Arch. Pharm. Res. (2014) 37:88–95 DOI 10.1007/s12272-013-0285-1
REVIEW
Overview of current standpoints in profiling of circulating tumor cells Kyobum Kim • Kwan Hyi Lee • Jongmin Lee Jonghoon Choi
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Received: 9 September 2013 / Accepted: 29 October 2013 / Published online: 9 November 2013 Ó The Pharmaceutical Society of Korea 2013
Abstract The goal of this review is summarizing current technologies developed as the in vitro prognostic/diagnostic systems that can rapidly separate and detect circulating tumor cells (CTCs) from cancer patient’s blood (1–10 CTCs of 1 billion red blood cells) by labeled and non-labeled method. The review is focused on three major areas of CTC research (1) Summary of previous research on capturing of CTCs, (2) New development of the in vitro prognostic diagnosis system of cancer that is capable of rapid separation of CTCs, (3) Future direction on development of new technologies for CTC profiling. Current CTC researches have helped on identifying patients who may benefit from chemotherapy before treatment, patients who may benefit from continued chemotherapy, and leading to clinical development of CTC-guided chemotherapy strategies. We analyze the feasibility of clinical application of these current CTC research for the ultimate goal of increasing the survivability of cancer patient. The biomolecular assays of viable CTCs from cancer patient may
K. Kim Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA K. H. Lee Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, South Korea J. Lee Department of Oral and Maxillofacial Surgery, Division of Dentistry Graduate School, Kyung-Hee University, Seoul, South Korea J. Choi (&) Department of BioNano Engineering, Hanyang University, ERICA Campus, Ansan, South Korea e-mail:
[email protected]
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elucidate the mechanism of metastasis and tumor initiating cells and also may have high impact on the development of personalized medicine to overcome the incurable diseases. Keywords Circulating tumor cell Nanotechnology Cell characterization
Introduction Importance and remarkable history of researches in circulating tumor cells Recently, many researchers in biomedical engineering and medical science have had interests in circulating tumor cells (CTCs). Since Cristofanilli and coworkers revealed that the number of CTCs could be utilized as an indicator of a breast cancer in 2004, CellSearchÒ device has been approved by Food and Drug Administration (FDA) and released in a market for biomedical researches. CTC contains genetic information of a patient’s cancer and the amount and/or number of CTCs indicates the progress status of the cancer. Based on one of the recent reports (Joosse and Pantel 2013), it has also been revealed that CTCs participate in cancer metastasis and stimulate the proliferation of oncocytes (or tumours cells) once they encounter primary tumors that are originated from CTC itself (Conteduca et al. 2013). As CTC counting rate in blood cells of a patient with a metastasis cancer is very low (i.e., one CTC in 10 million blood cells), a series of extensive researches to investigate effective separation and concentration of CTCs have been initiated in American and Europe about 10 years ago (Issadore et al. 2012). In medicine, CellSearchÒ (Janssen Diagnostics, https:// www.cellsearchctc.com/) is conducting a clinical trial to test
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with a prognosis. In a case of lung cancer study, it has also been reported that the number of CTCs decreases when using Tyrosine kinase inhibitor for epidermal growth factor receptors (EGFRs) and increases as T790 M mutation occurs in EGFRs with augmented resistance (Huang et al. 2013; Hiltermann et al. 2012). Currently available tools for CTC separation/isolation
Scheme 1 Schematic composition of EpCAM (Winter et al. 2003)
a feasibility of CTCs as prognostic/diagnostic marker by separation techniques using epithelial cell adhesion molecule (EpCAM) antibody of CTCs (Bao et al. 2013). In cancer biology, various studies to investigate the mechanism of cancer metastasis by using CTCs that involve in the intermediate stage between invasion, intravasation, extravasation, and metastasis (Hiltermann et al. 2012; Punnoose et al. 2012). Especially, clinical results of recent subgroup analysis demonstrated that HER2 in CTCs in a HER2-positive breast cancer is about 38 %, which is higher than one in a primary tumor. Several clinical trials are ongoing to investigate the functionality and effectiveness of Herceptin in HER2-negative cancer since it was reported that Herceptin properly works using Herceptin in HER2-negative breast cancer (Jiang et al. 2013). Likely, a clinical report shows that the number of CTCs in a colon and prostate cancer is related
