The goal of this manuscript is to describe the KitV558Δ/+ mouse model and techniques for successful dissection and processing of mouse specimens.
Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor types, numerous mouse models have been developed in order to investigate the next generation of cancer therapies. However, in GIST, most in vivo studies use xenograft mouse models which have inherent limitations. Here, we describe an immunocompetent, genetically engineered mouse model of gastrointestinal stromal tumor harboring a KitV558Δ/+ mutation. In this model, mutant KIT, the oncogene responsible for most GISTs, is driven by its endogenous promoter leading to a GIST which mimics the histological appearance and immune infiltrate seen in human GISTs. Furthermore, this model has been used successfully to investigate both targeted molecular and immune therapies. Here, we describe the breeding and maintenance of a KitV558Δ/+ mouse colony. Additionally, this paper details the treatment and procurement of GIST, draining mesenteric lymph node, and adjacent cecum in KitV558Δ/+ mice, as well as sample preparation for molecular and immunologic analyses.
GIST is the most common sarcoma in humans with an incidence of about 6,000 cases in the United States of America1. GIST appears to originate from the gastrointestinal pacemaker cells named the interstitial cells of Cajal, and is typically driven by a single mutation in the tyrosine kinase KIT or PDGFRA2. Surgery is the mainstay of treatment for GIST and can be curative, but patients with advanced disease may be treated with the tyrosine kinase inhibitor (TKI), imatinib. Since its introduction over 20 years ago, imatinib has transformed the treatment paradigm in GIST, improving the survival in advanced disease from 1 to over 5 years3,4,5. Unfortunately, imatinib is rarely curative due to acquired KIT mutations, so new treatments are needed for this tumor.
Mouse models are an important research tool in the investigation of novel therapies in cancer. Multiple subcutaneous xenograft and patient-derived xenograft models have been developed and investigated in GIST6,7. However, immunodeficient mice do not fully represent human GIST since GISTs harbor differential immune profiles depending on their oncogenic mutation, and altering the gastrointestinal tumor microenvironment improves upon the effects of TKI therapy8,9. The KitV558Δ/+ mouse has a heterozygous germline deletion in Kit exon 11, which encodes the juxtamembrane domain, the most commonly mutated site in human GIST10. KitV558Δ/+ mice develop a single cecal GIST with 100% penetrance, and tumors have similar histology, molecular signaling, immune infiltration, and response to therapy as human GIST8,11,12,13. Here, we describe breeding, treatment, and specimen isolation and processing in KitV558Δ/+ mice for use in molecular and immunologic research in GIST.
All mice were housed under pathogen-free conditions at the University of Pennsylvania according to NIH guidelines and with approval of the University of Pennsylvania IACUC. Euthanasia was performed following the University of Pennsylvania Laboratory Animal Resources standard operating procedures.
1. KitV558Δ/+ mouse breeding
2. KitV558Δ/+ mouse treatment
3. KitV558Δ/+ mouse organ harvest
4. Western blot analysis of GIST tissue
5. Immunohistochemistry of GIST tissue
6. Single cell suspension of mesenteric lymph node
7. Single cell suspension of GIST
8. Single cell suspension of cecum
The KitV558Δ/+ mouse model allows for the investigation of therapeutics in an immunocompetent mouse model. KitV558Δ/+ mice have an average lifespan of 8 months due to progressive bowel obstruction (Figure 4). Tumors from KitV558Δ/+ mice express canonical markers of GIST including the tyrosine kinase KIT and the transmembrane channel DOG1 (Figure 5), as well as the transcription factor ETV1 (not shown). Tumors can be studied for changes in the KIT signaling pathway, such as the downstream markers ERK and AKT (Figure 6), or immune microenvironment which closely mimics human GIST8,11,12,13. MRI8 or CT (Figure 7) can also be used to track tumor volume as an accurate measurement of tumor response. An untreated KitV558Δ/+ mouse was given 200 µL of gastrograffin orally 1 h prior to imaging. CT imaging was completed on a vivaCT 80 platform. 3D reconstruction was performed with Fiji software, which can also measure tumor volume.
Figure 1: Comparison of tumor weights in male and female KitV558Δ/+ mice. Tumors from 9-week-old untreated KitV558Δ/+ male and female mice were isolated and weighed (n = 15 mice/group). Data represents mean ± standard error of mean (SEM); p values were calculated using a student's t test; * = P < 0.05. Please click here to view a larger version of this figure.
