Filter Characteristics Influencing Circulating Tumor Cell Enrichment from Whole Blood

A variety of filters assays have been described to enrich circulating tumor cells (CTC) based on differences in physical characteristics of blood cells and CTC. In this study we evaluate different filter types to derive the properties of the ideal filter for CTC enrichment. Between 0.1 and 10 mL of whole blood spiked with cells from tumor cell lines were passed through silicon nitride microsieves, polymer track-etched filters and metal TEM grids with various pore sizes. The recovery and size of 9 different culture cell lines was determined and compared to the size of EpCAM+CK+CD45−DNA+ CTC from patients with metastatic breast, colorectal and prostate cancer. The 8 µm track-etched filter and the 5 µm microsieve had the best performance on MDA-231, PC3-9 and SKBR-3 cells, enriching >80% of cells from whole blood. TEM grids had poor recovery of ∼25%. Median diameter of cell lines ranged from 10.9–19.0 µm, compared to 13.1, 10.7, and 11.0 µm for breast, prostate and colorectal CTC, respectively. The 11.4 µm COLO-320 cell line had the lowest recovery of 17%. The ideal filter for CTC enrichment is constructed of a stiff, flat material, is inert to blood cells, has at least 100,000 regularly spaced 5 µm pores for 1 ml of blood with a ≤10% porosity. While cell size is an important factor in determining recovery, other factors must be involved as well. To evaluate a filtration procedure, cell lines with a median size of 11–13 µm should be used to challenge the system.     

Isolation of Rare Tumor Cells from Blood Cells with Buoyant Immuno-Microbubbles

Circulating tumor cells (CTCs) are exfoliated at various stages of cancer, and could provide invaluable information for the diagnosis and prognosis of cancers. There is an urgent need for the development of cost-efficient and scalable technologies for rare CTC enrichment from blood. Here we report a novel method for isolation of rare tumor cells from excess of blood cells using gas-filled buoyant immuno-microbubbles (MBs). MBs were prepared by emulsification of perfluorocarbon gas in phospholipids and decorated with anti-epithelial cell adhesion molecule (EpCAM) antibody. EpCAM-targeted MBs efficiently (85%) and rapidly (within 15 minutes) bound to various epithelial tumor cells suspended in cell medium. EpCAM-targeted MBs efficiently (88%) isolated frequent tumor cells that were spiked at 100,000 cells/ml into plasma-depleted blood. Anti-EpCAM MBs efficiently (>77%) isolated rare mouse breast 4T1, human prostate PC-3 and pancreatic cancer BxPC-3 cells spiked into 1, 3 and 7 ml (respectively) of plasma-depleted blood. Using EpCAM targeted MBs CTCs from metastatic cancer patients were isolated, suggesting that this technique could be developed into a valuable clinical tool for isolation, enumeration and analysis of rare cells.     

High Immunogenicity of the Human Leukocyte Antigen Peptidomes of Melanoma Tumor Cells

Human leukocyte antigens (HLA) bind peptides generated by limited proteolysis in cells and present them at the cell surfaces for recognition by T cells. Through this antigen presentation function they control the specificity of T cell responses and thereby adaptive immune responses. Knowledge of HLA-bound peptides is thus key to understanding adaptive immunity and to the development of vaccines and other specific immune intervention strategies. To gain insight into the antigenicity of melanomas, peptides were extracted from HLA isolated from the tumor cells, separated by two-dimensional HPLC, and sequenced by mass spectrometry. The spectra were analyzed by database-dependent MASCOT searches and database-independent de novo sequencing and, where required, confirmed with synthetic peptides, which were also used to determine their immunogenicity. Comparing four different melanoma cell lines, little overlap of the HLA-bound peptides was found, suggesting a high degree of individualization of the HLA peptidomes. This notwithstanding, the peptidomes were highly immunogenic in the patients from whom the tumor cells had been established and in unrelated patients. This broad cross-patient immunogenicity was only exceptionally related to individual peptides. The majority of the identified epitopes were derived from low to medium abundance proteins, mostly involved in sensitive cellular processes such as cell cycle control, DNA replication, control of gene expression, tumor suppressor function, and protein metabolism. The peptidomes thus provide insights into processes potentially related to tumorigenesis. Furthermore, analyses of the peptide sequences yield information on the specificity of peptide selection by HLA applicable to the developing prediction algorithms for T cell epitopes.     