The major researches in CTC separation investigate EpCAM antibody that are expressed in CTCs. EpCAM is a cell surface antigen with a universal expression in epithelial cells, but cells in blood stream do not express EpCAM since they are originated from mesenchyme. This antigen is a type I transmembrane glycoprotein with a size of 40 kDa. EpCAM is composed of two epidermal growth factor-like extracellular domains (Scheme 1), a cysteine poor region, a transmembrane domain, and a short intracellular cytoplasmic tail (Litvinov et al. 1994; Winter et al. 2003). EpCAM is overexpressed in various epithelial carcinomas and thus has a potential for diagnostic and prognostic oncogenic marker (van der Gun et al. 2010). One of the in vitro studies demonstrated that silencing EpCAM expression decreased the proliferation, migration, and invasion of breast cancer cell lines, by enhancing E-cadherin mediated cell–cell adhesion (Osta et al. 2004). The several outcomes for CTC separation technique are reported: Immunomagnetic separation such as a CellSearchÒ device, microfluidics CTC chp, and isolation by size. CellSearchÒ device is the only FDA-approved CTC diagnostic tool. This device includes steps of a pre-treatment of a sample and an analysis of the treated samples (Yu et al. 2011). In a pre-treatment, anti-EpCAM antibody is conjugated with magnetic particles, mixed with a patient’s blood sample, and separated by magnetic filed. For negative selection of CTCs, a fluorescent antibody to CD45 specific to leukocytes is also used (Mostert et al. 2009). Combination of anti-cytokeratin and anti-EpCAM specific to epithelial cells is also used for CTC enrichment (Mostert et al. 2011). However, epithelial-specific antigens can label non-tumor epithelial cells by specific labeling as well as non-tumor non-epithelial cells by non-specific labeling (Paterlini-Brechot and Benali 2007). Therefore, a presence of non-tumor epithelial cells in peripheral blood results in false positive results of actual number of tumor cells. Subsequently, a low capturing frequency of CTC in the pre-treatment step may reduce a diagnostic sensitivity. In addition, some tumorous cells do not express EpCAM or the expression level is low (Lianidou and Markou 2011). A recent study showed various levels of EpCAM expression in tumor cells (Spizzo et al. 2011). For example, MCF7 cell lines express EpCAM while the other breast cancer cell line, MDA-MB-231, does not. It might be also possible that
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majority of tumor cells in blood does not express EpCAM as it has been known that epithelial–mesenchymal transition (EMT) occurs during metastasis. In addition, a microfluidic CTC chip with anti-EpCAM antibody-coated 78,000 microposts was reported by a group in MIT-Harvard in Nature in 2007 (Nagrath et al. 2007). By using EpCAM antigen–antibody reaction, the number of CTCs attached to microposts is analyzed with immunofluorescence staining. In order to increase this interaction between EpCAM-expressing target CTCs and antibody-coated chip surface, a high-throughput mixing device is also utilized (Stott et al. 2010a). However, CTC attachment onto microposts is a technical limitation to pursue a further biological research using CTCs. Therefore, it is necessary to develop a separation technique to isolate cells without EpCAM expression. EpCAM independent label-free CTC isolation is an alternative tool to isolate/analyze a variety of CTCs in Patient’s bloodstream and improve ascertaining prognosis for patients. For the intensive cancer researches in a metastasis mechanism, highly sensitive EpCAM negative separation technique is required. ISETÒ (Isolation by Size of Epithelial/Throphoblastic Tumor Cells; Rarecells Diagnostics, http://www.rarecells. com/) is a physical separation by filtration to separate peripheral blood leukocytes and relatively larger CTCs (Vona et al. 2000). However, the separation sensitivity might not be improved significantly due to the size overlap between leukocytes and CTCs and a shear stress on filtering may influence a viability of separated cell populations (Hosokawa et al. 2013). Another approaches for CTC isolation utilize a realtime RT-PCR to qualify and quantify CTCs. Despite its analytical sensitivity and specificity, this method requires a selection of appropriate mRNA target markers. Cell disruption during the process inhibits a further separation or characterization of CTCs in biomedical researches. In addition, variation in numbers of expressed transcripts in different cells may lead to inaccurate estimation of the number of CTCs in a blood sample (PaterliniBrechot and Benali 2007; Lianidou and Markou 2011).
