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Figure S1. Production of Recombinant LIGHT/LIGHT-RGR Proteins, RGR Peptide Tumor Homing and Survival Analysis, Related to Figure 1 (A) Purification steps for murine LIGHT and LIGHT-RGR proteins: Left: M, protein marker; Sup, fusion protein expression in bacterial lysate; flow through (F/T), unbound bacterial lysate; Wash 1 and 3: column washing steps; Eluate, fraction released from Ni-NTA column; Dialysed: protein after overnight dialysis; Cut, released protein after tobacco edge virus (TEV) protease cleavage; TEV, band size of TEV. Middle: Representative image showing purified LIGHT at approximately 17 kDa (boxed); bovine serum albumin (BSA) was added for stability. Right: representative image of purified LIGHT-RGR at 20 kDa (boxed); BSA was added for stability. BSA; band size of BSA. (B) Specific vascular (red, CD31+) binding of FAM-RGR peptide (green) in tumors of 27-week-old RIP1-Tag5 mice as compared to normal pancreas. Quantification of RGR specific vessel binding in normal pancreas and tumor tissue, n = 3 mice. Scale bar, 100 µm. (C) Kaplan-Meier graph for RIP1-Tag5 mice treated with LIGHT (n = 6), LR (n = 17), AdT (n = 12), or LR in combination with AdT (n = 15). Mice were sacrificed at a pre-defined end point of 30 weeks. For statistical analysis see Figure 1 and Methods.
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Figure S2. Pericyte Markers, LIGHT Receptor Expression, and Vascular Pericyte Coverage in RIP1-Tag5 Tumors, Related to Figure 2 (A-C) Pericyte markers in untreated 27 week-old RIP1-Tag5 mice. (A) αSMA+ cell co-localization with CD31+ (red) tumor vessels. (B) Co-staining of αSMA+ cells with the pericyte marker NG2. (C) Quantification of vessel-associated αSMA+ cells (expressed as % αSMA+
CD31+ cells in relation to total αSMA+ cells), and αSMA+ co-localization with NG2 (% in relation to total αSMA+ cells), n = 3 mice. Scale bar, 100 μm. (D) FACS analysis of RIP1-Tag5 tumor stromal components: IC (immune cells, CD45+ CD31- NG2-), EC (endothelial cells, CD45- CD31+ NG2-), PC (pericytes, CD45- CD31- NG2+). (E) LIGHT receptor (LTRβ and HVEM) expression (in %) gated on ICs, ECs, PCs and TCs (tumor cells, CD45- CD31- NG2-). DAPI+ dead cells were excluded; AF, autofluorescence. (F) Vessel coverage by αSMA+ cells in untreated RIP1-Tag5 tumors (Untr), or after 2 weeks of LIGHT-RGR (LR) treatment, and quantification of % αSMA+ CD31+ cells in relation to total CD31+ cells. (G) Analysis as in (F) performed with the pericyte marker NG2, n=3 mice; ns, not statistically significant. Arrows indicate CD31+ vessels which lack coverage by αSMA+ or NG2+ cells. Scale bars, 100 µm.
Figure S3. Long Term LIGHT-RGR Treatment Does Not Induce Local Tumor Invasiveness or Vessel Death, Related to Figure 3 (A) RIP1-Tag5 mice were left untreated (Untr) or treated long term (8 weeks) with LIGHT-RGR (LR), or a combination of cyclophosphamide (Cyclo) + LR. Histology (H&E staining) shows tumors with intact margins (dotted line). Scale bar, 500 μm. (B) Histology pictures depict CD31+ vessels in all groups and quantification of % total vessels/field, not statistically significant. Scale bar, 100 μm.
