|Year : 2014 | Volume
| Issue : 4 | Page : 973-978
Study on application of ultrasound microbubbles in the diagnosis of buccal carcinoma and metastatic cervical lymph nodes in mice
Xin Zhao1, Liang Pang2, Zhi Gao3, Kai Zhu1, Guijie Li1, Rui Wang1, Yu Qian1, Lihua Qiu2
1 Department of Biological and Chemical Engineering, Chongqing University of Education, Chongqing, China
2 Department of Oral and Maxillofacial Surgery, The Affiliated Hospital of Stomatology; Chongqing Research Center for Oral Diseases and Biomedical Science, Chongqing Medical University, Chongqing, China
3 Department of Stomatology, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, China
|Date of Web Publication||9-Jan-2015|
Department of Oral and Maxillofacial Surgery, The Affiliated Hospital of Stomatology, Chongqing Medical University, Chongqing 401147
Source of Support: This study was supported by the Chongqing Education Committee (Yukejiao (2010) No. 6 KJ100322), People’s Republic of China, Conflict of Interest: None
Background: This project's aim was to explore the enhancing effect of contrast media on primary cancer and cervical metastatic lymph nodes of cheek carcinoma by local injection.
Materials and Methods: Lymphatic metastasis cheek carcinoma mice models were established, then self-made contrast media was administered into the primary cheek carcinoma and harmonic mode imaging was performed to observe the enhancement of primary lesions and cervical metastatic lymph nodes.
Results: The echo intensity (EI) was compared. The detection rate of metastatic lymph nodes and inflammatory lymph nodes was 61% (17/28) and 50% (6/12) before contrast-enhanced ultrasound (CEUS) imaging and 82% (23/28) and 75% (9/12) after CEUS imaging. After CEUS imaging, the EI of primary cancer and cervical metastatic lymph nodes was significantly increased, (P < 0.01).
Conclusion: After injection of self-made contrast media via the primary lesion, it can enter the lymph duct and obviously enhance the effect of ultrasonography on cervical metastatic lymph nodes of cheek carcinoma.
Keywords: Cervical, imprinting control region mice, lymph nodes, metastatic, microbubble
|How to cite this article:|
Zhao X, Pang L, Gao Z, Zhu K, Li G, Wang R, Qian Y, Qiu L. Study on application of ultrasound microbubbles in the diagnosis of buccal carcinoma and metastatic cervical lymph nodes in mice. J Can Res Ther 2014;10:973-8
|How to cite this URL:|
Zhao X, Pang L, Gao Z, Zhu K, Li G, Wang R, Qian Y, Qiu L. Study on application of ultrasound microbubbles in the diagnosis of buccal carcinoma and metastatic cervical lymph nodes in mice. J Can Res Ther [serial online] 2014 [cited 2019 Nov 17];10:973-8. Available from: http://www.cancerjournal.net/text.asp?2014/10/4/973/138009
| > Introduction|| |
Oral squamous cell carcinoma is a type of epithelial malignancy, which occurs in the oral mucosa and is the most common oral maxillofacial malignancy. It is characterized by fast growth, strong local aggression and easy metastasis to cervical lymph nodes, which exert great influence on prognosis. , Timely diagnosis and treatment of metastatic cervical lymph nodes is an important element to improve patients' survival rate. Ultrasound microbubbles (UM) are a new type of ultrasound contrast agents (UCA), which have been developed in recent years. By changing the tissues' ultrasonic characteristics, UCA can produce contrast effects and can be applied to contrast imaging of human blood and solid organs. 
UMs, after entering the general blood circulatory system through peripheral intravenous injection, can change the interaction between sound waves and tissues by altering acoustic attenuation, sound speed and by enhancing back scattering, thereby intensify the bloodstream's echo signal of the examined area. However, as normal tissues and focal tissues differ in their intake of contrast agents, those contrast agents can not only enhance bloodstream signal, but can also improve the signal-to-noise ratio at the same time, thus intensifying the tissues' gray scale visibility. , When microbubbles containing gas enter into bodies through intravenous injection, UMs usually stay in the blood-vascular system. In addition, they can also pass through microcirculation. Microbubbles, whose hemodynamic characteristics are similar to that of red blood cells, are relatively stable in blood vessels. When their diameter is larger than 7 μm, those detained in lungs mechanically may be destroyed by the lungs; while others whose diameter is smaller than 7 μm, can reach any other parts of the body through the lungs and will be ultimately devoured by white blood cells. As UMs are able to pass the blood brain barrier, they can enter into the brain to kill tumor cells. However, they are regarded as foreign matter by the organism. A small amount of microbubbles, <0.02 mL/kg/min, can be detected when they enter into human the human body but will not cause physiological disorder. However a large amount of microbubbles' entry into microcirculation will cause ischemia of relative microcirculation. The fatal dose is 2 mL/kg/min.  Recently, some scholars have found that a dose of 2-3 μm microbubbles, especially those which were soft-shelled and flexible, injected subcutaneously could enter into lymph vessels, mainly through intercellular space between lymphatic endothelium or via endocytosis or exocytosis. As ultrasound is very sensitive to microbubbles, lymphangion's absorption of them could be detected. If the injection site was massaged once or twice, contrast agents could fill lymph nodes again. Microbubbles were found to remain at the injection site after autopsy, which proved that low-dose microbubbles could also enter into lymph tracts in the later stages and broadbrand harmonic imaging technology could obviously improve their visibility.  Finally microbubbles were swallowed by the reticuloendothelial system cells (mainly Kupffer cells and spleen's phagocytes), however the swallowed microbubbles were still intact and still had the ability to against acoustic sources. 
