Evaluation of parietal pleural invasion in subpleural lung cancer by VEGFR2 targeted microbubble-based ultrasound molecular imaging

Introduction

Lung cancer is the leading cause of cancer-related death worldwide (1). Invasion of the chest wall by peripheral lung cancer accounts for 5–8% of cases, which is an independent indicator of T3 stage, indicating a poorer prognosis and more complex treatment (2-4). The depth of invasion can be classified into two subtypes: those limited to the parietal pleura, designated pleural invasion 3a (PL3a), and those penetrating the parietal pleura, designated pleural invasion 3b (PL3b) (5,6). Lesion mobility and direct signs of invasion may be obvious and easy to detect (7); thus, several noninvasive medical imaging techniques, such as computed tomography (CT), magnetic resonance imaging (MRI) and ultrasound (US) (8-10), have demonstrated relatively high and comparable diagnostic accuracy rates for PL3b lung tumors. However, in PL3a tumors, early superficial invasion may not significantly affect lung lesion mobility, and signs of direct invasion may be too slight to be observed via common medical imaging techniques. One study reported that four-dimensional dynamic ventilation CT was superior to conventional CT in identifying parietal pleural invasion by assessing the abnormal regional motion of the lung surface, but with more radiation. In our previous research (11), abnormally enhanced areas between lesions and the parietal pleura were observed in 17 of 20 subpleural lung cancer patients with parietal pleura invasion via contrast-enhanced ultrasound (CEUS) imaging, whereas only nine of those 20 lesions were detected via B-mode US. The remaining three PL3a lung tumors with abnormally enhanced areas between the lesions and the parietal pleura were confirmed as the nodular invasive lesions on the parietal pleura discovered during the operation and pathological examinations. These invasive lesions simultaneously increased and diminished along with the normal pleura on CEUS, which may be the reason for the difficulty in detection.

Neoangiogenesis is essential for tumor growth, invasion, and metastasis (12-14). During the process of angiogenesis, vascular endothelial growth factor receptor 2 (VEGFR2) is an important regulatory factor that is more highly expressed on vascular endothelial cells of tumors than on those of normal tissue (15,16), making it a good target for VEGFR2-targeted microbubbles (VEGFR2-MBs) via US molecular imaging for detecting early lesions in cervical cancer and breast cancer (17,18). However, its value in evaluating parietal pleural invasion in patients with peripheral lung cancer remains uncertain.

Therefore, in our study, VEGFR2-MBs were first constructed via the streptavidin (SA)-biotin bridge method, and their basic characteristics were detected. A rabbit subpleural VX2 lung cancer model with early parietal pleura invasion was subsequently established via the method described in our previous study. Finally, the value of targeted microbubbles and blank microbubbles in evaluating parietal pleural invasion in patients with lung cancer was compared and analyzed. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1758/rc).

Methods

Preparation and characterization of VEGFR2-MBs

The VEGFR2-MBs in our study were prepared via the SA-biotin bridge method as previously described in the literature. Briefly, formulations composed of dipalmitoyl phosphatidylcholine (DPPC), 2-stearoyl-phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), DSPE-PEG2000-biotins and dipalmitoyl phosphatidic acid (DPPA) (Avanti Research, Alabaster, Alabama, USA) at a molar ratio of 30:3:1:1 were dissolved in 5 mL dipropylene glycol in a water bath at 60 ℃ for 30 min, and then a mixture of 5 mL glycerol and 40 mL ultrapure water was added with nitrogen introduced from the bottom of the liquid simultaneously to avoid an oxidation reaction with oxygen. Equal amounts of SA were added to DSPE-PEG2000-biotins, after which the phospholipid dispersion solution was cooled. Next, 2 mL of the phospholipid dispersion solution was added to the vial, and 20 mL of perfluoropropane gas (C3F8) (Guangqi Gas Co., Ltd., Guangzhou, China) was used to replace the above mixture, followed by mechanical vibration at a frequency of 3,000 Hz for 50 s to obtain SA-biot-MBs. Then, 20 µL of biot-VEGFR2 antibody (1 mg/mL) (Cell Signaling Technology, USA) was added to the SA-biot-MBs, which were incubated for 30 min on ice. After being centrifuged at 400 ×g for three minutes to remove the free SA and biot-VEGFR2 antibody and resuspended in 5% glycerol solution, the VEGFR2-MBs were prepared successfully. Control microbubbles (Con-MBs) were prepared via the same protocol without DSPE-PEG2000-biotins, SA, and biot-VEGFR2 antibodies.

