ctc are usually detected in the peripheral circulation , but we can find ctc in other body fluids like the cerebrospinal fluids or the urines . the limitations to discover the ctc in these fluids are the same than in the blood circulation .
however , it is possible to extract a relatively big amount of blood without harming the patient and much easier .
we will focus on the methods of ctc detection in the blood . as we describe above , ctc in the peripheral circulation
occur at an estimated number of one ctc per 10 peripheral blood mononuclear cell or pbmc .
because of the scarcity of the target cells , it is necessary to concentrate the sample .
since enrichment will inevitably be accompanied by some loss of ctc , irrespective of the method , some essays are performed directly in whole blood .
two different groups of techniques can be used to enrich samples , the nonspecific and the specific enrichment techniques .
the nonspecific enrichment techniques use physicochemical ctc properties ( size , density , etc . ) .
advantages of the nonspecific and specific enrichment techniques are summarized in the table 2 and described in the following paragraphs .
the tumor cells , epithelial cells , platelets , and low - density leukocytes from leukocytes and erythrocytes can be separated by the propriety of their particular density ( table 2 ) .
density gradient centrifugation is the preferred method to purify cells , subcellular organelles , and macromolecules .
density gradients can be generated by placing layer after layer of gradient media such as sucrose in a tube with the heaviest layer at the bottom and the lightest at the top in either a discontinuous or continuous mode .
the cell fraction to be separated is placed on top of the layer and centrifuged .
density gradient separation can be classified into two categories : ( 1 ) rate - zonal ( size ) separation .
rate - zonal separation takes advantage of particle size and mass instead of particle density for sedimentation . in isopycnic separation ,
a particle of a particular density will sink during centrifugation until a position is reached where the density of the surrounding solution is exactly the same as the density of the particle . once this quasi - equilibrium is reached , the length of centrifugation does not have any influence on the migration of the particle . a common example for this method
a variety of gradient media can be used for isopycnic separations [ 157161 ] . in the context of the ctc enrichment by centrifugation
the cells that have a density higher than the density of the buffer will stay in the bottom of the tube .
if the density of the cells is lower than the buffer , they will remain on the top of the liquid , forming a ring . on the contrary , if the density of the cells is the same than the buffer , the cells will form a ring in the middle of the tube .
a well - known example of the method is the commercial buffer ficoll tube or ficoll - paque plus ( ge healthcare bioscience , amersham biosciences ab ) or lymphoprep ( nicomed ) to separate the red blood cells from the other cells including ctc ( table 2 ) .
oncoquick ( greiner ) method uses a specific buffer able to isolate the ctc [ 47 , 139 , 140 ] .
these methods are usually fast but expensive and found in a context of clinical laboratory used in routine diagnosis ( table 2 ) .
alternative and cheaper methods can be used by preparing in the laboratory the gradient / density buffers .
the same tube can contain one , two or three gradient buffers to increase the specificity of the separation between the different cells present in the blood [ 27 , 47 , 162 , 163 ] .
it is possible to expose the samples to buffer(s ) that can be hypo - or hyperosmotic to any cell different to the target cells .
after the lysis step the mix is centrifuged and the pellets will contain the ctc .
after lysis , the next step is the extraction of dna or rna ( e.g. , red lysis buffer from qiagen or panomics ) or the extracted cells can be purified by immunomagnetic beads enrichment [ 147149 ] .
however , methods using lysis buffer can induce the death of a lot of cells including the ctc and it is not appropriate if the sample contains few ctc leading to false negative results .
the cytocentrifugation was designed for hypocellular fluids ; it spins at lower speeds and has more gradual acceleration and deceleration than normal centrifuges
cytocentrifugation could be used in research purposes and is also widely used in the routine surgical pathology practice .
this method is fast and affordable [ 65 , 144 , 145 , 164 ] . methods to identify ctc can after be used ( see below ) . as it occurs with magnetic beads , cytospin increases mortality of the target cells ( table 2 ) . because enrichment by cytocentrifugation is a critical step ,
addition of 10% buffered formaldehyde solution added to the blood sample can preserve morphology of the cells and will certainly preserve nucleic acids integrity , but the disadvantage of this method is that formaldehyde kills the cells ( table 2 ) .
liquid - based cytology ( lbc ) using a filtration process and computer assisted thin layer deposition of cells has been developed as a replacement for cytocentrifugation and/or smearing , owing to its improved cell recovery capabilities and better cell preservation . in
one is that it produces a thin layer of cells which is easier to evaluate than a thick smear .
