Blood Supply of Early Lung Adenocarcinomas in Mice and the Tumor-supplying Vessel Relationship: A Micro-CT Angiography Study

Lung cancer is among the most frequently occurring malignancies and is the leading cause of death worldwide. Adenocarcinoma is the most frequent histologic type, accounting for approximately 40% of lung cancers (1, 2). Because the 5-year survival rate is significantly higher for patients with stage IA lung cancer than for those with advanced lung cancer (71.1% vs. 16.8%; refs. 3, 4), early detection, accurate diagnosis, and treatment are critical for improving the prognosis of lung cancer. The blood supply, which plays a pivotal role in oncogenesis, development, and metastasis, is useful for the early detection and diagnosis of lung cancer (5). It is well known that the blood supply of lung cancer mainly originates from the bronchial artery, especially in the advanced stage of cancer (6). However, the blood supply of early lung cancers has not been completely elucidated. Our previous clinical studies showed that the pulmonary artery and vein were the main related vessels of early lung adenocarcinomas, and their morphologic abnormalities could be used to differentiate minimally invasive adenocarcinomas (MIAs) and preinvasive lesions from invasive adenocarcinomas appearing as ground-glass nodules (7, 8). Other studies investigated the nodule–vessel relationship and found that it was helpful for distinguishing malignant nodules from benign nodules (9–11). These studies suggest that vessel morphology markedly changes along with the growth of lung cancer. Therefore, the evaluation of vessel morphology and its change law with invasive statues will be helpful for the qualitative and quantitative (such as invasiveness) diagnosis of lung cancer.

However, early lung adenocarcinomas are too small to be analyzed for CT studies of small vessels after being sampled for frozen and paraffin section diagnoses. Primary lung cancers in mice have morphologic, histogenic, and molecular features similar to those of human lung adenocarcinomas. In this study, we established a mouse model of early lung adenocarcinomas based on our previous studies and performed pulmonary and bronchial micro-CT arteriography to investigate the blood supply of early lung adenocarcinomas and the relationship between early lung adenocarcinomas and their supplying vessels. We thought that our observations in mice would confirm our hypothesis and would have significance in translational medicine.

This study was conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Science and Technology Committee of China, and with the approval of the Institutional Review Board of Jinshan Hospital, Fudan University (Shanghai, China) Institutional Animal Care and Use Committee. Every effort was made to minimize suffering and the number of animals used in this experiment. Three- to 4-week-old female KM mice (Jiesijie Laboratory Animal Company) weighing 18–22 g were housed under a temperature of 23°C with a 12-hour light/dark cycle. Food and water were available ad libitum. The mice were randomly assigned to experimental and control groups. As described previously (12), an early lung adenocarcinoma model was established in 10 mice by subcutaneously injecting 0.2 mL 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) solution (2.0 mg/mL; Ruji Biotech Company) once weekly for 4 weeks. The control group comprised of 5 mice that were subcutaneously injected with 0.2 mL normal saline once weekly for 4 weeks.

On the 90th day after the first injection, mice in the experimental and control groups were deeply anesthetized with 0.02 mL ketamine solution (10 mg/mL) and heparinized. A thoracotomy was performed via a midline incision, and the heart and lungs were exposed. The bilateral superior and inferior vena cava were ligatured. Through the right ventricle, the pulmonary artery was punctured with a 26-G needle. A 30-cm long polyethylene tube connected to a 26-G needle was used as a catheter and inserted into the aorta to perfuse the bronchial artery. To flush the blood out of the pulmonary artery and bronchial artery, diluted heparin sodium (50 U/mL) was pumped at a rate of 0.5 and 2 mL/minutes, respectively, using a continuous syringe pump (Longer Precision Pump, Co.). Paraformaldehyde was pumped at the same flow rate as above to fix the pulmonary artery and bronchial artery. Microfil (Flow Tech, Inc.), a silicone polymer casting compound, was mixed with a diluent at a 5:4 (diluent:compound) volume ratio and added to a 5% (by volume) curing agent. This freshly mixed silicone polymer casting material was then pumped into the pulmonary artery and bronchial artery at rates of 0.1 and 0.5 mL/minutes, respectively. The perfusion was stopped when the polymer was uniformly visible at the lung surface or incision in the inferior vena cava. After complete polymerization at 4°C for 24 hours, the tissues were fixed with 10% formalin for 24 hours.

