- Top of page
- MATERIALS and METHODS
- LITERATURE CITED
- Supporting Information
The majority of cancer-related deaths result from metastasis, which has been associated with the presence of circulating tumor cells (CTCs). It has been shown that CTC cut-off values exist that predict for poorer overall survival in metastatic breast (≥5), prostate (≥5), and colorectal (≥3) cancer based on assessment of 7.5 ml of blood. Development of the CellSearch® system (Veridex) has allowed for sensitive enumeration of CTCs. In the current study, protocols were developed and optimized for use with the CellSearch system to characterize CTCs with respect to user-defined protein markers of interest in human blood samples, including the cancer stem cell marker CD44 and the apoptosis marker M-30. Flow cytometry (FCM) experiments were initially carried out to assess expression of CD44 and M-30 on MDA-MB-468 human tumor cells. Human blood samples were then spiked with MDA-MB-468 cells and processed with the appropriate antibody (CD44/M-30) on the CellSearch. Detailed optimization of CD44 was carried out on the CellSearch using various antibody concentrations, exposure times, and cell lines with varying CD44 expression. Troubleshooting experiments were undertaken to explain observed discrepancies between FCM and CellSearch results for the M-30 marker. After extensive optimization, the best CD44/M-30 concentrations and exposure times were determined to be 1.5/3.5 μg/ml and 0.2/0.8 s, respectively. The percentage of CD44+ tumor cells was 99.5 ± 0.39% by FCM and 98.8 ± 0.51% by the CellSearch system. The percentage of M-30+ tumor cells following paclitaxel treatment was 17.6 ± 1.18% by FCM and 10.9 ± 2.41% by CellSearch. Proper optimization of the CD44 marker was achieved; however, M-30 does not appear to be a suitable marker for use in this platform. Taken together, the current study provides a detailed description of the process of user-defined protein marker development and optimization using the CellSearch, and will be an important resource for the future development of protein marker assays by users of this platform. © 2012 International Society for Advancement of Cytometry
It has been estimated that 1,596,670 new cases of cancer will be diagnosed in the United States and 571,950 individuals will die from this disease in 2011 (1). The majority of these deaths are as a result of the development of metastases (2). These deaths are mainly due to the ineffectiveness of current therapies in treating metastatic disease and a general lack of understanding of the metastatic cascade. Metastatic disease has been correlated with the presence of circulating tumor cells (CTCs) in the blood (3). Detection of very small numbers of these rare cells has been shown to be predictive of overall survival in metastatic breast (4), prostate (5), and colorectal (6) cancer, where patients with ≥5 (breast and prostate) or ≥3 (colorectal) CTCs in 7.5 ml of blood have a poorer prognosis then those with fewer or no detectable CTCs.
Several methods have been utilized to enrich and detect CTCs, including density-gradient centrifugation (7, 8), immunomagnetic selection (9, 10), polymerase chain reaction (PCR)-based assays (11, 12), and flow cytometry (FCM) techniques (13, 14). All of these approaches have unique advantages and disadvantages; however, one commonality they all share is a lack of standardization; a necessity for use in the clinical setting. The development of the U.S. Food and Drug Administration (FDA) cleared CellSearch® system by Veridex provides a standardized method for the sensitive detection and quantification of these rare CTCs in human blood using fluorescence microscopy and immunology based techniques (4–6). This system is currently considered the gold standard in CTC enumeration and is the only CTC platform approved for in vitro diagnostic (IVD) use in the clinic at the present time.
The CellSearch system consists of two components, (i) the CellTracks® AutoPrep® system, which automates the blood sample preparation, and (ii) the CellTracks Analyzer II®, which scans the prepared samples. The CellTracks AutoPrep system uses an antibody mediated, ferrofluid-based magnetic separation technique and differential staining with fluorescent particles to distinguish CTCs from contaminating leukocytes in blood samples. Initially, the system performs a positive epithelial cell adhesion molecule (EpCAM) selection for CTCs using antiEpCAM antibodies conjugated to iron nanoparticles incubated in a magnetic field. The remainder of the fluid is then aspirated, selected tumor cells are resuspended, and fluorescently labeled antibodies and nuclear stain are added. This sample is then incubated in a magnetic cartridge, called a Magnest™, and scanned using the CellTracks Analyzer II.
