Microenvironment crosstalk and immune evasion of circulating tumor cells: From mechanism to clinical significance

Abstract

Circulating tumor cells (CTCs), shed from the primary malignancies, are regarded as the “seeds” of tumor metastasis. They employ sophisticated strategies to evade immune detection during blood circulation by engaging with various blood components, ultimately facilitating their colonization in distant organs. Elucidating the mechanisms underlying CTC immune evasion may unlock novel immunotherapeutic strategies to prevent tumor metastasis. Recent advancements in CTC isolation and single-cell sequencing have provided insights into their complex microenvironment and immune evasion mechanisms. Many strategies for targeting either CTCs or their associated blood cells have been explored. Here, we systematically delineated the interaction network between CTCs and the diverse blood cells. By elucidating the microenvironmental and biological characteristics of CTCs, we summarize several potential immune evasion mechanisms, including immune checkpoint modulation, CTC clustering, platelet interactions, etc. Additionally, we highlight recent advances in intervention strategies targeting CTCs and the clinical application of CTCs in cancer liquid biopsy.

Introduction

Tumor metastasis remains the most lethal manifestation of cancer progression, accounting for over 90% of cancer-related mortality worldwide.^[1,2] Circulating tumor cells (CTCs), shed from the primary lesions, are regarded as the “seeds” of tumor metastasis.^[3] The advent of advanced CTC capture technologies has enabled their utilization as liquid biopsy markers for monitoring therapeutic response and predicting clinical outcomes.^[4] While substantial efforts have been devoted to optimizing CTC capture technologies and correlating CTC counts with clinical outcomes,^[5–8] there are still several critical challenges in current CTC research, including a predominant focus on diagnostic applications rather than therapeutic targeting and a limited understanding of the immune evasion mechanism during circulation. Central to these challenges is the unresolved question of how CTCs systematically evade immune surveillance to establish metastases.

Recent technological breakthroughs, particularly single-cell multi-omics profiling, have unveiled unprecedented heterogeneity in CTC populations and their dynamic crosstalk with circulatory components. Emerging evidence reveals that CTCs orchestrate a sophisticated “immune defense network” through: (1) dynamic recruitment of circulatory components, such as platelet, to create immune suppressive niches;^[9,10] (2) real-time adaptation via interactions with blood cells, such as neutrophils, macrophages, and myeloid-derived suppressor cells (MDSCs);^[11–13] (3) hijacking of immune checkpoint through heterotypic clustering.^[11,14–17] For instance, single-cell transcriptomic analyses have unveiled that CTCs engage platelets to upregulate the immune checkpoint histocompatibility leukocyte antigen (HLA)-E and HLA-C to escape the immune surveillance of natural killer (NK) cells.^[14] These adaptive mechanisms support intravascular survival and facilitate extravasation and colonization at distant sites. Consequently, a systematic understanding of the interactions between CTCs and blood-derived cells, along with their immune evasion mechanism, is imperative for the development of innovative clinical treatment approaches.

Here, we systematically depict the interaction network between CTCs and various blood-derived cells, including platelets, NK cells, T cells, neutrophils, macrophages, etc. [Figure 1]. By elaborating on the microenvironment and biological characteristics of CTCs, we summarize several immune evasion mechanisms of CTCs. According to the immune evasion mechanism, we propose several therapeutic strategies that target CTCs. Furthermore, we explore the application of CTCs in tumor diagnosis and liquid biopsy. Overall, this review presents the opportunities and challenges involved while providing novel insights for tumor therapy by comprehensively describing the essential role of CTCs in tumor metastasis.

Figure 1.

The interactions between CTCs and blood cells in the circulation. (A) CTCs activate platelets via direct contact or soluble factors. Activated platelets transfer inhibitory molecules (e.g., TGF-β, P-selectin, MHC-I) to CTCs, impairing T and NK cell surveillance. (B and C) CTCs suppress NK (B) and CD8⁺ T cell (C) functions via immune checkpoints (e.g., PD-L1, HLA-E) and induce Treg differentiation through proinflammatory factors, further dampening immune responses. (D) CTC-neutrophil clusters, formed via integrins, promote CTC proliferation, dormancy escape, survival, and extravasation. Neutrophils release ARG-1, NO, ROS, and PGE2 to aid immune evasion. (E) Via CD47-SIRPα, CTCs evade macrophage phagocytosis. Macrophages enhance CTC invasiveness by secreting CXCL8, CXCL2, and EGF. (F) CTC-MDSC interaction through Nodal-Cripto promotes CTC growth and suppresses CD8⁺ T cells via ROS and ARG-1. (G) CTCs intravasate via MMP1-PAR1 and Ca²⁺-MLC pathways; extravasation involves E-selectin/sLe^x and CD44, followed by endothelial contraction via ERK and PI3K signaling. ARG-1: Arginase-1; CXCL2: C-X-C motif chemokine ligand 2; CXCL8: C-X-C motif chemokine ligand 8; CTCs: Circulating tumor cells; EGF: Epidermal growth factor; ERK: Extracellular signal-regulated kinase; HLA-E: Human leukocyte antigen-E; MDSC: Myeloid-derived suppressor cell; MHC-I: Major histocompatibility complex class I; MLC: Myosin light chain; MMP-1: Matrix metalloproteinase-1; NO: Nitric oxide; NK: Natural killer; PAR1: Protease-activated receptor 1; PD-L1: Programmed death-ligand 1; PGE2: Prostaglandin E2; PI3K: Phosphoinositide 3-kinase; ROS: Reactive oxygen species; SIRP α: Signal-regulatory protein α; sLe^x: Sialyl Lewis X; TGF-β: Transforming growth factor-β; Treg: Regulatory T cell.

Historical Perspective on CTCs

CTC is a kind of tumor cell that sheds from the primary lesion, plays a pivotal role in tumor metastasis. However, the exploration of CTCs spans over a century, marked by germination period, emergence period, and rapid development period [Figure 2]. The budding period began in 1869 when Ashworth first observed tumor-like cells in the blood of a breast cancer patient, suggesting a potential link to metastasis.^[18] However, technical limitations hinder a deeper understanding of the properties of these cells. The rise period saw significant progress in 1959, with the first successful isolation of CTCs using a combination of centrifugation, incubation, filtration, and staining, which allowed for cellular-level analysis.^[19] The research indicated that patients with detectable tumor cells in their blood succumbed within a month, emphasizing the clinical relevance of these cells. Until the late 1990s, immunomagnetic separation techniques significantly enhanced the ease and accuracy of CTC isolation, and defined them as cytokeratin/epithelial cell adhesion molecule (CK/EpCAM)⁺, nuclear⁺, and CD45^– cells.^[20]

Figure 2.

The historical milestones in research related to CTC. With a deeper understanding of CTCs over time, their development can be divided into three periods: the germination period (1869–1958, pink), the emergence period (1959–2003, purple), and the rapid development period (2004–present, yellow). AJCC: American Joint Committee on Cancer; ASCO: American Society of Clinical Oncology; CTC: Circulating tumor cell; EMT: Epithelial-to-mesenchymal transition; FDA: Food and Drug Administration; HLA: Human leukocyte antigen; HTMSU: High throughput microsampling unit; KIR: Killer-cell immunoglobulin-like receptor; NCCN: National Comprehensive Cancer Network; NK: Natural killer (cells); NKG2A: Natural killer group 2A; PMMA: Polymethyl methacrylate.

In recent years, the advancement of single-cell sequencing technologies has promoted the transformative leap of CTC research.^[21] Single-cell sequencing enables unlocked multi-omic profiling (genomic, transcriptomic, epigenomic, and proteomic) of individual CTCs, revealing unprecedented insights into their heterogeneity, metastatic mechanisms, and interactions with the blood microenvironment.^[22,23] For example, our prior work leveraged single-cell RNA sequencing (scRNA-seq) to map immune checkpoint dynamics on CTCs, exposing vulnerabilities for immunotherapy and metastasis blockade.^[14,24] These advances have catalyzed the discovery of CTC-targeted therapeutic markers and predictive signatures for metastasis, propelling CTC research into an era of clinical translation.

Crosstalk between CTCs and Circulatory Microenvironment

Upon entering the bloodstream, CTCs become susceptible to immune system attacks. However, some CTCs evade immunosurveillance and disseminate to distant organs, forming metastatic foci. The primary mechanism enabling this immune evasion is the crosstalk between CTCs and various circulating blood cells. Consequently, a comprehensive understanding of the microenvironment in which CTCs circulate is essential for elucidating the process of tumor metastasis and for devising innovative approaches to hinder metastatic spread.