Leukocyte Culture and Chromosome Preparations from Adult Dog Blood

Plasma is obtained from dog blood after 3 hr settling in a syringe. Portions of the plasma (0.5-1.0 ml) are added to 4 ml of a medium consisting of 17 parts of BME Spinner, 3 parts of calf serum, 0.5 parts of glutamine, 0.5 parts of penicillin-streptomycin, and 0.1-1.0 parts of Scarlet Runner bean phytohemagglutinin. Colchicine, 0.1 ml of 10:1 stock solution, is added after 72 hr and incubation continued for 2 hr, then centrifuged 5 min at 700 rev/min. The supernatant is discarded, 3 ml of distilled water added, and the cell suspension centrifuged again. The supernatant is discarded and the fixative, consisting of 45% glacial acetic acid allowed to act for 0.5 hr. Acetic-orcein stains of smears were very satisfactory.     

Circulating Tumor Cells from Prostate Cancer Patients Interact with E-Selectin under Physiologic Blood Flow

Hematogenous metastasis accounts for the majority of cancer-related deaths, yet the mechanism remains unclear. Circulating tumor cells (CTCs) in blood may employ different pathways to cross blood endothelial barrier and establish a metastatic niche. Several studies provide evidence that prostate cancer (PCa) cell tethering and rolling on microvascular endothelium via E-selectin/E-selectin ligand interactions under shear flow theoretically promote extravasation and contribute to the development of metastases. However, it is unknown if CTCs from PCa patients interact with E-selectin expressed on endothelium, initiating a route for tumor metastases. Here we report that CTCs derived from PCa patients showed interactions with E-selectin and E-selectin expressing endothelial cells. To examine E-selectin-mediated interactions of PCa cell lines and CTCs derived from metastatic PCa patients, we used fluorescently-labeled anti-prostate specific membrane antigen (PSMA) monoclonal antibody J591-488 which is internalized following cell-surface binding. We employed a microscale flow device consisting of E-selectin-coated microtubes and human umbilical vein endothelial cells (HUVECs) on parallel-plate flow chamber simulating vascular endothelium. We observed that J591-488 did not significantly alter the rolling behavior in PCa cells at shear stresses below 3 dyn/cm². CTCs obtained from 31 PCa patient samples showed that CTCs tether and stably interact with E-selectin and E-selectin expressing HUVECs at physiological shear stress. Interestingly, samples collected during disease progression demonstrated significantly more CTC/E-selectin interactions than samples during times of therapeutic response (p=0.016). Analysis of the expression of sialyl Lewis X (sLe^x) in patient samples showed that a small subset comprising 1.9-18.8% of CTCs possess high sLe^x expression. Furthermore, E-selectin-mediated interactions between prostate CTCs and HUVECs were diminished in the presence of anti-E-selectin neutralizing antibody. CTC-Endothelial interactions provide a novel insight into potential adhesive mechanisms of prostate CTCs as a means to initiate metastasis.     

A Method for the Increased Extraction of Cholesterol from Human Red Blood Cells by Modified Plasma Lipoprotein

The in vitro extraction of cholesterol from erythrocytes by plasma lipoproteins of reduced cholesterol content would be expected to be free of cholesterol-unrelated alterations of the cell membrane. The earlier application of this method utilized whole blood plasma in which the major part of the lipoprotein cholesterol was esterified by the plasma enzyme lecithin-cholesterol acyl transferase (LCAT) in a preliminary incubation. Because of the cholesterol remaining unesterified in the plasma, only 35% of the cell cholesterol could be removed. The method reported here uses HDL., a plasma lipoprotein which is the preferred substrate for LCAT, instead of whole plasma for the extraction. Multiple extractions with LCAT treated HDL, resulted in the removal of up to 77% of the erythrocyte cholesterol with only minor hemolysis.     

