B16-F10 melanoma cells and cell culture supernatant enhance angiogenesis in mouse ischemic limb

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1 Acta Physiologica Sinica, April 25, 2009, 61 (2): Research Paper B16-F10 melanoma cells and cell culture supernatant enhance angiogenesis in mouse ischemic limb ZHOU Tao 1, HU Zhao-Hui 2, ZHOU Bo 3,4, FU Wei-Guo 1,*, WANG Yu-Qi 1 1 Department of Vascular Surgery; 2 Department of Cardiology, Zhongshan Hospital Affiliated to Fudan University, Shanghai , China; 3 Department of Neurosurgery, Second Affiliated Hospital, Zhejiang University, Hangzhou , China; 4 College of Pharmacy and Biomedical Research and Integrative Neuro-Imaging Center, University of New Mexico, Albuquerque, New Mexico 87131, USA Abstract: Generation of therapeutic angiogenesis to enhance vascularization in the ischemic tissues is a method for treating ischemic tissues in atherosclerotic cardiovascular artery disease. The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor (CXC chemokine receptor 4, CXCR4) play a critical role in the process of post-natal neovascularization. The SDF-1-CXCR4 axis is a potential mechanism for the treatment of ischemic limb. Here, we investigated the role of CXCR4 in bone marrow cells (BMCs) in neovascularization induced by tumor cells and the supernatant of culture media in a murine hind-limb ischemia model which was made by resecting femoral artery and vein. After the injection of mouse melanoma cells B16-F10 ( cells in 0.1 ml at the operation day, s.c.) into the abdomen or the cell culture supernatant (0.1 ml/d for 21 d after operation, i.m.) into the ischemic abductor muscle, the CXCR4 positive BMCs were analyzed by flow cytometry. The perfusion of the ischemic limb was evaluated by laser Doppler perfusion imaging (LDPI) on 7, 14 and 21 d after vascular injury operation. Capillary endothelial alkaline phosphatase (AP) was stained to quantify the presence of capillaries, and histological method was used to evaluate the capillary density as a measure of neovascularization in ischemic tissues. The proportions of CXCR4 positive BMCs were notably higher in ischemic limb injected with tumor cells or the supernatant compared to those in the control group (P<0.05). Injection of tumor cells or the supernatant resulted in significantly improved perfusion as measured by LDPI perfusion ratios on 7, 14 and 21 d after femoral artery and vein resection in mice, compared to the controls (P<0.05). Tissue samples harvested from the lower calf muscle at day 21 demonstrated increased capillary densities in mice receiving tumor cells (0.81±0.13) or the supernatant (0.63±0.05), compared with those in control group (0.44±0.09, P< 0.05). In conclusion, the injection of B16-F10 tumor cells or the supernatant induces the increase of CXCR4 positive cells in BMCs and the improvement of in vivo neovasculogenesis in mouse ischemic limb. Key words: ischemia; neovascularization; chemokine stromal cell-derived factor-1; CXC chemokine receptor 4; laser Doppler perfusion imaging 黑色素瘤细胞及细胞培养液促进小鼠缺血后肢血管新生周涛 1, 胡朝晖 2 3,4 1,* 1, 周波, 符伟国, 王玉琦 1 复旦大学中山医院血管外科 ; 2 心内科, 上海 ; 3 浙江大学附属第二医院神经外科, 杭州 ; 4 美国新墨西哥州大学药学院及生物医学和神经影像联合研究中心, 阿尔伯克基 摘要 : 本文旨在探讨小鼠黑色素瘤细胞及培养提取液对小鼠后肢缺血组织血管新生的影响, 以及细胞趋化因子基质细胞衍生因子 1 (stromal cell derived factor 1, SDF-1) 及其特异性趋化因子受体 (CXC chemokine receptor 4, CXCR4) 是否在这种影响中发挥作用 制作小鼠后肢缺血模型, 在腹部皮下注射小鼠黑色素瘤细胞 B16-F10 或在缺血后肢肌肉注射细胞培养上清液, 分别于 7,14 和 21 天行激光多普勒扫描 (laser Doppler perfusion imaging, LDPI) 评估后肢缺血组织血流灌注情况,21 天后流式细胞术分析小鼠骨髓细胞 (bone marrow cells, BMCs) 中的 CXCR4 表达, 碱性磷酸酶染色后组织学分析缺血后肢血管新生情况 结果 Received Accepted This work was supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China (No ). * Corresponding author. Tel: ; drfuweiguo@gmail.com