Figure 2: Effect of imatinib on tumors from KitV558Δ/+ mice. KitV558Δ/+ mice were treated with vehicle or 600 mg/L imatinib in drinking water for 1 or 4 weeks. Tumors were isolated and weighed (n = 4-5 mice/group). Data represents mean ± SEM; p values were calculated using a one-way ANOVA comparison with a Bonferroni post-test for comparison of individual groups; * = P < 0.05. Please click here to view a larger version of this figure.
Figure 3: KitV558Δ/+ tumor with cap. Representative photo of a tumor from a KitV558Δ/+ mouse with a cecal cap containing serous fluid. Please click here to view a larger version of this figure.
Figure 4: Lifespan of KitV558Δ/+ mice. Survival of untreated KitV558Δ/+ mice was tracked for > 400 days (n = 43 mice). Please click here to view a larger version of this figure.
Figure 5: Immunohistochemical analysis. Representative histology of a KitV558Δ/+ tumor where scale bar is 40 µm. Abbreviations: H&E = hematoxylin and eosin staining; Kit = a canonical mark of GIST and receptor tyrosine kinase; Dog1 = a canonical marker of GIST with role in anion transport. Please click here to view a larger version of this figure.
Figure 6: Molecular signaling analysis. KitV558Δ/+ mice were treated with vehicle or 600 mg/L imatinib in drinking water for 1 week. Mice were given a single i.p. injection of vehicle or 45 mg/kg imatinib 6 h prior to analysis. Protein lysates from KitV558Δ/+ tumors were examined by western blot (n = 2 mice/group). Abbreviations: P Kit = phosphorylated Kit receptor tyrosine kinase; T kit = total Kit receptor tyrosine kinase; P ERK = phosphorylated mitogen activated protein kinase; T ERK = total mitogen activated protein kinase; P AKT = phosphorylated serine-threonine protein kinase; T AKT = total serine-threonine protein kinase. Please click here to view a larger version of this figure.
Figure 7: CT imaging analysis. 3D CT reconstruction of an untreated KitV558Δ/+ mouse demonstrating a tumor (arrow) in the pelvis. Please click here to view a larger version of this figure.
The KitV558Δ/+ mouse model is a powerful research tool in the molecular and immunologic analysis of GIST. Although the breeding strategy requires a single cross, using KitV558Δ/+ mouse cohorts in experiments analyzing tumor response requires extensive breeding. Mice should be age- and sex-matched to ensure similar tumor weights, and 10% of mice die before 8 weeks of age when tumors are established. Less extensive breeding strategies are possible if using advanced imaging techniques such as CT or MRI to track tumor volume within individual mice. Nonetheless, KitV558Δ/+ mice have been successfully crossed to other knock-out or inducible mouse models, revealing important immune and molecular mechanisms12.
Tumor cells from KitV558Δ/+ mice are easily isolated by column sorting for KIT (CD117) or by flow cytometry18. Isolated tumor cells from KitV558Δ/+ mice can be grown in vitro12. At early passages, isolated tumor cells from KitV558Δ/+ mice retain KIT expression and can be used for in vitro studies. However, after several passages, these cell lines lose KIT expression, limiting their applicability. Like most mouse models, KitV558Δ/+ tumors do not metastasize, which limits the study of extraintestinal GIST. Similarly, tumors from KitV558Δ/+ mice only occur in the cecum, and cells isolated from KitV558Δ/+ tumors do not grow when isolated and injected into the liver or spleen, which limits the evaluation of the tumor microenvironment from different sites of disease. Also, the KitV558Δ/+ mutation does not have any known impact on the hematological development of cells in this mouse model.
The immune microenvironment in tumors from KitV558Δ/+ mice contains mostly macrophages followed by T cells, which closely mimics human GIST11. However, it is imperative that the cecal cap be removed completely from the tumor prior to assessing tumor weight or immune infiltrate, as distinct immune populations exist in the tumor versus the cecum. In our experience, tumor weights are comparable even among those with caps, and there is no significant association between tyrosine kinase inhibitor therapy and the development of a cecal cap. Furthermore, we have found no significant difference in the tumor microenvironment comparing tumors with and without cecal caps.