Forces on a Wall-Bound Leukocyte in a Small Vessel Due to Red Cells in the Blood Stream

As part of the inflammation response, white blood cells (leukocytes) are well known to bind nearly statically to the vessel walls, where they must resist the force exerted by the flowing blood. This force is particularly difficult to estimate due to the particulate character of blood, especially in small vessels where the red blood cells must substantially deform to pass an adhered leukocyte. An efficient simulation tool with realistically flexible red blood cells is used to estimate these forces. At these length scales, it is found that the red cells significantly augment the streamwise forces that must be resisted by the binding. However, interactions with the red cells are also found to cause an average wall-directed force, which can be anticipated to enhance binding. These forces increase significantly as hematocrit values approach 25% and decrease significantly as the leukocyte is made flatter on the wall. For a tube hematocrit of 25% and a spherical protrusion with a diameter three-quarters that of the vessel, the average forces are increased by ∼40% and the local forces are more than double those estimated with an effective-viscosity-homogenized blood. Both the enhanced streamwise and wall-ward forces and their unsteady character are potentially important in regard to binding mechanisms.     

Red Blood Cells Protein Profile Is Modified in Breast Cancer Patients

Metastasis is the primary cause of death for most breast cancer (BC) patients who succumb to the disease. During the hematogenous dissemination, circulating tumor cells interact with different blood components. Thus, there are microenvironmental and systemic processes contributing to cancer regulation. We have recently published that red blood cells (RBCs) that accompany circulating tumor cells have prognostic value in metastatic BC patients. RBC alterations are related to several diseases. Although the principal known role is gas transport, it has been recently assigned additional functions as regulatory cells on circulation. Hence, to explore their potential contribution to tumor progression, we characterized the proteomic composition of RBCs from 53 BC patients from stages I to III and IV, compared with 33 cancer-free controls. In this work, we observed that RBCs from BC patients showed a different proteomic profile compared to cancer-free controls and between different tumor stages. The differential proteins were mainly related to extracellular components, proteasome, and metabolism. Embryonic hemoglobins, not expected in adults’ RBCs, were detected in BC patients. Besides, lysosome-associated membrane glycoprotein 2 emerge as a new RBCs marker with diagnostic and prognostic potential for metastatic BC patients. Seemingly, RBCs are acquiring modifications in their proteomic composition that probably represents the systemic cancer disease, conditioned by the tumor microenvironment.     

Tumor-Associated Endothelial Cells Promote Tumor Metastasis by Chaperoning Circulating Tumor Cells and Protecting Them from Anoikis

Tumor metastasis is a highly inefficient biological process as millions of tumor cells are released in circulation each day and only a few of them are able to successfully form distal metastatic nodules. This could be due to the fact that most of the epithelial origin cancer cells are anchorage-dependent and undergo rapid anoikis in harsh circulating conditions. A number of studies have shown that in addition to tumor cells, activated endothelial cells are also released into the blood circulation from the primary tumors. However, the precise role of these activated circulating endothelial cells (CECs) in tumor metastasis process is not known. Therefore, we performed a series of experiments to examine if CECs promoted tumor metastasis by chaperoning the tumor cells to distal sites. Our results demonstrate that blood samples from head and neck cancer patients contain significantly higher Bcl-2-positive CECs as compared to healthy volunteers. Technically, it is challenging to know the origin of CECs in patient blood samples, therefore we used an orthotopic SCID mouse model and co-implanted GFP-labeled endothelial cells along with tumor cells. Our results suggest that activated CECs (Bcl-2-positive) were released from primary tumors and they co-migrated with tumor cells to distal sites. Bcl-2 overexpression in endothelial cells (EC-Bcl-2) significantly enhanced adhesion molecule expression and tumor cell binding that was predominantly mediated by E-selectin. In addition, tumor cells bound to EC-Bcl-2 showed a significantly higher anoikis resistance via the activation of Src-FAK pathway. In our in vivo experiments, we observed significantly higher lung metastasis when tumor cells were co-injected with EC-Bcl-2 as compared to EC-VC. E-selectin knockdown in EC-Bcl-2 cells or FAK/FUT3 knockdown in tumor cells significantly reversed EC-Bcl-2-mediated tumor metastasis. Taken together, our results suggest a novel role for CECs in protecting the tumor cells in circulation and chaperoning them to distal sites.     