The morphology of the microbubbles was observed via optical microscopy (Nikon, Tokyo, Japan), and their size was evaluated via dynamic light scattering (DLS) (Malvern Zetasizer Nano, Malvern, U.K.) at 25 ℃.

In vitro specific targeting ability of VEGFR2-MBs

To verify the ability of VEGFR2-MBs to target cells in vitro, human umbilical vein endothelial cells (HUVECs, ScienCell, San Diego, California, USA) highly expressing VEGFR2 were cultured in endothelial cell medium (ECM, ScienCell) supplemented with 5% fetal bovine serum (FBS), 1% endothelial cell growth supplement and 1% penicillin/streptomycin in a 5% CO₂ humidified incubator at 37 ℃.

First, the expression of VEGFR2 in HUVECs was verified via immunofluorescence staining. HUVECs were seeded in 6-well plates at a density of 1×10⁶ cells/well and incubated overnight. The next day, the culture media were discarded; the cells were washed with 1 mL of phosphate-buffered saline (PBS) and then fixed with 1 mL of methanol for 30 min at 37 ℃. After washing, the cells were incubated in 1 mL of VEGFR2 antibody solution diluted 1:500 with PBS for 60 min in a shaking incubator at room temperature. Then, all the cells were washed with PBS three times and stained with an AF555-labeled mouse anti-rabbit secondary antibody in the dark for 30 min. After being washed, the cells were stained with Hoechst333342 (Beyotime, Shanghai, China) and observed under an inverted fluorescence microscope (200×).

The ability of VEGFR2-MBs to target HUVECs was subsequently tested. HUVECs were seeded in four-well plates and incubated overnight. VEGFR2-MBs and Con-MBs were diluted 1:20 with PBS. After washing with PBS three times, the cells were incubated with one mL of VEGFR2-MBs, Con-MBs or VEGFR2-MBs after preincubation with VEGFR2 antibody for 30 min. The plates were filled with PBS at a depth of 2 cm to raise the uncombined microbubbles and observed under an optical microscope (200×). The number of microbubbles and cells was counted. The above experimental procedures were repeated three times each, and the average values were taken.

Model construction and confirmation of subpleural lung tumors

SPF-level New Zealand white rabbits (10 male and 10 female) with body weights of 2.5–3 kg were obtained from Guangdong Mingzhu Biotechnology Co., Ltd. (Guangzhou, China), and maintained under standard environmental conditions. A protocol was prepared before the study without registration. The experimental operations were conducted in Guangzhou Huateng Biomedical Technology Co., Ltd. All animal experiments were performed under a project license (No. HTSW221023) granted by the Animal Care Committee of Guangzhou Huateng Biomedical Technology Co., Ltd., in compliance with institutional guidelines for the care and use of animals.

A model of subpleural lung cancer with early parietal pleura invasion was established via our previous study (11). The VX2 tumor tissue frozen in a −80 ℃ refrigerator was rapidly resuscitated in a 37 ℃ water bath and cut into pieces in saline. After routine anesthesia induction, skin preparation and disinfection, the needle tip of a 1 mL syringe containing 0.5 mL of VX2 tumor tissue block suspension was guided to the pleura under real-time ultrasonic guidance and then inserted 3–5 mm into the subpleural lung parenchyma. In the end, the VX2 tumor tissue was injected at two points of the lung parenchyma beneath the pleura and pleura line where the lung tissue contacted the pleura, for a more rapid occurrence of parietal pleura invasion. The complications were examined with US. Penicillin (200,000 U/d) was administered via intramuscular injection for one day to prevent pulmonary infection. The lung US examination was performed seven days after tumor implantation. The model was considered to be successfully established if a subpleural lung tumor was detected by US in the injection area. Two weeks after the subpleural lung tumors were observed via US, CEUS examination was performed, and the animal models were sacrificed to obtain tumor tissue.

