A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.
Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.
Colorectal cancer (CRC) continues to be one of the leading causes of cancer-related death in western countries.1 While the prognosis of patients with early stage disease is good, many tumors are diagnosed at later stages in which, despite numerous treatment options, the prognosis is limited.2,3,4,5
The majority of current mouse models of CRC are based on the implantation of tumor cells derived from cell lines or patient tumors into immunodeficient mice.6,7,8 This leads to local and, depending on the injection site and the tumor cells used for injection, sometimes metastatic tumors.9,10 However, the resulting xenograft models have major limitations. They must be established in immunodeficient mice, thus eliminating the complex interaction between the tumor and the host immune system. In addition, as the tumor stroma is derived from host cells, the interaction between human tumor parenchyma and murine stroma is defective and therefore not representative of the disease. These deficiencies can be avoided by the use of murine cell lines for injection. However, only few murine CRC cell lines are available and, similar to most available human CRC cell lines, are monoclonal and highly anaplastic.11 In summary, most currently available CRC mouse models are highly artificial and not fully representative of the human disease.
Genetically engineered mouse models (GEMMs) of CRC can avoid these drawbacks as they feature genuine mouse tumors which are created via induction of key mutations of CRC in the colon.12,13,14 This can be achieved by the activation of conditional (floxed) germline mutations by cre recombinase within the colorectal mucosa. While in GEMMs of many other tumor entities germline (inducible) cre expression driven by tissue-specific promoters is used, germline cre cannot be used in the colon as this leads to a great number of adenomas throughout the colon causing death by benign tumor load at a very young age. Therefore, in the here described model an adenoviral vector expressing cre is used to infect a short colon segment. This leads to the induction of tumorigenesis within this segment of the mucosa at a time point defined by the investigator, resulting in adenomas ultimately progressing to invasive and metastatic carcinoma. The tumors are genuine mouse tumors, grow in an intact microenvironment and are therefore able to simulate the entirety of colorectal oncogenesis including tumor–host interaction and the metastatic cascade. This model is therefore an attractive platform for studies of cancer biology and preclinical therapeutic trials.
A major disadvantage of genetically engineered mouse models of CRC is their technical complexity. Local cre delivery using rectal adeno-cre enemas in mice carrying floxed Apc alleles has been described before; however, the incidence, multiplicity and location of the intestinal tumors can be highly variable with this technique.15 Therefore, the technique of confining the adeno-cre infection by surgical clamping of the segment to be induced has been developed.13 We have modified this procedure in order to improve animal welfare, as well as reduce mortality and the number of resulting tumors. With this protocol, all labs with experience in small rodent surgery should be able to reproduce the model and to produce tumors which are highly reproducible and easily accessible to colonoscopy. Depending on the conditional mutations used for tumorigenesis, the full spectrum of adenoma, invasive carcinoma and metastases can be observed. As the tumors are located in the distal colon, serial endoscopic assessment is easily possible in this model.
The animal experiments presented here were independently reviewed and approved by an institutional and a governmental Animal Care and Use Committee and were conducted according to Federation of Laboratory Animal Science Associations (FELASA) guidelines. All possible measures were taken to minimize suffering including anesthesia and analgesia or, if necessary, premature euthanasia.
1. Local Tumor Induction via Surgical Adeno-cre Infection
2. Colonoscopy
NOTE: Depending on the conditional mutations used, adenoviral infection leads to endoscopically visible tumors within 2 – 4 weeks. Therefore, perform the first postoperative colonoscopy 2 weeks after the adenoviral induction and repeat every 2 weeks. A commercially available system is recommended for murine colonoscopy.20
If performed adequately, > 85% of the animals develop tumors. The mortality of the here presented surgical procedure is < 5%, mortality of the colonoscopy is virtually non-existent. In the majority of mice, a single lesion is detected; in about 30% 2 – 3 small adenomas can be detected which usually fuse to a single tumor within 2 – 3 weeks after tumor induction.
