In vivo optical imaging of tumor stromal cells with hypoxia‐inducible factor activity

Tumors contain various stromal cells, such as immune cells, endothelial cells, and fibroblasts, which contribute to the development of a tumor‐specific microenvironment characterized by hypoxia and inflammation, and are associated with malignant progression. In this study, we investigated the activity of intratumoral hypoxia‐inducible factor (HIF), which functions as a master regulator of the cellular response to hypoxia and inflammation. We constructed the HIF activity‐monitoring reporter gene hypoxia‐response element‐Venus‐Akaluc (HVA) that expresses the green fluorescent protein Venus and modified firefly luciferase Akaluc in a HIF activity‐dependent manner, and created transgenic mice harboring HVA transgene (HVA‐Tg). In HVA‐Tg, HIF‐active cells can be visualized using AkaBLI, an ultra‐sensitive in vivo bioluminescence imaging technology that produces an intense near‐infrared light upon reaction of Akaluc with the D‐luciferin analog AkaLumine‐HCl. By orthotopic transplantation of E0771, a mouse triple negative breast cancer cell line without a reporter gene, into HVA‐Tg, we succeeded in noninvasively monitoring bioluminescence signals from HIF‐active stromal cells as early as 8 days after transplantation. The HIF‐active stromal cells initially clustered locally and then spread throughout the tumors with growth. Immunohistochemistry and flow cytometry analyses revealed that CD11b(+)F4/80(+) macrophages were the predominant HIF‐active stromal cells in E0771 tumors. These results indicate that HVA‐Tg is a useful tool for spatiotemporal analysis of HIF‐active tumor stromal cells, facilitating investigation of the roles of HIF‐active tumor stromal cells in tumor growth and malignant progression.