Circulating tumor microemboli: Progress in molecular understanding and enrichment technologies

Circulating tumor cells (CTCs) and their clusters, also known as circulating tumor microemboli (CTM), have emerged as valuable tool that can provide mechanistic insights into the tumor heterogeneity, clonal evolution, and stochastic events within the metastatic cascade. However, recent investigations have hinted that CTM may not be mere aggregates of tumor cells but cells comprising CTM exhibit distinct phenotypic and molecular characteristics in comparison to single CTCs. Moreover, in many cases CTM demonstrated higher metastatic potential and resistance to apoptosis as compared to their single cell counterparts. Thus, their evaluation and enumeration may provide a new dimension to our understanding of cancer biology and metastatic cancer spread as well as offer novel theranostic biomarkers. Most of the existing technologies for isolation of hematogenous tumor cells largely favor single CTCs, hence there is a need to devise new approaches, or re-configure the existing ones, for specific and efficient CTM isolation. Here we review existing knowledge and insights on CTM biology. Furthermore, a critical commentary on current and emerging trends in CTM enrichment and characterization along with recently developed ex-vivo CTC expansion methodologies is presented with the aim to facilitate researchers to identify further avenues of research and development.     

Sensitive and Specific Biomimetic Lipid Coated Microfluidics to Isolate Viable Circulating Tumor Cells and Microemboli for Cancer Detection

Here we presented a simple and effective membrane mimetic microfluidic device with antibody conjugated supported lipid bilayer (SLB) “smart coating” to capture viable circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) directly from whole blood of all stage clinical cancer patients. The non-covalently bound SLB was able to promote dynamic clustering of lipid-tethered antibodies to CTC antigens and minimized non-specific blood cells retention through its non-fouling nature. A gentle flow further flushed away loosely-bound blood cells to achieve high purity of CTCs, and a stream of air foam injected disintegrate the SLB assemblies to release intact and viable CTCs from the chip. Human blood spiked cancer cell line test showed the ~95% overall efficiency to recover both CTCs and CTMs. Live/dead assay showed that at least 86% of recovered cells maintain viability. By using 2 mL of peripheral blood, the CTCs and CTMs counts of 63 healthy and colorectal cancer donors were positively correlated with the cancer progression. In summary, a simple and effective strategy utilizing biomimetic principle was developed to retrieve viable CTCs for enumeration, molecular analysis, as well as ex vivo culture over weeks. Due to the high sensitivity and specificity, it is the first time to show the high detection rates and quantity of CTCs in non-metastatic cancer patients. This work offers the values in both early cancer detection and prognosis of CTC and provides an accurate non-invasive strategy for routine clinical investigation on CTCs.     

Detection of Circulating Tumor Cells and Circulating Tumor Microemboli in Gastric Cancer

PURPOSE: Gastric cancer studies indicated a potential correlation between circulating tumor cells (CTCs) in peripheral blood and tumor relapse/metastasis. The prevalence and significance of circulating tumor microemboli (CTM) in gastric cancer remain unknown. We investigated the prevalence and prognostic value of CTCs and CTM for progression-free survival (PFS) and overall survival (OS) in gastric cancer patients. METHODS:Eighty-one gastric cancer patients consented to provide 5 ml of peripheral blood before systematic therapy. CTCs and CTM were isolated using isolation by size of epithelial tumor cells and characterized by cytopathologists. For 41 stage IV gastric cancer patients, CTM was investigated as a potential biomarker to predict prognosis. RESULTS:CTCs were detected in 51 patients; the average count was 1.81. In clinical stage I, II, III, and IV patients, the average CTC counts were 1.40, 0.67, 1.24, and 2.71, respectively. CTM were detected in 3 of 33 clinical stage I to IIIb patients, at an average of 0.12 (0-2). CTM were detected in 13 of 53 clinical stage IIIc to IV patients, at an average of 1.26 (0-22). In stage IV patients, CTM positivity correlated with the CA125 level. PFS and OS in CTM-positive patients were significantly lower than in CTM-negative patients (P < .001). CTM positivity was an independent factor for determining the PFS (P = .016) and OS (P = .003) of stage IV patients in multivariate analysis. Using markers of the epithelial-mesenchymal transition, single CTCs were divided into three phenotypes including epithelial CTCs, biphenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs. For CTM, CK?/Vimentin+/CD45? and CK+/Vimentin+/CD45? phenotypes were observed, but the CK+/Vimentin?/CD45? CTM phenotype was not. CA125 was detected in gastric cancer cell lines BGC823 and MGC803. CONCLUSIONS: In stage IV patients, CTM positivity was correlated with serum CA125 level. CTM were an independent predictor of shorter PFS and OS in stage IV patients. Thus, CTM detection may be a useful tool to predict prognosis in stage IV patients.     

