# The Correlation between Targeted Contrast-Enhanced Ultrasound Imaging and Tumor Neovascularization of Ovarian Cancer Xenografts in Nude Mice

Authors: Rong Hu; Qianqian Zeng; Xiaoling Su; Wenxia Feng; Hong Xiang
Journal: Journal of Healthcare Engineering
 (2021)

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## Abstract

In order to explore the correlation between targeted contrast-enhanced ultrasound imaging and tumor neovascularization of ovarian cancer xenografts in nude mice, a total of 49 nude mice were selected and randomly divided into 1-week group, 2-week group, 3-week group, 4-week group, 5-week group, 6-week group, and 7-week group according to their ovarian cancer xenografts’ growth time, with 7 ovarian cancer xenografts in each group. After preparing antibody-carrying targeted contrast agent, each group of xenografts performed normal and targeted contrast ultrasound examinations to obtain peak intensity, time to peak, and other imaging parameters; then, those ovarian cancer xenografts were sacrificed for pathological analysis: the neovascular density and antibody expression of the cancer xenografts at different stages were observed and counted, and the correlation between targeted contrast-enhanced ultrasound parameters and tumor neovascular densities of the ovarian cancer xenografts was analyzed. The results show that the peak intensities of targeted contrast ultrasound imaging are greater than that of ordinary ultrasound imaging in the 2-, 3-, 4-, and 5-week groups with statistically significant differences (P<0.05); the time to peak of targeted contrast ultrasound imaging is shorter than that of ordinary ultrasound imaging in the 2-, 3-, 4-, and 5-week groups with statistically significant differences (P<0.05); there is a positive correlation between the peak intensities of targeted contrast ultrasound imaging and tumor neovascular densities of the ovarian cancer xenografts in the 2-, 3-, and 4-week group (r2 = 0.645, r3 = 0.668, and r4 = 0.693, P<0.05); there is a negative correlation between the time to peak of targeted contrast ultrasound imaging and tumor neovascular densities of the ovarian cancer xenografts in the 2-, 3-, and 4-week groups (r2 = −0.669, r3 = −0.692, and r4 = −0.704, P<0.05). Therefore, the targeted contrast-enhanced ultrasound imaging parameters have a certain correlation with tumor neovascular density of ovarian cancer xenografts in nude mice and this correlation is more significant in the early stage of ovarian cancer; hence, targeted contrast-enhanced ultrasound imaging may provide a new method, new idea, and new basis for the diagnosis of early ovarian cancer.


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## Body

## 1. Introduction

Ovarian cancer has become one of the gynecological malignancies with a high fatality rate due to its insidious incidence, easy invasion, and metastasis, and its incidence is getting younger and younger; therefore, early diagnosis is the key to reducing the fatality rate and is also an urgent problem to be solved [1]. The growth, metastasis, and malignancy of ovarian cancer are related to angiogenic mimicry, which is a tumor blood supply pattern that has been formed before the appearance of neovascular structure in solid tumors. If the tumor neovascular structure in ovarian cancer can be detected, the purpose of early diagnosis can be achieved and antibody is an important member of matrix enzyme and plays an important role in tumor growth and metastasis [2]. According to the specific expression of antibody in tumor angiogenesis, it can be used as a targeted contrast agent for tumor angiogenesis mimicry site of action and neovascularization plays a key role in tumor growth, invasion, metastasis, and prognosis. Therefore, targeted ultrasound molecular imaging for tumor neovascularization is of great significance to the early diagnosis and treatment of tumors [3]. Compared with ordinary blood pool imaging, ultrasound molecular-targeted imaging is a new technology for noninvasive evaluation of diseased tissues in the body at the molecular level and it can better enhance the echo intensity of diseased tissues to achieve the purpose of early diagnosis of diseases [4].With the increasing application of contrast-enhanced ultrasound in the diagnosis of ovarian cancer, improving the ability of early diagnosis of ovarian cancer has become the direction of further research. Targeted contrast ultrasound agents with special factors can carry drugs not only for targeted treatment of tumors but also for malignant tumors [5]. By destroying tumor blood vessels or inhibiting the formation of tumor neovascularization, tumor tissues can be necrotic due to ischemia and hypoxia and the growth and metastasis of ovarian cancer tumors are inseparable from the formation of blood vessels. Previous studies have shown that the capitation effect caused by the destruction of ultrasound microbubbles can damage the microvascular endothelium, thereby significantly reducing blood perfusion in the tumor area, which is expected to become a new method for the treatment of tumors [6]. With the continuous innovation of contrast-enhanced ultrasound technology, clinical diagnosis and treatment and ultrasound imaging have gradually integrated and achieved leapfrog development. The growth of ovarian tumors depends on oxygen and other nutrients, neovascularization is needed to increase blood supply, and neovascularization can express a large number of specific antigens, which can further promote tumor tissue growth [7]. Targeted contrast-enhanced ultrasound technology is currently a cutting-edge topic worldwide, early diagnosis is even more important, and antibody prevents cancer cells from spreading further by decomposing various influencing factors in the extracellular matrix and plays a key role in tumor invasion and metastasis [8].In order to explore the correlation between targeted contrast-enhanced ultrasound imaging and tumor neovascularization of ovarian cancer xenografts in nude mice, a total of 49 nude mice were selected and randomly divided into 1-week group, 2-week group, 3-week group, 4-week group, 5-week group, 6-week group, and 7-week group according to their ovarian cancer xenografts’ growth time, with 7 ovarian cancer xenografts in each group. After preparing antibody-carrying targeted contrast agent, each group of xenografts performed normal and targeted contrast ultrasound examinations to obtain peak intensity, time to peak, and other imaging parameters; then those ovarian cancer xenografts were sacrificed for pathological analysis: the neovascular density and antibody expression of the cancer xenografts at different stages were observed and counted, and the correlation between targeted contrast-enhanced ultrasound parameters and tumor neovascular densities of the ovarian cancer xenografts was analyzed. The results of this paper provide a reference for further researches on the correlation between targeted contrast-enhanced ultrasound imaging and tumor neovascularization of ovarian cancer xenografts in nude mice. The detailed chapters are arranged as follows: Section2 presents research data and methods; Section 3 performs result analysis; Section 4 explores the correlation between targeted contrast-enhanced ultrasound imaging and tumor neovascularization of ovarian cancer xenografts in nude mice; Section 5 present the discussion; Section 6 presents the conclusion.


## 2. Materials and Methods

### 2.1. General Information

A total of 49 nude mice were chosen, and they were female (6–8 weeks old; weighing 15–20 g). Those 49 nude mice were selected and randomly divided into 1-week group, 2-week group, 3-week group, 4-week group, 5-week group, 6-week group, and 7-week group according to their ovarian cancer xenografts’ growth time, with 7 ovarian cancer xenografts in each group. Those experimental animals were raised in an environment and given sterile full-price nutritional pellets and sterile distilled water. Some other materials are as follows: human ovarian cancer cell line, ultrasound contrast agent, anti-mouse monoclonal antibody, immunohistochemistry reagent, phosphate buffer, goat anti-mouse biotin-labeled secondary antibody, fetal bovine serum, color-developing solution, and staining solution. The general information of nude mice in each group is shown in Table1.Table 1
General information of nude mice in each group (x¯ ± s).
Group1 week2 weeks3 weeks4 weeks5 weeks6 weeks7 weeksn7777777GenderFemaleFemaleFemaleFemaleFemaleFemaleFemaleAge/week7.23 ± 0.116.89 ± 0.037.45 ± 0.377.22 ± 0.096.94 ± 0.177.37 ± 0.586.77 ± 0.25Weight/g16.82 ± 1.5317.31 ± 1.2418.39 ± 1.1618.41 ± 1.6619.55 ± 1.0618.58 ± 1.4619.07 ± 1.88P＞0.05＞0.05＞0.05＞0.05＞0.05＞0.05＞0.05


### 2.2. Model Establishment of Ovarian Cancer Xenografts in Nude Mice

The cultured human ovarian cancer cells in logarithmic growth were digested with 0.20% suspended in serum-free solution. The concentration of viable cells was adjusted to 4 × 107 cells/mL, the number of cells inoculated subcutaneously on the back of nude mice was 1 × 107/mouse, and each nude mouse was inoculated subcutaneously at 1 point. After all tumor-bearing mice were inoculated on the third week, the treatment was started and the cells in the logarithmic phase with the most active growth and the strongest cell viability are cultivated in a specialized cell culture laboratory in a sterile environment. After the concentration of the cell suspension is diluted, an experienced animal laboratory required the teacher to inject 0.2 mL of cell suspension subcutaneously into the buttocks of nude mice one by one. Starting from the second day of subcutaneous injection of the cell suspension, it was recorded as the first day after tumor transplantation. The nude mice were sacrificed in accordance with the ethical requirements of experimental animals and pathological sections were made.


### 2.3. Ultrasound Contrast Agent Preparation

To prepare the targeted microbubble contrast agent, 6 mL of normal saline is injected into the container and shaken well. The preparation of antibody-carrying targeting contrast agent uses the biotin bridge method to observe the fluorescence of targeted microbubbles by indirect immune-fluorescence. The typical area of the neovascular density is first found under a low power microscope and then counted under a high power microscope, and the average value of the neovascular density in multiple fields is calculated. The placement time of the two contrast agents from the beginning of preparation to the beginning of injection remains the same.


