Ultrasonics 54 (2014) 451454

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Ultrasonics
journal homepage: www.elsevier.com/locate/ultras

A study of ovarian cancer biomarker amplication using ultrasound for early stage detection q
Danhong Peng a,b, Tian Xu a, Timothy J. Mason c, Wei Wu a,
a

Key Laboratory of Environment Medicine and Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing 210009, PR China Department of Obstetrics and Gynecology, Zhongda Hospital, Southeast University, Nanjing 210009, PR China c Sonochemistry Centre, Faculty of Health and Life Science, Coventry University, Coventry CV1 5FB, UK
b

a r t i c l e

i n f o

a b s t r a c t
The application of serum biomarker to ovarian tumors for early stage detection and clinical diagnosis is a rapidly expanding research area. The problem with conventional markers is that they are often released too late or at too low a level to be detected in time to trigger effective treatment. Ultrasound has been used to inuence bio-effects in living cells, but there is only one reported case of the use of ultrasound to enhance the release of a biomarker (Carcinoembryonic antigen CEA). In this study we report the use of ultrasound to enhance the release of a combination of ovarian cancer biomarkers (CA125 and CA199) to help in the diagnosis of ovarian cancer at an early stage. The results indicated that after 5 min sonication at a frequency of 1 MHz and intensity of 0.3 W cm2, the CA125 and CA19-9 levels were increased by 2.02 and 4.21-fold respectively. These ndings suggest that ultrasonic treatment can be used to enhance the release of serum biomarkers from ovarian tumors. 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Article history: Received 29 July 2012 Received in revised form 30 May 2013 Accepted 30 May 2013 Available online 20 June 2013 Keywords: Ovarian cancer cell line Biomarker Amplication Ultrasound

1. Introduction Ovarian cancer is one of the leading causes of cancer deaths among women but if the disease is treated at an early stage there is an excellent prognosis for survival following treatment. However some 70% of patients are diagnosed at an advanced stage and in this case there is a poor survival rate of only 1030% therefore it is important to detect ovarian cancer at early stage [1,2]. Current diagnosis methods for ovarian cancer are the CA-125 blood test and transvaginal ultrasound [3]. But neither of these tests are sufciently sensitive or specic [4]. Recently, biomarkers such as CA125 and CA19-9 have been used to help in the early screening and diagnosis of ovarian cancer i.e. at a stage when it is the most treatable and before it has had a chance to grow and spread [58]. CA125 is the most widely used tumor marker in ovarian cancer but its sensitivity and specicity are not ideal because the levels of this marker are raised to approximately 80% of all epithelial ovarian cancers (EOC) and in only 50% of stage I EOC. The sensitivity and specicity rates of another biomarker CA19-9 are both lower than those of CA125. From this it can be argued that the results from individual existing markers are not specic

q This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike License, which permits noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Corresponding author. Tel.: +86 13073420218. E-mail address: 101005800@seu.edu.cn (W. Wu).

enough because they can be affected by other malignancies, benign conditions such as diverticulitis, liver cirrhosis and also by physiological conditions including menstruation and pregnancy. In addition the concentration of a biomarker signal in the blood is often very low and so is difcult to detect. In order to be used in screening for early stage cancer the tumor marker must be sufciently detectable to provide a positive result. Research [916] suggests that the use of a combination of biomarkers improves sensitivity for the detection of ovarian cancer. Ultrasound has been shown to be effective in a range of cancer therapies including the direct killing of cancer cells, enhanced drug delivery and for improving membrane permeability for the uptake of drugs. In 2009, researchers [17] reported the use of ultrasound to increase the release of biomarker Carcinoembryonic antigen (CEA). In this study, ultrasound at 1 MHz with an intensity of 0.3 W cm2 was applied in cell culture experiments using the human colon cancer cell line LS174T. The biomarker CEA concentration was measured before and after exposure of the tumor cells to ultrasound. After 30 min treatment there was a 4-fold increase in the CEA levels when compared with untreated control samples. Mouse tumor xenograft models were also tested using 1 MHz ultrasound at an intensity of 2 W cm2 applied directly to the site of the tumor for 6 min. The CEA level was increased by more than 10-fold, which may have been due to the somewhat higher intensity used. The ultrasonic transducer applied in this work was a commercial device (Sonitron 2000, Belgium), which can provide a well collimated beam penetrating deep into the tissue. This work indicated the possibilities that the concentration of tumor

0041-624X/$ - see front matter 2013 The Authors. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ultras.2013.05.014