The phenotype and biological behavior of the resulting tumors is highly dependent on the conditional mutations of the animals. The local cre-mediated knockout of Apc is sufficient to induce tumorigenesis and initially leads to adenoma, which can be detected 2 – 4 weeks after adenoviral infection and which progresses to invasive adenocarcinoma within 12 – 16 weeks. This process can be accelerated by adding a conditional oncogenic Kras mutation (Kras G12D), the resulting tumors quickly progress to invasive adenocarcinoma. Upon the addition of oncogenic Tp53 R172H the tumors rapidly progress to invasive and metastatic carcinoma. Survival strongly depends on the genotype of the tumors; median survival is usually about 80 – 200 days after tumor induction. The cause of death in the great majority of cases is large bowel obstruction secondary to tumor growth.
The tumors can be easily and repeatedly monitored via colonoscopy (Figure 1) without major stress for the animals.
The disease manifestations are very similar to human CRC. The animals develop adenomas and ultimately adenocarcinomas of the distal colon (Figures 2B, 2C, 2F). Depending on the conditional genotype of the mice, the tumors also metastasize to the peritoneum (Figure 2D) the livers (Figure 2E) and rarely the lungs (not shown). If a cre reporter allele (e.g., ZSGreen19) is used, all tumor manifestations can be easily identified. The ZSGreen reporter allele features a fluorescent protein expression bright enough to be visible at daylight (Figure 1B, Figure 2C).
Histologically, 95% of the developing tumors are adenocarcinomas. About 5% of tumors are of mesenchymal origin, e.g., fibrosarcoma or leiomyosarcoma, presumably developing from stromal cells which have been accidentally infected with adeno-cre. The histomorphology of the adenocarcinomas closely resembles human CRC, featuring the entire spectrum from non-invasive adenoma to adenocarcinoma invading surrounding structures (Figure 3A – 3C).
Figure 1. Colonoscopic Images of Colorectal Tumors (Conditional Alleles of the Given Animal in Brackets).
A. Normal distal colon. B. Early adenoma 2 weeks after adeno-cre infection. Note the green color due to a GFP reporter allele in this mouse. (Apctm2Rak, Krastm4Tyj, Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze). C. Late adenoma (Apctm2Rak, Krastm4Tyj). D. Colorectal adenocarcinoma (as diagnosed by pathology after the colonoscopy images were obtained; Apctm2Rak, Krastm4Tyj, Tp53tm2Tyj). Please click here to view a larger version of this figure.
Figure 2. A. Intraoperative Situs. Note the large, rubberized Fogarty clip at the bottom and the transanally inserted tube for adeno-cre injection (red arrow). B. Colorectal tumor (white arrow) with consecutive large bowel obstruction 8 weeks after adeno-cre infection (Apctm2Rak, Krastm4Tyj, Tp53tm2Tyj). C. Colorectal tumor (white arrow) 10 weeks after adeno-cre infection (Apctm2Rak, Krastm4Tyj, Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze). Note the greenish color due to a green fluorescent protein (GFP) reporter allele in this mouse. D. Peritoneal carcinosis (black arrows) in the GEMM (Apctm2Rak, Krastm4Tyj, Tp53tm2Tyj). Liver and intestine have been removed to expose the kidneys and the diaphragm. E. Gross hepatic metastases in the GEMM 12 weeks after adeno-cre infection (Apctm2Rak, Krastm4Tyj). F. Colon with tumor after removal from the animal depicted in Figure 2C. Please click here to view a larger version of this figure.
Figure 3. Hematoxylin /Eosin (H/E) Stained Sections of Colorectal Tumors from the GEMM.
A. Transitional zone from normal mucosa to adenocarcinoma with infiltration of mucosa, submucosa and muscularis mucosae (Apctm2Rak, Krastm4Tyj). B. Transitional zone from normal colonic mucosa to invasive adenocarcinoma (Apctm2Rak, Krastm4Tyj). C. High grade adenocarcinoma with infiltration of surrounding tissue (Apctm2Rak, Krastm4Tyj, Tp53tm2Tyj). Please click here to view a larger version of this figure.
While they are generally easy to generate and maintain, classical CRC mouse models based on cell line injection are artificial and are not able to fully recapitulate the human disease. As a consequence, GEMMs have been developed. The first CRC GEMM was the ApcMin mouse, which harbors a heterozygous null mutation in the Apc gene, therefore mimicking the human hereditary disease familial adenomatous polyposis (FAP).21 However, ApcMin mice invariably develop multiple intestinal adenomas not limited to the colon; also, the time and exact location of adenoma formation is random and the tumors rarely develop into malignant lesions as the mouse dies of benign tumor load before the adenomas can progress. Therefore, the ApcMin mouse is a model of FAP, not sporadic CRC. Other models present mutations in DNA mismatch repair genes.22,23 Although some of these mice are excellent models for hereditary gastrointestinal cancer, they do not represent sporadic colorectal cancer.