### 2.4. Targeted Contrast-Enhanced Ultrasound Examination

Those 7 groups of nude mice were examined by contrast-enhanced ultrasound after reaching the growth cycle. First, the nude mice were anesthetized and restrained, and the tumor shape, size, and internal echo were observed by two-dimensional ultrasound using an ultrasound system; then the largest section was selected and transferred to humans for comparison pulse sequence contrast mode; during the inspection period, the probe emission frequency is maintained at 8 MHz, the mechanical index is 0.15, and the recording frame frequency is 7 Hz; each mouse is injected with 0.3 mL contrast agent through the tail vein; and then ultrasound contrast examination was performed at an interval of 1 h. After the contrast agent is cleared, a targeted contrast inspection is performed, and the dynamic image is recorded from the moment the contrast agent is injected until the moment the contrast agent is cleared. The contrast perfusion characteristics of ovarian cancer were compared and observed after contrast between the two and all the contrast-enhanced images were saved for offline analysis. The contrast-enhanced ultrasound software was used to analyze the peak intensity and time to peak of contrast ultrasound parameters. Figure1 shows the analytical framework of correlation between targeted contrast-enhanced ultrasound imaging and tumor neovascularization of ovarian cancer xenografts in nude mice.Figure 1
Analytical framework of correlation between targeted contrast-enhanced ultrasound imaging and tumor neovascularization of ovarian cancer xenografts in nude mice.


### 2.5. Developed Image Analysis

The image with an ultrasound image quantifier was analyzed, and a certain time point from the recorded images was selected to analyze the video intensity of the tumor tissue, observe its change over time, and draw the corresponding time-intensity curve. A quantitative instrument was used to perform digital subtraction and color coding on the image after 30 minutes of contrast in order to visually observe the difference in the development intensity of the two contrast agents.


### 2.6. Pathological Examination

After angiography, the nude mice were killed immediately, the tumors were taken out completely and fixed in formaldehyde solution, the angiogenic mimicry was detected by the immunohistochemical double staining method, and the antigen was detected by antibody immunohistochemistry. The neovascular density result judged the purple-red spindle or tubular structure surrounded by adjacent tumor cells, and the antigen-positive result judged that the cell membrane or cytoplasm had clear brown deposits. The typical area of the neovascular density is first found under a low power microscope and then counted under a high power microscope, and the average value of the neovascular density in multiple fields is calculated. The density of blood vessels in the first group was higher than that in the second group. The experiment uses low-frequency ultrasound for treatment, which has a longer wavelength, which allows microbubbles to have more time to diffuse during the half-period of expansion. The immune-histochemical method determines the expression of antigens in isolated specimens by scoring the percentage of positive cells under the microscope and the intensity of staining, using semiquantitative results.


### 2.7. Statistical Methods

The SPSS 19.0 software was used for statistical analysis of experimental results. After the normal distribution and homogeneity of variance test, the contrast parameter comparison between the ordinary ultrasound contrast agent and the targeted contrast ultrasound agent group adopts a paired designt-test, and the comparison of the overall difference within the ordinary and targeted contrast ultrasound agent groups adopts repeated measurement variance; the significant difference test within the group was further analyzed by the modified least significant difference method. P<0.05 means that the difference is statistically significant.


## 2.1. General Information

A total of 49 nude mice were chosen, and they were female (6–8 weeks old; weighing 15–20 g). Those 49 nude mice were selected and randomly divided into 1-week group, 2-week group, 3-week group, 4-week group, 5-week group, 6-week group, and 7-week group according to their ovarian cancer xenografts’ growth time, with 7 ovarian cancer xenografts in each group. Those experimental animals were raised in an environment and given sterile full-price nutritional pellets and sterile distilled water. Some other materials are as follows: human ovarian cancer cell line, ultrasound contrast agent, anti-mouse monoclonal antibody, immunohistochemistry reagent, phosphate buffer, goat anti-mouse biotin-labeled secondary antibody, fetal bovine serum, color-developing solution, and staining solution. The general information of nude mice in each group is shown in Table1.Table 1
General information of nude mice in each group (x¯ ± s).
Group1 week2 weeks3 weeks4 weeks5 weeks6 weeks7 weeksn7777777GenderFemaleFemaleFemaleFemaleFemaleFemaleFemaleAge/week7.23 ± 0.116.89 ± 0.037.45 ± 0.377.22 ± 0.096.94 ± 0.177.37 ± 0.586.77 ± 0.25Weight/g16.82 ± 1.5317.31 ± 1.2418.39 ± 1.1618.41 ± 1.6619.55 ± 1.0618.58 ± 1.4619.07 ± 1.88P＞0.05＞0.05＞0.05＞0.05＞0.05＞0.05＞0.05


## 2.2. Model Establishment of Ovarian Cancer Xenografts in Nude Mice

The cultured human ovarian cancer cells in logarithmic growth were digested with 0.20% suspended in serum-free solution. The concentration of viable cells was adjusted to 4 × 107 cells/mL, the number of cells inoculated subcutaneously on the back of nude mice was 1 × 107/mouse, and each nude mouse was inoculated subcutaneously at 1 point. After all tumor-bearing mice were inoculated on the third week, the treatment was started and the cells in the logarithmic phase with the most active growth and the strongest cell viability are cultivated in a specialized cell culture laboratory in a sterile environment. After the concentration of the cell suspension is diluted, an experienced animal laboratory required the teacher to inject 0.2 mL of cell suspension subcutaneously into the buttocks of nude mice one by one. Starting from the second day of subcutaneous injection of the cell suspension, it was recorded as the first day after tumor transplantation. The nude mice were sacrificed in accordance with the ethical requirements of experimental animals and pathological sections were made.


## 2.3. Ultrasound Contrast Agent Preparation

To prepare the targeted microbubble contrast agent, 6 mL of normal saline is injected into the container and shaken well. The preparation of antibody-carrying targeting contrast agent uses the biotin bridge method to observe the fluorescence of targeted microbubbles by indirect immune-fluorescence. The typical area of the neovascular density is first found under a low power microscope and then counted under a high power microscope, and the average value of the neovascular density in multiple fields is calculated. The placement time of the two contrast agents from the beginning of preparation to the beginning of injection remains the same.


## 2.4. Targeted Contrast-Enhanced Ultrasound Examination

Those 7 groups of nude mice were examined by contrast-enhanced ultrasound after reaching the growth cycle. First, the nude mice were anesthetized and restrained, and the tumor shape, size, and internal echo were observed by two-dimensional ultrasound using an ultrasound system; then the largest section was selected and transferred to humans for comparison pulse sequence contrast mode; during the inspection period, the probe emission frequency is maintained at 8 MHz, the mechanical index is 0.15, and the recording frame frequency is 7 Hz; each mouse is injected with 0.3 mL contrast agent through the tail vein; and then ultrasound contrast examination was performed at an interval of 1 h. After the contrast agent is cleared, a targeted contrast inspection is performed, and the dynamic image is recorded from the moment the contrast agent is injected until the moment the contrast agent is cleared. The contrast perfusion characteristics of ovarian cancer were compared and observed after contrast between the two and all the contrast-enhanced images were saved for offline analysis. The contrast-enhanced ultrasound software was used to analyze the peak intensity and time to peak of contrast ultrasound parameters. Figure1 shows the analytical framework of correlation between targeted contrast-enhanced ultrasound imaging and tumor neovascularization of ovarian cancer xenografts in nude mice.Figure 1
Analytical framework of correlation between targeted contrast-enhanced ultrasound imaging and tumor neovascularization of ovarian cancer xenografts in nude mice.


## 2.5. Developed Image Analysis

The image with an ultrasound image quantifier was analyzed, and a certain time point from the recorded images was selected to analyze the video intensity of the tumor tissue, observe its change over time, and draw the corresponding time-intensity curve. A quantitative instrument was used to perform digital subtraction and color coding on the image after 30 minutes of contrast in order to visually observe the difference in the development intensity of the two contrast agents.


## 2.6. Pathological Examination

After angiography, the nude mice were killed immediately, the tumors were taken out completely and fixed in formaldehyde solution, the angiogenic mimicry was detected by the immunohistochemical double staining method, and the antigen was detected by antibody immunohistochemistry. The neovascular density result judged the purple-red spindle or tubular structure surrounded by adjacent tumor cells, and the antigen-positive result judged that the cell membrane or cytoplasm had clear brown deposits. The typical area of the neovascular density is first found under a low power microscope and then counted under a high power microscope, and the average value of the neovascular density in multiple fields is calculated. The density of blood vessels in the first group was higher than that in the second group. The experiment uses low-frequency ultrasound for treatment, which has a longer wavelength, which allows microbubbles to have more time to diffuse during the half-period of expansion. The immune-histochemical method determines the expression of antigens in isolated specimens by scoring the percentage of positive cells under the microscope and the intensity of staining, using semiquantitative results.