in addition , the entire cell sample is captured in the fixative vial which leads to a more representative smear being prepared .
one of the most important advantages of this test is that the material that is left over after a smear has been prepared can be used for adjunctive testing .
computer software  reads  the smears and registers the coordinates of the fields with what it regards as the most abnormal cells .
on review , the system directs the cytotechnologists to these fields where they are evaluated .
there are also some disadvantages , which include increased manpower needed to prepare the smears and the dependence of smear preparation on the instrument .
the red blood cells need to be eliminated ( see table 2 ) and after the sample can be processed by this technique ( table 2 ) . however , optimization of cell capture and fixation can be achieved by methods other than cytyc thinprep lbc , particularly while using meticulous modern cytocentrifugation methods in the study of hypocellular fluids [ 166 , 167 ] . in their study ,
conclude that cytyc thinprep lbc and modern cytocentrifugation techniques are appropriate methods for cytology - based molecular studies . from an economical point of view (
standard cytocentrifugation are around $ 538 compared to cytyc thinpreph $ 1,278 ) , and taking into account the value of a meticulous technique , cytocentrifugation with disposable sample chambers remains the quality standard for current treatment of urinary samples for example
. a nonspecific method of enrichment using filters can captured the cells with a certain size .
the cells captured on the filter can after be transferred and analyzed on a slide . in this case
the samples can come from blood or body fluids ( urines , cerebrospinal fluid , and ascites ) .
we will describe tow kinds of methods using this technology and usually used to isolate ctc ( table 2 ) : the isolation by size of epithelial tumor cells ( iset ) method and the micro - electro - mechanical system ( mems ) .
( iset ) method ( metagenex , paris , france , http://www.metagenex.fr/ ) separates cells by size with a filter .
cells larger than 10  m , including tumor cells from carcinomas , are enriched from leukocytes ( erythrocytes are lysed , see above ) on a filter .
enriched cells are stained on the filter and ctc are precisely counted after cytopathological evaluation .
the cells on the filter can be also studied by immunolabelling , fish , tunel , and molecular analysis .
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 [ 47 , 68 , 141 ] .
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 .
the technique also avoids damaging the tumor cell which can be diagnosed using a simple pathologist analysis .
however , the pathologist should get use 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 . reported that microemboli and isolated ctc could be detected in hcc patient .
-catenin mutations were found in only 3 ctc that highlighted the weak impact of these mutations in the initial step of tumor cell invasion .
it 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 .
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 .
but the disadvantages of this technique are that morphology of the cells is lost , besides markers also and the capacity to count exactly the number of ctc ( table 2 ) .
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 , macroiron beads , ferrofluid(colloidal iron- ) based systems ) to enrich the sample have been used successfully . another approach to enrich the sample is to use the properties of ctc to grow - up in a specific culture cell medium . a method ( epispot ) that combines the capacity of ctc to secret specific markers and
grow - up in specific cell medium was developed ( see table 2 ) . 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 nonlabeled cells are containing in the supernatant and are discerned .
this use of magnetic beads to catch specifically ctc is called  positive selection  [ 40 , 47 , 49 ] . 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 ) [ 47 , 169 , 170 ] .
the problem of this procedure is that gets ride the majority of leukocytes but still hold in nonmalignant epithelial cells and loses tumor cells which whish do not express epithelial antigens and/or are lysed during the first step [ 47 , 171 ] .
the methods using antibodies like immune - magnetic methods ( macs system , deanabeads invitrogen , macroiron beads , ferrofluid(colloidal iron- ) based systems ) to enrich the sample will induce false - positive extraction . for example , antibodies against cytokeratin ( ck ) or other epithelial - specific antigens have been reported to bind both specifically and nonspecifically to macrophages , plasma cells and nucleated hematopoietic cells precursors .
the nonspecific binding of the antibodies involves fc receptor - bearing leukocytes and monocytes or illegitimate expression of epithelial antigens in normal hematopoietic cells .
variable numbers of epithelial cells have been found in peripheral blood of subject without malignancy in some physiopathological conditions like benign epithelial proliferative diseases , inflammation , surgeries , and tissue trauma [ 47 , 65 , 169 , 172174 ] .