Current status of CTC research Label-free isolation of CTC It is important to develop a label-free CTC isolation technique and keep a viability of CTCs during label-free isolation. Label-free isolation helps a genetic and molecular analysis of CTCs in various biological researches and it is essential to develop in-line label-free isolation system for a discovery of new markers. For the improved prognosis/diagnosis for cancer patients, it is of importance to
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develop (1) a system for CTC isolation and detection using a specific label such as EpCAM and (2) high performance in-line system that is capable of simultaneous CTC counting and sorting, with a higher sensitivity than a currently reported cell-attached CTC chip. Moreover, it is necessary to use a multiplex antibody and discover a specific marker in order to increase the sensitivity and specificity of EpCAM positive CTC isolation. Incomplete separation of CTC from other cells in blood stream and low stability of isolation systems prevent the development of commercially available systems. Therefore, development and discovery of receptors is inevitable for a selective isolation of various CTCs originated from different types of cancers. Aptamer is a common capturing agent due to its smaller size (i.e., one-sixth in size compared with a general antibody to recognize high molecular weight components) and easiness in handling and modulation. For the effective discovery of aptamers of extraordinarily rare CTCs in blood, it is essential to develop a microfluidic on-chip Systemic Evolution of Ligands by Exponential Enrichment (SELEX) system (Chang et al. 2013). This microfluidic system has a significantly high area to volume ratio due to a small dimension, and this will address the limited reaction sensitivity of conventional SELEX system. For the innovative system CTC isolation and detection, (1) enhanced purity and high cell viability, (2) a discovery of new markers by a biological investigation for CTCs, ascertaining metastasis and prognosis, patient-specific clinical trials, (3) the development of diagnostic chip for feed-back loop would be required. Highly efficient CTC analysis technique Current label-free methods of detecting CTCs include a physical separation of cancer cells from blood and following immunochemical staining of targets. Cancer markers such as EpCAM enable higher capturing efficiency of marker-positive cells applied together with physical separation of CTCs. However, heterogeneous population of physically separated cells often includes normal cells such as mesenchymal stem cells that down-regulate the efficiency of isolation. Furthermore, EpCAM marker has been reported to have limitations in accurate diagnosis of patient’s pathogenesis (Stott et al. 2010b; Arya et al. 2013). Low specificity of EpCAM marker often results in false positives from normal donors that highlight the need of advanced technologies for CTC detection. Current techniques on CTC capture Moon et al., have reported a high throughput hydrodynamic separation of CTCs from blood cells by using a microfluidic device without labeling (Moon et al. 2011). Based on this
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Fig. 1 Schematic description of a multi-orifice flow fractionation (MOFF) system and particle focusing phenomena by lateral migration forces in channels
filtration technique, presence of CTCs in a blood sample of patients with a breast cancer was identified. The feasibility of this separation technique is assumed to be between basic research and commercialization. A systemic micro-fluidic device enables a systemic and continuous analysis of CTCs from pre-treatment to a final diagnosis by using physical properties of CTCs such as size, density, and deformations in fluids. This system shows advantages of a molecular level analysis for biologists and a medical application for clinicians. A multi-orifice flow fractionation (MOFF) system is a multiorifice microchannel for size-based particle separation (Fig. 1). By tubular pitch effect, particles in the channels are located in a circular shape. Simultaneously, a lateral force is applied to particles by a secondary flow and particles are focused in both sides of a channel wall when closed to the outlet. This study demonstrated that a relative size of particles and channel regulated size separation of particles in channels and that blood cells and breast cancer cells are effectively separated (Moon et al. 2011). A parallel multi-orifice flow fractionation (p-MOFF) device successfully isolate real CTCs from a blood sample of breast cancer patient. Following immunofluorescence staining identified that MCF-7 cell line expressed EpCAM (EpCAM?/DAPI?/CD45–) while MDA-MB-231 does not.
In this system, a commercial cell counter can analyze the size of flowing particles through the microfluidic channels. Simultaneously, the presence of fluorescence in the particle can be identified by a fluorescence-activated cell sorter (FACS) system. By combining microfluidic devices and FACS technique, a feasible and reliable micro-FACS system can be developed (Dong et al. 2013). Various platforms of chipbased separation technologies have been theoretically suggested and experimentally demonstrated. In addition, applying a unique virus-based fluidic array system to this combinational sorting system will lead to develop a molecular detection and diagnostic devices for human samples. Rapid field-free electroosmotic micropump (RFEP) applied on the glass microchannel surface shows rapid and accurate responses of an electric sorting system (Fig. 2). Moreover, this system prevents a direct exposure of high electric filed to analytes, which might cause undesired changes in biological analytes including cells. Based on electroosmotics, a partial capillary network was selectively coated with an opposite surface charge. Therefore, particles are not directly exposed to the electric filed but flow direction can be modulated by electroosmotics.