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biomarkers in blood could be increased leading to a more sensitive and specic diagnosis. As an extension of this it would be of interest to determine whether ultrasonic treatment can enhance the release of two other ovarian tumor biomarkers CA125 and CA19-9 to a concentration enabling early stage screening and a consequent improvement in patient survival rate. One of the bio-effects of ultrasound is to induce a temporary increase in the permeability of the cell membrane. 1 MHz, continuous-wave ultrasound (8 W cm2) for 30 s treatment has been suggested to induce transient pores in the cell membranes of some surviving cells [18]. This was the driving force for our work which was to use this effect to provide a safe but effective ultrasonic treatment for the enhanced release of ovarian tumor biomarkers without causing permanent damage to the human cells involved. 2. Materials and methods 2.1. Cells Human ovarian carcinoma cell lines, SKOV3 were cultured in McCOYs 5A medium (12400024, Gibco, Life Technologies, USA) supplemented with 10% fetal calf serum (SV30087.02, Hyclone Laboratories Inc., USA) at 37 C and under 5% CO2 concentration. Cells grew in incubator (SERIES 8000 WJ, Thermo Scientic, USA). 2.2. Ultrasonic equipment The parameter settings of the HM-1 ultrasonic equipment (Jiangsu Hanmei Science and Technology Co., Ltd., Taizhou, China) were: frequency of 1 MHz, intensity of 0.3 W cm2 and 50% duty cycle. The diameter of the ultrasonic probe was 21 mm. An ultrasound power meter (Model UPM-DF-1E, Ohmic Co., Ltd., USA) was used for intensity measurement and an oscilloscope (model TDS1000B, Tektronix Co., Ltd., USA) for the measurement of frequency. 2.3. Ultrasonic treatment of cells Cells were seeded at 5 106 in culture asks (Gibco, USA), and grown overnight in complete media forming a 100% conuent monolayer of cells. On the following day, the media was removed and the cells were rinsed three times with 23 mL fresh media. 5 mL cells were then added into culture asks immediately before sonication. Ultrasonic treatment was applied from the bottom of the ask for 5 min. To achieve good sonication, ultrasound coupling gel (Shengyou, Taizhou, China) was used between the bottom of the asks and the ultrasonic probe, with the probe placed ush against the bottom of the asks (Fig. 1). Control samples were run in parallel without sonication. Immediately after treatment 0.5 mL samples of suspension were removed for analysis.

2.4. CA125/CA19-9 quantication CA125 and CA19-9 concentration in media were determined using a Roche automatic electrochemiluminescence immunoassay analyzer (COBAS6000, Switzerland). The sensitivity of this method was 0.6 U/mL. 2.5. Cell death detection Cell death was determined immediately after sonication. The media was removed, the cells were harvested and cell death was determined by adding 10% Trypan blue to the cell suspension. Only dead cells with disrupted cell membranes are stained by the dye. The percentage of dead cells was counted using an inverted microscope (1X51, OLYMPUS, Japan) within 3 min after staining. 2.6. Statistical analysis Statistical analyses were done with SPSS17.0. All data are presented as the mean standard deviation (SD). Analysis of variance (Univariate ANOVA) and t test were adopted. A signicance level of 0.05 was used. 3. Results and discussion 3.1. Time release of CA125 from ovarian cancer cells in culture treated with ultrasound To study the time release of biomarkers, cells were sonicated using the ultrasonic probe system described above. A signicant increase of CA125 was observed from a treatment time of 5 min and the biomarker concentration (P < 0.01) continued to increase slowly over the remaining 30 min sonication. The average concentration of CA125 after sonication was 6.26 U/mL, which was a 3fold increase in CA125 levels compared with control samples (2.15 U/mL). The release of CA125 was also seen to increase slightly (P > 0.05) without sonication but this was signicantly lower compared with sonicated samples (Fig. 2). Similar reports of ultrasonic effects on CEA were published by DSouza et al. [17]. In our study we found that the major increase occurred after 5 min treatment but after this the continued increase of CA125 concentration was slow (P > 0.05). Thus, we decided to set the sonication time in all further experiments for 5 min.

Fig. 1. Schematic diagram of experimental set-up.

Fig. 2. Time release of CA125 from ovarian cancer cells in culture in the presence and absence of ultrasound.

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Fig. 3. Comparisons on time release of CA125 and CA19-9 in the presence and absence of ultrasound.

3.2. Comparisons on time release of CA125 and CA19-9 after sonication CA125 and CA19-9 levels observed after sonication were higher than these without sonication (P < 0.01 in both cases). The initial concentration of CA125 and CA19-9 were 2.19 0.25 U/mL and 0.61 0.01 U/mL, respectively. After sonication, the CA125 concentration was 4.41 0.09 U/mL and CA19-9 concentration was 2.38 0.10 U/mL (Fig. 2). The results indicated that ultrasonic treatment (1 MHz frequency, 0.3 W cm2 intensity and 50% duty cycle) over a period of 5 min increased the concentration of both of these biomarkers, conrming that ultrasound can amplify tumor biomarker release (see Fig. 3). Ovarian cancer is the most frequent cause of death from gynecological cancer and the fourth most common cancer-related killer

