This work is dedicated to the memory of Professor Moritz Koch.

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
<ol>
	<li>Preparation of animals for surgery 
	NOTE: Virtually any conditional (&#34;floxed&#34;) mutation can be induced via the here described method. The use of mutations in genes relevant in colorectal cancer such as Apc, Kras or Tp53 is recommended. The efficiency of cre recombination is dependent on the size of the construct to be excised. Large floxed sequences are excised less efficiently. The recombination of all alleles should be confirmed in the tumors by PCR.
	<ol>
		<li>For the development of colorectal tumors, use a cross of the following conditional alleles for a basic model of CRC (MGI database number in brackets): 
		Apctm2Rak (MGI: 3688435)16 
		Krastm4Tyj (MGI: 2429948)17 
		Tp53tm2Tyj (MGI: 3039263)18</li>
		<li>If a fluorescence reporter allele is required (e.g., to detect micrometastases), use the following allele: 
		Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze (MGI: 3809522)19 
		NOTE: All above strains are available via the NCI Mouse Repository or the Jackson Laboratory. No fasting is required as all remaining fecal matter can be flushed out prior to the adenoviral infection. Preoperative fasting leads to higher perioperative mortality and constitutes tremendous stress for small rodents.</li>
		<li>Use sevoflurane at 3 - 3.5 vol% for general anesthesia. A loss of the toe pinch reflex indicates sufficient anesthesia.</li>
		<li>Prior to the first incision, inject 0.05 mg/kg of buprenorphine subcutaneously.</li>
		<li>Cover the eyes of the anesthetized mouse with ophthalmic ointment to avoid desiccation of the cornea.</li>
		<li>Place the mouse in a supine position on a small table. Use non-traumatic adhesive tapes to restrain the mouse.</li>
		<li>Shave the abdomen with an electric shaver (depilatory cream can be used alternatively) and disinfect with alcohol swabs or iodine. Use the contact time recommended by the manufacturer.</li>
		<li>Cover the surgical field with sterile drapes. 
		NOTE: The use of perioperative antibiotics is optional and subject to institutional guidelines.</li>
		<li>Use sterile single use or sterilized instruments for all surgical procedures.</li>
	</ol>
	</li>
	<li>Midline laparotomy and exposure of the colon
	<ol>
		<li>Use scissors (scalpels can be used alternatively) to make a midline incision (~ 15 mm) of the skin on the lower abdomen.</li>
		<li>Pick up the abdominal wall musculature with forceps and carefully incise it with scissors, thus opening the abdominal cavity.</li>
		<li>Identify the distal colon, only touch it with atraumatic forceps. Clamp the colon with a delicate clamp (e.g., a Micro Serrefine vascular clamp) approximately 15 mm proximally of the anus. 
		NOTE: Give special attention to the vulnerability of the colon at all times. Perforation inevitably leads to peritonitis and sepsis and requires euthanasia of the animal.</li>
	</ol>
	</li>
	<li>Segmental colon infection with Adeno-cre virus
	<ol>
		<li>Insert a flexible Teflon tube transanally and carefully advance it until it reaches the lumen occlusion achieved by the clamp previously placed at 15 mm from the anal verge. Do not use excess force as this may lead to perforation.</li>
		<li>Cannulate the tube with a 30G cannula, connect a standard 1 mL syringe and flush the colon with normal saline in order to evacuate remaining fecal matter. This may require several mL of saline.</li>
		<li>Once the distal colon is empty remove the tube and replace it with a fresh Teflon tube and again position it directly distal to the clamp as described above.</li>