Interplay between CTCs and platelets

Platelets, tiny anucleated blood cells with diameters ranging from 2 to 4 μm, are the second most prevalent cells in the bloodstream.^[25] Traditionally recognized for their crucial role in hemostasis and thrombosis, platelets have emerged as key contributors to the survival and metastasis of CTCs.^[26,27] Platelets act as the first responders to CTCs in the bloodstream.^[28] Many studies have demonstrated a complex interplay between CTCs and platelets: (1) CTCs induce platelet activation,^[26,29] (2) Platelets adhere to CTCs to protect them from blood flow shear and immunological surveillance, and (3) CTCs endocytose platelets and obtain platelet-derived genes to promote proliferation, increase stemness, and enhance immune evasion ability [Figure 1A and Figure 3].^[30,31]

Figure 3.

The crosstalk between CTCs and platelets. The CTC-platelet interaction follows the “4A” model: (A) Activation: CTCs activate platelets via soluble factors and direct contact, forming tumor-educated platelets. (B) Aggregation: Platelets aggregate through CTC interaction and MMP-2/CTSB secretion in tumor cell-induced platelet aggregation (TCIPA). (C) Acquirement: CTCs acquire platelet genes (e.g., PF4, CD41, CD61) via adhesion (GPIb-IX-V/GPIbα-vWF) or endocytosis (dynamin-/fusion-dependent). (D) Assistance: Platelets protect CTCs from shear stress and immune attack, support EMT (via TGF-β, PDGF), promote angiogenesis (VEGF), extravasation (P-selectin), and metastatic niche formation. Platelet-derived genes also help CTCs mimic normal cells to evade immunity. CTCs: Circulating tumor cells; CTSB: Cathepsin B; EMT: Epithelial-to-mesenchymal transition; GPIb-IX-V: Glycoprotein Ib-IX-V; GPIbα: Glycoprotein Ibα; MMP-2: Matrix metalloproteinase-2; PDGF: Platelet-derived growth factor; PF4: Platelet factor 4; TCIPA: Tumor cell-induced platelet aggregation; TGF-β: Transforming growth factor-β; VEGF: Vascular endothelial growth factor; vWF: von willebrand factor.

CTCs activate platelets through both direct and indirect mechanisms [Figure 3A]. Indirect activation involves the secretion of thrombin and tissue factor (TF), leading to platelet aggregation and external coagulation that promotes thrombosis.^[26,32] CTCs also induce platelet activation by releasing metabolites, such as adenosine diphosphate (ADP), P-selectin, and thromboxane A2 (TXA2).^[33] Other activators, such as interleukin (IL)-8, matrix metalloproteinase (MMP), the chromatin protein high mobility group box 1 (HMGB1), and tumor-derived TF-positive microvesicles, also activate platelets.^[26,34] Besides soluble regulators, CTCs directly activate platelets via receptor-ligand interactions, such as the interaction of the surface of podoplanin and disintegrin and metalloproteinase (ADAM) 9 on CTCs with C-type lectin-like receptor 2 (CLEC-2) and α6β1 on platelets.^[34] These interactions primarily lead to platelet activation and coagulation, resulting in increased thrombin generation known as tumor cell-induced platelet activation (TCIPA).^[10] The ultimate effect is to promote the survival and enhance the invasive capacity of CTCs. Moreover, tumor cells modify platelet behavior by inducing tumor-platelet aggregation [Figure 3B], releasing extracellular vesicles, altering platelet phenotypes and RNA profiles, and promoting thrombosis,^[10,26] giving rise to tumor-educated platelets (TEPs).^[35]

Platelets adhere to CTCs to promote their survival and metastasis. Activated platelets highly express adhesion molecules, such as integrins, P-selectin, and immunoglobulin superfamily proteins, which promote interactions with CTCs and further facilitate the formation of a protective layer [Figure 3C].^[34] The attachment of activated platelets provides physical protection by forming a thrombus envelope on the surface of CTCs, shielding them from shear stress in the blood [Figure 3C and D].^[36] Platelet-secreted mediators not only facilitate CTC-platelet adhesion but also enhance the interaction between CTCs and the vascular endothelium by upregulating the expression of P-selectin, E-selectin, integrin αIIbβ3, and α6β1 on the platelet surface.^[33,37] Increased adhesion facilitates the stagnation of CTCs in the vasculature, their extravasation, eventual arrival at the target organ, and colonization of the metastatic site. This process also promotes the formation of early metastatic niches. For instance, platelet-adhered CTCs secrete C-X-C motif chemokine ligand 5 (CXCL5) and CXCL7,^[38] which attract granulocytes and aid in the formation of early metastatic niches [Figure 3D].^[39]

CTCs acquire platelet-derived genes through endocytosed platelets to assist in their immune evasion. A large number of platelet-related genes, including PF4, CD41, and CD61,^[40] have been identified in CTCs, suggesting potential platelet fusion with CTCs [Figure 3C]. Our prior research revealed that platelets not only form a protective layer on the surface of CTCs but are also engulfed by CTCs.^[14] Numerous studies have corroborated these findings, demonstrating the ability of CTCs to phagocytose platelets, a process involving membrane fusion and dynamin [Figure 3C].^[31,41] Furthermore, CTCs demonstrate efficient uptake and utilization of platelet-derived lipids, nucleic acids, and proteins.^[31] Consequently, CTCs evade immune recognition via a mechanism termed platelet mimicry, which simultaneously enhances their proliferation and stemness and is instrumental in facilitating their successful immune evasion.^[42]

Interaction between CTCs and NK cells

NK cells play a vital role in the immune system, contributing to the body’s defense against various diseases, including cancer.^[43,44] Typically comprising about 5–15% of circulating lymphocytes,^[45,46] NK cells inhibit the dissemination of tumor cells by blocking their proliferation, migration, and colonization in new tissues without requiring prior sensitization.^[46,47] Studies have shown that NK cells can eliminate up to 80% of CTCs within 24 hours of their entry into the bloodstream.^[48] Our previous study analyzed the interaction between CTCs and blood-derived immunocytes by utilizing scRNA-seq technology and revealed that NK cells are the predominant immune surveillants of CTCs.^[14] Mego et al^[49] reported that patients with metastatic breast cancer harboring ≥5 CTCs per 7.5 mL of peripheral blood exhibit significantly reduced NK cell function compared to those with <5 CTCs per 7.5 mL. Moreover, several research studies have highlighted a strong correlation between the NK cell status and the prognosis of cancer patients.^[50] Thus, NK cells are considered the primary immune surveillance cells targeting CTCs, serving as an independent factor for the progression of CTCs.^[50,51]

CTCs exert immunosuppressive effects on NK cells through ligand-receptor interactions [Figure 1B].^[52] CTCs can interact with immune checkpoints on the surface of NK cells, thereby inhibiting NK cell activity and compromising their immune surveillance function.^[53–55] Specifically, CTCs modulate the expression levels of various MHC molecules on their surface to interact with inhibitory receptors on NK cells,^[56] such as natural killer group 2D (NKG2A) and killer-cell immunoglobulin-like receptor (KIR) protein families, or activating receptors like NKG2D, thereby impairing the immune surveillance function of NK cells.^[57] Moreover, CTCs can suppress the function and metabolism of NK cells through the secretion of inhibitory factors and the recruitment of other inhibitory immunocytes. For example, CTCs-derived nitric oxide synthase 2 (NOS2) impairs the metabolism and proliferation of NK cells.^[58] CTCs secrete inhibitory factors transforming growth factor-β (TGF-β), transforming NK cells into a noncytotoxic state that promotes metastasis and angiogenesis.^[59] Furthermore, CTC-derived TGF-β recruits regulatory T cells (Tregs) and MDSCs, further impeding NK cell activity.^[59,60] In conclusion, the interplay between NK cells and CTCs reduces the NK cell quantity and impairs their function, which further fuels cancer metastasis.