for female patients. It is not easy to diagnose at early stage although the cure/survival rate would be much higher if treatment could be applied at this stage. The use of tumor biomarkers can improve the accuracy of diagnosis and management of patients with ovarian cancer. Unfortunately at an early stage the biomarker levels are usually too low to be detected. The most widely used and extensively researched tumor marker in ovarian cancer is CA125 but it cannot be used alone for cancer detection because only 5060% of patients with stage I epithelial ovarian cancer will have levels high enough to register positive. CA19-9 is another tumor antigen. Although it is not used as widely as CA125 in ovarian cancer CA19-9 has been studied in the gynecological eld since the 1980s and shown to have prognostic signicance for tumor cells. Compared with CA125 alone (64%), a combination of CA19-9 and CA125 presents an increase in positivity rate to 100% [13]. Thus the combined use of CA125 and CA19-9 offers the possibility of improvements in sensitivity of ovarian cancer detection without signicant loss in specicity. Our results also suggest the possibility that ultrasonic treatment could also increase the levels of other markers e.g. HE4, CA72-4, SF and AFP. The ultrasonic technique has been shown to produce pores in the cell membrane without immediate killing effects and so can cause a temporary increase in permeability [19]. Direct observation of the effects of sonication at 1.075 MHz (intensity not reported) for 0.2 s on Xenopuslaevis oocytes revealed that 110 nm pores were generated in the membrane [20]. These transient pores in cell membranes provided channels through which macromolecules could pass. Based on this observation we deduce that this type of effect can also enhance the movement of biomarkers out of the cell. Although the mechanism of the formation of transient pores in biological membranes induced by ultrasonic cavitation is not fully understood, it is believed that they are associated with the generation of shockwaves and microjects produced on bubble

Fig. 4. Flow chart of experimental design.

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collapse together with the effects of stable bubble oscillations [19]. In addition, when an ultrasonic pressure wave is reected and meets the further transmitted waves, it will result in the formation of an ultrasound standing wave eld. Standing waves can help in achieving high sonoporation efciency, but it is difcult to induce the standing wave in vivo for a clinical application. Thus, the formation of a standing wave should be minimized for further work either by the movement of the probe or the use of pulsed waves [21,22]. Future investigations of these ultrasonic effects on biomarker release from human cell lines will involve the use of a wider range of parameter settings such as frequency, intensity, sonication time and duty cycle. 3.3. Cell death after sonication During 5 min sonication the percentage of cell death observed was 2.12% (P < 0.01) compared with only 0.54% (P < 0.01) in the control. An increase in the intensity of ultrasound would certainly increase the rate of cell death but as a consequence of this there might be some safety concerns for the patient. For this reason it is important to maintain the ultrasonic parameters at levels sufcient to achieve amplication of biomarker release but to minimize cell death. The American Federal Drugs Administration (FDA) has raised the limit of permitted spatial peak temporal average intensity for commercially available ultrasound devices from 0.094 W cm2 to 0.72 W cm2 with two safety-related indices, the thermal index (TI < 6.0) and the mechanical index (MI < 1.9) [23]. The ultrasonic intensity applied in our experiments was 0.3 W cm2 with TIS (MI) of 5 (1.43), which was within the current safety requirement. This study is at very early stage and the main achievement has been to demonstrate that insonation can improve the ovarian tumor biomarker level in blood. Further investigations in this work are outlined in the ow chart (Fig. 4). At the cell culture stage the concentration of ovarian cancer biomarkers will be tested under dened ultrasound parameters (frequency, power, duty cycle, time). For the second stage, living subjects will be treated using insonation at appropriate ultrasonic parameter settings identied from the rst stage. Living subjects will be observed before and after treatment to determine the mechanism of action of the insonation. The biological effects of ultrasound on the cell membrane will be studied. We will also use animal studies in lead optimization to avoid unwanted cell death. Treatments safety will be tested by cell survival rates. If data from animal models can predict that the treatment is likely to be safe and efcacious for tumor biomarker release the study will be carried onto clinical trials. 4. Conclusion The experiments revealed that using a frequency of 1 MHz at an intensity of 0.3 W cm2 over 5 min, CA125 and CA19-9 levels increased by 2.02 and 4.21-fold respectively compared with control experiments in the absence of ultrasound. There have been no previous reports about the use of ultrasound in connection with CA125 and CA19-9 expression and transfer process. The mechanism for enhanced release is not yet clear but the hypothesis is that ultrasonic treatment increases cell membrane permeability [21]. Biomarkers produced in or on the cell surface are then released into the blood. The results strongly suggest that early stage diagnosis of ovarian cancer may be possible using a combination of biomarkers in combination with sonication.

Acknowledgements This work was supported by Chinese Ministry of Education Open Foundation for Key Laboratory of Environment Medicine and Engineering, Ministry of Education (2011EME004). In addition, we greatly appreciate the constructive comments from the anonymous reviewers. References
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