4.1. History of CTC Identification
In the past decade, various CTC capturing methods have been explored and many, which were based on polymerase chain reaction (PCR), were problematic in their sensitivity and reproducibility. One of the many CTC capturing methods developed after the PCR-based methods, includes the CellSearch System®
established by Cristofanilli et al. [1
], using automated immunostaining. This method is based on direct observation of CTCs under a fluorescent microscope and was anticipated to be a new cancer biomarker.
The CellSearch System® was approved by the US Food and Drug Administration (FDA) for the prediction of progression-free survival (PFS) and overall survival (OS) in metastatic breast cancer in 2004. Its approval was further extended to monitoring treatment effect of metastatic breast cancer in 2006, and then prediction of PFS and OS in metastatic colon and prostate cancers in 2007 and 2008, respectively. However, as will be discussed below, several challenges have been reported since then.
Here, we discuss the principles of CTC identification used in the CellSearch System® and their problems. Only several to several tens of CTCs are present in 1 mL of blood, which contains approximately 5 billion cells, which are mostly red blood cells and PBMCs. Consequently, capturing and isolation of CTCs are extremely difficult.
4.2. Regarding the Weak Point of CellSearch System®
The CellSearch System®
, which was established by Cristofanilli et al. [2
] using automated immunostaining based on direct observation of CTCs under a fluorescent microscope, was anticipated to be a new cancer biomarker. The CellSearch System®
obtained FDA approval for predicting the prognosis of patients with metastatic breast and prostate cancers. Although CellSearch®
is the only FDA-approved method, its basic principle involves identifying CTCs as epithelial cellular adhesion molecule (EpCAM)-positive cells and it is difficult to identify EpCAM-negative CTCs.
Epithelial-mesenchymal transition (EMT) is the ability of cells to migrate and penetrate other tissues, losing their shape as epithelial cells and cell adhesion function to surrounding cells when cancer cells invade or metastasize. EMT is thought to be closely involved in the most important aspects of cancer treatment. Cancer is originally characterized by epithelial cells. However, in highly malignant cancers, EMT often occurs with the loss of epithelial cell characteristics. Furthermore, although EpCAM is an antigen expressed on epithelial cell surface, its expression is known to be attenuated in cancer cells, which causes EMT, leading to the inability to identify CTCs using CellSearch®.
Additionally, EpCAM has been reported to be an epithelial cell-specific marker, which is highly expressed in breast, prostate, and colon cancers but not RCC [3
The following sections describe the principle and problems of CTC identification, and the latest methods for identifying CTCs of RCC.
4.3. Microfluidic Chip of CelSee®
As an alternative to CellSearch®, new CTC detection methods are being developed sequentially, including a method using a microfluidic chip (microfluidic device method), which is thought to have the highest detection sensitivity. CelSee® is a microfluidic device method that was awarded “The Scientist’s Annual Top 10 Innovations of 2015.” Compared with CellSearch®, which has been accredited by the US FDA, Celsee® has been reported to have a high CTC capture rate. The basic principle of CelSee® is explained as follows.
The microfluidic chip method was designed based on the principle that the deformability and diameter of CTCs and peripheral blood mononuclear cells (PBMCs), as white blood cells (WBCs), differs greatly. Consequently, CTCs can be captured by the device, whereas PBMCs can pass through because their characteristic deformation is high even when they are larger than the pore channel, whereas that of CTCs is less, precluding permeation. CelSee®
is an apparatus used for the process of concentration of CTCs [6
]. The procedure involves placing a microfluidic chip on one manifold and the blood is allowed to flow through. Cells are subsequently capture in approximately 50,000 trapping chambers of the Microfluidic Chip.
4.4. On-chip Sort®
The cell sorter is a widely used device for detecting and collecting target cells, but its use is associated with some problems in detecting and collecting rare cells such as CTCs. For example, in a typical capillary type cell sorter, it is impossible to analyze the entire sample as a “dead volume” exists in the flow path, and contamination between samples is a concern because the same flow path is used. The rare cell-sorting method using the microchip type cell sorter On-Chip Sort® (On-chip Biotechnologies, Tokyo, Japan) substantially addresses these problems. On-chip Sort® is a novel benchtop cell sorter equipped with a disposable microfluidic device, allowing the detection and isolation of rare tumor cells for subsequent molecular analyses.