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Figure S4. Co-Localization of Contractile Markers with CD31+ Tumor Blood Vessels, Related to Figure 4 (A-E) 27 week-old RIP1-Tag5 mice were left untreated (Untr) or treated for 2 weeks with 0.2 ng LIGHT-RGR (LR). (A) Brightfield images depicting specific vascular staining of myocardin around vessels (detail analysis). L= lumen. Scale bar, 50 μm. (B-E) Histological analyses of vascular beds (CD31+) and contractile markers in untreated or LR treated tumors (overview analysis) and quantification. Vessel (CD31+) coverage with (B) myocardin, (C) caldesmon, (D) calponin and (E) collagen I (Col I). Representative images from three independent experiments are shown. (B-E) n = 5-10 mice, * P ≤ 0.04, **P = 0.006, Student’s t-test. Scale bar, 100 μm. (F-H) 27 week-old RIP1-Tag5 mice were left untreated (Untr) or treated with 2 or 4 LIGHT-RGR (2 x LR or 4 x LR) injections. (F) Injection of FITC-lectin i.v. into tumor bearing mice and quantification of ratio FITC-lectin/CD31+ vessels as surrogate marker for tumor perfusion, *P = 0.014, **P = 0.009. (G) Quantification of calponin induction in tumors, *P = 0.01 Untr versus 2 x LR, *P = 0.04 Untr versus 4 x LR. (H) Quantification of caldesmon signals in tumors, *P = 0.002. Student’s t-test. (F-H) n = 4-7 mice. Data are presented as mean ± s.d.
Figure S5. LIGHT-RGR Effects in Orthotopic Breast Cancer, Related to Figure 4 (A – E) Orthotopic breast cancer (4T1) in Balb/c mice. Mice with palpable tumors were left untreated or treated for 2 weeks with bi-weekly i.v. injections of 20 ng LIGHT-RGR (LR). (A) CD31+ vessels in untreated and LR treated tumors and quantification, ns, not statistically significant. Scale bar, 100 µm. (B) Vessel coverage with caldesmon and quantification of % caldesmon-positive vessels compared to all vessels/field. Scale bar, 50 µm. Arrows point at caldesmon signals aligned with CD31+ vessels. (C) Tumor perfusion assessed by i.v. injection of FITC-lectin and quantification of lectin/CD31 overlay. Scale bar, 50 µm. Arrows point at lectin signals associated with CD31+ vessels. (D) Hypoxia staining and quantification. Scale bar, 200 µm. (A-D) n = 5- 6 tumors, *P = 0.04, **P < 0.01, ***P < 0.001, Student’s t-test. (E)
Quantification of lung metastases (colonic assay, count of colony outgrowth in vitro) from orthotopic breast cancer in untreated and LR-treated mice, n = 7-8 mice. P = 0.1, not statistically significant, Student’s t-test.
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tubulin
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Figure S6. TGFβ is Specifically Upregulated in Peri-Vascular Macrophages and Induces Pericyte Contractile Marker Expression, but not Endothelial-Mesenchymal Transition, Related to Figure 5 (A) Mesenchymal precursor cells (10T1/2) express the LIGHT receptor LTβR, but not HVEM, representative FACS analysis (BD Accuri™ C6). (B) Western blot analysis of contractile protein (caldesmon, calponin, αSMA) expression in 10T1/2 cells incubated with or without LIGHT. Tubulin was used as a loading control. (C) LTβR and HVEM receptor expression in intratumoral CD45+ CD68+ F4/80+ macrophages; representative FACS analysis (BD FACS Aria II). (D) Histology images depicting intratumoral TGFβ staining and macrophages (CD68) in untreated RIP1-Tag5 tumors (Untr), or after 2 weeks of LIGHT-RGR treatment (LR). Arrows point at TGFβ-positive macrophages, arrowheads at TGFβ-positive peri-vascular cells which are not macrophages. (E) Quantification of TGFβ+ cells in relation to total cells (DAPI+ nuclei), n=4-5 mice, **P = 0.002, Student’s t-test. (F) Quantification of TGFβ-signals derived from macrophages (MØ) and cells which are not macrophages (non MØ), expressed as % total TGFβ+ cells in untreated and LR treated RIP1-Tag5 mice; n = 4-5 mice. *P = 0.03, Student’s t-test. Scale bar, 100 µm. (G) Western blot analysis of 10T1/2 cells stimulated with or without recombinant TGFβ, and histology images of caldesmon and myocardin expression in 10T1/2 cells stimulated with 4 ng/ml recombinant TGFβ. Representative images from three independent experiments are shown. Scale bars, 50 μm. (H) Histology images depicting mannose receptor (MRC1) positive cells in untreated and LR treated tumors (T) and normal pancreatic tissue (N). MRC1+ cells are exclusively located in the tumor-surrounding normal tissue but not tumor parenchyma. Scale bar, 100 μm. (I) Gene expression profile of ex-vivo purified intratumoral macrophages (CD45+ CD68+ F4/80+) for selective macrophage polarization markers. Fold regulation in macrophages isolated from LR-treated tumors as compared to untreated tumors (set at 1) is shown. *P = 0.04. (J) FSP1-specific signals in untreated and LR treated tumors and quantification of FSP1-positive endothelial cells (yellow, indicated by arrows) in relation to total FSP1+ cells; n = 4-5 mice, ns, not statistically significant. Scale bar, 25 μm. (K) VE-Cadherin signals in untreated and treated tumors and quantification of VE-Cadherin+ vessels in relation to total CD31+ vessels; n = 6 mice, P = 0.006. Scale bar, 50 μm.
