Role of circulating tumor cell clusters in breast cancer

Introduction

Breast cancer (BC) has been identified as the most frequent malignant disease in women worldwide (1). For nonmetastatic BC the 5-year survival rate is 99%, whereas in metastatic BC the rate is as low as 27%. Dissemination of BC, where metastatic cancer cells invade other organs, is considered a substantial cause of morbidity and represents the primary cause of mortality. Circulating tumor cells (CTCs) represent cancer cells that leave the primary or metastatic tumor tissue and intravasate to generate metastatic lesions (2). CTCs can travel alone or in small or medium-sized clusters and these groupings seem to have a higher metastatic potential and to be correlated to a poorer outcome than caused by single CTCs in BC patients. BC develops in the breast lobules, tubes, or connective tissue (3). In stage IV BC, metastatic lesions occur in lung, bones, or the brain (2,3). The process of BC metastasis comprises several succeeding discrete events. In detail, malignant cells attach to the endothelium and reach the circulation via lymph or blood vessels on their route to other organs. When the original or metastatic tumors release cancer cells, they circulate as CTCs that either deteriorate or are trapped in suitable niches to develop new lesions (4).

Metastatic cancer cannot be studied in cancer patients since tumor dissemination may have started years before a tumor is detected. Only 20% of the primary BC patients have detectable CTCs and studies deal mostly with the 50% of metastatic breast cancer (MBC) patients harboring large numbers of CTCs (2). In this case, CTCs may help to establish a prognosis and to identify drug sensitivity of cancer cells and their progress and development during the malignant disease. These investigations studying CTCs are hampered by technical difficulties, lack of standardized techniques and low sensitivity. Truly, metastasis-inducing cells are exceedingly rare and lack reliable markers; therefore, most isolated CTCs are not relevant to metastasis (5). As an alternative model to study the progress of metastasis, murine tumor models in the form of human patient-derived xenograft (PDX) samples are performed (6). However, the degree to which such an experimental animal model is representative for tumor dissemination in patients is severely limited. For different steps in the metastasis by CTCs, the participation of cell populations such as cancer stem cells (CSC) and cells transformed by epithelial-mesenchymal transition (EMT) has been proposed. However, involvement of such specialized tumor cells is not firmly established and subject of ongoing research.

CTCs

So far, following the course of cancer development and assess the response of treatment can be carried out using invasive tumor biopsies that are not easily repeated and may miss the heterogeneity of the tumor tissue (7,8). Cancer patients’ peripheral blood contains CTCs, whereas healthy people’s blood samples do not (8,9). CTCs are detectable in a multitude of cancer types including breast, prostate, ovarian, lung, liver, pancreatic, colorectal, gastric cancer and melanoma (10). Experimental and clinical investigations have indicated that the dissemination of CTC occur early in tumor development at small lesion sizes upon first ingrowths of vessels (11,12). As CTCs are as rare as one cell in 10⁷ white blood cells in 10 mL of blood, there are large difficulties in isolating and characterizing these cells (13,14). Technologies for the detection and isolation of CTCs rely either on cell surface markers or exploit differences in size, charge or rigidity of CTCs to separate them from normal blood cells (11). CTCs may express antigens such as the epithelial cell adhesion molecule (EpCAM) and are differentiated from CD45-positive normal blood leukocytes (15). CellSearch^® is based on the proof of EpCAM expression of the CTCs and was the first technique approved by the Food and Drug Administration (FDA) the for monitoring metastatic cancer (16). The Parsortix^® PC1 System is an alternative, marker-independent FDA-cleared technologies for isolating CTCs sorting by size discrimination. The confirmation of >5 EpCAM-positive and CD45-negative viable CTCs in 7.5 mL of blood by the CellSearch^® system correlates with both reduced progression-free survival (PFS) and reduced overall survival (OS) in patients (10,11,17). Furthermore, the detection of CTCs at the beginning of treatment and their sustained presence during therapy has been correlate to a poorer survival in MBC patients (18-20). Positive tests for CTCs indicate a higher metastatic risk, with median CTC counts increasing from n=2 in stage 1 up to n=1283 in stage IV BC (21).

In BC, dissemination may start during early stages of tumor initiation at small size and when the malignancy is still confined by a basement membrane (22,23). Dispersal of cancer cells continues until the source tumor is removed, as demonstrated by CTC counts in patients undergoing surgical resection (24). In the same patients, the circulating half-life of CTCs was only a few hours (24). Within a few minutes after breaking away from a tumor, CTCs may become trapped in the capillaries of distinct organs. CTCs can settle intact in capillaries for days until they either grow and eventually extravasate or disintegrate (25). However, further metastases may not become obvious until months or decades after initial diagnosis and removal of a primary tumor (26).