		<li>Occlude the colon with a second clamp ~ 3 mm distal to the proximal clamp (i.e., over the inserted tube, ~ 12 mm from the anal verge), resulting in a 3 mm isolated segment to be infected. 
		NOTE: For distal occlusion of the segment Fogarty coronary artery clips have proven most suitable as they are rubberized, leading to tight occlusion of the colon despite the intraluminal tube between the clamp&#39;s branches.</li>
		<li>Use a second syringe (standard 1 mL with a 30G cannula) to carefully inject 50 - 80 &#181;L of 0.25% Trypsin-EDTA into the clamped colon segment and incubate for 10 min. Leave the cannula and the syringe attached to the Teflon tube to prevent the fluid from leaking back. 
		NOTE: The colon must be inflated to break up the mucosal barrier and to reach the crypt stem cells, yet not too much inflated to avoid perforation of the clamped segment.</li>
		<li>First remove the distal clamp, and then the trypsin tube.</li>
		<li>Flush the distal colon with ~ 500 &#181;L of normal saline to remove remaining trypsin.</li>
		<li>Insert a new Teflon tube, put the distal clamp back in place and inflate the colon segment with 50 - 80 &#181;L of adenoviral solution (1011 plaque-forming units (PFU)/mL in phosphate-buffered saline) and incubate for 30 min (Figure 2A). 
		NOTE: Do not spill viral solution as contact with adeno-cre may lead to tumor development in any tissue of conditionally mutant mice.</li>
		<li>Remove the clamps and the tube.</li>
	</ol>
	</li>
	<li>Closure of the abdomen and postoperative recovery
	<ol>
		<li>Close the abdominal wall with 6-0 rapidly absorbable running sutures (e.g., polydioxanone (PDS)).</li>
		<li>Close the skin with surgical wound clips.</li>
		<li>Place the mouse on heating pad set to 38 &#176;C until it has fully recovered from the anesthesia.</li>
		<li>Administer another bolus of 0.05 mg/kg buprenorphine i.p. 12 h after surgery, followed by additional buprenorphine boluses every 12 h&#160;if needed.</li>
		<li>Monitor the mice at least once daily for signs of distress due to tumor growth.</li>
	</ol>
	</li>
</ol>
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
<ol>
	<li>Preparation of animals for colonoscopy 
	NOTE: No fasting is required. Remaining fecal matter is usually well-formed in the distal colon and can be pushed beyond the tumor during colonoscopy, thus making the stressful process of repeated fasting unnecessary.
	<ol>
		<li>Use sevoflurane at 3 - 3.5 vol% for general anesthesia. A loss of the toe pinch reflex indicates sufficient anesthesia.</li>
		<li>Cover the eyes of the anesthetized mice with ophthalmic ointment to avoid desiccation of the cornea.</li>
		<li>Restrain the mice in a supine position on a small table.</li>
	</ol>
	</li>
	<li>Colonoscopy
	<ol>
		<li>Insert the scope (diameter 1.9 mm; length 10 cm) into the intestinal tract through the anus and carefully insufflate air under visual control to distend the colon. Do not insufflate more air than required for the examination. 
		NOTE: For air insufflation, the anti-fog air pump of the colonoscopy system may be used. If no anti-fog air pump is available, any other air pump with very low pressure settings can be used, or pressurized air with a delicate pressure-reducing valve. Carbon dioxide (CO2) easily leads to acidosis in small rodents and must therefore be avoided.</li>
		<li>Carefully push the scope forward until a mucosal lesion in the distal colon can be identified (Figure 1A - 1D).</li>
		<li>Save endoscopic images for later evaluation. An endoscopic scoring system for intraluminal tumors has been described before.20</li>
		<li>Carefully remove the scope and place the mouse on heating pad set to 38 &#176;C until it has fully recovered from the anesthesia.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 ...

