Better together: circulating tumor cell clustering in metastatic cancer
Authors: Emma Schuster, Rokana Taftaf, Carolina Reduzzi, Mary K. Albert, Isabel Romero-Calvo, Huiping Liu
Journal: Trends in cancer (2021)

Circulating tumor cell (CTC) clusters are up to 100 times more metastatic than single CTCs in breast cancer, regardless of subtype or prior treatment.Recent advances in CTC detection technology using microfluidics, flow cytometry, and immunohistochemistry have allowed specific isolation and analysis of clusters.CTC clusters have enhanced stemness and plasticity properties driven by molecules such as CD44, intercellular adhesion molecule 1 (ICAM1), and epigenetic reprogramming. Other molecules, such as plakoglobin, strengthen CTC cluster adhesion.CTC clusters can be both homotypic and heterotypic, with heterotypic clusters forming with immune cells and cancer-associated fibroblasts (CAFs).Therapeutics are being developed and tested in clinical trials to specifically target CTC clusters. Circulating tumor cells (CTCs) are vital components of liquid biopsies for diagnosis of residual cancer, monitoring of therapy response, and prognosis of recurrence. Scientific dogma focuses on metastasis mediated by single CTCs, but advancement of CTC detection technologies has elucidated multicellular CTC clusters, which are associated with unfavorable clinical outcomes and a 20- to 100-fold greater metastatic potential than single CTCs. While the mechanistic understanding of CTC cluster formation is still in its infancy, multiple cell adhesion molecules and tight junction proteins have been identified that underlie the outperforming attributes of homotypic and heterotypic CTC clusters, such as cell survival, cancer stemness, and immune evasion. Future directions include high-resolution characterization of CTCs at multiomic levels for diagnostic/prognostic evaluations and targeted therapies. Circulating tumor cells (CTCs) are vital components of liquid biopsies for diagnosis of residual cancer, monitoring of therapy response, and prognosis of recurrence. Scientific dogma focuses on metastasis mediated by single CTCs, but advancement of CTC detection technologies has elucidated multicellular CTC clusters, which are associated with unfavorable clinical outcomes and a 20- to 100-fold greater metastatic potential than single CTCs. While the mechanistic understanding of CTC cluster formation is still in its infancy, multiple cell adhesion molecules and tight junction proteins have been identified that underlie the outperforming attributes of homotypic and heterotypic CTC clusters, such as cell survival, cancer stemness, and immune evasion. Future directions include high-resolution characterization of CTCs at multiomic levels for diagnostic/prognostic evaluations and targeted therapies. Although metastasis accounts for 90% of solid tumor–related mortality, it currently evades targeted treatments and demands a better understanding. Metastasis is a multistep process during which cancer cells spread from the primary tumor site to distant organs, starting with the primary tumor formation, where tumor cells gradually expand and locally invade the surrounding stroma and tissues, including the blood and lymphatic vessels. At this point, the intravasated tumor cells, now called 'circulating tumor cells' (CTCs) and a vital component of liquid biopsy [1.Pantel K. Alix-Panabieres C. Liquid biopsy and minimal residual disease – latest advances and implications for cure.Nat. Rev. Clin. Oncol. 2019; 16: 409-424Crossref PubMed Scopus (401) Google Scholar,2.Keller L. Pantel K. Unravelling tumour heterogeneity by single-cell profiling of circulating tumour cells.Nat. Rev. Cancer. 2019; 19: 553-567Crossref PubMed Scopus (224) Google Scholar], develop adaptive mechanisms that favor their survival in the harsh microenvironment of the circulatory system. CTCs may disseminate to distant parts of the body before they finally extravasate or get trapped within the capillaries in certain organs, form metastatic niches, and regenerate secondary tumors [3.Aceto N. et al.Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis.Cell. 2014; 158: 1110-1122Abstract Full Text Full Text PDF PubMed Scopus (1380) Google Scholar, 4.Toss A. et al.CTC enumeration and characterization: moving toward personalized medicine.Ann. Transl. Med. 2014; 2: 108PubMed Google Scholar, 5.Kantara C. et al.Methods for detecting circulating cancer stem cells (CCSCs) as a novel approach for diagnosis of colon cancer relapse/metastasis.Lab. Investig. 2015; 95: 100-112Crossref PubMed Scopus (55) Google Scholar]. The presence of CTCs in the blood of patients with cancer was first detected in 1869 by Thomas Ashworth [6.Ashworth T.R. A case of cancer in which cells similar to those in the tumours were seen in the blood after death.Med. J. Aust. 1869; 14: 146-147Google Scholar], but, because of advances in technology, CTCs have only recently attracted great attention for their key role in tumor metastasis [6.Ashworth T.R. A case of cancer in which cells similar to those in the tumours were seen in the blood after death.Med. J. Aust. 1869; 14: 146-147Google Scholar,7.Aceto N. et al.En route to metastasis: circulating tumor cell clusters and epithelial-to-mesenchymal transition.Trends Cancer. 2015; 1: 44-52Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar]. Many studies have shown that CTCs may be used to predict disease progression and prognosis in patients with metastatic cancer [4.Toss A. et al.CTC enumeration and characterization: moving toward personalized medicine.Ann. Transl. Med. 2014; 2: 108PubMed Google Scholar]. In metastatic cancers such as breast cancer, the currently accepted level of CTCs that correlates with worse overall survival and progression-free survival is five or more CTCs in 7.5 mL of blood [8.Cristofanilli M. et al.The clinical use of circulating tumor cells (CTCs) enumeration for staging of metastatic breast cancer (MBC): International expert consensus paper.Crit. Rev. Oncol. Hematol. 2019; 134: 39-45Crossref PubMed Scopus (124) Google Scholar]. CTC enumeration can be used to better stratify patients with stage IV breast cancer as stage IV aggressive, with more than five CTCs, and stage IV indolent, with fewer than five CTCs [8.Cristofanilli M. et al.The clinical use of circulating tumor cells (CTCs) enumeration for staging of metastatic breast cancer (MBC): International expert consensus paper.Crit. Rev. Oncol. Hematol. 2019; 134: 39-45Crossref PubMed Scopus (124) Google Scholar]. Stage IV indolent disease is associated with significantly longer overall survival, regardless of disease subtype and prior treatment [8.Cristofanilli M. et al.The clinical use of circulating tumor cells (CTCs) enumeration for staging of metastatic breast cancer (MBC): International expert consensus paper.Crit. Rev. Oncol. Hematol. 2019; 134: 39-45Crossref PubMed Scopus (124) Google Scholar]. The presence of CTCs in early breast cancer was also demonstrated to have prognostic impact [9.Rack B. et al.Circulating tumor cells predict survival in early average-to-high risk breast cancer patients.J. Natl. Cancer Inst. 2014; 106dju066Crossref PubMed Scopus (262) Google Scholar,10.Bidard F.C. et al.Circulating tumor cells in breast cancer patients treated by neoadjuvant chemotherapy: a meta-analysis.J. Natl. Cancer Inst. 2018; 110: 560-567Crossref PubMed Scopus (136) Google Scholar]. Thus, CTC analysis in patients with cancer is a minimally invasive, clinically informative method of predicting disease stage and survival that is not dependent on cancer subtype or previous treatment. Although most CTCs are single cells, a small portion circulate as a group of clustered cells (two or more nuclei) [7.Aceto N. et al.En route to metastasis: circulating tumor cell clusters and epithelial-to-mesenchymal transition.Trends Cancer. 2015; 1: 44-52Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar,11.Liu X. et al.Homophilic CD44 interactions mediate tumor cell aggregation and polyclonal metastasis in patient-derived breast cancer models.Cancer Discov. 2019; 9: 96-113Crossref PubMed Scopus (140) Google Scholar]. CTC clustering has been shown to be an adaptive mechanism that enhances CTC survival in the harsh bloodstream and promotes their metastatic potential [3.Aceto N. et al.Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis.Cell. 2014; 158: 1110-1122Abstract Full Text Full Text PDF PubMed Scopus (1380) Google Scholar,11.Liu X. et al.Homophilic CD44 interactions mediate tumor cell aggregation and polyclonal metastasis in patient-derived breast cancer models.Cancer Discov. 2019; 9: 96-113Crossref PubMed Scopus (140) Google Scholar]. In multiple cancer types, such as breast and prostate cancer, CTC clusters metastasize at 20–100 times greater efficiency and are associated with worse prognosis and lower overall survival of patients with cancer than single CTCs [3.Aceto N. et al.Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis.Cell. 2014; 158: 1110-1122Abstract Full Text Full Text PDF PubMed Scopus (1380) Google Scholar,7.Aceto N. et al.En route to metastasis: circulating tumor cell clusters and epithelial-to-mesenchymal transition.Trends Cancer. 2015; 1: 44-52Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar,11.Liu X. et al.Homophilic CD44 interactions mediate tumor cell aggregation and polyclonal metastasis in patient-derived breast cancer models.Cancer Discov. 2019; 9: 96-113Crossref PubMed Scopus (140) Google Scholar, 12.Grillet F. et al.Circulating tumour cells from patients with colorectal cancer have cancer stem cell hallmarks in ex vivo culture.Gut. 2017; 66: 1802-1810Crossref PubMed Scopus (115) Google Scholar, 13.Massague J. Obenauf A.C. Metastatic colonization by circulating tumour cells.Nature. 2016; 529: 298-306Crossref PubMed Scopus (1002) Google Scholar]. Moreover, the detection of CTC clusters in patients with metastatic cancer showed an additional prognostic value confirming the increased aggressiveness of clusters compared with single CTCs observed in mouse models [3.Aceto N. et al.Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis.Cell. 2014; 158: 1110-1122Abstract Full Text Full Text PDF PubMed Scopus (1380) Google Scholar,14.Mu Z. et al.Prospective assessment of the prognostic value of circulating tumor cells and their clusters in patients with advanced-stage breast cancer.Breast Cancer Res. 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Studies on the clinical significance of CTC clusters in early-stage breast cancer are indeed lacking, but recent studies showing the detection of CTC clusters in patients with nonmetastatic breast cancer open a new opportunity to investigate the role of CTC clusters in the early steps of tumor dissemination and further emphasize the need to understand the molecular drivers across all subtypes and stages of breast cancer [18.Silvestri M. et al.Detection of genomically aberrant cells within circulating tumor microemboli (CTMs) isolated from early-stage breast cancer patients.Cancers (Basel). 2021; 13: 1409Crossref PubMed Scopus (5) Google Scholar, 19.Krol I. et al.Detection of clustered circulating tumour cells in early breast cancer.Br. J. Cancer. 2021; 125: 23-27Crossref PubMed Scopus (14) Google Scholar, 20.Reduzzi C. et al.Circulating tumor cell clusters are frequently detected in women with early-stage breast cancer.Cancers (Basel). 2021; 13: 2356Crossref PubMed Scopus (9) Google Scholar]. Over the years, many technologies have been developed for CTC detection, which have been employed for CTC cluster analysis as well [21.Ferreira M.M. et al.Circulating tumor cell technologies.Mol. Oncol. 2016; 10: 374-394Crossref PubMed Scopus (317) Google Scholar,22.Batth I.S. et al.CTC analysis: an update on technological progress.Transl. Res. 2019; 212: 14-25Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar]. Most of the studies focusing on the clinical significance of CTC clusters in breast cancer have used the CellSearch platform and reported clusters in about 17–20% of patients with metastatic breast cancer, associated with worse prognosis and high CTC burden (five or more CTCs) [14.Mu Z. et al.Prospective assessment of the prognostic value of circulating tumor cells and their clusters in patients with advanced-stage breast cancer.Breast Cancer Res. Treat. 