## 2.7. Statistical Methods

The SPSS 19.0 software was used for statistical analysis of experimental results. After the normal distribution and homogeneity of variance test, the contrast parameter comparison between the ordinary ultrasound contrast agent and the targeted contrast ultrasound agent group adopts a paired designt-test, and the comparison of the overall difference within the ordinary and targeted contrast ultrasound agent groups adopts repeated measurement variance; the significant difference test within the group was further analyzed by the modified least significant difference method. P<0.05 means that the difference is statistically significant.


## 3. Result Analysis

### 3.1. Comparative Analysis of Contrast-Enhanced Ultrasound Results

Ultrasound microbubble contrast is to inject an ultrasound targeted contrast agent carrying a specific agent into the body to specifically gather at the target tissue site, which can locate the target tissue in the body and has a better advantage in the specificity of early diagnosis of diseases. In this experiment, a targeted contrast ultrasound agent carrying a monoclonal antibody was used to investigate the angiogenesis mimicry of tumor-bearing mice at different periods and the targeted contrast agent can clearly show the blood supply boundary of the tumor [9]. The experimental curve shows that the peak intensity and area of the two groups of tumors are measured and there is a significant correlation with the blood vessel count. The effect caused by the destruction of microbubbles by ultrasound can also increase the permeability of local tissue capillaries and cell membranes, which is conducive to the entry of drugs into tumor cells and can further enhance the antitumor effect of drugs. The results of the double staining method showed that the vascular structure can be seen in the central area of ovarian cancer. The density of blood vessels in the first group was higher than that in the second group. It can be speculated that blood vessels are an important source of blood supply in the early stage of ovarian cancer, which is consistent with the results of previous studies. The blood supply in the early stage of the tumor comes from the blood vessels; the more the neovascular density, the higher the density of expressing specific antigens. On the basis of this study, the characteristics of tumor density and targeted contrast perfusion were further analyzed in different periods, hoping to discover more characteristics of the ovary. The comparison of peak intensity in the normal and targeted contrast ultrasound examinations is shown in Table 2. Figure 2 shows the peak intensity of ovarian cancer xenografts at different cancer stages.Table 2
Comparison of peak intensity in the normal and targeted contrast ultrasound examinations (x¯ ± s, dB).
Group1 week2 weeks3 weeks4 weeks5 weeks6 weeks7 weeksNormal54.17 ± 4.3556.18 ± 5.6657.28 ± 5.1558.75 ± 6.5759.27 ± 3.4458.48 ± 3.3857.40 ± 2.11Targeted70.57 ± 2.9471.33 ± 3.0373.94 ± 4.0775.45 ± 3.7677.66 ± 2.6474.03 ± 5.4372.27 ± 4.29t value7.3235.8336.4154.0222.2843.1396.733P＜0.05＜0.05＜0.05＜0.05＜0.05＜0.05＜0.05Figure 2
Peak intensity of ovarian cancer xenografts at different cancer stages.Previous studies have confirmed that although the effect can cause damage to the neovascularization in the tumor, the blood perfusion in the tumor tissue can be basically restored to its state before the treatment after a few hours, and recanalization is formed. Therefore, the experimental design is set to perform ultrasound irradiation every two days, which can repeatedly destroy the formation of new blood vessels in the tumor tissue for two weeks, thereby minimizing the possibility of restoring blood perfusion. The experiment uses low-frequency ultrasound for treatment, which has a longer wavelength, which allows microbubbles to have more time to diffuse during the half-period of expansion, has less sound energy absorption, has a strong ability to penetrate tissues, and has a smaller effect on normal tissues. Neovascularization in the tumor maintains the growth of the tumor, but compared with normal blood vessels under the stimulation of high expression of vascular growth factors, these blood vessels have weaknesses such as loose endothelial cell connections, basement membrane rupture or lack of basement membrane, poor tube wall elasticity, and insufficiency. The ultrasonic effect is a series of dynamic processes in which the bubbles in the liquid are unstable due to vibration, expansion, contraction, and finally rupture under the action of ultrasound, which can be divided into steady-state and transient capitation [10].Because the effect of chemotherapy is related to the penetration of chemotherapy drugs by residual cancer foci in the abdominal cavity, previous studies have found that the depth of administration of drugs to the tumor tissue is only a few millimeters, which affects the effect of drug chemotherapy, and drug-carrying microbubbles are caused by the rupture of microbubbles. The targeted microbubbles carrying monoclonal antibodies develop in the blood vessel area and continue to accumulate to the target, so the peak intensity value of ultrasound contrast is higher; the antibody-carrying targeted contrast ultrasound agent can better assess the angiogenic mimicry of ovarian cancer. The biological effect produced can theoretically increase the drug concentration in the tumor, and the chemotherapy route can avoid the resistance encountered by the targeted drug passing through the normal capillary endothelial gap, which makes ovarian cancer in the drug-loaded microvesicles, and there are advantages in targeted chemotherapy. Previous studies have found that ultrasound irradiated cells can increase the permeability of the cell membrane, thereby causing an increase in the drug concentration in tumor cells, enhancing the killing effect of chemotherapy drugs on tumor cells, and inducing tumor cell apoptosis. Vascular endothelial growth factor is a highly specific vascular endothelial agent, which specifically acts on vascular endothelial cells, induces vascular endothelial cells to divide and proliferate, participates in tumor angiogenesis, and is the migration of vascular endothelial cells, and the metastasis of tumor cells provides a substrate.


### 3.2. Comparison of Normal and Targeted Contrast Ultrasound Imaging

The thin and soft outer membrane of the contrast agent makes the microbubbles have good resonance characteristics under the action of low sound pressure, can generate stronger harmonic signals, and can obtain real-time harmonic images with lower noise. This low mechanical and exponential sound beam can effectively preserve the microbubbles in the tumor, which is conducive to scanning each section for a longer time. Neovascularization is characterized by relatively thin wall, lack of smooth muscle, and only a layer of endothelial cells. Compared with normal blood vessels, its blood flow is rich, it is less elastic, vascular flexure is disordered, and the intercellular space of the vessel wall is widened; there are many gaps. The establishment of tumor microcirculation is affected by comprehensive factors such as the external growth environment and its own characteristics. At present, there is no unified conclusion on the occurrence and development of microcirculation in different malignant tumors. Neovascular density plays a major role in blood supply in the early stage of ovarian cancer, making it more malignant, more invasive, and metastatic. The expression of neovascularization in the late stage of ovarian cancer gradually increases, which is due to the rapid growth and blood supply of ovarian cancer. The main methods all provide new directions for the treatment of ovarian cancer, that is, combined treatment of ovarian cancer and key treatments at different stages. The comparison of time to peak in the normal and targeted contrast ultrasound examinations is shown in Table3. Figure 3 shows the time to peak of ovarian cancer xenografts at different cancer stages.Table 3
Comparison of time to peak in the normal and targeted contrast ultrasound examinations (x¯ ± s, s).
Group1 week2 weeks3 weeks4 weeks5 weeks6 weeks7 weeksNormal60.48 ± 6.1858.27 ± 8.3456.39 ± 4.2751.64 ± 4.6644.88 ± 2.2731.23 ± 4.4424.11 ± 5.68Targeted47.69 ± 3.5338.01 ± 3.9635.55 ± 1.3531.17 ± 7.4827.74 ± 2.3322.45 ± 2.1720.35 ± 4.03t value2.1040.9357.7396.3573.8861.4875.984P＜0.05＜0.05＜0.05＜0.05＜0.05＜0.05＜0.05Figure 3
Time to peak of ovarian cancer xenografts at different cancer stages.The apoptotic state of tumor tissue is the main indicator to evaluate whether the treatment is effective and is roughly divided into two ways of cell necrosis and cell apoptosis. Cell necrosis is more common in pathological conditions, such as necrosis and liquefaction necrosis, while apoptosis is an active and orderly death method. The entire process of apoptosis is regulated by multiple genes and apoptosis is better for the body of active death occurring due to the adaptation to the environment. When tumors occur, the apoptosis of cancer cells is blocked, and cell death cannot be eliminated normally. The treatment of tumors needs to rebuild the apoptosis signal transduction pathway of the cells, inhibit growth genes, and activate the expression of death genes [11]. Compared with the normal saline control group, the apoptosis of each treatment group increased to different degrees and the microbubble combined with the ultrasound group and the normal group had the highest apoptosis rate. Compared with other groups, the difference was statistically significant and the apoptosis rate of the combined ultrasound group was significantly higher than that of the normal group. This may be related to the destruction of microbubbles by ultrasound, which improves the local hypoxic microenvironment of tumor tissues while increasing targeted deposition and increases the sensitivity of tumor tissues. Ultrasound-stimulated microbubbles have a good antitumor effect on ovarian cancer subcutaneous xenografts in nude mice and the mechanism may be related to inhibiting tumor expression and promoting cell apoptosis.After the contrast agent with strong blood vessel penetrating power is injected into the body to reach the target area, the surface-linked antibody binds to the receptor on the surface of the ovarian cancer cell, and then it can selectively aggregate and reside in the target tissue of ovarian cancer for a long time. The contrast agent has the characteristics of agglomeration imaging; a large number of agents with extremely low echo reflectance accumulate in the target area to produce a significantly enhanced signal while maintaining the lowest possible background noise. Therefore, the contrast agent is an ideal molecule imaging contrast agents that can significantly improve the detection and resolution capabilities of ultrasound for ovarian cancer and the experiment environment is single; there are few interference factors. When the contrast agent enters the internal circulation, it is firstly diluted by blood to reduce its concentration and is also necessary to overcome the strong vascular shear force in the high-speed blood flow. The body’s immune response and many other damages to its stability factors make the concentration in the body very small, and the concentration after entering the tissue through the blood vessel is even lower, so there is no obvious difference in imaging. Although targeted microbubbles have good targeting in vitro, there is still a lot of work to be done in order to achieve real high efficiency and targeting in experiments, so as to achieve clinical applications [12].


