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

Abstract

tumor cells (CTCs) have been in the focus ofcancer research for decades. The presence of these cells in theblood of patients with primary and metastatic cancer is asso-ciated with poor prognosis (1). CTCs have a central role in theprocess of metastasis formation: To become a CTC, cancercells must shed from the primary tumor by loosing cell–cellcontacts with other cancer cells. Furthermore, cancer cellsmust have the ability to migrate through the extracellular ma-trix and to invade the surrounding tissue. After intravasationinto the bloodstream, cancer cells became CTCs. In the blood-stream, a large fraction of the CTCs die quickly because of kill-ing 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, CTCsdo accumulate lots of properties enabling them to survive inthe bloodstream and to extravasate the blood vessels. SomeCTCs extravasate into distant organs and persist there as dor-mant cells or are capable of dividing and forming micrometas-tases and later metastases (2).In the past decade, several CTC detection and enumera-tion methods have been developed. Immunolabeled cancercells from the blood can be enriched by different approachesincluding immunomagnetic separation and physical methods(filtration and density gradient). After enrichment, the iso-lated cancer cells can be enumerated and analyzed using differ-ent technical approaches (e.g., flow cytometer, laser scanningcytometer, and fluorescence microscope). Reverse transcrip-tion (RT-PCR)-based methods are also widely used. These arehighly sensitive for the expression of candidate genes specificto cancer cells or to the normal tissues from which the tumorcells originate (3). Unfortunately, these valuable methods arebarely usable to describe differences between CTC subpopula-tions.In a heterogenic CTC population, several CTC subpopu-lations might exist with different properties. Because cancercells having the ability to form micrometastases and metasta-ses are likely emerge from CTCs, a high medical need exists todevelop new methods to characterize the genotype and pheno-type of CTCs. According to our opinion, investigation of CTCsubpopulations might open new ways in anticancer research.Methods capable of studying CTC subpopulations might bevaluable tools not only for basic cancer research but also forcancer diagnostic, evaluation of prognosis, or following up theefficacy of anticancer therapy.In this issue, Takao and Takeda (page 107) describe a sim-plified protocol to enumerate CTCs without membrane-per-meabilization using a novel cross contamination free flow cy-tometer apparatus. The device has included a separate micro-fluidic chip, so using the new method CTCs can not only beenumerated with a cytometric multicolor analysis but also thedevice is able to collect both dead and viable circulating cancercells. The ratio of dead and live CTCs might have an impor-tance in clinical practice, for example, an increase in the ratiomight suggest a successful therapeutic outcome. We agree withthe authors that monitoring dead/live CTC ratio in additionto CTC number after adjuvant or neoadjuvant therapy couldprovide clinically relevant information. Furthermore, mostmethods which enumerate CTCs are not able to collect the