# The Significance of PAX8-PPARγ Expression in Thyroid Cancer and the Application of a PAX8-PPARγ-Targeted Ultrasound Contrast Agent in the Early Diagnosis of Thyroid Cancer

Authors: Tianbing Fan; Wenbo Zhu; Min Kong; Xiaochan Yang; Cheng Wang; Min Wang; Zhaoyin Wang
Journal: Contrast Media & Molecular Imaging
 (2022)

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## Abstract

Objective. To investigate the significance of PAX8-PPARγ expression in thyroid cancer and the application of a PAX8-PPARγ-targeted ultrasound contrast agent in the early diagnosis of thyroid cancer. Methods. In this study, the expression of PAX8-PPARγ in thyroid cancer tissues, paracancer groups, and normal thyroid tissues was detected by western and immunohistochemical techniques; the effects of PAX8-PPARγ expression inhibition on thyroid cancer cell growth, clonogenic ability, and antiapoptosis were examined. The terminal carboxylactic acid/hydroxyacetic acid copolymer (PLGA-COOH) nanoparticles were prepared by the double emulsification solvent volatilization method. The in vitro cytotoxicity of the targeted contrast agent was detected by MTS and other methods; LD50 was used to evaluate its short-term in vivo toxicity after intraperitoneal injection in mice. Results. PAX8-PPARγ expression was significantly increased in thyroid cancer tissues, and the expression level of PAX8-PPARγ was closely correlated with TNM staging and lymph node metastasis (P < 0.05). In addition, PAX8-PPARγ was also expressed at high levels in thyroid cancer cell lines relative to normal thyroid cells. MTS experiments showed that the PAX8-PPARγ-targeted ultrasound nanocontrast agent had no significant toxic side effects on thyroid cells; countess observed that the contrast agent had no effect on cell survival and mortality; the LD50 assay showed that the targeted contrast agent had a wide safety range. Western blot showed the expression of caspase-3, BAX, and Bcl-2 in thyroid cancer cells, indicating that the nanocontrast agent has a good biosafety. In vitro targeting experiments showed that there were more nanospheres aggregated around the cells in the targeted contrast group. In vivo targeting imaging of nude mice revealed that the ultrasound signal was significantly enhanced in the targeted group compared with the nontargeted group after 20 min of LIFU irradiation. Conclusion. PAX8-PPARγ overexpression in thyroid cancer cell lines and thyroid cancer tissues promoted the proliferation and antiapoptotic ability of thyroid cancer cells and promoted the tumorigenic ability in nude mice in vivo. We successfully prepared a PAX8-PPARγ-targeted ultrasound nanocontrast agent, which has regular morphology, uniform size, and high stability, and its liquid-gas phase change can be promoted at lower temperature. Therefore, this contrast agent can achieve US-targeted imaging and temperature phase transition function, and may have enhanced ultrasound imaging potential.


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## Body

## 1. Introduction

Thyroid cancer is the most common malignancy of the endocrine system and although its mortality rate is low, especially in stage I or II of tumor development, the 10-year survival rate of patients is higher than 98%; however, it is of concern that when the tumor is detected in a more advanced stage or when distant metastases are present, it can reduce the survival rate to 56% [1, 2]. Currently, the clinical diagnosis of thyroid tumors is still based on pathological diagnosis as the gold standard, but surgical resection treatment is an invasive test. Ultrasound-guided thyroid aspiration cytology, which is widely used in clinical practice, has partially reduced the invasiveness, but many clinical challenges are often encountered, such as small tumors that are difficult to get to the location of the tumor, small amounts of tissue or tissue damage that makes pathology difficult to make a correct diagnosis, and surgical biopsy is an absolute contraindication for patients with bleeding tendency [3]. Therefore, it is important to study new methods for noninvasive and targeted diagnosis of thyroid tumors.In recent years, with the continuous research in the ultrasound neighborhood, drug- or gene-loaded microbubbles have been successfully prepared, which can be coupled with monoclonal antibodies to nanoparticles or microbubbles by various methods for targeted contrast, for drug- and gene-loaded purposes, providing new ideas for the diagnosis of malignant tumors [4, 5]. Leong-Poi et al. showed that targeted nanobubble contrast agents have good specificity by coupling specific antibodies on the surface of lipid microbubbles to achieve targeted visualization of the tumor vessels [6, 7]. Thus, molecular imaging based on targeted nanobubbles brings a new hope for the early noninvasive diagnosis of tumors and has become a hot topic of research in the biomedical neighborhood in recent years.It has been shown that the coding region of the paired cassette gene, chromosome 2q13, is fused to the coding region of the peroxisome proliferator-activated receptorγ, 3q13, to form a new gene, PAX8-PPARγ, which is characteristically expressed in follicular thyroid cancer (FTC) and also in other thyroid tumor tissue types; its positive expression rate varies among different ethnic and geographical populations. The positive rate of expression also varies among different ethnic and geographical groups [8, 9].In recent years, new methods based on mutations and other molecular markers have been partially applied in differential diagnosis of thyroid tumors (e.g., BRAF, TP53, CTNNB1, and other genes), and these molecular markers have significantly improved the diagnostic accuracy of cytology and have had a profound impact on the classification and management of patients with thyroid tumors. However, the application of nanotechnology combined with specific antibody targeting visualization technology in non-invasive imaging diagnosis of thyroid cancer has not been reported yet. Since our previous experiments confirmed that PAX8-PPARγ was expressed in solid tumor tissues of thyroid cancer and had an impact on its malignant biological behavior both in vivo and ex vivo, this would potentially provide a new molecular tumor marker for thyroid cancer and provide the necessary experimental basis for the next quantitative assessment of PAX8-PPARγ aggregation in thyroid cancer by molecular imaging methods in vivo. Therefore, we envision that a breakthrough point needs to be found for the early noninvasive diagnosis of thyroid cancer with PAX8-PPARγ coupled with an ultrasound nanobubble contrast agent.Based on the above theories, this project proposes to prepare PAX8-PPARγ-targeted ultrasound contrast agents, and it is our main goal to explore new methods based on nanomicrobubble targeting to mediate thyroid tumor diagnosis using the constructed cellular and animal models. By coupling the thyroid-specific recognition antibody PAX8-PPARγ and magnetic materials on the nanomicrobubbles, we can target PAX8-PPARγ on the cell membrane surface of thyroid cancer cells and target PAX8-PPARγ on the animal model. PPARγ and in vivo tracing of tumors in animals, which increases the sensitivity of ultrasound imaging of thyroid tumors and can confirm the microscopic pathological basis of thyroid cancer lesions based on the anti-PAX8-PPARγ-targeted polymeric contrast agent, improves the early, non-invasive, and accurate diagnosis of thyroid cancer, and helps to explore the next step of molecularly targeted therapy for thyroid cancer based on it.In summary, this project can provide a new theoretical basis and drug targets for the clinical targeting treatment of thyroid cancer through the development of a novel, efficient, and specific nanotargeted ultrasound contrast agent with clinical application prospects, which can diagnose thyroid cancer noninvasively and early, and has not been reported in the literature at home and abroad.


## 2. Method

### 2.1. Research Subjects

Sixty-five cases of surgically resected TC tissue and corresponding paracancerous tissue (5 cm from the edge of the cancer foci) were collected from May 2018 to July 2019 in our thyroid surgery, and the patients' ages ranged from 22 to 71 years, with a median age of 44 years; 18 of them were male and 47 were female. All patients were diagnosed with primary thyroid cancer preoperatively.Patients who had not yet undergone systemic antitumor therapy and who were included or their families signed the informed consent form for this study.


