1
Supplementary Materials and Methods
HSTS-26T human sarcomas and T-241 murine fibrosarcomas were injected as
single cell suspensions subcutaneously above the distal aspect of the gastrocnemius
muscle. Mice were injected once intraperitoneally with 1 µg of diphtheria toxin in 0.3
ml of PBS or with 0.3 ml of PBS alone as a negative control.
To identify functional blood vessels, biotinylated Lycopersicon esculentum lectin
(Vector Laboratories, Inc., Burlingame, CA) was injected intravenously (10 µg/gram
body weight). After 5 minutes, animals were sacrificed by perfusion fixation1. To
identify functional lymphatic vessels, ferritin microlymphangiography was performed
according to published methods2. Ferritin was detected by a 40 minute incubation in a
solution of 10% HCl and 5% potassium ferrocynate. Immunohistochemistry (IHC) for
lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) (ref. 3) was performed
according to published methods1. The anti-LYVE-1 antibody4 was the generous gift of
Dr. Erkki Ruoslahti of The Burnham Institute (La Jolla, CA). Biotinylated Lycopersicon
esculentum lectin was detected by Vector Elite ABC Kit. IHC for MECA-32 (BD
Biosciences Pharmingen, San Diego, CA; 1:200 dilution), -smooth muscle actin (SMA, DakoCytomation, Glostrup, Denmark; 1:400), proliferating cell nuclear antigen
(PCNA) (DAKO ready-to-use, 1:2), and collagen IV (Chemicon, Temecula, CA; 1:500)
was performed utilizing microwave antigen retrieval (DakoCytomation) and Envision
Plus Kit (DakoCytomation). Apoptosis (TUNEL) staining was performed using
ApopTag Peroxidase In Situ Apoptosis Detection Kit (Chemicon). IHC for collagen I
(LF-67 antibody5; the generous gift of Dr. Larry Fisher, National Institute of Dental
Research, Bethesda, MD; 1:100) was performed using heat antigen retrieval
(DakoCytomation), 0.5% trypsin retrieval, and Envision Plus Kit (DakoCytomation).
Northern blots for the molecular determinants of blood and lymphatic vessel
formation and remodelling were performed by fractionating total RNA (15 g/lane) on
a 1.0% denaturing formaldehyde/agarose gel, transferring to a nylon Gene Screen Plus
Hybridization Transfer Membrane (PerkinElmer Life Sciences, Boston, MA), and
UV-cross-linking with a UVCrosslinker (Fisher Scientific, Pittsburgh, PA). The cDNA
probes were synthesized by PCR (murine vascular endothelial growth factor (VEGF)
2
primers: forward, 5’-GTACCTCCACCATGCCAAGT-3’; reverse, 5’GCATTCACATCTGCTGTGCT-3’; murine VEGF-C primers: forward, 5’CAAGGCTTTTGAAGGCAAAG-3’; reverse, 5’-TGCTGAGGTAACCTGTGCTG-3’;
murine VEGF receptor (VEGFR)-2 primers: forward, 5’GCTTTCGGTAGTGGGATGAA-3’; reverse, 5’-GGAATCCATAGGCGAGATCA-3’;
human VEGF primers: forward, 5’-AAGGAGGAGGGCAGAATCAT-3’; reverse, 5’TTCTTGCGCTTTCGTTTTT-3’; human VEGF-C primers: forward, 5’CTACAGATGTGGGGGTTGCT-3’; reverse, 5’-CATCCAGCTCCTTGTTTGGT-3’;
murine -actin primers: forward, 5’-TGTATGCCTCTGGTCGTACC-3’; reverse, 5’CCACGTCACACTTCATGATGG-3’). Hybridization probes were prepared with the
Gene Images AlkPhos Direct Labelling and Detection System (Amersham Biosciences,
Piscataway, NJ). Blots were hybridized overnight at 55-60 C, detected with CDP-Star
detection reagent (Amersham Biosciences, Piscataway, NJ), and exposed on Pegasus
Blue Sensitive Film (Pegasus Scientific, Inc., Burtonsville, Maryland). The relative
expression levels in diphtheria toxin (DT)-treated and PBS-treated tumours were:
murine VEGF-A DT/PBS ratio = 1.3; murine VEGF-C DT/PBS ratio = 1.0; murine
VEGFR-2 DT/PBS ratio = 1.6; human VEGF-A DT/PBS ratio = 0.7; human VEGF-C
DT/PBS ratio = 1.0. Expression of angiopoietin-1 was near the detection limit in both
diphtheria toxin- and PBS-treated tumours. IHC showed no difference in the pattern of
angiopoietin-2 staining.
To assess vascular function intravitally, angiography and lymphangiography were
performed. Fluorescence microlymphangiography was performed by injecting
tetramethylrhodamine dextran (2 million MW) into the tumour at low pressure6.
Angiography was performed using intravenous injection of FITC dextran (2 million
MW)7. The vessels were imaged using multiphoton laser scanning microscopy7,8 and
vascular parameters quantified. Both intravital and ex vivo assessments of vascular
function were used to determine that there was an increase in the number and diameter
of perfused blood vessels and lack of functional lymphatics after diphtheria toxin
treatment.
To quantify vascular morphology, digital images were taken from
immunohistochemistry sections and vessels (MECA-32- or LYVE-1-positive structures)
3
were hand-traced using NIH Image software. Morphological parameters were measured
from these tracings. Tumour margin lymphatics were classified as those located within
150 m of the surface of the skin overlying the tumor2,6. Collagen I area was quantified
by taking digital images and determining the proper intensity threshold using NIH
Image software (method developed by Dr. Lance L. Munn, Massachusetts General
Hospital, Boston, MA). Cellular density was determined by manually counting cells.
The aspect ratio—the ratio of maximum to minimum diameters—of stained vessels was
calculated. The larger the aspect ratio, the more compressed the vessel. Endothelial cells
were rarely TUNEL- or PCNA-positive in these tumours. Published mathematical
models9,10 predict lower levels of solid stress in the tumour margin than in the tumour
centre. The fraction of LYVE-1-positive structures with open lumen in the tumour
margin was significantly greater than in the tumour itself. The aspect ratio of
LYVE-1-positive structures was also significantly smaller in the tumour margin than
inside the tumour.
All experiments were performed under the guidelines of the Massachusetts
General Hospital Institutional Animal Care and Use Committee. Supported by NIH
(Bioengineering Research Partnership Grant R24-CA85140). For further details on the
content of this report, please contact the authors.
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