The Role of Circulating Tumor Cells in the Metastatic Cascade: Biology, Technical Challenges, and Clinical Relevance

Abstract

Metastases and cancer recurrence are the main causes of cancer death. Circulating Tumor Cells (CTCs) and disseminated tumor cells are the drivers of cancer cell dissemination. The assessment of CTCs' clinical role in early metastasis prediction, diagnosis, and treatment requires more information about their biology, their roles in cancer dormancy, and immune evasion as well as in therapy resistance. Indeed, CTC functional and biochemical phenotypes have been only partially characterized using murine metastasis models and liquid biopsy in human patients. CTC detection, characterization, and enumeration represent a promising tool for tailoring the management of each patient with cancer. The comprehensive understanding of CTCs will provide more opportunities to determine their clinical utility. This review provides much-needed insights into this dynamic field of translational cancer research.

Keywords: cancer stem cells; circulating tumor cells; dormancy; immune escape; liquid biopsy; metastasis; therapy resistance.

Figure 1

Models of CTC intravasation and cluster formation. (a) EMT allows single CTCs to detach from the primary tumor lesion; then, VEGF expression increases endothelial permeability and promotes CTC intravasation and dissemination to distant organs. (b) TAMs contribute to metastasis formation by ECM remodeling, and CAFs physically pull away cancer cells from the primary tumor. Partial EMT and stemness features due to OCT4, NANOG, SOX2 upregulation in cancer cells within clusters enhance cell–cell junction formation and facilitate collective invasion, intravasation, dissemination, and metastasis initiation. (c) Clusters may be formed by intravascular aggregation of single CTCs that overexpress heparanase (HPSE) via HPSE-mediated induction of cell–cell adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and the focal adhesion kinase (FAK)-Src-paxillin pathway. Abbreviations: CTC, circulating tumor cell; EMT, epithelial-to-mesenchymal transition; VEGF, vascular endothelial growth factor; TAM, tumor-associated macrophages; ECM, extra cellular matrix; CAF, cancer-associated fibroblasts.

Figure 2

Cancer cell dormancy. Low shear stress and size limitation at vascular branches promote CTC extravasation. Disseminated tumor cells (DTC) dormancy is supported through different mechanisms: (a) Growth arrest-specific protein 6 (GAS6) activates AXL-associated dormancy signaling. Transforming growth factor-β2 (TGFβ2) and BMP7 signaling mediate the production of cell cycle inhibitors by dormant cells. (b) Regulatory T (Treg) cells-derived adenosine and tumor necrosis factor receptor 1 (TNFR1) signaling protect tumor cells against oxidative stress and induce dormancy signals, respectively. (c) The ECM integrin αvβ3 binds to urokinase plasminogen activator (uPA), and suppresses the dormancy effect of Extracellular signal-Regulated Kinase (ERK) signaling. (d) Stressful conditions result in PI3K-AKT survival pathway inhibition. (e) The GTPase RHEB-mTOR pathway suppresses apoptosis in DTCs. Endothelial cells produce thrombospondin 2 (TSP2) that maintains DTCs in a dormant state; however, TGFβ1 and periostin (POSTN) promote DTC proliferation.

Figure 3

CTCs evade the immune systems in several ways (a–d). (a) CTCs modulate NK cell activity by producing inhibitory cytokines and blocking NK cell activator receptors (e.g., NKG2D and KIRs). (b) CTCs induce production of tumor-supporting M2 type of macrophages (MQ). M2 macrophages induce matrix-remodeling, neoangiogenesis, and exert an immune suppressive phenotype, which all support tumor metastases [78]. Binding of CTC CD47 to macrophage SIRPα inhibits tumor cell phagocytosis by macrophages. (c) PDL-1 and Fas Cell Surface Death Receptor Ligand (FASL) upregulation at the CTC surface reduces T-cell anti-tumor responses and induces T-cell apoptosis, respectively. CTCs exploit Treg cells and myeloid-derived suppressor cells (MDSCs) to promote metastasis formation in patients with various malignancies. (d) Platelets interact with CTCs, and protect them from antigen recognition by immune cells. Platelets produce MHC-I-positive vesicles, thus sending self-signal to NK cells. Platelets produce also TGF-β, a factor that initiates and maintains EMT, and helps CTCs to evade immune attacks [79]. Abbreviations: NKG2D, NK cell receptor D; SIRPα, signal-regulatory protein α; PD-L1, programmed death ligand 1; Treg, regulatory T cells.

Figure 4

Methods for CTC enrichment based on their (a) biological, and (b) biophysical properties. Immunoaffinity-based CTC isolation using two strategies: (a1) Positive enrichment where CTCs or CTSCs are captured by immunomagnetic devices or CTC-chips by trapping their surface markers, such as EpCAM, CD133, HER2, CSV, prostate-specific membrane antigen (PSMA); and (a2) Negative enrichment by depletion of WBCs and platelets using anti-CD45 and -CD61 antibodies, respectively. (b1) Density: CTC density is lower than that of red blood cells and white blood cells; (b2) Size and deformability: filters with pores of 7- 8µm in diameter allow the passage of blood components, but not of CTCs; (b3) The distinct electrical charges of CTCs and blood cells allow separating these cell types; and (b4) Inertial focusing: CTCs are passively separated using microfluidic devices based on their size through the application of inertial forces (shear-gradient lift force and a wall effect lift force) that affect cell positioning within the flow channel.