Circulating tumor cells as therapy-related biomarkers in cancer patients

Carcinomas (tumors of epithelial origin) are responsible for most of all new cancers in the industrialized countries. Due to the high mortality rate caused by the metastatic spread of aggressive cancer cells, there is an urgent demand in finding new biomarkers, which should detect early formation of metastases and monitor efficacy of systemic adjuvant therapy in a timely manner. It has been considered that the molecular analysis of cells which are shed from tumors into the blood system (circulating tumor cells (CTCs)) might provide new insights for the clinical management of cancer, probably far earlier than using traditional high-resolution imaging technologies. Clinical trials indicated that CTCs can be deployed for diagnostic, monitoring, and prognostic purposes. Furthermore, these cells are discussed to be suitable as predictive markers. In any case, identification of CTCs requires innovative and challenging technologies as detection methods should be specific, sensitive, standardized, and highly reproducible. Although many different approaches have been developed until now, only the CellSearch? method has been cleared by the American Food and Drug Administration. Although the detection of CTCs has already shown to have a prognostic impact in many tumor entities including breast, prostate, lung and colon cancer, ongoing and future studies are aimed to explore whether CTCs can be used for an individual therapy decision making including novel immunotherapeutic approaches. This review discusses (1) different detection strategies for CTCs, (2) their clinical impact, and (3) the potential use of CTCs guiding the treatment of individual cancer patients.     

Circulating tumor cells in the diagnosis and management of pancreatic cancer

Pancreatic cancers are typically resistant to chemo and radiation therapy and are predisposed to distant metastases. Circulating tumor cells (CTCs) are tumor cells disseminated from primary and metastatic sites and can be isolated from peripheral blood. CTC may overcome the limitation of the current available tumor markers, CA19-9. As a surrogate for 'real-time biopsy', CTCs allow recurrent assessment of a tumor's biological activity. We review the current methodologies for CTC extraction and characterization including antibody-based immunological assays, PCR-based assays, and novel technologies based on the physical or biological characteristics of CTCs. CTCs also provide an accessible link to the existence of epithelial to mesenchymal transition, tumor stem cell markers, and ongoing clonal mutations and epigenetic changes in the tumor. We also explore the potential of using CTC profiling in diagnosis, selection of neoadjuvant and adjuvant therapy, detection of recurrent disease, examination of pharmacodynamic biomarkers, as well as in gene therapy and immunotherapy for pancreatic cancer. Ongoing CTC characterization not only has the potential to represent all cells shed from primary pancreatic tumor and each metastatic site, but also allows dynamic sampling at multiple time points during the clinical course to identify the subpopulations of CTCs and the specific molecules driving metastasis and chemo resistance. We predict that CTC genotyping and phenotyping will play an increasing role in personalized therapy and in identification of novel therapeutic targets as well as monitoring the course and status of the disease.     

Circulating tumor cell isolation,culture, and downstream molecular analysis

Circulating tumor cells (CTCs) are a major contributor of cancer metastases and hold a promising prognostic significance in cancer detection. Performing functional and molecular characterization of CTCs provides an in-depth knowledge about this lethal disease. Researchers are making efforts to design devices and develop assays for enumeration of CTCs with a high capture and detection efficiency from whole blood of cancer patients. The existing and on-going research on CTC isolation methods has revealed cell characteristics which are helpful in cancer monitoring and designing of targeted cancer treatments. In this review paper, a brief summary of existing CTC isolation methods is presented. We also discuss methods of detaching CTC from functionalized surfaces (functional assays/devices) and their further use for ex-vivo culturing that aid in studies regarding molecular properties that encourage metastatic seeding. In the clinical applications section, we discuss a number of cases that CTCs can play a key role for monitoring metastases, drug treatment response, and heterogeneity profiling regarding biomarkers and gene expression studies that bring treatment design further towards personalized medicine.     

