A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis

This protocol describes a series of techniques within a continuous workflow that can be used for assessing the in vivo effects of candidate regulators of melanoma metastasis, but also of metastasis of other solid malignancies. The main advantage of our systematic workflow is increased reproducibility of the data due to robust and standardized in vivo models and techniques. This pipeline offers a pathway for target discovery and therapy development by testing candidates inferred from Omics data or in vitro assays in an in vivo setting.

The learning curve associated with the techniques presented in this protocol is shortened by using the materials provided and following the detailed description when practicing the steps. Helping to demonstrate the procedure will be Orlando Aristizabal, a research scientist in The Preclinical Imaging Core and Ran Moubarak, an instructor in my lab. For intradermal injections, anesthetize and shave an eight to 10 week old mouse inside a biosafety cabinet while maintaining aseptic conditions.

Then grasp and retract the skin backwards against the trajectory of the needle stab and using a six millimeter long, 31 gauge insulin syringe needle, gently puncture the skin at an acute angle with the bevel facing upward. Inject 30 microliters of the tumor cell suspension slowly, until a dome shaped wheel is observed. Monitor the progression of the tumor growth, weight loss, and overall health status.

During these monitoring sessions, take measurements with calipers and use the length and width dimensions of the tumor to calculate the volume. For intracardiac injections, transfer an anesthetized mouse onto the heated platform of the ultrasound machine. Then using hypoallergenic tape, secure the mouse to the nose cone.

Shave the thorax with a straight razor blade or a scalpel blade, tilted at a 30 degree angle and clean the skin around the procedure area with 10%povidone iodine. Then, position the ultrasound probe in the middle of the thorax on the left side of the mouse to capture a horizontal window oriented to obtain a cross-sectional view of the left ventricle. With the long axis of the probe facing upwards, fix the probe at a 50 degree angle and the heated platform at a 20 degree angle, then lock the probe and the support frame in the position.

While working inside the safety cabinet, draw up the cell suspension in a tuberculin one milliliter syringe with a 30 gauge one inch needle. Remove any air bubbles present in the syringe. Lock the syringe in the stereotactic injector.

And then under ultrasound guidance, advance the needle through the thoracic wall into the left ventricle of the heart and inject 100 to 250 microliters of the tumor cell suspension slowly. For staged survival surgery, place the anesthetized mouse on the warming pad and clean the skin around the procedure area with a 10%povidone iodine solution. After donning sterile personal protection equipment and gloves, lay a sterile drape over the animal's body.

Next, using Iris scissors or a scalpel, incise the skin, maintaining a five to seven millimeter resection margin from the edge of the tumor. In case of intradermal tumors, resect the tumor along with the circumferential skin. For subcutaneous tumors, dissect and remove the tumor under the skin.

After tumor resection, close the wound with a nine millimeter stapling device. For in vivo imaging, administer d-luciferin substrate to the mouse by intraperitoneal injection with a one milliliter insulin syringe and a 28 gauge needle then anesthetize the mouse and place it into a nose cone inside the bioluminescence imaging scanner. Start the instrument by pressing initialize and set the exposure time to auto.

For capturing a blank image for background subtraction, click acquire and save the image after the acquisition sequence is completed. For data analysis and the same in vivo imaging software, navigate to the folder where the images are saved and open the images of all the mice from one experiment. Set the units to radiance and ensure that the checkbox indicating individual is not checked as this will preclude normalization of signal across groups.

Next, using the region of interest or ROI drawing tool, draw circular ROI's for the brain region and rectangular ROI's for the body, exclude the ears and nose in the brain ROI as they tend to emit unspecific luminance. To minimize bias, draw ROI's on the photographs of the mice without the luminescent signal overlaid, then select measure ROI's to quantify the signal and export the data to a spreadsheet. Analyze differences between groups by plotting total luminescent flux in body regions of interest.

After a NuMA staining using software, draw an ROI to include all NuMA stained cells within the organ tissue, except other organ parenchyma and empty spaces. Adjust the settings to categorize the NuMA positive and NuMA negative cells while using appropriate positive and negative controls for each organ. Use an established software algorithm to quantify the total number of percentage of NuMA positive cells for each sample.

Bioluminescence, bright field, ex vivo fluorescence and hematoxylin and eosin staining images illustrate the multi-pronged approach for the analysis of candidate genes effects on melanoma metastasis. Fucosyltransferase silencing impaired the metastatic dissemination of melanoma cells. The NuMA stained lung sections of melanoma cells infected with control Linda virus and with a metastasis suppressor expressing Linda virus are shown here.

The metastatic melanoma cells are labeled green and the organ area is delimitated by a green hatched line. Maintaining proper tension in the skin when performing intradermal injections, avoiding air embolism when preparing syringes and obtaining adequate resection margins that should not interfere with the animal's locomotion or predispose it to wound complications, help improve the success rate and reproducibility. Further employment of multiplex imaging to examine immune filtration, single cell RNA sequencing for gene expression analysis and spatial transcriptomics could all provide complimentary avenues to examine intratumoral heterogeneity.

The combination of techniques described in this protocol helped us identify novel regulators in melanoma brain metastasis, such as the role of melanoma secreted amyloid beta in suppressing neuroinflammation and promoting brain metastasis.