**1. Introduction**

With estimated 700,000 deaths each year, colorectal carcinoma (CRC) continues to be the fourth leading cause of cancer-related deaths in both sexes worldwide [1]. The 5-year relative survival rate for stage IV metastatic CRC is about 11%, while in stage I this number rises to nearly 90%. These figures reflect the fact that despite the high incidence and mortality rate of CRC, its mortality is among the most avertable ones. In addition, the fact that liver metastasis is the cause of most deaths from CRC [2], underlines the significance of (early) metastasis prevention. In other words, it is of great importance to develop new approaches for more accurate and early diagnosis of primary CRC on one hand and of its metastasis on the other; including screening programs as well as genetic, molecular and biomarker tests.

Colorectal cancer progression is driven by increasing or recurring growth of the primary carcinoma as well as hematogenic and lymphatic spread. For hematogenic spread, the liver is most important as it constitutes the first vascular bed in which disseminating CRC cells can be trapped after their dissemination. Hence, this organ is affected in up to 10–20% of CRC patients at the time of presentation. Another 20–25% will develop overt liver metastasis during the course of their illness [3, 4].

The main purpose of our experimental studies was first to develop a suitable model for investigating the efficacy of novel drugs [5–7]. One of the few wellcharacterized animal models for hepatic CRC benefits from the rat CC531 cell line. After injecting the cells, liver metastases develop and their growth has been frequently used for studying effects of various anti-cancer treatments [8–10].

The second aim was to identify genes, which are instrumental in the survival and metastasis formation of disseminated CRC cells. In addition, we reasoned that there are genes, which are necessary for the primary tumor as well as those, which are essential for metastasis initiation and formation. We furthermore hypothesized that the latter genes would be modulated in expression during the cells' colonization of the liver. Consequently, temporal changes in gene expression of CRC cells homing into the liver were investigated using an *in vivo* rat model, which is characterized by a definite metastatic proliferation-onset in rat liver after intra-portal inoculation of CC531 rat colorectal cancer cells. This model relies on the successful reisolation of CC531 cells at various time intervals after their injection into the mesenteric vein of syngeneic rats and allows exploring the chronological modulation of gene expression, from the very beginning of cancer cell homing into the liver to their final colonization of the whole organ. Based on this procedure, a cDNA microarray was performed to analyze gene expression profiles of several thousand genes in the reisolated CC531 cells. Upon analysis of microarray's data, candidates from gene families being significantly up- or down-regulated were chosen for further study by using different *in vitro* models. These candidate genes included claudins and insulin like-growth factor binding proteins. It was hoped that the emerging genes or their products would be useful as target of a specific therapy or as a biomarker.

The National Institutes of Health defined a biomarker as "a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention [11]. From a therapeutic point of view, genome variations are recognized as the main cause of variable response to and side effects of drugs and a "one size fits all" approach is not the best solution any more. The individual's genetic and molecular makeup will be devoted to improve and develop more specific and "personal" diagnostic and therapeutic approaches.

Claudins (CLDNs) are tight junction (TJ) proteins that serve an intercellular adhesion function. The aberrant expression of individual claudins is well documented in different stages of various human cancers [12]. In addition, some claudins were proven to be useful as biomarkers [13, 14].

The insulin-like growth factor (IGF) axis attracts increasing attention since it is involved in several stages of cancer [15–17], and for its vital role in regulating cell survival and growth [18, 19] as well as the possible use of constituents of this axis as tumor and/or metastasis markers, which is becoming the focus of increasing research activities.