Prognostic impact of circulating tumor cells in patients with ampullary cancer

Circulating tumor cells (CTCs) are an important topic of investigation for both basic and clinical cancer research. In this prospective study, we evaluated the clinical role of CTCs in ampullary cancer. We analyzed blood samples from 62 consecutively diagnosed patients with ampullary adenocarcinoma and 24 healthy controls for their CTC content. Combined data from immunostaining of CD45, 4′,6‐diamidino‐2‐phenylindole (DAPI), and fluorescence in situ hybridization with a chromosome 8 centromere (CEP8) probe were used to identify CTCs; cells that were CD45‐/DAPI+/CEP8>2 were considered CTCs. The Cox proportional hazards model was used to assess the relationship between CTCs, clinical characteristics, and patient outcomes. We detected ≥2 CTCs/3.2 ml whole blood in 43 of 62 patients (69.4%), as well as ≥5 CTCs/3.2 ml in 16 of these patients (25.8%). A CTC cutoff value of 2 cells/3.2 ml achieved 69.4% sensitivity and 95.8% specificity as a diagnostic tool; CTCs were associated with tumor burden. CTC levels ≥3/3.2 ml (hazard ratio [HR]: 2.5, 95% confidence interval [CI]: (1.2–5.2), p = 0.014) and ≥5/3.2 ml (HR: 3.5, 95% CI: 1.7–7.3, p < 0.001) were both associated with shorter disease‐free survival. Moreover, ≥3 CTCs/3.2 ml (HR: 2.7, 95% CI: 1.2–6.3, p = 0.019) and ≥5 CTCs/3.2 ml (HR: 3.8, 95% CI: 1.8–8.5, p < 0.001) were predictive of shorter overall survival. CTC assessment may help identify patients with ampullary cancer who are at high risk of an unfavorable outcome.     

循环肿瘤细胞在女性实体器官肿瘤中的研究进展

循环肿瘤细胞(circulating tumor cells,CTCs)指的是从实体的肿瘤或转移的病灶进入外周血液循环的恶性肿瘤细胞。自发现以来,随着其检验技术日趋成熟,循环肿瘤细胞(CTCs)日渐成为肿瘤学炙手可热的研究对象。因为它将通过外周血的检验来实现监测肿瘤的发生、发展、转移、复发等情况,相对于肿瘤实体活检,"液体活检"不仅让患者易于接受,更有利于医务工作者监测病情变化。本文综述了循环肿瘤细胞(CTCs)的检测方法并综述了循环肿瘤细胞在女性实体肿瘤--乳腺癌、卵巢癌、宫颈癌、子宫内膜癌中的研究进展。其中着重介绍了其在早期乳腺癌及复发转移性乳腺癌中的重大意义以及在评价治疗效果中的分子学特征。实践表明,循环肿瘤细胞(CTCs)与HE-4、CA125的联合应用在评估卵巢癌化疗敏感性中也具有重要的临床意义。     

Patterns and functional implications of platelets upon tumor “education”

While platelets are traditionally recognized to play a predominant role in hemostasis and thrombosis, increasing evidence verifies its involvement in malignancies. As a component of the tumor microenvironment, platelets influence carcinogenesis, tumor metastasis and chemotherapy efficiency. Platelets status is thus predictable as a hematological biomarker of cancer prognosis and a hot target for therapeutic intervention. On the other hand, the role of circulating tumor cells (CTCs) as an inducer of platelet activation and aggregation has been well acknowledged. The cross-talk between platelets and CTCs is reciprocal on that the CTCs activate platelets while platelets contribute to CTCs’ survival and dissemination. This review covers some of the current issues related to the loop between platelets and tumor aggression, including the manners of tumor cells in “educating” platelets and biofunctional alterations of platelets upon tumor “education”. We also highlight the potential clinical applications on the interplay between tumors and platelets. Further studies with well-designed prospective multicenter trials may contribute to clinical “liquid biopsy” diagnosis by evaluating the global changes of platelets.     

