Enrichment, Isolation and Characterization of CTCs

Circulating tumor cells (CTCs) are considered to be an important factor that takes part in the dissemination of metastases in patients with cancer. Current technologies allow for the isolation of CTCs from the peripheral blood of patients, which can be subsequently analyzed at the genetic and molecular level for the detection of tumor-associated genes and/or antigens.

1. Circulating Tumor Cells (CTCs) Circulating tumor cells (CTCs) are considered to be an important factor that takes part in the dissemination of metastases in patients with cancer. Current technologies allow for the isolation of CTCs from the peripheral blood of patients, which can be subsequently analyzed at the genetic and molecular level for the detection of tumor-associated genes and/or antigens.

2. Enrichment, Isolation and Characterization of CTCs CTCs are very rare and have a short half-life in the bloodstream. Usually, between 1 to 10 CTCs can be detected per milliliter of peripheral blood of patients with metastatic stage cancer. For this reason, peripheral blood-derived CTCs fractions require an enrichment before they can be analyzed. Methods for enrichment and analysis are based on physical and/or biological properties of CTCs, such as size, deformability, density, polarity and electrical charge, epithelial cell adhesion molecule ( EpCAM ), cytokeratins (CKs) and tumor-associated markers expression.

3. CTCs Enrichment Separation based on cell size Negative enrichment by immunomagnetic beads Positive enrichment by immunomagnetic beads Magnetophoretic mobility-based separation Negative enrichment Density-dependent cell separation Microfluidic devices

4. This method for CTCs enrichment utilizes an inert polysucrose termed Ficoll, which was originally produced for the isolation of mononuclear blood cells from whole blood. The Ficoll protocol relies on differential migration properties that derive from diverse cell-type dependent buoyant densities. A flaw of the density-based cell separation method is the possible loss of some CTCs. Density-dependent cell separation

5. Separation based on cell size This type of CTCs enrichment is based on size and deformability of epithelial cancer cells. It is carried out with 10-cm silicon wafer devices, crescent-shaped isolation wells and micro-filters made of Parylene . Microdevices are engineered to enhance hydrodynamic efficiency and to facilitate the optimal retrieval of CTCs. Tumor heterogeneity exhibits significant variations in malignant cells size. This may result in loss of CTCs during the analysis.

6. A negative enrichment technique consists of the depletion of most of the leukocytes and erythrocytes by means of RosetteSep , which utilizes antibodies that target hematopoietic cells of human whole blood and crosslink them to multiple erythrocytes. The crosslink, in turn, leads to the formation of immunorosettes . Centrifugation over the Ficoll- Paque buoyant density medium allows for the precipitation of immunorosettes and unbound red blood cells, while CTCs fractions can be recovered from the medium. This protocol is limited by the possible loss of some CTCs. Negative enrichment

7. Ferromagnetic beads coated with anti-CD45 antibodies can be utilized as a further purification step of CTCs fractions derived from density gradient centrifugation procedures. Anti-CD45 immunomagnetic beads bind only blood cells, which can be subsequently removed with a magnetic field. The capture efficiency of this protocol ranges from 52% to 88%. Negative enrichment by immunomagnetic beads Positive enrichment by immunomagnetic beads This method is also known as magnetic activated cell sorting (MACS). Ferromagnetic beads coated with anti- EpCAM antibodies can be used to isolate CTCs, following a density gradient centrifugation step. EpCAM -positive CTCs can be automatically isolated with the MagSweeper , which utilizes a magnetic arm to harvest the ferromagnetic beads. VS

8. Negative enrichment cells sorting. Cells are labelled with magnetic nanoparticles targeting CD34(+) CD45(+) hematopoietic cells, which are removed with a magnetic field of the Quadruple Magnetic Cell Sorter (QMS). QMS can sort 10 million cells per second, with a 99% depletion efficiency of CD34(+) CD45(+) peripheral blood cells. This system is commercially available. This system allows only for the depletion of unwanted cells. After the enrichment step, other detection techniques must be applied for the isolation of pure CTCs populations. Magnetophoretic mobility-based separation

9. In microfluidic devices, the blood flows on so-called CTC-chips that are made of equilateral triangle structures, which contain an assortment of anti- EpCAM antibody-coated microposts or chips. Microfluidific devices exhibit an efficacy of capture rate that is comparable among cancer cell lines with different levels of EpCAM expression. T he sensitivity for the capture of cell lines with low EpCAM expression might not be sufficient for the selection of biopsy-derived CTCs that have undergone advanced EMT Microfluidic devices A more advanced microfluidic system, CTC- iChip , combines size-based separation of nucleated cells from red blood cells, platelets and plasma, and depletion of magnetically labeled white blood cells to enrich CTCs.