Interaction between CTCs and T cells

T cells, integral components of the adaptive immune system, play a pivotal role in tumor progression and metastasis. Mego et al^[49] first proposed that patients with at least one CTC in 7.5 mL of peripheral blood exhibit adaptive immune abnormalities. This research established a link between CTCs and various T cell subsets, aiming to elucidate the mechanisms by which CTCs evade immune surveillance. Chalfin et al^[61] subsequently reported that patients with programmed death-ligand 1 (PD-L1)-positive CTCs and diminished CD8⁺ T cell counts exhibit reduced survival rates. Moreover, diverse T cell populations have been implicated in either facilitating or impeding CTC activity.^[62] Specifically, CTCs alter their surface molecule expression to escape recognition by cytotoxic T cells.^[61] Additionally, CTCs can attenuate the function of effector T cells or induce a transition towards an immunosuppressive phenotype through the secretion of specific regulatory molecules.^[63]

CD8⁺ T cells are regarded as the predominant immunocytes for regulating tumor growth and metastasis.^[64] In the early stages of metastasis, CTCs employ multiple strategies to evade the cytotoxic effects of CD8⁺ T cells, including modulating MHC-I expression and immune checkpoint signaling [Figure 1C].^[64,65] Tregs, a subset of CD4⁺ T cells defined by the expression of FOXP3, are recruited by CTCs via the release of chemokines, including chemokine ligand (CCL) 5, CCL22, CCL17, CXCL9, and CXCL10 [Figure 1C].^[66] Tregs bolster CTCs’ viability by inhibiting CD8⁺ T cells’ proliferation and attenuating the cytotoxic actions of NK cells and dendritic cells (DCs).^[67] Taken together, the interactions between Tregs and CTCs are associated with augmented metastatic potential.^[63]

Crosstalk between CTCs and neutrophils

Neutrophils, which originate from bone marrow precursors, are widely acknowledged as the predominant innate immune cells in bone marrow and peripheral blood and exhibit notable plasticity and diversity.^[68] In tumor environments, the activation and conversion of neutrophils into prometastatic forms by tumor cells suggest that CTCs may similarly influence neutrophils through unidentified mechanisms, highlighting a potential avenue for future investigation.

Recent advancements in CTC capture techniques have facilitated the identification of CTC-neutrophil clusters in the bloodstream.^[69] CTCs express intercellular cell adhesion molecule-1 (ICAM-1), which binds to lymphocyte function-associated antigen-1/macrophage-1 antigen (LFA-1/MAC-1) receptors on neutrophils, thereby facilitating their interaction [Figure 1D].^[43] Szczerba et al^[70] conducted a cellular analysis of blood samples from 70 breast cancer patients and tumor-bearing mouse models and revealed the presence of CTC-white blood cell (WBC) clusters, with neutrophils being the predominant type of white blood cell in most clusters. Moreover, their experiments demonstrated that CTC-neutrophil clusters represent the most efficient subset of cells for metastasis formation within breast cancer CTCs. The existence of these clusters in patients’ blood was associated with an unfavorable prognosis compared to other forms of CTCs. The interaction between neutrophils and CTCs plays a crucial role in facilitating the metastasis of CTCs at various stages: (1) Neutrophils protect CTCs from immune surveillance and elimination. For example, CTC-derived TGF-β stimulates neutrophils to produce Arginase 1 (ARG1), leading to reduced arginine levels in target cells and inhibition of CD3 molecule synthesis, thereby suppressing T cell activation and proliferation.^[71] (2) Neutrophils facilitate CTC extravasation and seeding by agitating the premetastatic niche. For instance, activated neutrophils in the premetastatic niche secrete IL-1β, tumor necrosis factor-α (TNF-α), IL-6, and cyclooxygenase-2 (Cox-2), leading to the degranulation of azurophilic granules and the seeding of CTCs.^[72] (3) Neutrophils awaken dormant CTCs and facilitate their proliferation. Neutrophils also secrete neutrophil elastase (NE) and MMP-9, which degrade extracellular matrix (ECM) components, further disrupting the dormant state of tumor cells, driving cell cycle progression, and promoting the intravasation of CTCs.^[73,74]

Interaction between CTCs and macrophages

Macrophages, which originate from myeloid progenitor cells, exhibit multiple functions and engage in processes crucial for immune defense and homeostasis, including phagocytosis, immune regulation, tissue repair, remodeling, and antigen presentation.^[75] In recent years, emerging elegant studies have illuminated the existence of CTC-macrophage clusters within the circulating bloodstream, a phenomenon characterized by the interaction of CTCs and macrophages [Figure 1E].^[76] CTCs exhibit high levels of microtentacles on their cell membranes, which are microtubule-forming cellular protrusions supported by vimentin and various forms of tubulin.^[77] The presence of microtentacles not only enables CTCs to withstand shear stress and anoikis but also facilitates their aggregation with macrophages in the bloodstream.^[76,78]

In addition, the ligands and receptors on the surfaces of CTCs and macrophages play a critical role in mediating their interaction. For example, the CD47/signal regulatory protein α (SIRPα) axis functions as a myeloid-specific immune checkpoint that is essential for evading phagocytosis [Figure 1E].^[11] Macrophages also secrete protumor factors, epidermal growth factor (EGF), and CXCL8 (also known as IL-8), which can efficiently promote the CTC-mediated tumor metastasis.^[79,80] Moreover, macrophages contribute to tumor metastasis through phenotypic transformation, exemplified by the emergence of cancer-associated macrophage-like cells (CAMLs). CAMLs are tumor-associated macrophages (TAMs) after phagocytosis of tumor cells and dissemination into the peripheral blood.^[81,82] CAMLs thus acquire stemness and surface molecular markers similar to those of tumor cells.^[76,83] Adams et al^[76] identified CAMLs bound to CTCs in the peripheral blood of patients with metastatic tumors. They also hypothesized that CAMLs and CTCs may bind at the primary site and disseminate into the peripheral blood by a similar endocytosis. This suggested that CAMLs possessed the ability to promote CTCs’ migration across the endothelium and lead to metastasis.

Overall, macrophages play critical roles in CTCs dissemination, intravasation, and cluster formation. Illustrating their interplay mechanism may provide valuable insights for developing targeted therapies against cancer metastasis.

Interactions between CTCs and myeloid-derived suppressor cells

MDSCs, characterized by a phenotype of CD11b⁺CD33⁺HLA-DR^–/low,^[84] possess immunosuppressive capabilities.^[85] Specifically, MDSCs contribute to tumor progression through two main mechanisms: (1) facilitating the evasion of immune surveillance by T and NK cells for CTCs,^[86] and (2) directly interfering with CTC/CTC clusters, thereby promoting their tumorigenic potential [Figure 1F].^[87,88]

On one hand, MDSCs suppress antitumor immune response of T cells by depleting essential nutrients, hindering migration to tumor sites, triggering apoptosis in antitumor T cells, and promoting the transformation of initial CD4⁺ T cells into protumor Tregs.^[89] MDSCs also decrease the population of NK cells and impede their function through membrane contact-dependent mechanisms.^[89,90]

On the other hand, the inducible nitric oxide synthase-mediated production of reactive oxygen species (ROS) is a fundamental function of MDSCs.^[91] Studies have shown that distinct subsets of MDSCs in mice express varying levels of inducible nitric oxide synthase (iNOS) and ARG1. These enzymes inhibit the function of immune effector cells by generating ROS, thereby promoting tumor cell proliferation and metastasis.^[89] Due to their interaction with CTCs, MDSCs facilitate the proliferation and metastasis of CTCs by activating survival signaling pathways, including the Notch signaling pathway, in response to ROS generated by MDSCs.^[88] These factors enhance the survival and effective dissemination of CTCs, as well as the stimulation of the aggregation of CTCs and MDSCs.^[87,88]

Interaction between CTCs and other immunocytes

Beyond the major immune populations discussed earlier, emerging evidence reveals that CTCs dynamically engage with B cells, DCs, and mast cells to orchestrate immune evasion.

Recent studies have progressively elucidated the multifaceted roles of B cells in tumor progression. While certain B cell subsets (e.g., plasma cells) may exert anti-metastatic effects via antibody-dependent cellular cytotoxicity (ADCC), some B cells can enhance CTC invasiveness through multiple mechanisms: (1) secretion of pro-angiogenic factors (VEGF-C, IL-8) and MMP-2/9 to remodel the ECM; (2) induction of a Th2-polarized cytokine milieu (IL-4, IL-10) that dampens cytotoxic T cell activity; and (3) differentiation of regulatory B cells (Bregs) that directly suppress NK cell-mediated CTC clearance.^[92] Strikingly, CTCs reciprocally modulate B cell function by overexpressing CD40L and BAFF, which promote Breg expansion while inhibiting antigen-presenting B cell maturation.^[93]

DCs, as professional antigen-presenting cells, are subverted by CTCs to establish immune tolerance. Direct contact between CTCs and DCs induces downregulation of MHC-II and co-stimulatory molecules (CD80/86) on DCs, impairing their ability to activate tumor-specific T cells.^[94] Furthermore, CTC-DC clusters exhibit upregulated adhesion molecules (ICAM-1, VCAM-1), facilitating vascular arrest and extravasation.^[95] ScRNA-seq reveals that CTC-educated DCs adopt a tolerogenic phenotype characterized by PD-L1^hi, IDO1^hi, and IL-10^hi signatures, which recruit Tregs to establish metastatic niches.^[96,97]

While the functional roles of mast cells in tumor progression have been gradually unveiled in recent years,^[98,99] their low abundance in circulation has hindered investigations into CTC-mast cell interactions. Although no direct evidence currently exists, mast cells are known to modulate vascular permeability and immune suppression in metastatic niches,^[100,101] suggesting a plausible but understudied role in regulating CTC behavior. However, the rapid advancement of single-cell technologies promises to elucidate the regulatory effects of mast cells on CTC functional states, potentially revealing novel mechanisms of immune evasion during hematogenous spread.