In vitro generation of effector cells cytotoxic to autologous targets from chronic myeloid leukemia patients in remission

Summary Peripheral blood lymphocytes (PBL) from chronic myeloid leukemia (CML) patients in remission were stimulated in vitro, in a 3-cell assay with autologous leukemic cells or autologous bone marrow (BM) cells alone, or each in combination with allogeneic PBL. The responder cells were used as effectors in a 4-h ⁵¹Cr release cytotoxicity assay using autologous targets such as leukemic cells, BM cells, phytohemagglutinin-induced lymphoblasts, and allogeneic K562 (erythroblastoid leukemic cell line) target cells. Sensitization of lymphocytes from CML patients with either autologous leukemic cells or BM cells generated cytotoxic cells (CTCs) capable of killing both the targets. These results suggested that in CML, the PBL may have been sensitized to myeloid maturation-related antigens in vivo, which, on secondary stimulation in vitro, may result in differentiation of CTCs cytotoxic to immature myeloid cells, either from autologous leukemic cells or autologous BM. The inability of PBL from patients with oral cancers to lyse autologous BM cells upon in vitro stimulation, supported this possibility. Clonogenic assays conducted to assess the colony forming potential of BM cells which had interacted with CTCs indicated that there was about 37% reduction in committed granulocyte stem cell colony formation without an appreciable change in committed granulocyte/monocyte stem cell units and clusters. Therefore, since the BM toxicity of the CTCs is not very high, these cells may have a potential clinical use in CML.     

Mutational Analysis of Circulating Tumor Cells from Colorectal Cancer Patients and Correlation with Primary Tumor Tissue

Circulating tumor cells (CTCs) provide a non-invasive accessible source of tumor material from patients with cancer. The cellular heterogeneity within CTC populations is of great clinical importance regarding the increasing number of adjuvant treatment options for patients with metastatic carcinomas, in order to eliminate residual disease. Moreover, the molecular profiling of these rare cells might lead to insight on disease progression and therapeutic strategies than simple CTCs counting. In the present study we investigated the feasibility to detect KRAS, BRAF, CD133 and Plastin3 (PLS3) mutations in an enriched CTCs cell suspension from patients with colorectal cancer, with the hypothesis that these genes` mutations are of great importance regarding the generation of CTCs subpopulations. Subsequently, we compared CTCs mutational status with that of the corresponding primary tumor, in order to access the possibility of tumor cells characterization without biopsy. CTCs were detected and isolated from blood drawn from 52 colorectal cancer (CRC) patients using a quantum-dot-labelled magnetic immunoassay method. Mutations were detected by PCR-RFLP or allele-specific PCR and confirmed by direct sequencing. In 52 patients, discordance between primary tumor and CTCs was 5.77% for KRAS, 3.85% for BRAF, 11.54% for CD133 rs3130, 7.69% for CD133 rs2286455 and 11.54% for PLS3 rs6643869 mutations. Our results support that DNA mutational analysis of CTCs may enable non-invasive, specific biomarker diagnostics and expand the scope of personalized medicine for cancer patients.     

Clinical Implications and Future Perspectives of Circulating Tumor Cells and Biomarkers in Clinical Outcomes of Colorectal Cancer

Colorectal cancer (CRC) is a major public health problem. Early CRC detection, pretherapeutic responsiveness prediction, and postoperative micrometastasis monitoring are the hallmarks for successful CRC treatment. Here, the methodologies used for detecting circulating tumor cells (CTCs) from CRC are reviewed. In addition to the traditional CRC biomarkers, the persistent presence of posttherapeutic CTCs indicates resistance to adjuvant chemotherapy and/or radiotherapy; hence, CTCs also play a decisive role in the subsequent relapse of CRC. Moreover, the genetic and phenotypic profiling of CTCs often differs from that of the primary tumor; this difference can be used to select the most effective targeted therapy. Consequently, studying CTCs can potentially individualize treatment strategies for patients with CRC. Therefore, CTC detection and characterization may be valuable tools for refining prognosis, and CTCs can be used in a real-time tumor biopsy for designing individually tailored therapy against CRC.     