Non-invasive qualitative tests for cervical lymph nodes in patients with oral carcinoma have always caused great attention. However, there hasn't been one comparatively reliable and objective imaging examination method able to distinguish tumor metastasis, inflammatory hyperplasia and normal cervical lymph nodes. According to some reports, UM contrast agents injected percutaneously could show cervical lymph nodes and detect tumor-metastatic sentinel nodes.  Scholars, such as Yang et al., found that in the part of a body injected with UM contrast agents, one could clearly distinguish tumor metastasis, inflammatory and normal cervical lymph nodes by making full use of UM sonographic echo intensity (EI), which provided iconographic basis for researchers on whether cervical lymph nodes of oral carcinoma have spread.  Until now, there haven't been any reports about UMs applied to the diagnosis and treatment of metastatic lymph nodes of oral carcinoma. By using self-made UMs, the effects were observed in the subject and cervical lymph nodes in the form of harmonic imaging after those self-made surfactant UMs were injected into primary foci and massaged locally. Meanwhile echo intensities were compared to explore the transformation, contrast effects and image features of oral carcinoma and cervical lymph nodes before and after ultrasound contrast.
| > Materials and methods|| |
A 7-week-old male ICR (Institute of Cancer Researcch) mice (n = 40) were maintained in a temperature-controlled facility (temperature 23 ± 1°C, relative humidity 50 ± 5%) with a 12-h light/dark cycle. The mice had unlimited access to a standard mouse chow diet and water.
Cancer cell preparation
U14 squamous cell carcinoma cells were obtained from the Chinese Academy of Medical Sciences (Beijing, China). The cancer cells were cultured in RPMI (Roswell Park Memorial Institute)-1640 medium (Gibco Services Inc., Birmingham, MI, USA) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco Services Inc.) at 37°C in a humidified atmosphere containing 5% CO 2 (model 311, Thermo Forma Inc., Waltham, MA, USA). The medium was changed 2-3 times each week. The in vitro-cultured U14 cells (5 × 10 6 cells/mouse) were inoculated into the abdominal cavity of 7-week-old female ICR mice. After 1 week, the carcinoma ascites were collected and diluted in sterile saline at a concentration of 1 × 10 7 cells/mL. 
Induction of buccal mucosa cancer
To establish the buccal mucosa cancer animal model, 50 mice were inoculated with 0.05 mL U14 cancer cell suspension (1 × 10 7 cells/mL) on the buccal mucosa. Ten mice were bred as normal and acted as controls. Mice that succumbed to natural causes were collected and their buccal mucosa and lymph node tissues were examined. 
Take the proper amount of surfactant contrast agents: UMs diameter of 0.8-4.0 μm (the diameter of 95% UMs below 2.0 μm), concentration 7.2 × 10 9 /mL. Span-60 was mixed with Tweens-80 in a certain proportion and a certain amount of media solution was added to it and heated until Span-60 and Tweens-80 were dissolved completely. 40 mL of the miscible liquids was taken and vibrated with sonicvibrator. Then leave the suspension static. At ½ h later, it was separated into three layers. Extract the middle layer of the suspension and wash it with media solution repeatedly. Finally, pour it into a small beaker and conserve it in a refrigerator at temperature−5°C after sealing. 
Specifications of the diasonograph were as follows: 12 L linear probes, 5.2 MHz of second harmonic emissive frequency and 10.5 MHz of receiving frequency. The mouse was anaesthetized with ether and fixed horizontally. Before the contrast, scan the ICR mice inoculated the U14 cells 20 days ago ultrasonically with conventional fundamental waves. The scan can fully show their pars buccalls' primary foci and cervical lymph nodes and store ideal imaging sections. Then same dose of 0.5 mL UCA was injected into the primary foci of every ICR mouse twice. The contrast agent must be injected beneath buccal mucosa in the form of bolus injection. After injection, the injected part should be massaged for 30 s. Moreover the time interval between the two injections must be at least 30 min. Observe the whole ultrasound imaging process in real time. Analysis and dermintation of sonographic EI: Use ultrasoundgraphic quantity analysis system (Chongqing Medical University, Chongqing, China); the analyzed area can be designed in any form; take the section sonograph of the primary foci and lymph nodes as the testing area and examine the mean gray-scale EI.