The lungs were inflated until the bottom reached the diaphragm. The micro-CT (Quantum GX, PerkinElmer, Inc.) scan was performed using the following protocol: voltage, 90 kV; current, 88 mA; field of view (FOV), 36 × 25 mm; acquisition time, 14 minutes; camera mode, high resolution; matrix size, 512 × 512; and spatial resolution, 50 μm. After identifying all tumors on the original images, each tumor was set in the center, and an FOV of 4.6 × 4.6 × 4.6 mm was reconstructed to obtain 9 μm high-resolution images.

The original images and high-resolution images were reviewed, and the number, diameter, margin, and solid component of the tumors were analyzed. The supplying vessels of the tumor and their relationships with the tumor (hereafter referred to as the tumor–vessel relationship) were specifically observed.

According to the latest National Comprehensive Cancer Network guidelines for non–small cell lung cancer that suggest surgical resection should achieve resection margins greater than 2 cm, and considering the size ratio of 20:1 for human to mouse lungs (12), the pulmonary arteries and bronchial arteries within the region of interest (ROI), which included the tumor and surrounding lung (1 mm), were used for quantitative and texture analyses with 3D Slicer Software (4.10.2, NIH, Bethesda, MD). In addition, four nontumor ROIs were randomly selected for comparisons in each mouse. The lungs of the control mice were scanned using the same micro-CT protocol. The same quantitative and texture analyses were also employed for four randomly selected ROIs in each control mouse. The sizes of the nontumor ROIs and control ROIs were set between the minimum and maximum tumor ROIs. Quantitative and texture analyses were performed on the basis of five quantitative parameters and 18 textural features (Supplementary Table S1).

The lung lobes of each mouse were separated and fixed in neutral formalin. On the basis of the tumor location on micro-CT images, the lobes were sampled and embedded in paraffin. The whole paraffin block was cut into 50-μm sections from one end, and when a tumor was identified, a series of 3-μm sections at an interval of 50 μm were cut to obtain at least 10 tumor sections. The sections were stained with hematoxylin and eosin (H&E) and microscopically analyzed to determine the histology, diameter, shape, margin, and growth pattern of the tumors, especially the tumor–vessel relationship. The histopathologic findings were compared with the micro-CT findings.

Statistical analyses were performed using SPSS 22.0 Statistical Software (SPSS, Inc.). Pearson χ² test was conducted to compare the relationships between tumor size and tumor–vessel relationship patterns and between the solid component and tumor–vessel relationship patterns. Pairwise comparisons of quantitative parameters and texture features among the tumor, nontumor, and control groups were performed with the Mann–Whitney U test. Spearman correlation analysis was performed to analyze the correlation between the sizes of the tumor and solid component and the quantitative and texture features of the vessels. A correlation coefficient |r| = 0–0.5 was considered a weak correlation, 0.5–0.8 was considered a moderate correlation, and |r| > 0.8 was considered a high correlation. A P value less than 0.05 was considered statistically significant. Quantitative and texture parameters were expressed as medians and quartiles.

Micro-CT revealed tumor formation in all 10 mice in the experimental group. The number of tumors in each mouse ranged from eight to 36, with a total of 167 tumors in 10 mice. All the tumors were lung adenocarcinomas, as confirmed by histology. The tumor sizes ranged from 0.17 to 1.95 mm, with a mean diameter of 0.55 mm. The lung adenocarcinomas were classified into three types based on the proportion of the solid component on micro-CT: nonsolid (NS; n = 44, 27%), totally solid (TS; n = 49, 29%), and partly solid (n = 74, 44%). These types corresponded to lepidic, hilic (solid), and mixed growth patterns in histopathology, respectively (Supplementary Figs. S1–S3). The control group had no tumor formation.