2015; 154: 563-571Crossref PubMed Scopus (84) Google Scholar, 15.Jansson S. et al.Prognostic impact of circulating tumor cell apoptosis and clusters in serial blood samples from patients with metastatic breast cancer in a prospective observational cohort.BMC Cancer. 2016; 16: 433Crossref PubMed Scopus (86) Google Scholar, 16.Wang C. et al.Longitudinally collected CTCs and CTC-clusters and clinical outcomes of metastatic breast cancer.Breast Cancer Res. Treat. 2017; 161: 83-94Crossref PubMed Scopus (108) Google Scholar, 17.Larsson A.M. et al.Longitudinal enumeration and cluster evaluation of circulating tumor cells improve prognostication for patients with newly diagnosed metastatic breast cancer in a prospective observational trial.Breast Cancer Res. 2018; 20: 48Crossref PubMed Scopus (52) Google Scholar,23.Paoletti C. et al.Circulating tumor cell clusters in patients with metastatic breast cancer: a SWOG S0500 translational medicine study.Clin. Cancer Res. 2019; 25: 6089-6097Crossref PubMed Scopus (24) Google Scholar]. Even though CellSearch is highly standardized and is the only FDA-approved CTC detection platform, it was not developed or tested (as were most of the other CTC detection technologies) to specifically detect CTC clusters. Hence, an underestimation of CTC clusters may result from using CellSearch because of a possible inefficiency in the enrichment of large-size clusters using ferrofluids [24.Krebs M.G. et al.Analysis of circulating tumor cells in patients with non-small cell lung cancer using epithelial marker-dependent and -independent approaches.J. Thorac. Oncol. 2012; 7: 306-315Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar]. Moreover, CellSearch only detects epithelial CTCs [expressing both epithelial cell adhesion molecule (EpCAM) and cytokeratin] and might miss CTCs undergoing dedifferentiation and/or epithelial-to-mesenchymal transition (EMT). This limitation could be particularly important for some CTC clusters that express enriched basal lineage and stem cell markers along with low expression of epithelial markers, as demonstrated in a few studies [11.Liu X. et al.Homophilic CD44 interactions mediate tumor cell aggregation and polyclonal metastasis in patient-derived breast cancer models.Cancer Discov. 2019; 9: 96-113Crossref PubMed Scopus (140) Google Scholar,25.Yu M. et al.Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition.Science. 2013; 339: 580-584Crossref PubMed Scopus (1720) Google Scholar, 26.Khoja L. et al.A pilot study to explore circulating tumour cells in pancreatic cancer as a novel biomarker.Br. J. Cancer. 2012; 106: 508-516Crossref PubMed Scopus (197) Google Scholar, 27.Gkountela S. et al.Circulating tumor cell clustering shapes DNA methylation to enable metastasis seeding.Cell. 2019; 176: 98-112.e14Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar]. In recent years, technologies were developed to specifically detect CTC clusters, mostly based on microfluidic approaches. Among these are the cluster chip [28.Sarioglu A.F. et al.A microfluidic device for label-free, physical capture of circulating tumor cell clusters.Nat. Methods. 2015; 12: 685-691Crossref PubMed Scopus (493) Google Scholar], the deterministic lateral displacement (DLD) chip [29.Au S.H. et al.Microfluidic isolation of circulating tumor cell clusters by size and asymmetry.Sci. Rep. 2017; 7: 2433Crossref PubMed Scopus (119) Google Scholar], and the 3D scaffold chip [30.Cheng S.B. et al.Three-dimensional scaffold chip with thermosensitive coating for capture and reversible release of individual and cluster of circulating tumor cells.Anal. Chem. 2017; 89: 7924-7932Crossref PubMed Scopus (46) Google Scholar] (for a detailed review, see [31.Pineiro R. et al.Relevance of CTC clusters in breast cancer metastasis.Adv. Exp. Med. Biol. 2020; 1220: 93-115Crossref PubMed Scopus (11) Google Scholar]). These technologies allow whole blood processing (thus avoiding red blood cell lysis and centrifugation steps) and reduced shear stress to preserve CTC cluster integrity, resulting in an increased sensitivity compared with other techniques. However, they require specialized instruments and equipment that are not commonly available in many clinical research centers, possibly constituting a limit to their widespread use. Recently, Smart BioSurface CTC technology was developed to attain shear stress–free CTC cluster detection by directly analyzing blood specimens transferred onto nanostructured, titanium oxide–coated slides (after red blood cell lysis) [19.Krol I. et al.Detection of clustered circulating tumour cells in early breast cancer.Br. J. Cancer. 