Cytokine Levels Correlate with Immune Cell Infiltration after Anti-VEGF Therapy in Preclinical Mouse Models of Breast Cancer

Abstract

The effect of blocking VEGF activity in solid tumors extends beyond inhibition of angiogenesis. However, no studies have compared the effectiveness of mechanistically different anti-VEGF inhibitors with respect to changes in tumor growth and alterations in the tumor microenvironment. In this study we use three distinct breast cancer models, a MDA-MB-231 xenograft model, a 4T1 syngenic model, and a transgenic model using MMTV-PyMT mice, to explore the effects of various anti-VEGF therapies on tumor vasculature, immune cell infiltration, and cytokine levels. Tumor vasculature and immune cell infiltration were evaluated using immunohistochemistry. Cytokine levels were evaluated using ELISA and electrochemiluminescence. We found that blocking the activation of VEGF receptor resulted in changes in intra-tumoral cytokine levels, specifically IL-1β, IL-6 and CXCL1. Modulation of the level these cytokines is important for controlling immune cell infiltration and ultimately tumor growth. Furthermore, we demonstrate that selective inhibition of VEGF binding to VEGFR2 with r84 is more effective at controlling tumor growth and inhibiting the infiltration of suppressive immune cells (MDSC, Treg, macrophages) while increasing the mature dendritic cell fraction than other anti-VEGF strategies. In addition, we found that changes in serum IL-1β and IL-6 levels correlated with response to therapy, identifying two possible biomarkers for assessing the effectiveness of anti-VEGF therapy in breast cancer patients.

Myeloid Derived Suppressor Cells and Their Role in Diseases

“…PGE2 is also often associated with inflammatory responses and tumour-related diseases [32,51,55]. Tumour cells were found to express PGE2, which can directly interact with HSCs through PGE2 receptors to induce proliferation into MDSCs, thus promoting tumour survival [32,51,55].…”

“…Tumour cells were found to express PGE2, which can directly interact with HSCs through PGE2 receptors to induce proliferation into MDSCs, thus promoting tumour survival [32,51,55]. PGE2 can also induce indirect MDSC proliferation and accumulation by inducing secretion of factors such as VEGF, cyclooxygenase 2 (COX2) and IL-6 [32].…”

The VEGF family in cancer and antibody-based strategies for their inhibition

“…Bleeding events are also a concern with angiogenesis and immune cell infiltration in vivo. [108][109][110] Research with 2C3 and r84 demonstrate that inhibition of VEGF-mediated VEGFR2 activation is sufficient to control tumor growth and calls into question the pro-tumorigenic function of VEGFR1. In addition, because r84 binds mouse and human VEGF, it facilitates evaluation of the effects of blocking tumor-derived (human) and stromal (mouse) VEGF in preclinical xenografts models.…”

“…A mouse chimeric version of r84 (mcr84) has been generated and is a useful tool for studying angiogenesis in immunocompetent animals and in syngenic tumor models. 109 To date, treatment of tumor-bearing mice with r84 has not induced anti-VEGF-related toxicities that have been characterized in other preclinical models using different inhibitors of the VEGF pathway. [111][112][113] Therefore, the potential efficacy of r84 as a cancer therapeutic for treating cancer patients is highly anticipated.…”

“…147 Macrophages in tumor bearing animals express VEGFR2, and blockade of this receptor has been demonstrated to reduce macrophage migration and infiltration in tumors. 60,109 Therefore, targeting VEGFR2 directly can negatively affect tumor growth and metastasis by reducing the population of tumor-promoting immune cells within the tumor microenvironment.…”