Biology, vulnerabilities and clinical applications of circulating tumour cells

Abstract

In recent years, exceptional technological advances have enabled the identification and interrogation of rare circulating tumour cells (CTCs) from blood samples of patients, leading to new fields of research and fostering the promise for paradigm-changing, liquid biopsy-based clinical applications. Analysis of CTCs has revealed distinct biological phenotypes, including the presence of CTC clusters and the interaction between CTCs and immune or stromal cells, impacting metastasis formation and providing new insights into cancer vulnerabilities. Here we review the progress made in understanding biological features of CTCs and provide insight into exploiting these developments to design future clinical tools for improving the diagnosis and treatment of cancer.

Fig. 1. Stepwise progression of the metastatic cascade.

a, Invasion of cancer cells. The formation of invasive features (for example, invadopodia) and hypoxic conditions (indicated by a blue haze) favour release of cancer cells away from the primary tumour site via upregulation of hypoxia inducible factor 1α (HIF1α), NMYC downstream-regulated gene 1 protein (NDRG1) and vascular endothelial growth factor A (VEGFA). This is further enhanced by metastasis-promoting features, including the expression of CXC-chemokine receptor 4 (CXCR4) and angiopoietin-like protein 4 (ANGPTL-4), the secretion of matrix metalloproteinases (MMPs), decreased expression of phosphoglycerate dehydrogenase (PHGDH) and cytoskeletal rearrangements. External conditions conducive to spreading are further provided by physical factors (for example, fluid pressure and stiffness) and surrounding cells (for example, cancer-associated fibroblasts and endothelial cells) in the tumour microenvironment. Mechanical stimuli from the tumour microenvironment promote pro-metastatic conditions by activating Yes-associated protein 1 (YAP)–transcriptional co-activator with PDZ-binding motif (TAZ) in cancer associated fibroblasts, favouring cancer cell invasion. Further, paracrine factors secreted by endothelial cells may reduce PHGDH levels in cancer cells, potentiating cell migration and invasion. Intravasation (part b) and circulation (part c). Circulating tumour cells (CTCs) and their clusters have a short half-life in circulation, due to hostile conditions, including physical forces (that is, shear stress) and anoikis. CTCs can escape the immune system via downregulation of major histocompatibility complex class I (MHC-I), expression of immune checkpoint molecules (for example, programmed cell death protein 1 (PD1) ligand 1 (PDL1) and CD47) or through support from platelets and neutrophils. Cell-intrinsic factors (for example, expression of anti-apoptotic factors) enhance CTC survival and successful transit, while circadian rhythmicity (and related hormone fluctuations) dictates the timing of CTC intravasation events, reaching a peak during the rest phase. d, Extravasation. The efficiency of extravasation relies upon the expression of adhesion molecules (for example, CD44, mucin 1 (MUC1) and sialyl-Lewis A (sLeA)/sialyl-Lewis X (sLeX), chemokine release, physical properties (for example, CTC cluster size and deformability) and supporting cells (for example, neutrophils via the formation of neutrophil extracellular traps (NETs)). Signalling via transforming growth factor-β (TGFβ)/SMAD family member 3 (SMAD3) leads to the upregulation of various adhesion-related molecules and facilitates CTC vascular adhesion. e, Successful homing into a new environment is dependent on niche factors (that is, various organ-specific cell types), but is also influenced by the preset metastatic potential of CTCs. f, CTCs may spread from the primary tumour or from metastatic lesions to either seed new metastasis (metastasis-to-metastasis dissemination) or return to the primary tumour site (tumour self-seeding). DTC, disseminated tumour cell; HIFPH2, hypoxia-inducible factor prolyl hydroxylase 2; NK natural killer; N-WASP, neural Wiskott–Aldrich syndrome protein.

Fig. 2. Biological features of circulating tumour cell clusters.