The CellTracks Analyzer II utilizes a 10× objective lens to scan samples using different filters, each with the exposure time optimized to the appropriate fluorescent particle. CTCs are identified by binding anti-EpCAM, anti-pan-cytokeratin (CK)-phycoerythrin (PE) (CK8, 18, and 19), and the DNA stain 4′,6-diamidino-2-phenylindole (DAPI). Leukocytes are identified by staining with anti-CD45-allophycocyanin (APC) and DAPI. After the scan is complete, a gallery of computer-defined potential tumor cells is presented, from which the user must select, via qualitative analysis, which cells are CTCs based on the differential staining discussed earlier. The CellSearch system has been primarily utilized for the detection and enumeration of these rare cells. However, this platform does allow for single-cell characterization of CTCs for user-defined markers of interest, using an additional fluorescein isothiocyanate (FITC) fluorescence channel not required for CTC identification and enumeration (15). However, the detailed process for user-defined protein marker assay development and optimization using this platform is not well-defined.
Tumor profiling of metastatic lesions is not routine practice in the clinic. In fact, this profiling is often impractical or even impossible depending on the location and size of the metastatic tumors. Therefore, CTCs could act as a real-time, minimally invasive liquid biopsy, and the characterization of these rare cells could inform clinical decision-making. For example, human epidermal growth factor receptor 2 (HER2) is over-expressed in a subset of breast cancer patients, and has been exploited as a marker for targeted therapy using the HER2 receptor interfering monoclonal-antibody Herceptin® (16). However, this therapy has only been shown to be effective in patients whose primary tumor expresses sufficient levels of HER2. Fehm et al., demonstrated that approximately one-third of breast cancer patients with metastases whose primary tumors were HER2− had HER2+ CTCs (17, 18). Whether or not these patients with HER2+ CTCs would benefit from treatment with Herceptin still requires investigation; however, CTCs hold great promise for improving personalized cancer treatment.
Two general categories of protein markers are available for exploration on CTCs; markers that reflect tumor biology (tumor phenotyping) and may act as target molecules for therapy, and those that reflect cellular response to therapy. Currently, Veridex has developed and optimized two tumor phenotyping reagents for assessing well characterized therapeutic targets, and these are commercially available for research use only (RUO) applications on the CellSearch system. Using these reagents, CTCs can be analyzed for expression of either HER2/neu or epidermal growth factor receptor (EGFR). The development and optimization of CellSearch system assays for other user-defined markers of interest on tumor cells could identify new targets for novel therapies and enable a better understanding of the mechanisms that allow these cells to escape into the circulation, extravasate into distant tissue and form clinically relevant macrometastases.
The aim of this study was therefore to develop and optimize protocols for characterization of CTCs on the CellSearch platform for two proteins of interest, CD44 and M-30. CD44 has been associated with metastasis and has shown to be expressed by “cancer stem cells” (CSCs), a subpopulation of tumor cells that are believed to be the cells responsible for tumor initiation and metastasis (19). The ability to characterize and track CD44+ cells would therefore be an important tool for understanding the metastatic cascade. The M-30 CytoDeath antibody recognizes a neoepitope of CK18 that is exposed following caspase cleavage at residue 396 during the early events of apoptosis (20). This marker could be utilized as a measure of therapy effectiveness, and potentially indicate the necessity for a change in treatment much earlier than standard clinical evaluation techniques such as imaging. For the first time in the literature, the current study provides a detailed description of the process of user-defined protein marker development and optimization using the CellSearch system, and will be an important resource for the future development of protein marker assays by users of this platform.
- Top of page
- MATERIALS and METHODS
- LITERATURE CITED
- Supporting Information
The majority of cancer-related deaths are because of ineffective treatment of metastatic disease and an incomplete understanding of the biology of metastasis. Advances in the area of CTC detection and enumeration allows for investigation of the early stages of metastasis that until recently was limited by technological challenges. The characterization of CTCs could be a powerful clinical tool, acting as a real-time, minimally invasive liquid biopsy that would inform clinical decision-making and help direct tailored, individualized therapy. In the present study, our aim was therefore to develop protocols that would allow for the characterization of CTCs using the U.S. FDA-cleared CellSearch system. To the best of our knowledge, this is the first study in the literature to describe the detailed process of protocol development and optimization using this platform. We have demonstrated the appropriate steps that must be taken for proper optimization of user-defined protein marker assays on this system, including comparison of results with a well validated protein expression technology (FCM); appropriate troubleshooting; and detailed optimization techniques using cell lines with various target marker antigen densities. In addition, we have demonstrated that not all markers are ideal candidates for use with the CellSearch system.
