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Introduction: The characterization of circulating tumor cells (CTC) and circulating tumor microemboli (CTM) has emerged as both a challenge to the standard view of metastasis, and as a valuable means for understanding genotypic and phenotypic variability shown even within the same cancer type. However, in the case of salivary gland neoplasms, limited data are available for the role that CTCs and CTMs play in metastasis and secondary tumor formation.ru.AQ1 In response to this, we propose that similarities between in vitro clusters of cultured salivary gland cancer cells may act as a surrogate model for in vivo CTCs and CTMs isolated from patients. Materials and Methods: Using techniques in immunofluorescence, immunoblotting, and 2-dimensional migration, we isolated and characterized a group of cohort cells from a commercially available cell line (HTB-41). Results: Here, cells exhibited a hybrid phenotype with simultaneous expression of both epithelial and mesenchymal markers (E-cadherin, vimentin, and α-SMA). Cohort cells also exhibited increased migration in comparison to parental cells. Conclusion: Data suggest that these isolated cell clusters may fucntion as a potential in vitro model of CTCs and CTMs. [ABSTRACT FROM AUTHOR]

Circulating tumor cells (CTCs) and/or circulating tumor microemboli (CTM) from non-small cell lung cancer (NSCLC) patients may be a non-invasive tool for prognosis, acting as liquid biopsy. CTCs interact with platelets through the transforming growth factor-β/transforming growth factor-β receptor type 1 (TGF-β/TGFβRI) forming clusters. CTCs also may express the Cluster of Differentiation 47 (CD47) protein, responsible for the inhibition of phagocytosis, the "don't eat me" signal to macrophages. Objectives: To isolate, quantify and analyze CTCs/CTMs from metastatic NSCLC patients, identify TGFβRI/CD47 expression in CTCs/CTMs, and correlate with progression-free survival (PFS). Methods: Blood (10 mL) was collected at two time-points: T1 (before the beginning of any line of treatment; T2 (60 days after initial collection). CTCs were isolated using ISET®. Immunocytochemistry was conducted to evaluate TGFβRI/CD47 expression. Results: 45 patients were evaluated. CTCs were observed in 82.2% of patients at T1 (median: 1 CTC/mL; range: 0.33–11.33 CTCs/mL) and 94.5% at T2 (median: 1.33 CTC/mL; 0.33–9.67). CTMs were observed in 24.5% of patients and significantly associated with poor PFS (10 months vs. 17 months for those without clusters; p = 0.05) and disease progression (p = 0.017). CTMs CD47+ resulted in poor PFS (p = 0.041). TGFβRI expression in CTCs/CTMs was not associated with PFS. Conclusion: In this study, we observed that CTC/CTM from NSCLC patients express the immune evasion markers TGFβRI/CD47. The presence of CTMs CD47+ is associated with poor PFS. This was the first study to investigate CD47 expression in CTCs/CTM of patients with NSCLC and its association with poor PFS. [ABSTRACT FROM AUTHOR]

Introduction: Cancer-associated thrombosis (CAT) is a major cause of morbidity and mortality in oncology patients. There are no accurate risk assessment tools to predict venous thromboembolism (VTE). Circulating tumor cells (CTCs), circulating tumor microemboli (CTM), and high platelet-lymphocyte ratio (PLR) may predispose to VTE. Objective: To evaluate correlations of CTCs, CTM, and PLR with VTE and recurrence-free survival (RFS) in gastric cancer patients. Material and Methods: Patients with gastric cancer (localized and metastatic disease) were recruited (March 2016 to April 2017). CTCs were analysed by ISET at two timepoints: before neoadjuvant treatment (CTC1) and after surgery/before adjuvant therapy (CTC2) for patients with localized disease, and before first-line chemotherapy (CTC1) and after 6 months (CTC2) for patients with metastases. VTE incidence was determined retrospectively. RFS was estimated by Kaplan-Meier analysis. Results: We evaluated 93 patients. According to Khorana scores, 63 (67.7%) patients were at intermediate and 30 (32.3%) were at high risk for VTE. VTE incidence was 20.4% and CTM were found in 39.8%. VTE developed in 7/37 (18.9%) CTM-positive and in 11/50 (22%) CTM-negative patients (p=0.93). When PLR >288, VTE occurred in 7/14 patients (p=0.005). PLR also associated with poor RFS (p

