In vivo Tumor Growth and Spontaneous Metastasis Assays Using A549 Lung Cancer Cells

Materials and Reagents

1. 10 cm cell culture dishes (Corning, catalog number: 430167 )
2. 1.5 ml Eppendorf tubes
3. Pipet tips
4. Mouse (NOD.CB17-Prkdc^scid/J, The Jackson Laboratory, catalog number: 00 1303 )
5. A549 cells (ATCC, catalog number: CCL-185 )
6. DMEM/F12 medium (Sigma, catalog number: D8437 )
7. Pen/Strep/Glutamine (Gibco, catalog number: 10378-016 )
8. Fetal Bovine Serum (FBS) (Sigma, catalog number: F0926 )
9. Phosphate-buffered saline (PBS) (Thermo Fisher, catalog number: 10010-049 )
10. Growth Factor Reduced Matrigel (Corning, catalog number: 354230 )
11. Analgesic (Buprenorphine HCl, 0.1 mg/kg SQ, ZooPharm)
12. Trypsin-EDTA (Gibco, catalog number: 25300-054 )
13. 70% ethanol (Decon Labs, catalog number: DSP-MD-43)
14. 10% neutral buffered formalin (Sigma, catalog number: HT501128 )
15. Hematoxylin (VWR, catalog number: 97062-216)
16. Eosin (VWR, catalog number: 97061-034)

Equipment

1. Sorvall Legend X1 Centrifuge(Thermo Scientific, catalog number: 75004221)
2. Vi-cell XR Cell Viability Analyzer (Beckman Coulter, catalog number: 1096573) or Hemocytometer
3. Operating Scissors (Fisher, catalog number: 13-806-4)
4. Exel International Sterile disposable scalpels with surgical steel blades size 11 (Fisher, catalog number: 14-840-01)
5. Dissecting forceps, fine tip, curved and straight (Fisher, catalog numbers: 08-875 and 08-880)
6. Biological safety cabinet
7. Surgical Autoclip System Kit (clip applier and remover are included) (Fine Science Tools, catalog number: 12120-00)
8. Ear tags (1005-1) and Applicator (1005s1) (National Band & Tag Company)
9. Digital Caliper 150 mm/6 (World Precision Instruments, catalog number: 501601 )
10. E·Z Anesthesia (Euthanex CORP., Serial NO: EZV2007 )
    

Software

1. Olympus cellSens Standard 1.17 image analysis software (Olympus, Center Valley, PA)
2. SAS9.4 (SAS Institute Inc., Cary, NC, USA)
3. R (Comprehensive R Archive Network, http://cran.us.r-project.org, 1997)
   

Procedure

1. Prepare cells
   1. Seed cells into 10 cm dishes (0.5 x 10⁶ cells/dish) three days before implantation.
   2. Change media (DMEM/F12 + 10% FBS + 1% Pen/Strep/Glutamine) the day before implantation.
   3. Harvest cells by trypsinization, rinse with PBS three times, count and resuspend cells in PBS at 2 x 10⁷/ml.
      Note: Total volume of cells prepared will depend on the number of mice needed.
   4. Prepare 1.5 ml Eppendorf tube per mouse and mix 60 µl cell suspension in PBS with 60 µl Matrigel (the ratio of cells:Matrigel is 1:1).
      Note: Since matrigel forms a gel above 10 °C, matrigel solution should be thawed overnight at 4 °C and kept on ice throughout the preparation. Tubes and tips for transferring Matrigel solution should also be chilled prior to implantation.
   
   
2. Implant cells and Matrigel mixture into NOD.CB17-Prkdc^scid/J (#00 1303 ; The Jackson Laboratory)
   1. Shave hair from the lateral body wall area.
   2. Administer analgesic (Buprenorphine HCl, diluted in sterile saline, 0.1 mg/kg SQ) 20-30 min before mouse surgery.
      Note: Buprenorphin HCl is a controlled substance and should be stored in a double locked cabinet.
   3. Anesthetize mouse by isoflurane inhalation until the mouse is unresponsive to toe pinch. Keep mouse warm and monitor breathing during procedure.
      Note: You should follow the IACUC protocol to administer the right dose of isoflurane.
   4. Disinfect surgical site with providone-iodine alternating with 70% ethanol for 3 times.
      
      

      Note: See Video 1 for Steps B5-B9.

   5. Lift the skin with sterilized dissecting forceps and make ~5 mm incision with operating scissors in the lateral body wall just posterior to the axillary area.
   6. Make a subcutaneous pouch by blunt dissection.
   7. (Optional) Transfer 100 µl of cells mixed with Matrigel (1 x 10⁶ cells) into the pouch using pipette with 200 µl wide bore Pipet tip to minimize the shearing of the cells.
   8. Place ear tag on mice.
   9. Close incision using clip applier with 9 mm surgical clips and place mouse in recovery cage. Mouse should recover from anesthesia within 1-2 min.
      
