Circulating tumor cell enumeration for improved screening and disease detection of patients with colorectal cancer.

BACKGROUND: The immunochemical fecal occult blood test (iFOBT) for colorectal cancer (CRC) screening and the serum carcinoembryonic antigen (CEA) assay for disease detection of CRC is associated with a high false-positive rate and a low detection sensitivity, respectively. There is an unmet need to define additional modalities to complement these assays. Different subsets of circulating tumor cells (CTCs) are present in the peripheral blood of cancer patients. Whether or not CTCs testing supplements these clinical assays and is valuable for patients with CRC was investigated. METHODS: CTCs were enriched from pre-operative patients with CRC (n = 109) and the non-cancerous controls (n = 65). CTCs expressing either epithelial cell adhesion molecule (EpCAM) or podoplanin (PDPN, the marker associated with poor cancer prognosis) were defined by immunofluorescence staining and were analyzed alone or in combination with iFOBT or serum CEA. RESULTS: Patients with early or advanced stage of CRC can be clearly identified and differentiated from the non-cancerous controls (p < 0.001) by EpCAM+-CTC or PDPN+-CTC count. The sensitivity and specificity of EpCAM+-CTCs was 85.3% and 78.5%, respectively, when the cutoff value was 23 EpCAM+-CTCs/mL of blood; and the sensitivity and specificity of PDPN+-CTCs was 78.0% and 75.4%, respectively, when the cutoff value was 7 PDPN+-CTCs/mL of blood. Combined analysis of iFOBT with the EpCAM+-CTC and PDPN+-CTC count reduced the false-positive rate of iFOBT from 56.3% to 18.8% and 23.4%, respectively. Combined analysis of serum CEA with the EpCAM+-CTC and PDPN+-CTC count increased the disease detection rate from 30.3% to 89.9% and 86.2%, respectively. CONCLUSION: CTC testing could supplement iFOBT to improve CRC screening and supplement serum CEA assay for better disease detection of patients with CRC.

Sequential Circulating Tumor Cell Counts in Patients with Locally Advanced or Metastatic Hepatocellular Carcinoma: Monitoring the Treatment Response.

Hepatocellular carcinoma (HCC) is among the most common causes of cancer death in men. Whether or not a longitudinal follow-up of circulating tumor cells (CTCs) before and at different time points during systemic/targeted therapy is useful for monitoring the treatment response of patients with locally advanced or metastatic HCC has been evaluated in this study. Blood samples (n = 104) were obtained from patients with locally advanced or metastatic HCC (n = 30) for the enrichment of CTCs by a negative selection method. Analysis of the blood samples from patients with defined disease status (n = 81) revealed that those with progressive disease (PD, n = 37) had significantly higher CTC counts compared to those with a partial response (PR) or stable disease (SD; n = 44 for PR + SD, p = 0.0002). The median CTC count for patients with PD and for patients with PR and SD was 50 (interquartile range 21-139) and 15 (interquartile range 4-41) cells/mL of blood, respectively. A longitudinal analysis of patients (n = 17) after a series of blood collections demonstrated that a change in the CTC count correlated with the patient treatment response in most of the cases and was particularly useful for monitoring patients without elevated serum alpha-fetoprotein (AFP) levels. Sequential CTC enumeration during treatment can supplement standard medical tests and benefit the management of patients with locally advanced or metastatic HCC, in particular for the AFP-low cases.

Hyaluronic Acid-Based Multilayer Films Regulate Hypoxic Multicellular Aggregation of Pancreatic Cancer Cells with Distinct Cancer Stem-Cell-like Properties.

In vitro spherical cancer models have been widely used in cancer stem cell (CSC) research, and the ability of CSCs to form multicellular colonies is recognized as a morphological marker. However, although several spherical/colony models share a common three-dimensional (3D) conformation, each model displays its own intrinsic properties. Thus, the CSC phenotypes with distinct multicellular aggregate morphologies must be defined and clarified. Here, a novel 3D model was designed to regulate the type of pancreatic CSC colonies that form using niche mimetic hyaluronic acid (HA)-based multilayer nanofilms and hypoxia. The multicellular aggregate morphology, CSC phenotypes, CSC-related marker expression, cell cycle, invasion, and drug resistance were determined. On the basis of the results of a cell morphology analysis, colonies formed on multilayer nanofilms in response to both normoxia and hypoxia, but round and island-type colonies, were investigated. Immunostaining results revealed a significantly higher expression of stem cell markers, such as OCT4, CXCR4, and CD44v6, in colonies that formed on multilayer nanofilms. These colonies also expressed higher levels of E-cadherin, hypoxia-inducible factor-1α, and vimentin, particularly the round-type colonies that formed on HA-based multilayer nanofilms, [poly(allylamine) (PAH)/HA]3, indicating that these colonies exhibit hybrid and metastable epithelial/mesenchymal phenotypes. Moreover, the cell cycle and invasion tests revealed that most of the cells in colonies growing on multilayer nanofilms showed a quiescent, slow cycling phenotype but displayed higher invasion after induction. Furthermore, a hypoxic environment strongly influences the drug resistance. This study describes a useful tool to investigate the diverse phenotypes of pancreatic CSC colonies and to study their regulatory factors that may benefit CSC research.

Circulating Epithelial Cell Characterization and Correlation with Remission and Survival in Patients with Thyroid Cancer.