Tumor metastasis is directly correlated to poor prognosis and high mortality. Circulating tumor cells (CTCs) play a pivotal role in metastatic cascades, of which CTC clusters is highly metastatic compared to single CTCs. Although platelets and neutrophils within the bloodstream could further exacerbate the pro-metastatic effect of single CTCs, the influence of platelets and neutrophils on CTC clusters mediated metastasis remains unclear. In this study, a pro-metastatic complex composed of CTC clusters, platelets and neutrophils, namely circulating tumor microemboli (CTM), was identified in vivo among different metastatic tumor, which was demonstrated with highly upregulation of hypoxia-inducible factor-1α (HIF-1α). While knock-out of HIF-1α or therapeutically downregulating of HIF-1α via HIF-1α inhibitor (BAY87–2243)-loaded neutrophil cyto-pharmaceuticals (PNEs) could efficiently restrain CTM mediated lung metastasis. The underlying mechanism of metastasis inhibition was attributed to the downregulation of HIF-1α-associated PD-L1, which would enhance immune response for inhibiting metastatic cells. Thus, our work here illustrates that hypoxia was an essential factor in promoting CTM colonization in lung. More importantly, we provide a promising strategy by targeted downregulation of HIF-1α in CTM via neutrophil cyto-pharmaceuticals for treatment of CTM mediated metastasis. Targeted downregulation of hypoxia-inducible factor-1α via neutrophil cyto-pharmaceuticals for restraining circulating tumor microemboli mediated metastasis [Display omitted] • CTC clusters, platelets and neutrophils constitute the pro-metastatic complex. • Circulating tumor microemboli was verified with upregulation of HIF-1α and PD-L1. • Neutrophil cyto-pharmaceuticals efficiently restrain CTM mediated lung metastasis. [ABSTRACT FROM AUTHOR]

This review presents a broad aspect of smart material‐integrated systems for isolating and profiling rare circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) to provide physiological, biological, and mechanistic insights into cancer research. In particular, CTCs/CTM have emanated as essential pieces of evidence that can reveal clonal evolution, tumor heterogeneity, and disease progression within the metastatic cascade. Morphologies, cellular compositions, and rarity of CTCs/CTM make them difficult to track and isolate for profiling distinct molecular characteristics in the case of metastatic potential. Accordingly, with the advanced‐characterization techniques, examining the aspects of the specific surface markers of CTCs/CTM, epithelial‐to‐mesenchymal (EMT), mesenchymal‐to‐epithelial (MET) transitions, and timing of tumor cell dissemination would assist us to understand cancer biology and metastatic characteristics. Existing clinical and research methods for the enrichment and isolation of these sporadic cells depend on mainly conventional methods with low‐yield and expensive features. Owing to their specialized functions and analytical performances, smart material‐based technologies hold an enormous impact not only on cell detection, but also on cell isolation for downstream analyses. Herein, the main reasons for cell isolation are discussed and the recent developments in CTCs/CTM approaches for identifying further methods and future perspectives are elaborated on. [ABSTRACT FROM AUTHOR]

Background. Breast cancer (BC) is the most common type of malignant neoplasm among women, with a high rate of metastasis. Early non-invasive diagnosis is required to increase the effectiveness of anticancer therapy.Objective: to determine the content of circulating tumor cells (CTCs) and their derivatives in the peripheral blood using the MDA-231 aptamer, compare the results obtained with the clinical and molecular characteristics of BC.Materials and methods. The study included 22 patients with BC. Detection of CTCs and circulating tumor microemboli was carried out in 3.5 ml of the blood of BC patients with the help of the MDA-231 aptamer which is affine for breast cancer cells, labeled with the fluorescent Cy3 dye. The count of CTCs in the blood samples was performed using fluorescent and laser scanning microscopy.Results and conclusions. The content of CTCs and circulating tumor microemboli in the peripheral blood of patients with BC of various molecular subtypes was analyzed using the MDA-231 aptamer. The relationship between the number of CTCs and the molecular biological subtype was revealed. The obtained results show the possible prognostic value of CTCs use for monitoring effectiveness of anticancer therapy and control of recurrence of BC.

Colorectal cancer is a common and often deadly cancer. Circulating tumor cells (CTCs) have been implicated as a potentially valuable prognosis factor. The detection of circulating tumor microemboli (CTM) and of simple blood component parameters that reflect inflammatory status, such as the platelet-to-lymphocyte ratio (PLR) and neutrophil-to-lymphocyte ratio (NLR), may provide information about tumor progression. The aim of this study was to explore the importance of CTCs, CTM, PLR, and NLR prospectively in non-metastatic colon cancer progression. CTCs were enriched using ISETⓇ (Isolation by SizE of Tumor cells) and identified by immunocytochemical exclusion of leukocytes. We evaluated CTCs and blood cell parameters in a cohort of 69 stage I–III colon cancer patients (52.2% men; median age, 61 years; age range, 19–87 years) at a baseline timepoint prior to resection surgery. The median of CTC levels at baseline was 20 cells/8 mL (0–94) and higher levels were associated with CTM presence (p = 0.02). CTM were found in 18 (26.1%) patients. Of 18 stage I patients, 33.3% had CTM and of 51 stages II or III patients, 13.7% had CTM (p = 0.08). Patients with a high PLR (>124) were mostly (75.6%) diagnosed with high-risk stages II/III cancer (stages I/low-risk II, 24.4%; p = 0.014). All 8 patients that had disease recurrence during follow-up had a high PLR (p = 0.02 vs. low PLR). NLR was not significantly associated with disease stage or recurrence. The present results indicate that CTCs and PLR analyses may be clinically useful for colon cancer management and risk stratification.