Figure S7. Tumor Homing of Adoptively Transferred Macrophages, Related to Figure 6 Histology image of CD11b+ macrophages in untreated RIP1-Tag5 tumors, tumors after adoptive transfer of control macrophages (PBS MØ AdT), or macrophages stimulated with LIGHT in vitro (LIGHT MØ AdT). Quantification of CD11b signals in all treatment groups, n= 3 mice; *P < 0.01, **P = 0.001, Student’s t-test. Scale bar, 100 µm.
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SUPPLEMENTAL EXPERIMENTAL PROCEDURES LIGHT/LIGHT-RGR Production and Tumor Homing
LIGHT or LIGHT-RGR expressing pET-44a plasmids were transfected in E-coli (Rosetta) for
optimal protein expression. After isopropyl-β-d-galactopyranoside (IPTG) induction for 6 hours
at 22°C in the presence of 5 mM EGTA, cultures were centrifuged, resuspended in lysis buffer
(50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 1 mM DTT, 1mM PMSF, 1 mM
EDTA/EGTA, 1% triton-X100, protease inhibitor cocktail (Sigma), 1µg/ml pepstatin
(Calbiochem), pH 8.0), followed by sonication, and subsequent purification using Ni-NTA beads
(Qiagen) following the manufacturer’s instructions. Recombinant fusion proteins were dialysed
overnight at 4°C in Tris buffer (50 mM Tris, 1 mM EDTA, 1 mM EGTA, 1mM PMSF, pH 8.0).
Nus•Tag/His•Tag was cleaved with tobacco etch virus (TEV) protease (Life Technologies) for
90 min at 30°C. After cleavage, LIGHT protein was re-purified using Ni-NTA beads in the
presence of protease inhibitors (1 mM PMSF, 1 mM EDTA, 1 mM EGTA, 1µg/ml pepstatin and
protease inhibitor cocktail), salts (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole) and
0.005% BSA. Purity was assessed on coomassie brilliant blue stained protein gels and the
concentration was determined by measuring protein intensity in comparison to a band of similar
size and known concentration. LIGHT and LIGHT-RGR were biotinylated following standard
procedures (EZ-Link Sulfo-NHS-LC-Biotin, Pierce), and dialysed against PBS overnight. For
tumor homing studies, 60 μg biotinylated protein or N-terminal 5(6)-carboxyfluorescein (FAM)-
RGR peptide were injected i.v. into 27 week-old RIP1-Tag5 mice. After 10 min circulation, mice
were perfused with 10% neutral–buffered formalin and tissue post-fixed in 10% formalin for 2 h,
followed by incubation in 10% (2 h) and 30% sucrose (overnight), and embedded in OCT. 10
µm sections were stained using streptavidin (SA)-FITC (Jackson Immuno Research), or FAM-
RGR directly visualised using a Nikon Ti-E microscope and NIS software.
Adoptive T cell Transfers, Vaccination Studies and Chemotherapy
Adoptive transfers of in vitro activated CD8+ T cells were performed as previously described
(Johansson et al., 2012). Briefly, CD8+ T cells were harvested from TagTCR8 lymph nodes and
spleen, and activated for 3 days in the presence of 10 U/ml IL2 (Peprotech) and 25 nM Tag
peptide 560-568 (SEFLIEKRI). Tumor-bearing RIP1-Tag5 mice received a total of 2.5 x 106
CD8+ T cells i.v. and i.p, at week 2 and 4 after start of LIGHT-RGR therapy. For vaccination
studies, mice were primed with a single subcutaneous injection at the tail base of 50 µg
recombinant SV40 Large T antigen (Tag) protein mixed with 50 µg CpG-ODN1668
(phosphorothioate-stabilized CpG-ODN 1668, TCCATGACGTTCCTGATGCT, Sigma) in 200
µl PBS. Thereafter, mice were injected with 50 µg Tag protein mixed with 50 µg CpG-ODN
1668 i.p. every second week. For cytotoxic therapy, cyclophosphamide was administered in
drinking water (10 mg/kg/day). Tumors were isolated from pancreatic tissue and measured using
microcalipers. Volume was determined using the following formula: Tumor volume = ½ (length
× width2).
