Castro-Giner and Aceto Genome Medicine (2020) 12:31 https://doi.org/10.1186/s13073-020-00728-3 REVIEW Open Access Tracking cancer progression: from circulating tumor cells to metastasis Francesc Castro-Giner1,2 and Nicola Aceto1* Abstract The analysis of circulating tumor cells (CTCs) is an outstanding tool to provide insights into the biology of metastatic cancers, to monitor disease progression and with potential for use in liquid biopsy-based personalized cancer treatment. These goals are ambitious, yet recent studies are already allowing a sharper understanding of the strengths, challenges, and opportunities provided by liquid biopsy approaches. For instance, through single-cell- resolution genomics and transcriptomics, it is becoming increasingly clear that CTCs are heterogeneous at multiple levels and that only a fraction of them is capable of initiating metastasis. It also appears that CTCs adopt multiple ways to enhance their metastatic potential, including homotypic clustering and heterotypic interactions with immune and stromal cells. On the clinical side, both CTC enumeration and molecular analysis may provide new means to monitor cancer progression and to take individualized treatment decisions, but their use for early cancer detection appears to be challenging compared to that of other tumor derivatives such as circulating tumor DNA. In this review, we summarize current data on CTC biology and CTC-based clinical applications that are likely to impact our understanding of the metastatic process and to influence the clinical management of patients with metastatic cancer, including new prospects that may favor the implementation of precision medicine. Background for disease prognosis, and their suitability for additional Circulating tumor cells (CTCs) are defined as those can- applications is being tested. In this short review, we cer cells that depart from a solid tumor lesion and enter summarize selected methods to achieve CTC isolation the bloodstream. CTCs are not the only tumor deriva- and molecular interrogation, and we discuss advances in tives in circulation, but they contain a population of understanding disease progression through CTC ana- metastatic precursors that are of paramount importance lysis, particularly focusing on those that aim at the de- for the accomplishment of disease progression [1, 2]. velopment of new diagnostic and therapeutic tools that The isolation of viable CTCs has been limited for several could be applied in the clinical setting. We also discuss years by technological challenges, mostly resulting from potential advantages and challenges associated with the rarity of CTCs compared to blood cells and from an CTC-related investigations and more generally within inability to isolate them in a viable state [3, 4]. Recently, the liquid biopsy field, and we speculate on future CTC- however, specialized technologies have been developed based applications and open questions. not only to capture and enumerate CTCs, but also to in- terrogate them at the molecular level and to test their CTC isolation technologies suitability for clinical applications. In the clinic, CTCs A plethora of specialized CTC isolation technologies has have already proven their value as predictive biomarkers been developed in the past 15 years. Essentially, these can be divided into two main groups: those that capture * Correspondence: [email protected] CTCs based on the expression of a particular antigen 1Department of Biomedicine, Cancer Metastasis Laboratory, University of Basel and University Hospital Basel, 4058 Basel, Switzerland (positive selection) and those that deplete all cells that Full list of author information is available at the end of the article are not CTCs in a given sample (negative selection). A © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Castro-Giner and Aceto Genome Medicine (2020) 12:31 Page 2 of 12 key example of positive CTC enrichment is provided by interrogation. This problem can be addressed by pooling the CellSearch system [5]. This technology is based on a cells from the same individual, but at the expense of destroy- two-step procedure, in which the first step consists of ing the resolution that would enable investigations of CTC sample centrifugation to eliminate plasma components molecular heterogeneity [12]. Functional characterization can while CTCs are captured with anti-epithelial cell adhe- be achieved through experimental solutions such as CTC- sion molecule (EpCAM)-conjugated magnetic ferro- derived explants and the development of CTC-derived cell fluids. In the second step, putative CTCs are further lines. These can be used for drug screening and can also be stained and identified using anti-cytokeratin antibodies exploited to increase the number of cells available for a given while contaminant white blood cells (WBCs) are identi- analysis [13–15]. Nevertheless, their use for patient stratifica- fied by means of CD45 staining. Currently, CellSearch is tion is challenging, especially because the development of the only CTC isolation device that is approved by the CTC-derived lines is a long and inefficient process (typically US Food and Drug Administration (FDA) [5], although lasting several months), requires large numbers of cells, and “sister” antigen-based CTC isolation technologies are is subject to possible biases during ex vivo culturing [16]. continuously emerging [6, 7]. CellSearch has been vali- Traditional approaches for molecular interrogation of dated in large patient cohorts, and its main use concerns CTCs include testing for genomic aberrations using im- CTC enumeration to identify high-risk patients with de- munostaining combined with fluorescence in situ creased progression-free survival and decreased overall hybridization (FISH) and real-time quantitative PCR survival in breast, prostate, and colorectal cancers [5, 8]. (RT-qPCR) to evaluate the transcript levels of specific Although positive CTC selection generally offers high genes [17]. Yet, it is the development of single-cell tech- capture rates of antigen-expressing CTCs and low blood nologies during the past decade that has enabled a com- cell carryover, CTCs may dynamically alter the expres- prehensive interrogation of CTCs from patients with sion of a given antigen under certain conditions, result- various cancer types. Combined with next-generation se- ing in an underestimation of the total CTC population quencing (NGS) and mass cytometry technologies, it is in a given sample. For this reason, antigen-independent now possible to characterize the genome, transcriptome, technologies have been developed to deplete red blood methylome [18], and proteome [19] of individual cells. cells and WBCs from a blood sample, leaving a CTC- The recent development of a single-cell RNA-seq plat- enriched, antigen-agnostic product for subsequent form based on a hydrodynamic barcoding technique downstream analysis. These technologies include size- (Hydro-Seq) also shows a promising efficiency for whole- based filtration of CTCs (taking advantage of their larger transcriptomeanalysisofCTCs[20]. As reviewed in the fol- diameter when compared to blood cells), CTC enrich- lowing sections, these approaches have already generated in- ment based on other physical parameters, and immuno- teresting observations, including the identification of magnetic depletion of WBCs [9]. A comprehensive list oligoclonality in CTCs, the definition of specific molecular of CTC isolation technologies is beyond the scope of this profiles in comparisons of primary and metastatic tumors, short review, but it is important to recognize that each and the discovery of gene expression signatures that technology may favor the enrichment of a particular characterize metastatic precursors or differences between CTC subtype (e.g., high antigen-expressing CTCs in the CTC subpopulations [21]. In addition, multi-omics ap- case of positive selection methods, larger CTCs in the proaches now enable the parallel interrogation of both RNA case of size-based filtration methods, among others) and and DNA from individual cells, aiming towards a more that possible technology-driven biases should be recog- comprehensive understanding of the cellular processes that nized and considered when interpreting downstream occur in cancer cells [22–25]. Despite the improvements in molecular results. single-cell sequencing protocols, major analytical challenges remain for proper data interpretation, including strong sto- Molecular analysis of CTCs