Background/Aim: Liquid biopsy is changing the diagnosis and treatment strategies of various neoplasms. However, the circulating tumor cells (CTCs) of gastrointestinal stromal tumor (GIST) patients with different disease process are not clear. To better understand the dynamic change of CTCs in GIST patients, we conducted a real-life setting study. Patients and Methods: One-hundred fifty GIST patients were included. The isolation by size of tumor cell (ISET) method was employed to detect the CTCs/circulating tumor microemboli (CTM). Imatinib (IM) plasma concentration was detected by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Multivariate and univariate analysis were used to analyze the effects of clinical characteristics on the positive rate of CTC and the number of CTCs/CTM. Results: The positive rate of CTCs was 72%. The median number of CTCs and CTM was 4 and 0. Logistic multivariate regression analysis showed that tumor diameter was the only independent factor of the positive rate of CTCs (P < 0.05). The numbers of CTCs and CTM had intensive linear correlation (P < 0.001). Tumor diameter, Ki 67 expression and mitotic were related to the number of CTCs (P < 0.05). Patients with higher Ki 67 expression tend to have more CTM (P < 0.05). IM plasma concentration showed no influence to the CTCs/CTM (P > 0.05). Conclusions: In the current study, we assessed the CTCs and CTM of GIST patients in various disease progressions and identified clinicopathological factors influencing the detection of CTCs and CTM. These results are instructive for clinicians to understand CTCs/CTM in GIST patients. [ABSTRACT FROM AUTHOR]

Serial examination of circulating tumor cell presence after renal cancer surgery could become an irreplaceable source of information reporting on tumor heterogeneity and evolution in time. Test for presence of circulating tumor cells belongs to the group of liquid biopsy tests and one of its main advantages is the relatively minimal invasiveness (blood withdrawal), which indeed enables tumor dynamics follow-up. Besides circulating tumor cells the pre sence in patients with renal cell carcinoma undergoing surgery, molecular character of circulating cells can be evaluated by gene expression analysis. [ABSTRACT FROM AUTHOR]

Objective: Circulating tumor cell (CTC) detection is a promising noninvasive technique that can be used to diagnose cancer, monitor progression, and predict prognosis. In this study, the authors aimed to investigate the clinical utility of CTCs in the management of diffuse glioma., Methods: Sixty-three patients with newly diagnosed diffuse glioma were included in this multicenter clinical cohort. The authors used a platform based on isolation by size of epithelial tumor cells (ISET) to detect and analyze CTCs and circulating tumor microemboli (CTMs) in the peripheral blood of patients both before and after surgery. Least absolute shrinkage and selector operation (LASSO) and Cox regression analyses were used to verify whether CTCs and CTMs are independent prognostic factors for diffuse glioma., Results: CTC levels were closely related to the degree of malignancy, WHO grade, and pathological subtypes. Receiver operating characteristic curve analysis revealed that a high CTC level was a predictor for glioblastoma. The results also showed that CTMs originate from the parental tumor rather than from the circulation and are an independent prognostic factor for diffuse glioma. The postoperative CTC level is related to the peripheral immune system and patient survival. Cox regression analysis showed that postoperative CTC levels and CTM status are independent prognostic factors for diffuse glioma, and CTC- and CTM-based survival models had high accuracy in internal validation., Conclusions: The authors revealed a correlation between CTCs and clinical characteristics and demonstrated that CTCs and CTMs are independent predictors for the diagnosis and prognosis of diffuse glioma. Their CTC- and CTM-based survival models can enable clinicians to evaluate patients' response to surgery as well as their outcomes.