Isolation of Peritoneal Macrophages and Adoptive Macrophage Transfers
4 % thioglycolate solution (Brewer modified, BD Pharmingen) was injected i.p. into C3H mice.
4 days later, the abdomen was rinsed with PBS, cells collected, washed, and incubated overnight
in cell culture media (Dulbecco's Modified Eagle's medium (DMEM), supplemented with 10%
FBS, glutamax, sodium pyruvate and penicillin/streptomycin (Life Technologies)). The next day,
attached macrophages were washed with PBS and culture media replaced with low serum media
(0.5% FBS) and 10 ng/ml LIGHT. After 24 hours stimulation, the supernatant was analyzed for
TGFβ1 expression using an ELISA for active TGFβ1 (R&D Systems) according to the
manufacturer’s protocol. Absorbance was read at 450 and 654 nm and a standard curve was used
to calculate TGFβ1 concentration (pg/ml). For adoptive macrophage transfers, macrophages
were isolated from C3H mice as described above and incubated overnight in DMEM (10% FBS).
Cells were washed in PBS, cultured in low serum media (0.5% FBS) and stimulated with PBS or
10 ng/ml LIGHT for 6 hours. Cells were trypsinized and resuspended in sterile PBS. 2 x 106
macrophages were i.p injected into tumor-bearing RIP1-Tag5 mice with or without 300 µg TGFβ
blocking antibody (clone 1D11.16.8 BioXCell). An additional 150 µg TGFβ blocking antibody
was administered 3 days after macrophage transfer. Mice were sacrificed on day 4 after transfer
and tumors analyzed.
Immunohistochemistry
For histology, mice were perfused with 2% formalin, tumors isolated and incubated in 10%
sucrose (2 h) followed by 30% sucrose (overnight). Tumors were embedded in OCT and 10 μm
sections stained. For immunohistochemical staining the following antibodies were used: CD8
(Ly-2, BD Pharmingen), CD31 (Mec 13.3, BD Pharmingen), CD68 (FA-11, BD Pharmingen),
Col I (rabbit polyclonal, Abnova), Col IV (rabbit polyclonal, Abcam), caldesmon (E89, Abcam),
calponin (EP798Y, Abcam), ICAM1 (YNI/1.7.4, ATCC), pMLC (phospho S20, rabbit
polyclonal, Abcam), αSMA-FITC (1A4, Sigma, amplified using the mouse on mouse (M.O.M.)
kit (Vector)), TGFβ (rabbit polyclonal, Abcam), FSP1/S100A4 (rabbit polyclonal, Millipore),
VE-Cadherin/CD144 (11D4.1, Pharmingen), MRC1/CD206 (MR5D3, Ad Serotec), and
chondroitin sulfate proteoglycan (NG2, rabbit polyclonal, Millipore). For secondary detection,
Cy3, 7-Amino-4-methylcoumarin-3-acetic acid (AMCA) or FITC-conjugated IgG F(ab’)2
fragments (Jackson Immuno Research) were used. Myocardin (goat polyclonal, Santa Cruz)
staining was further amplified using the TSATM Cy3 System (Perkin Elmer) according to the
manufacturer’s protocol. Primary antibodies raised in goat are not compatible with the M.O.M
kit required for αSMA detection. Hence pericyte-specific myocardin staining was assessed with
the marker NG2. Assessment of vascular function using FITC-labelled lectin (Lycopersicon
esculentum, Vector), or 70 kDa Texas Red dextran (Invitrogen) was performed as previously
described (Johansson et al., 2012). Apoptosis was assessed using the TUNEL staining kit
(Roche). Hypoxia was evaluated using the HypoxyprobeTM-1 kit (Hypoxiprobe, Inc.) on tissues
injected with pimonidazole hydrochloride according to the manufacturer’s protocol. A Nikon Ti-
E microscope was used for imaging. Images were quantified using NIS software modules.
