A Minimally Invasive Method for Generating a Syngeneic Orthotopic Mouse Model of Lung Cancer

Summary

A major challenge of developing new therapies for lung cancer includes modeling the tumor microenvironment as regional disease progresses to metastatic disease. Here, we describe a simple method of establishing an orthotopic syngeneic model of lung cancer that allows for in vivo tracking of disease over time.

Abstract

Non-small cell lung cancer (NSCLC) continues to be the number one cause of cancer-related death for both women and men worldwide. More information needs to be gathered to understand the interactions between cancer cells, the immune system, the microenvironment within each tumor, and the host tissue to develop more effective treatment modalities. Reported here is a simple, repeatable method for inducing cancer within the mouse lung, allowing for the monitoring of tumor growth from early to late-stage disease. The surgical procedure involves a small superficial incision in the skin and an injection of cancer cells through the intercostal muscles into the lung. In vivo imaging systems (IVIS) in combination with cells engineered to express luciferase provide a convenient way of evaluating tumor growth and disease progression over the course of a study. This technique is easily transferable across various models of lung cancer and can be used to test the feasibility and efficacy of novel therapeutics in a preclinical model of lung cancer that mimics the natural tumor microenvironment. This broadens the relevance of both efficacy and safety/toxicity studies using standard-of-care treatments, including surgical resection, radiation, and immunotherapies in conjunction with experimental therapeutics.

Introduction

Lung cancer continues to be one of the most diagnosed cancers and the number one cause of cancer-related death among men and women globally^1,2,3. Non-small cell lung cancer (NSCLC) accounts for nearly 90% of all lung cancer cases in the United States, and although the treatment landscape has greatly improved over the last few years, there continues to be much interest in developing new therapies that improve outcomes⁴. While there have been many advances in developing targeted therapies for specific driver mutations and immunotherapies for responsive NSCLC subtypes, the overall five-year survival rate continues to be low, sitting around 20% for all stages of lung cancer^2,5,6,7. Furthermore, although immunotherapy has made huge strides in improving survival in advanced disease, these improvements have only moved the needle from a five-year overall survival of 6.8% to 10.7% and increased survival time from 7 months to 8 months⁸. There is still a great need to understand the mechanisms of action of the disease and to develop new therapies that synergize with current standard-of-care therapies. Understanding the mechanisms at play of the natural progression of NSCLC and the response to new therapies require preclinical models that are reliably reproducible, easy to induce, and encapsulate the tumor microenvironment within which the cancer develops.

Over the last few decades, many different procedures have been developed to assess orthotopic models of lung cancer in rodents. Procedures range from very invasive, requiring a high level of expertise, such as intrapulmonary implantation or transplant procedures that open the chest cavity^{9,10,11,12,13}; moderately invasive like intratracheal and orotracheal procedures^14,15,16,17; to minimally invasive procedures, such as percutaneous injections, tail vein, or intranasal induction^18,19,20,21. All procedures offer different risks and benefits -- with more invasive techniques allowing for more precision but potentially higher attrition rates, and less invasive techniques lending to better survival but more variability of tumor implantation sites. Of these various protocols, some opt to not track tumor growth in vivo and only complete assessments of tumors ex vivo^9,11,12,16. Of the protocols that do track tumor growth over time, earlier protocols rely more on systems such as computed tomography (CT)^14,19, position emission tomography (PET)¹⁸, and magnetic resonance imaging (MRI)¹⁷, while later protocols, after bioluminescence imaging (BLI) systems and genetically-modified cells became more readily available, opt for tracking tumor growth over time with BLI^20,21.

The purpose of developing this protocol was to take the learnings from these other procedures and create a simple yet reliable method of inducing a solitary lung tumor to study the progression of NSCLC starting from early disease, where the tumor is confined to the lung. The overall goal of this paper is to describe the methods to generate a reliable and minimally invasive syngeneic orthotopic mouse model of lung cancer that can be monitored using in vivo imaging and can enable researchers to study lung cancer in the organ from which the disease arises. This method is effective for generating both xenograft orthotopic mouse models using human lung cancer cell lines and syngeneic orthotopic mouse models using mouse lung cancer cell lines in mice with intact immune systems, giving versatility to the method to study both human cancer types and the complex interplay of the immune system and NSCLC.