CTCs may circulate as single cells or in form of smaller or larger cell aggregates (27-29). Cluster formation appears to promote CTC aggressiveness by enhancing CTC proliferative capacity, resistance to anoikis and damage from the circulatory shear forces (27). Although CTC clusters represent only a minute subset of CTC events (2–5%) they are claimed to hold a 20–50-fold higher potency for developing metastatic lesions. CTC clusters can be homotypic or heterotypic in association with cells of mesenchymal or epithelial origin, cells of the immune system or pericytes, platelets, and cancer-associated fibroblasts (30). Several studies have suggested a correlation between the formation of CTC clusters, their protection as aggregates and a lower patient survival (31-33).

It has been discussed that the assessment of EpCAM and CD45 antigens alone may fail to detect and differentiate more diverse subpopulation of CTCs. The lack of surface expression of CTC epithelial markers may be due to their ability to undergo EMT (34). These cells do not appear in systems such as CellSearch^® and have been claimed to be present in as many as 35% of MBC patients and 60% of brain metastases (35). It was also postulated that CTCs that exhibit a partial mesenchymal state are required for metastasis since fully mesenchymal CTCs may not establish metastatic lesions (36). However, an evaluation of blood samples of 27 metastatic lung cancer patients showed that the presence of EpCAM(+) CTCs was associated with poor outcome, whereas the EpCAM(−) CTCs were not (37).

Their relative scarcity and difficulty of isolation also hinders the clinical use of CTCs as a routine diagnostic (11). Additionally, their use in early detection remains controversial (11). Several studies indicate that the long-term monitoring of CTC numbers in the same patient provide more valid prognostic data and should be incorporated into clinical practice (38). In early-stage BC there is insufficient clinical trial data for the use of CTS to guide therapy due to very low counts (39). Despite its promise, several challenges, including standardization, validation, and integration into clinical practice, remain to be addressed. Although the cultivation of CTCs in vitro can help for screening anti-metastatic drugs and studying CTC phenotypes, this approach is severely limited by the low efficiency of setup of in in vitro CTC cultures (40). An exception to the otherwise limited panel of CTC cultures is the establishment of several small cell lung cancer (SCLC) CTC lines, derived from patients with tumors shedding up to several thousand CTCs per 7.5 mL of blood and a correspondingly high content of metastasis-initiating cells (41).

EMT

The steps leading to metastasis, which accounts for over 90% of cancer-related fatalities, remain partially unresolved. EMT and the reverse process of mesenchymal-epithelial transition (MET) has been implicated in tumor metastasis and therapeutic resistance (42). EMT includes reorganization of the epithelial-type cell cytoskeleton with vimentin, detachment from the basement membrane and the expression of mesenchymal functionality (43). EMT is responsible for differentiating and de-differentiating cancer cells in the tumor microenvironment (TME). The epithelial phenotype demonstrates high proliferation capabilities, whereas the mesenchymal phenotype demonstrates enhanced migration and invasion capabilities (44). Collective migration is a cooperative phenomenon, where cells move as a cohesive formation, maintaining contact as they forge a way through tumor stroma (45). Mesenchymal cells maintain transient adherence junctions and migrate within loosely attached chains of cells. However, collective migration of tumor cells has been studied using various in vitro and in vivo model systems but has not been found in tumor stroma and is not accountable for the intravasation of thousands of CTCs in SCLC (46). Intratumoral cancer cell intravasation can occur independently of invasion into the adjacent stroma (47). A fully mesenchymal EMT may lead to a loss of tumor-initiating potential (48). A stochastic model of metastasis formation, comprising the intravasation of cells from a growing tumor, their extravasation at a distant site and their impact on patient outcome is described by Szczurek et al. (49). The process of intravasation is largely stochastic and the shed cells can then move randomly through the periphery and settle randomly in the body (50,51). In conclusion, collective migration may include cluster of tumor cells but intravasation via leaky tumor vessels allow for the stochastic release of a multitude of single cells and small clusters.