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Introduction: Soft tissue Sarcomas (STS) are rare malignances, with high mortality rates. Half of patients develop metastasis. The presence of isolated Circulating Tumor Cells (CTCs) and Circulating Tumor Microemboli (CTM) in the blood may be early markers of tumor invasion. Epidermal Growth Factor (EGF) family receptors can also influence this process. Objectives: to quantify CTCs and identify CTM as well as the EGF Receptor (EGFR) protein expression in these cells and correlate with clinical outcome in metastatic STS. Materials and methods: Approximately 8mL of blood was prospectively collected from patients with different types of high-grade STS, before the beginning of chemotherapy. The samples were processed and filtered by ISET (Rarecells, France) for the isolation and quantification of CTCs and CTMs. EGFR expression was analyzed by immunocytochemistry (ICC) on CTCs/ CTMs. Results: We analyzed 18 patients with median age of 49 years (18-77 y). The positivity for EGFR protein expression in CTCs was observed in 93.75% of the patients. This result shows that targeting EGFR positive CTCs from STS origen can be translated in clinical benefit for some patients. In addition, if target therapy is chosen, the EGFR expression in CTCs can be used in follow-up to measure treatment effectiveness. Conclusions: This is the first study to demonstrate the expression of EGFR protein in CTCs from sarcoma patients. It may open an area for future investigations. The next step is to characterize CTCs in a larger cohort of patients to better understand the role of EGFR in sustaining tumor metastasis in sarcomas. [ABSTRACT FROM AUTHOR]

The significance of circulating tumor microemboli (CTMs) in patients with pancreatic ductal adenocarcinoma (PDAC) is still unknown. Thus, the present study used a epithelial cellular adhesion molecule independent subtraction and immunostaining-fluorescence in situ hybridization (SET-iFISH) platform to enumerate circulating tumor cells (CTCs) and CTMs in a total of 86 peripheral blood samples from 19 patients with PDAC. The associations between CTCs and CTMs with clinicopathologic factors and prognoses were analyzed. Prior to treatment, CTCs were detected in all 19 patients, and CTMs were detected in 4 patients with different tumor-node-metastasis (TNM) stages. A total of 85 of the 86 peripheral blood samples had cytokeratin 18-negative CTCs. The number of CTCs and CTMs were significantly associated with tumor size and vascular invasion. Patients with CTMs had poorer overall survival and disease-free survival when compared with those without CTMs (7.3 vs. 25.40 months, P=0.001; and 1.80 vs. 18.97 months, P=0.037). The presence of CTMs in the peripheral blood prior to surgery was predictive of poor prognosis in PDAC patients. CTMs could be detected in patients of different TNM stage (II, III and IV). Surgery did not benefit patients with CTMs, thus, surgeons should take greater consideration when assessing the requirements for surgery in patients with CTMs. [ABSTRACT FROM AUTHOR]

Enumeration of circulating tumor cells (CTCs) in the bloodstream can predict prognosis and survival in cancer patients. However, CTC rarity and heterogeneity pose challenges in using them as biomarkers. Recent publications have reported new classes of circulating, non-cancerous tumor-derived cells present in cancer patients but not in healthy controls; these include cancer-associated macrophages, tumor-endothelial clusters (TECs), and cancer-associated fibroblasts (CAFs). Well-established marker-dependent CTC enrichment technologies will miss this group of circulating cells. To maximize our chance of finding useful circulating biomarkers in cancer patients, we propose the use of size-based enrichment technologies to isolate both cancerous and non-cancerous cells in circulation. We review their biological properties and discuss device features to consider in their enrichment. [ABSTRACT FROM AUTHOR]

Intraoperative manipulation causes circulating tumor cell (CTC) shedding into the blood and accelerates metastasis in non-small cell lung cancer (NSCLC). The present study was conducted to assess the degree of dissemination resulting from surgery and to explore the biological features of CTCs. In patients with NSCLC who underwent complete resection, the pulmonary vein (PV) was isolated and stapled following thoracotomy. The number of CTCs retained per 7.5 ml PV blood (CTC-PV) and peripheral blood were detected. Following hematopoietic cell depletion, a xenograft assay was performed using the CTC-PV. A total of 32 consecutive patients were enrolled in the study, the majority of whom had CTCs in their PV blood (n=29, 90.6%). Circulating tumor microemboli (CTM) were identified in 12 patients (37.5%). The CTC-PV and CTM-PV counts were positively correlated with tumor size (P=0.012 and P=0.028, respectively). Patients with small tumors (<3.0 cm) also had considerable CTC-PV and CTM-PV. A total of 8 patients received platinum-based chemotherapy prior to surgery. The CTC-PV and CTM-PV counts in patients with partial response were significantly lower than those in patients with stable disease or who did not receive induction therapy (P=0.025 and P=0.044, respectively). The enriched CTC-PV from 3 patients were injected into 3 immunodeficient mice, and 1 mouse developed a xenograft tumor. To conclude, the present study indicates that intraoperative manipulation contributes to the hematogenous dissemination of tumorigenic CTCs and CTM. Lobectomy is recommended for lung cancer of any tumor size and stage according to oncological principles, in addition to ligating the PV, if possible, prior to any other treatment. [ABSTRACT FROM AUTHOR]