We focused on optimizing this protocol in the immune-competent NSCLC mouse model, Lewis Lung Carcinoma (LLC), as this model enables researchers to better understand the immune system response with and without current therapies such as chemotherapy, radiation therapy, immunotherapy, and other targeted therapies^22,23,24,25. The protocol generates a model that is representative of early-stage disease at inoculation (tumor in the lung parenchyma) and metastasis towards the end of studies as the tumor will often outgrow the lung parenchyma and seed other tissues.

An important aspect of studying orthotopic models is the ability to track tumor growth repeatedly, with minimal stress to the animal, and quickly so that each animal in a study can be assessed at a single timepoint. While imaging techniques like MRI or CT allow for enhanced resolution of the tumor, these imaging modalities are expensive, time-consuming, and have difficulty tracking early tumor growth in small animal models such as mice^{26,27,28,29,30,31}. Therefore, we opted to monitor tumor growth through bioluminescence using cancer cells engineered with luciferase. In this protocol, we injected 25,000-50,000 LLC cells engineered with firefly luciferase (LLC-luc) and detected tumors as soon as the next day by BLI. The luciferin-luciferase reaction is a useful tool for tracking tumor growth in vivo using spectral imaging instruments. Due to the sensitivity of these imaging systems to track photons of light, cancer cells expressing luciferase can be tracked in vivo early, often before tumors can be detected in a mouse model by MRI or CT, and frequently^26,32,33,34.

Protocol

All procedures involving animals are reviewed and approved by the University of Iowa Institutional Animal Care and Use Committee (IACUC). C57BL6/N mice (age 4-6 weeks old) are purchased and acclimated for at least 1 week prior to surgeries. All animals are on a 12 h light/dark cycle. Animals are housed 4 per cage and have full access to food and water up to the time of anesthesia.

1. Selection and growth of cells for injection

1. Perform a luciferase assay to select a cell line as described in³⁵.
   1. Grow LLC-luc cells in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), L-glutamine, and penicillin/streptomycin in a 5% CO₂ incubator at 37 °C with high humidity. Perform the assay when the cells are 50%-100% confluent.
   2. Before performing the assay, wash cells with 1x PBS.
   3. Add 200 µL of lysis buffer/well (25 mM Tris/phosphate, 4 mM EGTA, 1% Triton X-100, 10% glycerol) in a 6-well plate. Scrape cells from the well and transfer to a microcentrifuge tube. Spin cells at 16,000 × g for 5 min at room temperature. Confirm a compact cell pellet forms at the bottom of the tube and collect the supernatant into a new microcentrifuge tube.
   4. Transfer half of the cell extract into a new microcentrifuge tube and save for protein quantification. Add 2 mM dithiothreitol to the remaining half and mix well.
   5. Combine 8 µL of cell extract containing 2 mM dithiothreitol with 42 µL of assay buffer (25 mM Tris/phosphate, 20 mM MgSO₄, 4 mM EGTA, 2 mM ATP, 1 mM dithiothreitol). Add 50 µL of 0.5-1 mM luciferin.
   6. Measure peak luminescence reported as relative light units (RLU) and assess protein content by a preferred protein quantitation method to report luminescence as RLU per mg of protein (RLU/mg).
2. Select a cell line with luminescence between 8,000-15,000 RLU/mg.
   NOTE: Avoid selection of a higher luciferase expression (>15,000 RLU/mg) as this may saturate the BLI instrument.

2. Cell preparation

1. Grow LLC-luc cells in DMEM supplemented with 10% FBS, L-glutamine, and penicillin/streptomycin in a 5% CO₂ incubator at 37 °C with high humidity for at least 1 week before use.
   NOTE: Monitor growth to limit confluency to 70%-80% and split using 0.25% trypsin-EDTA as needed to reach the desired quantity for injections as described earlier³⁶.