Evaluation of subpleural lesions and parietal pleura invasion

After anesthesia induction, the rabbits were placed laterally on the experimental table. B-mode US and CEUS examinations were performed via a transthoracic US instrument (Resona 7T; Mindray, Shenzhen, China) with an L9-3U probe (2.5–9.0 MHz). The subpleural lesions and parietal pleura were observed in detail via B-mode US. When the tumor penetrated the parietal pleura and invaded the muscles or ribs, exclusion was necessary. CEUS of VEGFR2-MBs and Con-MBs was subsequently performed by two sonographers with 5 years of experience in lung US and CEUS. First, the probe was fixed on the largest section of the tumor, and a 0.1 mL/kg bolus of Con-MBs was intravenously injected, followed by flushing with 2 mL of saline to evaluate the enhancement characteristics of the subpleural lesions by Sonographer 1. After an interval of 60 min to remove the remaining microbubbles, another 0.1 mL/kg bolus of Con-MBs was intravenously injected, and the probe was moved around the subpleural lesions to observe the parietal pleura. On the second day, the enhancement of VEGFR2-MBs on the subpleural lesions and parietal pleura was performed by sonographer 2, who was blinded to the results of the Con-MBs in the same way. All the data were recorded and saved in video form. The CEUS data of subpleural lesions were quantitatively analyzed through time-intensity curve (TIC). The data of the parietal pleura were reviewed by the two sonographers to make a consistent decision of parietal pleura invasion when abnormally enhanced areas were observed on the parietal pleura and/or between lesions and the parietal pleura.

The gold standard for detecting parietal pleura invasion of subpleural lung cancer is anatomical findings during surgery and pathological examinations. The parietal pleura was not invaded if the pleura was smooth and complete without adhering to the lesion. When parietal pleural invasion was suspected, a pathological evaluation of the subpleural lesion, visceral pleura, and parietal pleura was conducted for confirmation. All the anatomical samples were fixed in 10% formalin and subjected to hematoxylin and eosin (HE) staining. The pathological procedures were determined through consultation between two pathologists with at least 5 years of experience who were blinded to the sonographers’ evaluation results.

Statistical analyses

Statistical analyses were performed via SPSS version 22.0 (IBM Corp., Armonk, NY, USA). Continuous variables are expressed as the means with standard deviations, and categorical variables are expressed as frequencies or percentages. The accuracy, specificity, and sensitivity of VEGFR2-MBs and Con-MBs in diagnosing parietal pleura invasion of subpleural lung cancer were analyzed with paired χ² tests. A P value <0.05 in a two-sided test was considered statistically significant.

Results

The characteristics of VEGFR2-MBs

The preparation of VEGFR2-MBs in our study was carried out via the SA-biotin bridge method, which is common for linking microbubbles with antibodies firmly. Compared with Con-MBs, the VEGFR2-MBs visually appeared as a uniform spherical shape under the optical microscope, with an average particle size of 1.7±0.51 µm, which was not significantly different from that of Con-MBs, with an average particle size of 1.5±0.54 µm (Figure 1A).

Figure 1 The characteristics of VEGFR2-MBs. (A) The microbubbles of VEGFR2-MBs and Con-MBs visually appeared as a uniform spherical shape under the optical microscope, with an average particle size of 1.7±0.51 and 1.5±0.54 µm detected by DLS, respectively, without significant statistical difference. (B) The expression of VEGFR2 on HUVECs was confirmed with immunocytochemical method. (C) The adherence of Con-MBs and VEGFR2-MBs on HUVECs and VEGFR2-MBs on HUVECs preincubated with VEGFR2 antibody with the average number of 0.1±0.04, 7.6±1.6, and 2.1±0.8 bubbles per cell, with a significant statistical difference. *, P<0.05. Con-MBs, control microbubbles; DLS, dynamic light scattering; HUVECs, human umbilical vein endothelial cells; VEGFR2, vascular endothelial growth factor receptor 2; VEGFR2-MBs, vascular endothelial growth factor receptor 2-targeted microbubbles.

In vitro adhesion ability of VEGFR2-MBs

To verify the targeting ability of VEGFR2-MBs in vitro, we selected HUVECs with positive expression of VEGFR2 molecules for the experiment. First, the expression of VEGFR2 molecules on the surface of HUVECs was confirmed through immunofluorescence staining (Figure 1B). The cell nucleus was stained with Hoechst33342 and is shown in blue. AF555 is conjugated with VEGFR2 antibodies, which mark VEGFR2 molecules red and are distributed on the cell membrane surface around the cell nucleus.