Although previous studies have examined CTCs for the expression of CD44 (25–27), none of these previous studies have utilized the CellSearch system to do so. As this system is still the gold-standard and the only FDA-cleared instrument for CTC enumeration and clinical decision-making, the ability to characterize CTCs in combination with enumeration using this particular platform is more clinically applicable then the ability to do so using other techniques. Additionally, Rossi et al., 2010, is the only published study (to our knowledge) that has utilized user-defined, non-Veridex optimized protein markers assays (specifically M-30), on the CellSearch system (28). However, this manuscript fails to provide details of proper optimization of the protocol for the use of this protein marker on the CellSearch. This highlights the necessity for the current study, which provides a detailed description of the process of protein marker optimization in order for users of this instrument to develop properly optimized protein marker protocols that could be utilized in a clinical setting.
The advantages of utilizing the CellSearch platform include system standardization; clearance by the U.S. FDA for assessment of prognosis in metastatic breast, prostate, and colorectal cancer; and the ability to examine CTC heterogeneity at the single cell level. However, the CellSearch system does have a number of disadvantages, including the use of the epithelial marker EpCAM for CTC enrichment. Others have shown that many of the tumor cells found in the circulation are actually mesenchymal in phenotype and are therefore potentially undetectable by this system due to a lack of EpCAM expression (29–32). In addition, it has been demonstrated that the epithelial-to-mesenchymal transition (EMT), which may produce these undetectable CTCs, is associated with enhanced cell aggressiveness (33, 34). Therefore, cells that may potentially form metastatic lesions and are of great interest for characterization may be missed by the CellSearch system. New CTC platforms are currently under development, and hold great promise for enhanced CTC detection and characterization (35–36). However, the CellSearch system, although not a perfect detection and characterization platform, is currently the clinical gold standard for CTC analysis, and for this reason we explored the development of additional CTC characterization assays using this system.
We initially began protocol development for the CD44 marker using the CellSearch CTC kit, with limited success. We hypothesized that the low CD44 positivity results might be due to contamination with leukocytes expressing CD44, thereby simulating a situation in which cells appear to have a lower than expected antigen density. We tested this hypothesis by utilizing the CellSearch CXC kit, which is optimized for the visualization of lower antigen density markers. Utilization of this kit resolved this observed discrepancy, showing levels of CD44 expression that were not significantly different from those observed by FCM. Three cell lines with various CD44 expression levels were then chosen to optimize this protocol. This was a necessary step in the optimization process as the CD44 antigen density in patient CTCs is unknown, and likely to be quite variable across patient samples. As demonstrated, the 21NT cell line (low CD44-expressing) was unable to be adequately visualized at a low exposure time using 1.0 μg/ml of anti-CD44-PE, and therefore the concentration had to be increased to 1.5 μg/ml to ensure adequate sensitivity. In addition, the LNCaP cell line (CD44−) was utilized to ensure specificity of the assay protocol.
As with all assays, limitations do exist when utilizing the CellSearch system for the visualization of CD44. CD44 is a marker of cancer stem cells (19), a phenotype that has been associated with EMT (37). There is always the possibility that CTCs from patient samples may express CD44, but largely by those cells that are undetectable by the CellSearch system, due to a lack of or low level of EpCAM expression. In addition, we have demonstrated that leukocytes can affect adequate CD44 visualization. Therefore, this assay could be compromised in patients with exceptionally high levels of contaminating leukocytes. However, we are confident that this assay is appropriately optimized for use in future clinical studies of metastatic cancer patients.