3. Presurgical preparation

1. For mice with fur, shave 1 day prior to surgery.
2. Place the mouse in an induction chamber to anesthetize using isoflurane. Set the oxygen flow rate to 2.0 L/min and vaporizer at 2% isoflurane; adjust this based on the size of mouse being induced.
3. Monitor the breathing of the mice and employ a pedal withdrawal reflex to confirm appropriate anesthetization.
4. Place the mouse in a nose cone in the prone position on an IACUC approved warming device. Check to ensure the mouse is properly anesthetized using the pedal withdrawal reflex before continuing.
5. Apply ophthalmic ointment to both eyes.
6. When the mouse is appropriately anesthetized, use electrical clippers to remove a large rectangular area (2 cm x 3 cm) in the left dorsal portion of the mouse. Shave to the spine medially, the table laterally until no fur is visible, the last rib (13th) inferiorly, and the superior edge of the scapula towards the head.
7. Place the mouse in an empty cage on an IACUC approved warming device and observe the breathing of the mouse until the mouse is awake and alert. At this time, return the mouse to its home cage until the surgery.

4. Preparation of cell suspension for injection

1. On the day of cell harvesting (step 2.2), add fresh media with 50 mg/mL gentamycin 3-4 h prior to collection to remove dead and detached cells.
2. Aspirate the media and wash the cells with 1x PBS. Add trypsin-EDTA and incubate for 1-2 min at 37 °C.
3. Disperse the cells and add media to neutralize the Trypsin. Collect cells in a 50 mL conical tube.
4. Spin at 1,200 × g for 3 min at room temperature. Confirm a compact cell pellet forms at the bottom of the tube, aspirate the supernatant, and wash the pellet with 1x PBS. Repeat this step 3x.
5. Resuspend the cell pellet in 1x PBS at approximately 1-2 million cells/mL for counting. Filter through a 40 µm cell strainer prior to counting.
6. Combine 10 µL of cell suspension with 10 µL of Trypan Blue and count using a hemocytometer. Make a cell suspension 2x and count both to verify the cell counts. Calculate total cell count and viability.
7. Spin cells down at 1,200 × g for 3 min at room temperature for resuspension in the final injection volume. Aspirate the PBS.
8. Resuspend the cells in 0.5 mg/mL Matrigel in 1x PBS to the desired cell number per injection in a volume of 50 µL. Keep the final cell suspension on ice throughout the procedure to prevent solidification.
   NOTE: This protocol uses LLC-luc cells. Inject at 25,000-50,000 cells in 50 µL of volume per mouse.

5. Surgical preparation

1. Perform the procedure in an IACUC-approved surgical room for aseptic procedures. Clean the surgical table with a veterinary-approved disinfectant.
2. Place a disinfected IACUC-approved heating pad in the surgical area. Lay a sterile surgical drape over the heating pad.

6. Surgical anesthesia

1. Place the mouse in an induction chamber to anesthetize using isoflurane. Set the oxygen flow rate at 2.0 L/min and vaporizer at 2% isoflurane.
   NOTE: The isoflurane concentration can be adjusted based on the size of the mouse.
2. Monitor the breathing of the mouse and employ a pedal withdrawal reflex to confirm appropriate anesthetization.
3. Place the mouse in a nose cone in the right lateral recumbent position on the sterile surgical drape. Check to ensure the mouse is properly anesthetized using the pedal withdrawal reflex before continuing.
4. Give preoperative analgesic, meloxicam (2 mg/kg weight) subcutaneously, and ophthalmic ointment to both eyes.