Interaction between CTCs and endothelial cells

Endothelial cells, the specialized cells lining blood vessels, exert a crucial influence on the intricate mechanisms of cancer metastasis, particularly in facilitating the movement of CTCs into and out of blood vessels. In the metastatic cascade, these endothelial cells play a dual role, orchestrating both the ingress of CTCs into the bloodstream (intravasation) and their subsequent egress from vessels into distant tissues (extravasation). Through intricate molecular interactions and signaling pathways, endothelial cells regulate the adhesion, migration, and invasion of CTCs, thereby impacting the dissemination of cancer cells throughout the body.

The intravasation of CTCs and endothelial junction disruption are key steps for tumor cells to enter the blood. Pre-CTCs refer to the state before fully penetrating the endothelium. They traverse vascular endothelial layers during metastasis to become CTCs. This critical step involves two main processes: paracellular intravasation and transcellular intravasation [Figure 1G].^[102] Paracellular intravasation disrupts endothelial tight junctions, allowing pre-CTCs to migrate with MMP-1 assistance.^[103] Pre-CTCs also exploit the Notch receptor to engage with Notch ligands on endothelial cells, promoting metastasis through intercellular junctions.^[104] In transcellular transmigration, pre-CTCs trigger actomyosin contraction via Ca²⁺-calmodulin, forming pore-like structures for rapid passage.^[105]

Correspondingly, the mechanisms of CTCs extravasation and endothelial interaction are crucial for understanding how they exit the bloodstream and invade new tissues.^[106] CTCs must breach the vascular barrier and exit the circulation to establish metastatic colonies. Endothelial cells actively participate in this intricate phenomenon by engaging in adhesive interactions with cancer cells, ligands or receptors that can interact with molecules on the surface of CTCs [Figure 1G].^[102] Initial attachment is facilitated by E-selectin on endothelial cells binding to CD44 or sialyl Lewis a (sLe^a)/sLe^x.^[107,108] Stable adhesion involves integrins, CD44, and/or MUC1, while secreted factors influence endothelial functionality.^[109,110]

Taken together, these findings indicate that the interaction between endothelial cells and CTCs is crucial for metastatic lesion formation. Thoroughly underlying the mechanism of CTC-endothelial cell interactions will enhance our understanding of metastatic mechanisms and may unveil novel insights and strategies for cancer therapy.

Immune Evasion Mechanisms of CTCs

In light of the interaction between CTCs and the blood circulation microenvironment, we summarized the mechanisms by which CTCs evade immune surveillance. These mechanisms are broadly categorized into two intrinsic and extrinsic factors [Figure 4]. Intrinsic mechanisms refer to changes within CTCs to promote immune evasion, including upregulation of immune checkpoint ligands, alteration of MHC I, and upregulation of FasL expression. Extrinsic mechanisms encompass the capacity of CTCs to evade immune responses through the assistance of other circulating cells, including the formation of CTC clusters and engagement of platelets.

Figure 4.

Deciphering the immune evasion mechanisms of CTC. (A) CTCs express immune checkpoint ligands (e.g., HLA-E, PD-L1, CD47) to evade recognition by NK cells, T cells, neutrophils, macrophages, and dendritic cells. (B) CTCs downregulate classical MHC I (e.g., HLA-A, HLA-B), mimic platelet-derived MHC I to appear normal, reduce MICA/B (NKG2D ligands), and upregulate non-classical MHC I (e.g., HLA-E, HLA-G) to suppress NK and T cell activity. (C) CTCs avoid FasL-induced apoptosis by reducing Fas expression, increasing FasL, or limiting exposure to soluble FasL from immune cells. (D) CTC clusters, formed via intercellular adhesion or receptor–ligand interactions, resist anoikis, enhance invasiveness, and integrate supportive signals from immune cells, promoting survival and metastasis. (E) Platelets shield CTCs from immune detection, and platelet endocytosis enables CTCs to acquire genes that upregulate immune checkpoints and promote metastasis. CTCs: Circulating tumor cells; Fas: First apoptosis signal receptor; FasL: Fas ligand; HLA-A: Human leukocyte antigen-A; HLA-B: Human leukocyte antigen-B; HLA-E: Human leukocyte antigen-E; HLA-G: Human leukocyte antigen-G; MHC I: Major histocompatibility complex I; MICA/B: MHC I chain-related A/B; NK cells: Natural killer cells; NKG2D: Natural killer group 2D; PD-L1: Programmed death-ligand 1.

CTCs employ immune checkpoints to escape immune surveillance

Immune checkpoints are receptor-ligand pairs that inhibit or stimulate the immune response.^[111] CTCs camouflage themselves from recognition by immune cells through the upregulation of the expression of immune checkpoints [Figure 4A]. Several immune checkpoints have been identified on CTCs and immunocytes that present as potential drug targets, including PD-1/PD-L1,^[112,113] lymphocyte activation gene 3 (LAG-3)/fibrinogen-like protein 1 (FGL-1),^[114] and CD47/SIRPα [Figure 5].^[115]

Figure 5.

The immune checkpoints between CTCs and immunocytes in the blood circulation. The left panel illustrates the immune checkpoint molecular pairs between CTCs surface ligands and the receptors on T cells, macrophages, and DCs. The right panel shows the immune checkpoints between CTCs and NK/neutrophils. ARG-1: Arginase-1; CTCs: Circulating tumor cells; EGF: Epidermal growth factor; ERK: Extracellular signal-regulated kinase; HLA: Human leukocyte antigen; MDSC: Myeloid-derived suppressor cell; MHC-I: Major histocompatibility complex class I; MLC: Myosin light chain; MMP-1: Matrix metalloproteinase-1; NO: Nitric oxide; NK: Natural killer; PAR1: Protease-activated receptor 1; PD-L1: Programmed death-ligand 1; PGE2: Prostaglandin E2; PI3K: Phosphoinositide 3-kinase; ROS: Reactive oxygen Species; SIRP α: Signal-regulatory protein α; TGF-β: Transforming growth factor-β; Treg: Regulatory T cell.

Tumor cells escape T cell immune surveillance by upregulating PD-L1, which engages with PD-1 on T cells and induces their dysfunction. PD-1/PD-L1 is one of the most prevalent immune checkpoints involved in multiple malignancies.^[116] Mazel et al^[117] reported that 68.8% of ER⁺HER^– breast cancer patients included in the cohort had PD-L1⁺ CTCs. This percentage was 54.4% for head and neck squamous cell carcinoma and 64% for melanoma.^[118,119] Similar to PD-L1, other B7 family immune checkpoint molecules, such as CD276, have also been identified on CTCs.^[120] CD276 is commonly expressed on more than 50% of prostate cancer-transformed CTCs, and PD-L1 and PD-L2 are expressed on approximately 20% of prostate cancer-transformed CTCs.^[121] CTCs from colorectal and ovarian cancers have been shown to engage with the immune checkpoint T cell immunoglobulin and ITIM domain (TIGIT) via CD155/CD112, thereby inhibiting T cell activity.^[122]

Apart from T cell-mediated adaptive immune surveillance, innate immunocytes, such as NK cells and macrophages, are the main immune surveillants of CTCs in the blood circulation.^[51] In our previous study, we found that CTCs escape NK cell surveillance by hijacking the immune checkpoints HLA-E: CD94-NKG2A and HLA-C: KIR2DL1.^[14,15] Studies suggest that HLA-E binds to the NKG2A/CD94 receptor on NK cell surfaces, recruiting protein tyrosine phosphatases to the cytoplasmic membrane and thereby suppressing the cytotoxic function of NK cells. Moreover, IL-8 derived from CTCs upregulates the expression of PD-1 on NK cells and promotes NK cell-dependent tumor metastasis in a PD-1/PD-L1-mediated manner.^[16] Recently, it has been demonstrated that CD155 on CTCs can also inhibit the cytotoxic activity of NK cells through its interaction with the TIGIT receptor on NK cells.^[17] CTCs hijack the CD47/SIRPα checkpoint to evade phagocytosis by macrophages. By overexpressing CD47, CTCs deliver a “do not eat me” signal through SIRPα binding, effectively suppressing macrophage-mediated clearance.^[11] This mechanism is particularly critical in cancers, such as lung adenocarcinoma and prostate cancer, where CD47-positive CTCs exhibit enhanced metastatic potential.^[113] Collectively, these checkpoint interactions enable CTCs to circumvent both adaptive and innate immune surveillance, underscoring their role as pivotal mediators of immune evasion during hematogenous spread.

