodes, were cryosectioned (n=10). Tumors, lungs and lymph nodes from 6, 9 and 12 week old PyMT+ FVB and PyMT- FVB females were also cryopreserved (n=10). 5X10 5 LLC cells or B16 melanoma cells were injected subcutaneously into C57Bl6 or integrin α4y991a mice (n=10-12). Animals were sacrificed after 3 weeks of tumor growth. In treatment studies, C57Bl6 mice were subcutaneously implanted with 5X10 5 LLC or B16 melanoma cells. Animals were treated for three weeks by intraperitoneal injections every third day with 200µg per 25g body weight of functionblocking anti-integrin α4 (n=10), isotype matched control rat IgG1 (n=10) or saline (n=10). Tumor length and width were measured every third day with calipers and volume was determined using the formula (l X w 2 )/2. Tumors, lungs and lymph nodes were cryopreserved and analysed for the presence of lymphatic vessels, blood vessels and metastases. Experiments were performed three times. For orthotopic pancreatic carcinoma studies, the abdominal cavities of immunocompetent C57Bl6 mice and integrin α4y991a knockin mice (n=10) were opened and the tails of the pancreas were exteriorized. One million syngeneic Panc02 cells were injected into the pancreatic tail, the pancreas was placed back into the abdominal cavity and the incision was closed. Animals were sacrificed and tumours, lymph nodes, lungs and intraperitoneal metastases were excised after 30 days. Experiments were performed three times. Lymphangiogenesis assays Lymphangiogenesis assays were performed by subcutaneously injecting 400µl of cold Matrigel containing saline or 400ng of VEGF-C (R&D Systems, Minneapolis, MN) into wild-type C57Bl/6 or integrin α4y991a mice (n = 10) proximal to inguinal lymph nodes. Additionally, Matrigel containing VEGF-C or saline was injected subcutaneously into Tie2Cre+α4 loxp/loxp (n=4), Tie2Cre+α4 loxp/+ (n=4) or Tie2Cre-α4loxp/loxp (n=4) mice. In additional studies, VEGF-C implanted mice were then injected at day 1 and day 3 with 200µl of function-blocking rat anti-mouse anti-α4 (PS2) antibodies or isotype control antibodies (n=10). After 7 days, lymph nodes were removed, embedded in OCT, frozen and sectioned. In additional studies, mice were inoculated with Matrigel and treated with ELN476063, or inhibitors of p110α (PI3K2α) or p110γ (TG ). Alternatively mice were treated with intravascular injections of saline or VEGF-C (10 µg/kg/day) or were left untreated for seven days prior to removal of lymph nodes (n=7).

19 In all cases, 5µm sections were immunostained with anti-lyve-1 antibodies. At least five microscopic fields at X per tissue section were analyzed for quantification studies. Systemic delivery of anti-α4β1 or anti-vegfr3 antibodies C57BL/6 mice were inoculated subcutaneously in the dorsum with 5 X 10 5 LLC or B16 melanoma cells. Mice were systemically treated by intravenous tail vein injection every third day for 14 days with 200µl of sterile, endotoxin-free saline, anti-mouse α4 integrin (PS2), anti-vegf-r3, or isotype antibody control antibodies at 200µg/mouse/injection (n=8). Mice were sacrificed after 21 days and tumors, inguinal, brachial and mesenteric lymph nodes were removed and embedded in OCT for cryosectioning and histological analysis. Experiments were performed 3 times with equivalent results. Local delivery of anti-integrin α4β1 or anti-vegfr3 antibodies C57BL/6 mice were inoculated subcutaneously in the dorsum with 5 X 10 5 LLC cells. On the same day, 50µg in 50µl of sterile, endotoxin-free anti-mouse α4 integrin (PS2), anti-vegf-r3 (AFL4), isotype antibody control or 50µl saline was intradermally injected into the fatty tissue proximal to the inguinal lymph node. Intradermal injections were repeated every 3 days for up to 21 days (for a total of 7 injections per mice, n=10). Mice were sacrificed after 21 days. Inguinal, brachial and mesenteric lymph nodes were removed and embedded in OCT for cryosectioning and immunohistochemistry. Experiments were performed 3 times with equivalent results. Data from single experiments are shown in figures except where specified. Experimental lymphatic metastasis studies To test the role of lymphangiogenesis in experimental lymph node tumor metastasis, mice were intradermally injected above the left inguinal lymph node with a final volume of 50µl of sterile, endotoxin free saline or 50ng VEGF-C in 50µl saline (n=10). In other studies, mice were stimulated as described and intradermally injected above the left inguinal lymph node every three days with a final volume of 50µl of sterile, endotoxin free saline, 50µg rat anti-mouse α4 (PS2), 50µg rat anti-mouse VEGF-R3 (AFL4) or 50 µg control isotype-matched IgG every day for 7 days (n=8). Anesthetized mice were inoculated on the eighth day with 10 6 CMTMR LLC cells in a volume of 150µl saline by intradermal injection in the left footpad, with gentle massage of the footpad. Alternatively, C57Bl6 or α4y991a mice were intradermally injected above the left inguinal lymph node with a final volume of 50µl of sterile, endotoxin free saline or VEGF-C (50ng in 50 µl) each day for 7 days. Anesthetized mice were inoculated on the eighth day with 10 6 CMTMR labeled LLC cells in a volume of 150µl saline by intradermal injection in the left footpad, with gentle massage of the footpad. Mice were sacrificed 1 or 14 days after cell injection. Bilateral inguinal, brachial and mesenteric lymph nodes as well as lungs were removed from all mice and embedded in OCT for cryosectioning and immunofluorescence. Red fluorescent tumor cells and Lyve-1+ pixels were quantified on 5 different fields per lymph node and lung cryosection. At least