7. Orthotopic lung injection

1. Clean the surgical site 3x by alternating between 70% ethanol and betadine using sterile gauze. Place a sterilized fenestrated surgical drape with a 3 cm x 4 cm opening on the mouse. Place autoclaved sterilized instruments on the surgical drape.
2. Determine where to make the incision by locating the cranial edge of the scapula and the caudal edge of the thoracic rib cage using forceps. Ensure the skin is taut using the surgeon's thumb and forefinger and make a superficial 5 mm incision through only the skin directly below the scapula with a size 10 blade.
   NOTE: The surgeon must wear sterile gloves.
3. Use 115 mm straight scissors to blunt dissect the subcutaneous fat from the ribs and intercostal muscles. Do not cut through the intercostal muscles.
   NOTE: Be careful not to damage the thoracodorsal artery and deep branch of the transverse cervical artery, which are located near the scapula. Light bleeding may occur due to cutting capillaries within the skin and subcutaneous fat. Stop the bleeding by applying light pressure with a sterilized cotton-tipped applicator.
4. Use Micro-Adson forceps to hold the incision open. Starting from the seventh true rib, use curved medium point forceps to count upwards to identify ribs four and five. Note this area for injection.
5. Mix the cell suspension by inverting gently 3-4x. Immediately before injection, use a sterile 300 µL 31 G 8 mm insulin syringe to draw up 50 µL of the cell suspension and ensure no bubbles are present.
6. Add on a strip of sterilized 4 mm thick metal tape to the needle, flush to the syringe, to act as a stopper. This ensures the needle only goes 4 mm deep into the lung.
7. Inject the 50 µL cell suspension directly below to the fourth rib in the intercostal space between rib four and five at a 90° angle perpendicular to the lung. After injecting the cell suspension, hold the needle in place for 2-3 s to allow the Matrigel to harden and prevent the cells from exiting the injection site.
8. Slowly remove the needle at the same angle and dispose in a sharps container.
9. Suture the skin using 2-3 stitches, closing with square knots, and add veterinary surgical adhesive to ensure the wound is sufficiently closed.

8. Postsurgical procedures

1. Place the mouse in an empty cage on an IACUC-approved heating device. Monitor the mouse until it is fully recovered from anesthesia, which should take 1-2 min.
2. Once the mouse is alert and moving, place it in a cage with fresh bedding. Observe the mouse frequently within the first 24 h for any signs of pain or infection.
3. Give NSAID analgesic, meloxicam (2 mg/kg weight), via subcutaneous injection for 2 days following surgery.

9. Monitoring tumor growth by in vivo BLI

1. Monitor tumor growth in the lung using BLI as early as the day after surgery and as often as once per day.
2. Perform in vivo imaging using an in vivo imaging system (IVIS).
   1. Make up luciferin at 15 mg/mL in 1x PBS without magnesium or calcium. For a 20 g mouse, this equates to injecting 200 µL of the 15 mg/mL luciferin solution.
      NOTE: Protect prepared luciferin solution from light by covering with aluminum foil when not in use as it is light sensitive.
   2. Open software and set parameters for Bioluminescent Imaging. Set Exposure Time to Auto. Set Binning Factor to 4.
   3. Inject mice intraperitoneally with 150 mg of luciferin per kg weight.
   4. When imaging a new cancer cell model for the first time, perform a kinetic curve to identify the optimal luciferin uptake time, which is indicated by when the luminescence peaks before it starts to plateau.
      1. Inject mice with luciferin. Wait 3 min for luciferin to uptake.
      2. Image mice every 5-10 min for 60 min.
         NOTE: Optimal uptake time for different cell lines typically vary from 5-15 min.
   5. For all subsequent imaging sessions, use the optimal luciferin uptake time. Anesthetize the mice in preparation for imaging during this time. Place the mice into an induction chamber at 2% isoflurane with a flow rate of 2.0 L/min.
   6. After uptake is complete, take two images by selecting Acquire.
      1. First image the mouse laying in the right lateral recumbent position. This allows the camera to capture the injection site, which is located on the left lateral portion of the mouse. Use this view to quantify tumor growth. Images captured in this position will be denoted from here on as left lateral view.
      2. Immediately after the first image is complete, take a second image of the mouse in the supine position. This allows the camera to capture the photons emitted from the tumor in the chest cavity from the ventral view. Use this view (herein denoted as the ventral view) to estimate the location of the cancer within the chest cavity.
3. To analyze the images, change the units from Counts to Radiance.
4. Open the Tool Palate. Go to Image Adjust, select Manual. Choose a min and max for the color bar that best suits the images taken.
5. Open ROI Tools. Select the circle dropdown to add a region of interest (ROI) circle to the image. Move the circle over the incision for the left lateral view and over the center of the chest for the ventral view.
6. Select Measure ROIs.

