Monitoring Pulmonary Nodule Progression Using Micro-Computed Tomography and Blood Sampling in a Mouse Model

Summary

This protocol describes an efficient, simple, and minimally invasive method for studying pulmonary nodules. Submaxillary vein blood collection and micro-CT imaging are used as investigative techniques.

Abstract

Micro-computed tomography (micro-CT) is a real-time, intuitive, sensitive, and minimally invasive technique for monitoring changes from pulmonary nodules (PN) to lung cancer (LC). The integration of submandibular vein blood sampling enables rapid, stable, and straightforward detection of imaging and key target alterations during the progression of PN to LC. In this study, we administered a dosage of 100 mg/kg of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in A/J mice to develop a lung adenocarcinoma model. Disease progression in the experimental animals was then monitored through submandibular vein blood sampling and micro-CT assay. Experimental results showed the presence of nodular foci in the lungs of some animals by the 10^th week, with the development of lung adenocarcinoma images becoming evident by the 21^st week. In conclusion, micro-CT can effectively observe pathological changes in the lungs of mice and, when combined with submandibular vein blood sampling, can dynamically monitor changes in blood, protein, and targets. This method provides a highly specific, simple, and sensitive approach for drug screening, pharmacokinetic testing, toxicological experiments, and safety studies.

Introduction

Lung cancer (LC) is a severe neoplasm originating in the bronchial mucosa or lung glands. According to 2021 statistics, LC causes approximately two million fatalities globally each year, with incidence and mortality rates on the rise¹. Early diagnosis and intervention in LC contribute to higher cure rates, reduced mortality, and lower treatment costs. Pulmonary nodules (PN) are specific precursors to LC, characterized by localized, round, and denser solid or subsolid shadows ≤30 mm in diameter on radiological exams, without evidence of lung collapse, mediastinal lymph node enlargement, or pleural effusion². The National Comprehensive Cancer Network (NCCN) in 2022 categorized PN by number, diameter, and density, identifying combinations such as a 5 mm isolated ground-glass nodule in the right lung³. However, the NCCN guidelines indicate that the risk of malignancy in PN increases with the nodules' diameter and quantity. The widespread application of low-dose computed tomography has dramatically increased PN diagnoses, with millions of new cases identified each year⁴.

The combination of A/J mice with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most commonly used animal model for lung cancer (LC)^5,6. The use of micro-CT alongside submandibular vein blood sampling is an effective approach for real-time monitoring of changes from pulmonary nodules (PN) to LC. Chemical carcinogen induction, particularly with NNK and A/J mice, is the most prevalent method for lung cancer modeling and has proven to be an efficacious approach for establishing carcinoma in situ^7,8. This modeling method more accurately simulates the progression of PN to LC compared to the axillary inoculation method.

Previous studies have focused on statistical analysis of nodule morphology and pathological staining of tissue samples post-euthanasia⁹. However, these methods lack the capacity for real-time monitoring of the dynamic progression from PN to LC¹⁰. Micro-CT, as a non-invasive imaging technique, provides accurate longitudinal data with high resolution, fast imaging, a low radiation dose, and safety, making it suitable for detecting lung images in real time^11,12. Submandibular vein blood sampling is the latest, simplest, and fastest method for obtaining blood samples from mice¹³. This non-invasive technique requires minimal animal handling and allows for rapid recovery, aligning with the 3R principles that aim to reduce the number of animals used in research, minimize discomfort, and promote ethical treatment. The collected blood volume, approximately 0.2-0.5 mL, is sufficient for monitoring blood parameters with moderate requirements¹⁴.

The concurrent use of micro-CT and submandibular vein blood sampling allows for dynamic, real-time observation of PN-to-LC progression in imaging and the real-time detection of key targets within the bloodstream¹⁵. Additionally, this approach enables real-time investigation of metabolites and other biochemicals, which, when combined with techniques such as high-performance chromatography, advances our understanding of LC^16,17.

