**4.2.1 Magnetic separation**

342 Hepatocellular Carcinoma – Basic Research

FISH, TUNEL and molecular analysis. Molecular analysis can be performed specifically to CTC after laser microdissection. The filter can be also mounted between slide and coverslip for routine microscope observation and storage. Although promising, this method is expensive, time consuming and the filters are not easily available (Mejean et al., 2000; Paterlini-Brechot & Benali 2007; Pinzani et al., 2006). Cells should be better characterized using morphological methods that allow both detection and characterization. A second potential main advantage is that CTC could be compared to the primary tumor in order to better understand the mechanism of metastatic process. However, this approach has been rarely performed and neither firm recommendation nor conclusion could be drawn. This method has a high sensibility since one tumoral cell could be detect in 1 ml of blood. The technique also avoids damaging the tumor cell which can be diagnosed using a simple pathologist analysis. However, the pathologist should get used to this technique to avoid a misinterpretation with others types of cells. The use of ISET technology to detect and characterize CTC in HCC has been reported in one study. Vona et al., (Vona et al., 2004) reported that microemboli and isolated CTC could be detected in HCC patient. Presence of CTC was associated with a shorter survival. This work also showed the feasibility of molecular studies of individual circulating cells. Indeed, β-Catenin mutations were searched in samples of 60 single microdissected CTC. β-Catenin mutations were found in only 3 CTC that highlighted the weak impact of these mutations in the initial step of tumor cell invasion.

**The Micro-Electro-Mechanical System (MEMS**)-based is a parylene membrane microfilter device for single stage capture and electrolysis of circulating tumor cells in human blood, and the potential of this device to allow genomic analysis. After the CTC are captured in the filter, electrical lysis of cells on membrane filter is applied and the DNA as well as RNA can be extracted and analyzed by PCR or RT-PCR respectively. CTC enrichment is performed by either gradient centrifugation of CTC based on their buoyant density or magnetic separation of epithelial CTC, both of which are laborious procedures with variable efficiency, and CTC identification is typically done by trained pathologists through visual observation of stained cytokeratin-positive epithelial CTC. These processes may take hours, if not days. The Micro-Electro-Mechanical System (MEMS)-based makes the process simpler, faster and better to separate CTC (~90% recovery) from blood cells. Since enrichment will inevitably be accompanied by loss of CTC, irrespective of the exact method, some essays are performed directly in whole blood (Lu Y. et al., 2007). But the disadvantages of this technique are that morphology of the cells is lost, besides markers also and the capacity to count exactly the

The specific enrichment techniques can use specifically protein tumoral markers expressed by the CTC. These methods use antibodies against the protein tumoral markers coupled to steel beads, by applying a magnetic field the cells expressing the marker can be captured. Several immune-magnetic methods (MACS system, Deanabeads® Invitrogen, macro-iron beads, ferrofluid(colloidal iron)-based systems) to enrich the sample have been used successfully (Sergeant et al., 2008). We will describe the methods and their advantages and their disadvantages. Another approach to enrich the sample is to use the properties of CTC

Further studies are needed.

number of CTC (Table 2).

**4.2 The specific enrichment techniques** 

To use immune-magnetic detection system the first step is to deplete the whole blood of the red cells (by lysis buffers or density gradient) to obtain the PBMC. After, the magnetic particles coated and surrounded by a specific antibody are added to the PBMC supposing containing the CTC. Labeled cells are then collected by applying a magnetic force while non labeled cells are containing in the supernatant and are discerned. This use of magnetic beads to catch specifically CTC is called "positive selection" (Alix-Panabieres et al., 2008; Paterlini-Brechot & Benali 2007; Sleijfer et al., 2007).

Since a large number of leukocytes (potential source of false negative CTC) still remain trapped with the cells, some methods include a "negative selection" of leukocytes (with anti-CD 45 beads for example) followed with a "positive selection" with antibodies specific to epithelial cells (EpCAM, CK) (Allard et al., 2004; Paterlini-Brechot & Benali 2007; Smirnov et al., 2005). The problem of this procedure is that gets ride the majority of leukocytes but still hold in non-malignant epithelial cells and loses tumor cells which do not express epithelial antigens and/or are lysed during the first step (Paterlini-Brechot & Benali 2007; Zigeuner et al., 2003).

The methods using antibodies like immune-magnetic methods (MACS system, Deanabeads® Invitrogen, macro-iron beads, ferrofluid(colloidal iron)-based systems) to enrich the sample will induce false-positive extraction (Sergeant et al., 2008). For example, antibodies against cytokeratin (CK) or other epithelial-specific antigens have been reported to bind both specifically and non-specifically to macrophages, plasma cells and nucleated hematopoietic cells precursors. The non-specific binding of the antibodies involves Fc receptor-bearing leukocytes and monocytes or illegitimate expression of epithelial antigens in normal hematopoietic cells. Some of these positive cells are morphologically difficult to distinguish from CTC. Variable numbers of epithelial cells have been found in peripheral blood of subject without malignancy in some physio-pathological conditions like benign epithelial proliferative diseases, inflammation, surgeries and tissue trauma (Allard et al., 2004; Becker et al., 2005; Goeminne et al., 2000; Naume et al., 2004; Naume & Espevik 1991; Paterlini-Brechot & Benali 2007). Moreover, epithelial CTC may lose epithelial markers during dissemination through the process called epithelial-to-mesenchymal transition (EMT). Since the epithelial markers that get lost during EMT may include markers used for CTC measurement, underestimation of the actual CTC number may occur, inducing de facto false negative results (Christiansen & Rajasekaran 2006; Paterlini-Brechot & Benali 2007; Sleijfer et al., 2007; Wang J. Y. et al., 2006; Willipinski-Stapelfeldt et al., 2005). In the case that the method induce false positive, the problem can be diminished avoided using a second marker or a full panel of markers and techniques (see below) to characterize the CTC, like RT-PCR, immunocytochemistry or immunefluorescence, morphology by optical microscopy (Paterlini-Brechot & Benali 2007; Schuler & Dolken 2006; Sleijfer et al., 2007). In another hand, in the case of the false negative results the doubt persist, and only strict follow-up of the patient by repeating the detection of the CTC can potentially eliminate this doubt. No available antibodies are 100% tumor or tissue-specific (El-Serag 2004; Goeminne et al., 2000;

Hepatocellular Carcinoma: Methods of Circulating Tumor Cells (CTC) Measurements 345

tetrachloride (CCl4), choline deficient diet or thiacetamide (Heindryckx et al., 2009; Weylandt et al., 2011). Transgenic mouse models were also developed, for example mice that contained HBV or HCV viruses or expressed specifically oncogenes (c-myc, c-myc + E2F1) or growth factors (TGF-, TGF- + c-myc, EGF, FGF19, GMNT, PDGF, 1-antitrypsin) (Heindryckx et al., 2009). Circulating tumor cells were not looked for in any of these animal models. One reason is the huge differences between models and the presence of specific

In order to solve these problems, researchers developed ectopic implantation that is fast and easy to perform. However, there is still many differences between the cell lines, no direct interaction with the liver tissues and difficulty to export to humans (Heindryckx et al., 2009).

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

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

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

bone marrow could be early and permanently colonized by CTC (Scatton et al., 2006).

markers for each situation.

**5.2 Orthotopic implantation** 

correlated to the tumor size.

**5.3 Small imaging animal models** 

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