10. Assessment of inclusion and exclusion criteria

1. First, confirm the presence of tumor luminescence in the lung by observing the left lateral view image.
2. Next, confirm the presence of tumor luminescence in the left chest cavity in the ventral view image. Then, confirm there is only a single site of luminescence from the ventral view image.
3. If these two criteria (steps 10.1, 10.2) are met, then assess for exclusion criteria by ensuring the luminescence does not cross midline in the chest cavity in the ventral view image. If no luminescence is detectable in the left lateral view image after two consecutive IVIS images over the course of 4 days after surgery, exclude this mouse from further studies, as the cancer cells likely did not take.

11. Assessment of tumors at the end of studies

1. When the total flux of the tumor luminescence reaches 1 × 10⁸ photons/s (p/s) or greater, harvest the mice for endpoint assessments.
2. Collect the tumor for gross anatomical analysis or histology by performing the following steps for anesthetization and perfusion of the lung as described earlier³⁷.
   1. Place the mouse in an induction chamber to anesthetize using isoflurane. Set the oxygen flow rate at 2.0 L/min and vaporizer at 2% isoflurane. Monitor the breathing of the mouse and employ a pedal withdrawal reflex to confirm appropriate anesthetization.
      NOTE: The isoflurane concentration can be adjusted based on the size of the mouse.
   2. Place the mouse in a nose cone in the supine position. Check to ensure the mouse is properly anesthetized using the pedal withdrawal reflex before continuing.
   3. Open the chest cavity of the mouse to visualize the lungs and heart behind the diaphragm.
   4. Make a long vertical cut inferiorly along the midline of the mouse. Move the intestines to visualize the left renal artery.
   5. Cut through the diaphragm to visualize the heart.
   6. Cut the left renal artery.
   7. Inject approximately 5-10 mL of 1x PBS through the right ventricle of the heart at a rate of approximately 5 mL/min.
   8. Make an incision along the neck. Clear any subcutaneous fat located above the trachea. Be careful not to cut the trachea.
   9. Pierce the trachea parallel with a needle to inject 10% formalin directly into the trachea to inflate and fix the lungs.
   10. Procure the lungs from the chest cavity by cutting the trachea above the apex of the lung, and cut the pleura and ligaments attaching the lung to the chest cavity.
   11. Place in 10% formalin to fix.

Results

In this study, C57BL6/N mice were injected per the protocol presented (Figure 1). LLC cells genetically engineered to express luciferase (LLC-luc) were used. Additionally, a sham mouse was injected with Matrigel without cancer cells. BLI on IVIS can be initiated as soon as 1 day post surgery. This LLC-luc study was optimized on BLI to assess early-stage lung cancer. The BLI system used in this paper has a minimum detectable radiance (MDR) of 10 photons∙s^-1∙cm^-2∙sr^-1 and a minimum image pixel resolution of 50 µm per manufacturer's technical specifications.

Figure 1: Schematic of minimally invasive orthotopic lung cancer injections. (A) Diagram shows the cell and mouse preparation steps. (B) Diagram indicating location of lung cancer injection site. (C) Diagram of post injection BLI. Abbreviation: BLI = bioluminescence imaging. Please click here to view a larger version of this figure.

To determine which mice to include in a study, inclusion and exclusion criteria were created to make assessments in a semi-quantitative fashion (Figure 2A). Mice were imaged by IVIS for at least two consecutive imaging sessions to ensure tumors were taking and were inoculated in the correct region of the lung. Two days post injection, the following images were taken for each mouse: 1) one where the mouse was placed in the right lateral recumbent position, capturing a left lateral view image and 2) another where the mouse was placed in the supine position, capturing a ventral view image. The left lateral view image was quantified for tumor luminescence, presented as total radiance flux in photons per second (p/s).

