Targeting the SHP2 phosphatase promotes vascular damage and inhibition of tumor growth

A, B

AMG386 (5.6 mg/kg s.c.; twice/week) and SHP099 (a: 200 mg/kg; b: 100 mg/kg orally/daily) were administered individually or together to B16F10 tumor‐bearing mice (A: control n = 7; SHP099: n = 8; AMG386: n = 8; SHP099+AMG386 n = 11; B: control, SHP099 and AMG386: n = 4; SHP099+AMG386: n = 8). Panels depict tumor growth from initiation of treatment to endpoint (left) and tumor weight (right) at endpoint; error bars ± SD; P values by analysis of variance with Dunnett’s multiple comparison test; **P < 0.01, ***P < 0.001.

C, D

p‐TIE2 in tumor vessels from control and AMG386‐treated mice; representative confocal images; arrowheads point to CD31⁺/p‐TIE2⁻ endothelial cells (scale bars:50 µm) (C), and quantification (D) of p‐TIE‐2/CD31⁺ in control (n = 3) and AMG386‐treated (n = 3) tumors (1,200 CD31⁺ cells/group). P values from two‐tailed Student’s t‐test; ***P < 0.001.

E, F

p‐EphrinB (E) in representative tumor vessels from control (n = 3) and AMG386‐treated (n = 3) tumors; confocal images (E), (scale bar: 50 µm) and quantification of p‐EphrinB/CD31⁺ (F); P values from two‐tailed Student’s t‐test; ***P < 0.001.

G

Vessel perfusion visualized by FITC‐dextran (green); CD31 (red) identifies the endothelium; DAPI (blue) identifies the nuclei; representative tumor confocal images (scale bar: 50 µm) of 3 mice/group; arrowheads point to FITC‐dextran extravasation; asterisks to extravasated erythrocytes.

H

Quantification of cleaved caspase‐3 in the tumor vasculature (CD31⁺ cells); n = 3 tumors/group; number of CD31⁺ cells counted: control (n = 311); SHP099 (n = 425); AMG386 (n = 451); combined SHP099+AMG386 (n = 484). Error bars: ± SD; P values by analysis of variance with Dunnett’s multiple comparison test; **P < 0.01, ***P < 0.001.

I

Quantification of tumor vascular area; n = 5/tumors group. Error bars: ± SD; P values by analysis of variance with Dunnett’s multiple comparison test; **P < 0.01, ***P < 0.001.

J

Quantification of “vascular sleeves” (CD31⁻Collagen IV⁺) n = 5/tumors group. Error bars: ± SD; P values by analysis of variance with Dunnett’s multiple comparison test; **P < 0.01, ***P < 0.001.

K, L

(K) % necrotic tumor area (n = 4/tumors group) and (L) % proliferating tumor cells (n = 3/tumors group) in control and treated groups. Error bars: ± SD; P values by analysis of variance with Dunnett’s multiple comparison test; *P < 0.05, **P < 0.01, ***P < 0.001.

M

Representative control and treated (n = 5/group) B16F10 tumor sections through the maximum diameter; H&E staining; (scale bars: 10mm).

N

Proliferating Ki67⁺ cells (green) in representative control and treated tumors (n = 3/group); CD31 identifies the vessels; DAPI identifies cell nuclei. Confocal images (scale bar: 50 µm).

Figure 6. SHP099 and AMG386 cooperatively reduce B16F10 tumor growth.