Regulation of the Tumor Microenvironment By High Molecular Weight Kininogen

Introduction

High molecular weight kininogen (HK) is a central component of the contact activation and kallikrein-kinin systems. We have previously reported that the cleaved, bradykinin-free form of HK (HKa) induces apoptosis of proliferating endothelial cells and inhibits angiogenesis, and that tumors grow larger and more quickly in kininogen deficient (mKng1-/-) mice. However, HK is known to interact with many different cell types, and the mechanism by which it inhibits tumor growth in vivo is uncertain. Here, we address this question by comparing protein expression and localization in wild type and mKng^1-/- mice harboring experimental tumors.

Methods

A syngeneic tumor model in which 1 x 10⁶ B16F10 melanoma cells cultured in log-growth phase were implanted subcutaneously in the flanks of wild-type and mKng1-/- mice was used in these studies. Tumor growth and animal condition were monitored daily. Parallel studies were performed in tumors from mKng^1-/- mice treated immediately prior to tumor implantation with a lentiviral vector that was either empty (control) or contained cDNA for murine Kng1. Mice were sacrificed approximately 17 days after tumor cell implantation, at which time the size of tumors in mKng^1-/- mice were ~ 1.5 cm, substantially greater than that in wild-type mice. Tumors were harvested at the time of sacrifice and processed for flow cytometry, immunohistochemistry, immunoblot and antibody array (Proteome Profiler, Angiogenesis Protein Array, RND Systems). Changes in protein levels seen on arrays were confirmed by immunoblotting.

Results

Tumors in mKng^1-/- mice grew more rapidly and had a volume 2-3-fold greater than those in wild-type mice by day 17; these tumors also demonstrated increased vascular density. Antibody array and immunoblot analysis demonstrated increased expression of several tumor and stroma associated proteins including MMP3, MMP9, VEGF, PlGF2, CD44 and MCP1 in tumors from mKng^1-/- mice; the same differences were observed between mKng^1-/- mice treated with an empty lentivirus when compared to mKng^1-/- mice in which kininogen expression was restored using lentivirus-HK. Cellular localization by immunohistochemistry and immunofluorescence staining further demonstrated expression of MMP3 and MMP9 primarily in the tumor compartment, while expression of VEGF was most prominent in the stroma. PLGF2 expression was highly localized to regions immediately surrounding tumor vasculature. Increased, diffuse expression of CD44, a stem cell marker, was evident in tumors from mKng^1-/- mice, localized primarily to the tumor compartment; increased CD44 expression was confirmed by flow cytometry and cell sorting, and may suggest increased numbers of tumor stem cells in tumors from mKng^1-/- mice, though expression of CD34⁺, FLK1⁺, and CD133⁺ was unaltered. Tumor associated macrophages were significantly reduced in tumors from mKng^1-/- mice as demonstrated by F4/80 immunostaining.

Conclusions

While the anti-angiogenic activity of HKa likely contributes to the increased growth of tumors in mKng^1-/- mice, these findings suggest that HK/HKa may also have profound effects on multiple cell types in the tumor microenvironment, including suppression of MMP, VEGF and PLGF2 expression. HKa may also inhibit the tumor stem cell compartment, though further characterization of the CD44 positive cell population in these tumors is needed. Additional studies are needed to better define the interactions of HK/HKa with these diverse cell types.