Comparative analysis of innate immune system function in metastatic breast,colorectal, and prostate cancer patients with circulating tumor cells

In recent years, circulating tumor cells (CTCs) in metastatic cancer patients have been found to be a promising biomarker to predict overall survival and tumor progression in these patients. A relatively high number of CTCs has been correlated with disease progression and poorer prognosis. This study was designed to assess innate immune system function, known to be responsible for the immune defense against developing neoplasms, in metastatic cancer patients with CTCs. Our aim is to provide a link between indication of poorer prognosis, represented by the number of CTCs to the cytotoxic activity of natural killer cells, an important component of the innate immune system, and to represent a promising expanded approach to management of metastatic cancer patients with CTCs. Seventy-four patients, with metastatic breast, colorectal, or prostate cancer, were recruited for this study. Using a flow cytometric assay, we measured natural killer (NK) cell cytotoxicity against K562 target cells; and CTCs were enumerated using the CellSearch System. Toll-like receptors 2 and 4 expression was also determined by flow cytometry.     

Case study of the morphologic variation of circulating tumor cells

We report a detailed cytomorphologic evaluation of the circulating component of widely metastatic breast carcinoma. A previously healthy 38-year-old woman was diagnosed with breast cancer. Wide local excision revealed a 1.7-cm infiltrating ductal adenocarcinoma, BSR score 7/9 with angiolymphatic invasion, and 4/20 lymph nodes positive for carcinoma. Five years later, a bone marrow biopsy revealed involvement of bone marrow by metastatic breast carcinoma, and shortly thereafter, metastases were identified in the liver and lung hilum. She enrolled in a clinical investigation for the detection of circulating tumor cells (CTCs) in breast carcinoma. A total of 659 CTCs were identified in a 10-mL blood sample using an immunofluorescent protocol targeting cytokeratins and detected using fiber-optic array scanning technology. The detected CTCs were subsequently stained with a Wright-Giemsa stain, and representative cells were evaluated in detail by light microscopy for morphologic evaluation. We find that the patient's CTCs exhibit a high degree of pleomorphism including CTCs with high and low nuclear-to-cytoplasmic ratios along with CTCs exhibiting early and late apoptotic changes. In addition, in comparison with her tumor cells in other sites, the full morphologic spectrum of cancer cells present in primary and metastatic tumor is also present in peripheral blood circulation.     

Circulating tumor cells in breast cancer: A tool whose time has come of age

Circulating tumor cells (CTCs) are isolated tumor cells disseminated from the site of disease in metastatic and/or primary cancers, including breast cancer, that can be identified and measured in the peripheral blood of patients. As recent technical advances have rendered it easier to reproducibly and repeatedly sample this population of cells with a high degree of accuracy, these cells represent an attractive surrogate marker of the site of disease.     

Liquid biopsy in breast cancer: A comprehensive review

Breast cancer is the most common cancer among women worldwide. Due to its complexity in nature, effective breast cancer treatment can encounter many challenges. Traditional methods of cancer detection such as tissue biopsy are not comprehensive enough to capture the entire genomic landscape of breast tumors. However, with the introduction of novel techniques, the application of liquid biopsy has been enhanced, enabling the improvement of various aspects of breast cancer management including early diagnosis and screening, prediction of prognosis, early detection of relapse, serial sampling and efficient longitudinal monitoring of disease progress and response to treatment. Various components of tumor cells released into the blood circulation can be analyzed in liquid biopsy sampling, some of which include circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), cell-free RNA, tumor-educated platelets and exosomes. These components can be utilized for different purposes. As an example, ctDNA can be sequenced for genetic profiling of the tumors to enhance individualized treatment and longitudinal screening. CTC plasma count analysis or ctDNA detection after curative tumor resection surgery could facilitate early detection of minimal residual disease, aiding in the initiation of adjuvant therapy to prevent recurrence. Furthermore, CTC plasma count can be assessed to determine the stage and prognosis of breast cancer. In this review, we discuss the advantages and limitations of the various components of liquid biopsy used in breast cancer diagnosis and will expand on aspects that require further focus in future research.     