Clustering of circulating tumour cells (CTCs) may occur exclusively between tumour cells (homotypic CTC clusters), as well as between tumour cells and other cell types (heterotypic CTC clusters). This results in enhanced proliferation and survival in the circulation, enabling superior metastatic proficiency. a, Homotypic clustering of CTCs leads to the creation of typically oligoclonal clusters, kept together by cell adhesion molecules (for example, plakoglobin, claudins and CD44). Expression of these molecules and cluster formation are promoted by hypoxic conditions. CTC clustering triggers epigenetic changes (for example, hypomethylation of binding sites for OCT4, NANOG and SOX2), leading to stem-like cell behaviour, which facilitates metastasis seeding. b, Heterotypic CTC clusters (for example, between tumour cells and neutrophils, cancer-associated fibroblasts or platelets) display increased proliferation, invasion and homing at the metastatic site, as well as protection against immune surveillance. E-cadherin, epithelial cadherin; GPIb-IX-V, glycoprotein Ib–IX–V; GPVI, glycoprotein VI; N-cadherin, neural cadherin; VCAM1, vascular cell adhesion molecule 1.

Fig. 3. Circulating tumour cell capture, analysis and clinical trial designs.

Available circulating tumour cell (CTC) detection technologies and examples of how CTCs can be included in clinical trial designs. a, CTC capture tools include antigen-dependent technologies (for example, immune capture via immobilized antibodies, antibody coated beads or coated intravascular guidewires) and antigen-independent technologies (for example, density gradient centrifugation, microfluidic systems based on deformability and size, size-based filtration systems, electrical charge-based technologies or cytapheresis). The latter do not require a priori knowledge of phenotypic profiles and are thought to capture more heterogeneous CTC populations compared with antigen-dependent methods. Downstream analysis of CTCs includes direct drug phenotyping, the creation of CTC-derived xenograft ‘avatar’ models and multi-omics interrogations at the single cell-level: epigenomic, proteomic, genomic and transcriptomic. b, Validation of the use of CTCs in the setting of innovative clinical trials includes randomizing and benchmarking CTCs against standard-of-care (SOC) diagnostic and therapeutic approaches or other liquid biopsy analytes (for example, circulating tumour DNA (ctDNA)) and testing of CTC-based treatment strategies. The figure shows various possibilities for CTC-based clinical trial design: randomization of CTC-based liquid biopsies versus tissue biopsies to guide the choice of therapy; randomization based on the presence of CTCs (positive versus negative) to guide the choice of therapy (that is, experimental or targeted versus SOC); randomization of patients who are positive for CTCs to treatment with different targeted or experimental drugs; randomization according to longitudinal or repeated CTC assessment to guide subsequent lines of therapy (for example, targeted or experimental treatment versus SOC); randomized trials which compare either use of CTCs alone with SOC diagnostic approaches (for example, medical imaging) or use of other liquid biopsy analytes (for example, ctDNA) or each individual modality with combined use of modalities.

Fig. 4. Circulating tumour cell-targeting strategies.

Outlined here are various potential circulating tumour cell (CTC)-targeting strategies that are proposed on the basis of recent experimental work. a, Inhibition of cancer cell intravasation via normalization of the hypoxic tumour microenvironment; for example, by ephrin B2 Fc chimaera protein (EpB2) treatment leading to modulation of vascular endothelial growth factor receptor (VEGFR) signalling and vascular normalization, blockade of intravasation-relevant proteins (for example, Polo-like kinase (PLK1) inhibition) or blocking of cellular interactions between cancer and endothelial cells (for example, integrin-targeted antibodies). b, Dissociation of CTC clusters or prevention of their formation, for example via Na⁺/K⁺-ATPase inhibition, heparanase (HPSE) inhibition, stimulation with the urokinase-type plasminogen activator (urokinase) or inhibition of platelet receptors on CTCs. c, Targeting CTC survival via metabolic interference by increasing oxidative stress and inhibition of pyruvate or proline metabolism, or by use of E-selectin/tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-coated nanoliposomes that mimic the activity of natural killer (NK) cells. d, ‘Demasking’ CTCs for immune clearance by targeting immune evasion via immune checkpoint inhibitors against programmed cell death protein 1 (PD1) and PD1 ligand 1 (PDL1), cytotoxic T lymphocyte-associated antigen 4 (CTLA4) or CD47. e, Use of engineered CTCs as therapeutic vehicles (for example, prodrug conjugates) or CTCs for tumour vaccine development. f, CTC-based chronotherapy (that is, delivering treatments to be maximally effective at the times of greatest CTC production). VCAM1, vascular cell adhesion molecule 1.