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 [ 46 , 47 , 49 , 175 , 176 ] . 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 [ 37 , 47 , 49 ] . in another hand , in the case of the false negative results the doubt persist , and only strict followup of the patient by repeating the detection of the ctc can potentially eliminate this doubt .
no available antibodies are 100% tumor or tissue specific [ 13 , 47 , 172 ] . to isolate ctc a method using a ligand biotinylated
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 promising approach has to be confirmed in a larger cohort of patients and still depends on the receptors expressed at the surface of the ctc .
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 .
the good conditions of culture growth and specially the culture medium leading the growth of the ctc , but not the other epithelial or nonepithelial cells , has to be determined through an experiment .
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 petri dish .
after few days , only the fibroblasts are present in the flask . 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 [ 40 , 43 , 151 ] .
it is a protein - secreting profiling based on the secretion or active release of specific marker proteins using an adaptation of the enzyme - linked 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 [ 40 , 150152 ] .
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 [ 40 , 151 ] .
after the enrichment and isolation of the ctc , the next step is to identify , characterize , and finally enumerate them .
the chemicals usually used are diethylnitrosamine ( den ) , peroxisome proliferators , aflatoxine , carbon tetrachloride ( ccl4 ) , choline deficient diet or thiacetamide [ 178 , 179 ] .
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 ) . circulating tumor cells
one reason is the huge differences between models and the presence of specific markers for each situation . 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 .
orthotopic implantation is a more suitable model because the cells are directly implanted in the liver tissue .
there are big differences between cell lines and the choice of the markers is still limited .
currently , there are few models of orthotopic implantation of human tumoral cells [ 180 , 181 ] . an experimental model of human orthotopic hcc transplantation in nod / scid ( nonobese diabetic / several compromise immunodeficient ) mice allows to generate and to modulate ctc [ 180 , 181 ] . 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 .
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 ) .
an important finding is that the bone marrow could be early and permanently colonized by ctc . 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 ,
this technique was applied to study the ctc in ectopic or orthotopic hcc cell lines implantation .
as we discuss below , 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 .
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 luciferase ( hcc - lm3 )   or red luminescence protein
this approach to study ctc in the context of hcc is very promising , but the major problem is the sensitivity of the bioluminescence machine .
this approach has not yet studied in the context of ctc in the blood or in organs other than liver . in vivo flow cytometry method to detect ctc from hcc has been developed .
this method combined the flow cytometry technology that can detect specific fluorochromes based on their wavelengths , the specificity of these fluorochromes attached to an antibody that detects the ctc or fluorescently labelled cells as described in li et al .
briefly , fluorescence signal from a given circulating cell population is recorded as the cells pass through the slit of light .
confocal detection of the excited fluorescence enables continuous monitoring of labeled cells in the upper layers of scattering tissue , such as the skin of a mouse ear .
based on algorithm and data analysis the computer is able to estimate the number of ctc in the blood stream .
this method is still used in animal models to detect ctc from hcc but as promising future to detect ctc in human patients .
the second problem is that few cells are present in the bloodstream . to overcome these problems ,
few years ago new approaches have been developed such as the techniques to study membrane proteins by mass spectrometry and the development of fluorescent hepatoma cells .
however , it is undeniable that early detection of tumors and metastasis is urgently needed in medicine and these new exciting techniques and findings are changing our point of view of carcinogenesis very fast . in the future
, ctc detection will certainly be an important diagnostic tool in cancer patients , providing a new and accurate assessment of lesion staging .
 liver cancer is the fifth most common cancer in men and the seventh in women . during the past 20 years 
 , the incidence of hcc has tripled while the 5-year survival rate has remained below 12% . 
 the presence of circulating tumor cells ( ctc ) reflects the aggressiveness nature of a tumor . 
 many attempts have been made to develop assays that reliably detect and enumerate the ctc during the development of the hcc . in this case , the challenges are ( 1 ) there are few markers specific to the hcc ( tumor cells versus nontumor cells ) and ( 2 ) they can be used to quantify the number of ctc in the bloodstream . 
 another technical challenge consists of finding few ctc mixed with million leukocytes and billion erythrocytes . 
 ctc detection and identification can be used to estimate prognosis and may serve as an early marker to assess antitumor activity of treatment . 
 ctc can also be used to predict progression - free survival and overall survival . 
 ctc are an interesting source of biological information in order to understand dissemination , drug resistance , and treatment - induced cell death . 
 our aim is to review and analyze the different new methods existing to detect , enumerate , and characterize the ctc in the peripheral circulation of patients with hcc . 
5. Enrichment of the Sample
6. The Nonspecific Enrichment Techniques
7. The Specific Enrichment Techniques
8. Experimental Models
9. Conclusion