BACKGROUND: Thyroid cancer is the most common endocrine tumor and generally has relatively good clinical outcomes. However, 15-20% of patients ultimately develop recurrence or disease-related death. The appropriate prognostic factors for thyroid cancer are still elusive. This study evaluated whether the number of circulating tumor cells/circulating epithelial cells (CECs) expressing either epithelial cell adhesion molecule (EpCAM), podoplanin (PDPN), or thyrotropin receptor (TSHR) is related to remission and disease-specific mortality (DSM) of patients with thyroid cancer. METHODS: Blood samples were collected from patients (n = 128) after thyroidectomy or radioactive iodide therapy. CECs were enriched by lysis of red blood cells and depletion of leukocytes. Subtyping and quantification of the enriched cells were performed with immunofluorescence staining using antibodies against EpCAM, TSHR, and PDPN, respectively. Whether the number of a specific subtype of CECs is related to remission and DSM of patients was determined by univariate and multivariate analyses. RESULTS: The EpCAM+-CECs, TSHR+-CECs, and PDPN+-CECs counts for patients in the non-remission group (n = 43) were significantly higher when compared to the remission group (n = 85; p < 0.001). Receiver operating characteristic analysis showed that the number of EpCAM+-CECs, TSHR+-CECs, and PDPN+-CECs was able to distinguish the status of remission from non-remission. The cutoff point for EpCAM+-CECs, TSHR+-CECs, and PDPN+-CECs was 40, 47, and 14 (cells/mL), with the accuracy of the assay equivalent to 80.4%, 76.6%, and 77.3%, respectively. On the other hand, the number of EpCAM+-CECs (p < 0.001), PDPN+-CECs (p = 0.013), and TSHR+-CECs (p < 0.001) for patients in the DSM group (n = 17) was significantly higher when compared to the patients who survived (n = 111). Receiver operating characteristic analysis showed that EpCAM+-CECs, TSHR+-CECs, and PDPN+-CECs counts were able to distinguish mortality from survival status. The cutoff point for EpCAM+-CECs, TSHR+-CECs, and PDPN+-CECs was 27, 25, and 9 (cells/mL), with the accuracy of the assay equivalent to 69.5%, 67.2%, and 68.5%, respectively. CONCLUSIONS: CEC testing is a useful tool for analysis of overall survival and remission status of patients with thyroid cancer. Implementation of CEC testing into routine clinical test may be worthy to consider for patient clinical care.

Disabled-2 is a negative immune regulator of lipopolysaccharide-stimulated Toll-like receptor 4 internalization and signaling.

Toll-like receptor 4 (TLR4) plays a pivotal role in the host response to lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria. Here, we elucidated whether the endocytic adaptor protein Disabled-2 (Dab2), which is abundantly expressed in macrophages, plays a role in LPS-stimulated TLR4 signaling and trafficking. Molecular analysis and transcriptome profiling of RAW264.7 macrophage-like cells expressing short-hairpin RNA of Dab2 revealed that Dab2 regulated the TLR4/TRIF pathway upon LPS stimulation. Knockdown of Dab2 augmented TRIF-dependent interferon regulatory factor 3 activation and the expression of subsets of inflammatory cytokines and interferon-inducible genes. Dab2 acted as a clathrin sponge and sequestered clathrin from TLR4 in the resting stage of macrophages. Upon LPS stimulation, clathrin was released from Dab2 to facilitate endocytosis of TLR4 for triggering the TRIF-mediated pathway. Dab2 functions as a negative immune regulator of TLR4 endocytosis and signaling, supporting a novel role for a Dab2-associated regulatory circuit in controlling the inflammatory response of macrophages to endotoxin.

The endocytic adaptor protein Disabled-2 is required for cellular uptake of fibrinogen.

Endocytosis is pivotal for uptake of fibrinogen from plasma into megakaryocytes and platelet α-granules. Due to the complex adaptor and cargo contents in endocytic vehicles, the underlying mechanism of fibrinogen uptake is not yet completely elucidated. In this study, we investigated whether the endocytic adaptor protein Disabled-2 (DAB2) mediates fibrinogen uptake in an adaptor-specific manner. By employing primary megakaryocytes and megakaryocytic differentiating human leukemic K562 cells as the study models, we found that fibrinogen uptake is associated with the expression of integrin αIIbß3 and DAB2 and is mediated through clathrin-dependent manner. Accordingly, constitutive and inducible knockdown of DAB2 by small interfering RNA reduced fibrinogen uptake for 53.2 ± 9.8% and 59.0 ± 10.7%, respectively. Culturing the cells in hypertonic solution or in the presence of clathrin inhibitor chlorpromazine abrogated clathrin-dependent endocytosis and diminished the uptake of fibrinogen. Consistent with these findings, 72.2 ± 0.2% of cellular DAB2 was colocalized with clathrin, whereas 56.4±4.1% and 54.6 ± 2.0% of the internalized fibrinogen were colocalized with clathrin and DAB2, respectively. To delineate whether DAB2 mediates fibrinogen uptake in an adaptor-specific manner, K562 stable cell lines with knockdown of the adaptor protein-2 (AP-2) or double knockdown of AP-2/DAB2 were established. The AP-2 knockdown cells elicited normal fibrinogen uptake activity but the uptake of collagen was diminished. In addition, collagen uptake was further reduced in DAB2/AP-2 knockdown cells. These findings thereby define an adaptor-specific mechanism in the control of fibrinogen uptake and implicate that DAB2 is the key adaptor in the clathrin-associated endocytic complexes to mediate fibrinogen internalization.