2021; 125: 23-27Crossref PubMed Scopus (14) Google Scholar]. In a pilot study, this platform was able to detect clusters in 5 of 28 early-stage patients, supporting its higher sensitivity than CellSearch and other conventional CTC detection methods. The detection of clusters in early-stage patients by using blood filtration was also recently reported. CTC clusters were detected in 26 of 37 patients before starting neoadjuvant treatment [20.Reduzzi C. et al.Circulating tumor cell clusters are frequently detected in women with early-stage breast cancer.Cancers (Basel). 2021; 13: 2356Crossref PubMed Scopus (9) Google Scholar], and the malignancy of clusters detected by filtration before neoadjuvant chemotherapy was confirmed for 46 of 48 clusters isolated from six patients by single-cluster isolation and genomic profiling [18.Silvestri M. et al.Detection of genomically aberrant cells within circulating tumor microemboli (CTMs) isolated from early-stage breast cancer patients.Cancers (Basel). 2021; 13: 1409Crossref PubMed Scopus (5) Google Scholar]. These results further support the ability of filtration techniques to detect CTC clusters, as already reported in metastatic breast cancer [32.Mu Z. et al.Detection and characterization of circulating tumor associated cells in metastatic breast cancer.Int. J. Mol. Sci. 2016; 17: 1665Crossref PubMed Scopus (52) Google Scholar] and other malignancies at different disease stages [24.Krebs M.G. et al.Analysis of circulating tumor cells in patients with non-small cell lung cancer using epithelial marker-dependent and -independent approaches.J. Thorac. Oncol. 2012; 7: 306-315Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar,26.Khoja L. et al.A pilot study to explore circulating tumour cells in pancreatic cancer as a novel biomarker.Br. J. Cancer. 2012; 106: 508-516Crossref PubMed Scopus (197) Google Scholar,31.Pineiro R. et al.Relevance of CTC clusters in breast cancer metastasis.Adv. Exp. Med. Biol. 2020; 1220: 93-115Crossref PubMed Scopus (11) Google Scholar, 32.Mu Z. et al.Detection and characterization of circulating tumor associated cells in metastatic breast cancer.Int. J. Mol. Sci. 2016; 17: 1665Crossref PubMed Scopus (52) Google Scholar, 33.Amantini C. et al.Expression profiling of circulating tumor cells in pancreatic ductal adenocarcinoma patients: biomarkers predicting overall survival.Front. Oncol. 2019; 9: 874Crossref PubMed Scopus (35) Google Scholar, 34.Sawabata N. et al.Cluster circulating tumor cells in surgical cases of lung cancer.Gen. Thorac. Cardiovasc. Surg. 2020; 68: 975-983Crossref PubMed Scopus (12) Google Scholar, 35.Abdallah E.A. et al.A higher platelet-to-lymphocyte ratio is prevalent in the presence of circulating tumor microemboli and is a potential prognostic factor for non-metastatic colon cancer.Transl. Oncol. 2021; 14: 100932Crossref PubMed Scopus (6) Google Scholar]. Size exclusion might in fact be the best strategy to enrich CTC clusters, considering that they are considerably larger than white blood cells (WBCs). Indeed, by direct comparison of filtration and the CellSearch platform with clinical samples, two studies demonstrated increased CTC cluster detection attained by using filters [20.Reduzzi C. et al.Circulating tumor cell clusters are frequently detected in women with early-stage breast cancer.Cancers (Basel). 2021; 13: 2356Crossref PubMed Scopus (9) Google Scholar,24.Krebs M.G. et al.Analysis of circulating tumor cells in patients with non-small cell lung cancer using epithelial marker-dependent and -independent approaches.J. Thorac. Oncol. 