EMT has been proposed to enable cells to evade shear stress, apoptosis, anoikis, cell senescence and immune surveillance (Figure 1). At least, EMT can enhance the resistance of tumor cells against chemo- and radiotherapy. Thus, after disseminating and infiltrating distant organs using the mesenchymal abilities acquired by EMT, the metastasis-inducing cells are supposed to undergo MET to reinitiate tumor lesions (52). However, the dynamic nature of epithelial-mesenchymal plasticity (EMP), the apparent requirement to reverse mesenchymal changes for macro-metastatic outgrowth, and the likelihood that only minor subpopulations exhibit EMP at any given time have made such evidence difficult to obtain in the clinical setting (42). The diversity of the EMT spectrum and the heterogeneity of tumor cells displaying different states along the spectrum in the same patient will likely undermine the development of EMT-targeting therapeutics (53).

Figure 1 Small primary tumors are typically vascularized by narrow blood vessels, allowing only individual cells to intravasate and enter the circulation as single CTCs. In contrast, larger tumors develop larger and leaky vessels, which can facilitate the intravasation of whole clusters of CTCs. These clusters can get preferentially stuck in smaller capillaries. Cancer cells within the primary tumor are exposed to interstitial pressures of about 5–40 mmHg of pressure (equivalent to 2.67–5.33 kPa), their environment changes markedly once they enter blood vessels, where CTCs are subjected to fluid shear stress generated by blood flow and a pressure of 80 mmHg (10.67 kPa), which goes down to 20 mmHg (2.67 kPa) in small capillaries. CTCs, circulating tumor cells; EMT, epithelial-mesenchymal transition.

EMT is described as phenotypic shift of epithelial cells into mesenchymal-like cells with increased mobility in cell culture: however, a fully mesenchymal conversion of epithelial cells in vivo is rare, with cells with mesenchymal features occasionally observed in tumors (54). EMT is not rate-limiting for invasion and metastasis, but seem to be important in resistance to chemotherapy. Such transient, reversible and yet partially controlled EMT-MET plasticity is considered to be unlikely, in the light of the genetic and/or epigenetic disruptions of the tumor cells that may led to initial tumor formation (55). Studies have either failed to observe increased tumor metastasis following induction of EMT, or have described tumor dissemination in the absence of EMT-associated gene expression, thus leading to the rating that EMT is not a precondition for metastasis (56).

Intravasation of tumor cells

Angiogenesis (or neovascularization) is defined by the formation of new blood vessels from endothelium of the existing vasculature. When a new tumor reaches a 1–2 mm, its further growth requires the induction of new and irregular blood vessels, which may consequently lead to the development of metastases via the penetration of malignant cells into the circulation. Experimental and clinical evidence support the mechanism of the intravasation of single or clustered CTCs to occur via leaky neoangiogenetic vessels in the tumor core and not by transmigation of EMT-type cells through the adjacent tumor stroma (57). Furthermore, in lung cancer studies only EpCAM-positive CTCs lacking EMT features have been found to have predictive power. The rate-limiting step of CTC shedding is most likely the presence of irregular and leaky tumor vessels or through vessels formed by vasculogenic mimicry. Expression of vascular endothelial growth factor (VEGF) and the micro vessel density (MVD) are key prognostic indicators for tumors and ultimately, CTCs counts seem to be correlated to neoangiogenetic vascular supply of tumors and prognosis (58). Cancer cell intravasation seems not to take place along the whole cancer-associated vasculature, but may restricted to specialized intravasation doorways, called tumor microevironment of metastasis (TMEM) (59,60). In good accordance, the number of TMEM doorways in primary BC predicts distant metastasis (DM), independently of other prognostic factors. Mechanistically, perivascular macrophages within TMEM doorways are capable of secreting VEGF-A, which results in the breakdown of underlying endothelial-specific junctions transiently enhancing localized vascular permeability, which allows for the passing of highly-invasive cancer cells into the circulation. A case-control study of 30 patients who developed MBC and 30 patients without metastatic disease revealed that TMEM density was greater in the group of patients who developed systemic metastases compared with the patients with only localized BC (61). The ability of TMEM to predict DM was independent of lymph node status and other currently used prognosticators (61). Although tumor cells utilize TMEM doorway-associated transient vascular openings to intravasate, increasingly, mechanisms are described that demonstrate novel ways of tumor cell intravasation such as the formation of mosaic vessels containing endothelial and tumor cells (60,62).