Next, the targeting specificity and stability of VEGFR2-MBs were verified in HUVECs. A considerable number of VEGFR2-MBs were bound to the surface of the HUVECs, with an average number of 7.6±1.6 bubbles per cell in the VEGFR2-MBs group, and 0.1±0.04 bubbles per cell in the Con-MBs group. In the preincubated VEGFR2 antibody group, the average number of 2.1±0.8 bubbles per cell bound to the cells was significantly lower than that in the targeted microbubble group (Figure 1C). When observed under a microscope, the cell culture dish was filled with PBS solution to a depth of 20 mm. Owing to their buoyancy, the unbound microbubbles floated on the surface, whereas the VEGFR2-MBs still managed to bind to the cells, which indicated that our targeted microbubbles have good targeting specificity and stability.

Construction of the animal model

Two rabbits with muscle tissue invasion observed by B-mode US and confirmed by anatomical findings were excluded, and 18 rabbits with subpleural lung tumors were included in the study. One patient developed a large amount of pleural effusion and died during the CEUS examination. A total of 17 rabbits with tumors were subjected to all the examinations within 10–21 days after tumor injection. On the basis of the anatomical findings or pathological results, there were eleven and six cases of subpleural lesions with and without parietal pleura invasion, respectively.

CEUS characteristics of VEGFR2-MBs and Con-MBs in subpleural lung tumors

During the 180-second CEUS process, all the lung tumors showed rapid high enhancement in the early phase, followed by gradual weakening in the middle and later phases. The weakening speed in the VEGFR2-MB group was slower than that in the Con-MB group (Figure 2). The analysis of the TIC revealed statistically significant differences in half time of descent (HT), descending slope (DS), and area under the curve (AUC) between the VEGFR2-MB group and the Con-MB group (P<0.05), whereas there were no differences in the arrival time (AT), time to peak (TTP) or peak intensity (PI) (Table 1). This finding indicated that VEGFR2-MBs remained in the tumor tissue for a longer period of time than Con-MBs did.

Figure 2 CEUS characteristics of VEGFR2-MBs and Con-MBs in subpleural lung tumors. (A) The CEUS of subpleural lung tumors with VEGFR2-MBs and Con-MBs at different time points. (B) The TIC of subpleural lung tumors with VEGFR2-MBs and Con-MBs. CEUS, contrast-enhanced ultrasound; Con-MBs, control microbubbles; TIC, time-intensity curve; VEGFR2-MBs, vascular endothelial growth factor receptor 2-targeted microbubbles.

CEUS characteristics of VEGFR2-MBs and Con-MBs on parietal pleura

During the CEUS examination, the enhanced pattern of the normal parietal pleura was the same as that of subpleural lung tumors. When the parietal pleura was infiltrated, the abnormally enhanced areas between the lesions and the parietal pleura were observed in the early phase and simultaneously weakened with the parietal pleura in the middle and later phases in both VEGFR2-MBs and Con-MBs groups. However, the abnormally enhanced areas on the parietal pleura were shown as higher enhancement than that of the normal parietal pleura in the middle and later phases of VEGFR2-MBs group, while not observed in the entire phase of Con-MBs group. The above results were all confirmed by pathology (Figure 3).

Figure 3 CEUS characteristics of VEGFR2-MBs and Con-MBs on parietal pleura. Red circle shown the abnormally enhanced areas on the parietal pleura as higher enhancement than that of the normal parietal pleura in the middle and later phases of VEGFR2-MBs group, while not observed in the entire phase of Con-MBs group. Green square shown the abnormally enhanced areas between the lesions and the parietal pleura were observed in the early phase of CEUS with both VEGFR2-MBs and Con-MBs. They were all confirmed by pathology. Hematoxylin and eosin staining is used for the pathological tissue. CEUS, contrast-enhanced ultrasound; Con-MBs, control microbubbles; VEGFR2-MBs, vascular endothelial growth factor receptor 2-targeted microbubbles.

Comparison of VEGFR2-MBs and Con-MBs in the diagnosis of parietal pleura invasion

The characteristics of CEUS images of VEGFR2-MBs and Con-MBs with parietal pleura invasion are shown in Table 2. There were 4, 3, 2 and 6, 0, 0 cases with abnormally enhanced areas observed between the lesions and the parietal pleura, on the parietal pleura and both in the VEGFR2-MBs and Con-MBs groups, respectively. The sensitivity, specificity, and accuracy of the VEGFR2-MBs and Con-MBs groups for diagnosing parietal pleura invasion were 81.8%, 100.0%, and 88.2% and 54.5%, 100.0%, and 70.6%, respectively. There was a statistically significant difference between the two groups in terms of accuracy (P=0.153) and specificity (P=0.109) according to Fisher’s exact test. The κ values of the VEGFR2-MB and Con-MB groups were 0.761 (95% CI: 0.458–1.000) and 0.459 (95% CI: 0.210–0.896), respectively.