### 2.2. Expression Levels of PAX8-PPARγ mRNA in Tissues and Cells by qPCR

RNAiso Plus was used to extract total RNA from tissues or cells, and the concentration of RNA was measured spectrophotometrically. 1μg of RNA was diluted to 1 ml of nuclease-free water and used as a template for reverse transcription. Primer sequences were as follows: PAX8-PPARγ forward: 5′-TTCGATGTCTCTAGGGTGGC-3′ and reverse: 5′-GATCCACCTCTCTCGCTCTCTCAG-3′. 10 μl of the SYBR Green nucleic acid fluorescent dye system was used for the qPCR, and three parallel wells were set up, after predenaturation at 95°C for 10 min, denaturation at 95°C for 15 s, annealing at 60°C for 30 s, extension at 72°C for 45 s, and 40 cycles. The expression levels of UCHL5 and BRAF mRNA in tissues and cells were calculated by the 2-ΔΔCT method using β-actin as the internal reference gene.


### 2.3. WB Detection of UCHL5 and BRAF Protein Expression Levels in TC Tissues and Cells

The total protein of TC tissues and cells was extracted and the BCA protein quantification kit was used to detect protein concentration, take 40μg protein line, 10% SDS-PAGE, 120 V constant pressure for 2 h, 260 mA constant flow wet transfer to the PVDF membrane, Add UCHL5 and BRAF antibodies (1 : 1 000) and incubate it overnight in a refrigerator at 4°. The film was exposed to the ChemiDoc XRS + imaging system (Bio-Rad, USA), and the grayscale values of the bands and the relative protein expression were calculated using ImagePlus.


### 2.4. Characterization of Targeted Ultrasound Contrast Agents

#### 2.4.1. NPs-PAX8-PPARγ General Properties Assay

(1)
Contrast agent morphology and distribution were observed under light microscopy(2)
Morphology and distribution under scanning electron microscopy(3)
Measurement of nanoparticle size and a zeta potential analyzer to detect its particle size


### 2.5. Flow Cytometry Detection of Binding Rate

The other one was incubated with a fluorescently labeled secondary antibody for 30 min and was mixed every 10 mins by using a gun tip, and then the binding rate was detected by flow cytometry in each group.


#### 2.5.1. NPs-PAX8-PPARγ In Vitro Thermal Phase Change Experiments

(1)
Adjust the heating plate put in position, so that the microscope observation is simple and easy to operate(2)
Several drops of nanoparticles were placed on the heating plate(3)
The heating plate temperature was set to 60°C


#### 2.5.2. In Vitro Experiments on Ultrasound-Induced NPs-PAX8-PPARγ Phase Transition and Ultrasound Development

Nanospheres of NPs and phase-change targeting nanospheres of NPs-PAX8-PPARγ were added to preprepared agarose gel wells and irradiated in advance with LIFU (acoustic power 1.60 w/cm for 1 min). Images were acquired before and after the irradiation.


### 2.6. Targeted Ultrasound Contrast Agent Cytotoxicity Test

Nthy-ori3-1 cells were obtained at the logarithmic growth stage. The cells were routinely digested into cell suspensions and the cell concentration was adjusted to 5 × 103 cells in 96-well culture plates and incubated for 24 hours at 37°C and 5% CO2. Different concentrations of nanocontrast agent nanoparticles were added in 4 gradients (5 pmol/mL, 10 pmol/mL, 15 pmol/mL, and 20 pmol/mL), and a negative control and a positive control were set up. After incubation for 48 h, the liquid in the wells was discarded and 10μL/well of MTS was added, and the light absorption values were measured after incubation at 37°C for 3 h. The data were statistically analyzed and plotted using Excel software.


### 2.7. In Vitro Cell Targeting Experiments

(1) Cell membranes were stained by adding 0.05 mg/ml DiO per cell crawl in each well of a six-well plate. (2) The stained cells were incubated for 30 min, and then the culture medium was drained and washed three times with PBS. (3) 150μL of the targeted polymer contrast agent (labeled with Dil) was added to each of the three wells, and 150 μL of the normal polymer contrast agent (labeled with Dil) was added to each of the other three wells. (4) The cells were incubated in the incubator for 1 h, then each coverslip was washed with PBS and the cells were fixed; the binding of the contrast agent to the cells was observed by light microscopy.


### 2.8. Establishment of Nude Mouse Model

SW579 cells at the logarithmic growth stage were digested with conventional trypsin and resuspended in a serum-free medium.The cells were then centrifuged, resuspended in PBS and placed on ice, and inoculated on the right side of the back of nude mice under the skin for about 4 weeks for subsequent experiments.


### 2.9. In Vivo Observation of the Target-Seeking Ability of Targeted Contrast Agents in Animal Experiments

#### 2.9.1. Experimental Grouping

Five mice from each of the established PAX8-PPARγ SW579 nude mouse models were used and grouped according to the type of the intravenous contrast agent injected into the following two groups;Group I (model + normal contrast group),n = 5Group II (model + dual-modality-targeted contrast group),n = 5


### 2.10. Ultrasonic Imaging

The Vevo 770 UBM small animal ultrasound scanning system (VisualSonics Inc., Toronto, Canada) was used for tumor imaging and analysis. After the injection of various types of ultrasonic microbubble contrast agents into the tail vein of the rats, LIFU was used for irradiation (1.40 w/cm2 for 20 min) to observe the changes in the echogenic intensity of the tumors after the injection of different contrast agents.


### 2.11. Statistical Processing

SPSS23.0 statistical software was used for analysis, and the normally distributed measurement data were expressed asxˉ ± s, and count data were expressed as percentages. The t-test was used for comparison between groups for measurement data, and χ2 test was used for comparison between groups for count data, with P < 0.05 or P < 0.01 indicating that the differences were statistically significant.


## 2.1. Research Subjects

Sixty-five cases of surgically resected TC tissue and corresponding paracancerous tissue (5 cm from the edge of the cancer foci) were collected from May 2018 to July 2019 in our thyroid surgery, and the patients' ages ranged from 22 to 71 years, with a median age of 44 years; 18 of them were male and 47 were female. All patients were diagnosed with primary thyroid cancer preoperatively.Patients who had not yet undergone systemic antitumor therapy and who were included or their families signed the informed consent form for this study.


## 2.2. Expression Levels of PAX8-PPARγ mRNA in Tissues and Cells by qPCR

RNAiso Plus was used to extract total RNA from tissues or cells, and the concentration of RNA was measured spectrophotometrically. 1μg of RNA was diluted to 1 ml of nuclease-free water and used as a template for reverse transcription. Primer sequences were as follows: PAX8-PPARγ forward: 5′-TTCGATGTCTCTAGGGTGGC-3′ and reverse: 5′-GATCCACCTCTCTCGCTCTCTCAG-3′. 10 μl of the SYBR Green nucleic acid fluorescent dye system was used for the qPCR, and three parallel wells were set up, after predenaturation at 95°C for 10 min, denaturation at 95°C for 15 s, annealing at 60°C for 30 s, extension at 72°C for 45 s, and 40 cycles. The expression levels of UCHL5 and BRAF mRNA in tissues and cells were calculated by the 2-ΔΔCT method using β-actin as the internal reference gene.


## 2.3. WB Detection of UCHL5 and BRAF Protein Expression Levels in TC Tissues and Cells

The total protein of TC tissues and cells was extracted and the BCA protein quantification kit was used to detect protein concentration, take 40μg protein line, 10% SDS-PAGE, 120 V constant pressure for 2 h, 260 mA constant flow wet transfer to the PVDF membrane, Add UCHL5 and BRAF antibodies (1 : 1 000) and incubate it overnight in a refrigerator at 4°. The film was exposed to the ChemiDoc XRS + imaging system (Bio-Rad, USA), and the grayscale values of the bands and the relative protein expression were calculated using ImagePlus.