Background Circulating tumor microemboli (CTM) are clusters of circulating tumor cells (CTCs), involved in metastasis, as also transforming growth factor-β (TGF-β). The purpose of this study was to verify their role in progression-free survival (PFS). Methods Blood from patients with locally advanced head and neck squamous cell carcinoma (HNSCC; n = 53) was analyzed in 2 moments. TGF-β receptor I (TGF-βRI) expression was evaluated by immunocytochemistry. Results Comparing CTM1 (baseline) with CTM2 (first follow-up), patients with CTM1-positive disease who became CTM2-negative were classified as favorable (PFS 20 months). Patients with unfavorable evolution (CTM1-negative/CTM2-positive), had PFS of 17.5 months. Patients always CTM-negative showed PFS of 22.4 months, those always positive, 4.7 months ( P < .001). The TGF-βRI expression in the first follow-up correlated with poor PFS (12 × 26 months; P = .007), being an independent prognostic factor (hazard ratio [HR] = 6.088; P = .033). Conclusion CTM1/2, TGF-βRI expression, and unfavorable CTM kinetics may represent poor prognosis in locally advanced HNSCC. [ABSTRACT FROM AUTHOR]

Background: Circulating tumor cells (CTCs) detected in peripheral blood (PB) of cancer patients can be identified as isolated CTCs and circulating tumor microemboli (CTM). This study aimed to evaluate the prognostic value of CTM detection and CTC phenotype in advanced colorectal cancer (CRC) patients during chemotherapy. Methods: A size-based platform for CTC isolation was applied. PB samples (5 ml) from 98 advanced CRC patients during 2-6 cycles chemotherapy were collected for CTC detection, and CTC count was correlated to patient's clinicopathological characteristics and clinical outcome. And CTC phenotype was measured by immunofluorescent staining and evaluate the predictive significance on survival in 32 CTCs-positive patients with advanced CRC. Results: Forty-eight of 98 patients were CTCs-positive, including 18 CTM-positive patients, and CTC detection was positively correlated with lymphatic invasion (P = 0.049), TNM stage (P = 0.023), and serum CEA level (P = 0.014). Moreover, Kaplan-Meier survival and Cox regression analyses revealed that the presence of CTCs was an independent factor for poor PFS and OS (P < 0.05) in advanced CRC patients during chemotherapy, and CTM-positive patients had shooter survival than isolated CTCs-positive patients (P < 0.05). Furthermore, patients with vimentin+ isolated CTCs/ CTM had shorter PFS and OS compared with CK+ CTCs (P < 0.05). Conclusions: This study provided evidence that the presence of CTCs was positively correlated with poor prognosis, and furthermore, CTM and vimentin+ CTCs predicted poorer survival, which indicated that CTM and vimentin+ CTCs detected by a sensitive platform could be used to improve prognostic value of CTCs in advanced CRC patients under treatment. [ABSTRACT FROM AUTHOR]

Circulating tumors cells ( CTCs) can be detected in the blood of metastatic melanoma patients ( MMPs) both as isolated circulating tumor cells ( iCTCs) and circulating tumor microemboli ( CTMs), but their clinical significance remains unknown. The aim of this work was to evaluate the prognostic impact in metastatic cutaneous melanoma of CTMs and iCTCs identified by a cytomorphological approach using the isolation by size of tumor cell ( ISET) method. We characterized the phenotype of CTCs using anti- PS100, anti- SOX10, anti- CD10, and anti- TRF2 antibodies. 128 MMPs and 37 control healthy individuals with benign nevi were included in this study. Results were compared to the follow-up of patients. 109/128 (85%) MMPs showed CTCs, 44/128 (34%) with 2 to 6 CTMs and 65/128 (51%) with 4 to 9 iCTCs. PS100 expression was homogeneous in iCTCs and heterogeneous in CTMs. SOX10, CD10, and TRF2 were mainly expressed in CTMs. None of the control subjects demonstrated circulating malignant tumor cells. Overall survival was significantly decreased in patients with CTMs, independently of the therapeutic strategies. In conclusion, the presence of CTMs is an independent predictor of shorter survival from the time of diagnosis of MMPs. [ABSTRACT FROM AUTHOR]