Mouse numbers (n) per group are provided in all figure legends. At least 6 tumors per treatment
group were analysed. At least two slides per tumor and marker were assessed, and 5-8 images
per slide were recorded for quantification. Vessel diameters were determined by dividing vessel
area by its length (Albrecht et al., 2010). All experimental groups which are summarized in one
graph were stained and imaged simultaneously with standardized threshold intensity. Positively
stained features are expressed as % marker expression compared to total tumor surface area in
one image (surface area %). Alternatively, co-localization was measured as fluorescence
intensity ratio between red and green channels or % overlay of red/green fluorescence.
RNA Analysis
RNA from snap frozen tumor tissue or cell lines was prepared using trizol (Life Technologies)
and qPCR was performed as previously described (Johansson et al., 2012). Gene expression was
normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT). Primer sequences:
ANG1: FP: TGGAAGAATGAAAGTGTAGGCACAT, RP: TTCTTCGCTGCCATTCTGACT;
CCL2: FP: TTAAAAACCTGGATCGGAACCAA, RP: GCATTAGCTTCAGATTTACGGGT;
CCL3: FP: CAAGCTTTCTCAGCGCCATATG, RP: ACGATGAATTGGCGTGGAAT; CCL4:
FP: CCCGAGCAACACCATGAAG, RP: AGCCCATTGGTGCTGAGAAC; Col1α: FP:
GCTCCTCTTAGGGGCCACT, RP: CCACGTCTCACCATTGGGG; Crbp1: FP:
CTGAGCAATGAGAATTTCGAGGA RP: GCGGTCGTCTATGCCTGTC; CXCL9: FP:
GAGGAACCCTAGTGATAAGGAATGC, RP: TCTTCAGTGTAGCAATGATTTCAGTTT;
CXCL10: FP: CCTTCACCATGTGCCATGC, RP:
TCTTACATCTGAAATAAAAGAGCTCAGGT; HPRT: FP:
ACACCTGCTAATTTTACTGGCAACA, RP: TGGAAAAGCCAAATACAAAGCCTA; IL2:
FP:TGAGCAGGATGGAGAATTACAGG, RP: GTCCAAGTTCATCTTCTAGGCAC; IL10:
FP: CCTCAGGATGCGGCTGAG, RP: GACACCTTGGTCTTGGAGCTTATT; IL12: FP:
CCCTGACCATCACTGTCAAAGA, RP: TTGTGGAGCAGCAGATGTGAGT; iNOS: FP:
GTTCTCAGCCCAACAATACAAG, RP: GTGGACGGGTCGATGTCAC; Myocardin: FP:
GATGGGCTCTCCAGATCAG, RP: GGCTGCATCATTCTTGTCACTT; PDGFB: FP:
AAGTTTAAGCACACCCATGACAAG, RP: ATTAAATAACCCTGCCCACACTCT; SM22α:
FP: CCTCGGAACTTCTCGGACAA, RP: TCTGCAACCCAATCACGTTCT; TIE2: FP:
TTTACAACAGCGTCTATCGGACTC, RP: TCGCCTCTCGACTTCCACAT; TGFβ: FP:
GCCCTTCCTGCTCCTCATG, RP: TTCTCTGTGGAGCTGAAGCAATAG. Primers for
calponin, IL1β and Myh11 were obtained from primer bank
(http://pga.mgh.harvard.edu/primerbank/).
Fluorescence Activated Cell Sorting (FACS) and in vitro Cell Stimulation
For FACS analysis, tumors were enzymatically digested (0.2 mg/mL collagenase P (Roche), 0.8
mg/mL dispase (Invitrogen), 0.1 mg/mL DNase I (Worthington)) in sterile filtered PBS in a
37°C waterbath for 25 min, at 150 rpm, and filtered through a 70 μm membrane. Digestion was
terminated by adding 3% FBS. Cells were resuspended in FACS buffer (PBS with 1% FBS).