A microchip filter device incorporating slit arrays and 3-D flow for detection of circulating tumor cells using CAV1-EpCAM conjugated microbeads

Circulating tumor cells (CTCs) are rare cells and the presence of these cells may indicate a poor prognosis and a high potential for metastasis. Despite highly promising clinical applications, CTCs have not been investigated thoroughly, due to many technical limitations faced in their isolation and identification. Current CTC detection techniques mostly take the epithelial marker epithelial cell adhesion molecule (EpCAM), however, accumulating evidence suggests that CTCs show heterogeneous EpCAM expression due to the epithelial-to-mesenchymal transition (EMT). In this study, we report that a microchip filter device incorporating slit arrays and 3-dimensional flow that can separate heterogeneous population of cells with marker for CTCs. To select target we cultured breast cancer cells under prolonged mammosphere culture conditions which induced EMT phenotype. Under these conditions, cells show upregulation of caveolin1 (CAV1) but down-regulation of EpCAM expression. The proposed device which contains CAV1-EpCAM conjugated bead has several tens of times increased throughput. More importantly, this platform enables the enhanced capture yield from metastatic breast cancer patients and obtained cells that expressed various EMT markers. Further understanding of these EMT-related phenotypes will lead to improved detection techniques and may provide an opportunity to develop therapeutic strategies for effective treatment and prevention of cancer metastasis.     

Minimal residual disease in breast cancer: an overview of circulating and disseminated tumour cells

Within the field of cancer research, focus on the study of minimal residual disease (MRD) in the context of carcinoma has grown exponentially over the past several years. MRD encompasses circulating tumour cells (CTCs)—cancer cells on the move via the circulatory or lymphatic system, disseminated tumour cells (DTCs)—cancer cells which have escaped into a distant site (most studies have focused on bone marrow), and resistant cancer cells surviving therapy—be they local or distant, all of which may ultimately give rise to local relapse or overt metastasis. Initial studies simply recorded the presence and number of CTCs and DTCs; however recent advances are allowing assessment of the relationship between their persistence, patient prognosis and the biological properties of MRD, leading to a better understanding of the metastatic process. Technological developments for the isolation and analysis of circulating and disseminated tumour cells continue to emerge, creating new opportunities to monitor disease progression and perhaps alter disease outcome. This review outlines our knowledge to date on both measurement and categorisation of MRD in the form of CTCs and DTCs with respect to how this relates to cancer outcomes, and the hurdles and future of research into both CTCs and DTCs.     

Combination of antibody-coated,physical-based microfluidic chip with wave-shaped arrays for isolating circulating tumor cells

Circulating tumor cells (CTCs) are found in the peripheral blood of patients with metastatic cancers, which have critical significance in cancer prognosis and diagnostics. Enumeration is significantly valuable since number of CTCs is strongly correlated to severity of disease. This article is proposed and demonstrated an antibody-coated, size-based microfluidic chip with wave-shaped arrays could efficiently capture CTCs combining two separation methods of both size- and deformability-based and affinity-based segregation. Utilizing immunocapture of capture chemistry of Epithelial Cell Adhension Molecule (EpCAM), tumor cells could be captured by narrow gaps or have a friction with microposts edges to realize both immune-affinity and size capture. This wave-shaped layout of microfluidic chip with varying gaps between adjacent circular microposts can generate perpendicular velocities to the fluidic direction. This oriented fluidic direction will carry cells to next smaller neighboring gap and then be captured gradually. The experiment results indicate capture efficiency is ~90% and viability is ~95% after extracted and cultured 3 days. Furthermore, this chip has been validated for whole blood with cancer cell lines and mimic patient blood. This study demonstrates feasibility using our microfluidic chip for CTCs research, monitoring cancer progress and evaluating therapeutic treatment.     