CTCs agitate MHC I molecules to escape immune surveillance

MHC class I molecules, also referred to as HLA class I molecules, are glycoproteins that are extensively present on the surface of human cells. They consist of classical (Ia) antigens, HLA-A, HLA-B, and HLA-C, and nonclassical (Ib) antigens, HLA-E, HLA-F, and HLA-G.^[123] Comprising heavy (α) and light (β2 microglobulin) chains linked by noncovalent bonds, these molecules play a pivotal role in the immune system. The presentation of antigens by MHC I molecules is crucial for activating CD8⁺ T cells and NK cells.^[124–126] However, CTCs dynamically manipulate MHC I expression to evade immune detection.^[11,127]

On the one hand, CTCs impair antigen presentation to CD8⁺ T cells and allow CTCs to escape CTL-mediated lysis by downregulating classical MHC class I molecules,^[124] such as HLA-A and HLA-B [Figure 4B].^[128] On the other hand, CTCs escape the immune surveillance of NKs by regulating nonclassical MHC class I molecules [Figure 4B]. Tumor cells often show increased levels of nonclassical MHC class I molecules to suppress NK cell cytotoxicity, such as HLA-G and HLA-E.^[125] In addition, MICA/MICB also mediated the immune evasion of CTCs. In the absence of MHC I molecules, NK cells can induce cytotoxicity against cancer cells by interacting with NKG2D and MICA/MICB ligands, which are predominantly expressed on virus-infected and/or tumor cells. Decreasing MICA/MICB expression can enhance resistance to NK cell-mediated cytotoxicity.^[129]

CTCs induce immunocyte apoptosis via the Fas/FasL axis

FasL is a type II transmembrane protein of the TNF family known for its role in extrinsic apoptosis through Fas binding.^[130] FasL is widely expressed in activated T cells, NK cells, macrophages, and certain tumor cells. Its main function is to initiate apoptosis upon binding to Fas receptors. The interaction between FasL and Fas results in the formation of a death-inducing signaling complex (DISC), triggering a cascade of reactions that culminate in cell death. Hence, FasL holds significant promise for tumor immune surveillance and immunotherapy. However, in specific scenarios, CTCs evade FasL-induced apoptosis by downregulating Fas expression, upregulating FasL expression, or secreting soluble FasL (sFasL), thus evading immune attacks and fostering tumor progression [Figure 4C]. This evasion strategy enables continued tumor proliferation and dissemination, leading to disease progression.^[131]

CTCs can induce apoptosis in Fas-expressing immune cells by upregulating FasL, while downregulating Fas reduces their recognition by FasL-expressing immune cells.^[132] This dual alteration in Fas and FasL expression enables CTCs to evade immune-mediated apoptosis and facilitates their metastatic potential.^[133] Specifically, upon interaction, NK cells express FasL, which induces caspase-dependent apoptosis in CTCs.^[134] The binding of Fas-expressing T cells to FasL on tumor cells triggers T cell apoptosis, shifting tumor cells toward a proapoptotic phenotype and increasing susceptibility to tumor-induced immune suppression. Furthermore, immune cells express a soluble form of FasL, sFasL, which retains its ability to interact with Fas but exhibits lower cytotoxicity than membrane-bound FasL, explaining the absence of systemic tissue damage despite elevated levels of circulating sFasL in certain cancers.^[135]

CTC clusters accelerate their immune evasion and metastasis

CTC clusters are multicellular aggregates of two or more cells held together through intercellular junctions. Initially detected at necropsy, these clusters are believed to be intravascular tumor microemboli.^[136,137] CTC clusters have been found in a wide variety of tumor types, including breast cancer,^[138] lung cancer,^[139] prostate cancer,^[140] hepatocellular carcinoma,^[141] and colorectal cancer.^[142] CTC clusters exhibit decreased susceptibility to immune cell-mediated destruction, increased resistance to diverse stimuli and attacks, and heightened metastatic capabilities.

There were two types of CTC clusters: homozygous (composed purely of CTCs) and heterozygous (consisting of CTCs interacted with other cell types) [Figure 4D].^[143] While homozygous clusters are relatively simpler in composition, heterozygous clusters exhibit greater biological complexity due to their heterogeneous cellular makeup, making them a focus of recent research. Aceto et al^[144] demonstrated that CTC clusters arise from the aggregation of CTCs rather than from the proliferation of a single CTC. Heterozygous clusters, in contrast, involve dynamic interactions between CTCs and stromal components. When CTC clusters were captured using the Cluster-Chip, it was found that CTC clusters contain both actively proliferating and quiescent types of CTCs, along with tumor-associated macrophages.^[145] Additionally, in 2001, Duda et al^[146] showed that circulating endothelial cells and fibroblasts contribute to the aggregation of CTCs. Moreover, leukocytes and platelets have also been shown to be involved in the formation of CTC clusters.^[70,147]

CTC clusters are less abundant in the circulation but more metastatic than single CTCs. In Aceto et al’s^[144] mouse experiments, single CTCs accounted for 97.4% of the total, while CTC clusters represented 2.6%. Remarkably, the metastatic capacity of CTC clusters was 23–50 times greater than that of a single CTC, suggesting a potential link between the presence of CTC clusters and adverse outcomes. There are several reasons why CTC clusters are more metastatic than single CTCs [Figure 4D]: (1) The collaboration of cells within CTC clusters bolsters immune evasion capabilities, ultimately promoting the formation of metastatic sites.^[148] (2) The formation of CTC clusters induces enhanced stemness and mobility abilities that are absent in single CTCs. Gkountela et al^[149] reported that CTC cluster aggregation results in hypomethylation of OCT4, NANOG, SOX2, and SIN3A binding sites, predominantly active in embryonic stem cells, that confer on CTC clusters the ability to regulate self-renewal and proliferation. CTC clusters are characterized by highly variable cell morphology and greater deformability than single CTCs.^[69] They move more slowly in circulation, closer to the vascular endothelium,^[150] and more rapidly extravasate.^[69] (3) CTC clusters show greater tolerance to blood flow shear stress and lower apoptosis rates than single CTCs due to increased cell number and size.^[148] To be precise, several mechanisms protect CTC clusters from anoikis, including the interaction between circulating galectin-3 and the transmembrane mucin MUC1 on CTC clusters,^[150] and the overexpression of the antiapoptotic protein Bcl2.^[151] In addition to anoikis, NK cells are also more inclined to recognize and clear single CTCs, with a significantly diminished killing effect of CTC clusters.^[152] (4) The cell-cell junctions required for the formation of CTC clusters are not only the basis of adhesion but also important for the manifestation and maintenance of stemness in CTC clusters.^[149] Various cell-cell junction proteins, including plakoglobin, E-cadherin, Ig superfamily cell-adhesion molecules (CAMs), integrins, and heparin sulfate proteoglycans, aggregate cells to form CTC clusters.^{[149,150,153]} Plakoglobin, identified as a critical component of adherens junctions and bridging granules, contributes to intercellular adhesion and metastatic foci formation of CTCs in the study by Aceto et al.^[144] The presence of intercellular adhesion mechanisms not only aggregates cells, but also protects the pluripotency and retains stem cell-like properties that facilitate the metastasis initiation.

In conclusion, the formation of CTC clusters increases their invasiveness while weakening the effectiveness of various damage effects from others, enabling their survival in circulation and promoting metastasis.

CTCs engage platelets to evade immune surveillance

CTCs exploit the support provided by adhered or engulfed platelets to regulate immunosuppression effects from both internal and external perspectives [Figure 4E].

Activated platelets create a physical barrier on the surface of CTCs, rendering them cloaked to evade detection by circulating immune cells.^[154,155] This defense mechanism involves platelets secreting TGF-β and platelet-derived growth factors (PDGFs).^[154,156] Additionally, platelets transfer their own MHC I molecules to the surface of CTCs while suppressing the expression of CTC-derived MHC I.^[32] This dual-action mask CTCs from T cell recognition by mimicking host cell surface markers.