Histological analysis of buccal mucosa cancer
Buccal mucosa and lymph node tissues were removed and embedded in paraffin for histological analysis with hematoxylin and eosin staining, as previously described. 
Data are presented as the mean ± standard deviation Differences between the mean values for individual groups were assessed with a one-way analysis of variance with Duncan's multiple range test. Differences were considered significant when P < 0.05. Statistical analysis system (SAS) version 9.1 (SAS Institute Inc., Cary, NC, USA) was used for statistical analyses.
| > Results|| |
The primary foci showed image enhancement immediately after injection with UMs. Lymph nodes showed image enhancement about 5 s after injection and showed the highest density within 30 s. The image then faded away gradually. The enhancement usually appeared first around the lymph nodes and then would quickly fill up from the periphery to the center of the lymph nodes. The contrast was comparatively uniform when it was at the highest density. The echo of capsule intensified as well. No accumulation of contrast agents or rear contrast could be detected. It usually took 20-30 min for contrast sites to return to the original contrast state when injected every time [Figure 1],[Figure 2] and [Figure 3]. Visual observation of the fundamental wave before contrast: The tumor-metastatic lymph nodes were larger than the swollen ones, but the difference did not have any statistical significance (P < 0.05). The vertical section of hyperplastic lymph nodes was oval in shape with regular forms, clear boundaries and smooth surfaces. The EI was low, but with uniform distribution. The center of the lymph nodes was medulla with a dense echo and the rest was cortex with a low echo [Figure 4]. Moreover, the echo of tumor-metastatic lymph nodes was lower than that of hyperplastic ones. The internal echo and corticomedullary differentiation were unclear [Figure 3]. Sonographic observation after contrast: Compared with hyperplastic lymph nodes, the ultrasonographic enhancement effect of tumor-metastatic nodes was weaker and non-uniform and some small filling defects were visible. Period strong echo contrast agents could be seen deposit in the rear of individual lymph nodes with clear acoustic shadow in the later stages [Figure 5], and [Figure 6].
|Figure 1: Primary tumor ultrasound imaging before injecting ultrasound microbubbles into the mice|
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|Figure 2: Primary tumor ultrasound imaging after injecting ultrasound microbubbles into the mice|
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|Figure 3: Metastatic lymph node imaging before injecting ultrasound microbubbles into the mice|
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|Figure 4: Metastatic lymph node imaging after injecting ultrasound microbubbles into the mice|
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|Figure 5: Hyperplastic lymph node imaging before injecting ultrasound microbubbles into the mice|
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|Figure 6: Hyperplastic lymph node imaging after injecting ultrasound microbubbles into the mice|
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It is a video frequency method of ultrasound tissue characterization. Copy the image file directly to a computer and analyze it with DFY ultrasound graphic quantity analysis system. Take lymph node section as the test area. Set consistent gray scale sample frame areas and test their gray scale values [Table 1]. The EI value of the primary foci was (83.78 ± 1.26) generalized series (GS) before contrast and (127.65 ± 6.93) GS after contrast; the EI value of the hyperplastic lymph nodes was (74.57 ± 3.78) GS before contrast and (120.96 ± 8.17) GS after contrast; the EI value of metastatic lymph nodes was (69.23 ± 3.54) GS before contrast and (106.19 ± 2.98) GS after contrast; there was obvious statistical difference (P < 0.05) in EI values before and after contrast; hyperplastic lymph nodes were bigger than metastatic ones after contrast.
|Table 1: The EI of the primary tumor and lymph nodes before and after inject UM|
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All 40 mice left faces became swollen and formed tumors. In addition, their necks also became swollen. All the mice were euthanized after the contrast experiment and a microscope was used to observe their buccual and cervical lymph node tissues after HE stain. The result showed that all buccal tissues contained primary tumors [Figure 7]. 28 of the mice's pathological section showed that the centers of their lymph nodes became necrotic and occurred metastasis occurred [Figure 8] and there were hyperplastic lymph node occurred in 12 mice [Figure 9]. Before ultrasound contrast, the positive rate of metastatic lymph node was 60.7% (17 mouse/28 mouse), the positive rate of hyperplastic lymph node was 50.0% (6 mouse/12 mouse); after ultrasound contrast, the positive rates were increased to 82.1 (23 mouse/28 mouse) and 75.0 (9 mouse/12 mouse).
| > Discussion|| |
U14 cells are a squamous cell carcinoma cell line  that are ectopically induced by treating the uterine cervix with methylcholanthrene thread. They are able to induce cancer by implantation into the subskin of adult mice.  In the early stages, its structure is similar to a carcinosarcoma. It is considered an undifferentiated carcinoma, with a metastasis rate of 95% in the lymph node and 80% in the lungs. Moreover, it is a bidirectional metastasis tumor strain and an optimum model that is widely used in studies on tumor metastasis, invasion, recurrence and drug screening. This tumor strain is characterized by low cell differentiation, high proliferation, extremely strong infiltration and metastasis. In addition, its animal inoculation survival rate is capable of reaching 100%.  The U14 cells injected mice model can used in medical research on the buccal mucosa cancer and cervical lymph node metastasis.