The insulin-like growth factors IGF-I and –II orchestrate their roles through the interaction with other members in this system, namely their receptors IGF-IR and -IIR, their binding proteins (IGFBPs) and the IGFBP proteases including matrix metalloproteinases (MMPs), cathepsins, and kallikreins [20]. Type I receptor mediates the growth promoting effects of IGFs [21], which are further modulated by 6 binding

**21**

*a*

*b*

**Table 1.**

*CC531 cells.*

*Experimental Results Help Shape the Development of Personalized Medicine...*

**2.1 Modulation of selected genes in reisolated CC531 tumor cells**

low affinity [23, 24], also known as IGFBP-related proteins (IGFBP-rp-1-4).

in addition to these IGF-dependent actions, IGFBPs were found to exert IGFindependent effects, as was reported for IGFBP1 [25], IGFBP3 [26–28], IGFBP5

In this report, we have used the technique of cancer cell reisolation from rat liver, which permitted to monitor for the first time the expression profile of numerous candidate genes in a time-dependent manner. Based on these results we summarize our knowledge on claudins and IGFBP members and delineate their potential as

For identifying genes, which enable tumor cells to metastasize and colonize the liver, the CC531 cells were reisolated from rats, which had been implanted intraportally with these tumor cells. After various periods following tumor cell implantation, the CC531 cells were reisolated with a specific technique [33]. In subsequent experiments, these cells were used for mRNA and protein isolation and the mRNA screened by cDNA microarray and RT-PCR, and the proteins by Western blot. As shown in **Table 1**, the microarray analysis revealed a significantly increased expression of insulin like growth factor binding proteins (*Igfbp3* and *Igfbp7)* and significantly decreased expression levels of claudins (*Cldn1* and *Cldn4*) in the beginning of liver colonization (days 3 and 6 after tumor implantation). These results were further confirmed by RT-PCR (for all four genes) and Western blot (for

To investigate the knockdown effect(s) of each gene on various functions of colorectal cancer cell lines, siRNA experiments for transient knockdown were

*Igfbp3* 6.88b 13.62 6 18.03 17.29 1.56 *0.9 Igfbp7* 90.02 101.57 38.62 49.13 42.03 19.46 1.47 *Cldn1* 0.16 0.12 0.31 0.63 0.55 1.29 1.53 *Cldn4* 0.15 0.09 0.87 1.11 1.37 1.47 1.11

*Gene expression profiles from members of two gene families, chosen from the microarray analysis of reisolated* 

**3 6 9 14 21 14 (***in vitro)* **22** *(in vitro)*

proteins (IGFBPs 1–6) with high affinity for IGFs [22] as well as at least 4 IGFBPs with

Based on the observation that the increased expression of IGFBPs attenuates the proliferative and anti-apoptotic effects of IGFs, they have been long considered as tumor suppressors, mostly due to their IGF-dependent roles. Interestingly, however,

*DOI: http://dx.doi.org/10.5772/intechopen.80752*

[29] and IGFBP7 (or IGFBP-rp1) [30–32].

tumor and/or metastasis markers.

the two claudins and igfbp7) (**Figure 1**).

**Gene Time point of cell reisolation (days)a**

*The day of tumor cell implantation was counted day 0.*

*The number denotes the fold change in expression versus an in vitro control.*

**2.2 Effects of genes' knockdown in colorectal cancer cells**

**2. Results**

performed.

*Experimental Results Help Shape the Development of Personalized Medicine... DOI: http://dx.doi.org/10.5772/intechopen.80752*

proteins (IGFBPs 1–6) with high affinity for IGFs [22] as well as at least 4 IGFBPs with low affinity [23, 24], also known as IGFBP-related proteins (IGFBP-rp-1-4).

Based on the observation that the increased expression of IGFBPs attenuates the proliferative and anti-apoptotic effects of IGFs, they have been long considered as tumor suppressors, mostly due to their IGF-dependent roles. Interestingly, however, in addition to these IGF-dependent actions, IGFBPs were found to exert IGFindependent effects, as was reported for IGFBP1 [25], IGFBP3 [26–28], IGFBP5 [29] and IGFBP7 (or IGFBP-rp1) [30–32].

In this report, we have used the technique of cancer cell reisolation from rat liver, which permitted to monitor for the first time the expression profile of numerous candidate genes in a time-dependent manner. Based on these results we summarize our knowledge on claudins and IGFBP members and delineate their potential as tumor and/or metastasis markers.
