Morphology of hypoxia following cryoablation in a prostate cancer murine model: Its relationship to necrosis, apoptosis and, microvessel density q Masaki Kimura a , Zahid Rabbani b , Vladimir Mouraviev a , Matvey Tsivian a , Zeljko Vujaskovic b , Takefumi Satoh c , Shiro Baba c , John M. Baust d , John G. Baust d , Thomas J. Polascik a, * a Duke Prostate Center and Division of Urologic Surgery, Department of Surgery, Duke University Medical Center, Durham, NC, USA b Department of Radiation Oncology, Duke University Medical Center, Durham, NC, USA c Kitasato University School of Medicine, Department of Urology, Kanagawa, Japan d Institute of Biomedical Technology, State University of New York, Binghamton, New York, NY, USA article info Article history: Received 15 June 2010 Accepted 22 June 2010 Available online 30 June 2010 Keywords: Prostate cancer Cryoablation Cryosurgery Hypoxia Apoptosis Microvessel density abstract The aim of this study is to investigate the tumor tissue changes in terms of hypoxia and demonstrate its relationship to vascularity and apoptosis following therapeutic cryoablation in a prostate tumor murine model. Total 67 male C57BL/J6 mice were assigned into sham-operation group and cryoablation group. Murine prostate tumors (RM-9) were inoculated subcutaneously in a right hind leg and treated with cryotherapy. Of 30 mice, tumor volumes were measured for 12 days following operation. Of 37 mice, tumor tissues were harvested in 24 h following operation, and histological/molecular changes were ana- lyzed. Hematoxylin and eosin or immunohistochemical staining were utilized to quantify tumor necrosis, hypoxia (pimonidazole), vascularization (CD31), and apoptosis (cleaved caspase-3). The results showed that cryoablated tumors demonstrated significant delayed growth following treatment compared to con- trols. Pathological analysis revealed that the severity of hypoxia increased in the cryoablation arm com- pared to controls. Necrotic and apoptotic populations were also found to be increased in the cryoablation arm (P = 0.028 and 0.021). Hypoxia demonstrated a positive correlation with necrosis (r = 0.520, P = 0.001) and apoptosis (r = 0.474, P = 0.003), while showing negative correlation with microvessel den- sity (MVD) (r = À0.361, P = 0.021). We concluded that in the peripheral areas from the cryoneedle impact site, strong hypoxic responses were found, which may play important role in tumor freezing injury. To our knowledge, this is the first report describing cryoablation-mediated changes of hypoxia at a molec- ular level in the prostate cancer murine model. Ó 2010 Elsevier Inc. All rights reserved. Introduction Cryoablation, a minimally invasive surgical strategy for localized prostate cancer, has the potential to provide an equivalent oncolog- ic outcome when compared with other treatment options, providing for lower treatment-related complications and morbidity compared to other procedures, such as radical prostatectomy, external beam radiation therapy, and brachytherapy [5,21,22, 7,17]. On the contrary, cryoablation alone is still a controversial treatment for the patients with locally advanced or loco-regional disease [6]. One technical issue surrounding cryoablation is the po- tential for insufficient ablation in the peripheral zone of the iceball distant from the cryoneedle energy source. In the peripheral area of freezing zone (hemi-ablated area), it is estimated that temperatures are not low enough to kill all of the cells, leading to a residual mix- ture of dead cells, apoptotic cells, and living cells allowing for the potential of re-growth [3]. This is due to the technological based challenge of creating lethal freezing temperatures (À40 to À50 °C) within the entire tumor while sparing surrounding tissue [2]. In or- der to overcome these problems, studies are required to investigate tumor tissue changes after cryotherapy. The in vitro changes of prostate tumor cells with respect to cel- lular death after cryoablation have been previously reported. These studies demonstrated that freezing injury includes direct physical cellular damage, necrosis and apoptotic signaling, which eventu- ally lead to cell death [3,1,14,15]. In vivo studies of the tumor microenvironment following cryoablation have not been reported in prostate cancer. In regards to the morphological relationship be- tween necrosis and apoptosis, a few articles have demonstrated that necrotic tissue is uniform in the central region of the freeze zone proximal to a cryoprobe [1,12]. Apoptotic cells have been re- ported in the border zone at the periphery of the previously frozen tissue in a lung cancer model [12]. While these studies serve as a 0011-2240/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2010.06.010 q There is no source of extra-institutional funding. * Corresponding author. Address: Duke University Medical Center, Yellow Zone, Room #1080, 200 Trent Drive, Durham, NC 27710, USA. Fax: +1 919 684 5220. E-mail address: polas001@mc.duke.edu (T.J. Polascik). Cryobiology 61 (2010) 148–154 Contents lists available at ScienceDirect Cryobiology journal homepage: www.elsevier.com/locate/ycryo