Expressions of MVD, VEGF, Ki67 in Residual Prostate Cancer after Cryoablation

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1 Clin Oncol Cancer Res (211) 8: DOI 1.17/s y 27 Expressions of MVD, VEGF, Ki67 in Residual Prostate Cancer after Cryoablation Yong LI Zhi GUO Yan-ping HAN Xiu-ying GUO Tianjin Key Laboratory of Cancer Prevention and Therapy; Tianjin Medical University Cancer Institute and Hospital, Tianjin 36, China. Correspondence to: Zhi GUO and Xiu-ying GUO OBJECTIVE To analyze the effects of cryoablation on the mice bearing Rm-1 prostate cancer through detecting tumor angiogenesis and cancer cell proliferation in the mice after cryoablation, and to explore the effects of cryoablation on vascular endothelium growth factor (VEGF), Ki67 protein expression and microvessel density (MVD) in the mice bearing prostate cancer. METHODS Sixty Rm-1 mouse models of prostate cancer were established.experimental mice were randomized into 2 groups: the cryoablation group (n = 3) and the control group (n = 3). A er the therapy, tumor tissues of the mice in group A and B were obtained at day (without cryoablation), 1st, 3rd, 5th, 7th, 14th day, respectively, a er cryoablation, and the expressions of MVD, VEGF and Ki67 proteins were detected at the same time points. RESULTS The expressions of MVD, VEGF and Ki67 proteins in group A were decreased.the lowest values of the factors were detected on the 3rd day a er cryoablation, and increased slowly after that. The expressions of MVD, VEGF and Ki67 proteins in the control group were not changed. Significant changes of the expressions of MVD, VEGF and Ki67 proteins in the group A were found at different time points. Correlation analysis suggested a positive correlation between the expressions of VEGF and MVD proteins (r =.8793), a positive correlation between the expressions of Ki67 and MVD proteins (r =.7614), and a positive correlation between the expressions of VEGF and ki67 proteins (r =.6921). CONCLUSION A er argon-helium cryoablation treatment for the mice bearing prostate cancer, the expressions of MVD, VEGF and Ki67 proteins in local tumor were reduced on the 1st day. The lowest values of the factors were detected on the 3rd day after cryoablation, and then increased a er that. Cryoablation combined with other modalities of treatment may effectively improve the treatment effects of cryoablation for prostate cancer. KEY WORDS: cryoablation, prostate cancer, MVD, VEGF, Ki67. Copyright 211 by Tianjin Medical University Cancer Institute & Hospital and Springer Introduction Received January 12, 211; accepted March 1, cocr@gmail.com Tel (Fax): Cryoablation has emerged as one of many tools available for the treatment of prostate cancer (PCa) [1,2]. This procedure utilizes the destructive forces of extreme cold to destroy disease tissues. The tumor tissues surrounding the center (nearest the cryoprobe) of the cryogenic legion are rendered complete necrosis, less is known about