Micro-CT revealed that 129 (77%) tumors connected to or entered the pulmonary arteries, 12 (7%) tumors adjoined but did not connect to the pulmonary arteries, and the remaining 26 (16%) tumors had no relationship with the pulmonary arteries. Histopathology showed that 117 (70%) tumors directly connected to or entered the pulmonary arteries, which contained microfil in their lumina. Instead of destroying the pulmonary arteries, most tumors grew along the pulmonary arteries, forming a perivascular cuff around the pulmonary arteries. No bronchial arteries were identified inside or around any early lung adenocarcinomas (Supplementary Figs. S4–S7).

On micro-CT, the spatial relationships between the tumors and the pulmonary arteries were classified into four patterns: type I (n = 45, 32%), the pulmonary artery was interrupted at the margin of the tumor; type II (n = 38, 27%), the pulmonary artery penetrated into the tumor with a tapered interruption; type III (n = 29, 21%), the pulmonary artery penetrated into the tumor with an intact lumen; and type IV (n = 110, 78%), the pulmonary artery ran along the border of the tumor with an intact or compressed lumen (Figs. 1–4). The statistical analyses found that with an increase in the size of the tumor or the solid component, the prevalence of type I and II tumor–pulmonary artery relationships increased (P < 0.001), and type III was observed mostly in tumors larger than 1 mm (P < 0.001), but there was no significant difference among the tumor patterns (P = 0.169); type IV was observed mostly in NS tumors (P < 0.05), but no significant difference was observed among different sized tumors (P = 0.635). There were significant differences in the prevalence of tumor–pulmonary artery patterns among different sizes of tumors and solid components (both P < 0.001; Supplementary Tables S2 and S3).

The tumor–pulmonary artery relationship was identified pathologically in 71 tumors: type I was observed in 14 (20%) tumors, type II in 18 (25%) tumors, type III in 29 (41%) tumors, and type IV in 47 (66%) tumors. The statistical analyses found that with an increase in the size of the tumor or solid component, the prevalence of type I, II, and III tumor–pulmonary artery relationships increased (P < 0.001); type IV was observed mostly in large tumors (P < 0.001), and the incidence decreased as the size of the solid component increased. There were significant differences in the prevalence of tumor–pulmonary artery patterns among different sizes of tumors and solid components (P < 0.001; Supplementary Tables S2 and S3).

A comparative analysis of imaging and pathology findings showed that the interrupted pulmonary artery sign in the margin or inside of the tumors was not caused by direct destruction but was mainly caused by tumor compression, and the wall of these pulmonary arteries remained intact, without obvious erosion and destruction. This sign was more common in large TS tumors than in NS tumors. In contrast, arterioles were found in the thickened alveolar septa in NS tumors (Supplementary Fig. S8).

The pairwise comparisons of pulmonary artery quantitative parameters and texture features among the tumor, nontumor, and control groups are summarized in Supplementary Table S4. Significant differences were found in vessel volume (VV), vessel volume fraction (VVF), vessel number (VN), vessel separation (VS), energy, entropy, inverse difference moment (IDM), inertia, cluster shade (CS), cluster prominence (CP), Haralick correlation (HC), gray level nonuniformity (GLN), low gray level run emphasis (LGLRE), high gray level run emphasis (HGLRE), short run low gray level emphasis (SRLGLE), short run high gray level emphasis (SRHGLE), long run low gray level emphasis (LRLGLE), and long run high gray level emphasis (LRHGLE) between the tumor group and the nontumor group (all P < 0.05) and in VV, VVF, VN, VS, energy, entropy, correlation, IDM, HC, GLN, and run length nonuniformity (RLN) between the tumor group and the control group (all P < 0.05); in addition, significant differences in VV, VVF, VN, VS, correlation, CS, CP, GLN, RLN, LGLRE, SRLGLE, and LRLGLE were found between the no-tumor group and the control group (all P < 0.05).

The correlations between tumor size and pulmonary artery quantitative parameters and texture features are presented in Table 1. There were moderate positive correlations between tumor size and VV, VVF, GLN, and RLN; weak positive correlations between tumor size and VT, VN, VS, and IDM; and weak negative correlations between tumor size and inertia, SRE, and LRE. The correlations between the size of the solid component of the tumor and pulmonary artery quantitative parameters and texture features are presented in Table 2. There were weak positive correlations between the size of the solid component of the tumor and VV, VVF, VT, VN, GLN, and RLN and weak negative correlations between the size of the solid component of the tumor and VS, CS, and SRE.