20 2-3 cryosections per tissue were examined. Experiments were performed 3 times with equivalent results. In vitro tumor cell to lymphatic endothelium adhesion assay Human LECs were cultured in EBM-2 on 24 well plates until eighty percent confluent. Murine LLC cells were removed from culture dishes with versene and were wellwashed. 300,000 cells were incubated in basal culture medium (DMEM) containing either no additives, 25 µg/ml rat anti-mouse VCAM-1 or isotype matched rat IgG for 1 hour on ice. Antibody solutions were removed, cells were washed and restored to culture medium, and aliquots of 100,000 cells were added per triplicate well of LEC. Plates were incubated for 90 minutes at 37ºC. Nonadherent cells were removed by gentle aspiration, then cell monolayers were washed and fixed in PBS containing 0.5% PFA, before counting the number of red fluorescent adherent cells. Alternatively, human LEC monolayers were incubated in the presence of basal culture medium (DMEM), 25 µg/ml anti-human α4β1 (HP2/1) or isotype-matched mouse IgG for one hour at 37ºC. Antibody solutions were removed, cells were washed and restored to basal culture medium, then incubated together for 90 minutes at 37ºC. Cell monolayers were washed and fixed in PBS 0.5% PFA before counting the number of adherent red fluorescent tumor cells. The dose dependency of antibody concentration was determined in quadruplicate by serial dilution; thereafter studies were performed using antibodies at 25 µg/ml to achieve maximal inhibition. Experiments were performed 3-5 times. In vivo tumor cell adhesion In one group of animals, inguinal lymph node lymphangiogenesis was stimulated by injecting mice intradermally above the left inguinal lymph node with saline or VEGF- C (50ng) in a final volume of 50 µl every day for 7 days. On the eighth day, animals were treated by intradermal injection into the fatty tissue proximal to the left inguinal lymph node with 50µl saline, 25µg in 50µl of anti-murine α4β1 (PS-2) or isotype matched control antibody (n=10). One hour later, 1 X 10 6 CMTMR-labelled LLC cells in a volume of 150µl were injected into the left footpad. In another group of animals, on the eighth day, 1 X 10 6 CMTMR- labeled LLC cells in a volume of 150µl were pre-incubated for one hour with 25µg rat anti-murine VCAM-1 or isotype matched control antibody, then injected into the left footpad in a final volume of 150µl (n=10). Animals were sacrificed after 4h and inguinal, brachial and mesenteric lymph nodes were removed and embedded in OCT for cryosectioning and immunofluorescence. Tumor cell and Lyve-1+ pixels were quantified in 5 different fields per node. Experiments were performed twice with equivalent results. sirna mediated knockdown Cultured human lymphatic endothelial cells were transfected using an AMAXA Nucleofection kit with 100 nm of sirna for ITGA4 (HS_ITGA4_1 or HS_ITGA4) or p110α (Hs_PIK3CA_5 or Hs_PIK3CA_8,) and cultured murine lung carcinoma cells (LLC) were transfected using an AMAXA Nucleofection Kit with 100 nm of sirna for VCAM-1 (Mm_Vcam1_2 or Mm_Vcam1_4) or non-silencing sirna (Ctrl_AllStars_1)

21 purchased from Qiagen. After transfection, cells were cultured for h in media containing 20% serum. Each sirna was tested individually for efficient knockdown of protein expression and for inhibition of adhesion. Integrin and VCAM-1 expression levels were analyzed by flow cytometry or Western blotting using anti-α4 (R1-2) and anti-vcam (M/K2) antibodies from ebioscience. Integrin, VCAM and PI3-Kinase p110α mrna expression levels were determined by RT-PCR, essentially as previously described (1). Similar results were achieved for each of two sirna oligonucleotides per gene, as listed above. Cell lysates were also evaluated by Western blotting. Results are shown only for HS_ITGA4_1, Mm_Vcam1_2 and Hs_PIK3CA_5. Statistical analysis Statistical analyses of in vivo tumor studies were performed with one-way ANOVA and all in vitro studies were analyzed with Student's t-test. Incidence of metastasis was analyzed using Fisher's exact test. All experiments were performed three times, and the data from a single representative experiment is presented. References 1. Schmid MC, et al (2011) Receptor tyrosine kinases and TLR/IL1Rs unexpectedly activate myeloid cell PI3kγ, a single convergent point promoting tumor inflammation and progression. Cancer Cell 19: Garmy-Susini B, et al (2010) Integrin alpha4beta1 signaling is required for lymphangiogenesis and tumor metastasis. Cancer Res. 70: Féral CC, et al (2006) Blocking the alpha 4 integrin-paxillin interaction selectively impairs mononuclear leukocyte recruitment to an inflammatory site. J Clin. Invest. 116: Scott, L.M., Priestley, G.V. & Papayannopoulou, T. Deletion of alpha4 integrins from adult hematopoietic cells reveals roles in homeostasis, regeneration, and homing. Mol. Cell. Biol. 23, (2003).