In Vivo Bioluminescence Imaging Monitoring of Hypoxia-Inducible Factor 1α, a Promoter That Protects Cells, in Response to Chemotherapy

Abstract

Bioluminescence imaging is a powerful technique that has shown that hypoxia-inducible factor 1 (HIF-1), a transcription factor that protects tumor cells from hypoxia, is up-regulated in tumors after radiation therapy. We tested the hypothesis that bioluminescence imaging would successfully and noninvasively depict an increase in HIF-1 in the novel therapeutic environment of chemotherapy and that, as in radiation therapy, the underlying mechanism involves inducible nitric oxide synthase originating in macrophages. Active HIF-1 consists of alpha and beta subunits that bind to promoter sequences in many genes, including those that protect endothelial cells, promote angiogenesis, and alter metastasis and tumor cell metabolism. We grew 4T1 murine breast carcinoma cells with an HIF-1alpha luciferase reporter construct to 7 mm in the right rear flanks of 18 Balb-C mice. The mice were evenly randomized to receive one of the following single intraperitoneal doses: maximum tolerated dose cyclophosphamide (231.5 mg/kg), maximum tolerated dose paclitaxel (10 mg/kg), or control saline solution. Immunohistochemical analysis of tumor sections from the cyclophosphamide and control groups was performed 10 days after treatment to assess the intensity and distribution of HIF-1alpha expression, hypoxia, macrophage infiltration, and expression of macrophage-derived inducible nitric oxide synthase in tumor tissues treated with maximum tolerated dose cyclophosphamide compared with control tumors. Cyclophosphamide, but not paclitaxel, significantly inhibited tumor growth and caused a significant increase in HIF-1alpha protein levels, which peaked at a 10-fold increase from baseline on day 10 after administration. In contrast, paclitaxel did not have an antitumor effect in this model and did not cause a significant increase in HIF-1alpha. Immunohistochemical analysis showed increased and more evenly dispersed levels of HIF-1alpha protein, macrophage infiltration, and expression of inducible nitric oxide synthase originating in macrophages after cyclophosphamide treatment. We successfully monitored increased expression of a tumor protective protein in a noninvasive manner. Such monitoring may be a means of detection of resistance to therapy, and it may be possible to use the monitoring findings to alter treatment strategies in real time. The tumor microenvironment seen at immunohistochemical analysis supports the hypothesized mechanism that the cytotoxic effects of radiation therapy that attract macrophages, causing the release of macrophage-derived inducible nitric oxide synthase and production of HIF-1alpha under aerobic conditions, also underlie chemotherapy. Such noninvasive imaging may be a means to development of therapeutic strategies that prevent HIF-1 up-regulation after chemotherapy treatments.