Discussion

Accurate preoperative diagnosis of chest wall invasion in subpleural lung cancer patients is essential for formulating treatment plans and assessing patient prognosis. However, early imaging diagnosis of parietal pleural invasion (PL3a) is often challenging. Our study demonstrated that parietal pleural invasion in subpleural lung cancer could be distinguished from surrounding normal tissues by US molecular imaging with VEGFR2-MBs. This easy-to-form, noninvasive imaging method may be a useful diagnostic tool for subpleural lung cancer with parietal pleural invasion.

Several meaningful indirect signs have been used to predict parietal pleural invasion or adhesion in patients with subpleural lung cancer. The relative distance between the lung cancer and the chest wall is shorter because of adhesion. By tracking the movements of the cancer and an adjacent structure of the chest wall, four-dimensional dynamic ventilation CT was reported to be significantly superior to conventional CT in the assessment of parietal pleural invasion or adhesion in subpleural lung cancer, although only 16 cases were investigated (19). On the basis of the advantages of real-time evaluation of lesion movement, B-mode US has been reported to be more accurate than CT in diagnosing wall invasion in patients with lung cancer (8,9). However, distinguishing between fibrous adhesion and invasive adhesion was difficult, and the decrease in movement distance was affected by the degree of adhesion. Blood vessels are essential for the growth and metastasis of tumors (20). If the adhesion between lung cancer and the chest wall is confirmed to have a blood supply, then the accuracy of predicting parietal pleural invasion by lung cancer may be greater. Abnormally enhanced areas between lesions and the parietal pleura in the CEUS examination in our previous research were used to evaluate pleural invasion or adhesion. The diagnostic sensitivity, specificity, and accuracy are 85.0%, 100.0%, and 91.2%, respectively, which are superior to those of B-mode US (11). Unfortunately, tiny lesions on the parietal pleura were missed, possibly because the lesions and surrounding normal tissues were both enhanced and receded simultaneously on CEUS. Similarly, in this study, six rabbits with parietal pleural adhesion were detected by both VEGFR2-MBs and Con-MBs. Interestingly, five of the seven rabbits with tiny invasive lesions on the parietal pleura were detected in the VEGFR2-MBs group but not in the Con-MBs group. The specific signs manifested as abnormally enhanced areas on the parietal pleura in the middle and late stages of the CEUS examination. This may be attributed to the targeted microbubbles specifically binding to VEGFR2 molecules on the surface of tumor blood vessel endothelial cells, remaining in the tumor area, thereby causing their echo intensity to be greater than that of the surrounding normal tissues. The TIC of lung cancer also supported the idea that targeted microbubbles could remain in the tumor for a longer period of time. Similar VEGFR2-MBs have been applied to the diagnosis of microinvasive lesions in cervical cancer and breast cancer (17,18,20,21).

Targeted microbubbles are composed of microbubbles, specific antibodies, or molecular peptides (22,23). The film hydration method is a commonly used approach for preparing microbubbles. Chloroform is an important solvent in the process of preparing microbubbles. However, it is volatile and toxic and needs to be carried out in a fume hood (24,25). In our research, propylene glycol and glycerol were used as solvents, which demonstrated better safety. The targeted microbubbles prepared via this method had a regular spherical shape and uniform particle size, with an average diameter of approximately 1.7±0.51 µm. The targeting effect of VEGFR2-MBs on HUVECs expressing VEGFR2 was verified. The petri dish in our study was filled with PBS to a depth of 20 mm. Free microbubbles or those that are not firmly attached floated on the surface of the liquid. More microbubbles were observed under the microscope than in the Con-MBs group. When the HUVECs were preincubated with the VEGFR2 antibody, the number of microbubbles bound to the cell surface significantly decreased, which indicated that the binding of targeted microbubbles to the target was blocked. Although we did not use the immunofluorescence method to show that the VEGFR2 antibody was attached to the microbubbles through the SA-biotin bridge, the good targeting properties of the VEGFR2-MBs in this study were confirmed by the specific targeted binding, which was comparable to those reported in the literature (Figure 1).