Aside from the underlying genetic alteration it is the location of the genetic lesion which sets apart models of sporadic and syndromal colorectal cancer. All of the above-mentioned models feature mutations which are constitutively active or induced throughout the colon or even the entire gastrointestinal tract. This results in the formation of multiple adenomas, which is not representative of human sporadic CRC, is hardly assessable by colonoscopy and, in addition, usually does not leave the tumors enough time to develop before the animal succumbs to the extensive tumor load. Therefore, a mouse model for sporadic colorectal cancer must incorporate local activation of colorectal tumorigenesis.
The tumors arising in the here presented GEMM are strictly confined to the surgically clamped and adeno-cre-infected segment of the colon. This results in the formation of a single tumor which is easily accessible to detection and surveillance by colonoscopy. In addition, the distal location makes bowel obstruction a late complication of tumor growth, allowing the tumors to develop into invasive and, depending on the genotype, metastatic carcinoma before the animal requires euthanasia. Another advantage of segmental cre infection is the unaffected environment. While in ApcMin and other hereditary mouse models, the surrounding mucosa and even the stromal cells suffer the same genetic aberrations as the tumors, the tumors in the here proposed model develop within normal colonic epithelium and stroma. This enables more realistic tumor-host interaction and molecular studies without restrictions. Furthermore, the time point of tumor induction is well-defined in this model. While in the ApcMin mouse, tumors develop after the "second hit", during which the second Apc allele is stochastically lost at a random time point, all desired mutations are activated simultaneously in the here described model, thus allowing studies of very early lesions or consecutive biopsies in a very defined manner.
However, this model also comes with limitations. The crossing of multiple alleles into one model requires tremendous time and resources; in addition, often not all offspring can be used for the experiments due to an unsuitable genotype. The procedure itself requires practice and is time-consuming. The production of adeno-cre virus suspension with sufficiently high titers and in large amounts is cost-intensive. Therefore, this model is not suitable to replace all other CRC mouse models and will be limited to laboratories with a distinct focus on GEM models.
The protocol itself is technically demanding, yet with some training non-surgical personnel can perform the procedures adequately. The critical step during the adeno-cre infection is the inflation of the clamped bowel segment – excess intraluminal pressure leads to perforation, insufficient pressure reduces the rate of successful infection. This step requires training.
A protocol for adenoviral infection of the distal colon has been published by Hung et al. before.13 The here presented protocol differs from the aforementioned protocol in several key aspects. As it frequently causes bowel perforation in unexperienced hands, mechanical abrasion of the mucosa before adeno-cre incubation is skipped in the present protocol. This results in a reduced rate of tumor formation, which however can be counteracted by increased viral titers. This way, the rate of mesenchymal tumors can also be reduced as without mucosal abrasion less mesenchymal cells within the bowel wall are exposed to adeno-cre.
Also, in contrast to the above mentioned protocol13 the here described protocol does not recommend overnight fasting prior to surgery or colonoscopy. Instead, flushing of the bowel or, in case of colonoscopy, simple mechanical manipulation is used to remove remaining fecal matter. This refinement of the protocol dramatically improves animal welfare. Overnight fasting is a highly stressful procedure for small rodents and known to strongly influence the murine metabolism, therefore also influencing experimental results.24,25 These unwanted effects of overnight fasting can be avoided in the here presented manuscript.
Another important difference between the here presented protocol and the protocol by Hung et al. is the length of the clamped (and thus infected) segment. While the here described protocol recommends a short length (~3 mm), Hung et al. recommend 20 mm of the distal colon to be infected. The shorter segment length has been chosen to reduce the number of recombined crypts and thus resulting tumors. As the majority of patients with sporadic CRC develop a single tumor rather than multiple tumors, this measure increases the clinical relevance of the model. The rate of tumor formation does not seem to be affected by the reduced surface of infected colon.