## 3.1. Comparative Analysis of Contrast-Enhanced Ultrasound Results

Ultrasound microbubble contrast is to inject an ultrasound targeted contrast agent carrying a specific agent into the body to specifically gather at the target tissue site, which can locate the target tissue in the body and has a better advantage in the specificity of early diagnosis of diseases. In this experiment, a targeted contrast ultrasound agent carrying a monoclonal antibody was used to investigate the angiogenesis mimicry of tumor-bearing mice at different periods and the targeted contrast agent can clearly show the blood supply boundary of the tumor [9]. The experimental curve shows that the peak intensity and area of the two groups of tumors are measured and there is a significant correlation with the blood vessel count. The effect caused by the destruction of microbubbles by ultrasound can also increase the permeability of local tissue capillaries and cell membranes, which is conducive to the entry of drugs into tumor cells and can further enhance the antitumor effect of drugs. The results of the double staining method showed that the vascular structure can be seen in the central area of ovarian cancer. The density of blood vessels in the first group was higher than that in the second group. It can be speculated that blood vessels are an important source of blood supply in the early stage of ovarian cancer, which is consistent with the results of previous studies. The blood supply in the early stage of the tumor comes from the blood vessels; the more the neovascular density, the higher the density of expressing specific antigens. On the basis of this study, the characteristics of tumor density and targeted contrast perfusion were further analyzed in different periods, hoping to discover more characteristics of the ovary. The comparison of peak intensity in the normal and targeted contrast ultrasound examinations is shown in Table 2. Figure 2 shows the peak intensity of ovarian cancer xenografts at different cancer stages.Table 2
Comparison of peak intensity in the normal and targeted contrast ultrasound examinations (x¯ ± s, dB).
Group1 week2 weeks3 weeks4 weeks5 weeks6 weeks7 weeksNormal54.17 ± 4.3556.18 ± 5.6657.28 ± 5.1558.75 ± 6.5759.27 ± 3.4458.48 ± 3.3857.40 ± 2.11Targeted70.57 ± 2.9471.33 ± 3.0373.94 ± 4.0775.45 ± 3.7677.66 ± 2.6474.03 ± 5.4372.27 ± 4.29t value7.3235.8336.4154.0222.2843.1396.733P＜0.05＜0.05＜0.05＜0.05＜0.05＜0.05＜0.05Figure 2
Peak intensity of ovarian cancer xenografts at different cancer stages.Previous studies have confirmed that although the effect can cause damage to the neovascularization in the tumor, the blood perfusion in the tumor tissue can be basically restored to its state before the treatment after a few hours, and recanalization is formed. Therefore, the experimental design is set to perform ultrasound irradiation every two days, which can repeatedly destroy the formation of new blood vessels in the tumor tissue for two weeks, thereby minimizing the possibility of restoring blood perfusion. The experiment uses low-frequency ultrasound for treatment, which has a longer wavelength, which allows microbubbles to have more time to diffuse during the half-period of expansion, has less sound energy absorption, has a strong ability to penetrate tissues, and has a smaller effect on normal tissues. Neovascularization in the tumor maintains the growth of the tumor, but compared with normal blood vessels under the stimulation of high expression of vascular growth factors, these blood vessels have weaknesses such as loose endothelial cell connections, basement membrane rupture or lack of basement membrane, poor tube wall elasticity, and insufficiency. The ultrasonic effect is a series of dynamic processes in which the bubbles in the liquid are unstable due to vibration, expansion, contraction, and finally rupture under the action of ultrasound, which can be divided into steady-state and transient capitation [10].Because the effect of chemotherapy is related to the penetration of chemotherapy drugs by residual cancer foci in the abdominal cavity, previous studies have found that the depth of administration of drugs to the tumor tissue is only a few millimeters, which affects the effect of drug chemotherapy, and drug-carrying microbubbles are caused by the rupture of microbubbles. The targeted microbubbles carrying monoclonal antibodies develop in the blood vessel area and continue to accumulate to the target, so the peak intensity value of ultrasound contrast is higher; the antibody-carrying targeted contrast ultrasound agent can better assess the angiogenic mimicry of ovarian cancer. The biological effect produced can theoretically increase the drug concentration in the tumor, and the chemotherapy route can avoid the resistance encountered by the targeted drug passing through the normal capillary endothelial gap, which makes ovarian cancer in the drug-loaded microvesicles, and there are advantages in targeted chemotherapy. Previous studies have found that ultrasound irradiated cells can increase the permeability of the cell membrane, thereby causing an increase in the drug concentration in tumor cells, enhancing the killing effect of chemotherapy drugs on tumor cells, and inducing tumor cell apoptosis. Vascular endothelial growth factor is a highly specific vascular endothelial agent, which specifically acts on vascular endothelial cells, induces vascular endothelial cells to divide and proliferate, participates in tumor angiogenesis, and is the migration of vascular endothelial cells, and the metastasis of tumor cells provides a substrate.


## 3.2. Comparison of Normal and Targeted Contrast Ultrasound Imaging

The thin and soft outer membrane of the contrast agent makes the microbubbles have good resonance characteristics under the action of low sound pressure, can generate stronger harmonic signals, and can obtain real-time harmonic images with lower noise. This low mechanical and exponential sound beam can effectively preserve the microbubbles in the tumor, which is conducive to scanning each section for a longer time. Neovascularization is characterized by relatively thin wall, lack of smooth muscle, and only a layer of endothelial cells. Compared with normal blood vessels, its blood flow is rich, it is less elastic, vascular flexure is disordered, and the intercellular space of the vessel wall is widened; there are many gaps. The establishment of tumor microcirculation is affected by comprehensive factors such as the external growth environment and its own characteristics. At present, there is no unified conclusion on the occurrence and development of microcirculation in different malignant tumors. Neovascular density plays a major role in blood supply in the early stage of ovarian cancer, making it more malignant, more invasive, and metastatic. The expression of neovascularization in the late stage of ovarian cancer gradually increases, which is due to the rapid growth and blood supply of ovarian cancer. The main methods all provide new directions for the treatment of ovarian cancer, that is, combined treatment of ovarian cancer and key treatments at different stages. The comparison of time to peak in the normal and targeted contrast ultrasound examinations is shown in Table3. Figure 3 shows the time to peak of ovarian cancer xenografts at different cancer stages.Table 3
Comparison of time to peak in the normal and targeted contrast ultrasound examinations (x¯ ± s, s).
Group1 week2 weeks3 weeks4 weeks5 weeks6 weeks7 weeksNormal60.48 ± 6.1858.27 ± 8.3456.39 ± 4.2751.64 ± 4.6644.88 ± 2.2731.23 ± 4.4424.11 ± 5.68Targeted47.69 ± 3.5338.01 ± 3.9635.55 ± 1.3531.17 ± 7.4827.74 ± 2.3322.45 ± 2.1720.35 ± 4.03t value2.1040.9357.7396.3573.8861.4875.984P＜0.05＜0.05＜0.05＜0.05＜0.05＜0.05＜0.05Figure 3
Time to peak of ovarian cancer xenografts at different cancer stages.The apoptotic state of tumor tissue is the main indicator to evaluate whether the treatment is effective and is roughly divided into two ways of cell necrosis and cell apoptosis. Cell necrosis is more common in pathological conditions, such as necrosis and liquefaction necrosis, while apoptosis is an active and orderly death method. The entire process of apoptosis is regulated by multiple genes and apoptosis is better for the body of active death occurring due to the adaptation to the environment. When tumors occur, the apoptosis of cancer cells is blocked, and cell death cannot be eliminated normally. The treatment of tumors needs to rebuild the apoptosis signal transduction pathway of the cells, inhibit growth genes, and activate the expression of death genes [11]. Compared with the normal saline control group, the apoptosis of each treatment group increased to different degrees and the microbubble combined with the ultrasound group and the normal group had the highest apoptosis rate. Compared with other groups, the difference was statistically significant and the apoptosis rate of the combined ultrasound group was significantly higher than that of the normal group. This may be related to the destruction of microbubbles by ultrasound, which improves the local hypoxic microenvironment of tumor tissues while increasing targeted deposition and increases the sensitivity of tumor tissues. Ultrasound-stimulated microbubbles have a good antitumor effect on ovarian cancer subcutaneous xenografts in nude mice and the mechanism may be related to inhibiting tumor expression and promoting cell apoptosis.After the contrast agent with strong blood vessel penetrating power is injected into the body to reach the target area, the surface-linked antibody binds to the receptor on the surface of the ovarian cancer cell, and then it can selectively aggregate and reside in the target tissue of ovarian cancer for a long time. The contrast agent has the characteristics of agglomeration imaging; a large number of agents with extremely low echo reflectance accumulate in the target area to produce a significantly enhanced signal while maintaining the lowest possible background noise. Therefore, the contrast agent is an ideal molecule imaging contrast agents that can significantly improve the detection and resolution capabilities of ultrasound for ovarian cancer and the experiment environment is single; there are few interference factors. When the contrast agent enters the internal circulation, it is firstly diluted by blood to reduce its concentration and is also necessary to overcome the strong vascular shear force in the high-speed blood flow. The body’s immune response and many other damages to its stability factors make the concentration in the body very small, and the concentration after entering the tissue through the blood vessel is even lower, so there is no obvious difference in imaging. Although targeted microbubbles have good targeting in vitro, there is still a lot of work to be done in order to achieve real high efficiency and targeting in experiments, so as to achieve clinical applications [12].


