Prospects for Comprehensive Analyses of Circulating Tumor Cells in Tumor Biology

Abstract

The comprehensive analysis of biological and clinical aspects of circulating tumor cells (CTCs) has attracted interest as a means of enabling non-invasive, real-time monitoring of cancer patients and enhancing our fundamental understanding of tumor metastasis. However, CTC populations are extremely small when compared to other cell populations in the blood, limiting our comprehension of CTC biology and their clinical utility. Recently developed proteomic and genomic techniques that require only a small amount of sample have attracted much interest and expanded the potential utility of CTCs. Cancer heterogeneity, including specific mutations, greatly impacts disease diagnosis and the choice of available therapeutic strategies. The CTC population consists primarily of cancer stem cells, and CTC subpopulations are thought to undergo epithelial-mesenchymal transition during dissemination. To better characterize tumor cell populations, we demonstrated that changes in genomic profiles identified via next-generation sequencing of liquid biopsy samples could be expanded upon to increase sensitivity without decreasing specificity by using a combination of assays with CTCs and circulating tumor DNA. To enhance our understanding of CTC biology, we developed a metabolome analysis method applicable to single CTCs. Here, we review-omics studies related to CTC analysis and discuss various clinical and biological issues related to CTCs.

Keywords: circulating tumor cells (CTCs); circulating tumor dna (ctDNA); genomics; heterogeneity; metabolomics; single cell.

Figure 1

Combined analysis of genomic alterations in circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) using targeted next-generation sequencing. (A) Genomic alterations in CTCs of head and neck cancer, esophageal cancer, gastric cancer, and colorectal cancer patients. The number of CTCs is indicated in the columns. * The number of CTCs could not be determined in 4 patients. (B) Genomic alterations in ctDNA from patients with head and neck cancer, gastric cancer, and colorectal cancer. ctDNA could not be extracted from 2 patients with colorectal cancer. Blue, yellow, orange, green, purple, and black spaces represent missense mutations, nonsense mutations, synonymous mutations, intronic mutations, frameshift deletions, and frameshift insertions, respectively [62].

Figure 2

Schematic illustration of the live single-cell mass spectrometry (LSC-MS) method. Blood samples were collected from patients with gastric cancer and colorectal cancer. A microfluidics technique was used to enrich circulating tumor cells (CTCs). Single CTCs were sampled and analyzed using the LSC-MS system [64]. PCA-DA, principal component analysis–discriminant analysis; EDTA, ethylenediaminetetraacetic acid; RBC, red blood cell.

Figure 3

Profiling of gastric cancer (GC) and colorectal cancer (CRC) circulating tumor cells (CTCs) at the single-cell level. (A) Principle component analysis–discriminant analysis to distinguish GC CTCs, CRC CTCs, and blank cells. Each dot represents a single cell. (B) Histogram of the frequency of peak distribution across the m/z scale for GC and CRC [64].

Figure 4

Graphical overview of tumor heterogeneity and circulating tumor cell (CTC) analysis. Heterogeneity is caused by (1) subclones present within the primary lesion, (2) selected cancer cells shed from the primary lesion that invade the blood vessels (e.g., interaction with the microenvironment surrounding tumor). CTC analysis is a useful tool for characterizing this heterogeneity.