**5.2 Orthotopic implantation**

344 Hepatocellular Carcinoma – Basic Research

Paterlini-Brechot & Benali 2007). To isolate CTC a method using a ligand biotinylated was used. Biotinylated asialofetuin, a ligand of asialoglycoprotein receptor, was experimented and followed by magnetic separation or density gradient (Ficoll-Paque PLUS; GE Healthcare). The cells were identified by microscopy, FISH, immunofluorescence staining, flux cytometry and RT-PCR. This technique shows 81% specificity and 20 cells/5ml for the sensitivity (Xu et al., 2011). This promising approach has to be confirmed in a larger cohort

After isolation of the CTC by the different methods described above, to increase the number of CTC, the primary tumor cells can be cultured in the specific culture medium (Allard et al., 2004). The optimal conditions of culture growth and specially the culture medium leading the growth of the CTC, but not the other epithelial or non epithelial cells, have to be determined through an experiment. Some companies propose commercial kits. For example, The Cancer Cell Isolation Kit® from Panomics includes lysis buffer to increase the number of CTC. One of the main problems is that cultured cells can lose their original markers and derive. Mimicry of tumoral microenvironment *in vitro* is particularly difficult because for most tumors it is largely unknown. Another problem is that the samples containing the CTC are usually contaminated by stromal cells like fibroblasts, which create competition in the

A technique that allows the detection of only viable cells after a CD45+ cell depletion was introduced for CTC analysis from bone marrow aspirates and blood samples (Alix-Panabieres et al., 2008; Alix-Panabieres et al., 2007; Braun et al., 2005). This technique was designated EPISPOT (epithelial ImmunoSPOT). It is a protein-secreting profiling based on the secretion or active release of specific marker proteins using an adaptation of the enzymelinked immunospot technology. As immunospots are the protein fingerprint left only by the viable releasing epithelial cells, a cell culture is needed to accumulate a sufficient amount of the released marker proteins (Table 2). The dying cells do not secrete adequate amounts of protein and are not detected (Alix-Panabieres et al., 2005; Alix-Panabieres et al., 2008; Alix-Panabieres et al., 2007; Czerkinsky et al., 1983). This assay can also provide important information on the profile of secreted proteins potentially relevant for metastasis formation. However, this technique has still to be validated in large-scale clinical studies on cancer patients (Alix-Panabieres et al., 2008; Alix-Panabieres et al., 2007). After the enrichment and isolation of the CTC, the next step is to identify, characterize and finally enumerate them. The CTC can be identified by indirect or direct methods. But these important steps need

Chemically induced HCC-models are diverse and not always reproducible. The chemicals usually used are diethylnitrosamine (DEN), peroxisome proliferators, aflatoxine, carbon

of patients and still depend on the receptors expressed at the surface of the CTC.

Petri dish. After few days, only the fibroblasts are present in the flask.

**4.2.3 Epithelial immunoSPOT (EPISPOT)** 

tumor markers specific to the CTC seeked.

Over decades, different HCC mouse models have been developed.

**5. Experimental models 5.1 HCC mouse models** 

**4.2.2 Culture of CTC** 

Orthotopic implantation is a more suitable model because the cells are directly implanted in the liver tissue. Nevertheless, the procedure is challenging. There are big differences between cell lines and the choice of the markers is still limited (Heindryckx et al., 2009). Mechanisms leading to tumoral cells spreading are ill known. Currently, there are few models of orthotopic implantation of human tumoral cells (Scatton et al., 2008; Scatton et al., 2006). An experimental model of human orthotopic HCC transplantation in NOD/SCID (non-obese diabetic/several compromise immuno-deficient) mice allows to generate and to modulate CTC (Scatton et al., 2008; Scatton et al., 2006). In this mouse model, tumoral spreading is an early event during tumoral development and the number of CTC is directly correlated to the tumor size.

When injected under the liver capsule, a primary tumor develops and continuously yields circulating tumor cells. In addition, the CTC could be modulated after tumor removal. Liver tumor removal led to a very low level of tumoral cells in blood 30 days later. After complete tumor removal, the number of CTC significantly decreases but still remains detectable even at a low level. The FACS was used to detect CTC (detection of human HLA marker in mouse bloodstream). The reality of CTC was then demonstrated. An important finding is that the bone marrow could be early and permanently colonized by CTC (Scatton et al., 2006).

#### **5.3 Small imaging animal models**

With the recent development of the small imaging apparatus (example: IVIS Lumina II XR Imaging System, positron emission tomography) to study development and the progression of diseases in the live animals like rheumatism, a new area to study CTC in live animals is open. This technique was applied to study the CTC in ectopic or orthotopic HCC cell lines implantation. As we discuss above, the lack of specific HCC markers makes CTC studies very challenging. The idea is to bind luminescence tag (luciferase, yellow fluorescence or red fluorescence proteins) in the hepatoma cell lines injected in the liver that be detected by bioluminescence machine. For example, thymidine kinase-luciferase was placing under the transcriptional control of endogenous AFP promoter to develop a transgenic mouse model that injected with DEN will develop HCC (Lu X. et al., 2011). The development of the HCC was followed in the live animal by bioluminescence and PET analyses. The inconvenient of this method is that the HCC model has to express AFP. To avoid this problem hepatoma cell lines where engineered with luciferease (HCC-LM3) (Ma et al., 2011) or red luminescence

Hepatocellular Carcinoma: Methods of Circulating Tumor Cells (CTC) Measurements 347

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