**3.1. Colon cancer cell lines**

It is worth mentioning that most of our understanding of the molecular mechanisms in‐ volved in CRC come from studies done on mouse or human cell lines that represent only a highly selected fraction of the original tumor and that may have acquired *in vitro* additional genetic abnormalities. Moreover, isolated cells grown on plastic dish flooded with growth factors appear retrospectively as a very poor model system to elucidate human CRC biolo‐ 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 maximize their utility in large scale drug discovery programs.

*3.1.4. Biomimetic cell culture models*

available to improve 2D or 3D cultures.

*3.1.5. Colon cancer stem cell models*

representation for CRC.

**3.2. Multicellular Spheroid models**

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

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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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

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.

#### *3.1.1. NCI-60 cancer cell lines collection*

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

#### *3.1.2. The Cancer Genome Project*

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