## 4. Analysis of the Correlation between Targeted Ultrasound Imaging and Neovascularization

### 4.1. Correlation between Targeted Contrast Ultrasound Parameters and Neovascular Density

The targeted ultrasound microbubbles prepared by the biotin bridge connection method have higher peak intensity than the microbubbles prepared by the direct connection method, and the continuous imaging time of the contrast examination is longer. This confirms that the biotin bridge connection law contrast-enhanced ultrasound imaging with targeted microbubbles is better [13]. The direct connection method does not add any chemical components and directly connects the targeting agent to the microbubble through its own electrostatic adsorption. Therefore, the microbubble and the agent are low in binding degree, the charge adsorption is not strong, and it circulates with the blood in the body easy to fall off. Another important component has a strong binding force to biotin and can connect any kind of agent to the surface of the microbubble, and acid, alkali, and denaturant do not affect its binding, especially suitable research in various fields of biomedicine. Targeted microbubbles can carry drugs and deliver anticancer drugs directly to cancer cells in tumor neovascularization, which can control tumor growth at an early stage. Observed by a microscope, it is found that, on the microvascular endothelium, the targeted ultrasound microbubbles adhere tightly to it and are not easily cleared by the circulatory metabolism. Since targeted ultrasound can reside in target tissues for a long time, they are effective in the observation of blood vessels inside tumors, targeted delivery of anticancer and antithrombotic drugs. The correlation between targeted contrast ultrasound parameters and neovascular density is shown in Table 4.Table 4
Correlation between targeted contrast ultrasound parameters and neovascular density (x¯ ± s, s).
Group1 week2 weeks3 weeks4 weeks5 weeks6 weeks7 weeksPIr0.4550.6450.6680.6930.6110.4840.268P＜0.05＜0.05＜0.05＜0.05＜0.05＜0.05＜0.05TTPR−0.414−0.669−0.692−0.704−0.576−0.348−0.284P＜0.05＜0.05＜0.05＜0.05＜0.05＜0.05＜0.05Note: PI: peak intensity; TTP: time to peak.The research on ultrasound molecular imaging has brought a new dawn to the diagnosis and treatment of ovarian cancer and the most important step is the preparation and research of tumor ultrasound molecular probes. However, tumor cells themselves lack specific targets. At present, tumor-targeting studies are mostly focused on the targeting effect of tumor vascular endothelium, and there is a lack of studies that really target tumor cells; ordinary ultrasound microbubbles not only are limited by particle size that is large but also cannot penetrate the vascular endothelium to achieve molecular imaging, and the characteristics of extremely unstable and short cycle time also limit practical applications. The microbubbles are not only complicated in production technology but also caused by weak harmonic signals. Using the visualization function of microbubbles, combined with targeted antibodies, ultrasound molecular imaging is expected to achieve early diagnosis of diseases. At present, the use of air-containing microbubbles as ultrasound contrast agents has become an indispensable tool for clinical ultrasound diagnosis. With the deepening of the development of contrast agents, the research on ultrasound molecular imaging has occurred. Moreover, the effect produced by the targeted destruction of microbubbles by ultrasound can produce a new targeted drug delivery system, which can deliver drugs to target tissues and locally release drugs for treatment, which is important for malignant tumors and cardiovascular diseases, and the diagnosis and treatment of others brought new breakthroughs [14]. The correlation coefficients between peak intensity and time to peak and neovascular density at different cancer stages are shown in Figure 4.Figure 4
Correlation coefficients between peak intensity (a) and time to peak (b) and neovascular density at different cancer stages.
(a)(b)Since the antibody binds to the nuclide in the body, the antibody and the nuclide are administered separately, and the antibody is injected into the body first, which can extend the residence time of the antibody on the tumor cells. When the antibody is fully bound to the tumor cell, the remaining unbound antibody is large. Part of it is excreted from the body, the tumor-binding antibody and the normal tissue-unbound antibody reach the maximum ratio, and then the nuclide is injected for in vivo labeling, which can significantly increase the tumor or nontumor radioactivity ratio and reduce the radioactive background and at the same time enable the application of short half-life nuclide to become possible. Prepositioning imaging technology can not only reduce the background and improve the imaging quality but also shorten the retention time of the marker in the blood circulation and advance the imaging time, so it can reduce the dose and reduce the damage to the liver and bone marrow [15]. The prepositioning technology can use short half-life nuclides, simplify the labeling of nuclides, and facilitate clinical operations and is expected to make imaging kits into a kit. Coupled with the selective effect of chemotherapeutic drugs on drug-resistant mutant cell lines, resistance to chemotherapeutic drugs is often developed after several courses of treatment; vascular endothelial cells are normal cells that have stable genes and are not prone to drug resistance. The development of drug resistance in tumor cells is closely related to the instability of their genes and drugs targeting vascular endothelial cells can be used to treat chemotherapy resistance and recurrent cancer.


### 4.2. Correlation between Neovascular Density and Antibody Expression

Compared with the nonspecificity and nonselectivity of ordinary ultrasound contrast agents, targeted contrast ultrasound agents carry antibodies or corresponding receptors to specifically bind to recognizable targets, and the biological binding is tight and firm, making targeted ultrasound. The contrast agent can stay in the blood pool for a longer time and with a relatively stable and higher concentration, so that the target disease area can be identified. Contrast-enhanced ultrasound uses the image basis provided by two-dimensional ultrasound to determine the blood supply and blood flow distribution of the ovarian tumor, and the richness of blood flow when the contrast agent fills the tumor. Filling speed, retreat speed, and general malignant tumors are rich in blood flow, the contrast signal is relatively bright, the contrast enhancement mode is fast, and the benign tumors caused by insufficient blood flow will fill up quickly and return slowly with high contrast contrast. The image intensity is relatively dark, and the contrast-enhanced ultrasound mode is mostly slow, uniform, and low-enhanced. The filling speed is slow, the regression is relatively fast, and two-dimensional ultrasound can provide the basis for the performance of ovarian tumors because benign and malignant tumors have rich blood flow and whether the vascular structure is heterogeneous. In the first step of tumorigenesis, high levels of strong angiogenesis inducers are released into the tumor ecosystem [16]. Figure 5 shows the neovascular density and antibody expression rate of ovarian cancer xenografts at different stages.Figure 5
Neovascular density rate (a) and antibody expression rate (b) of ovarian cancer xenografts at different stages.
(a)(b)A small amount of blood flow signals can be seen around and inside the tumor tissue through ultrasound examination and two different ultrasound contrast agents are injected from the tail vein of nude mice. In contrast to pulse sequence imaging technology, the ultrasound image shows that the contrast agent is rapidly filling from the base of the tumor to the inside, and the tumor is clearly and completely strengthened. The contrast agent gradually fades from the center of the tumor to the periphery and finally disappears completely and the contrast-enhanced ultrasound characteristics of the two are characterized by fast in and slow out. Because the imaging characteristics of different microbubbles in different tissues are different, ultrasound microbubbles are mainly divided into ordinary microbubbles and targeted microbubbles. Studies have pointed out that ultrasound microbubbles can enhance the imaging intensity of human tissues and various organs and can also significantly increase the imaging intensity of tumor tissues, so it is of great significance for the diagnosis of benign and malignant diseases. With the development of targeted therapy technology, more explorations need to be made on the path of simultaneous diagnosis and treatment of ultrasound microbubbles. It can specifically bind to ovarian cancer cells in vitro, can enhance the image development in vivo, and can stay in the target tissue for a long time and slowly fade away and bring new perspectives and ideas to the early diagnosis of ovarian cancer.After the targeted microbubbles specifically bind to the receptors on the membrane of ovarian cancer cells, targeted sound hole formation is realized under ultrasound positioning irradiation, which widens the intercellular space of tumor tissues and greatly improves membrane permeability. This is conducive to the uptake of surrounding substances by tumor cells, allowing more substances to enter the cell to exert antitumor effects. In addition, ultrasound microbubbles have a certain apoptosis-inducing effect on tumor cells, so this group inhibits cell proliferation and induces apoptosis effects that are the most obvious [17]. This group of substances can only diffuse into tumor cells by themselves without the effect of ultrasound microbubbles, and this ovarian cancer cell is resistant to it, so the effect of its single action is significantly worse than this group. The drug concentration in the tumor tissue of the tumor-bearing mice in each group also confirmed the above speculation. The concentration of the substance in the tumor tissue was the highest in the ultrasound group targeting microbubbles. Low-dose ultrasound and targeted microbubbles did not cause significant damage to tumor tissue cells, and there was no significant difference compared with the control group. Although ultrasound microbubbles had a weaker apoptosis-inducing effect on tumor cells, it was compared with the control group and the difference is still significant. Death is a type of programmed cell death that occurs in the body’s cells through the regulation of intracellular genes and their products under physiological or pathological conditions, which is closely related to the occurrence, development, and treatment of tumors.