Thrombotic events can herald the diagnosis of cancer, preceding any cancer-related clinical symptoms. Patients with cancer are at a 4- to 7-fold increased risk of suffering from venous thromboembolism (VTE), with ~7,000 patients with lung cancer presenting from VTEs. However, the physical biology underlying cancer-associated VTE remains poorly understood. Several lines of evidence suggest that the shedding of tissue factor (TF)-positive circulating tumor cells (CTCs) and microparticles from primary tumors may serve as a trigger for cancer-associated thrombosis. To investigate the potential direct and indirect roles of CTCs in VTE, we characterized thrombin generation by CTCs in an interactive numerical model coupling blood flow with advection-diffusion kinetics. Geometric measurements of CTCs isolated from the peripheral blood of a lung cancer patient prior to undergoing lobectomy formed the basis of the simulations. Singlet, doublet, and aggregate circulating tumor microemboli (CTM) were investigated in the model. Our numerical model demonstrated that CTM could potentiate occlusive events that drastically reduce blood flow and serve as a platform for the promotion of thrombin generation in flowing blood. These results provide a characterization of CTM dynamics in the vasculature and demonstrate an integrative framework combining clinical, biophysical, and mathematical approaches to enhance our understanding of CTCs and their potential direct and indirect roles in VTE formation. [ABSTRACT FROM AUTHOR]

Background: Single circulating tumor cells (CTCs) or circulating tumor microemboli (CTMs) are potential biomarkers of renal cell cancer (RCC), however studies of CTCs/CTMs in RCC are limited. In this pilot study we aimed to evaluate a novel blood filtration technique suited for cytomorphological classification, immunocytochemical and molecular characterization of filtered, so called circulating non-hematologic cells (CNHCs) - putative CTCs/CTMs - in patients with RCC. Methods: Blood of 40 patients with renal tumors was subjected to ScreenCell® filtration. CNHCs were classified according to cytomorphological criteria. Immunocytochemical analysis was performed with antibodies against CD45, CD31 and carbonic anhydrase IX (CAIX, a RCC marker). DNA of selected CNHCs and respective primary tumors was analysed by array-CGH. Results: CNHC-clusters with malignant or uncertain malignant cytomorphological features - putative CTMs - were negative for CD45, positive for CD31, while only 6% were CAIX positive. Array-CGH revealed that 83% of malignant and uncertain malignant cells did represent with a balanced genome whereas 17% presented genomic DNA imbalances which did not match the aberrations of the primary tumors. Putative single CTCs were negative for CD45, 33% were positive for CD31 and 56% were positive for CAIX. Conclusions: The majority of CNHC-clusters, putative CTMs, retrieved by ScreenCell® filtration may be of endothelial origin. Morphological criteria seem to be insufficient to distinguish malignant from non-malignant cells in renal cancer. [ABSTRACT FROM AUTHOR]

A 77-year-old man was admitted at our hospital due to "generalized increase in the number of masses and enlargement of the masses observed for one month". Combined assessment of the imaging (computed tomography and magnetic resonance imaging) findings and results of lung centesis biopsy and liquid biopsy suggest that the patient had small cell lung cancer of the left upper lobe, with right hilar, mediastinal, bilateral axillary, abdominal and retroperitoneal lymph node metastases, as well as widespread subcutaneous soft tissue, liver, bilateral adrenal, bilateral kidneys and multiple brain metastases (extensive stage). In order to obtain an evaluation of the development of the disease as soon as possible, the circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) in 6 mL peripheral blood were examined by subtraction enrichment-immunostaining fluorescence in situ hybridization (SE-iFISH) technology. A total of 919 epithelial cell adhesion molecule (EpCAM)-positive CTCs and 61 EpCAM-positive CTM were identified. Among them, there were 14 haploid CTCs (1.52%), 788 diploid CTCs (85.75%), 44 triploid CTCs (4.79%), 70 tetraploid CTCs (7.62%) and 3 pentaploid or higher-fold polyploid CTCs (0.33%). Herein, we reported a rare case with extremely high accounts of CTCs and CTM and positive findings for tumor markers, which was identified for the first time. The examination of CTCs by SE-iFISH contributed to the diagnosis, prognosis and treatment evaluation of cancer and facilitated the formulation of precise and individualized therapeutic regime., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Sciences).)