Diagnostic technologies for circulating tumour cells and exosomes

Circulating tumour cells (CTCs) and exosomes are promising circulating biomarkers. They exist in easily accessible blood and carry large diversity of molecular information. As such, they can be easily and repeatedly obtained for minimally invasive cancer diagnosis and monitoring. Because of their intrinsic differences in counts, size and molecular contents, CTCs and exosomes pose unique sets of technical challenges for clinical translation–CTCs are rare whereas exosomes are small. Novel technologies are underway to overcome these specific challenges to fully harness the clinical potential of these circulating biomarkers. Herein, we will overview the characteristics of CTCs and exosomes as valuable circulating biomarkers and their associated technical challenges for clinical adaptation. Specifically, we will describe emerging technologies that have been developed to address these technical obstacles and the unique clinical opportunities enabled by technological innovations.     

Circulating tumor cells: the 'leukemic phase' of solid cancers

It is well known that malignant cells circulate in the bloodstream of patients with solid tumors. However, the biological significance of circulating tumor cells (CTCs) and the clinical relevance of their detection are still debated. Besides technical issues regarding CTC-detection methods, discontinuous shedding of CTCs from established cancer deposits, genomic instability and metastatic inefficiency might underlie the conflicting results currently available. Nevertheless, technological advances and recent clinical findings are prompting researchers to dissect CTC biology further. Here, we review these recent findings, and discuss the prospects for the identification and molecular characterization of the CTC subset that is responsible for metastasis development. This would provide a formidable tool for prognosis evaluation, anticancer-drug development and, ultimately, cancer-therapy personalization.     

Epithelial-mesenchymal transition: a hallmark in metastasis formation linking circulating tumor cells and cancer stem cells

The occurrence of either regional or distant metastases is an indicator of poor prognosis for cancer patients. The mechanism of their formation has not yet been fully uncovered, which limits the possibility of developing new therapeutic strategies. Nevertheless, the discovery of circulating tumor cells (CTCs), which are responsible for tumor dissemination, and cancer stem cells (CSCs), required for tumor growth maintenance, shed light on the metastatic cascade. It seems that CTCs and CSCs are not necessarily separate populations of cancer cells, as CTCs generated in the process of epithelial-mesenchymal transition (EMT) can bear features characteristic of CSCs. This article describes the mechanisms of CTC and CSC formation and characterizes their molecular hallmarks. Moreover, we present different types of EMT occurring in physiological and pathological conditions, and we demonstrate its crucial role in providing CTCs with a CSC phenotype. The article delineates molecular changes acquired by cancer cells undergoing EMT that facilitate metastasis formation. Deeper understanding of those processes is of fundamental importance for the development of new strategies of early cancer detection and effective cancer treatment approaches that will be translated into clinical practice.     

Associations between the cyclooxygenase-2 expression in circulating tumor cells and the clinicopathological features of patients with colorectal cancer

While previous studies have shown that the number of circulating tumor cells (CTCs) alone is not sufficient to reflect tumor progression and that cyclooxygenase-2 (COX-2) expression is correlated with colorectal cancer (CRC) metastasis, COX-2 expression status and its potential functions in CTCs of CRC patients are unknown. Here, epithelial-mesenchymal transition (EMT) phenotype-based subsets of CTCs and the COX-2 expression status in CTCs were identified and their potential clinical values were assessed in 91 CRC patients. CTCs were enumerated in peripheral blood and subsets of CTCs (epithelial [eCTCs], mesenchymal [mCTCs], and biophenotypic [bCTCs]) and the COX-2 expression status were determined using the RNA in situ hybridization method. CTCs were detected in 80.2% (73 of 91) patients. Neither the total CTC nor eCTC numbers were found to significantly associate with any of the clinicopathological features. However, the number of mCTCs was significantly associated with distance metastasis (P = 0.035) and had a trend of being associated with lymph node metastasis ( P = 0.055). Among the 73 patients enrolled for evaluating COX-2 expression, 52.5% (38 of 73) were found to express COX-2 in CTCs, and COX-2 expression in CTCs was not found to associate with the clinicopathological factors. However, COX-2 expression in mCTCs tended to have a higher rate in patients with metastasis compared with those without metastasis (72.0% vs 42.8%; P = 0.072). Furthermore, COX-2 expression and mCTC marker expression correlated positively ( R = 0.287; P = 0.017). Further studies are required to investigate the clinical value of the expression of COX-2 in mCTCs, especially in CRC patients with the advanced tumor stage and distant metastasis.     