Our previous studies (12, 13) have established a mouse model for early lung adenocarcinomas. The optimal dose and time of carcinogen exposure have been extensively investigated and determined. On the 90th day of carcinogen exposure, micro-CT and pathologic studies show that the induced early lung adenocarcinomas include tumors of different sizes, invasiveness, and shapes. Therefore, we adopted this mouse model to investigate the blood supply of early lung adenocarcinomas, the morphologic changes of vessels, and their relationships with tumors.

This study showed that the mouse model of early lung adenocarcinomas manifested a close correlation with pulmonary arteries instead of bronchial arteries; therefore, we infer that the blood supply of early lung adenocarcinomas mainly originates from pulmonary arteries. However, bronchial arteries have long been recognized as the primary supplying vessels of lung cancers (14), and we believe that the different results were due to the different stages of lung cancers examined. Previous studies mainly included advanced and highly invasive lung cancers, which often destroy the originally existing pulmonary arteries and lead to angiogenesis in the systemic circulation to compensate for the blood supply, whereas early lung cancers are noninvasive or less invasive and have been confirmed to be able to efficiently grow by vessel cooption (15–18). Vessel cooption is a mechanism in which lung cancers exploit the originally existing vascular network of the lung and migrate along the vessels of the host organ or fill alveolar spaces to obtain blood supply rather than destroy the vessels. This study also demonstrated that early lung adenocarcinomas proliferated around the pulmonary arteries or migrated toward the adjacent pulmonary arteries without destroying the pulmonary arteries. In addition, it is well known that lung adenocarcinomas originate from alveolar epithelial cells and proliferate along the alveolar septa or extend inside the alveoli (19). The alveolar septa and walls are rich in capillaries, which allow early lung adenocarcinomas to obtain adequate nutrients from the pulmonary arteries (20, 21). Another study confirmed that bronchial arteries supply blood only to the trachea and mainstem bronchi but do not penetrate into the parenchymal airways in mice (22). Therefore, we concluded that early lung adenocarcinomas were mainly fed by pulmonary arteries in mice.

As pulmonary arteries are accompanied by bronchi in the center of the pulmonary lobes, segments, subsegments, and lobules (23), and the spatial relationships between pulmonary arteries and early lung adenocarcinomas were classified into four patterns according to the tumor–bronchus relationships described in our previous clinical and animal studies (13, 24). Wang and colleagues (25) adopted a similar classification to analyze the relationship between peripheral lung cancers and pulmonary arteries, type I relationship was more often observed in large (≥2.0 cm), solid, and stage II–IV tumors. In contrast, the mean tumor diameter in our study was only 0.55 mm, which is equivalent to a mean diameter of approximately 11 mm in human lung tumors (12). Small tumors were frequently less invasive and rarely destroyed pulmonary arteries, leading to fewer type I and II relationships. As tumor size increased, the prevalence of type I and II relationships increased. In addition, our previous clinical study (8) showed that abnormal pulmonary artery changes were more frequently found in invasive adenocarcinomas than in adenocarcinomas in situ (AIS) and MIAs. We inferred that with increases in invasiveness and angiogenesis and subsequent increases in the blood supply in lung adenocarcinomas, the differences in abnormal pulmonary arteries would increase between the AIS-MIA and invasive adenocarcinoma groups. To obtain an adequate blood supply, early lung adenocarcinomas migrate to or surround the pulmonary arteries; therefore, regardless of the sizes of the tumor and solid component and regardless of the methodology used (i.e., micro-CT or histopathology), type IV was the most common relationship observed in our study. The above results suggested that pulmonary artery morphology and tumor–pulmonary artery relationship could serve as biomarkers for the invasiveness of indeterminate nodules detected during lung cancer screening.

The correlation analyses between micro-CT and pathology showed that micro-CT could precisely display tumor and tumor–pulmonary artery relationships. Furthermore, owing to the micron-scale resolution and three-dimensional imaging ability of micro-CT, micro-CT could reveal the tumor–pulmonary artery relationship more comprehensively than pathology, which might explain the differences in the type III and IV relationships observed between micro-CT and pathology.