## 2.4. Characterization of Targeted Ultrasound Contrast Agents

### 2.4.1. NPs-PAX8-PPARγ General Properties Assay

(1)
Contrast agent morphology and distribution were observed under light microscopy(2)
Morphology and distribution under scanning electron microscopy(3)
Measurement of nanoparticle size and a zeta potential analyzer to detect its particle size


## 2.4.1. NPs-PAX8-PPARγ General Properties Assay

(1)
Contrast agent morphology and distribution were observed under light microscopy(2)
Morphology and distribution under scanning electron microscopy(3)
Measurement of nanoparticle size and a zeta potential analyzer to detect its particle size


## 2.5. Flow Cytometry Detection of Binding Rate

The other one was incubated with a fluorescently labeled secondary antibody for 30 min and was mixed every 10 mins by using a gun tip, and then the binding rate was detected by flow cytometry in each group.


### 2.5.1. NPs-PAX8-PPARγ In Vitro Thermal Phase Change Experiments

(1)
Adjust the heating plate put in position, so that the microscope observation is simple and easy to operate(2)
Several drops of nanoparticles were placed on the heating plate(3)
The heating plate temperature was set to 60°C


### 2.5.2. In Vitro Experiments on Ultrasound-Induced NPs-PAX8-PPARγ Phase Transition and Ultrasound Development

Nanospheres of NPs and phase-change targeting nanospheres of NPs-PAX8-PPARγ were added to preprepared agarose gel wells and irradiated in advance with LIFU (acoustic power 1.60 w/cm for 1 min). Images were acquired before and after the irradiation.


## 2.5.1. NPs-PAX8-PPARγ In Vitro Thermal Phase Change Experiments

(1)
Adjust the heating plate put in position, so that the microscope observation is simple and easy to operate(2)
Several drops of nanoparticles were placed on the heating plate(3)
The heating plate temperature was set to 60°C


## 2.5.2. In Vitro Experiments on Ultrasound-Induced NPs-PAX8-PPARγ Phase Transition and Ultrasound Development

Nanospheres of NPs and phase-change targeting nanospheres of NPs-PAX8-PPARγ were added to preprepared agarose gel wells and irradiated in advance with LIFU (acoustic power 1.60 w/cm for 1 min). Images were acquired before and after the irradiation.


## 2.6. Targeted Ultrasound Contrast Agent Cytotoxicity Test

Nthy-ori3-1 cells were obtained at the logarithmic growth stage. The cells were routinely digested into cell suspensions and the cell concentration was adjusted to 5 × 103 cells in 96-well culture plates and incubated for 24 hours at 37°C and 5% CO2. Different concentrations of nanocontrast agent nanoparticles were added in 4 gradients (5 pmol/mL, 10 pmol/mL, 15 pmol/mL, and 20 pmol/mL), and a negative control and a positive control were set up. After incubation for 48 h, the liquid in the wells was discarded and 10μL/well of MTS was added, and the light absorption values were measured after incubation at 37°C for 3 h. The data were statistically analyzed and plotted using Excel software.


## 2.7. In Vitro Cell Targeting Experiments

(1) Cell membranes were stained by adding 0.05 mg/ml DiO per cell crawl in each well of a six-well plate. (2) The stained cells were incubated for 30 min, and then the culture medium was drained and washed three times with PBS. (3) 150μL of the targeted polymer contrast agent (labeled with Dil) was added to each of the three wells, and 150 μL of the normal polymer contrast agent (labeled with Dil) was added to each of the other three wells. (4) The cells were incubated in the incubator for 1 h, then each coverslip was washed with PBS and the cells were fixed; the binding of the contrast agent to the cells was observed by light microscopy.


## 2.8. Establishment of Nude Mouse Model

SW579 cells at the logarithmic growth stage were digested with conventional trypsin and resuspended in a serum-free medium.The cells were then centrifuged, resuspended in PBS and placed on ice, and inoculated on the right side of the back of nude mice under the skin for about 4 weeks for subsequent experiments.


## 2.9. In Vivo Observation of the Target-Seeking Ability of Targeted Contrast Agents in Animal Experiments

### 2.9.1. Experimental Grouping

Five mice from each of the established PAX8-PPARγ SW579 nude mouse models were used and grouped according to the type of the intravenous contrast agent injected into the following two groups;Group I (model + normal contrast group),n = 5Group II (model + dual-modality-targeted contrast group),n = 5


## 2.9.1. Experimental Grouping

Five mice from each of the established PAX8-PPARγ SW579 nude mouse models were used and grouped according to the type of the intravenous contrast agent injected into the following two groups;Group I (model + normal contrast group),n = 5Group II (model + dual-modality-targeted contrast group),n = 5


## 2.10. Ultrasonic Imaging

The Vevo 770 UBM small animal ultrasound scanning system (VisualSonics Inc., Toronto, Canada) was used for tumor imaging and analysis. After the injection of various types of ultrasonic microbubble contrast agents into the tail vein of the rats, LIFU was used for irradiation (1.40 w/cm2 for 20 min) to observe the changes in the echogenic intensity of the tumors after the injection of different contrast agents.


## 2.11. Statistical Processing

SPSS23.0 statistical software was used for analysis, and the normally distributed measurement data were expressed asxˉ ± s, and count data were expressed as percentages. The t-test was used for comparison between groups for measurement data, and χ2 test was used for comparison between groups for count data, with P < 0.05 or P < 0.01 indicating that the differences were statistically significant.


## 3. Results

### 3.1. PAX8-PPARγ Is Highly Expressed in Thyroid Cancer Tissues and Thyroid Cancer Cell Lines

PAX8-PPARγ expression was detected by western blotting using normal thyroid cell line Nthy-ori3-1 cells and thyroid cancer cell lines SW579, IHH-4, FTC-133, TPC-1, DRO, TA-K, and ML-1 cells. The results showed that PAX8-PPARγ was highly expressed in thyroid cancer cell lines (Figure 1) compared to normal thyroid cell lines (Figures 1(a) and 1(b)). Immunohistochemistry and western blotting showed that PAX8-PPARγ was significantly highly expressed in thyroid cancer tissues compared to normal thyroid tissues (P < 0.001; Figures 1(c)–1(e)). = 0.8994; Table 1).Figure 1
Overexpression of PAX8-PPARγ in human thyroid cancer cell lines and tumor tissues.
(a)(b)(c)(d)(e)Table 1
PAX8-PPARγ expression in normal, peritumoural, and thyroid cancer tissues.
Pathologic diagnosisnPAX8-PPARγ expression−++++++Positive rate (%)Thyroid cancer6537342184.6Peritumoural cancer402019102.5Normal40336000


### 3.2. PAX8-PPARγ Expression Was Positively Correlated with the Degree of Thyroid Tumor Differentiation and TNM Staging

All thyroid cancer specimens were analyzed by immunohistochemistry. PAX8-PPARγ expression was not found to differ between genders. High PAX8-PPARγ expression was associated with thyroid tumor differentiation, TNM staging, and lymph node metastasis (P < 0.05; Tables 1 and 2). Therefore, it is speculated that PAX8-PPARγ may have a positive role in thyroid tumor initiation and progression.Table 2
Correlations of PAX8-PPARγ expression in human thyroid cancer with clinical pathological parameters.
Number (n)PAX8-PPARγ overexpressionχ2P valueAge (years)<4036290.64>0.05≥402922GenderMale18140.71>0.05Female4737Tumour (cm)<11290.48>0.051–44535>487Pathological typePapillary thyroid carcinoma31240.56>0.05Follicle carcinoma2217Medullary carcinoma119Undifferentiated carcinoma11TNM stageI-II39276.39<0.01III-2624Differentiation gradeI20117.08<0.01II2621III1919Lymph node metastasisPresent23228.17<0.01Absent4229Capsular invasionPresent20199.25<0.01Absent4532