Selection of mice for early-stage lung cancer were made with the following criteria: 1) location of the tumor bioluminescence, 2) total radiance flux from the left lateral view (LV), 3) total radiance flux from the ventral view (VV), and 4) the tumor luminescence relative to the mouse's midline (Figure 2A). An included mouse would have attributes that fulfilled all the inclusion criteria on two consecutive BLI sessions, such as having only one single focus of tumor luminescence that ranged from a total flux of 7 × 10⁴ p/s-1 × 10⁷ p/s from the LV and 5 × 10⁴ p/s-5 × 10⁶ p/s from the VV and with no tumor luminescence crossing the midline of the mouse (Figure 2B). An excluded mouse would either have no tumor luminescence (not pictured) or have too much non-specific tumor luminescence. For a mouse to be excluded for no tumor luminescence, it would have a total radiance below the amount detected from a sham mouse, typically <7 × 10⁴ p/s from the LV and <5 × 10⁴ p/s from the VV. For a mouse to be excluded for too much non-specific tumor luminescence, it would have multiple luminescence foci noted in the LV or VV, or tumor luminescence that crosses the midline to the right side of the mouse (Figure 2C).

Figure 2: Inclusion and exclusion criteria for early-stage lung cancer. (A) Criteria for determining inclusion and exclusion based on IVIS images. (B) Representative IVIS images of an included mouse showing the left lateral and ventral views of the mouse from two sequential IVIS sessions at 2 and 4 days post injection. (C) Representative LLV and VV images of an excluded mice at 2 and 4 days post injection; arrows show multiple foci of tumor luminescence and crossover of luminescence over midline into the right side of the chest. Abbreviations: LLV = left lateral view; VV = ventral view; IVIS = in vivo imaging system; DPI = days post injection. Please click here to view a larger version of this figure.

The growth of LLC-luc tumors was monitored via IVIS for the entirety of a study that lasted 21 days. For these LLC-luc cells, with an inoculation of 25,000 or 50,000 cells per mouse, studies typically last for 14-21 days before animals begin succumbing to the disease. Monitoring bioluminescence allows for tracking the progress of disease early and frequently, with tumor-associated bioluminescence appearing as early as 1 day after injection. As the tumors grow, the visible bioluminescence increases (Figure 3A). The increase in bioluminescence values correlated to tumor size for the duration of the study, reaching exponential growth towards the latter half of the study (Figure 3B). Endpoint criteria of a total flux between 1 × 10⁸ p/s and 1 × 10⁹ p/s from the LV was selected so that mice would still have viable lung and tumor tissue at the time of collection for downstream gross anatomical and histological assessment.

Figure 3: Tumor growth of LLC-luc tumors using BLI. (A) Representative IVIS images of mice injected with 25k, 50k, or no cells (sham). (B) Quantified average total flux of tumors over the course of a study, n=6 mice/group for 25k and 50k, n = 2 mice/group for sham. Data presented as mean ± SEM. Abbreviation: BLI = bioluminescence imaging. Please click here to view a larger version of this figure.

The endpoint criteria, tumors with an average luminescence of >1 × 10⁸ p/s from the left lateral view, was met at 21 days post-surgery in this study. At the end of the study, mice were imaged one last time by IVIS and then euthanized and lung tissue was collected. Dissected lung and tumor were immediately imaged on IVIS after dissection to confirm tumor formation in the lung by luminescence. The left lateral view image and dissected lung from a sham mouse showed no luminescence, and no gross anatomical signs of tumor were noted in the dissected lung (Figure 4A). The left lateral view IVIS image from an experimental mouse showed a tumor by bioluminescence and was confirmed after dissection of the lung (Figure 4B). Perfusion was performed to preserve the architecture of the lung and tumor tissue for assessment. A representative sham mouse, injected only with Matrigel, shows lungs with no signs of tumors (Figure 4C). After 21 days, this stage is representative of early disease, where the mice do not show physiological signs, and dissected lungs show tumors only in the left lobe (Figure 4D). A representative excluded mouse shows a large nodule located in the left lung in addition to tumor surrounding the heart and ventral portion of the left lung (Figure 4E). If advanced disease is desired for the beginning of a study, the exclusion criteria can be used to identify these mice.

Figure 4: Terminal BLI and gross anatomical images. (A) IVIS images of representative sham and (B) experimental mice, with respective IVIS and light images of dissected lungs. Images of isolated lungs from representative (C) sham, (D) included, and (E) excluded mice. Dashed lines outline the heart. Arrows point to tumor. Please click here to view a larger version of this figure.