Future applications of this technique include all protocols requiring local cre-recombination within the colon mucosa. Most applications in cancer research will include conditional oncogenes, leading to the formation of colorectal tumors; however, all other applications such as inducing reporter constructs within the colon mucosa can be achieved with the here presented protocol as well.
In conclusion, the here presented combination of a highly sophisticated genetically engineered mouse model of colorectal cancer along with the option of high-resolution colonoscopy for the detection and surveillance of the developing tumors provides an excellent setting to study the biology and treatment of CRC.
The authors have nothing to disclose.
This work is dedicated to the memory of Professor Moritz Koch.
Reagents / consumables | |||
Dulbecco's Phosphate Buffered Saline | Life Technologies GmbH | 14190169 | |
Trypsin-EDTA (0.25%, Phenol-Red) | Life Technologies GmbH | 25200072 | |
Normal saline 0.9% (E154) | Serumwerk Bernburg AG | 10013 | |
Aqua ad injectabilia | B. Braun Melsungen AG | 235144 | |
Ad5CMV-Cre (adenovirus, c = 2E+11 PFU/mL) | Gene Transfer Vector Core University of Iowa |
||
15 mL, 50 mL centrifuge tubes | Greiner Bio-One GmbH | 188271/227270 | |
Eppendorf tubes 1.5 mL/ 2 mL | Sarstedt AG & Co. | 72,695,400 | |
Petri dish PS 100/15 mm (sterile, Nuclon) | Fisher Scientific GmbH | 10508921/ NUNC150350 | |
1 mL Syringe (without dead volume) – Injekt-F SOLO | Braun/neoLab | 194291661 | |
30G injection needle | BECTON DICKINSON | 304000 | |
Name | Company | Catalog Number | Comments |
Analgesia / anesthesia | |||
Sevoflurane (Sevoflurane AbbVie) | AbbVie Germany GmbH & Co. KG | – | |
Medical oxygen | Air Liquide Medical GmbH | – | |
Buprenorphine (Temgesic) | Indivior Eu Ltd. | – | |
Bepanthen – ophthalmic ointment | Bayer Vital GmbH | 10047757 | |
Table Top Research Anesthesia Machine x/O2 Flush w/ Sevoflurane Vaporizer | Parkland Scientific | V3000PS/PK | |
Name | Company | Catalog Number | Comments |
Surgical Equipment | |||
Cellulose swabs | Lohmann & Rauscher Deutschland | 13356 | |
Insulin syringe EMG 1 mL (with 30G cannula) | B. Braun Melsungen AG | 9161627S | |
Fine Bore Tubing (bore: 0.28 mm/ diameter: 0.61mm) | Smiths Medical Deutschland | 800/100/100 | |
Micro-Adson Forceps | Fine Science Tools | 11018-12 | |
Iris Scissor – ToughCut | Fine Science Tools | 14058-11 | |
Olsen-Hegar Needle Holder | Fine Science Tools | 12002-12 | |
AutoClip Kit | Fine Science Tools | 12020-00 | |
PDS Z1012H 6/0 C1 (surgical suture) | Johnson & Johnson Medical GmbH | Z1012H | |
Curved Micro Serrefine Vascular Clamp | Fine Science Tools | 18055-05 | |
Fogarty Spring Clips | Edwards | CDSAFE 6 | |
Hot Plate 062 | Labotect | 13854 | |
Isis – Hair shaver | Aesculap – Braun | – | |
Name | Company | Catalog Number | Comments |
Colonoscopy | |||
Cold Light Fountain XENON 175 SCB | Karl Storz | 20132101-1 | Karl Storz Coloview System Mainz |
Fiber Optic Light Cable | Karl Storz | 69495NL | Karl Storz Coloview System Mainz |
TRICAM Three-Chip Camera Head | Karl Storz | 20221030 | Karl Storz Coloview System Mainz |
TRICAM SLII Camera Control Unit | Karl Storz | 20223011-1 | Karl Storz Coloview System Mainz |
15" Flat Screen Monitor EndoVue | Karl Storz | 9415NN | Karl Storz Coloview System Mainz |
HOPKINS Straight Forward Telescope diameter 1.9 mm; length 10 cm autoclavable fiber optic light transmission incorporated |
Karl Storz | 64301AA | |
Protection and Examination Sheath | Karl Storz | 61029C |