Next we investigated a different type of marker, one that measures cellular death in response to therapy. We attempted to optimize the integration of the early apoptosis marker M-30 with the CellSearch system. However, after extensive experimentation, we have demonstrated that this marker is unlikely to ever capture all early apoptotic cells in patient samples, as these results were unachievable even under highly controlled conditions. The lack of optimal M-30 positivity on the CellSearch system did not appear to be as a result of a decrease in either EpCAM or CK MFI in our paclitaxel-treated cells. However, when nuclear staining using DAPI was investigated by FCM, there did appear to be a significant decrease in the percentage of overall DAPIhi and M-30+DAPIhi cells, which could affect adequate visualization of DAPI positivity, and therefore CTC classification, on the CellSearch system. When only the M-30+DAPIhi FCM results were compared to our M-30+DAPI+ CellSearch data this appeared to rectify the observed difference. To validate this observation, reanalysis of the CellSearch data was performed to determine if inclusion of those cells that were not originally classified as a CTC due to poor DAPI staining would increase the number of M-30+ cells to that originally observed by FCM. However, reanalysis only marginally increased the percentage of M-30+ cells on the CellSearch system. This increased value was still significantly different from the M-30+ data obtained by FCM and therefore could not be a plausible explanation for why M-30 positivity using the CellSearch system was lower than that observed by FCM. It is possible that spiking cells that are in the process of cell death could reduce CTC recovery as some of these cells may be damaged during the prespiking preparation and others during CellSearch sample processing. Therefore, experiments would need to be performed to determine M-30+ CTC recovery, using spiked samples with high, medium, and low numbers of M-30+ CTCs, and to demonstrate that these recovery results are reproducible across laboratories before this protocol could be considered for clinical use. However, based on the results obtained in our study and our primary aim of demonstrating proper protocol development and troubleshooting, we will not be moving forward with this marker as we believe that there are other markers that are likely better suited to identifying therapy response.
In previous work by Rossi et al. (2010), attempts were made to utilize the M-30 assay on the CellSearch system (28). However, direct comparison between M-30 positivity by FCM and the CellSearch system was never performed; instead CellSearch results were compared to Annexin V positivity, with somewhat discordant results. The authors attempt to explain this difference as a result of nonclassification of some events as CTCs on the CellSearch system; however, experimentation was not undertaken to confirm this hypothesis. In addition, optimal M-30 antibody concentration (2 μg/ml) and exposure time (0.4 s) were chosen based on results from three patients with probable M-30+ CTCs, not in treated control spiked blood samples, as shown in this study.
The M-30 assay has potential for utilization as a measure of therapy effectiveness and as an early indicator of the necessity for a change in treatment. However, this assay is only able to identify apoptosis, one of many known mechanisms of cellular death (necrosis, autophagy, and mitotic catastrophe) (20). This presents a potential problem when examining CTC death as a marker of therapy effectiveness, as it is likely that not all therapy-induced cellular death will be apoptotic (38–40). Therefore, the discrepancies observed in the visualization of this marker in the present study could be a result of the overall complexity and lack of a complete understanding of cellular death in response to therapy.
Research in this area has led many in the field to believe that there is much overlap in the mechanisms that underlie these cellular death processes (38–40) and that cell death proceeds along a pathway in which many possible outcomes can occur. Therefore, many cells undergoing cellular death may be missed using this assay. An ideal apoptotic marker would measure all types of cell death; however, such a marker does not yet exist. The next ideal candidate would measure the most prevalent form of cellular death and be present for the longest detectable period of time. However, even this may be difficult to achieve. Others have demonstrated that the relative proportion of cells that undergo apoptotic cell death can change quite dramatically based on the stressor applied, stressor intensity (concentration and/or length of application), and the cells induced to undergo apoptosis (38). Problems therefore arise when examining patient samples as proper timing may be necessary to capture cell death in the appropriate state. Even if ideal conditions were satisfied, highly vascularized versus poorly vascularized /hypoxic tumors may respond differently (i.e., different cellular death pathways) to antitumor agents due to differences in drug concentrations received. The question then becomes whether the identification of all early apoptotic CTCs is necessary for prediction of therapeutic efficacy and clinical decision making. Instead, could the identification of any apoptotic cells represent a favorable prognosis for patients? In the study by Rossi et al. (2010) (28), CTC analysis was performed in blood from eight breast cancer patients using the integrated M-30 assay on the CellSearch system (2 μg/ml and exposure time 0.4 s). The change in the number of live verses dead (apoptotic) CTCs was determined and found to correlate with radiologic findings of disease status (progressive versus stable disease/partial response), as determined by radiology, with 100% concordance. Obviously, larger follow-up studies will have to be performed before any meaningful conclusions can be drawn from these data, but the results do appear promising.
In summary, we have demonstrated the detailed process of optimization that is required for the development of a user-defined marker on the CellSearch system. In addition, we have shown that not all markers are suitable candidates for use with this platform and the necessary troubleshooting that must be performed when dealing with markers that might alter CTC identification characteristics. This study will act as an important troubleshooting guide for the future development of protein marker assays by users of this platform.