Terstappen model of clinical BC

Terstappen and colleagues designed a mathematical model for metastasis in apparently tumor-free BC patients. The pooled analysis of individual data from 3,173 patients with nonmetastatic (stage I–III) BC showed one or more CTCs in 20.2% of the patients (63). CTC-positive patients had larger tumors, increased lymph node involvement, and a higher histological tumor grade than CTC-negative patients. Multivariate analysis, which included parameters comprising tumor size, nodal status, histologic tumor grade, and hormone receptor/human epidermal growth factor receptor 2 (HER2) status, confirmed that the CTCs count was an independent prognostic factor for disease-free survival, distant disease-free survival, BC-specific and OS.

Median CTC size assessed in a large cohort of BC and prostate cancer (PC) patients revealed that in BC, the median diameter CTCs was 12.4 vs. 18.4 µm for cultured BC cell line (64). For PC, median diameters of CTCs were 10.3 vs. 20.7 µm for cultured PC cell lines and the mean diameter of leukocytes were 9.4 µm. Therefore, typical CTCs are significantly smaller than corresponding tumor cell line cells. Although the frequency of CTCs was reported for distinct cancers, the knowledge of the proportion of viable CTCs found is incomplete. In a study to obtain viable CTCs, blood cells were subjected to microseeding and distributed to individual culture wells for enumeration and propagation. This procedure in MBC and PC patients revealed a CTC recovery of only 0–5% compared to CTC counts measured with the CellSearch^® system (65). Viability of the detected CTCs ranged from 0–36% (mean 13%), corresponding to common apoptotic features in CTCs observed with the CellSearch^® system. Some of the viable CTCs found remained intact for a several days but lacked to show cell division.

Women of the study cohort including 3,173 patients were diagnosed with pathological stage T1ANXM0–T2NXM0 primary invasive BC treated by mastectomy and analyzed to calculate the probability of DM within 5 years. While metastases are often found years after resection of the primary tumor, probably at least one metastasis was already present at the time of surgery. This cascade is an inefficient process, generating metastasis by sending large numbers of malignant cells into the circulation (2). The number of cells disseminated and the efficiency of metastasis formation contribute to the probability that a metastasis has formed, termed metastatic efficiency. Due to metastatic inefficiency, the presence of CTC does not imply that metastases already exist.

To generate a DM, cancer cells must break away from the primary tumor and endure several barriers to pass the metastatic cascade (5). The mathematical model for BC metastasis estimated tumor size and CTC load from a primary BC tumor before the first metastasis formed, relying on the relationship between tumor size and probability of DM for 38,715 patients with surgically removed TXNXM0 primary BC. Calibration of the model simulation used primary tumor size, probability of DM and time to establish distant metastases for 1,489 patients (25%) with stage T1BNXM0. For these 38,715 BC patients a tumor doubling time (DT) of 1.7±0.9 months was calculated and fitting the data for 25% of T1B patients revealed a metastatic efficiency of one metastasis formed per 60 million CTCs, resulting in good agreement between model and epidemiological data. These estimates are based on CTC counts for MBC patients of 3.0 CTC/mL (range, 0.02–417 CTC/mL) (17,66,67) and for patients with early-stage BC at 0.03 CTC/mL (range, 0.01–0.05 CTC/mL) (68-71). The product of dissemination rate and metastatic efficiency was fit to the probability of DM for 25% of patients with stage T1B. Tumor cell shedding begins well before the primary tumor is clinically detectable by imaging (72). The relationship between tumor diameter and the number of disseminated cells was assumed to be linear according to murine data (73,74).

The relationship between the number of CTCs and the number of macroscopic metastases was assumed to be linear (75). Only a minute number of CTCs are truly metastasis-inducing effectors and the migration from the primary tumor to the suspected metastatic site occurs within days. In detail, 3,550 patients (9.2%) developed DM within 5 years after surgery with an overall time to DM of 32.0±17.2 months. Thus, the DT for metastasis of 1.7±0.9 months and approximately 60 million disseminated cells per established macro-metastasis were calculated (5). In comparison, in a murine model metastasis model, a median estimate of 1 metastasis in 14,000 disseminated cells is found. The large difference of metastatic efficiency between a murine model and Terstappen et al. model may be attributed to the use of experimental cell lines with extremely high metastatic efficiency, the difference in size between human and mouse and the immunodeficiency of most mouse models. Murine models suggest that disseminated cells have an unusual high survival in circulation and are extremely efficient at extravasation. The most effective therapy to treat BC is the resection of a primary tumor before metastasis has occurred but fails due to the lack of available technology to detect the presence of small DM.