循环肿瘤细胞在肺癌诊疗中的临床研究进展

肺癌现已成为发病率及致死率最高的恶性肿瘤,且呈逐年增高趋势,给人类的身体健康及生命安全带来严重威胁。肺癌的复发与转移是导致患者死亡的最主要原因,可能与循环肿瘤细胞（CTCs）进入血液存在一定关系,CTCs在肺癌患者的早期诊断、治疗指导、疗效评估及预后等方面具有十分重要的作用。本文就CTCs在肺癌诊疗中临床应用及研究进展进行综述。     

Circulating tumor cells as a window on metastasis biology in lung cancer

Circulating tumor cell (CTC) number in metastatic cancer patients yields prognostic information consistent with enhanced cell migration and invasion via loss of adhesion, a feature of epithelial-to-mesenchymal transition (EMT). Tumor cells also invade via collective migration with maintained cell-cell contacts and consistent with this is the circulating tumor microemboli (CTM; contiguous groups of tumor cells) that are observed in metastatic cancer patients. Using a blood filtration approach, we examined markers of EMT (cytokeratins, E-cadherin, vimentin, neural cadherin) and prevalence of apoptosis in CTCs and CTM to explore likely mechanism(s) of invasion in lung cancer patients and address the hypothesis that cells within CTM have a survival advantage. Intra-patient and inter-patient heterogeneity was observed for EMT markers in CTCs and CTM. Vimentin was only expressed in some CTCs, but in the majority of cells within CTM; E-cadherin expression was lost, cytoplasmic or nuclear, and rarely expressed at the surface of the cells within CTM. A subpopulation of CTCs was apoptotic, but apoptosis was absent within CTM. This pilot study suggests that EMT is not prosecuted homogeneously in tumor cells within the circulation of lung cancer patients and that collective migration and enhanced survival of cells within CTM might contribute to lung cancer metastasis. Multiplex analysis and further detailed exploration of metastatic potential and EMT in CTCs/CTM is now warranted in a larger patient cohort.     

Toll-like receptor (TLR) expression of immune system cells from metastatic breast cancer patients with circulating tumor cells

The risk posed by breast cancer represents a complex interaction among factors affecting tumor immunity of the host. Toll-like receptors (TLRs) are members of the innate immune system and generally function to attract host immune cells upon activation. However, the good intentions of TLRs are sometimes not transferred to positive long-term effects, due to their involvement in exacerbating inflammatory effects and even contributing to continued inflammation. Chronic inflammatory states are considered to favor an increased predisposition to cancer, with continuous activation of inflammatory cytokines and other hallmarks of inflammation exerting a deleterious effect. Circulating tumor cells (CTCs) are neoplastic cells present in the peripheral blood circulation that have been found to be an indicator of disease progression and long-term survival. In the present study, we examined the expression of TLRs on dendritic cells, which play a major role in eliciting anti-tumor immunity, in metastatic breast cancer patients with CTCs. Flow cytometric data showed significant differences between circulating tumor cell (CTC) positive patients and CTC negative patients in their expression of TLR2 by CD8 positive cytotoxic T cells and TLR2, TLR4, TLR3, and TLR8 by CD11c positive dendritic cells (p < 0.05). Expression of TLR2, TLR4, and TLR8 was increased in CTC positive patients, whereas TLR3 expression was decreased in the dendritic cell population.     

Microdevice for the isolation and enumeration of cancer cells from blood

Cancer metastasis is the main attribute to cancer-related deaths. Furthermore, clinical reports have shown a strong correlation between the disease development and number of circulating tumor cells (CTCs) in the peripheral blood of cancer patients. Here, we present a label-free microdevice capable of isolating cancer cells from whole blood via their distinctively different physical properties such as deformability and size. The isolation efficiency is at least 80% for tests performed on breast and colon cancer cells. Viable isolated cells are also obtained which may give further insights to the understanding of the metastatic process. Contrasting with conventional biochemical techniques, the uniqueness of this microdevice lies in the mechanistic and efficient means of isolating viable cancer cells in blood. The microdevice has the potential to be used for routine monitoring of cancer development and cancer therapy in a clinical setting. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Simultaneous isolation and detection of circulating tumor cells with a microfluidic silicon-nanowire-array integrated with magnetic upconversion nanoprobes