### 3.3. Effect of Inhibiting PAX8-PPARγ Expression on the Proliferation Ability of Thyroid Cancer Cells

Thyroid cancer cells were transfected with a PAX8-PPARγ antisense oligonucleotide siRNAPAX8-PPARγ to block the expression of PAX8-PPARγ, and the effect of blocking on the clonal growth of thyroid cancer cells was examined and evaluated. The SW579 thyroid cancer cell line is a PAX8-PPARγ high-expression cell, and after treating the cells with different concentrations of the PAX8-PPARγ antisense oligonucleotide, the change of PAX8-PPARγ expression in the treated cells was detected by western blot. After treatment with different concentrations of the PAX8-PPARγ antisense oligonucleotide, the PAX8-PPARγ expression changes in the treated cells were detected by western blot. As shown in Figures 2(a) and 2(b), the clonal growth of thyroid cancer cells was significantly inhibited after receiving PAX8-PPARγ antisense oligonucleotide treatment for 4 days. In addition, after receiving different concentrations of the PAX8-PPARγ antisense oligonucleotide treatment (Figures 2(c)–2(e)), an increase in apoptosis of thyroid cancer cells was also observed with the decrease of PAX8-PPARγ protein expression, suggesting a positive role of PAX8-PPARγ in the survival and proliferation of thyroid cancer cells.Figure 2
PAX8-PPARγ antisense oligonucleotides induce growth inhibition and apoptosis of the thyroid cancer clonogenic growth. (a) SW579 cells were plated in 6-well culture plates in 0.7% soft agar with 0 μM and 3 μM for 21 days, and then colonies (>40 microphotographs of thyroid colonies after exposure to antisense oligonucleotides). (b) PAX8-PPARγ antisense oligonucleotide exposure decreases the plate colony formation rate of the SW579 cells. (c) Thyroid cancer cells were cultured in DMEM with 10% serum, treated with different doses (0 μ M-5 μM) of PAX8-PPARγ antisense oligonucleotides for 4 days, and then collected for the analysis of PAX8-PPARγ expression by western blot, and apoptosis with FCM. ∗P < 0.05; ∗∗P < 0.01.
(a)(b)(c)(d)(e)


### 3.4. Effect of Inhibition of PAX8-PPARγ Expression on the Tumorigenic Ability in Nude Mice In Vivo

Compared with the control group, the tumorigenic ability of SW579 thyroid cancer cell in nude mice was significantly inhibited after PAX8-PPARγ expression was blocked by interference. When nude mice were executed on the 50th day of inoculation, tumor formation was found in the nude mice with undisturbed PAX8-PPARγ expression, and the tumor weight and growth curve are shown in Figures 3(a) and 3(b).Figure 3
PAX8-PPARγ promotes tumor growth and tumor angiogenesis in a mouse xenograft model. (a) The solid tumors were weighed after the mice were sacrificed. (b) The tumor size was monitored every three days. The data are presented as means ± standard errors of the means (SEMs) (n = 5 per group) (∗∗P < 0.01).
(a)(b)Figure 4
Characterization of NPs-PAX8-PPARγ: (a) macrograph of NPs-PAX8-PPARγ; (b) bright field optical microscopy image (×400); (c, d) Size distribution and zeta potential.
(a)(b)(c)(d)


### 3.5. Preparation and Characterization of the NPs-PAX8-PPARγ Contrast Agent

(1)
The NPs-PAX8-PPARγ contrast agent was observed as a milky white suspension after preparation and could be stored stably at 4°C. The contrast agent prepared by adding DiI dye in advance was a red suspension (Figure 4(a)).(2)
The prepared nanocontrast agent was uniform in size and well dispersed as observed by using an ordinary light microscope (Figure4(b))(3)
The average particle size of NPs-PAX8-PPARγ was 535.7.2 ± 40.7 nm (Figure 4(c)) and the average surface potential was −14.23 ± 8.69 mV (Figure 4(d))


#### 3.5.1. Flow Cytometry Detection of Binding Rate

The PAX8-PPARγ antibody was bound to the nontargeted nanocontrast agent and flow cytometric detection showed 67.2% binding of the targeted nanocontrast agent to the secondary antibody (Figure 5(b)) and 0.02% binding of the nontargeted nanocontrast agent to the secondary antibody, as shown in Figure 5(a).Figure 5
NPs-PAX8-PPARγ binding with the secondary antibody.
(a)(b)Figure 6
Optical microscope image of NPs-PAX8-PPARγ (×200) after heating to 47°C.


### 3.6. In Vitro Thermal Phase Change Experiments of NPs-PAX8-PPARγ

(1)
The phase transformation of NPs-PAX8-PPARγ in the heated plate was observed by ordinary light microscopy (Figure 6).(2)
When the temperature of the heating plate increased, the nanoparticles began to show changes, and when the temperature reached 45.9°C, bubbles began to be generated. When the temperature rises to 47°C, more bubbles were gradually produced.


### 3.7. Ultrasound-Targeted Contrast Agent in In Vitro Phase Change and Contrast Development Experiments

Before irradiation with the LIFU instrument, the nanocontrast agent exhibited no echo in the ultrasound mode (mechanical index 0.08). After irradiation with the LIFU instrument, the targeted nanocontrast agent solution exhibited uniform strong echoes in the form of fine dots (Figure7).Figure 7
Characterization of NPs-PAX8-PPARγ : In vitro ultrasound image. Contrast-enhanced ultrasound imaging of NPs-PAX8-PPARγ before (a) and after (b) LIFU.
(a)(b)


### 3.8. In Vitro Cytotoxicity Assay with Targeted Contrast Agents

#### 3.8.1. MTS Method to Detect the Effect of Targeted Contrast Agents on the Proliferative Capacity of Thyroid Cells

The normal thyroid cell lines were treated with different concentrations of nanocontrast agents, and four gradients of nanoparticles (5 pmol/mL, 10 pmol/mL, 15 pmol/mL, and 20 pmol/mL) were set up in this experiment, with negative and positive controls, respectively. The effect of receiving targeted nanocontrast agent treatment on the proliferation ability of normal thyroid cell lines was observed by the MTS method. As shown in Figure8, there was no significant change in the growth and proliferation trend of thyroid Nth-ori3-1 cells between the groups treated with the nanocontrast agent and the blank control group, and the growth and proliferation ability of the positive control group treated with Adriamycin was significantly inhibited, which suggested that the targeted nanocontrast agent had no significant effect on the survival and proliferation of thyroid cancer cells.Figure 8
The lethal effect of Nthy-ori3-1 cell line in different terms.


#### 3.8.2. Staining Cell Nuclei with DAPI to Observe the Cytotoxicity of Targeted Contrast Agents

Cells were treated with different concentrations of nanocontrast agents for 72 h. Four gradients (nanoparticle concentrations of 5 pmol/mL, 10 pmol/mL, 15 pmol/mL, and 20 pmol/mL, respectively) were set up in this set of experiments, with negative and positive controls, respectively. The nuclei of the cells were stained with DAPI to observe the effect of the targeted contrast agent on the morphology of the nuclei to reflect the cytotoxicity of the targeted contrast agent, as shown in Figure9; the cells in the Adriamycin-treated group, which showed nuclear fragmentation, while the nuclei of the blank control and nanotargeted contrast agent groups remained intact and unaffected, which to some extent indicates that the targeted nanocontrast agent made in this study has in vitro cell safety.Figure 9
The lethal effect of SW579 cells in different term: (a) control; (b–e) NPs-PAX8-PPARγ5 pmol/mL, 10 pmol/mL, 15 pmol/mL, and 20 pmol/mL; (f) DOX 0.5 umol/mL.


#### 3.8.3. Countess Observation of Cell Survival and Mortality

Cells were treated with different concentrations of nanocontrast agents for 72 h. Four gradients (5 pmol/mL, 10 pmol/mL, 15 pmol/mL, and 20 pmol/mL) of nanoparticles were set up in this experiment, with negative and positive controls, respectively. The cells were stained with TissueBlue to observe the effect of targeting contrast agent treatment on the viability of the cells. As shown in Figure10, there was no significant effect on cell viability in the blank control and nanotargeted contrast agent groups, while the number of dead cells in the Adriamycin-treated group was significantly increased.Figure 10
The lethal effect of SW579 cells in different terms. (a) Control; (b–e) NPs-PAX8-PPARγ5 pmol/mL, 10 pmol/mL, 15 pmol/mL, and 20 pmol/mL; (f) DOX 0.5 umol/mL.


