Role of CTC in Cancer Metastasis and Relapse:  Circulating tumor cells (CTCs) are tumor cells released from the primary tumor mass. Following circulating in  blood, some of CTCs are apoptotic, and some of those survived CTCs may reach a target organ and attach to the endothelial cell. After invading the endothelium, a secondary tumor mass  will develop. The similar process is shared by cancer relapse.

Proposed Clinical and Scientific Significance of CTC
Cytelligen has developed an unique integrated platform of subtraction enrichment (SE) and immunostaining-FISH (iFISH) to efficiently detect circulating rare cells, including CTC shed from various epithelial solid tumors, circulating endothelial cell (CEC), and stem cell, etc.

Diagram of Cytelligen Integrated Platform to Isolate, Detect and Analyze CTC

Collecting Blood
  • Patient: 7.5 ml or any volume
  • Mouse: 50-200 μl
Additional Analyses
  • Plasma: protein and nucleic acid assay
  • WBCs: protein and nucleic acid assay as well as functional analyses
Subtraction Enrichment (SE) or Ultraclean SE to Isolate Circulating Rare Cell
  • Non-hemolytic removal of RBC and immunomagnetic removal of WBC
  • Non-hematopoietic cell separation matrix for separation of tumor cell by centrifugation
  • No damage to enriched CTC
  • Less than 1.5 hrs for sample processing
Primary Tumor Cell Culture of Enriched CTC
  • Monoclonal or polyclonal primary CTC cell culture
  • Single cell analysis
  • Establishment of monoclonal or polyclonal CTC cell lines
  • Establishment of CTC Biobank
Establishment of CTC Derived Xenograft (CDX)
  • Building monoclonal or polyclonal CDX tumor mouse models from
    cultured different subtypes of CTC
Identification and Analysis of Enriched CTC
Enriched single or pooled CTCs are suitable for protein expression, gene mutation and morphological analyses, etc.
Non-fixed CTC in Solution for Immunofluorescence Staining
  • Less than 2 hrs for entire procedure
  • Brighter fluorescence signal
  • Suitable for enumeration of immunostained CTC
  • Minimal interference for subsequent analysis of CTC
  • Collecting single non-fixed CTC from solution using an electronic vacuum micro-cell manipulator for single cell genomic or proteomic analyses
CTC Fixed on Slide for Immunofluorescence Staining or  i•FISH
  • Special cell fixative; special coated and formatted CTC slide
  • Ideal for long term storage
  • Suitable for enumeration of CTC and its subtype identified by iFISH
to Identify Non-hematopoietic CTC
  • Examination of non-diploid chromosome in tumor cells by FISH
  • Integrated immunostaining of WBC, CK, EpCAM, HER2, Vimentin, CD133 and endothelial cell marker, etc on the same sample
  • Less than 3.5 hrs for FISH and immunostaining
Acquiring Image
  • Collecting images manually or by Metafer - i•FISH automated scanning microscopy
Image analysis
  • Analyzing intact or necrotic CTC and its subtypes – CK, EpCAM, HER2, Vimentin, CD133 and/or other proteins as well as chromosome ploidy, etc.
  • Analyzing CEC and other rare cells, etc.
Single cell analysis
  • Single CTC or its subtype identified by either iFISH or immunofluorescence staining on the CTC slide is selected by non-laser microscopic single cell manipulator (NMSCM, Cytelligen)
  • Suitable for gene mutation, genomic or proteomic analyses, etc.

Subtraction Enrichment (SE)

Expression of tumor cell surface molecule such as EpCAM on many epithelia solid tumors  is very heterogeneous or even undetectable (such as melanoma) ,  resulting  in insufficiency, limitation, and even restriction of clinical application of such EpCAM-dependent strategy to directly capture CTC. Moreover, following antibody cross-linking cell surface antigens,  CTCs captured by anti-EpCAM antibody are no longer the unstimulated native cells, rendering intracellular  instability of the isolated CTCs which may not be suitable for subsequent protein,  molecular and genomic analysis.   
We have invented a novel  non-hemolytic and target cell surface molecule (such as EpCAM) - independent subtraction enrichment (SE). SE specializes in isolation of circulating rare cells from any volume of human or mouse peripheral blood via rapidly and efficiently depleting majority of white blood cells, red blood cells, and plasma proteins. Enriched rare cells include epithelial tumor cells from all different solid tumors and stem cells, etc. Obtained native cells, neither covered with magnetic beads nor touched by anti-cell surface molecule antibody which avoids triggering intracellular signaling pathways, are suitable for various downstream morphological and functional analyses, such as immunostaining, integrated immunofluorescence staining - chromosome fluorescence in situ hybridization (iFISH), protein expression profiling, and single or multiple cell gene or genomic analysis.
               Experiment Data
Characterization of specificity of Cytelligen immunomagnetic beads:
Highly heterogeneous expression and localization of EpCAM on different types of cancer cell:
Immunofluorescent staining of EpCAM was performed on colon adenocarcinoma cells SW480, pancreatic adenocarcinoma cells PANC-1 and non-small cell lung cancer (NSCLC) adenocarcinoma cells A549. SW480 cells show high expression of EpCAM on the plasma membrane. Whereas most of PANC-1cells have very low amount of EpCAM localized in cytoplasm and vesicles, and 1out of 5 cells shows EpCAM in nucleus (green arrow). All of A549 cells, except 2 in the middle showing negative staining (white arrows), have weak nucleus staining of EpCAM. Plasma membrane localization of EpCAM is not visualized on both PANC-1 and A549 cells [Lin, Clin Transl Med 4(38):1-7 (2015)]
Flow cytometry analysis shows high, low and non-expression of EpCAM on breast, bladder and melanoma cancer cells, respectively. Recovery rate of EpCAM-dependent strategy (CellSearch) correlates with the amount of expressed EpCAM. However, subtraction enrichment (SE) maintains high recovery rate of cancer cells despite heterogeneous or non-expression of EpCAM.
Unique Features of SE
  • Cytelligen immuno-magnetic beads suitable for circulating rare cell enrichment with low non-specific binding to non-hematopoietic cells and high specific binding to hematopoietic leukocytes
  • Cytelligen non-hematopoietic cell separation matrix
  • Cytelligen fixative suitable for iFISH and immunostaining
  • Cytelligen formatted CTC coated slide
  • any volume of human  or mouse blood  sample (as small as 100 μl)