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92 Fig. 2 Rapid field-free electroosmotic micropump (RFEP) based sorter microchip. This system can be applied to a cell sorting due to the absence of direct electric field to particles (Joo et al. 2007)
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Fig. 3 EpCAM evaluation as a CTC capturing marker. a EpCAM expression in breast cancer cell lines grouped by molecular subtype. b Expression of EpCAM in relation to other epithelial and
mesenchymal markers in breast cancer cell lines. c EpCAM expression in different tumor types and in white blood cells (Punnoose et al. 2010)
Researches for surface marker proteins in cancer cell lines
cells and closely involved in inhibiting spontaneous apoptosis of these cells, and thus contributes pancreatic cancer progression (Takano et al. 2008). Recent clinical proteomic profiling studies reported a surface-enhanced lase desorption/ionization (SELDI) mass spectrometry analysis to identify APOC1 biomarkers using surfaceenhanced lase desorption/ionization (SELDI) mass spectrometry in colorectal cancer (Ward et al. 2006) and endometrial cancer (Takano et al. 2010). In addition, APOC1 was also identified in an early postoperative serum from breast cancer patients (Goncalves et al. 2006).
For a metastasis of original tumor mass, tumor invasion into a blood vessel must be initiated and cancer cells tends to migrate to blood vessels through EMT. Currently available EpCAM-based enrichment systems contain a drawback of possible EpCAM downregulation during EMT. Therefore, it is necessary to target other epithelial antigens to develop more reliable a sorting/diagnostic system. EpCAM expression level was analyzed from a selective series of cancer cell lines (Punnoose et al. 2010) (Fig. 3). One of the overexpressed surface marker antibodies, apolipoprotein C1 (APOC1), was selected among cell lines without EpCAM expression from a basic microarray data analysis. APOC1 is overexpressed in pancreatic cancer
Genomic profiling techniques Currently, a gene expression profiling after RNA isolation and amplification from a small number of cells (about
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104–105 cells) in a minute cytology specimen, predicts a response of anti-cancer chemotherapy. Since cancer tumor cells infiltrate into normal cells (i.e., white blood cells) in gastric cancer endoscopic biopsy sample, laser capture microdissection (LCM) is the only available technique to obtain pure cancer-derived RNA for a further quantitative genomic analysis. During a tissue processing, microdissection was performed using PixCell II (Arcturus Bioscience) to isolate gastric cancer cells (There is no information about the numbers of 104–105 in this reference.) from cryosections of endoscopic biopsy sample from patients treated with cisplatin/5-FU anti-cancer chemotherapy. Biotinylated, antisense cRNA target was obtained by Affymetrix two-cycle labeling. Principal component analysis demonstrated distinguishable genomic data, depending on phenotype (Chun et al. 2004; Kim et al. 2011). Genomic data from rare cancer cells can be acquired from either (1) RNA isolation and amplification from cytology specimens of patients or (2) microdissection from a small number of cancer cells. This new approach can be developed into an important cancer diagnostic research since current cytological genomic analysis is based only on bulk tumor tissues and lack of sensitive collection and processing methods.
Future directions In order to overcome a cancer, efforts have been given on researches in cancer prevention, early diagnosis, and effective treatment but it still requires new methodologies that can accurately predict prognosis and diagnosis of disease in a clinical practice. Imaging approaches or biopsy for this purpose are still extremely difficult to determine the presence of CTC in the blood. Therefore, new diagnostic tools such as the characterization of CTC in the test liquid biopsy are required. This may serve as a gold standard to observe a step-by-step progression of cancer and prognosis of drug resistance during treatment, essential for the conquest of cancer. Commercially available CTC counting machines (for example, CellSearchÒ from Janssen Diagnostics) still lack in accurate diagnosis of cancer progression matching to the clinical data that opens up the need in new approaches and strategies to capture and characterize CTCs. Especially, development of accurate CTC isolation and counting without antibodies, a cancer prognostic test protocol for establishing clinical applications, and new markers from the studies of metastasis would be important(Alix-Panabieres and Pantel 2013). Recovery efficiency of CTCs in the reports has not been over 50 % as in Tonner’s group that should be enhanced further (Arya et al. 2013; Nagrath et al.
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2007; Joosse and Pantel 2013). In addition to the good separation of CTCs, the survival rates should also be acquired. Integrative technologies may help complete characterization of CTCs and their capture that will result in correlation of CTCs and progression and prognosis of cancer patients in clinics. Acknowledgments This work was supported by KIST CTP Program (#2V03130) and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2008-0061891).
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