Cells were analysed using a BD Accuri™ C6 flow cytometer, and sorted using the BD FACS
Aria II. For cell sorting, single cell suspensions were blocked with Fc block (CD16/CD32, clone
2.4G2, BD Pharmingen) for 15 min on ice. CD45+ CD68+ F4/80+ (all Biolegend) macrophages
were sorted from pools of tumors as previously described (Johansson et al., 2012). For ex vivo
stimulation, 1.3 x 105 macrophages were resuspended in growth factor reduced media (RPMI
supplemented with 0.5% FBS, glutamax, penicillin/streptomycin, sodium pyruvate) for 24 hours.
Media was collected and added with or without TGFβ neutralizing antibody (9016, R&D
Systems) or Rho kinase inhibitor Y27632 (10 µM, Calbiochem) to serum-starved 10T1/2 cells
(RPMI supplemented with 0.5% FBS). Recombinant murine TGFβ (R&D systems) was used as
positive control. After 24 hours, 10T1/2 cells were fixed in ice-cold aceton:methanol, or zinc
fixative for 5 min on ice and stained for caldesmon and myocardin, respectively, followed by
Cy3-conjugated secondary antibody. For LIGHT receptor analysis, cell suspensions were
blocked with Fc block and stained with biotinylated NG2 (1E6.4, Miltenyi) followed by
streptavidin brilliant blue 515 (BD Biosciences), CD45-PECy7 (30F-11, BD Biosciences),
PDGFRβ-APC (APB5, Biolegend), CD31-PECy7 (390, eBioscience) and either LTβR-PE
(ALY7, eBioscience) or HVEM-PE (LH1, eBioscience).
Western Blot Analysis
10T1/2 cells were serum starved for 24 hours followed by stimulation with LIGHT. Cells were
washed twice with PBS and lysed in RIPA buffer containing protease inhibitor cocktail (Roche).
Protein concentration was quantified using the BCA kit (Thermo Fisher Scientific). A total of 20
µg protein was separated on a 10% SDS-PAGE gel and transferred onto a PVDF membrane
(Millipore). The membrane was incubated with blocking buffer (20 mM Tris, 150 mM NaCl,
0.1% Tween 20, 5% milk powder, pH 7.6) for 1 h at RT, followed by incubation with primary
antibodies αSMA, calponin or caldesmon (see Immunohistochemistry) and tubulin (B-5-1-2,
Sigma) in blocking buffer overnight at 4 °C. Membranes were incubated with horseradish
peroxidase (HRP)-conjugated secondary antibodies, signals developed with enhanced
chemiluminescence solution (Thermo Fisher Scientific) and visualized using the ChemiDoc™
MP Imaging System (Bio-Rad).
Breast Cancer and Metastasis Assay
Murine, 6-thioguanine resistant 4T1 cells (ATCC) were injected orthotopically (5 x 106) into the
mammary fat pad of Balb/c mice. Once tumors were palpable, mice were injected bi-weekly i.v.
with 20 ng LIGHT-RGR in 100 µl PBS for 2 weeks. Prior to sacrifice mice were injected with
pimonidazole hydrochloride and FITC-lectin to assess hypoxia and vascular function,
respectively. Spontaneous metastases were measured following a published procedure (Pulaski
and Ostrand-Rosenberg, 2001). Briefly, after 2 weeks of LIGHT-RGR treatment, lungs were
removed from tumor-bearing mice, finely minced and digested in 1x HBSS containing 1 mg/ml
collagenase IV and 6 U/ml elastase for 75 min at 4 °C under gentle agitation. Digested cells were
washed, filtered and plated in serial dilutions on 10 cm culture dishes containing growth medium
and 60 µM 6-thioguanine. After 10 day, 6-thioguanine-resistant colonies were fixed in methanol,
stained with 0.03% (w/v) methylene blue and counted (colonies/lung).
SUPPLEMENTAL REFERENCES
Albrecht, I., Kopfstein, L., Strittmatter, K., Schomber, T., Falkevall, A., Hagberg, C.E., Lorentz,
P., Jeltsch, M., Alitalo, K., Eriksson, U., et al. (2010). Suppressive effects of vascular endothelial
growth factor-B on tumor growth in a mouse model of pancreatic neuroendocrine tumorigenesis.
PLoS One 5, e14109.
Pulaski, B.A., and Ostrand-Rosenberg, S. (2001). Mouse 4T1 breast tumor model. Curr Protoc
Immunol Chapter 20, Unit 20.2.
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