Molecular and Immunologic Techniques in a Genetically Engineered Mouse Model of Gastrointestinal Stromal Tumor

Our protocol describes the breeding, maintenance, dissection, and specimen processing of the only immunocompetent mouse model, GIST, currently in use. We hope that future researchers will be able to utilize this mouse model in order to further translational research in GIST. Traditional therapies in GIST involve targeted molecular therapy with tyrosine kinase inhibitors.

This model enables researchers to test immunotherapies in GIST. To begin, sterilize all instruments, wear gloves throughout the procedure, and maintain a sterile field. Prepare the area of incision with 70%ethanol.

Using scissors, make a two-centimeter midline vertical incision and enter the abdominal cavity. Sharply lyse any intra-abdominal adhesions. To remove the draining mesenteric lymph node, identify the cecum and lift its mesentery superiorly.

Approximately midway to the base of the colonic mesentery, identify the mesenteric lymph node and sharply dissect it. As needed, divide lymph node tissue into thirds for protein isolation, histology, and single cell suspension. Place lymph node tissue in 20 milliliters of RPMI medium for single-cell suspensions and keep on ice.

The cecum is mostly replaced by a GIST in the Kit558 mice. For isolation of the GIST and cecum, carefully divide the ileocolic junction from the base of the tumor. Then, divide the cecum again, two centimeters distal to the base of the tumor.

In 50 to 60%of the Kit558 mice, the head of the tumor contains a cap of cecal tissue, which typically contains serous fluid, but may rarely contain pus. Sharply dissect the cap tissue away from the tumor tissue. Divide the tumor tissue and cecum into thirds for protein isolation, histology, and single-cell suspension.

For single-cell suspensions, place tumor tissue or cecum in HBSS with 2%FCS, enough to cover the sample, and keep on ice. Pour RPMI media with the lymph node specimen over a 100-micrometer filter. Move the filter to a new 50-milliliter conical, and mash the lymph node with the soft end of a three-milliliter plastic syringe.

Wash filter with 20 milliliters of RPMI media. Centrifuge the samples and aspirate the supernatant. Resuspend the pellet in 20 milliliters of bead buffer and pour over a 40-micrometer filter.

Collect the cell filtrate. Count cells using a hemocytometer. Centrifuge the samples and discard the supernatant.

Then, resuspend in bead buffer for flow cytometry. Prepare collagenase buffer as described in the text manuscript. Place GIST in a sterile dish and add 2.5 milliliters of collagenase buffer.

Using a sterile scalpel and scissors, mince the tumor until it is in fine fragments. Use a large-bore pipette to aspirate the tumor and collagenase into a 50-milliliter tube. Incubate it at 37 degrees Celsius for 30 minutes at 100 rotations per minute, then quench the reaction with two milliliters of FBS.

Pour collagenase with GIST specimen over a 100-micrometer filter and mash the tumor with the soft end of a three-milliliter plastic syringe and collect in a 50-milliliter tube. Wash filter with 20 milliliters of HBSS. Centrifuge the filtrate at 450 RCF for five minutes at four degrees Celsius, then discard the supernatant.

Resuspend the pellet in 20 milliliters of bead buffer and pour over a 40-micrometer filter Collect the filtrate and count cells using a hemocytometer. Centrifuge and aspirate the supernatant, then resuspend in bead buffer for flow cytometry. Using scissors, split the cecum longitudinally to expose the inner mucosa.

Cut it into 0.5-centimeter sections and place it in a 50-milliliter tube with five milliliters of HBSS and 2%FBS. Shake vigorously for 30 seconds and centrifuge at 450 RCF for 20 seconds. Then, discard the supernatant.

Add five to 20 milliliters of HBSS with two-millimolar EDTA. Incubate it at 37 degrees Celsius at 100 rotations per minute for 15 minutes. Centrifuge and discard the supernatant, then resuspend the pellet in five to 20 milliliters of HBSS.

Shake the mixture vigorously for 30 seconds, then centrifuge at 450 RCF for 20 seconds and discard the supernatant. Repeat this process once more. Resuspend the pellet in five milliliters of collagenase buffer, then incubate it at 37 degrees Celsius for 30 minutes at 100 rotations per minute.

Shake vigorously every 10 minutes and quench the reaction with two milliliters of FBS. Pour collagenase with cecum specimen over a 100-micrometer filter and mash with the soft end of a three-milliliter plastic syringe. Wash the filter with 20 milliliters of HBSS and collect the filtrate.

Centrifuge and discard the supernatant. Resuspend the pellet in 20 milliliters of bead buffer and pour over a 40-micrometer filter. Collect filtrate and count cells using a hemocytometer.

Repeat the centrifugation process once more and resuspend the pellet in bead buffer for flow cytometry. The Kit558 mice had an average lifespan of eight months due to progressive bowel obstruction. Tumors from the Kit558 mice expressed canonical markers of GIST, including the tyrosine kinase KIT, the transmembrane channel Dog1, and the transcription factor ETV1.

Tumors were studied for changes in the KIT signaling pathway such as the downstream markers ERK and AKT or immune microenvironment, which closely mimicked human GIST. MRI or CT was used to track tumor volume as an accurate measurement of tumor response. Dissecting the tumor away from the ileocolic junction and away from the cecal cap is important to capture true tumor weight and immune and molecular analysis.

Within GIST, this model has allowed for the investigation of the tumor microenvironment, as well as immunotherapies.