## 4.1. Correlation between Targeted Contrast Ultrasound Parameters and Neovascular Density

The targeted ultrasound microbubbles prepared by the biotin bridge connection method have higher peak intensity than the microbubbles prepared by the direct connection method, and the continuous imaging time of the contrast examination is longer. This confirms that the biotin bridge connection law contrast-enhanced ultrasound imaging with targeted microbubbles is better [13]. The direct connection method does not add any chemical components and directly connects the targeting agent to the microbubble through its own electrostatic adsorption. Therefore, the microbubble and the agent are low in binding degree, the charge adsorption is not strong, and it circulates with the blood in the body easy to fall off. Another important component has a strong binding force to biotin and can connect any kind of agent to the surface of the microbubble, and acid, alkali, and denaturant do not affect its binding, especially suitable research in various fields of biomedicine. Targeted microbubbles can carry drugs and deliver anticancer drugs directly to cancer cells in tumor neovascularization, which can control tumor growth at an early stage. Observed by a microscope, it is found that, on the microvascular endothelium, the targeted ultrasound microbubbles adhere tightly to it and are not easily cleared by the circulatory metabolism. Since targeted ultrasound can reside in target tissues for a long time, they are effective in the observation of blood vessels inside tumors, targeted delivery of anticancer and antithrombotic drugs. The correlation between targeted contrast ultrasound parameters and neovascular density is shown in Table 4.Table 4
Correlation between targeted contrast ultrasound parameters and neovascular density (x¯ ± s, s).
Group1 week2 weeks3 weeks4 weeks5 weeks6 weeks7 weeksPIr0.4550.6450.6680.6930.6110.4840.268P＜0.05＜0.05＜0.05＜0.05＜0.05＜0.05＜0.05TTPR−0.414−0.669−0.692−0.704−0.576−0.348−0.284P＜0.05＜0.05＜0.05＜0.05＜0.05＜0.05＜0.05Note: PI: peak intensity; TTP: time to peak.The research on ultrasound molecular imaging has brought a new dawn to the diagnosis and treatment of ovarian cancer and the most important step is the preparation and research of tumor ultrasound molecular probes. However, tumor cells themselves lack specific targets. At present, tumor-targeting studies are mostly focused on the targeting effect of tumor vascular endothelium, and there is a lack of studies that really target tumor cells; ordinary ultrasound microbubbles not only are limited by particle size that is large but also cannot penetrate the vascular endothelium to achieve molecular imaging, and the characteristics of extremely unstable and short cycle time also limit practical applications. The microbubbles are not only complicated in production technology but also caused by weak harmonic signals. Using the visualization function of microbubbles, combined with targeted antibodies, ultrasound molecular imaging is expected to achieve early diagnosis of diseases. At present, the use of air-containing microbubbles as ultrasound contrast agents has become an indispensable tool for clinical ultrasound diagnosis. With the deepening of the development of contrast agents, the research on ultrasound molecular imaging has occurred. Moreover, the effect produced by the targeted destruction of microbubbles by ultrasound can produce a new targeted drug delivery system, which can deliver drugs to target tissues and locally release drugs for treatment, which is important for malignant tumors and cardiovascular diseases, and the diagnosis and treatment of others brought new breakthroughs [14]. The correlation coefficients between peak intensity and time to peak and neovascular density at different cancer stages are shown in Figure 4.Figure 4
Correlation coefficients between peak intensity (a) and time to peak (b) and neovascular density at different cancer stages.
(a)(b)Since the antibody binds to the nuclide in the body, the antibody and the nuclide are administered separately, and the antibody is injected into the body first, which can extend the residence time of the antibody on the tumor cells. When the antibody is fully bound to the tumor cell, the remaining unbound antibody is large. Part of it is excreted from the body, the tumor-binding antibody and the normal tissue-unbound antibody reach the maximum ratio, and then the nuclide is injected for in vivo labeling, which can significantly increase the tumor or nontumor radioactivity ratio and reduce the radioactive background and at the same time enable the application of short half-life nuclide to become possible. Prepositioning imaging technology can not only reduce the background and improve the imaging quality but also shorten the retention time of the marker in the blood circulation and advance the imaging time, so it can reduce the dose and reduce the damage to the liver and bone marrow [15]. The prepositioning technology can use short half-life nuclides, simplify the labeling of nuclides, and facilitate clinical operations and is expected to make imaging kits into a kit. Coupled with the selective effect of chemotherapeutic drugs on drug-resistant mutant cell lines, resistance to chemotherapeutic drugs is often developed after several courses of treatment; vascular endothelial cells are normal cells that have stable genes and are not prone to drug resistance. The development of drug resistance in tumor cells is closely related to the instability of their genes and drugs targeting vascular endothelial cells can be used to treat chemotherapy resistance and recurrent cancer.


## 4.2. Correlation between Neovascular Density and Antibody Expression

Compared with the nonspecificity and nonselectivity of ordinary ultrasound contrast agents, targeted contrast ultrasound agents carry antibodies or corresponding receptors to specifically bind to recognizable targets, and the biological binding is tight and firm, making targeted ultrasound. The contrast agent can stay in the blood pool for a longer time and with a relatively stable and higher concentration, so that the target disease area can be identified. Contrast-enhanced ultrasound uses the image basis provided by two-dimensional ultrasound to determine the blood supply and blood flow distribution of the ovarian tumor, and the richness of blood flow when the contrast agent fills the tumor. Filling speed, retreat speed, and general malignant tumors are rich in blood flow, the contrast signal is relatively bright, the contrast enhancement mode is fast, and the benign tumors caused by insufficient blood flow will fill up quickly and return slowly with high contrast contrast. The image intensity is relatively dark, and the contrast-enhanced ultrasound mode is mostly slow, uniform, and low-enhanced. The filling speed is slow, the regression is relatively fast, and two-dimensional ultrasound can provide the basis for the performance of ovarian tumors because benign and malignant tumors have rich blood flow and whether the vascular structure is heterogeneous. In the first step of tumorigenesis, high levels of strong angiogenesis inducers are released into the tumor ecosystem [16]. Figure 5 shows the neovascular density and antibody expression rate of ovarian cancer xenografts at different stages.Figure 5
Neovascular density rate (a) and antibody expression rate (b) of ovarian cancer xenografts at different stages.
(a)(b)A small amount of blood flow signals can be seen around and inside the tumor tissue through ultrasound examination and two different ultrasound contrast agents are injected from the tail vein of nude mice. In contrast to pulse sequence imaging technology, the ultrasound image shows that the contrast agent is rapidly filling from the base of the tumor to the inside, and the tumor is clearly and completely strengthened. The contrast agent gradually fades from the center of the tumor to the periphery and finally disappears completely and the contrast-enhanced ultrasound characteristics of the two are characterized by fast in and slow out. Because the imaging characteristics of different microbubbles in different tissues are different, ultrasound microbubbles are mainly divided into ordinary microbubbles and targeted microbubbles. Studies have pointed out that ultrasound microbubbles can enhance the imaging intensity of human tissues and various organs and can also significantly increase the imaging intensity of tumor tissues, so it is of great significance for the diagnosis of benign and malignant diseases. With the development of targeted therapy technology, more explorations need to be made on the path of simultaneous diagnosis and treatment of ultrasound microbubbles. It can specifically bind to ovarian cancer cells in vitro, can enhance the image development in vivo, and can stay in the target tissue for a long time and slowly fade away and bring new perspectives and ideas to the early diagnosis of ovarian cancer.After the targeted microbubbles specifically bind to the receptors on the membrane of ovarian cancer cells, targeted sound hole formation is realized under ultrasound positioning irradiation, which widens the intercellular space of tumor tissues and greatly improves membrane permeability. This is conducive to the uptake of surrounding substances by tumor cells, allowing more substances to enter the cell to exert antitumor effects. In addition, ultrasound microbubbles have a certain apoptosis-inducing effect on tumor cells, so this group inhibits cell proliferation and induces apoptosis effects that are the most obvious [17]. This group of substances can only diffuse into tumor cells by themselves without the effect of ultrasound microbubbles, and this ovarian cancer cell is resistant to it, so the effect of its single action is significantly worse than this group. The drug concentration in the tumor tissue of the tumor-bearing mice in each group also confirmed the above speculation. The concentration of the substance in the tumor tissue was the highest in the ultrasound group targeting microbubbles. Low-dose ultrasound and targeted microbubbles did not cause significant damage to tumor tissue cells, and there was no significant difference compared with the control group. Although ultrasound microbubbles had a weaker apoptosis-inducing effect on tumor cells, it was compared with the control group and the difference is still significant. Death is a type of programmed cell death that occurs in the body’s cells through the regulation of intracellular genes and their products under physiological or pathological conditions, which is closely related to the occurrence, development, and treatment of tumors.