2 140 Acta Physiologica Sinica, April 25, 2009, 61 (2): 显示, 与对照组相比, 肿瘤细胞和细胞培养液均可使 BMCs 中 CXCR4 阳性表达细胞数增加 (P<0.05), 小鼠缺血后肢血流灌注情况明显改善 (P<0.05), 小鼠缺血后肢肌肉毛细血管密度显著增加 (P<0.05) 以上结果提示, 黑色素瘤细胞及细胞培养液可以促进 BMCs 中 CXCR4 的表达, 通过 SDF-1/CXCR4 途径促进缺血组织的血管新生 关键词 : 局部缺血 ; 血管新生 ; 基质细胞衍生因子 1 ; 特异性趋化因子受体 ; 激光多普勒血流扫描中图分类号 :R543 Angiogenesis is a strategy for the therapy of myocardial ischemia [1] and limb ischemia [2]. Neovascularization is an important part in the recovery of the perfusion in ischemic tissues and the tumor metastasis. In both pathologic conditions, the chemokine stromal cell derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) system plays a critical role in the process of post-natal neovascularization [3]. Prior studies, primarily using adult bone marrow (BM)-derived mononuclear cells (MNCs) [4], point to the efficacy of cultured endothelial progenitor cells (EPCs) in mediating neovascularization in vascular ischemic animal models [1,5]. Initial animal studies suggest that such cells could potentially also be used to repair ischemic tissues [6, 7]. Clinical trials using autologous BM- or circulating blood-derived progenitor cells have illustrated the safety of cell infusions in human cardiac patients [8, 9]. The cellular mechanism underlying vasculogenesis, a process in which EPCs differentiate in situ into mature endothelial cells [10], has been further verified over the past 7 years with the identification of BM-derived circulating EPCs shown to migrate to the areas of ischemia and participate in neovascularization [11,12]. SDF-1, also known as CXCL12, is a constitutively expressed and inducible chemokine that regulates multiple physiological processes, including embryonic development and organ homeostasis. This pleiotropic chemokine is expressed in several organs including lung, liver, skin and BM. The cognate receptor for SDF-1, CXCR4, also known as CD184, is widely and constitutively expressed by numerous tissues, including hematopoietic and endothelial cells. CXCR4 + proangiogenic cells are composed of immature and mature hematopoietic cells, EPCs, and smooth muscle cell (SMC) progenitors, which all have direct or indirect pro-angiogenic properties [13]. Currently, we lack the understanding of the process of angiogenesis that occurs in animals and man. Cancer cells are the most known pro-angiogenic cells, and produce their effects through multiple complex pathways using multiple mediators. However, no experiments using tumor cells to mediate vasculogenesis in response to ischemia have been performed to date. In this study, we directly used tumor cells and the supernatant to promote neovascularization in a mouse limb ischemic model. Rather than trying to duplicate what cancer does to produce new blood vessels, we investigated whether cancer itself could release a set of pro-angiogenic growth factors, which could promote angiogenesis in the ischemic limb. Our hypothesis is that tumor growth releases a set of pro-angiogenic growth factors which can promote angiogenesis in ischemic limb through SDF-1/CXCR4 pathway. 1 MATERIALS AND METHODS 1.1 Cell culture and supernatant collection Highly metastatic murine melanoma cell line B16-F10 was purchased from the Cell Bank of the Shanghai Institute of Cell Biology. Cells were cultured and maintained in Dulbecco s modified Eagle s medium (DMEM; Sigma, St Louis, MO, USA) containing 10% fetal bovine serum (FBS) at 37 C in a humidified atmosphere containing 5% CO 2 in air. B16-F10 cells were harvested at day 7 of culture and injected into the study animals. Cell culture supernatant was collected by centrifuge and stored at -20 o C before use. 1.2 Murine hind-limb ischemia model Throughout the experiment, mice were maintained with free access to pellet food and water in plastic cages at (21±2) C and kept on a 12 h:12 h light-dark cycle. All animals were treated in accordance with the National Care and Use of Laboratory Animals and the related ethical regulations of Fudan university. All efforts were made to minimize the animals suffering and to reduce the number of animals used. Male mice (C57BL/6J, Shanghai Laboratory Animal Center), 8 to 10 weeks old and weighing 18 to 22 g, were anesthetized with Ketamine (100 mg/kg) and Xylazine (10 mg/kg) intraperitoneally (i.p.) prior to surgery [14]. The entire right superficial femoral artery and vein (from just below the deep femoral arteries to the popliteal artery and vein) as well as the deep femoral and circumflex arteries and veins were ligated, cut and excised. Immediately after surgery, the mice were divided into 3 groups randomly (6 mice per group). Group 1 was injected with 0.1 ml of saline (0.9% NaCl) into the ischemic muscle