The rabbit subpleural lung cancer model was constructed following the methods described in our previous research, with some improvements. During the operation, we injected VX2 tumor tissue at the locations of the lung parenchyma and the pleural line under US guidance, with the aim of enabling the animal model to develop tumors simultaneously in the lungs and the parietal pleura as soon as possible. Compared with blind insertion or CT guidance (26), this method is more accurate, simple and safe. Although this method does not conform to the biological characteristics of lung cancer invading the parietal pleura, it could meet the experimental requirements of our study to verify the value of targeted microbubbles in the diagnosis of parietal pleural invasion. A method for constructing a rabbit model of subpleural lung cancer with parietal pleura invasion, which was highly successful and reproducible, has been presented.

There are some limitations in this research. First, the stability and biological safety of VEGFR2-MBs have not been verified, which is a challenge in clinical translation. Second, the expression of VEGFR2 is influenced by various factors such as tumor heterogeneity and pathological subtypes, and it has certain limitations in terms of universality. Furthermore, owing to experimental constraints, ultrasonography was not compared with CT or MRI and the pathological gold standard. Finally, an animal model was employed, so further clinical verification is needed. And owing to the limited sample size, we could not analyze the factors influencing diagnostic accuracy.

Conclusions

VEGFR2-MBs-based US molecular imaging has great potential for clinical application in early detection of parietal pleura invasion in peripheral lung cancer patients.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1758/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1758/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1758/prf