Experimental modeling and biophysical context of CTCs

Since it is not possible to study details of the metastatic cascade in patients, experimental animal tumor models are used as surrogates. PDX/cell line-derived xenograft (CDX) models are created by implanting a human cancer cell line or tumor directly into an immunocompromised mouse (76). PDX tumors are created by implanting human tumor tissue into immune-compromised mice [e.g., NOD scid gamma (NSG), non-obese diabetic/severe combined immunodeficient (NOD/SCID)]. Instead of tumor biopsies or cell lines, blood samples can be employed to establish CDXs (57). PDXs are genetically similarly to the parent tumors but need serial passaging that is increasingly associated with altered genetics and partial changes of human characteristics under the control of the murine microenvironment.

PDX models preserve tumor heterogeneity of the original patient tumor for personalized drug testing and biomarker discovery. Disadvantages of metastasis PDX models include a low take rate, the slow development of spontaneous metastases, the rarity of overt metastasis, and the need for immuno-compromised mice, which prevents studying immune interactions crucial for metastasis. The period necessary for tumor “take” varies depending on tumor type, implantation site, and recipient strain. Still, in general, it is between 2 and 4 months, with failure of engraftment not being determined until at least 6 months (77).

Other drawbacks are the high cost, time commitment, and technical challenges of developing these models, as well as potential differences between the PDX and patient metastatic phenotypes due to host selection and tumor evolution. The chosen implantation site (e.g., subcutaneous) can limit the ability to study all stages of metastasis, such as the extravasation and homing processes. PDX models have low success rates for early-stage cancers and the prolonged tumor latency of 4–6 months, limits the use for guiding therapy (78). Creation of heterotopic and orthotopic metastatic models as well as injecting patient-derived tumor cells have limitations. Spontaneous metastasis in PDX models is slow but a high a metastatic rate obtained by injecting tumor cells results in a possibly nonphysiological model of tumor dissemination.

The host tumor environment in PDXs is a mixture of human and mouse cells, which differs significantly from the native human TME, affecting tumor progression and metastasis. The host mouse stroma replaces some of the original patient stromal cells in the tumor, altering the tumor’s microenvironment. The quantification of murine cells contributing to PDXs in lung cancer lines, showed a proportion of a few percentages to more than 95% in lung adenocarcinoma and small cell lung carcinoma. After 3–5 passages in animals, the human tumor-associated stroma is almost entirely replaced by a murine-derived extracellular matrix (ECM) and fibroblasts (79). While the tumor cells remain human, the stroma (supporting tissue), including blood vessels, fibroblasts, immune cells (if any) is gradually replaced or derived from the mouse host. So, the vascularization of the tumor in PDX models is typically driven by the murine angiogenesis system and mouse endothelial cells build the vessels (76,80). Humanized PDX models successfully recreated tumor-immune interactions in triple-negative breast cancer (TNBC) (81). Differential tumor growth rates and gene expression patterns highlighted the complexities of the immune response in the TME of humanized PDX models. Distinct types of tumors employed for PDX may exhibit aggressive metastasis as observed, for example, for TNBC. The CDX model metastasis can be supercharged by using a highly migratory and metastatic cell lines such as the TNBC line MDA-MB-231 driven by a KRAS G13D oncogenic mutation. Genetic drift, poor tumor heterogeneity and a murine background limits the CDX model’s clinical relevance (82). The tumor size can be readily monitored upon subcutaneous implantation but the subcutaneous microenvironment may differ from the tissue of origin for most tumor types. Furthermore, the subcutaneous application results in variable vascularization and low metastasis tendency (77). In conclusion, the use of xenograft models is a very limited proxy for the metastatic process in patients.

CTCs are released from tumors and enter the bloodstream. They experience blood pressure and shear stress arising from flow through the arterial circulation. For most adults, normal blood pressure is below 120/80 mmHg. The disseminated tumor cells are transported to the arterial end of the capillary being exposed to a pressure of 30–35 mmHg. At the venous end of the capillaries the pressure amounts to 10–15 mmHg, yielding an average capillary pressure of 20 mmHg.