in situ Phenotyping and Karyotyping of CTC Subtypes (in situ PKCs) by  i•FISH


Majority of CTCs express intracellular epithelial protein - cytokeratins (CK), and show aneuploidy or polyploidy of chromosome(s). However, recent studies indicated that not all CTCs express CK and not all chromosomes are abnormal in tumor cells (some CTCs may have normal diploidy of certain chromosomes), suggesting that CK immunostaining or FISH alone has its inevitable limitation and is incapable of  identifying all the isolated CTCs.  

We have developed an unique iFISH CTC identification platform via integrating immunofluorescence staining and chromosome fluorescence in situ hybridization (FISH) performed with centromere probe. CTCs are distinguished from hematopoietic leukocytes by immunostaining, and identified as  non-hematopoietic abnormal cells with aneuploidy or heteroploidy of chromosome(s). To increase sensitivity or upon additional requirement, immunofluorescence staining with desired antibodies against either extracellular or intracellular (such as cytokeratin, Her2, CEA, CA19-9 etc.) target proteins in/on CTCs could be performed simultaneously. In contrast to either conventional CK immunofluorescence staining or FISH alone to identify CTCs, in situ phenotypic and karyotypic characterization of CTCs (in situ PKCs) performed by iFISH enables classifying CTCs into diversified subtypes with higher sensitivity as well as higher specificity. Each CTC subtype may have distinct clinical significance relative to drug resistance or sensitivity, tumor metastasis and recurrence, respectively.

Hematopoietic and non-hematopoietic cells are distinguished by CD45 staining. CTCs are identified from those non-hematopoietic cells with abnormal number of chromosomes identified by FISH performed with chromosome centromere probe (CEP) and/or expression of cytokeratins  or other tumor markers such as Her2, CEA and CA19-9, etc.

Fully Automated and High Throughput Metafer - i•FISH

 Scanning System

Cytelligen, Carl Zeiss and MetaSystems (Germany) have successfully co-developed a novel automated imaging system (Metafer-iFISH



specializing in scanning and analyzing CTCs identified by i•FISH



Unique Features of Automated Metafer - i•FISH
 CTC Scanning and Image Analysis System
  • High Throughput - Automated slide loading system ensures 24/7 non-stop scanning
  • Full Automation - Automatically save all the raw data and digital coordinates of every acquired CTC image
  • No Human Error Interference – Standardized systematic CTC identification criteria eliminates inevitable human errors introduced by individual researchers
  • Comprehensive Imaging and Statistical Analysis - The system is able to classify acquired CTCs into diverse categories of both single cell (large and small cells, respectively) and clusters of CTCs; to quantify mono-, dual- and triple-tumor marker protein co-expression as well as chromosome aneuploidy in CTCs; and to statistically analyze all the obtained results
  • High Efficiency – Non-stop scanning, coordinates recording, imaging analysis and automatic CTC detection report could be finished in real-time

Clinical Utility of CTC Subtypes

Comparative analysis of CTC subtypes detected in either single or classified cancer patients or tumor animal models before and after treatment will find out how the specific CTC subtype correlates to the clinical endpoints, such as prognosis, metastasis, drug sensitivity or resistance and cancer relapse, etc. in situ Phenotyping and karyotyping of  CTC subtypes (in situ PKCs) performed by SE-iFISH will help  guide  more specific and significant genomic, proteomic and functional analyses performed on the targeted single tumor cell.

Applications of SE - i•FISH

  • suitable for both human and mouse blood samples.
  • applicable for enrichment of CTCs from all different  solid tumors including melanoma from peripheral blood, pleural effusions, ascites, bone marrow, cerebrospinal fluid and urine, etc.
  • suitable for subsequent primary tumor cell culture.
  • suitable for enrichment of stem cells, etc. and analyzing all components in blood, including plasma proteins, nucleic acids, and WBC, etc.
  • applicable for detection and characterization of different subtypes of CTC derived from various solid tumors.
  • useful for further protein, molecular and genomic analysis on single or multi-enriched tumor cells.
  • no potential interference from aneuploid tumor-associated endothelial cells (hTECs) which are depleted during the process of enrichment.
, the real solutions for circulating rare cells......
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