3 ZHOU Tao et al: Tumor Cells and Ischemia Therapeutic Angiogenesis 141 daily for 7 d. Group 2 was injected subcutaneously (s.c.) into the abdomen with B16-F10 melanoma cells ( cells in 0.1 ml) at the operation day. Group 3 was injected intramuscularly (i.m.) with B16-F10 cell culture supernatant (0.1 ml) into the ischemic abductor muscle daily for 21 d immediately after the surgery. Mice were sacrificed at day 21 after ischemic induction. 1.3 Laser Doppler perfusion images (LDPI) LDPI was performed to record blood flow over a course of three weeks postoperatively. The mice were anesthetized with Ketamine and Xylazine, and then affixed supinely on a cork plate. The hind-limbs were shaved. Limb blood flow was measured using an LDPI analyzer (Periscan PIM II Laser Doppler Perfusion Imager, Perimed AB, Sweden). Quantitative analysis of blood flow on the limb of the LDPI images was performed using the LDPI Win 2.5 program. To minimize the variability in perfusion, the LDPI perfusion data were expressed as the ratio of the ischemic (right) to normal (left) limb blood flow. All the measurements were performed in a double-blind manner by two independent researchers. 1.4 Capillary density Capillary endothelium alkaline phosphatase (AP) was stained to quantify the presence of capillaries and used as an additional evaluation of neovascularization [11]. The adductor muscle was dissected from mice at day 21 after surgery and frozen in liquid nitrogen-cooled isopentane. Frozen sections were fixed in acetone for 10 min at -20 C following staining for AP with an AP Chromogen Kit (BCIP/ NBT, Biomeda Co., Foster City, CA). Afterwards, samples were kept in post-fixation sucrose-buffered formalin (4%, ph 7.3), and muscle fiber and AP-positive spots were counted in randomly selected fields (5 fields per section, 20 fields per animal; 3 animals per group). The capillary density was expressed as a ratio of capillaries number and muscle fiber number. 1.5 Assessment of CXCR4 positive BM cells (BMCs) by flow cytometry Whole BMCs were harvested from the mice by flushing the tibias and femurs with phosphate-buffered saline (PBS). Red blood cells were lysed with red blood cell lysis buffer (150 mmol/l NH 4 Cl, 10 mmol/l KHCO 3, 0.1 mmol/l EDTA, ph 7.2) at 4 C for 20 min. The cells ( ) were washed three times with PBS, resuspended in 2 ml PBS, and used as freshly isolated BMCs. Flow cytometric analysis was performed using phycoerythrin-conjugated anti-cxcr4 (clone 2B11; BD Biosciences). The cells were examined by flow cytometry (FACSCalibur; BD Biosciences) and analyzed using CellQUEST software ver. 3.3 (BD Biosciences). Nonviable cells were excluded based on forward/side scatter, and no additional gating was applied. Positive-staining quadrants were set on non-stained samples as a result of the significant autofluorescence of cells. 1.6 Statistical analysis Data were summarized using mean±sd. Statistical significance was determined between the indicated values by 2- tailed, non-paired, unequal variance Student t tests to determine the differences between the control and study group mean values. P<0.05 was considered to be statistical significant. 2 RESULTS 2.1 CXCR4 expression in BMCs BMCs were harvested 21 d after the injection of tumor cells. The surface expressions of CXCR4 were analyzed by fluorescence-activated cell sorting (FACS). The CXCR4 positive BMCs in mice injected with tumor cells or the supernatant were increased compared to the control group (P<0.05). There was no significant difference (P>0.05) in CXCR4 positive BMCs between mice injected with tumor cells and the supernatant (Fig. 1). These data indicate that melanoma cells may secrete some cytokines to upregulate the expression of CXCR4 in BMCs. Fig. 1. Quantitve analysis of CXCR4 positive bone marrow cells (BMCs) in mice injected with melanoma cells B16-F10 or the supernatant by flow cytometry. n=6. Mean±SD. * P<0.05 vs control group. There was no significant difference between tumor cells and supernatant groups (P=0.45). 2.2 Perfusion in the ischemic limbs Blood perfusion was measured by LDPI. Injection of tu-