Background: This study compared whole blood dilution versus density gradient centrifugation for pre-processing blood samples prior to circulating tumor cell (CTC) capture on the efficiency of CTC separation by size-based isolation., Materials and Methods: Whole blood from a healthy volunteer spiked with SKBR3 cells was used to optimize the whole blood dilution protocol for sample volume, dilution ratio, and paraformaldehyde (PFA) concentration. Whole blood from healthy volunteers spiked with SKBR3, A549, or PC3 cells, and whole blood from patients with advanced gastric, esophageal, or liver cancer, was used to compare pre-processing by the optimal whole blood dilution protocol with density-gradient centrifugation. All statistical evaluations were performed using Student t test of the Statistical Package for Social Sciences (SPSS version 17.0)., Results: In blood samples from healthy volunteers, spiked SKBR3 cell recovery rates were highest in 5 ml of whole blood, diluted with 2.5 ml buffer, and fixed with 0.2% PFA, and spiked SKBR3, A549, and PC3 cell recovery rates from 5 ml whole blood were significantly greater when using the optimized whole blood dilution protocol (87.67% ± 1.76%, 79.50% ± 0.50% and 71.83% ± 1.04%, respectively) compared to density-gradient centrifugation (46.83 ± 1.76%, 37.00 ± 1.50% and 41.00 ± 1.50%, respectively)., (© 2022 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Simple Summary: With estimated numbers of 1–10 per mL of blood, circulating tumor cells (CTCs) are extremely rare compared to white (a few million) or red (billions) blood cells. Given their critical role in metastasis, CTCs have enormous potential as a biomarker for cancer diagnosis, prognosis, and monitoring of treatment response. There are now efforts to characterize CTCs more precisely through molecular and functional analysis, expanding the CTC effort from one of diagnosis and prognosis to now include the use of CTCs to specifically target cancers and discover therapeutic solutions, establishing CTCs as critical in precision medicine. This article summarizes current knowledge about CTC isolation technologies and discusses the translational benefits of different types of downstream analysis approaches, including single-CTC analysis, ex vivo expansion of CTCs, and characterization of CTC-associated cells. Circulating tumor cells (CTCs) have been recognized as a major contributor to distant metastasis. Their unique role as metastatic seeds renders them a potential marker in the circulation for early cancer diagnosis and prognosis as well as monitoring of therapeutic response. In the past decade, researchers mainly focused on the development of isolation techniques for improving the recovery rate and purity of CTCs. These developed techniques have significantly increased the detection sensitivity and enumeration accuracy of CTCs. Currently, significant efforts have been made toward comprehensive molecular characterization, ex vivo expansion of CTCs, and understanding the interactions between CTCs and their associated cells (e.g., immune cells and stromal cells) in the circulation. In this review, we briefly summarize existing CTC isolation technologies and specifically focus on advances in downstream analysis of CTCs and their potential applications in precision medicine. We also discuss the current challenges and future opportunities in their clinical utilization. [ABSTRACT FROM AUTHOR]

Simple Summary: Metastases cause the majority of breast cancer-related deaths. Circulating tumor cells (CTCs), and in particular CTC-clusters, are considered the seeds of metastasis, but their analysis in the early-stages of the disease has so far been limited by the fact that, by using conventional and epithelial-based technologies (as the FDA-approved CellSearch platform), they are more often detected in the metastatic setting. It is known, however, that cancer cells are heterogeneous and can downregulate the expression of epithelial markers, thus limiting the detection capability of epithelial-based technologies. Here, we show that it is possible to increase CTC-cluster detection by using an epithope-independent technology based on blood filtration, and in particular that this strategy allows to detect a high number of CTC-clusters in stage II-III breast cancer patients, before and during neoadjuvant treatment. Our results therefore offer a new opportunity to deepen our understanding of the cancer dissemination process in its early steps. The clinical relevance of circulating tumor cell clusters (CTC-clusters) in breast cancer (BC) has been mostly studied using the CellSearch®, a marker-dependent method detecting only epithelial-enriched clusters. However, due to epithelial-to-mesenchymal transition, resorting to marker-independent approaches can improve CTC-cluster detection. Blood samples collected from healthy donors and spiked-in with tumor mammospheres, or from BC patients, were processed for CTC-cluster detection with 3 technologies: CellSearch®, CellSieve™ filters, and ScreenCell® filters. In spiked-in samples, the 3 technologies showed similar recovery capability, whereas, in 19 clinical samples processed in parallel with CellSearch® and CellSieve™ filters, filtration allowed us to detect more CTC-clusters than CellSearch® (median number = 7 versus 1, p = 0.0038). Next, samples from 37 early BC (EBC) and 23 metastatic BC (MBC) patients were processed using ScreenCell® filters for attaining both unbiased enrichment and marker-independent identification (based on cytomorphological criteria). At baseline, CTC-clusters were detected in 70% of EBC cases and in 20% of MBC patients (median number = 2, range 0–20, versus 0, range 0–15, p = 0.0015). Marker-independent approaches for CTC-cluster assessment improve detection and show that CTC-clusters are more frequent in EBC than in MBC patients, a novel finding suggesting that dissemination of CTC-clusters is an early event in BC natural history. [ABSTRACT FROM AUTHOR]

