TβRIIfl/fl mice  were crossed with Prox1-CreERT2 mice  to generate TβRIIfl/fl; Prox1-CreERT2 mice. Tamoxifen (Tx) (Sigma-Aldrich, St. Louis, MO T5648) dissolved in corn oil (20 mg/mL) was intraperitoneally administrated to mice (40 mg/kg/day) for consecutive 5 days into control (TβRIIfl/fl) mice and TβRIIfl/fl; Prox1-CreERT2 mice, where TβRII gene in LECs is deficient (TβRII iΔLEC). For embryonic analysis, pregnant mice were administrated with Tx (40 mg/kg/day) 10.5 and 11.5 days after mating. Then, mice were sacrificed to analyze embryos at E13.5. The lymphatic structures of adult mouse ear and tail were examined 4 weeks after the first injection of Tx. The mice were housed in the animal facilities of the Laboratory Animal Resource Center at the Tokyo University of Pharmacy and Life Sciences under specific pathogen-free (SPF) conditions at constant temperature and humidity and fed a standard diet. Treatment of the mice was in accordance with the institutional guidelines of the Animal Care and Use Program of the Tokyo University of Pharmacy and Life Sciences (L18-3, L19-18, L20-5).
Establishment LLC cells expressing eGFP
LLC cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nakalai Tesuque, Kyoto, Japan) containing 10% fetal calf serum (FCS; Invitrogen, Carlsbad, CA, USA), 1× MEM nonessential amino acids (NEAA; Sigma-Aldrich) and 100 U/mL penicillin/streptomycin (FUJIFILM Wako, Osaka, Japan) . To obtain the eGFP stable transformants, LLC cells were co-transfected with pEGFP-N1 (GenBank Accession #U55762) and CS-CDF-CG-PRE (RDB04379) including the Zeocin-resistant gene. For selection of stable transformants, LLC cells were maintained in DMEM containing 600 mg/mL of Zeocin (Invitrogen).
In vivo lymphatic drainage of FITC-dextran
Four weeks after Tx administration, male TβRIIfl/fl and TβRII iΔLEC mice (7-12 weeks old) were subcutaneously injected with 50 μl of 10 mg/mL FITC-dextran (MW 2,000,000, Sigma-Aldrich, FD2000S) into their rear footpads. Five minutes later, the transport of FITC-dextran was visualized with MVX10 fluorescence stereomicroscope (Olympus, Tokyo, Japan).
In vivo xenografts
Male TβRIIfl/fl and TβRII iΔLEC mice (7–12 weeks old) were subcutaneously implanted with LLCs (2.5 × 105 cells) in 100 μl of PBS. Tumor volumes (V) were calculated using the following formula: length × width × width × 0.5 . The tumors were surgically removed and embedded into a frozen section compound (Leica). Lymphatic metastasis was measured by the expression of eGFP when LLC-GFP cells implanted into mouse footpad were metastasized. In brief, the legs were clarified with CUBIC 1 week after the LLC-GFP transplantation  and tumor metastasis to the popliteal lymph nodes (PLN) was evaluated with the fluorescent stereomicroscope (Olympus).
Immunofluorescence analysis and quantification
Antibodies were obtained from the following sources: Rabbit polyclonal anti-LYVE-1 (ab14917) and anti-Ki-67 (ab15580) antibodies from Abcam (Cambridge, UK); rat monoclonal anti-PECAM-1 (550274) and anti-VE-cadherin (550548) antibodies from BD Transduction Laboratories (Franklin Lakes, NJ); goat anti-VEGFR3 antibody (35917) from R&D systems (Minneapolis, MN). Ear skins from adult mice were dissected, fixed in 4% paraformaldehyde (PFA) in PBS for overnight at 4 °C, and washed three times with PBS. After connective tissues and hairs were removed, the samples were permeabilized in 0.1% Triton X-100 (Sigma-Aldrich) and incubated in blocking reagent (Dako, Glostrup, Denmark) before staining. The tumors and 1 cm from buttock of the tail were surgically removed and embedded into a frozen section compound (Leica). Fresh frozen sections (5 μm) were cut with a CM1850 cryostat (Leica Camera AG, Wetzlar, Germany), mounted on Cryofilm (Leica), and fixed in 100% ethanol and 4% PFA. The films were washed three times with PBS, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with Blocking Reagent for 1 h at 37 °C. First antibodies in Blocking Reagent were added and incubated overnight at 4 °C. The films were washed three times with PBS and then incubated with Alexa488-conjugated donkey anti-rabbit IgG (A21206, Thermo Fisher Scientific, Waltham, MA), Alexa488-conjugated goat anti-rabbit IgG (A11008, Thermo Fisher Scientific), Alexa594-conjugated goat anti-rat IgG (A21208, Thermo Fisher Scientific) antibody, Alexa594-conjugated donkey anti-rat IgG (A21209, Thermo Fisher Scientific) antibody, or Alexa647-conjugated donkey anti-goat IgG (A11058, Thermo Fisher Scientific) at 1:200 for 1 h at room temperature . After the nuclei as needed were stained with 2 μg/mL DAPI (Dojindo Laboratories, Kumamoto, Japan) for 5 min, the samples were washed three times with PBS, and the fluorescence signals were visualized by BZ-9000 fluorescence microscope (Keyence, Osaka, Japan). For mouse ear skin, the LYVE-1-positive area and the number of branches were analyzed with 12 μm2 image from each of the 4 mice used. For tumor sections, the immunostaining-positive area was measured using three independent images at 10x magnification from each mouse (3 control and 6 TβRIIiΔLEC mice). For tail sections, the immunostaining-positive area was measured using one image including tissues around the artery at × 10 magnification from each of the 4 mice. These analyses were carried out using BZ-X analyzer imaging software (Keyence) or ImageJ. The analysis of proliferative lymphatic endothelial cells from each mouse (n = 3) were measured the rate of Ki-67-positive cells from at least 100 VEGFR3-positive LECs per mouse.
RNA isolation and RT-PCR
Total RNA was extracted using a NucleoSpin® RNA Plus kit (Takara Bio Inc., Shiga, Japan). Reverse transcription was performed with a PrimeScript II 1st strand cDNA Synthesis Kit (Takara). qPCR was performed with a KAPA SYBR Fast qPCR kit (Nippon Genetics, Tokyo, Japan). All reactions were carried out on a LightCycler®96 (Roche). Each sample was analyzed in triplicate at least twice for each PCR measurement. Melting curves were checked to ensure specificity. Relative quantification of mRNA expression was calculated using the standard curve method with the GAPDH level. Before qPCR, the DNA fragment amplified using each primer set was detected to be a single band with the correct size by agarose gel electrophoresis. The following primer sets were used to amplify cDNAs; 5′-CAGCCCACCCTCAATACCAG-3′ and 5′-AGAAGGTGTTTGTGGCTGCT-3′ for mouse VEGF-C , 5′-TGCAGTGGCAAAGTGGAGATT-3′ and 5′-TGCCGTTGAATTTGCCGT-3′ for mouse GAPDH .
Numerical results were expressed as means ± standard deviation. Significance was assessed using the unequal variances t test and the chi-square test. Probability values below 0.05 were considered significant.