*3.3.3. Genetically Engineered Mouse models (GEMs)*

The main purpose for developing genetically engineered mice is to model the human dis‐ ease in order to first better understand the biological processes underlying normal and ma‐ lignant cell physiology, and second to establish a reliable preclinical model mimicking the true biology of human cancer and useful for drug discovery. In an attempt to accurately model the phenotype observed in FAP or sporadic CRC patients, a constellation of Apc ge‐ netically engineered mice, all based on the loss of the wild-type Apc allele, have been creat‐ ed (see [68] for review). To date, GEMs have been extensively used to demonstrate the function of candidate genes in CRC tumorigenesis, and the fact that tumors occur and devel‐ op naturally in the host constitutes undeniably an advantage of transgenic models compare to xenograft models.

The main disadvantage, except the time and the cost required to generate and maintain such animals, lies in the fact that none of these Apc mouse models consistently display metasta‐ sis, while treating metastasis is the current challenge.

### **3.4. Xenograft Models**

Consequently, spheroids strategy has been used for the screening of new anticancer agents,

Standardized spherical microtissue production in a 96 or 384-well hanging-drop multiwell plate format on robotic platform has been successfully achieved by 3D Biomatrix and In‐ sphero AG. Formation of standardized spheroids rely on the use of A431.H9, a human epi‐ thelial carcinoma cells, [62] or the colon cancer cell line HCT116 [63]. Interestingly, loss of cancer drug activity in HCT-116 cells during spheroid formation in a 3D spheroid cell cul‐ ture system has been reported [64]. Spheroid cell models also enable the study of colon can‐

Colon cancer can be induced in mouse by specific carcinogens like 1,2-dimethylhydrazine (DMH) and azoxymethane (AOM). Exposure of the mouse intestine to these chemicals trig‐ gers rapid and reproducible tumor induction which recapitulates the adenoma-carcinoma sequence that occurs in human sporadic CRCs, with the notable exception however of the invasive and metastatic stage. The application of colon carcinogenesis treatment to CRC mouse model, like the Apcmin/+ animals, results in an increased tumor incidence by up to 6 fold. Interestingly, differences in genetic mutations that arise in chemically induced colon tumor models are largely carcinogen specific. K-Ras mutations are predominant in the DMH model, while AOM treated mice exhibit tumors with activating mutations in the β-catenin gene [66]. These models could therefore be useful to assess therapies targeting specific CRC

The multiple intestinal neoplasia (Min) mouse was identified following random mutagene‐ sis with ethylnitrosourea [67]. A mutation in the Apc gene was identified as the cause of the disease, like in the human Familial Adenomatous Polyposys (FAP). However, Although Apcmin/+ mice spontaneously form a large number of benign adenomas in the small intestine, colon tumors develop in fewer than half of the animals, in contrast to human FAP patients

The main purpose for developing genetically engineered mice is to model the human dis‐ ease in order to first better understand the biological processes underlying normal and ma‐ lignant cell physiology, and second to establish a reliable preclinical model mimicking the true biology of human cancer and useful for drug discovery. In an attempt to accurately model the phenotype observed in FAP or sporadic CRC patients, a constellation of Apc ge‐ netically engineered mice, all based on the loss of the wild-type Apc allele, have been creat‐

like compounds that modulate apoptosis pathways [61].

cer chemoresistance and metastasis [65].

**3.3. Colon cancer animal models**

signaling axis.

446 Drug Discovery

*3.3.2. Apc min/+ mice*

*3.3.1. Chemically induced animal models*

which routinely develop invasive carcinomas.

*3.3.3. Genetically Engineered Mouse models (GEMs)*

The development of cancer xenograft models allows *in vivo* testing required for the predic‐ tive assessment of the clinical tolerability and efficacy of therapeutic agents. For decades, xenografts have been generated from human tumor cell lines that have been selected by *in vitro* culture.

#### *3.4.1. Subcutaneous xenografts*

As standard, tumor cells are implanted subcutaneous in the hindflank region of immunodefi‐ cient mice (e.g. Nude, NSG) to prevent rejection. Tumor growth during the treatment period is monitored either by measuring the tumor mass on the animals using Vernier calipers or by recording the activity of specific markers, like luminescent (Luciferase) or fluorescent (GFP) reporters, using non invasive imaging. At the end of the experiment, animals are euthanized and tumors are collected for histological or genetic analyses. Many applications are possible: complex growth competition assays can be performed inside a same tumor by injecting a mix of genetically modified tumor cell population, each expressing a specific reporter (Red/ Green assay). These assays allow the identification of new oncogenic targets, revealed by growth advantage, and therefore critical for tumor development [69]. Subcutaneous xenografts are useful for the study of tumor / stroma / vascular network interactions, which is not possible in cell lines. Nonetheless, this heterotypic human/mouse model has its limitations since some murine ligands are not able to activate human receptors (e.g. HGF/MET, [70]). In addition, some CRC cell lines, even if implanted subcutaneous, can produce distant metastasis to the lung or the lymphatic nodes, allowing to study the effect of therapies specifically designed against metastatic dissemination and growth (C. Mas, pers. comm.).

Here it is interesting to note that at the preclinical level, the *in vivo* antiangiogenic activity of Sunitinib (see "New anti-angiogenesis therapies" section before) was evaluated in sub-cuta‐ neous xenograft tumor models derived from HT29 and Colo205 human colon carcinoma cell lines implanted in athymic mice [71-72]. However, thereafter no advantage in anti-tumor ef‐ ficacy could be shown in Phase III trial. Although the reasons for this failure are not clearly established, the genetic heterogeneity observed in primary CRC patient tumors could ex‐ plain this lack of efficacy: *in vitro* selected cell lines are not enough representative of CRC patient's tumors. This observation suggests that new models including large tumor panels able to recapitulate the biological heterogeneity of patient's populations appear necessary for an accurate evaluation of molecular targeted agents.
