Af1320

Two days after the therapy, 3–4 mice from each experimental group were sacrificed and the tumors were excised. The tumors were fixed in IHC zinc fixative (BD Biosciences) and embedded in paraffin. Two consecutive 2-μm thick sections were cut from each paraffin block. The first section was used to estimate the percent of necrosis by 4 independent observers after staining with hematoxylin and eosin. The second section was used for immunohistochemical (IHC) staining of blood vessels. It was incubated with primary goat polyclonal antibodies against mouse CD105 (AF1320, R&D Systems, Minneapolis, MN, USA) at dilution 1:1800. As the colorogenic reagent, a peroxidase-conjugated streptavidin–biotin system (CTS008, Anti-Goat HRP-DAB Cell & Tissue Staining Kit, R&D Systems) was used and followed by hematoxylin counterstaining.
The IHC stained slides were observed under light microscopy and at least 5 images of viable tumor tissue from each slide were captured with a DP72 CCD camera (Olympus) connected to a BX-51 microscope (Olympus) under 40x magnification (numerical aperture 0.85). On the acquired images the number of CD105 positive blood vessels was determined.

Five micrometer mouse uterine sections were stained with haematoxylin and eosin (H&E) and stage of breakdown/repair graded by two masked independent observers using a previously published scoring system^{41 (link)} (Supplementary Fig. 2). Mouse uterine sections were also stained for endoglin (1:50, R&D Systems, AF1320, Abingdon, UK), alpha smooth muscle actin (αSMA, 1:250, C6198, Sigma, Dorset, UK), Ly6G (BioLegend, 127601, 1:1000. London, UK), F4/80 (Bio-Rad, MCA497GA, 1:50. Oxford, UK) and carbonic anhydrase IX (Abcam, ab184006, 1:2000, Cambridge, UK). Proliferating cells were identified using anti-bromodeoxyuridine antibody (BrdU, 1:1500, Fitzgerald, Acton, MA, USA) and hypoxia detected using anti-pimonidazole antibody as per manufacturer’s instructions (4.3.11.3 mouse MAb, Hypoxyprobe, Burlington, USA).

Brain tissue was fixed in 4% paraformaldehyde for 24 h at 4°C and then stored in PBS with 0.1% NaN₃. Next, the brain was embedded in 4% ultrapure low melting point agarose (16520050, Invitrogen) in PBS. The samples were cut into 40 µm coronal sections. Free-floating sections were stained with Lycopersicon Esculentum (Tomato) Lectin DyLight^TM 488 (1:1000, L32470, ThermoFisher). Mouse cardiac and liver tissue were fixed in 4% paraformaldehyde for 24 h at 4°C, paraffin embedded and cut into 4 µm (cardiac) or 7 µm (liver) sections. Cardiac sections were stained with Griffonia Simplicifolia Lectin I Isolectin B4 Dylight^TM 649 (1:100, DL-1208, Vector Laboratories). Liver sections were stained with primary Mouse endoglin CD105 (1:100, AF1320, R&D Systems) and secondary Cyanine 3 (Cy3 1:50, NEL704A001KT, Perkin Elmer) antibody. The eyes of the mouse were enucleated and fixed in 4% paraformaldehyde for 20 min at room temperature and then washed with PBS. The retina was dissected from the eye and further fixed in 4% paraformaldehyde for 24 h at 4°C. The free-floating retinas were stained with Griffonia Simplicifolia Lectin I Isolectin B4 Dylight^TM 649 (1:100, DL-1208, Vector Laboratories) and Collagen IV polyclonal antibody (1:200, 2150–1470, Bio-Rad) with secondary Rabbit IgG (1:400, A-31572, ThermoFisher).