2 28 Clin Oncol Cancer Res (211) 8: the mechanisms of cell death and survival. This method, based on the cytotoxic effects of low temperature, is safe, efficient, inexpensive and easy to perform [3,4]. Moreover, this technique does not cause any serious side effect, and it could also be described as minimally invasive ablative method or minimally invasive surgery [5-7]. The mechanisms of this minimally invasive ablative method are different: one is that cryoablation directly induces cell death and the other is the time-dependent effects of the cryoablation induced by its dual function: an immediate physical effect and a delayed vascular effect. VEGF and MVD are the common markers of tumor angiogenesis, and closely related with the development and prognosis of tumors. Ki67 is a molecular marker of tumor proliferation, and is used to examine the changes of the tumor proliferation. Therefore, we chose these markers as targets in this study. The aim of the study was to analyze the effect of cryoablation on the mice bearing Rm-1 prostate cancer through detecting tumor angiogenesis. The study also explored the influences of cryoablation on the tumor angiogenesis and VEGF protein expression in prostate cancer. Moreover, the correlation between these proteins was investigated. Materials and Methods Experimental model Briefly, 6 C57 mice were inoculated subcutaneously the Rm-1 cells (1 1 7 cells in.2 ml) into the left groin of each mouse. After 2 weeks, the tumors grew large enough to be operated on (1 cm 3 ). The mice were randomly separated into 2 groups, with 3 in each group. One was a study group (group A), in which the mice received cryoablation, and the other was control group without giving cryoablation. All operative procedures and animal care were carried out strictly in the same way. All the experimental procedures and animal maintenance confirmed to the strict guidelines of institutional animal ethics committee. Treatments Cryoablation was performed with an Argon-Helium cryoprobe, 2 mm in diameter. The cryoprobe was placed into the tumor at the same depth for each mouse. The temperature was measured with a thermometry probe. Two cycles of rapid freezing/slow thawing (6 s each) were performed only on the mice in group A. During the cryoablation process (1 s), the temperature in the tumor center was between -8 C and -7 C, and the temperature in tumor margin was between -2 C and -1 C, and the temperature of thaw (5 s) was up to 1 C. Tumor growth The volumes of the tumors and the surgical procedures between the 2 groups were compared. Tumor volume (TV) was calculated using the following formula: TV = L w h, (L: tumor length, w: tumor width, h: tumor height). Angiogenesis Mice were euthanized at time points of, 1st, 3rd, 5th, 7th, 14th day after the treatment (n = 5 for each group). Tumors were fixed in formalin and then embedded with paraffin to make specimen sections which were stained with H&E. Then immunohistochemical staining of the sections was conducted to assess the expressions of MVD (CD31), VEGF and Ki-67 proteins (CD31 antibody, VEGF antibody, Ki-67 antibody from Boster Biological Technology Ltd.). Each slide was analyzed by 3 independent observers. Statistical analysis Friedman non-parametric tests were used to analyze group trends at all time points. Paired samples were analyzed by t-tests. The Spearman s rank test was used to assess correlations among several markers, and to calculate the Spearman s correlation coefficient. P <.5 was considered statistically significant. Results Tumor growth A constant tumor growth in control group was observed, whereas tumors in group A presented a decreased volume (Fig.1). The volume of the tumors treated with cryoablation kept decreasing for 3 days after the treatment, and then kept stable from the 4th day to the 7th day. Because of the proliferation of surviving cells, the tumor volume increased again from the 8th day to the 1th day. There was statistical difference in tumor growth between the group A and the control group (P =.1) Tumor volume Fig.1. The tumor volume of the mice in each group was associated with the number of days after the treatment. Angiogenic state in the tumors Group A: The observation was kept from day (without Cryoablation) to day 14. The MVD value of the tumor tissue started to decrease on the 1st day after cryoablation. It reached the minimum size on the 3rd day, and then started to increase gradually. The change

3 Clin Oncol Cancer Res (211) 8: Fig.2 The comparison of the expressions of MVD (CD31) in the 2 groups at variable time points of analysis after immunohistochemical staining Fig.3. The expression of MVD (CD31) in the 2 groups on the 1st, 3rd and 7th day (1,2, 3 for the control group; 4, 5, 6 for the group A, 2). was statistically significant (P =.11). : The value of MVD increased respectively over time, and the results showed that there was no statistically significant difference (P =.167) among every collected data. However, comparisons of the data collected at the same time between the 2 groups showed that the value of MVD in the group A was reduced considerably, and the difference between the 2 groups was significant (P =.6) (Fig.2,3). There was not statistically significant difference (P =.268) in the expression of VEGF (optical density) in the control group at each time point. The expression of VEGF (optical density) in the group A started to decrease on the 1st day after cryoablation, and the tumors reached the minimum size on the 3rd day, and then started to increase gradually, and the change was statistically significant (P =.2). The expression of VEGF in cryoablation group was significantly different from those in control group (P =.1). Figs.4,5 summarizes the expressions of VEGF in the 2 groups at all time points. There was no significant difference in the expression of Ki67 in the control group at each time point (P =.82). The expression of Ki67 in tumors treated by cryoablation also decreased from the 1st day to the 3rd day after cryoablation, and then increased from the 3rd day. The change was also statistically significant (P =.12). Fig.6,7 summarizes the expressions of Ki67 in the 2 groups at all time points. Relationship among the MVD, VEGF and Ki67 The linear equations were made to show the correlation among the MVD (CD31), VEGF and Ki67. The analyses showed a positive correlation between the expressions of the VEGF protein and MVD (correlation coefficient r =.8793, P =.32), a positive correlation between the expressions of the ki67 and MVD (r =.7614, P =.41) and a positive correlation between the expressions of the VEGF and ki67 (r =.6921, P =.36, Fig.8).