Many genetically engineered mouse models have been developed for use in cancer research, especially since the advent of CRISPR gene editing. While numerous immunocompetent models rely on cre-loxP mediated activation of oncogenes or inactivation of tumor suppressor genes, the KitV558Δ/+ tumors are driven by their endogenous promoter. Accordingly, findings in the KitV558Δ/+ mouse model are highly translatable to human disease, particularly in the evaluation of KIT signaling19. Looking ahead, the KitV558Δ/+ mouse should continue to serve as a valuable model as novel treatments strategies including checkpoint blockade and CAR T therapy continue to be explored for the treatment of soft tissue tumors including GIST.
The authors have nothing to disclose.
KitV558Δ/+ mice were genetically engineered and shared by Dr. Peter Besmer10. This work was supported by NIH grants R01 CA102613 and T32 CA251063.
100 micron filter | EMSCO | 1194-2360 | |
1x RBC lysis buffer | Life Technologies | 00-4333-57 | |
3mL syringe | Thermo Fisher Scientific/BD Biosciences | 14823435 | |
4–15% Mini-PROTEAN TGX Precast Protein Gels, 10-well, 30 µl | Bio-Rad | 4561083 | |
4% Paraformaldehyde Solution | Thermo Fisher Scientific | AAJ19943K2 | |
40 micron filter | EMSCO | 1194-2340 | |
5M NaCl | Sigma Aldrich | S6546 | |
70 micron filter | EMSCO | 1194-2350 | |
AKT antibody (C67E7) | Cell Signaling | 4691 | |
C57BL/6J mice | The Jackson Laboratory | ||
Collagenase IV | Sigma Aldrich | C5138 | |
Complete mini edta free protease inhibitor | Thomas Scientific | C852A34 | |
Countess II Automated Cell Counter | Thermo Fisher Scientific | ||
Disposable Scalpels | Thermo Fisher Scientific/Exel International | 14-840-00 | |
Dnase I | Thomas Scientific | C756V81 | |
Dog1 antibody | abcam | ab64085 | |
EDTA | Sigma Aldrich | E9884 | |
ERK antibody (p44/42) | Cell Signaling | 9102 | |
FBS | Thomas Scientific | C788U23 | |
FIJI software | FIJI | https://imagej.net/software/fiji | |
Fisherbrand 850 Homogenizer | Thermo Fisher Scientific | 15-340-169 | |
HBSS | University of Pennsylvania Cell Center | ||
Imatinib mesylate | Selleck Chemicals | S1026 | |
KIT antibody (D13A2) | Cell Signaling | 3074 | |
KitV558Δ/+ Genotyping | Transnetyx | ||
Microcentrifuge tubes (1.5mL) | Thermo Fisher Scientific | 05-408-129 | |
Mouse on Mouse Immunodetection Kit, Basic | Vector Laboratories | BMK-2202 | |
Nitrocellulose Membrane, Precut, 0.45 µm | Rio-Rad | 1620145 | |
Nonfat Dry Milk | Thermo Fisher Scientific | NC9121673 | |
Nonidet P 40 Substitute | Sigma Aldrich | 74385 | |
p-AKT antibody (S473) | Cell Signaling | 4060 | |
p-ERK antibody (p44/42) | Cell Signaling | 9101 | |
p-KIT antibody (Y719) | Cell Signaling | 3391 | |
PMSF Protease Inhibitor | Thermo Fisher Scientific | 36978 | |
Proeinase K | Thermo Fisher Scientific | BP170050 | |
Round-Bottom Polystyrene Test (FACS) Tubes | Falcon/Thermo Fisher Scientific | 14-959-2A | |
RPMI | University of Pennsylvania Cell Center | ||
Sodium fluoride (NaF) | Sigma Aldrich | 201154 | |
Sodium orthovanadate (Na3VO4) | Sigma Aldrich | S6508 | |
SuperSignal West Dura Extended Duration Substrate | Thermo Fisher Scientific | 34076 | |
TBS buffer (10x) | University of Pennsylvania Cell Center | ||
Tissue culture dish (100mm2) | Thermo Fisher Scientific/Falcon | 08-772E | |
TrisHCL | Thermo Fisher Scientific | BP1757500 | |
Tween 20 | Rio-Rad | 1706531 | |
vivaCT 80 platform | Scanco medical |