Tumors typically lack functional lymphatic vessels, which are necessary to remove excess fluid, leading to its accumulation and pressure buildup. The elevated fluid pressure within solid tumors, the tumor interstitial pressure (TIP), ranges from 5 to 40 mmHg, and is significantly higher than in normal tissues (83). This high pressure is caused by a combination of hyperpermeable blood vessels, a lack of functional lymphatic drainage, and solid stress from proliferating cells (Figure 1). The rapid proliferation of tumor cells creates mechanical pressure on the surrounding tumor tissue, which contributes to interstitial hypertension. The mechanical stretch caused by high TIP can influence tumor cell proliferation, potentially stimulating growth. Reducing TIP strategies including anti-angiogenic therapies, vasodilators, and physical manipulations like hyperthermia. Leukemia cells float in the bloodstream, infiltrate bone marrow, and sometimes reside in organs. While they withstand normal blood pressure, very high or very low blood pressure can cause stress. At high blood pressure, blood flow velocity and shear stress increase. A number of studies demonstrate that CTCs are modified by the dynamic biophysical conditions in blood, in particular related to fluid shear stress (84). Leukemia cells are larger and less flexible than normal blood cells, so higher shear stress can damage or rupture some blasts. The Parsortix system enriches CTCs via size steps in a chip assembly. Under standard operating procedures, the pressure applied to pump the blood sample through the apparatus is approximately 74.3 mmHg (85). The CTCs circulate from the originating tumors to capillaries within minutes and, therefore, the cells are exposed to differences in pressure and shear force within a very short time that is not expected to lead to major adaptions in the CTCs.

CTCs and immunotherapy

In contrast to the adverse conditions in acidic and hypoxic tumor tissue with elevated interstitial pressure, CTCs are freely exposed to chemotherapeutics and immune checkpoint inhibitors (ICIs) in the circulation. CTCs that leave the specific TME of the primary tumor are confronted with immune surveillance in blood. During immunochemotherapy, CTCs decrease significantly during treatment, whereby a larger decrease in CTCs predicted a significantly longer PFS time. Durvalumab anti-programmed death-ligand 1 (PD-L1)-treated patients who progressed had sustained higher PD-L1-positive CTCs compared to resilient patients. Natural killer (NK) cells can eliminate CTCs in the blood and reduce their extravasation (86). Metastatic BC patients with >5 CTCs per 7.5 mL of blood exhibit defective NK cells compared to those from patients with ≤5 CTCs, revealing an inverse correlation between CTCs and PFS (87). Furthermore, in MBC, low circulating lymphocyte counts and high CTC levels were found to be poor predictive factors (88). In inflammatory BC patients, the CTCs counts correlated with a reduction of CD3⁺ T cells, CD4⁺ T cells, and CD8⁺ T cells (89). Furthermore, in these patients, CTC counts positively correlated with circulating regulatory T-cells (Tregs) that impair antitumoral immune responses and promote metastasis (89).

CTCs can be found as single cells or clusters, named circulating tumor microemboli (CTMs), that show adherence of CTCs to leukocytes, endothelial cells, fibroblasts, and other cells (90). Depending on the size of the CTMs, CTCs in their interior are protected from immune recognition and therapeutics. The downregulation or loss of surface MHC expression promotes the immune system escape of CTCs as well as tumor cells (91,92). The expression of PD-L1, which prevents T cell-mediated destruction has been shown on CTCs of several malignancies and is associated with a poor prognosis (93-95). Despite the different CTC detection platforms and PD-L1 assays, most studies relate a high PD-L1-positive CTC count with a worse OS and PFS (96,97). ICIs suppress inhibitory proteins and result in antitumor immune cell activation (98). Numerous drugs that block either cytotoxic T-lymphocyte antigen 4 (CTLA-4) or PDL1/programmed death-ligand1 (PD-1) interactions have been developed, including ipilimumab and tremelimumab for CTLA-4, nivolumab, pembrolizumab and cemiplimab for PD-1, and atezolizumab, durvalumab and avelumab for PD-L1. Since CTCs frequently express PD-L1, this observation may be employed as non-invasive means to assess PD-L1 status in real-time (93,97,99).

A predictive value of PD-L1 and PD-1 expression by CTC was reported in patients with metastatic lung cancer before treatment and following three cycles of chemotherapy (96). The presence of PD-1-positive CTCs correlated with shorter PFS. PD-L1-positive CTCs were detected in 25 out of 38 samples (69.4%). After initiation of the radiation therapy, the proportion of PD-L1-positive CTCs significantly increased, indicating an up-regulation of PD-L1 in tumor cells in response to the radiation. In a non-small cell lung cancer (NSCLC) study, CTCs were detected in 40% of the patients 76 patients and 80% of them were PD-L1-positive. One M0 (stage III) patient had PD-L1⁺ CTC cluster of three CTCs, while two-stage IV patients had each one CTC cluster with all cells positive for PD-L1, confirming the PD-L1-positivity of CTC clusters (100).