利用逆转录酶的聚合酶链式反应检测外周血中的肿瘤细胞

本文就RT－PCR技术检测外周血中肿瘤细胞的基本原理及其在黑色素瘤,肝癌,乳腺癌,结直肠癌,前列腺癌等检测中的应用加以综述。     

Internalizing Cancer Antibodies from Phage Libraries Selected on Tumor Cells and Yeast-Displayed Tumor Antigens

A number of approaches have been utilized to generate antibodies to cancer cell surface receptors that can be used as potential therapeutics. A number of these therapeutic approaches, including antibody-drug conjugates, immunotoxins, and targeted nucleic acid delivery, require antibodies that not only bind receptor but also undergo internalization into the cell upon binding. We previously reported on the ability to generate cancer cell binding and internalizing antibodies directly from human phage antibody libraries selected for internalization into cancer cell lines. While a number of useful antibodies have been generated using this approach, limitations include the inability to direct the selections to specific antigens and to identify the antigen bound by the antibodies. Here we show that these limitations can be overcome by using yeast-displayed antigens known to be associated with a cell type to select the phage antibody output after several rounds of selection on a mammalian cell line. We used this approach to generate several human phage antibodies to yeast-displayed EphA2 and CD44. The antibodies bound both yeast-displayed and mammalian cell surface antigens, and were endocytosed upon binding to mammalian cells. This approach is generalizable to many mammalian cell surface proteins, results in the generation of functional internalizing antibodies, and does not require antigen expression and purification for antibody generation.     

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.     

Circulating Angiogenic Cells can be Derived from Cryopreserved Peripheral Blood Mononuclear Cells

Background

Cell transplantation for regenerative medicine has become an appealing therapeutic method; however, stem and progenitor cells are not always freshly available. Cryopreservation offers a way to freeze cells as they are generated, for storage and transport until required for therapy. This study was performed to assess the feasibility of cryopreserving peripheral blood mononuclear cells (PBMCs) for the subsequent in vitro generation of their derived therapeutic population, circulating angiogenic cells (CACs).

Methods

PBMCs were isolated from healthy human donors. Freshly isolated cells were either analyzed immediately or cryopreserved in media containing 6% plasma serum and 5% dimethyl sulfoxide. PBMCs were thawed after being frozen for 1 (early thaw) or 28 (late thaw) days and analyzed, or cultured for 4 days to generate CACs. Analysis of the cells consisted of flow cytometry for viability and phenotype, as well as functional assays for their adhesion and migration potential, cytokine secretion, and in vivo angiogenic potential.

Results

The viability of PBMCs and CACs as well as their adhesion and migration properties did not differ greatly after cryopreservation. Phenotypic changes did occur in PBMCs and to a lesser extent in CACs after freezing; however the potent CD34⁺VEGFR2⁺CD133⁺ population remained unaffected. The derived CACs, while exhibiting changes in inflammatory cytokine secretion, showed no changes in the secretion of important regenerative and chemotactic cytokines, nor in their ability to restore perfusion in ischemic muscle.

Conclusion

Overall, it appears that changes do occur in cryopreserved PBMCs and their generated CACs; however, the CD34⁺VEGFR2⁺CD133⁺ progenitor population, the secretion of pro-vasculogenic factors, and the in vivo angiogenic potential of CACs remain unaffected by cryopreservation.     

Filtration Parameters Influencing Circulating Tumor Cell Enrichment from Whole Blood

Filtration can achieve circulating tumor cell (CTC) enrichment from blood. Key parameters such as flow-rate, applied pressure, and fixation, vary largely between assays and their influence is not well understood. Here, we used a filtration system, to monitor these parameters and determine their relationships. Whole blood, or its components, with and without spiked tumor cells were filtered through track-etched filters. We characterize cells passing through filter pores by their apparent viscosity; the viscosity of a fluid that would pass with the same flow. We measured a ratio of 5·10⁴∶10²∶1 for the apparent viscosities of 15 µm diameter MDA-231 cells, 10 µm white cells and 90 fl red cells passing through a 5 µm pore. Fixation increases the pressure needed to pass cells through 8 µm pores 25-fold and halves the recovery of spiked tumor cells. Filtration should be performed on unfixed samples at a pressure of ∼10 mbar for a 1 cm² track-etched filter with 5 µm pores. At this pressure MDA-231 cells move through the filter in 1 hour. If fixation is needed for sample preservation, a gentle fixative should be selected. The difference in apparent viscosity between CTC and blood cells is key in optimizing recovery of CTC.     