Discussion

This protocol describes an optimized, minimally invasive procedure that allows for quick recovery times and almost immediate tumor monitoring, which differs from more surgically intense methods previously described^{10,11,20,38,39,40,41,42,43,44,45}. Due to the simplicity of the procedure, researchers can induce high volumes of this model in a short period of time. Isoflurane allows for quick recovery and the ability to diligently monitor breathing, decreasing attrition of study animals due to anesthesia. No mice to date have died due to this procedure. Current available surgical models published in the literature often do not have strict criteria for cancer presenting as a solitary tumor contained in only one lung, which may decrease the ability to study a specific stage of lung cancer disease. For example, non-surgical models, like tracheal distillation, seed non-specifically throughout both lungs, which would only allow for the study of advanced disease. With the regional containment of the tumor in the left lobe of the lung and the minimally invasive procedure, standard of care therapy such as surgery may be performed on this model early to study disease recurrence. Many other orthotopic lung cancer models determine the success of the injection by confirming the non-specific presence of luminescence using IVIS. This model's specificity and strict inclusion/exclusion criteria allow for monitoring and treating early-stage lung cancer.

This protocol is a versatile method for studying lung cancer in a preclinical model. With appropriate modifications, this technique can be used for a large variety of study goals. If using different cell lines or studying other models of lung cancer than what is presented here, such as small cell lung cancer, alterations in the concentration and volume of cells injected may be needed to confirm tumors take. Similarly, different strains, ages, and sexes of mice will likely require minor adjustments in injection depth and incision size to visualize the lung and ensure delivery of cancer cells to the lung parenchyma. Each iteration will take optimization and practice to achieve consistency. To establish reliably reproducible injections, it is essential to inject between the same ribs for each mouse in a single study. Applying metal tape to the needle flush with the syringe will allow for achieving a constant and consistent depth within the lung. This protocol was optimized using the surgical instruments listed, but other tools can be used to accommodate for personal preference, animal size, and skill level.

The use of in vivo imaging makes for a simple, sensitive, and reliable tool for monitoring tumor growth over the course of a study. However, it is essential that mice stay properly shaved, as fur can obstruct the camera from accurately capturing photons. It is also important to consider IVIS assessment of tumor size is only an approximation of the location and size of the cancer growth. If endpoint BLI criteria are disproportionate to the size of the tumors, the luciferase activity may be too high and saturating the IVIS. A lower concentration of luciferin or a shorter imaging time can be used to prevent saturation when imaging. If the study requires more precise visualization of the tumor location and size in relation to other tissue architecture, such as lung or heart parenchyma, then an imaging method like CT or MR would provide greater resolution. A cell line that is not engineered with a bioluminescent marker, such as luciferase or fluorescent markers , can still be used but again would require CT or MRI for tumor monitoring. Luciferase expression can vary across clone and cell line. Construct a kinetic curve following luminescence over time for each new cell line to optimize uptake time prior to imaging.

Common issues that may be encountered when first using this protocol can be classified into two categories, where one relates to the procedural aspects of the induction and the second involves the optimization of the in vivo imaging. If tumors are grafting, but more than a solitary tumor is forming or tumors are forming in the right lung, an injection of 25,000-50,000 cells could be too high. Troubleshoot by injecting fewer cells to avoid too much seeding in the lung. In addition, this protocol was optimized on mice aged 5-8 weeks old. For a larger mouse, an injection lower in the chest cavity may prevent engraftment of multiple sites and the right lung. Assess the size of the mice and consider injecting between ribs five and six instead of between ribs four and five as outlined in this protocol. Regarding optimization of BLI, if saturation is happening, consider running a new kinetic curve (outlined in the protocol starting at step 9.2.4) and reducing the luciferin uptake time. If there are inconsistent flux rates noted in a single mouse longitudinally, consider whether fur is impeding light detection by the BLI imager. Frequent shaving of the fur will prevent impedance of flux across multiple imaging sessions. Other considerations to ensure reliable measurements are to weigh mice right before each imaging session to ensure the proper concentration of luciferin is being administered to each mouse.