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&#8211;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.

<table><tbody><tr><td>&lt;strong&gt;Reagents / consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Life Technologies GmbH</td><td>14190169</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%, Phenol-Red)</td><td>Life Technologies GmbH</td><td>25200072</td><td></td></tr><tr><td>Normal saline 0.9% (E154)</td><td>Serumwerk Bernburg AG</td><td>10013</td><td></td></tr><tr><td>Aqua ad injectabilia</td><td>B. Braun Melsungen AG</td><td>235144</td><td></td></tr><tr><td>Ad5CMV-Cre (adenovirus, c = 2E+11 PFU/mL)</td><td>Gene Transfer Vector Core&lt;br/&gt; University of Iowa</td><td></td><td></td></tr><tr><td>15 mL, 50 mL centrifuge tubes</td><td>Greiner Bio-One GmbH</td><td>188271/227270</td><td></td></tr><tr><td>Eppendorf tubes 1.5 mL/ 2 mL</td><td>Sarstedt AG &amp;amp; Co.</td><td>72,695,400</td><td></td></tr><tr><td>Petri dish PS 100/15 mm (sterile, Nuclon)</td><td>Fisher Scientific GmbH</td><td>10508921/ NUNC150350</td><td></td></tr><tr><td>1 mL Syringe (without dead volume) - Injekt-F SOLO</td><td>Braun/neoLab</td><td>194291661</td><td></td></tr><tr><td>30G injection needle</td><td>BECTON DICKINSON</td><td>304000</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Analgesia / anesthesia&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sevoflurane (Sevoflurane AbbVie)</td><td>AbbVie Germany GmbH &amp;amp; Co. KG</td><td>-</td><td></td></tr><tr><td>Medical oxygen</td><td>Air Liquide Medical GmbH</td><td>-</td><td></td></tr><tr><td>Buprenorphine (Temgesic)</td><td>Indivior Eu Ltd.</td><td>-</td><td></td></tr><tr><td>Bepanthen - ophthalmic ointment</td><td>Bayer Vital GmbH</td><td>10047757</td><td></td></tr><tr><td>Table Top Research Anesthesia Machine x/O2 Flush w/ Sevoflurane Vaporizer</td><td>Parkland Scientific</td><td>V3000PS/PK</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Surgical Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cellulose swabs</td><td>Lohmann &amp;amp; Rauscher Deutschland</td><td>13356</td><td></td></tr><tr><td>Insulin syringe EMG 1 mL (with 30G cannula)</td><td>B. Braun Melsungen AG</td><td>9161627S</td><td></td></tr><tr><td>Fine Bore Tubing (bore: 0.28 mm/ diameter: 0.61mm)</td><td>Smiths Medical Deutschland</td><td>800/100/100</td><td></td></tr><tr><td>Micro-Adson Forceps</td><td>Fine Science Tools</td><td>11018-12</td><td></td></tr><tr><td>Iris Scissor - ToughCut</td><td>Fine Science Tools</td><td>14058-11</td><td></td></tr><tr><td>Olsen-Hegar Needle Holder</td><td>Fine Science Tools</td><td>12002-12</td><td></td></tr><tr><td>AutoClip Kit</td><td>Fine Science Tools</td><td>12020-00</td><td></td></tr><tr><td>PDS Z1012H 6/0 C1 (surgical suture)</td><td>Johnson &amp;amp; Johnson Medical GmbH</td><td>Z1012H</td><td></td></tr><tr><td>Curved Micro Serrefine Vascular Clamp</td><td>Fine Science Tools</td><td>18055-05</td><td></td></tr><tr><td>Fogarty Spring Clips</td><td>Edwards</td><td>CDSAFE 6</td><td></td></tr><tr><td>Hot Plate 062</td><td>Labotect</td><td>13854</td><td></td></tr><tr><td>Isis - Hair shaver</td><td>Aesculap - Braun</td><td>-</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Colonoscopy&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cold Light Fountain XENON 175 SCB</td><td>Karl Storz</td><td>20132101-1</td><td>Karl Storz Coloview System Mainz</td></tr><tr><td>Fiber Optic Light Cable</td><td>Karl Storz</td><td>69495NL</td><td>Karl Storz Coloview System Mainz</td></tr><tr><td>TRICAM Three-Chip Camera Head</td><td>Karl Storz</td><td>20221030</td><td>Karl Storz Coloview System Mainz</td></tr><tr><td>TRICAM SLII Camera Control Unit</td><td>Karl Storz</td><td>20223011-1</td><td>Karl Storz Coloview System Mainz</td></tr><tr><td>15&quot; Flat Screen Monitor EndoVue</td><td>Karl Storz</td><td>9415NN</td><td>Karl Storz Coloview System Mainz</td></tr><tr><td>HOPKINS Straight Forward Telescope&lt;br/&gt; diameter 1.9 mm; length 10 cm&lt;br/&gt; autoclavable&lt;br/&gt; fiber optic light transmission incorporated</td><td>Karl Storz</td><td>64301AA</td><td></td></tr><tr><td>Protection and Examination Sheath</td><td>Karl Storz</td><td>61029C</td><td></td></tr></tbody></table>

a genetically engineered mouse model of sporadic colorectal cancer

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.

