Fig 2: LIF is induced by KAI1 through Src/p53 axis in PCs. a Src phosphorylation levels and positions and Lif immunoblotting results in left, WT primary PC and Kai1-/- primary PC and, right, WT 10T1/2 and Kai1-O/E 10T1/2. b Quantification of the obtained results (ns not significant, *p < 0.01, **p < 0.05, Means ± SEM, 3 technical replicates). c Top, scheme, showing the regions targeted with the chromatin immune-precipitation (ChIP) assay and mutagenesis. Bottom, binding of p53 and Pbx1 on Lif gene-regulating element or promoter region using ChIP assay. d Activation of mouse Lif gene-regulating element or promoter in Kai1-O/E 10T1/2 cells using luciferase assay. ns not significant, *p < 0.01, **p < 0.05, ***p < 0.001

Fig 3: Therapeutic potential of KAI1 to inhibit tumor angiogenesis and growth. a Left, In vitro matrigel tube formation assay using cellular spheroid consisting of MS1, 10T1/2 (WT and Kai1-KD), and B16 (melanoma cancer cell line) cells treated with 40 ng/mL of rhVEGF-A following the treatment with rhKAI1 (800 ng/mL). Scale bar, 100 µm. Right, Quantification of the obtained results (*p < 0.01, **p < 0.05, ***p < 0.001, Means ± SEM, 3 technical replicates). b Overview of experiment design applied to ELISA and western blot. c ELISA assay measuring the levels of mouse Lif and mouse Vegfa secreted into the media of three-hybrid spheres consisting of cancer (melanoma cells) and blood vessel cells, namely primary EC, as well as primary PC transfected with a mock vector (Group 1) and PC overexpressing Kai1 (Group 2). d Left, Vegfr2 phosphorylation levels and Kai1 immunoblotting results in group 1 and 2. Right, quantification of the obtained results (*p < 0.01, **p < 0.05, Means ± SEM, 3 technical replicates)

Fig 4: Therapeutic potential of KAI1 and peptide to inhibit angiogenesis in tumors and prevent OIR-induced neovascularization. a B16 cells were subcutaneously injected in C57BL/6 mice with or without KAI1 supplement [combination of [1] Kai1-O/E 10T1/2 supernatant to provide anti-angiogenic LIF and [2] rhKAI1 to directly bind and inhibit VEGF and PDGF]. Left, tumor growth images are presented. Scale bar, 5 mm. Right, quantification of the obtained results (Means ± SEM, 3 biological replicates). b PC3 (human prostate cancer cell line) cells were subcutaneously injected into NSG mice in combination with or without KAI1 supplement. Left, tumor growth images are presented. Scale bar, 5 mm. Right, quantification of the obtained results (Means ± SEM, 5 biological replicates). c CD31 (red) immunostaining, and DAPI (blue) staining of human prostate (PC3) tumors treated with or without KAI1 supplement. Scale bar, 100 µm. d Schematic figure of dual anti-angiogenic effect of KAI1 in tumors. e Neonatal C57BL/6 mice with OIR at postnatal day 15 (P 15) were treated or not treated with a single intravitreal injection (1 µL) of vehicle only [PBS] (n = 4 retinas), rhKAI1 (600 ng), wild-type peptide KAI WT (200 ng) (n = 4 retinas), or mutant peptide KAI M (200 ng) (n = 4 retinas). Whole-mount retinas were stained with isolectin-B4 conjugated to Alexa Fluor 594 red-fluorescent dye. Top, Representative confocal microscopy images of retinas of OIR neonatal C57BL/6 mice at P17. Bottom, quantification of neovascularization in the retinas of OIR neonatal C57BL/6 mice treated or not treated with rhKAI1, KAI WT or KAI M peptides. Scale bar, 500 µm. f Mice were injected with matrigel and MDA-MB 231 cells in the presence of PBS alone (vehicle) or 200 ng KAI WT or M peptide (n = 5). After 23 days, the plugs were removed and the angiogenic responses were evaluated. Top, tumor volume was presented and bottom, representative IF images of tumors are shown. g Quantitative results of tumor volume (left) and CD31 intensity (right). Endothelial cells were visualized with CD31 (red) immunostaining, and DAPI (blue) staining of human breast cancer (MDA-MB231) treated with KAI WT or KAI M peptides. Scale bar, 20 µm. ns not significant, *p < 0.01, **p < 0.05, ***p < 0.001

Fig 5: KAI1 was predominantly expressed in PCs rather than in ECs, and KAI1 expressed in PC has anti-angiogenic effects. a Left, retinal vasculature of the wild-type (WT) and Kai1 knockout (Kai1-/-) mice (postnatal day 2 and 5 days of age). Endothelial cells were stained with BS-I lectin. Scale bar, 100 µm. Right, quantification of the obtained results (Means ± SEM, 4–9 technical replicates). b Left, filopodia of retinal vessels in WT and Kai1-/- mice (postnatal day 4). Scale bar, 20 µm. Right, quantification of the obtained results (*p < 0.01, **p < 0.05, Means ± SEM, 3 or 4 technical replicates). c Tracking of Kai1 expressing cells in retina of the Kai1-GFP fusion knock-in mice. Confocal images of isolectin B4 (IB4, white), NG2 (red). Right, high-magnification images of the boxed area in the left panel Kai1 (green) was expressed in NG2 positive PCs (Arrow), but not ECs (Arrowhead). Asterisk indicates Kai1-expressing leukocyte or erythrocyte in vessel. Scale bar, 5 µm. d Kai1 mRNA expression in mouse primary ECs and cell lines (MS1, endothelial cell line), and mouse primary PCs and cell line (10T1/2, pericyte cell line). **p < 0.05. e KAI1 protein expression in the mouse primary ECs, mouse primary PCs, human cell lines, HUVECs and HBVPs. *p < 0.01. f Left, in vitro co-culture matrigel tube formation assay using GFP expressing mouse ECs (MS1) co-cultured with Kai1-/- or WT mouse primary PCs. Scale bar, 100 µm. MS1 cells were transduced by lentivirus encoding GFP. Right, quantification of the obtained results (Means ± SEM, 7 technical replicates). g Left, In vitro matrigel tube formation assay, performed using co-culture spheroids, consisting of primary ECs and PCs, transduced with Myc-Kai1 vector and observed using time-lapse microscopy at the indicated time, 0 and 107 h. Scale bar, 50 µm. Right, quantification of the obtained results (***p < 0.001, Means ± SEM, 3 technical replicates). h Left, in vitro tube formation assay in MS1 and 10T1/2 co-cultured cells, transduced with the indicated adenovirus. Scale bar, 200 µm. Right, quantification of the obtained results (*p < 0.01, **p < 0.05, ***p < 0.001, Means ± SEM, 4 technical replicates)