Regarding internal regulation, CTCs acquire platelet-associated genes, mRNAs, and other important components by endocytosis of platelets and regulate their cellular behavior accordingly.^[157] For instance, the uptake of platelet-derived RGS18 activates the AKT-GSK3β-CREB signaling pathway in CTCs, upregulating HLA-E expression.^[14] These intracellular changes are ultimately reflected in the inhibition of activation, proliferation, and immune surveillance of T/NK cells by CTCs.^[14,115] Furthermore, these genetic alterations modify lipid composition of the nuclear membrane and affect the cell cycle, genome regulation, and cell signaling.^[14,157] As a result, CTCs undergo a reprogramming process that facilitates immune evasion, proliferation, stemness, and metastasis.

CTC-based Cancer Treatment: From Bench to Clinic

Given the crucial involvement of CTCs in the cascade of tumor metastasis, targeting and killing CTCs is of significant importance in impeding metastatic spread. By elucidating the crosstalk between CTCs and blood cells, as well as understanding the immune evasion strategies employed by CTCs, it is possible to activate the patient’s immune system to target and kill CTCs, offering new therapeutic strategies for preventing tumor metastasis in clinical practice. Based on this, therapeutic approaches aimed at regulating CTCs’ microenvironment and hindering the immune evasion pathways encompass the utilization of antiplatelet medications, immune checkpoint blockade, along with the direct targeting of CTCs and disintegration of CTC clusters [Figure 6 and Supplementary Table 1, http://links.lww.com/CM9/C540].

Figure 6.

Strategies targeting CTCs. (A) Targeting platelets: CTCs interact with platelets to aid immune evasion and metastasis. Anti-platelet agents (e.g., COX-1 inhibitors, P2Y12/PAR1 antagonists, thrombin and P-selectin inhibitors, αIIbβ3 blockers) disrupt platelet activation, adhesion, and aggregation. (B) Immune checkpoint blockade: Inhibitors targeting PD-L1, CD47, and other checkpoints restore immune responses against CTCs. (C) Targeting oncogenic pathways: CTCs carry mutations (e.g., BRAF, EGFR, KRAS, PIK3CA); pathway-specific inhibitors have therapeutic efficacy. (D) Targeting CTC clusters: Na⁺/K⁺-ATPase inhibitors disrupt cell-cell adhesion; mobilizing immune cells with antibodies or inhibitors may weaken CTC support. BRAF: B-type rapidly accelerated fibrosarcoma; COX-1: Cyclooxygenase-1; CTC: Circulating tumor cell; EGFR: Epidermal growth factor receptor; KRAS: Kirsten rat sarcoma sarcoma viral oncogene homolog; Na⁺/K⁺-ATPase: Na⁺/K⁺-transporting adenosine triphosphatase; PAR1: Protease-activated receptor 1; PD-L1: Programmed death-ligand 1; PIK3CA: Phosphatidylinositol 3-kinase, catalytic alpha subunit; P2Y12: Purinergic 2Y 12 receptor.

Disrupting the protective function of platelets on CTCs

Platelets’ quantity and functional changes are among the most important indicators of tumor progression or metastasis. Considering the intricate interplay between platelets and CTCs, potential directions for antiplatelet drugs in treating tumor metastasis involve inhibiting platelet activation by CTCs and counteracting the protective effect of platelets on CTCs. Antiplatelet drugs mainly include COX antagonists, αIIbβ3 integrin antagonists, and P2Y12 antagonists, which can be broadly classified based on their impact on either platelet activation and amplification or platelet aggregation [Figure 6A and Supplementary Table 1, http://links.lww.com/CM9/C540].

As the earliest and most commonly utilized antiplatelet agent, Aspirin acts as an irreversible inhibitor of COX.^[158] By directly targeting the procarcinogenic activity of platelets through COX inhibition, Aspirin obstructs key enzymes involved in tumor proliferation.^[159] Yang et al^[160] demonstrated that Aspirin reduces the number of CTCs and blocks the acquisition of the epithelial-mesenchymal (EMT) phenotype in patients with metastatic colorectal and breast cancer. Furthermore, Aspirin can impede metastasis by inducing anoikis in CTCs through the thromboxane A2 (TXA2) pathway.^[161] In addition to Aspirin, three other types of antiplatelet drugs also with the potential to treat CTC-based cancer metastasis.^[10] Integrins, critical receptors for platelet adhesion, are involved in various steps of tumor metastasis and are associated with the development of drug resistance in tumor therapy.^[162] αIIbβ3 is one of the major platelet surface receptors involved in TCIPA.^[163] Therefore, the application of αIIbβ3 integrin antagonists disrupts the protective effect of platelets as a physical barrier around CTCs.^[164] Aside from integrins, the inhibition of TCIPA and subsequent antitumor effects have been demonstrated by blocking P-selectin and the P2Y12 receptor with specific blockers and antagonists. P2Y12, a receptor for platelet ADP, plays an important role in stabilizing platelet aggregation, activating integrins, and forming thrombi.^[165] Other studies have shown that P2Y12 deficiency would downregulate the EMT phenotype of CTCs by inhibiting the secretion of platelet-derived TGF-β.^[166] As a result, the invasiveness of CTCs is reduced, and lung metastasis of tumors is significantly decreased.^[167] Therefore, P2Y12 is important for antithrombotic therapy and serves as a promising target for inhibiting tumor metastasis. Available drugs for P2Y12 include Clopidogrel, Prasugrel, and Tegretol.

Furthermore, antiplatelet drugs not only inhibit the formation and metastasis of CTCs by reducing the platelet quantity and inhibiting platelet function. They also disrupt the interactions between CTCs and other immune cells that are mediated by platelets. This dual action leads to a more profound inhibitory effect on tumor progression.^[168] Additionally, research indicates that combining antiplatelet drugs with chemotherapy or immunotherapy can synergistically enhance the therapeutic effects of these treatments, resulting in improved clinical outcomes.^[31,169] Taken together, numerous studies of antiplatelet drugs have highlighted the intricate relationship between platelets and cancer, shedding light on the potential benefits of antiplatelet drugs in cancer treatment.

Treatment with immune checkpoint inhibitors

Observations indicate that CTCs can impede the surveillance and cytotoxic effects of immune cells by modulating the expression of surface immune checkpoint molecules, such as PD-L1, HLA-E, HLA-C, CD155, and CD47 [Figure 5]. Utilizing ICIs to alleviate the immunosuppressive impact of CTCs and restore immune clearance function offers a promising therapeutic approach to hinder metastasis by eliminating CTCs in circulation [Figure 6B and Supplementary Table 1, http://links.lww.com/CM9/C540].

By obstructing the interaction between PD-1 and PD-L1 or PD-L2, the proliferation of immune cells with cytotoxic capabilities will be improved. It can also reactivate the antitumor activity of T cells and other immune cells and inhibit tumor growth and metastasis.^[170,171] In a study of NSCLC patients treated with Pembrolizumab-targeted therapy, patients receiving Pembrolizumab experienced prolonged progression-free survival (PFS) along with decreased numbers of PD-L1⁺ CTCs.^[172] Besides PD-L1, we demonstrated that monalizumab blocks the NKG2A and HLA-E interaction, resulting in the restoration of antitumor activity in NK cells and CD8⁺ T cells in pancreatic ductal adenocarcinoma (PDAC) patients.^[14] Our mouse experiments indicated a higher activation ratio of NK cells, enhanced tumor cytotoxicity, and increased level of IFN-γ secretion when simultaneously blocking HLA-E: CD94/NKG2A and HLA-C: KIR2DL1/3 using monalizumab and lirilumab (a pan anti-KIR antibody) for metastasis prevention.^[15]

Ongoing studies are dedicated to creating novel medications, expanding the applications of existing drugs, and comparing the effectiveness of these agents when administered alone or in combination.^[111,173] Relatlimab, the first approved monoclonal antibody targeting LAG-3, when combined with a targeted PD-1 inhibitor, exhibited synergistic antitumor effects and enhanced efficacy.^[174,175] The utilization of anti-LAG-3 antibody has the potential to disrupt the tumor parenchyma, stimulate CD4⁺ and CD8⁺ T cells while decreasing the secretion of immunosuppressive factors, such as TGF-β and IL-10.^[176] Furthermore, monoclonal antibodies targeting the function of inhibitory receptors such as TIM-3,^[177] BTLA, and TIGIT have also shown considerable potential for clinical application,^[178] offering a novel approach for treating various tumor types that are inoperable or have metastasized.

Molecular targeted therapy for anti-CTC treatment

CTCs with multiple potential targets for targeted therapy. Current strategies aim to exploit vulnerabilities in CTCs through multiple approaches, including targeting genetic mutations, cell surface markers, and intracellular processes such as DNA repair or protein synthesis [Figure 6C and Supplementary Table 1, http://links.lww.com/CM9/C540].