4 142 mor cells enhanced angiogenesis. Reperfusion at the ischemic limb was better in the mice injected with tumor cells than that in those injected with supernatant or saline. Baseline perfusion ratio was 0.996±0.018 (n=17) before ligation. Immediately after femoral ligation, the perfusion ratio decreased to 0.32±0.02 in control group, 0.41±0.05 in tumor cells group, and 0.40±0.007 in tumor cell supernatant group, thus indicating reduced perfusion in all groups. As shown in Fig. 2, after 7 d, significantly higher perfusion ratio was noted in mice injected with tumor cells or the supernatant compared to control mice (P<0.05). Higher perfusion ratio was measured in animals injected with tumor cells compared to those injected with tumor cells supernatant (P<0.05) at this early time point. Sustained and Acta Physiologica Sinica, April 25, 2009, 61 (2): significant increase in blood flow at day 14 and 21 was measured in mice injected with either tumor cells or tumor cells supernatant compared with that in the control mice (P<0.05). Perfusion ratio in the control group remained low at day 14: 0.38±0.02, compared with 0.77±0.04 in the group that received tumor cell supernatant (P<0.05) and 0.81±0.04 in the group that received tumor cells (P< 0.05). It is important to note that at day 14 there was no significant difference in blood flow between the experimental groups (P>0.05), while perfusion ratios in tumor cells treated mice was higher than that in the supernatant treated group at day 21 (P<0.05) (Fig. 2). No limb loss was observed in any study animals during the 21 days of observation. Fig. 2. Effect of B16-F10 tumor cells and the supernatant on reperfusion in the ischemic hind-limb of mice. The blood flow of the lower limbs was measured using an LDPI analyzer, followed by calculation of the perfusion ratio of the ischemic (right) to normal limbs (left). A: Representative laser Doppler perfusion color images at indicated time points. B: Quantitative measurement of the perfusion ratio of the ischemic to normal limbs (n=6). Mean±SD. * P<0.05 vs control group, Δ P<0.05 vs supernatant group.

5 ZHOU Tao et al: Tumor Cells and Ischemia Therapeutic Angiogenesis Capillary density in ischemic tissues Data of capillary density from the 21-day time point are shown in Fig. 3. The number of capillaries per muscle fiber was increased in mice treated with tumor cells (0.81±0.13) and supernatant (0.63±0.05), compared with saline-treated mice (0.44±0.09, P<0.05) (Fig. 3). Fig. 3. Comparison of capillary density in experimental and control mice. At 21 days after isolation and ligation of the right femoral artery, lower calf muscle was harvested from the experimental and control mice. Tissue was fresh-frozen and stained for alkaline phosphatase by the indoxyl-tetrazolium method (A). Scale bar, 100 μm. Investigators counted AP-positive spots in 20 fields per sample with double-blind method, and capillary density was expressed as the number of capillaries per muscle fiber (B). Mean±SD. * P<0.05 vs control group. 3 DISCUSSION Transplantation of stem cells into injured myocardium has been used before [15]. Recently, therapeutic neovascularization has emerged as a novel strategy for the treatment of ischemic cardiovascular diseases such as peripheral arterial disease and myocardial ischemia. Many approaches have been used to generate therapeutic angiogenesis. Most are limited to modulating one, or at most a few, growth factors [16,17]. Overall, although statistically significant results have been documented, the therapeutic effect is underwhelming. It is clear that angiogenesis is a complex process requiring the interaction of many mediators, and requires accurate, appropriately timed expression of these mediator. However, no direct experiment on the effects of cancer cells to ischemic tissues has been performed to date. We therefore used cancer itself to generate angiogenesis in an in vivo murine model of hind-limb ischemia. Our studies directly compared infusions of blood flow and capillary density in the model of hind-limb vascular injury. We observed that both B16-F10 cells and the supernatant injections significantly increased blood flow in the ischemic leg by days 21 after injury/cell injection. To