In Vivo Bioluminescence Imaging of Tumor Progression in the Lewis Lung Carcinoma Orthotopic Mouse Model: A Comparison Between the Tail Vein Injection and Intranasal Instillation Methods

Abstract

Metastasis remains a leading cause of cancer-related mortality, yet its study has been constrained by the lack of reliable animal models that faithfully replicate this complex process. Syngeneic models for studying lung cancer metastasis are limited, with the Lewis lung carcinoma (LLC) model being the most commonly employed. The conventional LLC orthotopic model involves injecting LLC cells intravenously (i.v.) via the tail vein into syngeneic C57BL/6 mice. However, this model has significant drawbacks, such as tumor development in multiple anatomical sites, incomplete lung tumor penetrance, and challenges in monitoring lung tumor growth. This article highlights the advantages of using luciferase-expressing LLC cells combined with bioluminescence imaging (BLI) to quantify tumor progression in live animals. We demonstrate that both white- and black-furred C57BL/6 mice can be used for BLI. Finally, we propose that intranasal (i.n.) instillation of LLC cells offers a valuable alternative to the traditional i.v. tail vein injection method, particularly for its simplicity and improved reproducibility. Although the LLC i.n. model does not recapitulate the metastasis process via the blood vascular route, it is an effective model for studying tumor seeding within the lungs and is particularly useful for analyzing the impact of the lung microenvironment on tumor initiation and progression. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Lewis lung carcinoma intravenous injection method Support Protocol: In vivo bioluminescence imaging Basic Protocol 2: Lewis lung carcinoma intranasal instillation method.

Keywords: Lewis lung carcinoma; bioluminescence imaging; in vivo models; lung cancer; metastasis.

Figure 1.. Lateral tail vein injection technique.

A) Gently place the mouse in the restrainer and hold the tail at mid-length. Using your non-dominant hand, position your index finger and thumb around the tail just above the injection site to apply gentle pressure. Hold the syringe and needle in your dominant hand and position the needle at a shallow angle (around 10–20 degrees) to the tail. B) Insert the needle into the vein, parallel to the tail, moving towards the mouse’s body. You should feel little to no resistance when the needle enters the vein. C, D) If necessary, tape can be used to secure the restrainer to the bench to prevent movement during the injection procedure. Note: The two red marks on the mouse’s tail were made using a Sharpie marker for identification purposes and are not related to the injection procedure.

Figure 2.. Validation of the luciferase activity of LLC cells in vitro.

LLC-GLF cells were plated in triplicate in a 48-well plate and serially diluted as indicated. After adding D-luciferin to the cells at a final concentration of 150 μg/mL, allow the cells to incubate with luciferin for 5–10 minutes at 37°C. Measure luminescence by IVIS imaging using the appropriate exposure time (e.g., 45 seconds).

Figure 3.. Assessment of lung tumor growth by BLI in the LLC i.v. model.

A) Age- and sex-matched C57BL/6J albino mice were randomly assigned to three groups (n=6 mice/group). The mice were injected i.v. into the lateral tail vein with the indicated number of LLC-GLF cells on day 0, and tumor growth was monitored by BLI, as shown in this schematic overview of the study design. B) Representative photos of tumor signals quantified by BLI in the three experimental groups and at the different time points. The luminescence intensity is shown in a blue-to-red spectrum. C) IVIS radiance was recorded on day 28 after the LLC cell injection. Data are presented as mean ± SEM. D) Survival curves of mice in the three experimental groups. Values are expressed as a percentage of survival.

Figure 4.. Comparison of lung tumor growth in albino and wild-type (WT) C57BL/6J mice in the LLC i.v. model.

A) C57BL/6J albino and WT mice were injected i.v. into the lateral tail vein with the indicated number of LLC-GLF cells on day 0, and tumor growth was assessed by BLI, as shown in this schematic overview of the study design. B) Representative photos of tumor signals quantified by BLI on day 21 after the LLC cell injection in the three experimental groups (n=1 mouse/group is shown).

Figure 5.. BLI using the IVIS^® Lumina II and Living Image^® software.

A) Overview of the IVIS^® Lumina II Imaging System. B) Interior view of the imaging chamber. This system has three nose cones to provide anesthesia, enabling the simultaneous imaging of up to three mice. C) Open the Living Image^® software, and in the Acquisition Control Panel that appears, (1) click on “Initialize”. (2) The temperature box will turn green once the initialization process is complete, indicating that the imaging system is ready to use. D) Overview of the anesthesia system. (1) Check the isoflurane level and (2) refill the vaporizer with isoflurane if the level is low. (3) Turn the oxygen to the ON position. (4) Turn the evacuation pump button ON. (5) Set isoflurane to 2.5–3.5%. (6) Place the mice into the anesthesia chamber, and (7) turn the Induction Chamber button ON. (8) When the mice are anesthetized, turn the IVIS Flow Manifold button to ON and transfer the mice to the nose cones inside the imaging chamber. E) In the Acquisition Control Panel, (1) click on “Acquire”. (2) Select the sensitivity by choosing the appropriate exposure time, Binning, and F/stop. (3) Ensure “Overlay” is checked to acquire both luminescent and photographic images. F) In the Tool Palette, select the preferred ROI tool (e.g., oval) from the drop-down menu. G) Create an ROI of the desired size and right-click to duplicate it. Transfer the anesthetized mice to the nose cones inside the imaging chamber by placing them on their backs with ventral side up and confirm that the ROIs fit each mouse.

Figure 6.. i.n. instillation technique.

A) Place the anesthetized mouse in the palm of your hand. B) Position your thumb on the mouse’s jaw and throat to prevent a gag reflex or swallowing during the procedure. C) Use a P100 or P200 micropipette loaded with 50 uL of LLC cells in PBS and position the pipette tip gently over the mouse’s nostrils. D) Administrate the cells dropwise until E) the full dose is inhaled.

Figure 7.. Assessment of lung tumor growth by BLI in the LLC i.n. model.

A) Age- and sex-matched WT C57BL/6J mice (n=6 mice) were treated i.n. under light anesthesia (isoflurane) with the indicated number of LLC-GLF cells on day 0, and tumor growth was evaluated by BLI, as shown in this schematic overview of the study design. B) Representative photos of tumor signals quantified by IVIS at day 26 post LLC cell injection and after shaving. C) Representative photos of the 5 lung lobes for each mouse with visible macroscopic tumors. D) Representative photos of H&E-stained lung sections. The areas within the red borders were considered positive for lung tumors after image analysis using QuPath software. Scale bars, 2 mm.

Figure 8.. Potential problems encountered with the LLC i.v. model.

A) Tumor development in multiple anatomical sites including the injection site, B) the legs, C) the oral cavity, D) the liver, and E) the bladder as observed here in C57BL/6J albino mice by the naked eye (A and B) or BLI (C-E).