CTC clusters

In the blood of CTC-positive cancer patients, CTCs may circulate as single cells or clusters, consisting of 3–50 cells. Both CTCs and CTC clusters are regarded as metastatic initiators due to their ability to generate DM (101). CTCs must endure fluid shear forces and resist detachment-induced anoikis, therefore, only a minute fraction of the CTCs will result in metastasis (34). CTCs initiate their extravasation process by getting stuck within tiny capillaries and traversing the endothelium after physical blockage or selective adhesion (102). Krol et al. have reported the presence of CTC clusters in early non-metastatic BC patients that are three times more frequent than in MBC patients (103). The size of the CTC clusters varies, ranging from cell pairs to aggregates exceeding of up to 50 cells. In a meta-analysis involving 6,825 BC patients, CTCs have been demonstrated to have predictive power in early-stage malignancies and in metastatic patients (104). In 35–50% of MBC patients about 3.4 percent of all CTCs are found clustered. Clusters have a shorter half-life in blood with a half-life 6–10 min compared to 25–30 min for single CTCs (105). Several studies have claimed that CTC clusters have a greater metastatic potential than single CTCs. The metastatic potency increases when CTCs travel in conjunction with other cell types to form heterotypic CTC clusters, including neutrophils, myeloid-derived suppressor cells, macrophages, platelets, cancer-associated fibroblasts and red blood cells in circulation (104). Still, the degree of this disparity of the metastatic potency for cluster and single CTCs varies from 20 up to more than 100 times and in mouse models, CTC clusters were responsible for 50–97% of metastatic tumors (106-108).

In detail, the presence of CTC clusters was checked in blood specimens from a total of 79 BC patients with help of the herringbone (^HBCTC-Chip) that captures CTC clusters of all sizes (24,27). In microfluidic chambers CTCs bind to antibody-coated walls of the device (24). Furthermore, the ^negCTC-iChip depletes normal cells from blood specimens, leaving CTCs and CTC clusters in solution (109). Within 4 hours 3–12 mL of blood was processed through the chips resulting in the detection of clusters in 54/79 patients (68%). Among patients, 3 (5.6%) had CTC clusters on all time points, while 16 (29.6%) variable positivity and 35 (64.8%) had no detectable clusters. Mean PFS was 32.6, 134.8, 160.5 days for these three patient populations. Clusters consisting of dozens of tumor cells, may have difficulties to pass capillaries. In vivo flow cytometry studies indicate that clusters are more rapidly cleared from the circulation than single CTCs. Polyclonal BC metastases seem to arise from collective dissemination of keratin 14-expressing tumor cell clusters (110).

Either CTC clusters are shed from the primary tumor or they aggregate from single CTCs in the periphery (30,111). Although an experimental animal model shows direct release of clusters from tumors, tumor cell aggregation and polyclonal metastasis has been demonstrated by homophilic CD44 interactions in a PDX BC models employing patient-derived cancer cells (29). Homotypic CTC clusters represent a small percentage (1–30%) of all CTC events when detected in the peripheral circulation of patients or mice models, and their presence is linked to tumor size, disease stage, and molecular features (112). When the CTC clusters travel in blood, the outer layer of 2–50 cells is claimed to protect the “inner” CTCs from shear force (113). Remarkably, CTC clusters were shown reshape into a chain-like structures, which may enable their traverse through capillaries (63). Although CTC clusters seem to exhibit higher metastatic potential their clinical significance remains limited due to their rarity (27,33,38).

