Innate Immune Cells: Mediators of the Angiogenic Switch?

The role of Matrix metalloprotease type 9 (MMP-9) in the activation of Vascular Endothelial Growth Factor (VEGF), the induction and maintenance of chronic angiogenesis and early stage tumor growth has been well established. But what is the source of MMP-9 ? In a study published in 2006, Nozawa and co-workers, using the RIP1-Tag2 transgenic mouse as a model, identified two inflammatory cell types, neutrophils and macrophages, as the major sources of MMP-9.

The RIP1-Tag2 transgenic mouse is a well characterized model of multistep carcinogenesis involving the pancreatic islets. Nozawa and co-workers first isolated constituent cell types from a tumor and cell-sorted Gr-1⁺ and Mac-1⁺ cells and then carried out a semi-quantitative RT-PCR (see Fig.1)

Fig. 1: Semi-quantitative RT-PCR. MMP-9⁺ is expressed by Gr-1⁺ and Mac-1⁺cells innate immune cells (Adapted from Nozawa et. al, 2006)

Clearly, MMP-9 was expressed predominantly by innate immune cells positive for the Gr-1 and Mac-1 markers.

In order to determine the localization of these cells , a double label immunohistochemical staining of various leukocyte markers in normal and neoplastic tissue was performed. CD68 and F4/80 are two macrophage markers. Cells positive for both markers were observed inside as well as on the periphery of angiogenic islets and tumors (see Fig. 2). However, the cells inside were MMP-9^- while those along the periphery were MMP-9⁺ 

Fig. 2: MMP-9⁺ macrophages located along the periphery of the angiogenic islet.Macrophages inside the islet were MMP-9^- (Adapted from Nozawa et. al, 2006)

Along similar lines, an antibody against the "7/4" (a neutrophil marker) was used to localize neutrophils. Suprisingly, the situation, this time, was the opposite. 7/4⁺ cells inside the lesions were also MMP-9⁺ while 7/4⁺ cells along the periphery were not (see Fig. 3). All  7/4⁺ cells also displayed polymorphic nuclei, strongly indicating that these cells were neutrophils.

Fig. 3: MMP-9⁺ neutrophils located inside the angiogenic islet. Neutrophils along the periphery of the islet were MMP-9^- (Adapted from Nozawa et. al, 2006)

Neutrophils (7/4⁺ and Gr-1⁺) represented only 0.4% of the total tumor cells and expressed high levels of MMP-9. In contrast, macrophages (CD68⁺) represented 2% of the total tumor cells and expressed low levels of MMP-9.

The researchers then tried to determine the role of these MMP-9⁺ Gr-1⁺ cells that infiltrate angiogenic islets and tumors. An experimental regimen involving daily inoculation of anti-Gr-1 at 7 weeks (when hyperplastic islets start to undergo angiogenesis) was followed.

After 1 week of injection, most neutrophils disappeared from the pancreatic islets of the transgenic mice (see Fig.4)

Fig. 4: Left: Control; Right: Following 1 week of treatment with anti-Gr-1, MMP-9⁺ neutrophils located inside the angiogenic islet disappeared (Adapted from Nozawa et. al, 2006)

After two weeks of injection, the neutrophil population rebounded in neoplastic islets. Incredibly, none of the rebound neutrophils were MMP-9⁺ (see Fig. 5)!

Fig. 5: Left: Control; Right: Following 2 weeks of treatment with anti-Gr-1, neutrophils rebounded back into the islets. However, none of the rebound neutrophils were MMP-9⁺  (Adapted from Nozawa et. al, 2006)

In a short 2 week prevention trial (week7-week9), carried out to assess angiogenic switching frequency, the anti-Gr-1 antibody regimen, reduced the number of angiogenic islets by 57%!

Furthermore, immunostaining for VEGF:VEGF-R2 complex with GVM39 (red) and for endothelial cells with Meca-32 (green) showed a decrease in bioactive VEGF:VEGF-R2 interaction, consequent to the depletion of infiltrating MMP-9⁺ neutrophils (see Fig.6)

Fig. 6: Left: Control; Right: Short intervention trials lead to reduced VEGF:VEGF-R2 interactions. Endothelial cells are stained with an antibody directed against Meca-32 (a pan-endothelial cell marker)  (Adapted from Nozawa et. al, 2006)

Taken together, the results from this paper indicate that subtle infiltration by innate immune cells (such as neutrophils) may play a role in the progression of neoplasias towards angiogenic tumors.

Reference:

Nozawa H, Chiu C, & Hanahan D (2006). Infiltrating neutrophils mediate the initial angiogenic switch in a mouse model of multistage carcinogenesis. Proceedings of the National Academy of Sciences of the United States of America, 103 (33), 12493-8 PMID: 16891410