Steps in metastasis research: Analyzing, collecting, and culturing circulating tumor cells

Circulating tumor cells (CTCs) have been in the focus of cancer research for decades. The presence of these cells in the blood of patients with primary and metastatic cancer is associated with poor prognosis (1). CTCs have a central role in the process of metastasis formation: To become a CTC, cancer cells must shed from the primary tumor by loosing cell–cell contacts with other cancer cells. Furthermore, cancer cells must have the ability to migrate through the extracellular matrix and to invade the surrounding tissue. After intravasation into the bloodstream, cancer cells became CTCs. In the bloodstream, a large fraction of the CTCs die quickly because of killing by (i) the immune system; (ii) hemodynamic forces; and/or (iii) apoptosis evoked by loss of cell attachment and cell–matrix connections. Under a strong evolution pressure, CTCs do accumulate lots of properties enabling them to survive in the bloodstream and to extravasate the blood vessels. Some CTCs extravasate into distant organs and persist there as dormant cells or are capable of dividing and forming micrometastases and later metastases (2). In the past decade, several CTC detection and enumeration methods have been developed. Immunolabeled cancer cells from the blood can be enriched by different approaches including immunomagnetic separation and physical methods (filtration and density gradient). After enrichment, the isolated cancer cells can be enumerated and analyzed using different technical approaches (e.g., flow cytometer, laser scanning cytometer, and fluorescence microscope). Reverse transcription (RT-PCR)-based methods are also widely used. These are highly sensitive for the expression of candidate genes specific to cancer cells or to the normal tissues from which the tumor cells originate (3). Unfortunately, these valuable methods are barely usable to describe differences between CTC subpopulations. In a heterogenic CTC population, several CTC subpopulations might exist with different properties. Because cancer cells having the ability to form micrometastases and metastases are likely emerge from CTCs, a high medical need exists to develop new methods to characterize the genotype and phenotype of CTCs. According to our opinion, investigation of CTC subpopulations might open new ways in anticancer research. Methods capable of studying CTC subpopulations might be valuable tools not only for basic cancer research but also for cancer diagnostic, evaluation of prognosis, or following up the efficacy of anticancer therapy. In this issue, Takao and Takeda (page 107) describe a simplified protocol to enumerate CTCs without membrane-permeabilization using a novel cross contamination free flow cytometer apparatus. The device has included a separate microfluidic chip, so using the new method CTCs can not only be enumerated with a cytometric multicolor analysis but also the device is able to collect both dead and viable circulating cancer cells. The ratio of dead and live CTCs might have an importance in clinical practice, for example, an increase in the ratio might suggest a successful therapeutic outcome. We agree with the authors that monitoring dead/live CTC ratio in addition to CTC number after adjuvant or neoadjuvant therapy could provide clinically relevant information. Furthermore, most methods which enumerate CTCs are not able to collect the enumerated CTCs, the authors' device, however, allows collecting CTCs which are ready for further analysis (4-6). Importantly, the analyzed and collected CTCs are viable and can grow in a healthy manner. Comparison of gene expression profile of primary tumor and CTC and metastasis might reveal the cellular changes toward tumor progression, tumor recurrence, and metastasis development. The molecular events leading to the above-mentioned phenomena could be investigated based on CTC analysis. With the help of the new method one can investigate the in vitro effect of chemotherapeutic agents or antibody drugs on cell cultures emerged from CTCs isolated from human cancer patients. Although trastuzumab, a recombinant humanized monoclonal antibody against the extracellular domain of HER2 (7, 8) seems to be able to decrease the number of CTCs (9, 10), investigating the effect of trastuzumab and other anticancer agents on CTC cultures might be of great importance, because changes of HER2 status in CTCs and even loss of HER2 overexpression during the treatment containing trastuzumab has been observed (11) indicating that CTCs could be resistant to trastuzumab. Injecting the in vitro expanded and phenotypically selected CTCs into immunocompromised mice might answer the question, which CTC characteristics are needed for metastasis development. One possible answer to this question could be that those CTCs, which acquired STEM cell-like properties (12), have the potential to initiate micrometastases or metastases. Indeed, it was recently reported that some CTCs or CTC subpopulations have STEM cell-like properties (13). Similar questions can be addressed using CTC cultures. In preclinical experiments, it was previously shown that trastuzumab reduced the number of CTCs at a time when the primary tumor was already unresponsive to trastuzumab (9). This observation suggested that HER2 positive CTCs could be sensitive to trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC) even if when the primary tumor is already nonresponsive. The validity of this hypothesis can also be tested with the help of CTC cultures. In summary, because CTCs are rare cells, to isolate and culture them in vitro might be a valuable tool for further molecular and cellular characterization of CTCs. Further advantage of the new method presented by Takao and Takeda (page 107, this issue) is its applicability for conventional flow cytometers, which are standard equipments in many research and diagnostic institutes. In addition, it is clearly evident that there is a need to develop an easy to perform but reliable method, which can be standardized for the detection and characterization of these cells. The author's new method seems to fulfill these criteria. After all, we think that this novel cross-contamination free-flow cytometric method for enumeration, characterization, and collection of intact CTCs would be a valuable tool for both clinicians and basic cancer researchers. The next step is to apply the new method to clinical samples, and the detected CTCs could be used as “blood biopsy” providing and excellent opportunity for cancer diagnostics and characterization of the metastatic processes.