The development of sensitive and convenient methods for detection, enrichment, and analysis of circulating tumor cells (CTCs), which serve as an importance diagnostic indicator for metastatic progression of cancer, has received tremendous attention in recent years. In this work, a new approach characteristic of simultaneous CTC capture and detection is developed by integrating a microfluidic silicon nanowire (SiNW) array with multifunctional magnetic upconversion nanoparticles (MUNPs). The MUNPs were conjugated with anti-EpCAM antibody, thus capable to specifically recognize tumor cells in the blood samples and pull them down under an external magnetic field. The capture efficiency of CTCs was further improved by the integration with a microfluidic SiNW array. Due to the autofluorescence free nature in upconversion luminescence (UCL) imaging, our approach allows for highly sensitive detection of small numbers of tumor cells, which afterward could be collected for further analysis and re-culturing. We have further demonstrated that this approach can be applied to detect CTCs in clinical blood samples from lung cancer patients, and obtained consistent results by analyzing the UCL signals and the clinical outcomes of lung cancer metastasis. Therefore our approach represents a promising platform in CTC capture and detection with potential clinical utilization in cancer diagnosis and prognosis.     

CTCs hemodialysis: Can it be a new therapy for breast cancer?

Circulating tumor cells (CTCs) play an important role in Blood-borne distant metastasis, which is the leading cause of cancer-related death in breast cancer. So far, the impacts of CTCs as a tool for predicting or monitoring the efficacy of systemic therapy and that it is a independent prognostic factor have been confirmed. However, CTCs cannot be generally removed at primary surgery or by systemic therapy. In some EMT (epithelial mesenchymal transition)-related treatment fail, CTCs can be accumulated in the postoperative course of the patient which lead to a bad prognosis. In view of these, considering mature hemodialysis technology, we further propose CTCs hemodialysis (CHD), which filtrate CTCs out of blood, as a new therapy for the breast cancer.     

Advances in liquid biopsy using circulating tumor cells and circulating cell-free tumor DNA for detection and monitoring of breast cancer

Clinical and Experimental Medicine - Overview the progress of liquid biopsy using circulating tumor cells (CTCs) and circulating cell-free tumor DNA (cfDNA) to detect and monitor breast cancer....     

Epithelial-mesenchymal plasticity in circulating tumor cells

Epithelial-to-Mesenchymal Transition (EMT) is a complex process that supports the migratory capacity of epithelial tumor cells and is thought to play a crucial role in promoting cancer metastasis. Despite the wealth of experimental data, the exact role of EMT in cancer patients remains more controversial. Over the past 10 years, sensitive technologies that allow the detection and molecular characterization of circulating tumor cells (CTCs) in the peripheral blood of tumor patients have been developed. These analyses help to shed new light into the importance of EMT for human tumor cell dissemination. CTCs with mesenchymal features can be attributed in some clinical studies (in particularly on breast cancer) to higher disease stages, presence of metastases, and even to therapy response and worse outcome. However, the published studies addressing the impact of mesenchymal-like CTCs show heterogeneity with regard to assay specificity, size of cancer and control groups, and endpoint parameters. In the present review, we present the key features of the biology of CTCs in relation to epithelial-to-mesenchy-mal plasticity, describe the current technologies for enrichment and detection of CTCs with high epithelial-mesenchymal plasticity, and discuss the clinical studies that have assessed the relevance of mesenchymal CTCs in carcinoma patients.     

Assessment of circulating tumor cells (CTCs) in prostate cancer patients with low‐volume tumors

The objective of this study was to assess the incidence of circulating tumor cells (CTCs) in prostate cancer patients with low‐volume tumors (less than 0.5 cc) after radical prostatectomy (RP). Blood samples were collected from 64 RP patients to assess the incidence of CTCs following RP. The specimens were processed by whole‐mount section. Clinicopathological data (e.g. patient age, race, specimen weight, tumor volume, grade, stage and surgical margin status) and follow‐up PSA data were compared to CTC status. Of the 64 RP patients, nine had ‘low‐volume prostate cancer’. Seven of these patients had detectable levels of CTCs. In two of the seven patients with detectable CTCs, PSA elevation was also observed. Isolation and detection of circulating epithelial cells is possible in low‐volume prostate cancer patients. In the setting of low‐volume prostate cancer, CTCs may be associated with the presence of detectable PSA levels. However, the detection of CTCs did not predict PSA failure.     

