Effect of VEGF and VEGF Trap on vascular endothelial cell signaling in tumors

Abstract

Vascular endothelial growth factor (VEGF) A is a major promoter of tumor angiogenesis and a prime target of antiangiogenic cancer therapy. To examine whether endothelial cell signaling might provide histological biomarkers of angiogenesis and VEGF activity in vivo, normal mouse organs and multiple tumor models were studied immunohistochemically for endothelial expression of activated ERK, STAT3, and AKT. Phospho(p)-ERK and p-STAT3 expression was negligible in the endothelia of normal organs but was significantly elevated in tumor endothelium. p-AKT was present at significant and comparable levels in both tumor and normal endothelia. In K1735 tumors induced to express more VEGF, endothelial p-ERK, p-STAT3 and p-AKT increased accompanied by signs of accelerated angiogenesis. Treatment of K1735 and Colo-205 tumors with the VEGF inhibitor, VEGF Trap (aflibercept), decreased tumor endothelial p-ERK, p-STAT3 and p-AKT expression accompanied by signs of antiangiogenic effect. These results show that endothelial p-ERK and p-STAT3 (but not p-AKT) distinguish tumor from normal vessels and that the presence of these two signaling intermediates may be useful indicators of tumor angiogenic activity and angiogenesis inhibition by VEGF antagonist.

Figure 1

Induction of VEGF in K1735.VI4 tumor cells and effect of VEGF overexpression on MVD, tumor endothelial cell signaling and fate in vivo. K1735.M39 (parental), K1735.TRE (control) and K1735.VI4 (VEGF overexpressing) cells were exposed to DOX for 24 hours followed by VEGF analysis by ELISA performed on culture supernatants (A). K1735.M39, K1735.TRE and K1735.VI4 tumors were generated in C3H/HeN mice. DOX was added to their drinking water for 2 and 7 days, tumors were excised and VEGF levels in tumor lysates were assayed by ELISA (B). Paraffin embedded TRE Hygro and K1735.VI4 tumor sections were stained with anti-p-ERK (C), anti-p-STAT3 (D), anti-p-AKT (E) or anti-Ki67 followed by anti-CD34 staining. Microvessel densities were calculated and plotted (G) as were percentages of vessels that were positive for p-ERK (C), p-STAT3 (D), p-AKT (E) or Ki67 (F). Representative fields of CD34 staining in tumors not treated with doxycycline (left) or treated with doxycycline for 2 (middle) or 7 (right) days are shown at x200 magnification (H).

Figure 2

Inhibition of tumor growth and vasculature in VEGF-Trap treated K1735 and Colo-205 tumors. Female SCID mice bearing K1735 or Colo-205 tumors were subcutaneously injected twice a week with Fc or VEGF-Trap (25 mg/Kg) twice a week. Tumor growth was monitored and plotted as fold increase over the duration of therapy. Shown is the average volume fold increase for K1735 tumors (A) and Colo-205 (B) in each treatment group (± SD). Treated tumors with VEGF Trap or Fc, were excised after 3, 7 or 14 days of therapy for histological analysis. Tumor sections were stained with anti-CD34 antibody and microvessel densities (MVD) were calculated for K1735 tumors (C) and Colo-205 (D). Differences in MVD between the vehicle-treated and VEGF Trap-treated groups were analyzed by Student t-test. Representative fields of CD34 staining in tumors treated with vehicle (left) or treated with VEGF Trap (right) are shown at x200 magnification (E).

Figure 3

VEGF Trap induced necrosis in K1735 and Colo-205 tumors. Colo-205 (A) and K1735 (B) tumors after 7 and 14 days of vehicle or VEGF Trap treatment were stained with hematoxylin and eosin (x200).

Figure 4

VEGF Trap inhibits ERK, STAT3 and AKT activation in K1735 and Colo-205 tumors. Mice bearing K1735 or Colo-205 were treated sub-cutaneously with VEGF Trap (25 mg/Kg), or control Fc twice a week. The tumors were excised after, 3 days, 7 days and 14 days. Paraffin-embedded tumor sections were immunostained with anti-p-ERK, anti p-STAT3 and anti-pAKT, followed by anti-CD34 staining. Percentages of vessels p-ERK⁺ (A and B), p-STAT3⁺ (C and D) and p-AKT⁺ (E and F) were calculated and plotted in histograms. Differences were analyzed by Student's t-test.

Figure 5

VEGF Trap inhibits proliferation and promotes apoptosis in tumor vasculature. Paraffin-embedded sections of K1735 (A and C) or Colo-205 (B and D) tumors treated with VEGF Trap or control were immunostained for Ki67 or TUNEL followed by anti-CD34 staining to show proliferative and apoptotic vessels, respectively. Percentages of vessels that were Ki67⁺ (A and B) or TUNEL⁺ (C and D) were calculated and plotted. Differences were analyzed by Student's t-test.