4 3 Clin Oncol Cancer Res (211) 8: Fig.4. Expressions of VEGF in each group at variable time points of analysis. Results are expressed using the means of 5 independent experiments. Fig.6. The expressions of Ki67 in each group at variable time points of analysis. Results are expressed using the means of 5 independent experiments Fig.5. The expressions of VEGF on the 1st, 3rd and 7th day (a, b, c for the control group; e, f, g for the group A, 2) Fig.7. The expressions of Ki67 on the 1st, 3rd and 7th day (1, 2, 3 for the control group; 4, 5, 6 for the group A, 2).

5 Clin Oncol Cancer Res (211) 8: MVD Ki67 MVD VEGF VEGF Ki67 a b c Fig.8. The correlation between the expressions of (a) MVD and VEGF (r =.8793, P =.32); (b) VEGF and Ki67 (r =.7614, P =.41), (c) MVD and Ki67 (r =.6921, P =.36). Discussion In the present study, the volume size of the residual tumor demonstrated that the cryoablation could reduce the tumor burden and a relatively high rate of tumor cells were under-control in a period of time. The reason was probably related with the programmed necrosis and apoptosis of residual tumor cells after cryoablation [8-11]. The results of monitoring the changes of the tumor volume indicated that although the ice ball already covered the entire tumor, there might still be a certain amount of active residual tumor cells remained. The reason is supposed that active tumor may be left in the area between C and fatal temperature. In this area of the residual tumor, as a great amount of formed blood- supply systems are destroyed by cryoablation, obvious oxygen deficient would probably occur. However, under the oxygen deficient environment which is caused by cryoablation, a physical factor, for a certain period, which is able to change the microvessel density is generated [12-14]. It was observed that in group A, VEGF expression in the tumor kept a relatively low level for 1-3 days after the cryoablation, and then tended to rise on the 3rd day. Studies have shown that the diameter of nonvascular tumor increases from.2-2 mm, and afterwards it continues to increase on condition that new vessels are induced to generate. In the present study, the VEGF expression showed a steady tendency in the control group, while a gradually intensive in the group A. It was probably because that hypoxia stimulated the tumor to secrete hypoxia inducible factor-1α which led the high secretion levels of VEGF from macrophage and mastocyte in or around the residual tumor [14]. VEGF is capable to motivate multiplication and migration of vascular endothelial cells, to increase the vasopermeability and to facilitate the exosmose of fibrinogen, which is not only propitious to angiogenesis but also easy for tumor cells to shed into vessels or to diffuse into nearby fibrin and substance of connective tissue, which is relevant to invasion and metastasis of tumors [15]. Researches on MVD have verified abovementioned conclusion that under the inducement of VEGF, microvessel density shows an increased tendency, which actually provides a possible way in combination therapy. If blood supply to the residual tumor increases after cryoablation, the residual tumor cells receive more oxygenation and medicines can reach the residual tumor cells more easily, and as a result, the efficiency of chemotherapy or other combination therapies are increased [16-18]. The present study also investigated the expression of Ki67, generating a discouraging result, which showed that it had a correlation with the expressions of VEGF and MVD. However, it is commonly considered to be relevant to the proliferation activity of tumor and poor prognosis [19,2]. The conclusion of the present study was that the proliferation activity of the residual tumor cells were promoted in a certain time after cryoablation, which indicates that the capability of tumor invasion and metastasis are also possibly promoted. The necessity of combination therapy for the residual active tumor cells after cryoablation has been paid attention. Besides, the present study was to investigate the residual tumor tissue after cryoablation and the results of the study demonstrated that cryoablation is not a radical method in cancer treatment. It has also been reported that combined chemotherapy could receive a better treatment effects compare to cryoablation only [8]. Despite the existence of the abovementioned problems, the conclusion of the study interestingly indicates that it is needed to consider the necessity of radiation therapy, chemotherapy, anti-angiogenic treatment even immunotherapy when the cryoablation is performed for prostate carcinoma. Conclusion After the cryoablation for a certain period of time, angiogenesis and proliferation activity may be increased, which indicates that it is necessary and possible to apply combination therapy. However, further study to verify the relevant molecular mechanism of cryoablation is still needed.

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