confirm the neovascularization seen using blood-flow recovery by LDPI, we evaluated the capillary density in histological sections from experimental animals. At day 21, capillary density was significantly increased in animals that received B16- F10 cells or the supernatant. Although we observed significantly increased capillary density in animals treated with B16-F10 cell supernatant, it was lower than the tumor cells group. These observations suggest that components of the tumor microenvironment, possibly tumor stromal cells, contribute to augmentation of microvascular density. Previous studies have demonstrated that the SDF-1- CXCR4 axis could be a new mechanism for the treatment of ischemic cardiovascular diseases [1]. We also observed that the proportion of cells expressing CXCR4, the receptor for SDF-1, was markedly increased in BMCs. Expression of this chemokine receptor may have implications in the homing capacity of this cell population [18]. The SDF-1- CXCR4 axis supports vasculogenesis, contributes to in vivo neovascularization by augmenting cellular recruitment in ischemic tissues [2,18,19], and regulates endothelial cell branching morphogenesis [20], thereby serving a critical role in vascular remodeling in ischemic tissue [21]. In the context of local tissue ischemia in the mouse hind-limb ligation model, increased migration of BMCs homing to sites of hind-limb vascular injury may be expected to be facilitated

6 144 due to significantly increased CXCR4 positive cells in BMCs in tumor cells-injected mice compared to controls. We observed that 78% of BMCs expressed CXCR4 in the tumor cells-injected group and that 69% of BMCs expressed CXCR4 in the tumor cells supernatant-injected group, compared to only 47% in the control group. We also examined the expression of SDF-1 in the ischemic tissues, but there were no differences between the experiment groups and control groups (data not shown). As reported by others, the SDF-1 expression will increase shortly after the ischemia surgery [22]. Taken together, despite the differences in the CXCR4 positive cells in BMCs, the observed improvements in hind-limb blood flow in both tumor cellsand supernatant-injected mice may stem from the enhanced expression of the CXCR4 chemokine receptor in BMCs. Further studies are ongoing to determine the specific cell factors and their relative contributions in direct cell-mediated versus paracrine effects underlying the neovascularization in response to vascular injury. Further, the BM-derived CXCR4 + cells might be a promising source of therapeutic neovascularization for ischemic vascular diseases [23]. We showed that implantation of tumor cells accelerated capillary formation and improved blood flow. We also demonstrated that the injection of tumor cell supernatant markedly increased the proportion of CXCR4 positive cells in the BMCs. This, at least in part, suggesting that tumor cells may secrete certain factor(s) that affect CXCR4 expression in BMCs. Recently, it was reported that BM-derived circulating cells are retained in close proximity to angiogenic vessels by SDF-1 [23]. The precise mechanism by which neovascularization could be accelerated has not been fully understood. In addition, Grunewald et al. [22] demonstrated that vascular endothelial growth factor (VEGF) played an important role in neovascularization induced by the SDF-1/CXCR4 system. Taken together, the injected tumor cells might secrete cell factors into circulation, which then induce subsequent VEGF production in the ischemic limbs, thereby resulting in an acceleration of neovascularization. Finally, a recent study suggested that BM-derived CXCR4 + cells could promote neovascularization in ischemic tissue by the release of angiogenic factors [24]. There are several limitations in our study. Firstly, other angiogenic factors, such as IL-6 and MCP-1, which may participate in neovascularization, were not tested, and the expression of CCR7, CD3, Mac-1, CD34, CD31, Flk-1, Sca-1, and c-kit on BMCs was not examined. Secondly, the role of angiogenic factors induced by the tumor cells in response to ischemia also remains unclear. 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