Treatment of colorectal and hepatocellular carcinomas by adenoviral mediated gene transfer of endostatin and angiostatin-like molecule in mice

Abstract

Aim and method: In this study, we explored the responsiveness of different tumour entities (colorectal carcinoma (CRC), hepatocellular carcinoma (HCC), and the murine Lewis lung carcinoma (LLC)) to angiostatic antitumour treatment with two recombinant adenoviral vectors encoding angiostatin-like molecule (AdK1-3) and endostatin (Adendo).

Results: AdK1-3 and Adendo exerted inhibitory biological functions on endothelial cell proliferation, migration, and tube formation in vitro. AdK1-3 inhibited significantly endothelial cell infiltration in vascular endothelial growth factor embedded Matrigel plugs in mice whereas Adendo showed only minor effects. Both AdK1-3 and Adendo induced similar antitumour effects in the LLC tumour model in immune competent C57BL/6 mice but AdK1-3 had stronger inhibitory effects in athymic mice. Furthermore, AdK1-3 inhibited tumour growth in a murine CRC and human HCC model but was ineffective in a human CRC model. In contrast, Adendo did not reduce tumour progress in either of these tumour models although AdK1-3 and Adendo effectively reduced intratumoral microvessel density in LLC tumours.

Conclusion: Our data demonstrate that angiostatic gene therapy may form a feasible strategy for the treatment of established hepatocellular carcinomas and that in vivo antitumour efficacy of angiostatic proteins is tumour specific.

Figure 1

Western blot analysis for angiostatin (A) and ELISA for endostatin (B). Culture medium (CM) from uninfected and vector infected cells was subjected to western blot and ELISA, as described in the methods section. Lane 1, marker; lane 2, CM from cells infected with AdK1-3; lane 3, recombinant plasminogen. Angiostatin fragments were detected at 38 kDa in CM from AdK1-3. Endostatin levels are presented after infection of A549 cells at different multiplicities of infection. (B) In vitro data and (C) in vivo data are shown, seven days after systemic administration of Adendo (10¹⁰ plaque forming units/mouse; pooled serum of two Balb/c mice).

Figure 2

Inhibition of human umbilical vein endothelial (HUVE) cell proliferation (A), migration (B), and tube formation (C) by angiostatin-like molecule and endostatin expressed by adenoviral vectors. Data are given as mean (SEM) of 4–8 independent experiments. *p<0.05 (AdK1-3 and Adendo) compared with controls (control and Adluc). (B) Inhibition of HUVE cell tube formation by angiostatin expressed by adenoviral vectors. HUVE cells were preincubated with culture medium for 30 minutes in Matrigel coated culture wells and grown in culture medium supplemented with growth factor for an additional six hours. At this time point, HUVE cells started to form capillary-like structures. Intact tubes were quantified in the whole well under a microscope. Data are given as mean (SEM) of two independent experiments compared with control vector AdlacZ.

Figure 3

Suppression of vascular endothelial growth factor (VEGF) induced angiogenesis in vivo by systemic administration of Adendo and AdK1-3. Matrigel containing VEGF was injected subcutaneously into mice that had received intravenous administration of different adenoviruses six hours previously. After 14 days, Matrigel plugs were removed and stained with haematoxylin-eosin. Quantitative analysis of angiogenesis was made by counting the number of endothelium-like cells in an area (mm²). Data are expressed as mean (SEM), n = 4. *p<0.05 compared with control animals who received AdlacZ or saline (unpaired Student’s t test).

Figure 4

Inhibition of tumour growth on established Lewis lung carcinoma (LLC) tumours in C57BL/6 and athymic mice by systemic administration of AdK1-3 or Adendo. Established LLC tumours of approximately 50 mm³ in C57BL/6 mice (A) and athymic mice (B) were treated by intravenous injection of recombinant adenoviruses (10¹⁰ plaque forming units/mouse) or the control vector AdlacZ or saline. Tumour sizes were measured and are presented as mean (SEM), n = 7–13. *p<0.05 for AdK1-3 and Adendo (A) and for AdK1-3 (B) compared with the AdlacZ control (day 4, Mann-Whitney).

Figure 5

Inhibition of tumour growth in athymic mice with hepatocellular carcinoma tumours by systemic administration of AdK1-3 or Adendo. Ten days after implantation of Huh7 cells, mice were treated by intravenous injection of recombinant adenoviruses (10¹⁰ plaque forming units/mouse) or saline as control. Size of tumour was measured and presented as mean (SEM), n = 6–10. *p<0.05 for AdK1-3 day 3–day 13 compared with the control groups who received AdlacZ (Mann-Whitney).

Figure 6

Inhibition of tumour growth on established colorectal cancer (CRC) tumours by systemic administration of AdK1-3 or Adendo. Intratumoral vector administration (5×10⁹ plaque forming units/mouse) was initiated when tumours reached approximately 5 mm in mean diameter. Data are presented as mean (SEM), n = 7–11. *p<0.05 for AdK1-3 day 4–day 12 compared with the AdlacZ control (Mann-Whitney).

Figure 7

Inhibition of tumour angiogenesis by treatment with AdK1-3 or Adendo. Established Lewis lung carcinoma tumours of approximately 50 mm³ in athymic mice were treated by intravenous injection of recombinant adenoviruses (10¹⁰ plaque forming units/mouse) or saline as control. Animals were sacrificed on day 7 after treatment and tumour tissue was removed. Microvessel density was determined by anti-von Willebrand factor staining. Quantitative analysis of microvessel density was made by counting the positively stained cells in 10 high power fields (HPF, 400× magnification). Data are given as mean (EM) cell number/HPF, n = 3. *p<0.05 for Adendo compared with AdlacZ (Student’s t test).