The advantages of On-chip Sort®
are as follows. (1) It adopts a method of extruding the applied sample by air, making it is possible to analyze the whole sample in the chamber. (2) The flow path length is at the micrometer level and, so, the dead volume is ≤ 0.01 μL. (3) The flow path system is completely in the exchange type microchannel chip and, therefore, no contamination occurs between samples. (4) The sample flow occurs in the microchannel chip. (5) The compactness of the device allows it to be placed in a safety cabinet, and it can be sterilized using a sterilized chip. All these features make cell sorting is possible [7
As described above, when the EpCAM antigen is targeted, there are CTCs that cannot be identified and under extreme conditions, there is no antigen specific for all cancers. Since our goal is to identify renal CTCs in RCC, we decided to use a renal cancer-specific antibody that is specialized for identifying renal cancer cells. One such candidate antibody is the anti-G250 antibody.
4.5. G250 antigen
Because the expression of EpCAM antigen on RCC cell is low, other biomarkers have been explored for the detection of RCC-CTC. Although other biomarkers including p53, p21, hypoxia-inducing factor (HIF)-1α, caveolin-1 [8
], and survivin have been reported as potential prognostic biomarkers for RCC patients, they are not located on the cell membrane. Therefore, improving the efficiency of CTC capture in RCC patients by developing alternative cell surface biomarkers remains a challenge [9
]. Monoclonal antibody G250 (mAbG250) was isolated more than 25 years ago from a hybridoma produced from splenocytes of a mouse immunized with fresh human RCC cells [10
Subsequently, the cancer-associated antigen G250 antigen (MN/CA9) was reported in detail for the first time by Oosterwijk [11
Cancer-associated antigen G250 is glycoprotein present in cell membranes and nuclei and is considered a carbonic anhydrase isoenzyme. G250 antigen is expressed by virtually all ccRCC cells, but its expression in normal tissues is restricted. In addition, the most prominent known subtype of RCC is ccRCC at 70%. The mouse monoclonal antibody against human renal cell carcinoma mAb G250 specifically recognizes the ccRCC membrane antigen (G250).
G250 antigen has been shown to be expressed in 95% and 75% of primary tumors and metastatic lesions in immunohistological research, and its expression is hardly recognized in other normal tissues including the kidney. It is correct to describe the G250 antigen as a specific antigen of ccRCC [12
]. As shown in Figure 2
, RCC cells showed strong dyeability to the anti-G250 antibody, which has also been reported by other studies.
Therefore, we decided to use G250 antigen as a target in the detection of CTC instead of EpCAM antigen.
Bluemke et al. reported that the patient CTC count was an independent prognostic factor that correlated with lymph node invasion in RCC [15
]. However, CTC research in RCC has shown little progress, which is largely due to the lack of appropriate surface markers that can be used to capture antigens [16
]. Again, EpCAM is the major molecule used as the capture antigen for CTC research studies reported in the literature [17
]. EpCAM is an epithelial cell-specific marker, which is highly expressed in breast, prostate, and colon cancers but not RCC [3
]. Therefore, there is a need for another specific surface markers for capturing RCC-CTC to replace the EpCAM antigen.
One such candidate is G250 antigen. In this study, we showed, for the first time, the specificity of G250 to RCC. After CTC concentration using Celsee, we successfully discriminated between CTCs and PBMCs with high sensitivity using a combination of anti-CD45 and anti-G250 antibodies [18
The capture rate of RCC-CTC using our technology exceeded 90%. Although a small number was involved in the clinical investigation, we propose that the CTC test using patient samples indicates the appropriateness of using this test on patients with hematological distant metastasis such as that of the lung, liver, and bone. We also presented cases where this CTC test was positive and CTC disappeared in subsequent drug treatment. In such cases, this test would be very useful in determining the usefulness of the current drug therapy.
Furthermore, if the number of CTCs does not decrease after a certain drug treatment is commenced, it may suggest the drug needs to be changed. Presently, there are approximately 100 CTC identification methods worldwide. Many of them are based on capturing of EpCAM-positive cells. However as mentioned above, those methods are inaccurate. While limited only to RCC, we believe our technology is extremely accurate compared to previous techniques. Currently, clinical research using our technology is about to begin. If the clinical research proves the usefulness of our technology, we believe that it will contribute greatly to the treatment of kidney cancer without biomarkers, although there are various drug options. Because the clinical trial we conducted included a very small population, further studies with larger a number of cases are needed to determine parameters such as the cut-off value.