One key strategy involves targeting oncogenic mutations or dysregulated signaling pathways unique to CTCs. For instance, primary tumor mutations genes, such as BRAF, EGFR, K-RAS, and PIK3CA, have been identified in CTCs across various cancers.^{[35,179–183]} These mutations not only serve as prognostic markers but also guide therapeutic decisions.^[184,185] In NSCLC, the detection of BRAF^V600E mutations in CTCs has led to the successful use of BRAF inhibitors like vemurafenib, improving progression-free survival in clinical trials.^[186] Novel mutations, including HSP90AB1, KDM5B, and NEDD9, discovered through single-cell sequencing of CTCs, are being explored for drug development.^[187]

Another approach focuses on surface antigens or receptors overexpressed on CTCs. Prostate-specific membrane antigen (PSMA), for example, is targeted by ¹⁷⁷Lu-J591 radioligand therapies, which achieve an 89% clearance rate of CTCs in castration-resistant prostate cancer.^[188] In pancreatic cancer, CD44v6-targeted polymeric micelles loaded with niclosamide effectively suppress metastasis by inhibiting Wnt/β-catenin and STAT3 pathways in CTCs,^[189] reducing metastatic burden by 73% in animal models. These antigen-directed therapies minimize off-target effects while maximizing CTC-specific cytotoxicity.

Disruption of DNA replication and protein synthesis represents a third pillar of CTC-targeted therapy. Platinum-based agents like cisplatin induce DNA crosslinking damage in CTCs,^[190] significantly reducing their counts in metastatic breast and colorectal cancers. Topoisomerase inhibitors such as etoposide cause lethal DNA double-strand breaks during replication, selectively eliminating CTCs in circulation. However, CTCs often exhibit enhanced DNA repair mechanisms, necessitating combination therapies. Preclinical studies demonstrate that PARP inhibitors (e.g., olaparib) synergize with DNA-damaging agents to overcome resistance in BRCA-mutant CTCs.^[191] Additionally, protein synthesis inhibitors like homoharringtonine block eukaryotic elongation factor 2 (eEF2),^[192] impairing CTC proliferation, while ribosome-targeting agents such as omacetaxine suppress metastasis-initiating CTCs through ribosomal activity inhibition.^[193]

Emerging strategies also exploit epigenetic vulnerabilities in CTCs. DNA hypermethylation of genes, such as TIMP2 and CDH1, in CTCs correlates with increased metastatic potential and immune evasion.^[194] Moreover, the DNA methylation profiles of CTCs often differ significantly from those of primary tumors and are modulated by treatment, indicating a dynamic and adaptive epigenetic landscape.^[194] This epigenetic remodeling enhances metastatic capability and facilitates immune evasion, as shown by the association between abnormal gene methylation and aggressive CTC phenotypes.^[195] Hypomethylating agents such as azacitidine reverse these epigenetic modifications, reducing CTC intravasation capacity by 4.8-fold in NSCLC patients. Novel therapies like guadecitabine, a DNA methyltransferase inhibitor currently in clinical trials, further reduce CTC counts by modulating EMT and stemness pathways.^[196]

Disrupting CTC clusters

The cohesive bond within CTC clusters strengthens their journey through the bloodstream, enhancing resilience and facilitating metastatic spread. Inhibiting this intercellular adhesion could disrupt their pathogenic alliance, potentially impeding metastatic progression. There are two main strategies for disintegrating CTC clusters: (1) disrupting intercellular connections or communication may cause CTC clusters to break down into individual cells, and (2) targeting specific cell types within the cluster, such as neutrophils or platelets, could destabilize CTC clusters [Figure 6D and Supplementary Table 1, http://links.lww.com/CM9/C540].

Interruption of cell-cell connections and communication

Strategies for disrupting CTC clusters involve utilizing pharmacological agents and nanotechnology to target intercellular connections, modify DNA methylation patterns, and enhance immune recognition, ultimately hindering metastasis [Supplementary Table 1, http://links.lww.com/CM9/C540]. One method for disassembling CTC clusters involves the use of Na⁺/K⁺-ATPase inhibitors, such as Ouabain and Digitoxin.^[197,198] These inhibitors increase the Ca²⁺ concentration within CTCs, reducing intercellular connection formation and breaking down CTC clusters into individual cells. This approach can modify DNA methylation patterns within CTCs, decreasing their proliferative capacity and ultimately impeding metastasis. Similarly, cancer-specific calcium nanoregulators are nanodrugs developed by Li et al^[199] that employ poly (lactic-co-glycolic acid) (PLGA) nanoparticles loaded with the anticancer drugs doxorubicin (DOX) and digoxin (DIG) (CPDD). Enclosed in a layer derived from 4T1 cancer cell membranes, these nanoparticles exhibit structural and property homology with CTC clusters. Consequently, CPDD specifically recognizes and binds to CTC clusters in vivo before being internalized by them for DOX and DIG release. DOX inhibits DNA synthesis and cell proliferation while eliminating CTCs; DIG elevates intracellular Ca²⁺ levels to selectively dismantle CTC clusters, enabling individual cells to be more readily recognized by the immune system for clearance.^[200] Furthermore, TRAIL/E-selectin-based liposomes utilize liposomes conjugated with E-selectin to specifically target white blood cells near CTC clusters. Subsequently, the TRAIL protein induces apoptosis in tumor cells, reducing the number of CTCs present and inhibiting tumor metastasis.^[201]

Targeting the partner of CTC clusters

CTCs adhere to platelets, neutrophils, macrophages, and other cells in the bloodstream to form CTC clusters. These clusters withstand shear forces from blood flow and mitigate potential damage from circulating cells or molecules.^[202] The strategy involves targeting neutrophils within CTC clusters, as they aid immune evasion. Eliminating neutrophils restores immune function, leading to CTC eradication. For example, disrupting CTC-neutrophil clusters and delaying VCAM-1 shedding were achieved with neutralizing antibodies against Ly-6G in a mouse model of breast cancer, effectively dismantling the interaction between CTCs and neutrophils.^[70] Additionally, macrophages would phagocytose exogenous particles, rendering them promising therapeutic targets.^[203] For instance, bisphosphonates, commonly used for the treatment of bone metastasis, inhibit the activity of M2-like macrophages, thereby attenuating tumor growth and dissemination.^[204] Moreover, immunotherapy has the potential to modulate macrophage polarization and augment the proportion of M1-like macrophages, such as IL-12 and IFN-α.^[205] Immunomodulatory agents such as lenalidomide and thalidomide can impede protumor cytokine secretion by M2-like macrophages while stimulating antitumor cytokine secretion by M1-like macrophages.^[206]

In conclusion, targeting specific molecular markers or signaling pathways linked to CTC clusters holds promise for developing precise and effective anticancer medications, offering novel insights and approaches for cancer treatment and prevention.

Clinical Applications of CTCs

Apart from as a therapeutic target for cancer treatment, CTCs are ideal biomarkers for liquid biopsy. CTCs are extensively employed in the field of early diagnosis, staging evaluation, and prognostic assessment by monitoring quantitative and molecular changes in real time.

CTC detection and isolation technologies

The clinical utility of CTC-based liquid biopsies hinges on standardized detection methods with high reproducibility. Currently, two FDA-cleared platforms dominate clinical workflows: the CellSearch^® system,^[207] which enriches CTCs via anti-EpCAM antibody-coated magnetic beads and validates them through cytokeratin/CD45/DAPI staining (Janssen Diagnostics), and the CTC-iChip,^[208,209] a microfluidic platform combining size-based sorting and immunomagnetic depletion of hematopoietic cells. These systems achieve sensitivities of 1-10 CTCs per 7.5 mL blood in metastatic cancers, with CellSearch^® demonstrating 85–92% inter-laboratory concordance in multicenter trials for breast and prostate cancer monitoring.^[210,211]

However, standardization challenges persist due to biological heterogeneity (e.g., EMT-driven EpCAM loss) and technical variability in pre-analytical steps. For instance, blood collection tube types (CellSave^®vs. EDTA) alter CTC recovery rates by 15–30%,^[212] while time-to-processing beyond 72 hours degrades RNA signatures critical for molecular profiling.^[213] Recent efforts by the ISLB (International Society for Liquid Biopsy) established consensus protocols (ISO 20184-3:2021) specifying fixation methods, storage temperatures (4°C), and maximum processing delays (48h) to minimize pre-analytical variability.