In vitro culturing of viable circulating tumor cells of urinary bladder cancer

Objective: Approximately one third of patients diagnosed with muscle-invasive urinary bladder cancer (UBC) have undetected metastases at the time of treatment of the primary tumor. Currently there are no reliable specific serum markers for monitoring and evaluating risk profiles of urothelial cancers. Several studies suggest that detection of circulating tumor cells (CTCs) may correlate with the disease status and prognosis at baseline and early in the treatment of cancers. In this study a new way of isolation and in vitro cultivation of CTCs of urinary bladder cancer was introduced. Materials and methods: Peripheral blood (PB) samples from 53 patients who had undergone urological procedure were evaluated using the MetaCell device (MetaCell s.r.o., Ostrava, Czech Republic). The patients enrolled in the study were both oncological patients with UBC and non-oncological patients with inflammation (14 patients). The sensitivity and quantification of CTCs were evaluated. The separated CTCs were cultured in vitro. Results: 39 patients with confirmed UBC were enrolled in the study. CTCs were detected in 25 (64%) patients, and most of these patients had between 6 and 10 cells. The separated CTCs were successfully cultured in vitro. Conclusion: CTCs were detected in a higher percentage of patients than in other studies. This paper describes the first successful culturing of human UBC cells. The MetaCell approach used in this study enabled the capture of viable intact virgin CTCs (virgin CTC) suitable for next in vitro culturing, single cell analysis or drug testing.     

A mesofluidic multiplex immunosensor for detection of circulating cytokeratin-positive cells in the blood of breast cancer patients

We have recently reported the analytical performance of an immunosensor comprising one mm-scale parallel plate laminar flow chamber and applied to capture MCF7 breast cancer cells (Ehrhart et al., Biosens. Bioelectr. 24, 467, 2008). Herein we present a new multiplex immunosensor embodying four parallel plate laminar flow chambers that fit onto a standard, functionalized, microscopy glass slide. The four surfaces are coated with long alkyl chain spacers of 21-aminohenicosyl trichlorosilane (AHTS) and then grafted with a monoclonal anti-human epithelial cell adhesion molecule (EpCAM) antibody specific of target cells to immobilize. We first demonstrate a significantly (P < 0.01) improved capacity of each of the four flow chambers of the multiplex immunosensor to capture MCF7 cells compared to the previous single chamber device. Second, in addition to an increase of cell immobilization, the multiplex device offers a versatile tool easily grafted with various purified antibodies onto the four surfaces. Third, we obtained high cell capture rate and efficiency of various numbers of MCF7 cells spiked in buffer containing an equal number of background leukocytes. And fourth, we demonstrate isolation efficiency of circulating tumor cells (CTCs) from peripheral blood drawn from a small cohort of patients with localized or metastatic breast cancer. This new multiplex immunosensor could be tested for its potential to capture different subpopulations of CTCs.     

肝癌患者循环肿瘤细胞的检测及其临床应用研究进展

原发性肝癌发病率和死亡率均较高,其术后转移与复发已经成为影响患者预后的重要因素,临床上迫切需要能够有效预测和防治肝癌转移的检测手段。循环肿瘤细胞（circulating tumor cell,CTC）,作为近些年来新出现的肿瘤检测指标,因其能够实时、无创地监测肿瘤患者的病情状态,在早期诊断、早期治疗,判断预后和制定个体化治疗方案等方面具有重要意义而备受关注。CTC在乳腺癌、前列腺癌、结直肠癌、肺癌等疾病的应用已初步得到临床认可。最新的研究表明,肝癌患者病情状态与血液中CTC的数量也显示出极强的相关性。目前各种CTC检测体系主要包括富集纯化和鉴定两方面;但由于检测方法繁多,不同方法的敏感性、特异性不同限制了其临床应用。近年来,新的CTC检测方法层见叠出,涌现出了很多新技术用于肝癌CTC的临床研究。本文就其相关研究进展做一综述。