Retinoblastoma Tumor Burden: Effect of Macrophage Depletion on LHBETATAG Retinal Tumor Growth

Abstract

Purpose: : Macrophages are an integral part of the tumor microenvironment with supporting roles in tumor growth and angiogenesis. Since the eye is an immune priviledged site, they may behave differently in retinoblastoma tumors. The purpose of this study is to analyze the effect of macrophage depletion on LH_BETAT_AG retinal tumor growth.

Methods: : This study was approved by the IACUC and follows ARVO guidelines. Macrophage depletion was performed by subconjuctival delivery of chlodronate-encapsulated liposomes to 10-week-old mice. Post-injection, chlodronate is activated into a suicide toxin causing the depletion of the macrophages. Animals (n=7) were treated bi-weekly for 6 weeks. Eyes were analyzed at 16 weeks of age. The control group (n=7) received subconjunctival administrations of PBS-encapsulated liposomes. The effective depletion of macrophages was confirmed by flow cytometry of circulating monocytes and immunohistochemistry. Macrophages were detected with F4/80 or IBA-1 and M2 polarized macrophages were detected with CD-163.

Results: : Macrophages significantly decreased (p=0.0366) in the chlodronate treated group. Following macrophage depletion, tumor burden increased (p=0.0564), the density of both neovessels and mature vessels decreased but not significantly (p=0.137, p=0.130 respectively), the expression levels of the gelatinase matrix metalloproteinase-9 (MMP-9) decreased significantly (p=0.0142), and the density of the tumor promoting M2 polarized macrophages did not change (p=0.680).

Conclusions: : We have previously shown that the number of macrophages in LH_BETAT_AG retinal tumors is directly proportional to tumor size and that many of these macrophages are either activated microglia or bone-marrow derived macrophages recruited early in disease. In this study we show that the density of M2 polarized tumor promoting macrophages remains stable. Since there is a significant decrease in total macrophage density, it is possible that the remaining macrophage population is M1 polarized. Consistent with previous studies, the depletion of the M1 polarized macrophages would result in tumor growth, since the M1 population releases cytotoxic activity on tumor cells inducing tissue destructive reactions centered on the vascular endothelium.

Keywords: retina • tumors • immunomodulation/immunoregulation