Emerging technologies are revolutionizing the standardization of CTC detection by integrating multi-parametric approaches that synergistically enhance sensitivity, analytical depth, and clinical utility. A prime example is the EPISPOT assay, which combines functional profiling of tumor-specific protein secretion (e.g., FGF2 or Cathepsin D) with dual detection of EMT markers (EpCAM and vimentin).^[214] This strategy achieves 78% sensitivity in early-stage cancers—a 26% increase compared to the 52% sensitivity of conventional CellSearch^® systems.^[215] Complementing this, microfluidic platforms such as the Herringbone-Chip employ a dual-mode capture mechanism: size-based filtration (8–15 μm pore size) selectively isolates CTCs while immunomagnetic anti-CD45/CD66b antibody conjugation removes >99% of hematopoietic cells, resulting in 95% purity.^[69] Moreover, preserved viability of CTCs enables robust downstream single-cell RNA sequencing, as demonstrated by Liu et al^[24], who developed an integrated microfluidic system for simultaneous CTC capture and transcriptomic analysis. Their work revealed dynamic interactions between CTCs and circulating immune cells, identifying CD40LG, HLA-C, etc, as novel immune checkpoints upregulated in metastatic CTCs, thereby proposing actionable targets for immunotherapy. In addition, machine learning algorithms also complement these advances; a convolutional neural network (CNN) trained on 15,000 annotated CTC images reduced false-negative rates by 22% in pancreatic cancer screening through automated morphological classification.^[216] To standardize clinical reporting, the CANCER-ID consortium established tiered criteria: validated Tier 1 thresholds (≥5 CTCs/7.5 mL blood for breast/prostate cancer prognosis),^[217] exploratory Tier 2 biomarkers (EMT scores based on vimentin and TWIST1 mRNA levels, plus CTC clusters ≥3 cells), and investigational Tier 3 genomic markers (copy number variation burden assessed via low-pass whole-genome sequencing).^[218] These coordinated technological and analytical frameworks collectively bridge biological complexity with clinical practicality, paving the way for harmonized CTC-based diagnostics.

Application of CTC detection in cancer early diagnosis

Substantial evidence suggests that CTCs may appear early in the course of cancer development, potentially even before a clinical diagnosis, given that cancer cells can initiate localized invasion and infiltration rapidly, often within just a few hours.^[219] As technologies evolve to detect these rare cells, CTCs are now recognized as a characteristic of cancer across all stages. Several clinical studies have explored the potential of CTCs in cancer detection using liquid samples from diagnosed patients. For instance, Zhang et al^[220] investigated the epithelial markers EpCAM, HER2, and MUC1 in 109 patients with epithelial ovarian cancer (EOC) and reported a notably greater percentage of CTC-positive patients (93%) among stage IA-IB patients than among CA-125-positive patients (64%) in the same cohort. CTCs have also been identified in gastric cancer (GC) patients. While this study was conducted in already diagnosed patients, it suggests that CTCs may be more sensitive than CA-125 for identifying early-stage disease. Kang et al^[221] observed CTCs in 90.5% (105/116) of GC patients, with a CTC threshold of ≥2/7.5 mL blood indicating the presence of GC. Furthermore, the combined detection of CTCs and tumor markers enhances cancer diagnosis. For pancreatic cancer, CA19-9, particularly in conjunction with detection of folate receptors-positive CTCs and extracellular vesicles (EVs) GPC1, is a promising diagnostic approach.^[222]

Application of CTC detection in the staging of cancer

In the 2010 edition of the Cancer Staging Manual by the American Joint Committee on Cancer (AJCC), CTCs were introduced into the TNM staging system as a novel criterion for distant metastasis (M stage). They were categorized as cM0(i+), bridging between the M0 and M1 stages. The definition of cM0 (i+) staging entails the absence of clinical or radiographic signs of distant metastases in the presence of tumor cells measuring no more than 0.2 mm, detected either microscopically or using molecular techniques in circulating blood, bone marrow, or other nonregional lymph node tissue in asymptomatic patients.^[223]

Application of CTC detection in the evaluation of cancer prognosis and personalized treatment

The detection of CTCs is crucially tied to tumor prognosis, as supported by extensive research. Sun et al^[224] suggested that EpCAM-positive CTCs could serve as real-time indicators for monitoring hepatocellular carcinoma (HCC) recurrence, and the presence of Tregs enhances the sensitivity of EpCAM-positive CTCs in predicting HCC recurrence. According to the 2018 AJCC Guidelines, the detection of CTCs is considered an additional prognostic indicator for assessing breast cancer, in addition to the ER/PR ratio, HER2 expression, Ki67 expression, and tumor histological grade.

Another important application of CTC in assessing cancer progression is to monitor tumor dynamics and evaluate the efficacy of therapy. Beyond initial detection and baseline enumeration, dynamic changes in CTC counts and molecular characteristics during treatment have emerged as critical prognostic indicators. A decline in CTC count following surgery, chemotherapy, or targeted therapy is frequently associated with improved clinical outcomes, whereas persistently elevated or rising CTC levels may reflect minimal residual disease or early recurrence, indicating an unfavorable prognosis.^[225,226] Accordingly, serial CTC monitoring offers a non-invasive strategy for evaluating treatment response and guiding risk stratification promptly.^[225] Beyond quantitative fluctuations, therapy-induced phenotypic alterations in CTCs, such as changes in HER2, EGFR, or PD-L1 expression, have also been observed. Notably, discordance between molecular profiles of CTC and those of primary or metastatic tumors underscores tumor heterogeneity and may provide early indications of emerging drug resistance.^[227,228] These phenotypic shifts can inform treatment strategies, such as transitioning to alternative targeted therapies upon the emergence of resistance. Thus, dynamic CTC profiling not only improves prognostic accuracy but also offers new avenues for personalized cancer management.

Challenges and Perspectives

CTC, often termed the “seeds” of metastasis, hold immense potential as therapeutic targets. However, their dynamic interplay with the blood microenvironment, including immune evasion via platelet-mediated MHC-I transfer, Fas/FasL-induced immune cell apoptosis, and MDSC-mediated immunosuppression, complicates therapeutic strategies. A critical challenge arises from the divergent molecular profiles of CTCs compared to primary tumors. For instance, tumor-associated antigens (TAAs), such as MUC1 and HER2, which are overexpressed in primary lesions and serve as targets for CAR-T therapy and antibody-drug conjugates, exhibit significant heterogeneity on CTCs.^[229] This variability stems from EMT, platelet cloaking, and selective pressures during circulation. Specifically, EMT downregulates epithelial markers (e.g., EpCAM) while upregulating mesenchymal markers (e.g., Vimentin and Twist), rendering EpCAM-dependent immunotherapies ineffective against aggressive CTC subpopulations.^[230] Concurrently, platelet-derived TGF-β suppresses MHC-I-mediated TAA presentation, further shielding CTCs from immune surveillance.^[4] These adaptive mechanisms not only limit the efficacy of TAA-targeted therapies but also underscore the urgency of discovering CTC-specific antigens through integrated single-cell multi-omics (genomics, proteomics) and AI-driven epitope prediction.

Beyond therapeutic targeting, CTCs represent a promising liquid biopsy tool for real-time monitoring of tumor evolution and treatment response.^[231] However, clinical implementation faces three major hurdles: (1) low CTC abundance in early-stage patients reduces detection sensitivity; (2) EpCAM-dependent isolation methods (e.g., CellSearch^®) yield high false-negative rates due to EMT-induced EpCAM loss;^[4] and (3) the lack of broad-spectrum CTC-associated antigens.^[232] Emerging technologies may address these limitations. For example, microfluidics functionalized with antibodies against platelet markers (e.g., RG18, PPBP)^[14] or mesenchymal markers (e.g., Vimentin) improve CTC capture efficiency by targeting EMT-transformed subpopulations.^[4] Ex vivo CTC-immune cell co-culture models enable functional validation of CTC-specific antigens and immunotherapy efficacy, bridging bench-to-bedside translation.^[230] High-resolution single-cell sequencing profiling identifies novel capture markers.^[233]

In conclusion, while preventing metastasis via targeting CTCs is a promising strategy, the systemic heterogeneity of CTCs demands innovative approaches. By elucidating the spatiotemporal regulation of CTC phenotypes (e.g., platelet regulation, antigen dynamics) and leveraging multi-omics-driven insights, we may transform CTCs from passive biomarkers into active therapeutic targets. Collaborative efforts bridging single-cell technologies, functional immunology, and clinical trial design will be pivotal in advancing CTC research toward precision oncology.

Funding

This work was funded by the National Key Research and Development Program of China (No. 2022YFC2504700 [2022YFC2504703]), National Natural Science Foundation of China (Nos. 82473064, 22105137, and 82203539), and Natural Science Foundation of Sichuan Province, China (No. 2024NSFSC1919).

Conflicts of interest

None.

Supplementary Material

cm9-139-515-s001.docx ^{(242KB, docx)}