This protocol was developed to better understand NSCLC and the role of the immune system in the host tissue. It is advantageous for evaluating the entirety of tumor development, from early disease when it is restricted to the lung tissue to late disease where it has spread systemically. Standard of care, including radiation, surgical resection, and immunotherapies, is compatible with this model, making it extremely relevant in the context of developing lung cancer treatments preclinically. Future studies could use this model to evaluate novel treatment options for lung cancer in the host tissue, in combination with standard-of-care treatments, and evaluate the tumor microenvironment within the lung, as well as effects on nearby tissues. At the end of the study, tumor and lung tissues can be collected for downstream analysis, including histology, immunofluorescence, flow cytometry, and biochemical assays.

Lung cancer continues to kill millions annually, and there is a need to better characterize the tumor microenvironment in the development of effective therapies. This protocol provides clear instructions for generating a reliable orthotopic lung cancer model that is simple, minimally invasive, and efficient, allowing for generating large cohorts in a short period of time. We have also provided semi-quantitative inclusion and exclusion criteria for studying early-stage lung cancer in mice. This model will enable researchers to produce an easily reproducible orthotopic model, enhancing the ability for the lung cancer field to better understand the natural progression of NSCLC and to improve the development of novel cancer therapeutics.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA (1x)	Gibco	25200-056
10% Neutral Buffered Formalin	VWR	16004-112
100 mm x 20 mm Tissue Culture Treated Dishes	Dot Scientific Inc.	667621
190 Proof Pure Ethanol	Decon Labs	2801
3/10 mL Insulin Syringe with BD Ultra-Fine needle 31 G x 5/16 in.	Beckton Dickinson	328438
3M Vetbond	3M	1469SB
ATP	Sigma	A5394
Betadine Solution 10% Povidone-iodine Antiseptic	Arvio Health L.P.	67618-150-04
C57BL6/N Mice	Charles River Laboratories
Cell Strainer 40um Nylon	Falcon	352340
Compact Anesthetic Machine	Vetamac Anesthesia
Far Infrared Warming Pad with Controller	Kent Scientific Corporation
Conventional Surgical Blade Handle, Size 3	Brad-Parker	371030
Curved Medium Point General Purpose Forceps	Fisherbrand	16-100-110
Dithiothreitol	Research Products International	D11000
D-Luciferin (sodium salt)	Cayman Chemical	14682
Drape, Surgical 18” x 26” Non-Fenestrated, Sterile	Dealmed	783410
Dulbecco’s Modified Eagle Medium (1x)	Gibco	11965-092
Dulbecco’s Phosphate Buffered Saline (PBS) 1x	Gibco	14190-144
EGTA	Sigma	E3389
Fetal Bovine Serum	Gibco	A5670402
FL150 Fiber Optic Microscope Light Source	Meiji
Gentamycin Sulfate Solution	IBI Scientific	IB02030
Glycerol	Research Products International	G22025
Isoflurane	Primal Pharma Limited	66794-013-25
IVIS Lumina S5	PerkinElmer
Living Image Software	PerkinElmer
LL/2 (LLC1)	ATCC	CRL-1642	Use the cancer cell line that will best fit the needs of the study
Magnesium Sulfate	Fisher Scientific	M63
Matrigel Basement Membrane Matrix	Corning	354234
Mayo-Hegar Needle Holder, 6”, Serrated Jaws	Miltex	8-44TC
Micro-Adson Forceps	Fine Science Tools	10018-12
N5 Dog Paw Trimmer with Double Blades	Oneisall
Non-Sterile, 12 Ply Gauze Sponges	NDC	P157112
Optixcare Eye Lube	Aventix	5914304
Penicillin Streptomycin	Gibco	15140-122
Perma-Hand Silk Suture 6-0 1 x 18”	Ethicon	K802H
Phosphoric Acid 85%	Fisher Scientific	A242
Scalpel Blade Size 10, Stainless Steel, Sterile	Cincinnati Surgical	110
Screw Cap Tube Conical 50 mL	Sarstedt	62.547.100
Tissue Scissors 4.5” Staight Premium	Onsung	SCT115
Triton X-100	Sigma	T6878
Trizma Base	Sigma	T1503
Trypan Blue Stain (0.4%)	Gibco	15250-061
Utility Aluminum Foil Tape with Liner	IPG	ALF150L
Wooden Shaft Cotton Tipped Applicators	NDC	76200