The significance of CTC clusters was investigated in mouse CDX models with tagged mammary tumors that proofed that CTC clusters arise from the release of oligoclonal tumor cell groups and not from intravascular aggregation events in this system (27). The experimental study was done with MDA-MB-231-LM2 (LM2) cells, a highly lung-metastatic variant of the MDA-MB-231 human TNBC BC cells, carrying a KRAS G13D mutation (114,115). The shorter circulation half-life of CTC clusters reported is consistent with their more rapid entrapment within capillaries of distal organs, where they may initiate metastatic growth (74). The parent cell line MDA-MB-231 was derived from a pleural effusion of a BC patient suffering from widespread metastasis (116). NSG mice were injected with 2×10⁵ LM2 cells previously labeled with either green fluorescent protein (GFP) or mCherry fluorescent dyes (27). In mice, approximately 1 mL of blood was processed through the herringbone chip loaded with EpCAM/epidermal growth factor receptor (EGFR)/HER2 antibodies. Blood draw for CTC enumeration was performed 5 weeks after tumor onset and lung metastasis analysis 4 weeks after tumor onset. Of approximately 2,500 CTC events observed per mouse, a mean of 65 (2.6%) were CTC clusters and the rest were single cell CTCs. A mean of 323 lung foci was identified per mouse (n=5 mice), of which 171 (53%) were multicolor, and therefore derived from CTC clusters. Calculation of the frequency of lung metastases comparing the number of single CTCs and clusters revealed that a CTC cluster is ~50 times more likely to give rise to a metastatic lesion. Both LM2-single cell and LM2-cluster cells reached the lungs with equal efficiency (day 0), although it was claimed that CTC clusters are more likely than single CTCs to be trapped in small capillaries of the lung and distal organs with half-lives of 6–10 min for LM2-CL versus 25–30 min for LM2-SC. However, this model using an extremely aggressive TNBC cell line exhibiting metastases within weeks in a murine tumor environment via a high number of supposed metastasis-inducing CTCs, is of limited resemblance to the metastasis observed in patients with 1 in 60 million tumor cells causing a lesion within months or years. The highly selected aggressive TNBC cells seem not to truly resemble the rare metastasis-initiating cells in patients.

In BC, preclinical studies showed that inhibitors of the Na⁺/K⁺ ATPase suppress the formation of CTC clusters and block metastasis (117). An analysis of nine patients treated daily with a maintenance digoxin dose (0.7–1.4 ng·mL⁻¹ serum level) revealed a mean cluster size reduction of −2.2 cells per cluster upon treatment, meeting the primary endpoint of the study. A screen with 2,486 US FDA-approved drugs demonstrated that Na⁺/K⁺ ATPase inhibitors, such as cardiac glycosides, effectively dissolve CTC clusters into single cells, leading to metastasis suppression in mouse models of BC. However, this class of drugs is highly toxic for CTCs and the presence of clusters indicates that metastasis has occurred months to years in the past and disassembly of currently circulating clusters will not influence the generation of further metastatic lesions on short notice.

Conclusions

CTCs are an important tool to confirm cancer metastasis and to monitor the course of the disease and efficacy of treatment. Several publications point to an increased metastatic potential of CTC clusters in comparison to circulating single cells. Evidence for a higher rate of metastasis and poorer prognosis in the presence of CTC clusters is derived from clinical observations and animal experimental work. Investigations in CDX/PDX murine models confirm a high rate of CTC clusters giving rise to metastatic lesions. However, these models work with selected highly metastatic cell lines leading to metastasis within several weeks via CTCs that are most likely not representative of the extremely rare human metastasis-inducing cancer cells and the clinical time frame of metastasis. These xenografts have developed murine vessels and a corresponding TME, leaving solely the finding that clusters are more rapidly cleared from the circulation and lodge in capillaries with possibly increased survival rates compared to single cells. In contrast, in patients, homotypic aggregation of CTCs was reported. In BC patients CTC clusters were reported and their numbers correlated with poorer survival down to 32.6 days. Mathematical modeling of BC metastasis has suggested that actual metastasis-initiating cells are extremely rare and may not be part of the CTC clusters detected. Furthermore, the setup of a metastatic lesion requires years and is not visible within weeks and months. A very short survival time in MBC end stage disease with multiple metastases indicates that shed tumor cells will have no impact on patients’ survival by forming further metastases within the following years. The most likely explanation for the significance of CTC clusters in patients is the presence of larger originating tumors and metastases that hold a high number of leaky vessels and active pathways to enhance the release of tumor cells in aggregated form (Figure 1). The poor prognosis is not due to the increased metastatic potential of CTC clusters but due to the presence of large tumor lesions with irregular and leaky vessels that eventually limit the survival of the patients. The metastatic process is difficult to follow in patients and poorly simulated in CDX/PDX models and, therefore, to overcome the limitations of the models, extended investigations in the prognostic power of tumor vessel density and mechanisms of tumor cell release are necessary.

Acknowledgments

None.

Footnote

Peer Review File: Available at https://tbcr.amegroups.com/article/view/10.21037/tbcr-2025-1-60/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tbcr.amegroups.com/article/view/10.21037/tbcr-2025-1-60/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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