With the development and popularization of the UCA, tissue ultrasound contrast is developing rapidly. It has been an effective method for ultrasound diagnosis and treatment. UM contrast agent injection cannot only help detect the foci unable to be detected by two-dimensional ultrasound, but also can help identify different lesions. In general, a good contrast effect of blood vessels and solid organs can be achieved by injecting UM contrast agents into human bodies through blood vessels. However, it is difficult to get a satisfactory ultrasonoscopy through vessel injection as microbubbles cannot reach some organs and/or microbubble concentration has reduced before their arrival at their destination. Thus this kind of permeation restricts the sphere of ultrasound contrast study, especially the sphere of the lymphatic system study. As non-invasiveness has always been an advantage of ultrasonic tests, the indirect injection of the lymphatic system is an important research agenda. This kind of injection is also called injection in tissues, which injects colored injection into tissues and organs' interstitial spaces. Due to the existence of open gaps between lymphatic capillary endothelial cells, injection can enter into lymphatic capillaries with the help of injection pressure and injection diffusion, thus showing clearly showing lymphatic capillaries, collecting lymphatic vessels and lymph nodes. , In the experiment, self-made surfactant active agents could successfully enter into the homolateral popliteal lymph nodes through Palma injection. Lymph node EI of each group intensified in different extent after contrasts. However, the visual enhancement effect of the tumor-metastatic group was not as good as the other two groups, whose EI was lower. What's more, some irregular filling defects could be detected.  Strong echo contrast agents could be seen deposited in the rear of individual lymph nodes with clear acoustic shadow in the later stages.
Qualitative tests for lymph nodes have always caused great attention. Tests for identification of tumor-metastatic lymph nodes, in particular, are of great significance to tumor stage and prognosis. Clinically, an accurate result can generally be achieved by cutting off lymph nodes and by checking pathological sections. But how to detect sentinel nodes in an early phase and diagnose metastatic lymph nodes non-invasively has always been a research puzzle for oral and maxilofacial surgery and imageology. , Scholars such as Yang et al., have successfully built large animal models of inflammatory and tumor-metastatic lymph nodes. They used sonography to test EI changes and imaging effects of each model before and after ultrasound contrasts and discovered that sonographic EI could distinguish tumo-metastatic, inflammatory and normal lymph nodes before contrasts. However, the differences could be observed more clearly after microbubble contrasts. , The discovery laid the imageological foundation for the experiment.
| > Conclusions|| |
The experiment built animal models with buccal carcinoma and cervical lymph nodes metastasis based on the U14 cells. It then made ultrasonographic comparison of buccal primary foci and cervical hyperplastic lymph nodes before and after ultrasound contrasts. The results showed that the use of contrast agents clearly intensified the imaging of the primary foci, hyperplastic lymph nodes and metastatic ones. Before contrast, the vertical section of hyperplastic lymph nodes was oval in shape with clear capsule boudaries and low but uniform EI. However, the internal echo of metastatic lymph nodes and corticomedullary differentiation were both unclear. After contrast, compared with hyperplastic lymph nodes, the ultrasonographic enhancement effect of tumor-metastatic nodes was weaker and nonuniform and some small filling defects were visible. It proved that local injection of self-made acoustic contrast agent could intensify lymph node images and a good imaging effect could be achieved even in a harmonic condition. Cervical lymph nodes showed imaging enhancement 5s after UCA injection. The enhancement first appeared around lymph nodes and then filled up quickly from the periphery to the center of lymph nodes. Furthermore, the experiment could make a quantitative analysis with the help of EI of the video frequency method of ultrasound tissue characterization. It provided a new detection and diagnosis method for research into the lymphatic system, especially in pathological changes of lymph nodes. Meanwhile, it laid the foundation for UCA targeted detection and diagnosis of the lymphatic system as well as proving cervical lymphatic metastatic routes of buccal carcinoma. However, UCA used in the experiment is still at the experimental stagethus, the application of it to large animals and clinical treatment requires further studies.
| > Acknowledgment|| |
This study was supported by the Chongqing Education Committee (Yukejiao (2010) No. 6 KJ100322) and Program for Innovation Team Building at Institutions of Higher Education in Chongqing (KJTD201325), People's Republic of China.
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