Simple Summary: Distant metastases derive from the shedding and dissemination of single cancer cells (CTCs) or circulating tumor emboli (CTMs) into circulation. Previous studies on CTMs were mainly run in patients with metastatic disease; however, we observed that CTMs are more frequently detected in patients with early-stage breast cancer. Here, we collected single CTMs and their relative primary tumor tissue samples in early-stage patients. By studying genomic aberrations, present in tumors cells and absent in normal cells, we predicted the tumor fraction thanks to a statistical model developed from a calibration curve with breast cancer cell lines. The tumor fraction ranged from 8% to 48% and CTMs contained specific and shared alterations with respect to tissue. Thus, CTMs may derive from different regions of the primary tumor or from occult micrometastases. Moreover, CTM-private mutations may inform us about specific metastasis-associated functions of involved genes that should be further explored in follow-up and mechanistic studies. Circulating tumor microemboli (CTMs) are clusters of cancer cells detached from solid tumors, whose study can reveal mechanisms underlying metastatization. As they frequently comprise unknown fractions of leukocytes, the analysis of copy number alterations (CNAs) is challenging. To address this, we titrated known numbers of leukocytes into cancer cells (MDA-MB-453 and MDA-MB-36, displaying high and low DNA content, respectively) generating tumor fractions from 0–100%. After low-pass sequencing, ichorCNA was identified as the best algorithm to build a linear mixed regression model for tumor fraction (TF) prediction. We then isolated 53 CTMs from blood samples of six early-stage breast cancer patients and predicted the TF of all clusters. We found that all clusters harbor cancer cells between 8 and 48%. Furthermore, by comparing the identified CNAs of CTMs with their matched primary tumors, we noted that only 31–71% of aberrations were shared. Surprisingly, CTM-private alterations were abundant (30–63%), whereas primary tumor-private alterations were rare (4–12%). This either indicates that CTMs are disseminated from further progressed regions of the primary tumor or stem from cancer cells already colonizing distant sites. In both cases, CTM-private mutations may inform us about specific metastasis-associated functions of involved genes that should be explored in follow-up and mechanistic studies. [ABSTRACT FROM AUTHOR]

Simple Summary: Circulating tumor cells (CTCs) are the seeds that spread through the circulatory system and generate the metastatic sites. Among the various phenotypes exhibited by CTCs, epithelial-to-mesenchymal transition (EMT) has extensively gained attention because of its contribution to the acquisition of invasiveness and motility of CTCs, which is critical for the successful metastasis process. So far, many clinical studies have demonstrated that expressions of mesenchymal features in CTCs are associated with poor clinical outcomes in many types of solid cancer. In this review, the clinical significance of CTCs as a liquid biopsy is described in terms of the mesenchymal characteristics, expression of predictive biomarkers, and their correlations. Detailed analysis of these multivariate targets in CTCs could improve therapeutic decision-making. Metastasis-related events are the primary cause of cancer-related deaths, and circulating tumor cells (CTCs) have a pivotal role in metastatic relapse. CTCs include a variety of subtypes with different functional characteristics. Interestingly, the epithelial–mesenchymal transition (EMT) markers expressed in CTCs are strongly associated with poor clinical outcome and related to the acquisition of circulating tumor stem cell (CTSC) features. Recent studies have revealed the existence of CTC clusters, also called circulating tumor microemboli (CTM), which have a high metastatic potential. In this review, we present current opinions regarding the clinical significance of CTCs and CTM with a mesenchymal phenotype as clinical surrogate markers, and we summarize the therapeutic strategy according to phenotype characterization of CTCs in various types of cancers for future precision medicine. [ABSTRACT FROM AUTHOR]

Although metastasis is the primary cause of death in patients with malignant solid tumors, efficient anti-metastatic therapies are not clinically available currently. Sulfated glycosaminoglycans from marine sources have shown promising pharmacological effects, acting on different steps of the metastatic process. Oversulfated dermatan sulfates from ascidians are effective in preventing metastasis by inhibition of P-selectin, a platelet surface protein involved in the platelet-tumor cell emboli formation. We report in this work that the heparan sulfate isolated from the viscera of the ascidian Phallusia nigra drastically attenuates metastases of colon carcinoma cells in mice. Our in vitro and in vivo assessments demonstrate that the P. nigra glycan has very low anticoagulant and antithrombotic activities and a reduced hypotension potential, although it efficiently prevented metastasis. Therefore, it may be a promising candidate for the development of a novel anti-metastatic drug. [ABSTRACT FROM AUTHOR]

Evaluation of circulating tumor cells (CTCs) has demonstrated clinical validity as a prognostic tool based on enumeration, but since the introduction of this tool to the clinic in 2004, further clinical utility and widespread adoption have been limited. However, immense efforts have been undertaken to further the understanding of the mechanisms behind the biology and kinetics of these rare cells, and progress continues toward better applicability in the clinic. This review describes recent advances within the field, with a particular focus on understanding the biological significance of CTCs, and summarizes emerging methods for identifying, isolating, and interrogating the cells that may provide technical advantages allowing for the discovery of more specific clinical applications. Included is an atlas of high-definition images of CTCs from various cancer types, including uncommon CTCs captured only by broadly inclusive nonenrichment techniques.