2012; 7: 306-315Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar]. However, on the one hand, some concerns derive from the shear stress during filtration, which might damage CTC clusters, and from the presence of clusters of normal cells in the circulation, which could reduce the specificity of this approach if not coupled with additional phenotypic and/or genotypic analyses. On the other hand, blood filtration can easily be performed with disposable and commercially available devices, which could be easily transferred to clinical studies. Two recently developed, unbiased methods to detect putative CTCs in singles and clusters are (i) immunohistochemical staining to detect vascular CTCs in situ and (ii) flow cytometry of blood-derived CTCs with filtering based on cell/cluster sizes (side scatter and forward scatter channels) as well as molecular markers recognized by antibodies and/or binding ligands [11.Liu X. et al.Homophilic CD44 interactions mediate tumor cell aggregation and polyclonal metastasis in patient-derived breast cancer models.Cancer Discov. 2019; 9: 96-113Crossref PubMed Scopus (140) Google Scholar,36.Adorno-Cruz V. Liu H. Regulation and functions of integrin alpha2 in cell adhesion and disease.Genes Dis. 2019; 6: 16-24Crossref PubMed Scopus (51) Google Scholar,37.Liu X. et al.EGFR inhibition blocks cancer stem cell clustering and lung metastasis of triple negative breast cancer.Theranostics. 2021; 11: 6632-6643Crossref PubMed Google Scholar]. Although blood processing has the caveat of potentially introducing artificial effects on CTCs and cell clustering, the histology-based detection of vascular CTCs in tumor sections can certainly outperform and/or complement the other existing detection technologies in terms of specificity. However, the efficiency of immunohistochemistry-based detection of vascular CTCs is lower than blood-based CTC detection technologies. Combining multiple detection technologies is necessary not only to cross-validate but also to maximize the detection sensitivity and specificity. Overall, the perfect method for CTC cluster analysis (combining high sensitivity and specificity, technical standardization with high reproducibility, ease of use, wide availability, and transferability to clinical practice) has yet to be developed and optimized. Future technologies will be expected to provide profiles of single CTCs and/or clusters at the genomic, transcriptomic, and proteomic levels. Complementary models of patient CTC-derived cell lines, organoids, and xenografts have been generated for molecular and mechanistic studies [38.Yu M. et al.Cancer therapy. Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility.Science. 2014; 345: 216-220Crossref PubMed Scopus (657) Google Scholar, 39.Tsai C.F. et al.Surfactant-assisted one-pot sample preparation for label-free single-cell proteomics.Commun. Biol. 2021; 4: 265Crossref PubMed Scopus (6) Google Scholar, 40.Praharaj P.P. et al.Circulating tumor cell-derived organoids: Current challenges and promises in medical research and precision medicine.Biochim. Biophys. Acta Rev. Cancer. 2018; 1869: 117-127Crossref PubMed Scopus (78) Google Scholar]. A summary of the pros and cons of current CTC cluster detection technologies can be found in Table 1. To truly understand the clinical and biological significance of CTC clusters, more studies are needed to develop new technologies and to better evaluate the performance of the existing methods of detecting CTC clusters.Table 1CTC cluster detection technologiesaAbbreviation: IHC, immunohistochemistry.StandardizationSensitivitySpecificityAccessibility/availability for clinical practiceBlood-basedCellSearch√ FDA-approvedLowHigh√Microfluidics (cluster-specific)NoHighHighDifficult (low availability)FiltrationNoHighLowPossibleFlow cytometryNoHighLowPossibleTissue-basedIHC stainingNoLowHighPossiblea Abbreviation: IHC, immunohistochemistry. Open table in a new tab Multicellular CTC clusters can be composed of tumor cell–tumor cell homotypic clusters or tumor cell–blood cell heterotypic clusters with various formation mechanisms and functional advantages. Although 'homotypic CTC clusters' are a default term in most of the literature, we also summarize the recent discoveries on heterotypic CTC clusters and tumor cell–stromal cell clusters in metastasis. As discussed, CTCs can exist as both single cells and clusters. Although clusters are found less often, they offer up to 100-fold greater metastatic potential than single CTCs, making them a very clinically relevant population of cells to target [11.Liu X. et al.Homophilic CD44 interactions mediate tumor cell aggregation and polyclonal metastasis in patient-derived breast cancer models.Cancer Discov. 