In this study, A/J mice combined with NNK were used to create an in-situ lung cancer mouse model. Micro-CT scans were performed at 4, 10, and 20 weeks post-model induction to capture lung images, while blood was collected via submandibular vein sampling throughout the experiment. This study aims to establish a foundation for PN and LC research by combining submandibular vein blood sampling with micro-CT.

In oncology, micro-CT is a highly effective tool for detecting tumor growth, offering a high-resolution technique for measuring local shadow-focus changes at any time during such studies^18,19. However, it is essential to recognize that micro-CT alone does not provide information on shadow-focus characteristics, the physiological status of the animal, or levels of key biological factors. Therefore, submandibular vein sampling was utilized as a complementary method in this study.

Protocol

All animal experiments described in this study were approved by the Experimental Animal Welfare Ethics Committee of Chengdu University of Traditional Chinese Medicine and were conducted in accordance with relevant laws and ethical standards for animal research (review number: 2024035). Female inbred A/JGpt mice (7-8 weeks old) were maintained at a temperature of 20-24 °C with a relative humidity of 40%-70%. They were provided with standard animal feed and purified water ad libitum throughout a 12-h light-dark cycle. Before the experiment, each animal was acclimated to this environment for 7 days. Details of the reagents and equipment used are listed in the Table of Materials.

1. Reagents and animal preparation

1. Chemicals and reagents
   1. Dissolve NNK in saline to form a 10 mg/mL master mix²⁰. Administer a single intraperitoneal injection of 0.2 mL with a concentration of 100 mg/kg to the NNK group, while providing an equal volume of normal saline to the blank group.
      NOTE: Follow Jang et al.²¹ to determine the timing for micro-CT scanning and blood sampling.
2. Blood collection
   NOTE: To ensure the health of the mice, limit blood collection to no more than 0.2 mL at each time, and allow one week for recovery. Due to the abundance of hair in the submental region, take care to avoid contamination of the blood sample by hair during collection.
   1. Remove the animal's facial hair the day before the experiment using an appropriate shaver.
   2. Grasp the skin on the posterior aspect of the mouse's head firmly with the left hand to prevent any movement and to keep the mouse's head in a fixed position.
   3. Insert the blood collection needle swiftly into the submaxillary artery from the mandibular area behind the oblique eye socket. Keep the needle in place for at least 3 s to allow optimal blood flow. Collect 50-200 µL of blood.
   4. Collect the blood into an EDTA tube. Use a cotton swab to apply gentle pressure to the skin to stop bleeding. Once the bleeding has stopped, release the mouse and observe it for 30 s.
   5. Gently agitate the test tube to ensure that the blood is thoroughly mixed with the coagulant.
   6. For routine blood testing, place the collected blood in the veterinary blood routine testing instrument, press the collection button, and allow the instrument to collect the blood. Record the results displayed. Dispose of any remaining blood safely.

2. In vivo imaging by micro-CT

NOTE: Always remove metal objects, such as ear tags, from the test animal before using the micro-CT scan. Metal objects can cause severe artifacts in the image. Micro-CT emits a certain amount of radiation; ensure that other experimental results are not affected.