## 5. Discussions

### 5.1. Correlation Analysis of Targeted Contrast-Enhanced Ultrasound and Neovascular Density

The targeted contrast ultrasound agent binds to the agent through the receptor in the body, so that the targeted contrast agent stays in the specific target tissue and the application of ultrasound contrast technology can perform specific imaging of the tissue. Therefore, the targeted contrast agent in the preparation of and the choice of the target and the choice of the binding mode of the microbubbles and the target are very important. A large number of studies have shown that the high-speed, high-efficiency, high-affinity, and multistage amplification effect between biotin make the biotin system immune labeling and related tracer analysis more sensitive. The results of this study showed that the tumor cells of the nude mouse model of ovarian tumor transplantation were arranged in a solid sheet, the tumor cell nucleus was enlarged, the interstitium promoted the proliferation of fibrous tissue, and the inflammatory cell infiltration could be seen around it, suggesting the construction of the nude mouse model of ovarian cancer success [18]. Low-dose ultrasound and targeted microbubbles did not cause significant damage to tumor tissue cells, so there was no significant difference compared with the control group. Studies believe that the invasion and metastasis of ovarian cancer cells and the emergence of chemotherapy drug resistance are the main reasons for the poor prognosis and high mortality of ovarian cancer. The nude mouse model of ovarian transplantation tumor is an important means to explore the invasion and metastasis mechanism of ovarian cancer. Figure 6 shows the correlation between neovascular density and peak intensity in the 2-, 3-, 4-, and 5-week groups.Figure 6
Correlation between neovascular density and peak intensity in the 2-week (a), 3-week (b), 4-week (c), and 5-week (d) groups.
(a)(b)(c)(d)As a new type of drug carrier, liposome particles can be passively targeted to organs such as the liver and spleen after entering the body, but it is difficult to transport them to other organs. In this context, the surface of liposome is modified by specific antibodies or peptide chains to form new active targeting agents. Studies have found that luteinizing hormone-releasing hormone receptors are overexpressed in ovarian cancer, but in normal, ovarian tissue is almost not expressed, and the receptor has now become a new target for ovarian cancer-targeted preparation research. This may be related to the small particle size of the targeted oil liposome prepared in this experiment and the average particle size is liposome. After intravenous administration, it is easy to escape the capture of the system and can be widened. The tumor vascular endothelial space frees the blood vessels, binds specifically to the receptor on the tumor surface, enters the cell through receptor-mediated processes, and plays a targeted antitumor effect. It uses a thin film dispersion method combined with a biotin bridging method to prepare targeted oil liposome and observes its physical properties and in vivo targeting ability based on the establishment of a nude mouse ovarian cancer subcutaneous xenograft model. It is to systematically explore the therapeutic effects and possible mechanisms of the targeted liposome on subcutaneous xenografts of ovarian cancer in nude mice and provide a new method for targeted drug therapy for ovarian cancer [19].Vascular endothelial growth factor is the strongest and most specific vascular growth factor identified so far, which is closely related to tumor growth. Among the factors related to angiogenesis, studies have confirmed that vascular endothelial growth factor is overexpressed in most tumor cells. It is the most powerful proangiogenic factor in human tumor tissues, and the expression level of substances in tumor tissues is highly positively correlated with tumor microvessel density, degree of malignancy, and metastasis, while it is significantly negatively correlated with the prognosis of patients. Subcutaneous cancer xenografts nodules and surrounding tissues are clearly demarcated, the tumor tissue has a thin layer of fibrous tissue envelope, cancer nests are unclear, there are a few fibers and blood vessels between cancer nests, and cancer cells are of different sizes, staining depths, and cytoplasm. The coloration is lighter, a small number of cell nuclei shrink, occasionally, apoptotic cells, necrotic tissue, and inflammatory cells infiltrate the tumor tissue to form fibrosis, showing that the growth of tumor cells is inhibited. This model validates the working hypothesis of mouse cancer xenograft cell apoptosis, inhibits the growth of human ovarian cancer xenografts in nude mice, and explores its antitumor mechanism, trying to provide an experimental and theoretical basis for finding therapeutic drugs for ovarian cancer with high-efficiency and low-toxic side effect.


### 5.2. Correlation Analysis of Targeted Contrast Ultrasound Parameters and Angiogenic Mimicry

The antibodies carried are mostly concentrated in the extracellular matrix of the tumor and less concentrated in the neovascular density, suggesting that the neovascular density produces specific antigens in the early stage of ovarian cancer, which increases the invasiveness of ovarian cancer; and late ovarian cancer antigens produced by neovascularization in the extracellular matrix provide nutrition for ovarian cancer and accelerate tumor growth. In order to eliminate the echo from the tissue, digital subtraction is used when analyzing the picture; that is, the picture after the contrast is subtracted from the picture before the contrast, and the resulting picture is the intensity of the contrast enhancement. In order to visually observe the difference in imaging of the two contrast agents, color coding is adopted; that is, the enhanced part is expressed in color, and then the enhanced part in color is superimposed with the image before the contrast to obtain the image after the contrast. Ovarian tumors are rich in blood supply and grow rapidly and they are typical angiogenesis-dependent tumors; antiangiogenesis treatments should achieve good results and tumor angiogenesis mainly depends on the balance between vascular endothelial cell proliferation factors and inhibitors. In the research of tumor ultrasound molecular imaging, targeting tumor neovascularization has the most potential. By firmly connecting the agent and the contrast agent, the targeted visualization of tumor blood vessels can be achieved in vivo, which is expected to be used for early diagnosis and efficacy evaluation of tumors. Figure7 shows the correlation between neovascular density and time to peak in the 2-, 3-, 4-, and 5-week groups.Figure 7
Correlation between neovascular density and time to peak in the 2-week (a), 3-week (b), 4-week (c), and 5-week (d) groups.
(a)(b)(c)(d)The targeted contrast ultrasound parameters have been discovered that circadian clock genes are related to cell cycle regulation. They not only are necessary to maintain normal biological rhythms but also may participate in the growth and regulation of tumors and have an inhibitory effect on tumor growth, and breaking the rhythm may lead to female endocrine disorders, which may be related to tumor occurrence. Ovarian cancer is closely related to female endocrine, and female endocrine is regulated by the circadian rhythm of gene species. The activation of this signal pathway in malignant tumors not only stimulates the malignant proliferation of cells but also stimulates tumor angiogenesis, thereby further promoting the occurrence and development of tumors. The intuitive analysis of the image after the contrast is because the nontargeted contrast agent is basically completely metabolized in the body at this time, which is more conducive to comparing and observing the imaging effects of the two contrast agents. Angiogenesis is an important condition for tumor growth and metastasis, it is also common to all malignant tumors, and blocking tumor angiogenesis may prevent the further development of tumors. Vascular endothelial growth factor is the strongest known growth factor that directly affects vascular endothelial cells. Cooperation with other vascular permeability factors can promote the division and proliferation of endothelial cells from different sources and the construction of blood vessels, and increase the permeability of microvessels [20].Ascites xenografts reflect the characteristics of ovarian cancer cell growth in the abdominal cavity and the formation of ascites, which conforms to the clinical manifestations of human ovarian cancer to a certain extent and provides a reliable research carrier for drug selection and biological treatment of chemotherapy. The lesions often lack the appearance of solid tumors, and the survival time of the ascites xenografts model is significantly shorter than that of the other two models, which may be related to the model’s simulation of the formation of massive ascites in advanced ovarian cancer, water-electrolyte imbalance [6]. The advantage of this model is that it forms and grows in a single form at the inoculation site and the tumor continuously expands and grows to the surrounding tissues, which is clearly separated from the surrounding tissues, and it is easy to be surgically stripped by visual observation from outside [21]. It is often used for experimental observation and detection of cancer xenografts growth and as a basis for evaluation of drug efficacy, but in fact, the method of measuring subcutaneously implanted tumors with calipers is extremely inaccurate. Its limitation is that it can only measure the subcutaneous tumor at the edge of the muscle, and miss the part of the tumor that penetrates into the deep layer of the tissue. The measurement value takes into account the error between the inflammation part of the skin and the tissue surrounding the tumor [22]. Therefore, this model has certain limitations in displaying other biological characteristics of tumor cells, and its purpose is mainly used for chemotherapeutic susceptibility testing, imaging, and related studies of gene expression.