Funding: This study was supported by the Basic Research Program of the Science and Technology Bureau of Guangzhou City in 2023 (No. SL2023A03J00567), Guangzhou Joint Projects of University in 2024 (No. 2024A03J1226), Guangzhou Health and Wellness Science and Technology Project in 2023 (No. 20231A011085), Guangzhou Health and Wellness Science and Technology Project in 2024 (No. 20241A011076), the Science and Technology Program of Guangzhou (No. 202201020433), and Project for Cultivating and Promoting Technological Transformation of the First Affiliated Hospital of Guangzhou Medical University in 2018 (No. ZH201810).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1758/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments were performed under a project license (No. HTSW221023) granted by the Animal Care Committee of Guangzhou Huateng Biomedical Technology Co., Ltd., in compliance with institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Siegel RL, Kratzer TB, Giaquinto AN, et al. Cancer statistics, 2025. CA Cancer J Clin 2025;75:10-45. [Crossref] [PubMed]
2. Ettinger DS, Wood DE, Aisner DL, et al. NCCN Guidelines® Insights: Non-Small Cell Lung Cancer, Version 2.2023. J Natl Compr Canc Netw 2023;21:340-50. [Crossref] [PubMed]
3. Li H, Liu T, Sun Z, et al. Primary tumor resection of non-small cell lung cancer patients with ipsilateral pleural dissemination (M1a) in the era of targeted therapy. Thorac Cancer 2020;11:3213-22. [Crossref] [PubMed]
4. Kawaguchi K, Mori S, Usami N, et al. Preoperative evaluation of the depth of chest wall invasion and the extent of combined resections in lung cancer patients. Lung Cancer 2009;64:41-4. [Crossref] [PubMed]
5. Sakakura N, Mori S, Ishiguro F, et al. Subcategorization of resectable non-small cell lung cancer involving neighboring structures. Ann Thorac Surg 2008;86:1076-83; discussion 1083. [Crossref] [PubMed]
6. Sakakura N, Mizuno T, Kuroda H, et al. The eighth TNM classification system for lung cancer: A consideration based on the degree of pleural invasion and involved neighboring structures. Lung Cancer 2018;118:134-8. [Crossref] [PubMed]
7. Chansky K, Detterbeck FC, Nicholson AG, et al. The IASLC Lung Cancer Staging Project: External Validation of the Revision of the TNM Stage Groupings in the Eighth Edition of the TNM Classification of Lung Cancer. J Thorac Oncol 2017;12:1109-21.
8. Tahiri M, Khereba M, Thiffault V, et al. Preoperative assessment of chest wall invasion in non-small cell lung cancer using surgeon-performed ultrasound. Ann Thorac Surg 2014;98:984-9. [Crossref] [PubMed]
9. Bandi V, Lunn W, Ernst A, et al. Ultrasound vs. CT in detecting chest wall invasion by tumor: a prospective study. Chest 2008;133:881-6. [Crossref] [PubMed]
10. Akata S, Kajiwara N, Park J, et al. Evaluation of chest wall invasion by lung cancer using respiratory dynamic MRI. J Med Imaging Radiat Oncol 2008;52:36-9. [Crossref] [PubMed]
11. Zhang Y, Zhang Z, Liao H, et al. Evaluation of parietal pleural adhesion and invasion in subpleural lung cancer: value of B-mode ultrasound and contrast-enhanced ultrasound. Quant Imaging Med Surg 2024;14:3302-11. [Crossref] [PubMed]
12. Tian L, Goldstein A, Wang H, et al. Mutual regulation of tumour vessel normalization and immunostimulatory reprogramming. Nature 2017;544:250-4. [Crossref] [PubMed]
13. Viallard C, Larrivée B. Tumor angiogenesis and vascular normalization: alternative therapeutic targets. Angiogenesis 2017;20:409-26. [Crossref] [PubMed]
14. Chen J, Liu BX, Shen Q, et al. Limonin inhibits angiogenesis and metastasis of human breast cancer cells by suppressing the VEGFR2/IGFR1-mediated STAT3 signaling pathway. Transl Cancer Res 2020;9:6820-32. [Crossref] [PubMed]
15. Li G, Gao J, Ding P, et al. The role of endothelial cell-pericyte interactions in vascularization and diseases. J Adv Res 2025;67:269-88. [Crossref] [PubMed]
16. Liu ZL, Chen HH, Zheng LL, et al. Angiogenic signaling pathways and anti-angiogenic therapy for cancer. Signal Transduct Target Ther 2023;8:198. [Crossref] [PubMed]
17. Bzyl J, Palmowski M, Rix A, et al. The high angiogenic activity in very early breast cancer enables reliable imaging with VEGFR2-targeted microbubbles (BR55). Eur Radiol 2013;23:468-75. [Crossref] [PubMed]
18. Zhong J, Su M, Jiang Y, et al. VEGFR2 targeted microbubble-based ultrasound molecular imaging improving the diagnostic sensitivity of microinvasive cervical cancer. J Nanobiotechnology 2023;21:220. [Crossref] [PubMed]
19. Sakuma K, Yamashiro T, Moriya H, et al. Parietal pleural invasion/adhesion of subpleural lung cancer: Quantitative 4-dimensional CT analysis using dynamic-ventilatory scanning. Eur J Radiol 2017;87:36-44. [Crossref] [PubMed]
20. Pochon S, Tardy I, Bussat P, et al. BR55: a lipopeptide-based VEGFR2-targeted ultrasound contrast agent for molecular imaging of angiogenesis. Invest Radiol 2010;45:89-95. [Crossref] [PubMed]
21. Lee DJ, Lyshchik A, Huamani J, et al. Relationship between retention of a vascular endothelial growth factor receptor 2 (VEGFR2)-targeted ultrasonographic contrast agent and the level of VEGFR2 expression in an in vivo breast cancer model. J Ultrasound Med 2008;27:855-66. [Crossref] [PubMed]
22. Park D, Song G, Jo Y, et al. Sonophoresis Using Ultrasound Contrast Agents: Dependence on Concentration. PLoS One 2016;11:e0157707. [Crossref] [PubMed]
23. Li J, Zhou P, Xu H, et al. Antitumor activity of integrin α(V)β(3) antibody conjugated-cationic microbubbles in liver cancer. Transl Cancer Res 2019;8:899-908. [Crossref] [PubMed]
24. Luo W, Wen G, Yang L, et al. Dual-targeted and pH-sensitive Doxorubicin Prodrug-Microbubble Complex with Ultrasound for Tumor Treatment. Theranostics 2017;7:452-65. [Crossref] [PubMed]
25. Wang D, Luo W, Wen G, et al. Synergistic effects of negative-charged nanoparticles assisted by ultrasound on the reversal multidrug resistance phenotype in breast cancer cells. Ultrason Sonochem 2017;34:448-57. [Crossref] [PubMed]
26. Wang L, Che K, Liu Z, et al. Establishment and evaluation of the VX2 orthotopic lung cancer rabbit model: a ultra-minimal invasive percutaneous puncture inoculation method. Korean J Physiol Pharmacol 2018;22:291-300. [Crossref] [PubMed]