#### 3.8.4. LD50 Measurement Results

After intraperitoneal injection of different doses of targeted nanocontrast suspensions, the animals were observed daily until 15 d after administration. The mice in each experimental group did not show any death within 15 days of injection, and their body weight increased normally (Table3).Table 3
The results of acute toxicity testing of NPs-SHP2.
ControlDeath numberDeath rate (%)Survival (%)NPs00100NPs-SHP200100PBS00100


#### 3.8.5. Western Blot Detection of Caspase-3, BAX, and Bcl-2 Expression in Thyroid Cancer Cells

The thyroid cancer cell lines were treated with 1640, nontargeted contrast agent (20 pmol/mL), targeted contrast agent (20 pmol/mL), and adriamycin (0.5 umol/mL) for 72 hours, and then the effects on caspase-3, BAX, and Bcl-2 expression were observed. As shown in Figure11, the expression of caspase-3 and BAX was significantly higher in the adriamycin-treated cells compared with the blank control group and the targeted and nontargeted contrast agent-treated cells. In contrast, the expression of Bcl-2 protein was significantly lower in adriamycin-treated cells compared to the other three experimental groups. These results suggest that the targeted nanocontrast agent had no effect on cell growth and apoptosis.Figure 11
After treatment with NPs in SW579 cells, the expression of PAX8-PPARγ-NPs, and DOX, the BAX and caspase-3 was significantly increased while the Bcl-2 expression was lower than that in any other group.


### 3.9. Nude Rat Model Establishment

The animal model used in this experiment was a nude mouse, and 4 weeks after inoculation of tumor cells on the right side of the back, the tumor was successfully constructed by subcutaneous transplantation (Figure12); the size of the tumor was about 1.0 cm × 0.8 cm × 0.9 cm, ≤1 cm, measured under ultrasound.Figure 12
Mouse model for NPs-PAX8-PPARγ ultrasound imaging in in vivo experiments.


### 3.10. In Vitro Cell Targeting Assay

The prepared NPs-PAX8-PPARγ and NPs-control nanoparticles were coincubated with SW579 cells. The results showed that the number of nanoparticles attached per cell was significantly (P < 0.001) higher in the targeted contrast agent group (NPs-PAX8-PPARγ) than in the nontargeted contrast agent group (NPs-control), as shown in Figure 13.Figure 13
Representative results from vitro experiments after exposure to PAX8-PPARγ-targeted and nontargeted nanoparticles. Note. The specific attachment of PAX8-PPARγ-targeted nanoparticles and the substantial nanoparticles are shown as red dots. P < 0.01.
(a)(b)


### 3.11. In Vivo Target Finding Experiments in Nude Mice

The ultrasound enhancement imaging results showed that the ultrasound signal of the tumor in the tumor region was significantly increased in the targeted ultrasound contrast agent group (48.32 ± 2.9 a.u) and in the nontargeted ultrasound contrast agent group (6.03 ± 1.6 a.u). The ultrasound signal was also enhanced in the tumor region of the nontargeted contrast group (6.03 ± 1.6 a.u), but the signal enhancement was significantly weaker than that of the targeted group, as shown in Figure14.Figure 14
Target molecular imaging in in vivo experiments after LIFU irradiation 1.40 w/cm2 for 20 min.
(a)(b)(c)(d)Different ultrasound imaging modes were used following the injection of PAX8-PPARγ-signed contrast nanoparticles showing a high signal in thyroid tumour and showing only a background signal when the using nontargeted contrast nanoparticles (the region of interest were traced use blue).


## 3.1. PAX8-PPARγ Is Highly Expressed in Thyroid Cancer Tissues and Thyroid Cancer Cell Lines

PAX8-PPARγ expression was detected by western blotting using normal thyroid cell line Nthy-ori3-1 cells and thyroid cancer cell lines SW579, IHH-4, FTC-133, TPC-1, DRO, TA-K, and ML-1 cells. The results showed that PAX8-PPARγ was highly expressed in thyroid cancer cell lines (Figure 1) compared to normal thyroid cell lines (Figures 1(a) and 1(b)). Immunohistochemistry and western blotting showed that PAX8-PPARγ was significantly highly expressed in thyroid cancer tissues compared to normal thyroid tissues (P < 0.001; Figures 1(c)–1(e)). = 0.8994; Table 1).Figure 1
Overexpression of PAX8-PPARγ in human thyroid cancer cell lines and tumor tissues.
(a)(b)(c)(d)(e)Table 1
PAX8-PPARγ expression in normal, peritumoural, and thyroid cancer tissues.
Pathologic diagnosisnPAX8-PPARγ expression−++++++Positive rate (%)Thyroid cancer6537342184.6Peritumoural cancer402019102.5Normal40336000


## 3.2. PAX8-PPARγ Expression Was Positively Correlated with the Degree of Thyroid Tumor Differentiation and TNM Staging

All thyroid cancer specimens were analyzed by immunohistochemistry. PAX8-PPARγ expression was not found to differ between genders. High PAX8-PPARγ expression was associated with thyroid tumor differentiation, TNM staging, and lymph node metastasis (P < 0.05; Tables 1 and 2). Therefore, it is speculated that PAX8-PPARγ may have a positive role in thyroid tumor initiation and progression.Table 2
Correlations of PAX8-PPARγ expression in human thyroid cancer with clinical pathological parameters.
Number (n)PAX8-PPARγ overexpressionχ2P valueAge (years)<4036290.64>0.05≥402922GenderMale18140.71>0.05Female4737Tumour (cm)<11290.48>0.051–44535>487Pathological typePapillary thyroid carcinoma31240.56>0.05Follicle carcinoma2217Medullary carcinoma119Undifferentiated carcinoma11TNM stageI-II39276.39<0.01III-2624Differentiation gradeI20117.08<0.01II2621III1919Lymph node metastasisPresent23228.17<0.01Absent4229Capsular invasionPresent20199.25<0.01Absent4532


## 3.3. Effect of Inhibiting PAX8-PPARγ Expression on the Proliferation Ability of Thyroid Cancer Cells

Thyroid cancer cells were transfected with a PAX8-PPARγ antisense oligonucleotide siRNAPAX8-PPARγ to block the expression of PAX8-PPARγ, and the effect of blocking on the clonal growth of thyroid cancer cells was examined and evaluated. The SW579 thyroid cancer cell line is a PAX8-PPARγ high-expression cell, and after treating the cells with different concentrations of the PAX8-PPARγ antisense oligonucleotide, the change of PAX8-PPARγ expression in the treated cells was detected by western blot. After treatment with different concentrations of the PAX8-PPARγ antisense oligonucleotide, the PAX8-PPARγ expression changes in the treated cells were detected by western blot. As shown in Figures 2(a) and 2(b), the clonal growth of thyroid cancer cells was significantly inhibited after receiving PAX8-PPARγ antisense oligonucleotide treatment for 4 days. In addition, after receiving different concentrations of the PAX8-PPARγ antisense oligonucleotide treatment (Figures 2(c)–2(e)), an increase in apoptosis of thyroid cancer cells was also observed with the decrease of PAX8-PPARγ protein expression, suggesting a positive role of PAX8-PPARγ in the survival and proliferation of thyroid cancer cells.Figure 2
PAX8-PPARγ antisense oligonucleotides induce growth inhibition and apoptosis of the thyroid cancer clonogenic growth. (a) SW579 cells were plated in 6-well culture plates in 0.7% soft agar with 0 μM and 3 μM for 21 days, and then colonies (>40 microphotographs of thyroid colonies after exposure to antisense oligonucleotides). (b) PAX8-PPARγ antisense oligonucleotide exposure decreases the plate colony formation rate of the SW579 cells. (c) Thyroid cancer cells were cultured in DMEM with 10% serum, treated with different doses (0 μ M-5 μM) of PAX8-PPARγ antisense oligonucleotides for 4 days, and then collected for the analysis of PAX8-PPARγ expression by western blot, and apoptosis with FCM. ∗P < 0.05; ∗∗P < 0.01.
(a)(b)(c)(d)(e)