EpCAM-Independent Enrichment of Circulating Tumor Cells in Metastatic Breast Cancer

Circulating tumor cells (CTCs) are the potential precursors of metastatic disease. Most assays established for the enumeration of CTCs so far–including the gold standard CellSearch—rely on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM). But, these approaches may not detect CTCs that express no/low levels of EpCAM, e.g. by undergoing epithelial-to-mesenchymal transition (EMT). Here we present an enrichment strategy combining different antibodies specific for surface proteins and extracellular matrix (ECM) components to capture an EpCAM^low/neg cell line and EpCAM^neg CTCs from blood samples of breast cancer patients depleted for EpCAM-positive cells. The expression of respective proteins (Trop2, CD49f, c-Met, CK8, CD44, ADAM8, CD146, TEM8, CD47) was verified by immunofluorescence on EpCAM^pos (e.g. MCF7, SKBR3) and EpCAM^low/neg (MDA-MB-231) breast cancer cell lines. To test antibodies and ECM proteins (e.g. hyaluronic acid (HA), collagen I, laminin) for capturing EpCAM^neg cells, the capture molecules were first spotted in a single- and multi-array format onto aldehyde-coated glass slides. Tumor cell adhesion of EpCAM^pos/neg cell lines was then determined and visualized by Coomassie/MitoTracker staining. In consequence, marginal binding of EpCAM^low/neg MDA-MB-231 cells to EpCAM-antibodies could be observed. However, efficient adhesion/capturing of EpCAM^low/neg cells could be achieved via HA and immobilized antibodies against CD49f and Trop2. Optimal capture conditions were then applied to immunomagnetic beads to detect EpCAM^neg CTCs from clinical samples. Captured CTCs were verified/quantified by immunofluorescence staining for anti-pan-Cytokeratin (CK)-FITC/anti-CD45 AF647/DAPI. In total, in 20 out of 29 EpCAM-depleted fractions (69%) from 25 metastatic breast cancer patients additional EpCAM^neg CTCs could be identified [range of 1–24 CTCs per sample] applying Trop2, CD49f, c-Met, CK8 and/or HA magnetic enrichment. EpCAM^neg dual-positive (CK^pos/CD45^pos) cells could be traced in 28 out of 29 samples [range 1–480]. By single-cell array-based comparative genomic hybridization we were able to demonstrate the malignant nature of one EpCAM^neg subpopulation. In conclusion, we established a novel enhanced CTC enrichment strategy to capture EpCAM^neg CTCs from clinical blood samples by targeting various cell surface antigens with antibody mixtures and ECM components.     

“Sentinel” Circulating Tumor Cells Allow Early Diagnosis of Lung Cancer in Patients with Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a risk factor for lung cancer. Migration of circulating tumor cells (CTCs) into the blood stream is an early event that occurs during carcinogenesis. We aimed to examine the presence of CTCs in complement to CT-scan in COPD patients without clinically detectable lung cancer as a first step to identify a new marker for early lung cancer diagnosis. The presence of CTCs was examined by an ISET filtration-enrichment technique, for 245 subjects without cancer, including 168 (68.6%) COPD patients, and 77 subjects without COPD (31.4%), including 42 control smokers and 35 non-smoking healthy individuals. CTCs were identified by cytomorphological analysis and characterized by studying their expression of epithelial and mesenchymal markers. COPD patients were monitored annually by low-dose spiral CT. CTCs were detected in 3% of COPD patients (5 out of 168 patients). The annual surveillance of the CTC-positive COPD patients by CT-scan screening detected lung nodules 1 to 4 years after CTC detection, leading to prompt surgical resection and histopathological diagnosis of early-stage lung cancer. Follow-up of the 5 patients by CT-scan and ISET 12 month after surgery showed no tumor recurrence. CTCs detected in COPD patients had a heterogeneous expression of epithelial and mesenchymal markers, which was similar to the corresponding lung tumor phenotype. No CTCs were detected in control smoking and non-smoking healthy individuals. CTCs can be detected in patients with COPD without clinically detectable lung cancer. Monitoring “sentinel” CTC-positive COPD patients may allow early diagnosis of lung cancer.