DMXAA causes tumor site-specific vascular disruption in murine non-small cell lung cancer, and like the endogenous non-canonical cyclic dinucleotide STING agonist, 2'3'-cGAMP, induces M2 macrophage repolarization.

Charlene M Downey,Reto A Schwendener,Frank R Jirik,Mehrnoosh Aghaei,Chryso Kanthou

doi:10.1371/journal.pone.0099988

Abstract

The vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a murine agonist of the stimulator of interferon genes (STING), appears to target the tumor vasculature primarily as a result of stimulating pro-inflammatory cytokine production from tumor-associated macrophages (TAMs). Since there were relatively few reports of DMXAA effects in genetically-engineered mutant mice (GEMM), and models of non-small cell lung cancer (NSCLC) in particular, we examined both the effectiveness and macrophage dependence of DMXAA in various NSCLC models. The DMXAA responses of primary adenocarcinomas in K-rasLA1/+ transgenic mice, as well as syngeneic subcutaneous and metastatic tumors, generated by a p53R172HΔg/+; K-rasLA1/+ NSCLC line (344SQ-ELuc), were assessed both by in vivo bioluminescence imaging as well as by histopathology. Macrophage-dependence of DMXAA effects was explored by clodronate liposome-mediated TAM depletion. Furthermore, a comparison of the vascular structure between subcutaneous tumors and metastases was carried out using micro-computed tomography (micro-CT). Interestingly, in contrast to the characteristic hemorrhagic necrosis produced by DMXAA in 344SQ-ELuc subcutaneous tumors, this agent failed to cause hemorrhagic necrosis of either 344SQ-ELuc-derived metastases or autochthonous K-rasLA1/+ NSCLCs. In addition, we found that clodronate liposome-mediated depletion of TAMs in 344SQ-ELuc subcutaneous tumors led to non-hemorrhagic necrosis due to tumor feeding-vessel occlusion. Since NSCLC were comprised exclusively of TAMs with anti-inflammatory M2-like phenotype, the ability of DMXAA to re-educate M2-polarized macrophages was examined. Using various macrophage phenotypic markers, we found that the STING agonists, DMXAA and the non-canonical endogenous cyclic dinucleotide, 2′3′-cGAMP, were both capable of re-educating M2 cells towards an M1 phenotype. Our findings demonstrate that the choice of preclinical model and the anatomical site of a tumor can determine the vascular disrupting effectiveness of DMXAA, and they also support the idea of STING agonists having therapeutic utility as TAM repolarizing agents.

Highlights

Strategies targeting the tumor vasculature represent an attractive approach in cancer therapy, and as such there has been much interest in a class of drugs known as vascular disrupting agents (VDA) [1,2]
With the goal of gaining further insight into additional variables accounting for the differential effects of dimethylxanthenone-4-acetic acid (DMXAA) between preclinical and clinical trials, we examined the effects of this agent in several mouse models, including: (a) syngeneic subcutaneous and metastatic tumors due to a cell line (344SQ-ELuc) derived from the p53R172HDg/+ K-rasLA1/+ genetically engineered mutant mouse (GEMM) model of non-small cell lung cancer (NSCLC) [22,23,24]; (b) primary lung adenocarcinomas arising in the K-rasLA1/+ model of NSCLC; and (c) subcutaneous and metastatic tumors due to the human MDA-MB231 breast cancer cell line [25]
Consistent with the evidence that macrophages play an important role in the action of DMXAA [11,30], we found that 344SQ-ELuc subcutaneous tumors contained large numbers of infiltrating macrophages, as detected by ionized Ca2+-binding adaptor (Iba)-1 immunostaining

Summary

Introduction

Strategies targeting the tumor vasculature represent an attractive approach in cancer therapy, and as such there has been much interest in a class of drugs known as vascular disrupting agents (VDA) [1,2]. There is evidence that the actions of DMXAA on tumor vasculature involve both direct and indirect effects, via targeting of the endothelium, and macrophages, respectively. The latter appear to be the most important, and are the result of DMXAA-triggered release of tumor-associated macrophage (TAM)-derived factors, such as TNF-a and NO [5,7,10,11,12], together with contributions from various other cytokines and chemokines [2,6,7,8]. The finding that DMXAA was unable to activate human STING provided a salient explanation for the failure of this agent in the human clinical trials [20,21]

Methods

Results

Conclusion