## 3.4. Effect of Inhibition of PAX8-PPARγ Expression on the Tumorigenic Ability in Nude Mice In Vivo

Compared with the control group, the tumorigenic ability of SW579 thyroid cancer cell in nude mice was significantly inhibited after PAX8-PPARγ expression was blocked by interference. When nude mice were executed on the 50th day of inoculation, tumor formation was found in the nude mice with undisturbed PAX8-PPARγ expression, and the tumor weight and growth curve are shown in Figures 3(a) and 3(b).Figure 3
PAX8-PPARγ promotes tumor growth and tumor angiogenesis in a mouse xenograft model. (a) The solid tumors were weighed after the mice were sacrificed. (b) The tumor size was monitored every three days. The data are presented as means ± standard errors of the means (SEMs) (n = 5 per group) (∗∗P < 0.01).
(a)(b)Figure 4
Characterization of NPs-PAX8-PPARγ: (a) macrograph of NPs-PAX8-PPARγ; (b) bright field optical microscopy image (×400); (c, d) Size distribution and zeta potential.
(a)(b)(c)(d)


## 3.5. Preparation and Characterization of the NPs-PAX8-PPARγ Contrast Agent

(1)
The NPs-PAX8-PPARγ contrast agent was observed as a milky white suspension after preparation and could be stored stably at 4°C. The contrast agent prepared by adding DiI dye in advance was a red suspension (Figure 4(a)).(2)
The prepared nanocontrast agent was uniform in size and well dispersed as observed by using an ordinary light microscope (Figure4(b))(3)
The average particle size of NPs-PAX8-PPARγ was 535.7.2 ± 40.7 nm (Figure 4(c)) and the average surface potential was −14.23 ± 8.69 mV (Figure 4(d))


### 3.5.1. Flow Cytometry Detection of Binding Rate

The PAX8-PPARγ antibody was bound to the nontargeted nanocontrast agent and flow cytometric detection showed 67.2% binding of the targeted nanocontrast agent to the secondary antibody (Figure 5(b)) and 0.02% binding of the nontargeted nanocontrast agent to the secondary antibody, as shown in Figure 5(a).Figure 5
NPs-PAX8-PPARγ binding with the secondary antibody.
(a)(b)Figure 6
Optical microscope image of NPs-PAX8-PPARγ (×200) after heating to 47°C.


## 3.5.1. Flow Cytometry Detection of Binding Rate

The PAX8-PPARγ antibody was bound to the nontargeted nanocontrast agent and flow cytometric detection showed 67.2% binding of the targeted nanocontrast agent to the secondary antibody (Figure 5(b)) and 0.02% binding of the nontargeted nanocontrast agent to the secondary antibody, as shown in Figure 5(a).Figure 5
NPs-PAX8-PPARγ binding with the secondary antibody.
(a)(b)Figure 6
Optical microscope image of NPs-PAX8-PPARγ (×200) after heating to 47°C.


## 3.6. In Vitro Thermal Phase Change Experiments of NPs-PAX8-PPARγ

(1)
The phase transformation of NPs-PAX8-PPARγ in the heated plate was observed by ordinary light microscopy (Figure 6).(2)
When the temperature of the heating plate increased, the nanoparticles began to show changes, and when the temperature reached 45.9°C, bubbles began to be generated. When the temperature rises to 47°C, more bubbles were gradually produced.


## 3.7. Ultrasound-Targeted Contrast Agent in In Vitro Phase Change and Contrast Development Experiments

Before irradiation with the LIFU instrument, the nanocontrast agent exhibited no echo in the ultrasound mode (mechanical index 0.08). After irradiation with the LIFU instrument, the targeted nanocontrast agent solution exhibited uniform strong echoes in the form of fine dots (Figure7).Figure 7
Characterization of NPs-PAX8-PPARγ : In vitro ultrasound image. Contrast-enhanced ultrasound imaging of NPs-PAX8-PPARγ before (a) and after (b) LIFU.
(a)(b)


## 3.8. In Vitro Cytotoxicity Assay with Targeted Contrast Agents

### 3.8.1. MTS Method to Detect the Effect of Targeted Contrast Agents on the Proliferative Capacity of Thyroid Cells

The normal thyroid cell lines were treated with different concentrations of nanocontrast agents, and four gradients of nanoparticles (5 pmol/mL, 10 pmol/mL, 15 pmol/mL, and 20 pmol/mL) were set up in this experiment, with negative and positive controls, respectively. The effect of receiving targeted nanocontrast agent treatment on the proliferation ability of normal thyroid cell lines was observed by the MTS method. As shown in Figure8, there was no significant change in the growth and proliferation trend of thyroid Nth-ori3-1 cells between the groups treated with the nanocontrast agent and the blank control group, and the growth and proliferation ability of the positive control group treated with Adriamycin was significantly inhibited, which suggested that the targeted nanocontrast agent had no significant effect on the survival and proliferation of thyroid cancer cells.Figure 8
The lethal effect of Nthy-ori3-1 cell line in different terms.


### 3.8.2. Staining Cell Nuclei with DAPI to Observe the Cytotoxicity of Targeted Contrast Agents

Cells were treated with different concentrations of nanocontrast agents for 72 h. Four gradients (nanoparticle concentrations of 5 pmol/mL, 10 pmol/mL, 15 pmol/mL, and 20 pmol/mL, respectively) were set up in this set of experiments, with negative and positive controls, respectively. The nuclei of the cells were stained with DAPI to observe the effect of the targeted contrast agent on the morphology of the nuclei to reflect the cytotoxicity of the targeted contrast agent, as shown in Figure9; the cells in the Adriamycin-treated group, which showed nuclear fragmentation, while the nuclei of the blank control and nanotargeted contrast agent groups remained intact and unaffected, which to some extent indicates that the targeted nanocontrast agent made in this study has in vitro cell safety.Figure 9
The lethal effect of SW579 cells in different term: (a) control; (b–e) NPs-PAX8-PPARγ5 pmol/mL, 10 pmol/mL, 15 pmol/mL, and 20 pmol/mL; (f) DOX 0.5 umol/mL.


### 3.8.3. Countess Observation of Cell Survival and Mortality

Cells were treated with different concentrations of nanocontrast agents for 72 h. Four gradients (5 pmol/mL, 10 pmol/mL, 15 pmol/mL, and 20 pmol/mL) of nanoparticles were set up in this experiment, with negative and positive controls, respectively. The cells were stained with TissueBlue to observe the effect of targeting contrast agent treatment on the viability of the cells. As shown in Figure10, there was no significant effect on cell viability in the blank control and nanotargeted contrast agent groups, while the number of dead cells in the Adriamycin-treated group was significantly increased.Figure 10
The lethal effect of SW579 cells in different terms. (a) Control; (b–e) NPs-PAX8-PPARγ5 pmol/mL, 10 pmol/mL, 15 pmol/mL, and 20 pmol/mL; (f) DOX 0.5 umol/mL.