2019; 9: 96-113Crossref PubMed Scopus (140) Google Scholar,41.Cheung K.J. et al.Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumor cell clusters.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E854-63Crossref PubMed Scopus (395) Google Scholar]. Patients with breast cancer with five or more CTCs in one 7.5-mL blood draw have significantly worse overall survival and progression-free survival [16.Wang C. et al.Longitudinally collected CTCs and CTC-clusters and clinical outcomes of metastatic breast cancer.Breast Cancer Res. Treat. 2017; 161: 83-94Crossref PubMed Scopus (108) Google Scholar,42.Cristofanilli M. et al.Circulating tumor cells, disease progression, and survival in metastatic breast cancer.N. Engl. J. Med. 2004; 351: 781-791Crossref PubMed Scopus (3526) Google Scholar]. These prognostic values are even greater for patients with elevated numbers of CTC clusters [16.Wang C. et al.Longitudinally collected CTCs and CTC-clusters and clinical outcomes of metastatic breast cancer.Breast Cancer Res. Treat. 2017; 161: 83-94Crossref PubMed Scopus (108) Google Scholar]. CTC clusters are traditionally defined as groups of tumor cells with more than two nuclei that can shed as cohesive clusters from the primary tumor [3.Aceto N. et al.Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis.Cell. 2014; 158: 1110-1122Abstract Full Text Full Text PDF PubMed Scopus (1380) Google Scholar] but also occur through cellular aggregation based on intravital imaging studies on homotypic CTC cluster formation [11.Liu X. et al.Homophilic CD44 interactions mediate tumor cell aggregation and polyclonal metastasis in patient-derived breast cancer models.Cancer Discov. 2019; 9: 96-113Crossref PubMed Scopus (140) Google Scholar]. The CTC aggregation mechanism might be strengthened by or coordinate with cohesive intercellular interactions. Because CTC clusters have become a well-established clinical biomarker for disease severity and progression in breast cancer as well as other cancers, including lung cancer, the race to uncover the molecular mechanisms behind clustering is on [43.Murlidhar V. et al.Poor Prognosis indicated by venous circulating tumor cell clusters in early-stage lung cancers.Cancer Res. 2017; 77: 5194-5206Crossref PubMed Scopus (89) Google Scholar,44.Paoletti C. et al.Significance of circulating tumor cells in metastatic triple-negative breast cancer patients within a randomized, phase II trial: TBCRC 019.Clin. Cancer Res. 2015; 21: 2771-2779Crossref PubMed Scopus (63) Google Scholar]. In breast cancer, homotypic clustering of CTCs can be directed by CD44 homophilic interactions that upregulate OCT4, epidermal growth factor receptor (EGFR), and p21-activated kinase 2 (PAK2)/focal adhesion kinase (FAK) signaling pathways [11.Liu X. et al.Homophilic CD44 interactions mediate tumor cell aggregation and polyclonal metastasis in patient-derived breast cancer models.Cancer Discov. 2019; 9: 96-113Crossref PubMed Scopus (140) Google Scholar,37.Liu X. et al.EGFR inhibition blocks cancer stem cell clustering and lung metastasis of triple negative breast cancer.Theranostics. 2021; 11: 6632-6643Crossref PubMed Google Scholar] and also by a newly identified stemness-promoting adhesion molecule, intercellular adhesion molecule 1 (ICAM1) [45.Taftaf R. et al.ICAM1 initiates CTC cluster formation and trans-endothelial migration in lung metastasis of breast cancer.Nat. Commun. 2021; 12: 4867https://doi.org/10.1038/s41467-021-25189-zCrossref PubMed Scopus (18) Google Scholar] (Figure 1). Other studies characterizing CTC clusters have shown that intratumor hypoxia can trigger cluster formatio