1. Start the unit, launch the micro-CT software, and perform the probe calibration and warm-up. Use the mouse-specific tool bed for scanning.
2. Create a new database and name it for this scan, or connect it to an existing database.
3. Modify the parameters in the software setup window. Set the X-ray filter to Cu0.06 + Al0.5, with a voltage of 70 kV, a current of 80 µA, a field of view of 36 mm × 36 mm, 360° rotational scanning, and a scanning time of 4 min²².
4. Anaesthetise the mice with 3% isoflurane before scanning²³ (following institutionally approved protocols). Open the micro-CT viewing screen and secure the mice to the tool bed with adhesive tape. Maintain anesthesia continuously using a nasogastric tube placed within the micro-CT instrument on the tool bed.
5. Carefully guide the animal into the apparatus and monitor its position in real time. Use the appropriate buttons to adjust the mouse's position, ensuring that its chest is fully visible within the field of view.
6. Rotate the tool bed 90° to position the mouse. Use the buttons to adjust the position of the mouse, ensuring that the lung region is centrally located within the field of view. Then, return the tool bed to its original position.
7. To initiate the scan, select the Scan button. Allow the system to complete the scan without interruption, and avoid opening the viewing screen during the process. Observe the transaxial, coronal, and sagittal slices of the reconstruction through the software.
8. Evaluate the image quality immediately after the scan. If artifacts or blurred images appear, repeat the scanning procedure.
9. Remove the mice from the apparatus and monitor their health to ensure they are in stable condition before returning them to their cages.
10. At the end of the experiment, remove the adhesive tape from the tool bed, then clean the bed. Save the data and turn off the instrument.
11. Transfer the mice back to their cages gently to minimize stress. Ensure that the cages are clean and have appropriate bedding.
12. Monitor the mice for signs of recovery from anesthesia. Observe their behavior, mobility, and appetite. Provide food and water as needed.
13. Maintain a warm environment for the mice to prevent hypothermia after anesthesia. Use heating pads or blankets if necessary.
14. Conduct daily health checks for the next week. Look for signs of distress, unusual behavior, or any injuries. Document observations for each mouse.
15. If any mouse shows signs of illness or distress, consult with a veterinarian for appropriate intervention and care.
16. Ensure that the mice are returned to their normal housing conditions after they have fully recovered and are stable.

3. Data processing and analysis

1. Use statistics and graphing software as a valuable tool for data analysis and creating tables to present results.
2. Open the software. Select XY graphs from the newly created data table, input the weekly data for the NNK and control groups, and generate a chart displaying the changes in the weight of the mice.
3. Open the software again, select the contingency diagram from the newly created data table, input the blood routine data for the NNK group and the control group, and generate an icon.
4. Select the analysis options in software. Analyze the overall data using one-way ANOVA, followed by verification of the data through the implementation of a t-test.Mark significant differences.
5. Save the micro-CT data in SimpleViewer format or DICOM. Open the SimpleViewer software, and observe the imaging data under the guidance of a professional imaging physician. Label the nodules and quantify the shadow volume using the provided measurement tools.

Results

This study demonstrated the construction of a stable lung cancer model using NNK in combination with A/J mice. The experimental design is illustrated in Figure 1. The objective was to observe the real-time process of the transition from pulmonary nodules (PN) to lung cancer (LC) in mouse lungs, utilizing micro-CT and submandibular vein blood sampling. Accordingly, micro-CT scanning and blood sampling from the mouse lungs were conducted at the fourth, tenth, and twentieth weeks.

Discussion

It is important to reiterate several key points from this study. Firstly, although submandibular vein blood collection is a relatively low-injury procedure, it may still result in some degree of harm to the animals. Therefore, it is necessary to conduct multiple procedures to reduce the burden on the mice and complete the process in a timely manner²⁷. Secondly, the removal of hair prior to blood sample collection ensures the purity of the sample. Thirdly, it is imperative to use appropriate blood ...

Materials

Name	Company	Catalog Number	Comments
A/J mice	GemPharmatech LLC.	N000018
0.5 mL EDTA tubes	Labshark	130201070
1-Butanone,4-(methylnitrosoamino)-1-(3-pyridinyl)	Gu Shi Gong Yuan Medical Equipment Co.	N589770
75% ethanol	ChengDu Chron Chemicals Co,.Ltd	2023052901
Animal shaver	Codos	BM010220
Isoflurane	Shenzhen Reward Life Technology Co.	R510-22-16
medical tricorder	MedChemexpress	69652
Quantum GX2 microCT imaging system	PerkinElme	2020166501
Saline (medicine)	Beijing Biolabs Technology Co.	GL1736‌