### 3.8.4. LD50 Measurement Results

After intraperitoneal injection of different doses of targeted nanocontrast suspensions, the animals were observed daily until 15 d after administration. The mice in each experimental group did not show any death within 15 days of injection, and their body weight increased normally (Table3).Table 3
The results of acute toxicity testing of NPs-SHP2.
ControlDeath numberDeath rate (%)Survival (%)NPs00100NPs-SHP200100PBS00100


### 3.8.5. Western Blot Detection of Caspase-3, BAX, and Bcl-2 Expression in Thyroid Cancer Cells

The thyroid cancer cell lines were treated with 1640, nontargeted contrast agent (20 pmol/mL), targeted contrast agent (20 pmol/mL), and adriamycin (0.5 umol/mL) for 72 hours, and then the effects on caspase-3, BAX, and Bcl-2 expression were observed. As shown in Figure11, the expression of caspase-3 and BAX was significantly higher in the adriamycin-treated cells compared with the blank control group and the targeted and nontargeted contrast agent-treated cells. In contrast, the expression of Bcl-2 protein was significantly lower in adriamycin-treated cells compared to the other three experimental groups. These results suggest that the targeted nanocontrast agent had no effect on cell growth and apoptosis.Figure 11
After treatment with NPs in SW579 cells, the expression of PAX8-PPARγ-NPs, and DOX, the BAX and caspase-3 was significantly increased while the Bcl-2 expression was lower than that in any other group.


## 3.8.1. MTS Method to Detect the Effect of Targeted Contrast Agents on the Proliferative Capacity of Thyroid Cells

The normal thyroid cell lines were treated with different concentrations of nanocontrast agents, and four gradients of nanoparticles (5 pmol/mL, 10 pmol/mL, 15 pmol/mL, and 20 pmol/mL) were set up in this experiment, with negative and positive controls, respectively. The effect of receiving targeted nanocontrast agent treatment on the proliferation ability of normal thyroid cell lines was observed by the MTS method. As shown in Figure8, there was no significant change in the growth and proliferation trend of thyroid Nth-ori3-1 cells between the groups treated with the nanocontrast agent and the blank control group, and the growth and proliferation ability of the positive control group treated with Adriamycin was significantly inhibited, which suggested that the targeted nanocontrast agent had no significant effect on the survival and proliferation of thyroid cancer cells.Figure 8
The lethal effect of Nthy-ori3-1 cell line in different terms.


## 3.8.2. Staining Cell Nuclei with DAPI to Observe the Cytotoxicity of Targeted Contrast Agents

Cells were treated with different concentrations of nanocontrast agents for 72 h. Four gradients (nanoparticle concentrations of 5 pmol/mL, 10 pmol/mL, 15 pmol/mL, and 20 pmol/mL, respectively) were set up in this set of experiments, with negative and positive controls, respectively. The nuclei of the cells were stained with DAPI to observe the effect of the targeted contrast agent on the morphology of the nuclei to reflect the cytotoxicity of the targeted contrast agent, as shown in Figure9; the cells in the Adriamycin-treated group, which showed nuclear fragmentation, while the nuclei of the blank control and nanotargeted contrast agent groups remained intact and unaffected, which to some extent indicates that the targeted nanocontrast agent made in this study has in vitro cell safety.Figure 9
The lethal effect of SW579 cells in different term: (a) control; (b–e) NPs-PAX8-PPARγ5 pmol/mL, 10 pmol/mL, 15 pmol/mL, and 20 pmol/mL; (f) DOX 0.5 umol/mL.


## 3.8.3. Countess Observation of Cell Survival and Mortality

Cells were treated with different concentrations of nanocontrast agents for 72 h. Four gradients (5 pmol/mL, 10 pmol/mL, 15 pmol/mL, and 20 pmol/mL) of nanoparticles were set up in this experiment, with negative and positive controls, respectively. The cells were stained with TissueBlue to observe the effect of targeting contrast agent treatment on the viability of the cells. As shown in Figure10, there was no significant effect on cell viability in the blank control and nanotargeted contrast agent groups, while the number of dead cells in the Adriamycin-treated group was significantly increased.Figure 10
The lethal effect of SW579 cells in different terms. (a) Control; (b–e) NPs-PAX8-PPARγ5 pmol/mL, 10 pmol/mL, 15 pmol/mL, and 20 pmol/mL; (f) DOX 0.5 umol/mL.


## 3.8.4. LD50 Measurement Results

After intraperitoneal injection of different doses of targeted nanocontrast suspensions, the animals were observed daily until 15 d after administration. The mice in each experimental group did not show any death within 15 days of injection, and their body weight increased normally (Table3).Table 3
The results of acute toxicity testing of NPs-SHP2.
ControlDeath numberDeath rate (%)Survival (%)NPs00100NPs-SHP200100PBS00100


## 3.8.5. Western Blot Detection of Caspase-3, BAX, and Bcl-2 Expression in Thyroid Cancer Cells

The thyroid cancer cell lines were treated with 1640, nontargeted contrast agent (20 pmol/mL), targeted contrast agent (20 pmol/mL), and adriamycin (0.5 umol/mL) for 72 hours, and then the effects on caspase-3, BAX, and Bcl-2 expression were observed. As shown in Figure11, the expression of caspase-3 and BAX was significantly higher in the adriamycin-treated cells compared with the blank control group and the targeted and nontargeted contrast agent-treated cells. In contrast, the expression of Bcl-2 protein was significantly lower in adriamycin-treated cells compared to the other three experimental groups. These results suggest that the targeted nanocontrast agent had no effect on cell growth and apoptosis.Figure 11
After treatment with NPs in SW579 cells, the expression of PAX8-PPARγ-NPs, and DOX, the BAX and caspase-3 was significantly increased while the Bcl-2 expression was lower than that in any other group.


## 3.9. Nude Rat Model Establishment

The animal model used in this experiment was a nude mouse, and 4 weeks after inoculation of tumor cells on the right side of the back, the tumor was successfully constructed by subcutaneous transplantation (Figure12); the size of the tumor was about 1.0 cm × 0.8 cm × 0.9 cm, ≤1 cm, measured under ultrasound.Figure 12
Mouse model for NPs-PAX8-PPARγ ultrasound imaging in in vivo experiments.


## 3.10. In Vitro Cell Targeting Assay

The prepared NPs-PAX8-PPARγ and NPs-control nanoparticles were coincubated with SW579 cells. The results showed that the number of nanoparticles attached per cell was significantly (P < 0.001) higher in the targeted contrast agent group (NPs-PAX8-PPARγ) than in the nontargeted contrast agent group (NPs-control), as shown in Figure 13.Figure 13
Representative results from vitro experiments after exposure to PAX8-PPARγ-targeted and nontargeted nanoparticles. Note. The specific attachment of PAX8-PPARγ-targeted nanoparticles and the substantial nanoparticles are shown as red dots. P < 0.01.
(a)(b)


## 3.11. In Vivo Target Finding Experiments in Nude Mice

The ultrasound enhancement imaging results showed that the ultrasound signal of the tumor in the tumor region was significantly increased in the targeted ultrasound contrast agent group (48.32 ± 2.9 a.u) and in the nontargeted ultrasound contrast agent group (6.03 ± 1.6 a.u). The ultrasound signal was also enhanced in the tumor region of the nontargeted contrast group (6.03 ± 1.6 a.u), but the signal enhancement was significantly weaker than that of the targeted group, as shown in Figure14.Figure 14
Target molecular imaging in in vivo experiments after LIFU irradiation 1.40 w/cm2 for 20 min.
(a)(b)(c)(d)Different ultrasound imaging modes were used following the injection of PAX8-PPARγ-signed contrast nanoparticles showing a high signal in thyroid tumour and showing only a background signal when the using nontargeted contrast nanoparticles (the region of interest were traced use blue).