      
      
      Video 1. Subcutaneous implantation of cells and Matrigel mixture into mouse. (Animal study was performed according to the procedure approved by the University of Kentucky Institutional Animal Care and Use Committee (IACUC).)
      
      
3. Monitor tumor growth
   1. Check clips and mouse health daily until clips are removed using clip remover from mouse; wound clips should be removed 7-10 days after surgery.
   2. Measure tumor size using a caliper 3 times/week and weigh mouse until tumor size reaches around 1,000-1,500 mm³ (Note: endpoint will depend on the goal of the experiments). Three parameters of the tumor: the length (the longest diameter), the width (the diameter perpendicular to the length), and the height were recorded.
      
      
      
4. Harvest primary tumor tissues and organs
   1. Perform CO₂ euthanasia and maintain CO₂ flow for a minimum of 1 min after respiration ceases; then perform cervical dislocation.
      Note: You should follow your IACUC protocol to sacrifice mouse.
   2. Clean tumor area with 70% ethanol.
   3. Cut skin and remove tumor carefully and place tumor in a sterile tube containing DMEM/F12 media and keep it on ice.
   4. Perform necropsy to remove lungs or other organs such as liver, spleen, kidney or brain, and put them in tube containing DMEM/F12 media and keep the tube on ice.
   5. Rinse tissues with PBS.
   6. Fix tissues in 10% neutral buffered formalin overnight at 4 °C and replace solution with 70% ethanol before processing for paraffin embedding.
      Note: Histopathology Facility may be required to process the samples for this step.
   7. Stain tissues with hematoxylin and eosin (H&E).
   8. Examine H&E slides to identify foci of adenocarcinoma (for A549 cells) (Figure 1). These can be identified by recognizing characteristic architectural and cytologic features (e.g., glandular tumor formations, papillary growth, or lepidic tumor spread; loss of nuclear polarity, nuclei with fine chromatin and prominent nucleoli, anisonucleosis).
   9. Measure the diameter of each tumor foci in millimeters using a digital micrometer.
      Note: We used the Olympus cellSens Standard 1.17 image analysis software.
      
      
      Figure 1. H&E staining of lung section. Magnification = 40x for image in (A) and 200x for image in (B). Scales bar = 1 mm in (A) and 100 µm in (B). Arrows indicate tumor foci. Representative images are shown.

Data analysis

1. Record total number of foci and calculate the tumor foci incidence (total number of foci/observation time (day)).
2. Calculate tumor volume (V) based on the measurement from Step C2 as V = (L x W x H)/2 where V = tumor volume, L = tumor length, W = tumor width, and H = tumor height.
3. Data analysis: For in vivo experimental metastasis study, a linear mixed effect model is used to fit the longitudinal tumor volumes and test the significant difference of mean tumor volume profiles between treatment groups (Figure 2). The Welch two-sample t-test should be used to test the difference of the mean tumor foci size between groups. A Poisson model is used to model the number of tumor foci incidence per day and significant difference of tumor foci incidence rates between groups is tested using Poisson test. Statistical difference between groups will be considered significant at the level of 0.05.
   
   
   Figure 2. Tumor growth rate. Tumor growth curve was fitted and plotted using R software. The black line is fitted tumor volume profile and the blue line is the observed mean tumor volume.
   

Notes

1. Tumor tissues and organs from necropsy can be rinsed with PBS and fixed immediately or temporary stored in media without serum for less than 30 min on ice before further processing.
2. Assistance from a pathologist may be needed for slides assessment.
   

Acknowledgments

This study was supported by American Cancer Society Institutional Research Grant IRG-85-001-25 (MC); the Kentucky Lung Cancer Research program (MC); the National Center for Advancing Translational Sciences National Institutes of Health UL1TR000117 (MC); the University of Kentucky Center for Cancer and Metabolism [P20GM121327] for providing services through the Imaging Core and for the pilot funding [no grant number, to MC]); and the National Institutes of Health R01 CA109136 (KLO). The Markey Cancer Center Biospecimen Procurement and Translational Pathology, the Biostatistics and Bioinformatics and Cancer Research Informatics Shared Resource Facilities are supported by National Institutes of Health [P30 CA177558]. This protocol was briefly described and originally published in Liu et al., 2019. 

Competing interests

The authors report no conflict of interest.

Ethics

Animal study was performed according to the procedure approved by the University of Kentucky Institutional Animal Care and Use Committee (IACUC).
This protocol was modified from Liu et al., 2019 and Morton et al., 2007.

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