Array CGH Analysis of Paired Blood and Tumor Samples from Patients with Sporadic Wilms Tumor

Wilms tumor (WT), the most common cancer of the kidney in infants and children, has a complex etiology that is still poorly understood. Identification of genomic copy number variants (CNV) in tumor genomes provides a better understanding of cancer development which may be useful for diagnosis and therapeutic targets. In paired blood and tumor DNA samples from 14 patients with sporadic WT, analyzed by aCGH, 22% of chromosome abnormalities were novel. All constitutional alterations identified in blood were segmental (in 28.6% of patients) and were also present in the paired tumor samples. Two segmental gains (2p21 and 20q13.3) and one loss (19q13.31) present in blood had not been previously described in WT. We also describe, for the first time, a small, constitutive partial gain of 3p22.1 comprising 2 exons of CTNNB1, a gene associated to WT. Among somatic alterations, novel structural chromosomal abnormalities were found, like gain of 19p13.3 and 20p12.3, and losses of 2p16.1-p15, 4q32.5-q35.1, 4q35.2-q28.1 and 19p13.3. Candidate genes included in these regions might be constitutively (SIX3, SALL4) or somatically (NEK1, PIAS4, BMP2) operational in the development and progression of WT. To our knowledge this is the first report of CNV in paired blood and tumor samples in sporadic WT.     

Regeneration of Human Tumor Antigen-Specific T Cells from iPSCs Derived from Mature CD8+ T Cells

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Highlights? iPSCs generated from T cells specific for the MART-1 melanoma epitope ? Differentiation of iPSCs into T cells with a MART-1 specific T cell receptor ? MART-1-based stimulation of T cells demonstrates retained antigen specificity     

Generation of Dendritic Cells from Fresh and Frozen Cord Blood CD34Cells

Dendritic cells (DCs) are professional antigen-presenting cells that are required for the initiation of the immune response. DCs have been shown to be generated from CD34⁺pluripotent hematopoietic progenitor cells in the bone marrow and cord blood (CB), but relatively little is known about the effect of cryopreservation on functional maturation of DCs from hematopoietic stem cells. In this work we report the generation of DCs from cryopreserved CB CD34⁺cells. CB CD34⁺cells were cryopreserved at −80°C for 2 days. Cryopreserved CB CD34⁺cells as well as freshly isolated CB CD34⁺cells cultured with granulocyte—macrophage colony-stimulating factor (GM-CSF)/stem cell factor (SCF)/tumor necrosis factor-α (TNF-α) for 14 days gave rise to CD1a⁺/CD4⁺/CD11c⁺/CD14⁻/CD40⁺/CD80⁺/CD83⁺/CD86⁺/HLA-DR⁺cells with dendritic morphology. DCs derived from cryopreserved CB CD34⁺cells showed a similar endocytic capacity for fluorescein isothiocyanate-labeled dextran and lucifer yellow when compared with DCs derived from freshly isolated CB CD34⁺cells. Flow cytometric analysis revealed that two CC chemokine receptors (CCRs), CCR-1 and CCR-3, were expressed on the cell surface of DCs derived from both cryopreserved and freshly isolated CB CD34⁺cells, and these DCs exhibited similar chemotactic migratory capacities in response to regulated on activation normal T-cell expressed and secreted. DCs derived from cryopreserved as well as freshly isolated CB CD34⁺cells were more efficient than peripheral blood mononuclear cells in the primary allogeneic T-cell response. These results indicate that frozen CB CD34⁺cells cultured with GM-CSF/TNF-α/SCF gave rise to dendritic cells which were morphologically, phenotypically and functionally similar to DCs derived from fresh CB CD34⁺cells.