Systemic Administration of Hemoglobin Vesicle Elevates Tumor Tissue Oxygen Tension and Modiﬁes Tumor Response to Irradiation Manabu Yamamoto, M.D.,* Yotaro Izumi, M.D., Ph.D.,* ,1 Hirohisa Horinouchi, M.D., Ph.D.,* Yuji Teramura, Ph.D.,§ Hiromi Sakai, Ph.D.,§ Mitsutomo Kohno, M.D., Ph.D.,* Masazumi Watanabe, M.D., Ph.D.,* Masafumi Kawamura, M.D., Ph.D.,* Takeshi Adachi, M.D., Ph.D.,† Eiji Ikeda, M.D., Ph.D.,‡ Shinji Takeoka, Ph.D.,§ ,2 Eishun Tsuchida, Ph.D.,§ and Koichi Kobayashi, M.D., Ph.D.* *Division of General Thoracic Surgery, Department of Surgery; †Department of Biochemistry and Integrative Medical Biology; ‡Department of Pathology, School of Medicine, Keio University, Tokyo, Japan; and §Research Institute for Science and Engineering, Waseda University, Tokyo, Japan Submitted for publication September 22, 2007 Background. We have developed a phospholipid li- posome vesicle encapsulating concentrated human he- moglobin (hemoglobin vesicle, HbV) as an artiﬁcial oxygen carrier, as an alternative to red cell transfu- sion. We have veriﬁed its oxygen transporting capabil- ity in a variety of preclinical models. Recent evidence suggests that artiﬁcial oxygen carriers may also be applicable for better oxygenation of ischemic or hy- poxic tissues including tumors. To our knowledge, tu- mor oxygenation using a liposome-type artiﬁcial oxy- gen carrier has not been closely tested. In the present study, we tested whether systemic HbV administra- tion changes tumor tissue oxygen tension, and if it modiﬁes tumor response to irradiation. Materials and methods. Lewis lung carcinoma was grown subcutaneously in the left hindleg of C57BL/6 mice. Experiments were initiated when the tumors reached approximately 8 mm. All experiments were done under room air. Tumor tissue oxygen tension was measured by phosphorescence quenching up to 45 min after systemic sample administration (saline: n  5; HbV: n  5; HbV containing methemoglobin (metHbV): n  4; HbV with high oxygen afﬁnity (lowP50HbV): n  8) and compared between samples. To test the effects on irradiation response, samples (saline: n  7; HbV: n  7; metHbV: n  7; lowP50HbV: n  7) were administered prior to single 20-Gy irradi- ation, and tumor growth was compared. Results. Tumor tissue oxygen tension transiently in- creased approximately 2-fold after HbV administra- tion in comparison to other samples. Tumor growth was marginally delayed after irradiation by prior ad- ministration of HbV in comparison to other samples. HbV administration without irradiation did not affect signiﬁcant tumor growth delay. Conclusions. These results correlatively suggest that HbV augmented tumor growth delay following irradi- ation, at least in part, by affecting tumor tissue oxygen tension. © 2009 Elsevier Inc. All rights reserved. Key Words: hemoglobin vesicle; artiﬁcial oxygen car- rier; tumor oxygenation; radiosensitizer; liposome; HIF1alpha. INTRODUCTION Artiﬁcial oxygen carriers are currently being actively developed for use as transfusion alternatives. Artiﬁcial oxygen carriers do not have blood types, are free of potential infectious pathogens, and can be stored much longer than red blood cells (RBCs) [1]. Several preclin- ical studies indicate that they can be effectively applied as temporal resuscitative ﬂuids, and some are under- going clinical trials [2, 3]. Hemoglobin (Hb)-based oxygen carriers are classiﬁed into acellular chemically modiﬁed Hbs and encapsulated Hbs [4, 5]. We have developed a phospholipid liposome vesicle encapsulating concentrated human hemoglobin (hemoglobin vesicle, HbV) as an artiﬁcial oxygen carrier [1]. The cellular structure of HbV has characteristics that 1 To whom correspondence and reprint requests should be ad- dressed at Division of General Thoracic Surgery, Department of Surgery, School of Medicine Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: yotaro@sc.itc.keio. ac.jp. 2 Current afﬁliation: Consolidated Research Institute for Ad- vanced Science and Engineering, Waseda University, Tokyo, Japan. Journal of Surgical Research 151, 48 –54 (2009) doi:10.1016/j.jss.2007.12.770 48 0022-4804/09 $34.00 © 2009 Elsevier Inc. All rights reserved.