Method Article

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

DOI:

10.3791/68676

⸱

August 19th, 2025

 ,  ,  ,  ,  ,  , 
1Stead Family Department of Pediatrics, University of Iowa Health Care, 2Department of Neuroscience and Pharmacology, Carver College of Medicine, University of Iowa, 3Department of Radiation Oncology, Holden Comprehensive Cancer Center, University of Iowa, 4Free Radical Radiation Biology Program, Carver College of Medicine, University of Iowa, 5Geminii, Inc., 6Interdisciplinary Graduate Program in Molecular Medicine, Carver College of Medicine, University of Iowa, 7Department of Ophthalmology and Visual Sciences, University of Iowa Health Care

Corresponding Authors: Sunny C. Huang <sunny-huang@uiowa.edu>

* These authors contributed equally

In This Article

Summary

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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

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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

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Lung cancer continues to be one of the most diagnosed cancers and the number one cause of cancer-related death among men and women globally1,2,3. 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 cancer2,5,6,7. 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 cavity9,10,11,12,13; moderately invasive like intratracheal and orotracheal procedures14,15,16,17; to minimally invasive procedures, such as percutaneous injections, tail vein, or intranasal induction18,19,20,21. 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 vivo9,11,12,16. 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 BLI20,21.

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 therapies22,23,24,25. 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 mice26,27,28,29,30,31. 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 frequently26,32,33,34.

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Protocol

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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.

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Results

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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-...

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Discussion

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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 described10,11,20,38,39,40,41,42,

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Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

S.C.H. and C.S.C. are founders of Geminii, Inc. The terms of this arrangement have been reviewed and approved by the University of Iowa Conflict of Interest in Research Office. All other authors have no conflicts of interest to declare.

Acknowledgements

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We would like to thank members of the Carter and Sheffield labs, Susan Walsh, Bryan Allen, and Kranti Mapuskar for valuable discussions. We would also like to thank Samantha Eckley, senior clinical veterinarian in the Office of Animal Resources, for her guidance and advice. We would like to acknowledge the use of the IVIS Lumina S5 Imaging System at the Small Animal Imaging Core Facility, a core resource supported by the Department of Radiology at the University of Iowa. Graphical illustrations from figures generated using Biorender (https://biorender.com). This work was supported by National Institutes of Health grants NCI R21CA256301 (C.S.C., V.C.S.), Free Radical Radiation Biology Program NCI T32CA078586 (S.C.H.), IVIS Lumina S5 Imaging System purchased on 1S10OD026835.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.25% Trypsin-EDTA (1x)Gibco25200-056
10% Neutral Buffered FormalinVWR16004-112
100 mm x 20 mm Tissue Culture Treated DishesDot Scientific Inc.667621
190 Proof Pure EthanolDecon Labs2801
3/10 mL Insulin Syringe with BD Ultra-Fine needle 31 G x 5/16 in.Beckton Dickinson328438
3M Vetbond3M1469SB
ATPSigmaA5394
Betadine Solution 10% Povidone-iodine AntisepticArvio Health L.P.67618-150-04
C57BL6/N MiceCharles River Laboratories
Cell Strainer 40um Nylon Falcon352340
Compact Anesthetic MachineVetamac Anesthesia 
Far Infrared Warming Pad with ControllerKent Scientific Corporation 
Conventional Surgical Blade Handle, Size 3Brad-Parker371030
Curved Medium Point General Purpose ForcepsFisherbrand16-100-110
DithiothreitolResearch Products InternationalD11000
D-Luciferin (sodium salt)Cayman Chemical14682
Drape, Surgical 18” x 26” Non-Fenestrated, SterileDealmed783410
Dulbecco’s Modified Eagle Medium (1x)Gibco11965-092
Dulbecco’s Phosphate Buffered Saline (PBS) 1xGibco14190-144
EGTASigmaE3389
Fetal Bovine SerumGibcoA5670402
FL150 Fiber Optic Microscope Light SourceMeiji 
Gentamycin Sulfate SolutionIBI ScientificIB02030
GlycerolResearch Products InternationalG22025
IsofluranePrimal Pharma Limited66794-013-25
IVIS Lumina S5PerkinElmer 
Living Image SoftwarePerkinElmer 
LL/2 (LLC1)ATCCCRL-1642Use the cancer cell line that will best fit the needs of the study 
Magnesium SulfateFisher ScientificM63
Matrigel Basement Membrane MatrixCorning354234
Mayo-Hegar Needle Holder, 6”, Serrated JawsMiltex8-44TC
Micro-Adson ForcepsFine Science Tools10018-12
N5 Dog Paw Trimmer with Double BladesOneisall 
Non-Sterile, 12 Ply Gauze SpongesNDCP157112
Optixcare Eye LubeAventix5914304
Penicillin StreptomycinGibco15140-122
Perma-Hand Silk Suture 6-0 1 x 18”Ethicon K802H
Phosphoric Acid 85%Fisher ScientificA242
Scalpel Blade Size 10, Stainless Steel, SterileCincinnati Surgical110
Screw Cap Tube Conical 50 mLSarstedt62.547.100
Tissue Scissors 4.5” Staight PremiumOnsungSCT115
Triton X-100SigmaT6878
Trizma BaseSigmaT1503
Trypan Blue Stain (0.4%)Gibco15250-061
Utility Aluminum Foil Tape with LinerIPGALF150L
Wooden Shaft Cotton Tipped ApplicatorsNDC76200

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Lung Cancer ModelSyngeneic Mouse ModelMinimally Invasive SurgeryTumor MicroenvironmentBioluminescence ImagingTumor ImplantationCell Suspension InjectionPreclinical Cancer ModelEarly Stage Lung Cancer

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