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

Our current research focuses on understanding lung cancer disease and how to develop new treatment modalities that are compatible with standard of care therapy, including chemo, radiation, and immunotherapy. We could not find an orthotopic lung cancer model that established criteria to specifically study early stage lung cancer. We work to provide clear and replicable guidelines to address that gap with this protocol.

This protocol is minimally invasive and simple to perform. Additionally with the criteria we have provided, this protocol allows for screening of precise tumor implantation and enables early tumor detection through bioluminescent imaging. This protocol enables researchers to study early stage lung cancer in the host tissue, allowing for preclinical advancement of novel cancer therapeutics and investigation into the tumor microenvironment.

We will use this technique to evaluate the mechanisms and efficacy of exciting new lung cancer treatments pre-clinically and hope that it can lead to future patient care. To begin, place the anesthetized mouse in a nose cone in the prone position on a warming device, and apply ophthalm ointment to both eyes of the mouse. Once anesthesia is confirmed, use electrical clippers to remove fur from a two by three centimeter rectangular area on the left dorsal side of the mouse.

Shave from the spine medially, the table laterally, the last rib inferiorly and the superior edge of the scapula towards the head until no fur is visible. Place the mouse in an empty cage on a warming device. Observe the breathing pattern until the mouse regains alertness and then return it to its home cage until surgery.

On the day of cell harvesting, add fresh media containing 50 milligrams per milliliter of Gentamycin to the culture plate three to four hours before collection. Aspirate the media from the culture plate and wash the attached cells with 1x PBS. Then incubate the cells with trypsin EDTA solution for one to two minutes at 37 degrees Celsius.

Add fresh media to neutralize the trypsin. Disperse the cells by gently tapping or pipetting the plate. Then collect the cell suspension into a 50 milliliter conical tube.

Now, centrifuge the conical tube at 1, 200 G for three minutes at room temperature. After confirming the presence of a compact pellet at the bottom, aspirate the supernatant and wash the pellet with PBS. Resuspend the washed cell pellet in PBS to achieve a concentration of approximately one to 2 million cells per milliliter for counting.

Filter the suspension through a 40 micrometer cell strainer before proceeding. Next, combine 10 microliters of the cell suspension with 10 microliters of trypan blue. Load the stained sample into a hemocytometer and count the cells.

Calculate the total number of cells and the cell viability. Centrifuge the cells again at 1, 200 G for three minutes at room temperature and aspirate the PBS leaving the pellet. Resuspend the cell pellet in 0.5 milligrams per milliliter of metra gel in PBS.

Adjust to the desired cell number in a total injection volume of 50 microliters. Keep the cell suspension on ice to prevent matrigel solidification. Clean the surgical site on the anesthetized animal three times using sterile gauze, alternating between Betadine and 70%ethanol.

Places sterilized fenestrated surgical drape with a three by four centimeter opening over the mouse and lay out autoclave surgical instruments on top of the drape. Using forceps, identify the cranial edge of the scapula and the coddle edge of the thoracic rib cage. With the skin held taut between the surgeon's thumb and forefinger, make a superficial five millimeter skin incision directly below the scapula using a size 10 scalpel blade.

Then use 115 millimeter straight scissors to blunt dissect the subcutaneous fat away from the ribs and intercostal muscles without cutting through them. To stop any light bleeding caused by capillary damage, press a sterilized cotton tipped applicator lightly onto the bleeding area. Hold the incision open using micro ads and forceps.

Starting at the seventh true rib, use curved median point forceps to count upward and locate the fourth and fifth ribs. Mark this region as the injection site. Now, gently invert the cell suspension tube three to four times to mix.

Just before injection, draw 50 microliters of the suspension into a sterile 300 microliter 31 gauge eight millimeter insulin syringe, making sure no bubbles are present. Place a four millimeter thick strip of sterilized metal tape flushed to the syringe to act as a stopper and control injection depth. Inject the 50 microliter cell suspension at a 90 degree angle into the intercostal space between ribs four and five directly beneath the fourth rib, and hold the needle in position for two to three seconds.

Slowly withdraw the needle at the same angle as inserted and discard it in a sharps container. Finally, close the skin incision using two to three square knotted stitches and apply veterinary surgical adhesive over the sutures to reinforce wound closure. Bioluminescence imaging detected tumor associated signals in the experimental mice as early as one day post-injection and radiance visibly increased over time for both 25, 000 and 50, 000 cells per mouse groups, with greater intensity observed in the 50, 000 cell group.

Total tumor flux increased exponentially in both 25, 000 and 50, 000 cell groups, reaching over 10 times 10 to the power of eight photons per second by day 21 in the 50, 000 cell group. Sham mice showed no luminescence or visible tumor in dissected lungs while experimental mice showed high luminescence and red tumor masses in lung tissue. At 21 days post-injection, included mice showed tumors confined to the left lung lobe with no invasion into surrounding regions.