Role of macrophages in breast cancer angiogenesis in vivo

Background

The objective was to establish a murine model to study the role of macrophages in the initiation of angiogenesis by human breast tumour spheroids in vivo. Despite the increasing body of evidence, both experimental and clinical, implicating macrophages in breast tumour angiogenesis, there have been no previous in vivo studies demonstrating proangiogenic tumour activity.

Methods

Human breast tumour spheroids (600 μm) were infiltrated with human monocytes in vitro, allowed to differentiate into macrophages, coated with alginate to isolate from the host (murine) cells and implanted into dorsal skin-fold chambers on nude mice. The resultant angiogenesis surrounding the spheroids infiltrated with human macrophages prior to implantation was quantified using image analysis (Angiosys), and compared with that induced by spheroids consisting of tumour cells alone.

Results

The presence of macrophages resulted in at least a threefold upregulation in the release of vascular endothelial growth factor (VEGF) in vitro when compared with spheroids composed only of tumour cells. A homogeneous distribution of macrophages surrounding the hypoxic centre was observed in the majority of spheroid sections assessed. The angiogenic response measured around the spheroids 3 days after in vivo implantation was significantly greater in the spheroids infiltrated with macrophages; the number of vessels increased (macrophages vs no macrophages, 34 ± 1.9 vs 26 ± 2.5, P < 0.01), and were shorter in length (macrophages vs no macrophages, 116 ± 4.92 vs 136 ± 6.52, P < 0.008) with an increased number of junctions (macrophages vs no macrophages, 14 ± 0.93 vs 11 ± 1.25, P < 0.025), all parameters indicative of new vessel formation. By day 7 no significant differences were seen. Viable human but no murine macrophages were identified in the tumour spheroids at the end of the study, using immunohistochemistry.

Conclusion

This is the first in vivo study to demonstrate that macrophages modulate breast tumour angiogenesis, in the early stages of development, with an increased number of vessels and branches.