In addition to the above qualitative analysis, we also performed pulmonary artery quantitative and texture analyses to evaluate the relationship between the sizes of the tumor and solid component and supplying vessels. VV depicts the volume of vessels within the ROI. VVF represents the fraction of the specimen occupied by VV within the ROI. VS is defined as the mean interaxial distance between two vessels and is negatively correlated with VN (26, 27). VT is measured by calculating the average local voxel thickness within the vessel (27, 28). Our study showed that VV, VVF, and VN were significantly higher and that VS was significantly lower in the control group than in the tumor group and the nontumor group. We believe that the toxic effects of MNNG affected the growth of mice and the development of vessels (29), leading to this contradictory result (fewer vessels and decreased blood flow to the pulmonary arteries in the tumor group). Therefore, to make the study more objective, we used the nontumor group to serve as an authentic “control group” to eliminate the systematic effect caused by MNNG. Our data showed significantly higher VV, VVF, and VN values and lower VS values in the tumor group than in the nontumor group. The correlation analysis indicates that the pulmonary arteries increase in number and size with increases in tumor size to provide sufficient nutrients and oxygen for tumor growth. Moreover, this finding indirectly reflects the blood supply of early lung adenocarcinomas originating from the pulmonary arteries. However, further studies are needed to elucidate whether the increased blood supply originates from tumor-induced angiogenesis or from the reexpansion of reserved vessels.

Texture analysis provides an objective, quantitative assessment of the distribution and relationship of pixel or voxel gray levels in the image. Inertia and CS indicate the amount of skewness and asymmetry of the gray level cooccurrence matrix and the measure of variation in signal intensities (30, 31). SRE is expected to be large for fine textures, and LRE is expected to be large for coarse structural textures (30). GLN and RLN are expected to be small if the gray level values and the length of runs are similar throughout the image, respectively (32). IDM also represents the local homogeneity of the gray level (33). These results demonstrated that tumor growth and densification caused the texture and gray level values of pulmonary arteries to become coarse and heterogeneous. Therefore, in addition to using as a biomarker for tumor spatial heterogeneity, texture analysis also has potential in the imaging analysis of vessel-related disease and provides a deeper understanding of the complexities of vessel changes in lung cancer.

Although the model we built has a high rate of tumor formation, this study had some limitations. First, based on default pathologic criteria for early lung adenocarcinomas in mice, we simply assumed that the lesions were early lung adenocarcinomas according to the tumor size. Second, because the preneoplastic lesions were so small and inconspicuous, we could barely identify them on micro-CT and on gross pathologic specimens with the naked eye, making an image–pathology correlation unfeasible. Therefore, we only analyzed early lung adenocarcinomas rather than preneoplastic lesions.

This study confirmed that the blood supply of early lung adenocarcinomas originated from the pulmonary arteries in mice. Micro-CT angiography could clearly demonstrate the morphologic changes of pulmonary arteries and tumor–pulmonary artery relationships. The quantitative and texture analyses indicated that the sizes of early lung adenocarcinomas and solid components had positive correlations with pulmonary artery's size, number, coarse and heterogeneous texture, and gray level.

No potential conflicts of interest were disclosed.

L. Deng: Conceptualization, resources, data curation, software, formal analysis, investigation, visualization, methodology, writing-original draft. H. Tang: Data curation, software, formal analysis, supervision, investigation, visualization. J. Qiang: Conceptualization, resources, supervision, funding acquisition, validation, methodology, project administration, writing-review and editing. J. Wang: Data curation, software, formal analysis, investigation, visualization. S. Xiao: Supervision, validation, investigation, visualization, methodology.

This study was supported by grants from National Natural Science Foundation of China (grant no. 81171340) and Shanghai Municipal Health Commission (grant no. ZK2019B01). The authors are grateful to Ms. Junming Dong, Ms. Mengting Wu, and Ms. Xiao Shen from Department of Pathology, Jinshan Hospital of Fudan University for their support in making pathologic sections. The authors also thank Ms. Xiao Guo and Ms. Shengru Pang from the Joint Live Small Animal Imaging Laboratory of Fudan University Shanghai Medical College, PerkinElmer Company, for their technical support in the use of high-resolution micro-CT scanning.

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