## 4. Discussion

In recent years, numerous studies on the molecular biology and molecular mechanisms of tumors have confirmed that phosphatases play a key role in the regulation of tumorigenesis and development signaling; they are associated with abnormal cell proliferation and differentiation, and can be used as antitumor targets.PAX8-PPARγ, a member of the PTP family, has been shown to be widely expressed in mammalian tissues and can regulate several cellular processes, including cell growth, cell differentiation, and the mitotic cell cycle, and abnormal PAX8-PPARγ expression plays a key role in tumorigenesis, promoting the malignant biological process of several solid tumors, including breast, ovarian, lung, and liver cancer [10]. However, the role of PAX8-PPARγ in the malignant evolution of thyroid tumors has not been reported yet. Therefore, it is significant to investigate the role of PAX8-PPARγ in thyroid tumors and its mechanism for thyroid cancer. In the present study, we showed for the first time that the PAX8-PPARγ protein tyrosine phosphatase expression was significantly higher in thyroid cancer cell lines and thyroid cancer tissues relative to the normal thyroid cell lines and normal and paraneoplastic thyroid tissues, and this high expression of PAX8-PPARγ may be involved in the development of thyroid cancer and is closely associated with patient prognosis, TNM staging, as well as lymph node metastasis (Table 1). The expression level of PAX8-PPARγ was not significantly correlated with patient age and gender (P > 0.05). This suggests that high PAX8-PPARγ expression may be involved in the initiation of thyroid malignancy as well as invasion, metastasis, and further tumor evolution.In this study, we explored the relationship between the PAX8-PPARγ expression changes and malignant biological behavior of thyroid tumors from in vivo and ex vivo experiments, respectively, and the results showed that PAX8-PPARγ promoted the proliferation and antiapoptotic ability of thyroid cancer cells, and PAX8-PPARγ expression inhibition affected the tumorigenic ability of nude mice in vivo. These results suggest that PAX8-PPARγ could serve as a marker to predict the malignant biological behavior of thyroid cancer and potentially represent a novel target as thyroid cancer treatment. This therapy may promote tumor cell apoptosis, thereby inhibiting cancer cell growth and metastasis, and it may delay tumor recurrence and metastasis, and improve the overall survival of thyroid cancer patients. [11, 12].With the continuous development of medical technology, coupling microbubbles with specific antibodies or drugs for the purpose of targeted contrast, drug delivery, and gene delivery has provided new ideas for the diagnosis of malignant tumors. Some studies have shown that targeted nanomicrobubble contrast agents have good specificity, and some scholars have applied the contrast agent specifically targeting VEGFR2/KDR in clinics, and this targeted contrast agent can significantly improve the signal intensity of lesions in patients with breast and ovarian cancer, which improves the efficiency of clinical diagnosis and provides a new starting point for the clinical translation of targeted contrast agents [13–15]. Thus, molecular imaging based on targeted nanomicrobubbles brings a new hope for early noninvasive diagnosis of tumors and has become a hotspot for research in the biomedical neighborhood in recent years [16].In recent years, new methods based on mutations and other molecular markers have been partially applied in the differential diagnosis of thyroid tumors (e.g., BRAF, TP53, CTNNB1, and other genes), and these molecular markers have significantly improved the diagnostic accuracy of cytology and have had a profound impact on the classification and management of patients with thyroid tumors. However, the use of nanotechnology-based combined with specific antibody-targeted visualization for noninvasive imaging diagnosis of thyroid cancer has not been reported yet. Since our previous experiments confirmed that PAX8-PPARγ was specifically expressed in solid tumor tissues of thyroid cancer and had an impact on its malignant biological behavior both in vivo and ex vivo, this would potentially provide a new molecular tumor marker for thyroid cancer and provide the necessary experimental basis for the next quantitative assessment of PAX8-PPARγ aggregation in thyroid cancer in vivo by molecular imaging methods. Therefore, we envisioned that PAX8-PPARγ coupled with an ultrasound nanobubble contrast agent is needed to find a breakthrough point for early noninvasive diagnosis of thyroid cancer. In this study, we used PLGA nanospheres as a carrier and wrapped PFP inside to try to couple the PAX8-PPARγ antibody on nanobubbles, which is an FDA-approved carrier for in vivo delivery of drugs and microRNAs with unique advantages such as excellent biocompatibility, but its targeting is a difficult problem to be solved. In this experiment, PLGA was conjugated with the PAX8-PPARγ antibody by the carbodiimide method, and the surface of the prepared PLGA nanoparticles contained a large number of carboxyl groups. EDC/NHS was used as the coupling activator to activate the carboxyl groups of the nanoparticles. Then, it was combined with the amino group of streptavidin to form a stable covalent bond connection. In recent years, perfluorocarbons have been heavily used in various studies, and liquid fluorocarbons have a unique advantage, i.e., acoustic effect; however, the accumulation effect of liquid fluorocarbon emulsifiers in vivo alone to enhance ultrasound development is poor. In this study, perfluoropentane (PFP) with a boiling point of 29°C was used and wrapped in PLGA with a temperature threshold for the occurrence of liquid-gas phase transition which nearly doubled compared to the actual boiling point of PFP, and the targeted nanoparticles started to undergo phase transition when the temperature rose to 40–50°C. In the in vitro experiments, after the selected LIFU irradiation, the ultrasound image shows a significant acoustic enhancement effect on the nanoparticles of the PFP group, indicating that ultrasound can effectively induce the liquid fluorocarbon phase transition. The heating plate causes the liquid-gas phase change of nanoparticles only by temperature change regulation, while ultrasound can produce a cavitation effect and a mechanical effect in addition to the thermal effect, and these effects combined on PFP-targeted nanoparticles can lead to a greater possibility of phase change. In summary, the PFP-targeted nanoparticles prepared in this study can undergo liquid-gas phase transition in a short time under the excitation of LIFU, which provides a basis for the next in vivo experimental study.PAX8-PPARγ tyrosine phosphatase is encoded by the PTPN11 gene and regulates apoptosis, proliferation, and growth of hematopoietic stem cells in several signaling pathways. It has been shown that PAX8-PPARγ overexpression may increase the risk of tumor metastasis in different types of cancers, including hepatocellular carcinoma, breast cancer, and colon cancer. Our previous findings confirmed that the degree of PAX8-PPARγ expression in thyroid cancer tissues was positively correlated with tumor differentiation and progression, and that high PAX8-PPARγ expression in thyroid cancer tissues also increased thyroid cancer metastasis.Thyroid cancer is currently diagnosed clinically based on 2D ultrasound, and the gold standard for preoperative diagnosis mainly relies on fine needle aspiration cytology, but this is an invasive test. At present, ultrasonography disease is also used in the differential diagnosis of thyroid cancer, but its specificity and sensitivity are not high, which poses a problem for early diagnosis of thyroid cancer. Clinically used ultrasound contrast agents use lipid-encapsulated gas to enhance the ultrasound contrast by increasing the principle of backscatter, and some studies have reported that targeted ultrasound contrast agents have been used in the differential diagnosis of early pancreatic cancer, breast cancer, and ovarian cancer to improve the accuracy of ultrasound diagnosis. However, their micrometer diameter can only be used as a blood pool visualization within the blood vessels. Since PAX8-PPARγ was confirmed to be specifically highly expressed in thyroid cancer tumor tissues in our previous study, and had low or no expression in normal thyroid tissues, this suggests that PAX8-PPARγ may be a potential marker to identify thyroid tumors and may have some diagnostic specificity in ultrasound molecular imaging to increase the diagnostic accuracy.In this study, PAX8-PPARγ-targeted contrast agents were successfully prepared for in vitro and in vivo experiments to test the target binding specificity of targeted nanocontrast agents. The results of cellular assays showed that the targeted nanoparticles group (NPs-PAX8-PPARγ) had a higher affinity for thyroid cancer cells compared to the nontargeted group (NPs-Control). In vivo target-seeking experiments in nude mice showed that the tumor ultrasound signal was significantly enhanced in nude mice injected with targeted and nontargeted nanoultrasound contrast agents via the tail vein immediately after irradiation with LIFU, while the tumor ultrasound signal enhancement was not significant in the nontargeted group. These results suggest that PAX8-PPARγ has the potential to be used as a specific marker of the targeted contrast agent for real-time, non-invasive, and early diagnosis of thyroid tumors, and may provide a new theoretical basis and drug target for clinical targeted therapy of thyroid cancer, which has not been reported in the literature at home and abroad, and therefore will promote the further development of thyroid cancer diagnosis and treatment with high basic science and a high practical application value.



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*Source: 3265342-2022-06-23.xml*