If performed adequately, &#62; 85% of the animals develop tumors. The mortality of the here presented surgical procedure is &#60; 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 tumo...

Watch this Scientific Journal Video about A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer at JoVE.com

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

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 ...

a-genetically-engineered-mouse-model-of-sporadic-colorectal-cancer

Research

JoVE Journal

Cancer Research

9.4K Views.  Technische Universität Dresden. 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.

Video: A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Orthotopic Mouse Model of Colorectal Cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

Biology

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

Medicine

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

Genetics

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

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 KitV558&#916;/+ 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 KitV558&#916;/+ mouse colony. Additionally, this paper details the treatment and procurement of GIST, draining mesenteric lymph node, and adjacent cecum in KitV558&#916;/+ mice, as well as sample preparation for molecular and immunologic analyses.

The goal of this manuscript is to describe the KitV558&#916;/+&#160;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 ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Modeling Prostate Cancer in Genetically-engineered Mouse Models: A CRISPR/Cas9-mediated Localized Gene Editing Technique in Mouse Anterior Prostate Lobe Cells

Source: Riedel, M. et al. <a href="https://www.jove.com/video/57525" target="_blank">Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration.</a> J. Vis. Exp. (2018)
This video describes a method for editing specific genes in prostate gland cells using an adenovirus-based delivery system. The approach allows for orthotopic and localized alteration of gene expression utilizing CRISPR technology to develop novel mouse models for prostate cancer.

Source: Riedel, M. et al. Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration. J. Vis. Exp. (2018)
This video describes a method ...

JoVE Encyclopedia of Experiments

Prostate Cancer

Animal model studies

Segmental Adeno-Cre Infection: A Technique to Generate Isolated Colorectal Cancer using Genetically Engineered Mouse Models

Source: Betzler, A. M., et al. <a href="https://www.jove.com/v/55952"> A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer.</a>&#160;J. Vis. Exp. (2017).
This video describes the generation of a colorectal mouse model by segmental adeno-cre infection. The infected colon segment leads to tumorigenesis within this segment of the mucosa, resulting in adenomas ultimately progressing to invasive and metastatic carcinoma. This model is an attractive platform for studies of cancer biology and preclinical therapeutic trials.

Source: Betzler, A. M., et al.  A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer.&#160;J. Vis. Exp. (2017).
This video describes the ...

Colorectal Cancer

Spontaneous Metastasis Mouse Models: A Platform to Study the Metastatic Potential of Orthotopically Injected Colorectal Cancer Cells

Source: Okazawa, Y., et al. <a href="https://www.jove.com/v/57374"> High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor.</a>&#160;J. Vis. Exp.&#160;(2018).
This video describes the protocol to establish a spontaneous metastasis mouse model of colorectal cancer patient-derived xenografts or PDXs. These xenograft models partially mimic the features of human carcinomas and understand the critical aspects of human malignancies, including invasion and metastasis.&#160;

Source: Okazawa, Y., et al.  High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a ...

Generating PDOX Mouse Model of Colorectal Cancer: Studying Progression of Colorectal Cancer in a Murine Model

Source: &#160;Moret, R. et al. <a href="https://www.jove.com/v/59223"> Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis.</a> J. Vis. Exp. (2019).
This video describes the protocol for generating a patient-derived orthotopic xenograft colorectal cancer mouse model to study the progression of colorectal cancer cells in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. This murine model helps in understanding primary tumor growth and metastases pattern.

Source: &#160;Moret, R. et al.  Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and ...