## 5.1. Correlation Analysis of Targeted Contrast-Enhanced Ultrasound and Neovascular Density

The targeted contrast ultrasound agent binds to the agent through the receptor in the body, so that the targeted contrast agent stays in the specific target tissue and the application of ultrasound contrast technology can perform specific imaging of the tissue. Therefore, the targeted contrast agent in the preparation of and the choice of the target and the choice of the binding mode of the microbubbles and the target are very important. A large number of studies have shown that the high-speed, high-efficiency, high-affinity, and multistage amplification effect between biotin make the biotin system immune labeling and related tracer analysis more sensitive. The results of this study showed that the tumor cells of the nude mouse model of ovarian tumor transplantation were arranged in a solid sheet, the tumor cell nucleus was enlarged, the interstitium promoted the proliferation of fibrous tissue, and the inflammatory cell infiltration could be seen around it, suggesting the construction of the nude mouse model of ovarian cancer success [18]. Low-dose ultrasound and targeted microbubbles did not cause significant damage to tumor tissue cells, so there was no significant difference compared with the control group. Studies believe that the invasion and metastasis of ovarian cancer cells and the emergence of chemotherapy drug resistance are the main reasons for the poor prognosis and high mortality of ovarian cancer. The nude mouse model of ovarian transplantation tumor is an important means to explore the invasion and metastasis mechanism of ovarian cancer. Figure 6 shows the correlation between neovascular density and peak intensity in the 2-, 3-, 4-, and 5-week groups.Figure 6
Correlation between neovascular density and peak intensity in the 2-week (a), 3-week (b), 4-week (c), and 5-week (d) groups.
(a)(b)(c)(d)As a new type of drug carrier, liposome particles can be passively targeted to organs such as the liver and spleen after entering the body, but it is difficult to transport them to other organs. In this context, the surface of liposome is modified by specific antibodies or peptide chains to form new active targeting agents. Studies have found that luteinizing hormone-releasing hormone receptors are overexpressed in ovarian cancer, but in normal, ovarian tissue is almost not expressed, and the receptor has now become a new target for ovarian cancer-targeted preparation research. This may be related to the small particle size of the targeted oil liposome prepared in this experiment and the average particle size is liposome. After intravenous administration, it is easy to escape the capture of the system and can be widened. The tumor vascular endothelial space frees the blood vessels, binds specifically to the receptor on the tumor surface, enters the cell through receptor-mediated processes, and plays a targeted antitumor effect. It uses a thin film dispersion method combined with a biotin bridging method to prepare targeted oil liposome and observes its physical properties and in vivo targeting ability based on the establishment of a nude mouse ovarian cancer subcutaneous xenograft model. It is to systematically explore the therapeutic effects and possible mechanisms of the targeted liposome on subcutaneous xenografts of ovarian cancer in nude mice and provide a new method for targeted drug therapy for ovarian cancer [19].Vascular endothelial growth factor is the strongest and most specific vascular growth factor identified so far, which is closely related to tumor growth. Among the factors related to angiogenesis, studies have confirmed that vascular endothelial growth factor is overexpressed in most tumor cells. It is the most powerful proangiogenic factor in human tumor tissues, and the expression level of substances in tumor tissues is highly positively correlated with tumor microvessel density, degree of malignancy, and metastasis, while it is significantly negatively correlated with the prognosis of patients. Subcutaneous cancer xenografts nodules and surrounding tissues are clearly demarcated, the tumor tissue has a thin layer of fibrous tissue envelope, cancer nests are unclear, there are a few fibers and blood vessels between cancer nests, and cancer cells are of different sizes, staining depths, and cytoplasm. The coloration is lighter, a small number of cell nuclei shrink, occasionally, apoptotic cells, necrotic tissue, and inflammatory cells infiltrate the tumor tissue to form fibrosis, showing that the growth of tumor cells is inhibited. This model validates the working hypothesis of mouse cancer xenograft cell apoptosis, inhibits the growth of human ovarian cancer xenografts in nude mice, and explores its antitumor mechanism, trying to provide an experimental and theoretical basis for finding therapeutic drugs for ovarian cancer with high-efficiency and low-toxic side effect.


## 5.2. Correlation Analysis of Targeted Contrast Ultrasound Parameters and Angiogenic Mimicry

The antibodies carried are mostly concentrated in the extracellular matrix of the tumor and less concentrated in the neovascular density, suggesting that the neovascular density produces specific antigens in the early stage of ovarian cancer, which increases the invasiveness of ovarian cancer; and late ovarian cancer antigens produced by neovascularization in the extracellular matrix provide nutrition for ovarian cancer and accelerate tumor growth. In order to eliminate the echo from the tissue, digital subtraction is used when analyzing the picture; that is, the picture after the contrast is subtracted from the picture before the contrast, and the resulting picture is the intensity of the contrast enhancement. In order to visually observe the difference in imaging of the two contrast agents, color coding is adopted; that is, the enhanced part is expressed in color, and then the enhanced part in color is superimposed with the image before the contrast to obtain the image after the contrast. Ovarian tumors are rich in blood supply and grow rapidly and they are typical angiogenesis-dependent tumors; antiangiogenesis treatments should achieve good results and tumor angiogenesis mainly depends on the balance between vascular endothelial cell proliferation factors and inhibitors. In the research of tumor ultrasound molecular imaging, targeting tumor neovascularization has the most potential. By firmly connecting the agent and the contrast agent, the targeted visualization of tumor blood vessels can be achieved in vivo, which is expected to be used for early diagnosis and efficacy evaluation of tumors. Figure7 shows the correlation between neovascular density and time to peak in the 2-, 3-, 4-, and 5-week groups.Figure 7
Correlation between neovascular density and time to peak in the 2-week (a), 3-week (b), 4-week (c), and 5-week (d) groups.
(a)(b)(c)(d)The targeted contrast ultrasound parameters have been discovered that circadian clock genes are related to cell cycle regulation. They not only are necessary to maintain normal biological rhythms but also may participate in the growth and regulation of tumors and have an inhibitory effect on tumor growth, and breaking the rhythm may lead to female endocrine disorders, which may be related to tumor occurrence. Ovarian cancer is closely related to female endocrine, and female endocrine is regulated by the circadian rhythm of gene species. The activation of this signal pathway in malignant tumors not only stimulates the malignant proliferation of cells but also stimulates tumor angiogenesis, thereby further promoting the occurrence and development of tumors. The intuitive analysis of the image after the contrast is because the nontargeted contrast agent is basically completely metabolized in the body at this time, which is more conducive to comparing and observing the imaging effects of the two contrast agents. Angiogenesis is an important condition for tumor growth and metastasis, it is also common to all malignant tumors, and blocking tumor angiogenesis may prevent the further development of tumors. Vascular endothelial growth factor is the strongest known growth factor that directly affects vascular endothelial cells. Cooperation with other vascular permeability factors can promote the division and proliferation of endothelial cells from different sources and the construction of blood vessels, and increase the permeability of microvessels [20].Ascites xenografts reflect the characteristics of ovarian cancer cell growth in the abdominal cavity and the formation of ascites, which conforms to the clinical manifestations of human ovarian cancer to a certain extent and provides a reliable research carrier for drug selection and biological treatment of chemotherapy. The lesions often lack the appearance of solid tumors, and the survival time of the ascites xenografts model is significantly shorter than that of the other two models, which may be related to the model’s simulation of the formation of massive ascites in advanced ovarian cancer, water-electrolyte imbalance [6]. The advantage of this model is that it forms and grows in a single form at the inoculation site and the tumor continuously expands and grows to the surrounding tissues, which is clearly separated from the surrounding tissues, and it is easy to be surgically stripped by visual observation from outside [21]. It is often used for experimental observation and detection of cancer xenografts growth and as a basis for evaluation of drug efficacy, but in fact, the method of measuring subcutaneously implanted tumors with calipers is extremely inaccurate. Its limitation is that it can only measure the subcutaneous tumor at the edge of the muscle, and miss the part of the tumor that penetrates into the deep layer of the tissue. The measurement value takes into account the error between the inflammation part of the skin and the tissue surrounding the tumor [22]. Therefore, this model has certain limitations in displaying other biological characteristics of tumor cells, and its purpose is mainly used for chemotherapeutic susceptibility testing, imaging, and related studies of gene expression.


## 6. Conclusions

In order to explore the correlation between targeted contrast-enhanced ultrasound imaging and tumor neovascularization of ovarian cancer xenografts in nude mice, this study selected 49 ovarian cancer xenografts in nude mice and divided them into 7 groups for normal and targeted contrast-enhanced ultrasound examinations and then analyzed the correlation between targeted contrast-enhanced ultrasound parameters and tumor neovascular densities of the ovarian cancer xenografts. The results showed that the peak intensity of targeted contrast-enhanced ultrasound imaging for early-stage ovarian cancer was greater than that of ordinary contrast-enhanced ultrasound imaging and was positively correlated with tumor neovascular density; the peak time of targeted contrast-enhanced ultrasound imaging was shorter than that of normal contrast-enhanced ultrasound imaging and was negatively correlated with tumor neovascular density. Compared with the nonspecificity and nonselectivity of ordinary ultrasound contrast agents, targeted contrast ultrasound agents carry antibodies or corresponding receptors to specifically bind to recognizable targets, and the biological binding is tight and firm. The targeted contrast ultrasound agent binds to the agent through the receptor in the body, so that the targeted contrast agent stays in the specific target tissue. The application of ultrasound contrast technology can perform specific imaging of the tissue and the targeted contrast agent in the preparation of and the choice of the target and the choice of the binding mode of the microbubbles and the target are very important. Therefore, it is believed that the targeted contrast-enhanced ultrasound imaging parameters have a certain correlation with tumor neovascular density of ovarian cancer xenografts in nude mice and this correlation is more significant in the early stage of ovarian cancer; hence, targeted contrast-enhanced ultrasound imaging may provide a new method, new idea, and new basis for the diagnosis of early ovarian cancer. The results of this paper provide a reference for further researches on the correlation between targeted contrast-enhanced ultrasound imaging and tumor neovascularization of ovarian cancer xenografts in nude mice.



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