10. Nucleic acid-based systems Fluorescence in situ hybridization (FISH) Immunostaining-FISH, combined with subtraction enrichment (SE- iFISH ) Fluorescence assisted cell sorting (FACS) CTCs Detection

11. Quantitative RT-PCR is the preferred nucleic acid-based method for the analysis of CTCs associated markers. RT-PCR may be susceptible to provide false positives, because of sample contamination. There is also the possibility that the target gene is expressed in some normal cells. Another disadvantage of RT-PCR is that analyzed cells cannot be used for any other type of study. Nucleic acid-based systems

12. This technique utilizes fluorescent labeled DNA probes to detect specific DNA sequences within certain chromosomes. FISH analysis is very precise and can detect a variety of genetic abnormalities that are related to human illnesses. However, cells are no longer viable after FISH analysis and the experimental procedure requires highly trained personnel, is labor intensive and sometimes may not provide clear results. Fluorescence in situ hybridization (FISH)

13. Non-toxic matrix with a specific density to remove red blood cells from white blood cells and CTCs after a centrifugation. White blood cells are depleted with anti-CD45 antibodies conjugated with magnetic beads. Following SE, iFISH allows for the in situ phenotyping and karyotyping of CTCs, along with the classification of various CTCs subtypes, via detection of tumor biomarkers expression levels. Immunostaining-FISH, combined with subtraction enrichment (SE- iFISH )

14. Fluorescence assisted cell sorting (FACS) FACS relies on the use of antibodies and is commonly utilized for the separation of a specific cell type, which can be isolated with high purity from the general cell population. Forward scatter and side scatter allow for the characterization of physical properties such as cell size and internal cellular complexity, respectively. An electrostatic detection system leads to the accurate isolation of a determined cell fraction, based on the electric charge of that particular cell population. FACS is a versatile technique that has a wide range of applications, as several parameters can be simultaneously analyzed. FACS has a limited throughput, as cells are sorted individually. In some cases, flow sorting conditions may be detrimental to particular types of cells and, therefore, impede subsequent studies.

15. Fiber-optic array scanning technology (FAST) CellSearch ISET (isolation by size of epithelial tumor cells) AdnaTest Ariol system EPISPOT Simultaneous Enrichment and Detection of CTCs

16. Filtration through a membrane (8 mm diameter pores). Epithelial-derived CTCs stick to the membrane, whereas blood cells pass through. A comparative analysis revealed some discrepancies in the enumeration of CTCs between ISET and CellSearch . These discrepancies may be attributable to the heterogeneity of tumor cell size and/or to differential expression of EpCAM among cancer cells. ISET CellSearch Adna Test Magnetic separation of EpCAM -positive cells, followed by RT-PCR for the identification of tumor-associated transcripts. Produced same CTCs enumeration results as CellSearch . Cells are no longer viable after the assay, so they cannot be used for other types of analysis. RT-PCR may provide false positives, because of nucleic acid-related contaminations. A ferrofluid linked to anti- EpCAM antibodies is utilized for the initial enrichment of EpCAM (+) cells, which are isolated with a magnetic field. Isolation and detection take place in a single step. CellSearch can efficiently detect EpCAM (+) CK(+) cells. Not suitable for CTCs that do not express epithelial phenotypes and that have lost EpCAM and CKs expression because of advanced EMT. May underestimate the number of CTCs. A comparative analysis revealed some discrepancies in the enumeration of CTCs between ISET and CellSearch . These discrepancies may be attributable to the heterogeneity of tumor cell size and/or to differential expression of EpCAM among cancer cells. The latter is a limitation of CellSearch , whereas the former is a drawback of the isolation by cell size. ISET vs. CellSearch vs. Adna Test

17. A laser printing technique localizes rare subpopulations of immunofluorescent-labeled CTCs on glass substrates. Rapid detection and isolation of CTCs from large volumes of peripheral blood. The laser can excite 300,000 cells per second. This system is 500-fold faster than automated digital microscopy. FAST Detection of specific secreted proteins by enzyme-linked immuno-assays. Initial immunomagnetic depletion of CD45(+) cells, followed by immunomagnetic positive enrichment by selection of CXCR4(+) cells. This system is restricted to the analysis of CXCR4(+) cells. Not all malignant cells express CXCR4, because of tumor heterogeneity. EPISPOT Automated cell image capture for the analysis of CTCs placed on glass slides. Allows for the detection of EpCAM (+) CK(+) and EpCAM (-) CK(+) CTCs. Cells are not viable after the assay, so they cannot be used for other types of studies. Possible loss of CTCs that have undergone advanced EMT. Ariol system 1 2 3 FAST vs. EPISPOT vs. Ariol system

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