*3.1.4. Biomimetic cell culture models*

gy, especially with regard to the importance of growth signaling pathways (EGF/FGF) and tumor/stroma interactions in CRC progression. Clearly, the scientific community has taken into account these limitations, as shown by the growing interest for more complex models (e.g. 3D spheroids). However, although imperfect, colon cancer cell lines still represent a unique resource that can be extremely valuable in term of genetic manipulation and highthroughput screening, with cell viability, cell proliferation or promoter specific reporter ac‐ tivity being the usual endpoints followed. Several initiatives have been launched to

The NCI60 is a collection of 59 human cancer cell lines derived from diverse tissues, includ‐ ing colon (HT-29, COLO-205, HCT-15), which was established in the early 1990s by the Sanger institute (http://www.sanger.ac.uk/genetics/CGP/NCI). In an attempt to identify new active molecules, over 100,000 chemical compounds were pharmacologically tested in this cell line set. But disappointingly, most of the selected positive candidates were typical cyto‐ toxics, affecting cancer cells via general fundamental cellular processes, like cell cycle regu‐ lation. These cell lines are under further characterization by sequencing for mutations in known human oncogenes. Interestingly, this resource can be screened on demand for any chemical or biological agent. As an example, the NCI60 has been recently used to determine the permissivity of standard cancer cell lines to VACV infection and replication, with the

The emergence of tumor acquired resistance to pharmacological inhibitors linked to muta‐ tions in driver oncogenes has recently revived the interest for cancer cell lines. Indeed, an extensive characterization of cell lines at the genomic and genetic levels will allow determin‐ ing a genetic profile predictive of drug sensitivity. Such a signature will help to stratify pa‐ tient population and identify efficient therapeutics combination, as long as cell lines reflect real tumor biology. In this perspective, the Sanger Cancer Institute has started the genetic characterization of a panel of 800 cancer cell lines (The Cancer Genome Project, http:// www.sanger.ac.uk/genetics/CGP). Using current high throughput techniques this program intends to provide information on mutations, copy number variations, single nucleotide

Similarly, the cancer cell line encyclopedia project is a joint initiative between The Novartis Institutes for Biomedical research and scientists from the Broad Institute (http://www.broad‐ institute.org/ccle/home) to provide a detailed genetic and pharmacologic characterization of a panel of 1000 human cancer cell lines, including more than 60 CRC cell lines. Again, the ultimate purpose of this project is to establish genetic maps that would predict anticancer

polymorphisms (SNPs) and microsatellite instability of usual cancer cell lines.

maximize their utility in large scale drug discovery programs.

aim to better characterize viral oncolytic therapeutic strategies [54].

*3.1.1. NCI-60 cancer cell lines collection*

444 Drug Discovery

*3.1.2. The Cancer Genome Project*

*3.1.3. The Cancer Cell Line Encyclopedia*

drug sensitivity [55].

The derivation of a cancer cell line from the primary tumor is not an obvious process, and for many cancers, few if any cell line can be obtained. A success rate of less than 10% has been reported for the establishment of human colon cancer cell lines grew immediately *in vitro* from fresh tumors [56]. Elasticity of the surrounding microenvironment has been point‐ ed out as a critical parameter of *in vitro* cell growth. Indeed, culture plastic dishes are much more rigid than the epithelial wall of the intestine (10000 kPa vs 40 kPa). More importantly, depending on the stiffness of the substrate, cells can be differentially sensitive to drugs in term of spreading and apoptosis-induction, notably because of the expression and presenta‐ tion of surface receptors [57]. Therefore, the choice of an appropriate biomimetic substrate that will preserve the *in vivo* phenotype appears decisive not only for cell survival but also for clinical relevance. Soft polymer surface, with different degrees of stiffness reproducing the original tumor environment have been engineered (ExCellness Biotech) and are now available to improve 2D or 3D cultures.

#### *3.1.5. Colon cancer stem cell models*

Cancer stem cells (CSCs) are a discrete self-renewing tumor cell subpopulation that can dif‐ ferentiate into multiple lineages, drive tumor growth and metastasis. Moreover, CSCs are thought to be responsible for tumor recurrence after chemotherapy and radiotherapy. One of the characteristic of the CSCs is their ability to form spherical cell colonies when they are cultured in chemically defined serum-free medium at a relative low density [58]. This mod‐ el, also called colonospheres, constitutes a unique *in vitro* system to elaborate therapeutic strategies that specifically target colon CSCs, like oncolytic adenoviruses developed to target specific CSCs antigens (e.g. CD44 or LGR5). In addition, sorting of CSCs based on specific surface epitopes expression has also been used to enrich culture in tumor initiating cells in order to increase the success rate of cell line establishment and therefore improve cell line representation for CRC.

### **3.2. Multicellular Spheroid models**

Early stage development of novel anti cancer treatment requires *in vitro* methods able to de‐ liver fast, reliable and predictive results. To select the most active molecule lead in a library, pharmaceutical industry has turned its attention to High Throughput Screening (HTS) tests which mimic human tissues. Furthermore, 3-Dimensional (3D) test system has been widely accepted as being more informative and relevant than classical 2D cell systems. Combina‐ tion of HTS and 3D models such as the multicellular tumor spheroid model has been point‐ ed out having the potential to increase predictability of clinical efficacy from *in vitro* validation therefore contributing savings in both development cost and time [59]. Advantag‐ es of spheroids compared to classical 2D cell line culture have been reported [60]. Indeed, proteomic analysis of multicellular spheroids versus monolayers cultures identifies differen‐ tial protein expression relevant to tumor cell proliferation, survival, and chemoresistance. Consequently, spheroids strategy has been used for the screening of new anticancer agents, like compounds that modulate apoptosis pathways [61].

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

Colon Cancer: Current Treatments and Preclinical Models for the Discovery and Development of New Therapies

http://dx.doi.org/10.5772/53391

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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‐

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*

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

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

against metastatic dissemination and growth (C. Mas, pers. comm.).

to xenograft models.

**3.4. Xenograft Models**

*3.4.1. Subcutaneous xenografts*

*vitro* culture.

sis, while treating metastasis is the current challenge.

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‐ cer chemoresistance and metastasis [65].
