The significance of this protocol lies in the real time detectability of the lung tumors that developed spontaneously in this mouse model, from tumor initiation to tumor progression. The advantage of this technique are that is non-invasive, is require only basic skill and allow us directly monitor the effect of the drug and treatment of interest. Lung tumor progression can also be evaluated in the presence or absence of a gene of interest.
Using this analysis, novel treatment strategies can be potentially extended to the clinic. Seven and 18 weeks after lentiviral intubation, turn on the heating pump for the ultrasound gel and the temperature monitor and set up a 33 degrees celsius incubator. Place the 3D motor on the integrated rail system and confirm that the motor and transducer mounting system are secured in place.
Connect a 40 hertz frequency transducer to the 3D motor and open a new study in the ultrasound software. In the new study window, enter the study name. In the series name window, enter series name and any other relevant information.
After clicking done, select the transducer type. The program will change to B mode. Place a heating lamp in a convenient position above the animal platform and confirm a lack of response to pedal reflex in the anesthetized experimental mouse.
Place the mouse on the animal platform into cubitus ventral and apply ointment to the animal's eyes. Secure the limbs firmly to the platform with tape and apply a thin layer of the warmed ultrasound gel to the animal's chest. Use the height control knob to lower the acquisition probe until it touches the surface of the mouse chest and position the probe such that the heart of the mouse is approximately centered.
Then use the micro knobs to acquire images of the whole chest from both extremities in the transverse orientation. Ideally gathering 500 frames per mouse. When all of the images have been captured, clean the gel from the chest of the mouse and place the animal into the warming incubator.
For 2D analysis of the ultrasound images, open the acquired frames in the ultrasound software and manually scan the frames for tumors. For small initiating tumors, count the number of beelines periodically every 10 frames for the full length of the 500 frames acquired. For 2D measurements of large tumors use the linear tool to measure the width and length of the tumor present, then calculate the volume of the tumors using the formula.
Seven weeks after intra tracheal infection, ultrasound imaging is performed to visualize the various types of precursor lesions that occur in the experimental mouse model after the injection. These beeline identified precursor lesions can be counted by the eye and represent small tumors on the surface of the lungs. Scatter plotting of these data allows quantification of the tumors within the experimental mice to allow estimation of the relative tumor number per animal.
At 18 weeks post infection, large tumors appear as deep clefts interrupting the plural surface that can be measured within the ultrasound software. The relative tumor volume can then be quantified for statistical analysis. Hematoxylin and eosin staining on lung sections harvested at 20 weeks post infection confirms the formation of large tumors as well as the formation of adenoma and adenocarcinoma.
It is important not to confuse the lung tumors, which are dynamic, with false positives, which are static. Since the qualification of the tumor number of the volume is relative in this method, we suggest to use additional methods, such as petri staining. This model allows direct assessment of the effects of genetic aberrations or treatment strategies on lung cancer development in mice.
