Whole Blood Cells Research Articles

Ultrasensitive Nanopore Sensing of Mucin 1 and Circulating Tumor Cells in Whole Blood of Breast Cancer Patients by Analyte-Triggered Triplex-DNA Release.

The characterization of circulating tumor cells (CTCs) by liquid biopsy has a great potential for precision medicine in oncology. Here, a universal and tandem logic-based strategy is developed by combining multiple nanomaterials and nanopore sensing for the determination of mucin 1 protein (MUC1) and breast cancer CTCs in real samples. The strategy consists of analyte-triggered signal conversion, cascaded amplification via nanomaterials including copper sulfide nanoparticles (CuS NPs), silver nanoparticles (Ag NPs), and biomaterials including DNA hydrogel and DNAzyme, and single-molecule-level detection by nanopore sensing. The amplification of the non-DNA nanomaterial gives this method considerable stability, significantly lowers the limit of detection (LOD), and enhances the anti-interference performance for complicated samples. As a result, the ultrasensitive detection of MUC1 could be achieved in the range of 0.0005-0.5 pg/mL, with an LOD of 0.1 fg/mL. Moreover, we further tested MUC1 as a biomarker for the clinical diagnosis of breast cancer CTCs under double-blind conditions on the basis of this strategy, and MCF-7 cells could be accurately detected in the range from 5 to 2000 cells/mL, with an LOD of 2 cells/mL within 6 h. The detection results of the 19 clinical samples were highly consistent with those of the clinical pathological sections, nuclear magnetic resonance imaging, and color ultrasound. These results demonstrate the validity and reliability of our method and further proved the feasibility of MUC1 as a clinical diagnostic biomarker for CTCs.

Detection of cancer cells in whole blood using a dynamic deformable microfilter and a nucleic acid aptamer

Cancer cell count in the blood of cancer patients is extremely low. If these cells are easily detectable, cancer diagnosis may be possible by simply using a blood test, thus reducing patient burden. This study aimed to develop a cancer detection device by combining a microfilter that can be dynamically deformed and a nucleic acid aptamer that has a specific binding ability to cancer cells for easy detection. The cancer detection device was fabricated by photolithography, electroforming, and three-dimensional printing. The cancer cell detection ability of the fabricated device was evaluated using 1 mL of blood samples spiked with different concentrations of cancer cells. The lowest concentration of cancer cells in the blood was 5 cancer cells/1 mL blood. The fabricated microfilters specifically detected cancer cells in the blood successfully at exceedingly low concentrations. Moreover, the cancer detection experiment results using human whole blood revealed that cancer detection could be performed with higher accuracy using the fabricated cancer detection device compared to pre-existing cancer detection equipment (e.g., CellSearch system, Veridex). These findings provide important insights into the use of cancer cells in the blood as a diagnostic approach for cancer.

Toward the development of an optical technique for identifying and imaging white blood cells in whole blood

An optical-disk-based system was developed to obtain images of blood cells for healthcare applications. The blood cells on the disk are scanned using a laser, and the intensity data of reflected or transmitted light from the sample are collected and reconstructed to form an image. Images of red and white blood cells were successfully obtained and both types of blood cells were visually distinguishable in the transmitted-light image. Our method is promising for finding a specific cell, among many other types, since the optical-disk system can scan a large area at high speed.

The clinical characteristics of Mycoplasma pneumoniae pneumonia and its relationship between hypokalemia in west China.

BackgroundMycoplasma pneumoniae (MP) is one of the most common respiratory pathogens of community acquired pneumonia (CAP) in children. The purpose of this study was to identify epidemics of Mycoplasma pneumoniae pneumonia (MPP) and to determine the relationship between hypokalemia and MPP.MethodsThis study was retrospectively conducted from 1 August 2016 to 31 July 2018 at Bishan People’s Hospital in Chongqing, China. We analyzed the demographic characteristics (age, gender), extra-pulmonary manifestations, fever duration, length of hospital stay, whole blood cell (WBC) count, C-reactive protein (CRP) levels, and lactate dehydrogenase (LDH) levels.ResultsThe total number of children with pneumonia was 3,385, including 570 (16.84%) patients in the MP infection group. The male-to-female ratio was 1.34:1. The peak time of MPP incidence was in autumn. The MP infection group had a significantly higher ratio of fever, higher body temperature, and longer fever duration. The number of MP-infected patients with hypokalemia was highest in spring. The length of hospital stay in the mild hypokalemia group was longer than in the normokalemia group. Furthermore, severe hypokalemia cases were all in the severe MP infection group, and the severity of MP infection and hypokalemia were positively correlated.ConclusionsIn this study, we concluded that children infected with MP were more susceptible to fever, and had a longer duration of fever. The number of MP-infected patients with mild hypokalemia peaked in spring, and affected more boys than girls. Furthermore, patients with severe hypokalemia had longer fever duration, and a correlation was found between the severity of MP infection and hypokalemia.

Inertial focusing of circulating tumor cells in whole blood at high flow rates using the microfluidic CTCKey™ device for CTC enrichment.

Circulating tumor cells (CTCs) are extremely rare cells shed from tumors into the blood stream. These cells can provide valuable information about their tumor of origin and direct treatment decisions to improve patient outcomes. Current technologies isolate CTCs from a limited blood volume and often require pre-processing that leads to CTC loss, making it difficult to isolate enough CTCs to perform in-depth tumor analysis. Many inertial microfluidic devices have been developed to isolate CTCs at high flow rates, but they typically require either blood dilution, pre-processing to remove red blood cells, or a sheath buffer rather than being able to isolate cells directly from whole blood. To decrease the need for pre-processing while increasing CTC yield, we developed an inertial device, the CTCKey™, to focus CTCs in whole blood at high throughput yielding a concentrated product stream enriched for CTCs. The CTCKey™ consists of two sections to create CTC enriched blood that can be further processed using any CTC isolation device to selectively isolate the CTCs. A thorough analysis was performed using the MCF7 breast cancer cell line spiked into bovine serum albumin (BSA) solutions of varying concentrations, as well as whole blood to characterize the focusing patterns of the CTCKey™. At the optimal flow rate of 2.4 mL min-1, the CTCKey™ reduces the CTC containing blood volume by 78%; the CTCs from 1 mL of blood are now in 0.22 mL of blood. The CTCKey's™ ability to concentrate CTCs from a large original blood volume to a smaller, highly concentrated volume enables a much greater blood volume to be interrogated by downstream isolation and characterization methods despite their low volume input limitations.

Optimization of stimulation and staining conditions for intracellular cytokine staining (ICS) for determination of cytokine-producing T cells and monocytes

Cell-mediated responses to immunological stimuli are often localised in inflammatory sites and involve a number of cell types. These responses can be functionally characterised at the single-cell level on the basis of the types of cytokines expressed either in whole blood or PBMCs. The ability to measure antigen-specific cell responses at the single cell level is an important tool with a wide range of potential applications ranging from studies of disease pathogenesis to the evaluation of vaccines.A number of experiments were performed in this study in order to establish the optimal conditions for in vitro stimulation of cytokine production by T cells and monocytes in whole blood samples collected from healthy adult Malawian participants and the optimal staining conditions for various cytokine producing cells. Different stimulation methods and conditions, different culture tubes and incubators and different antibody labelling conditions were assessed in order to establish optimal conditions for detecting cytokine-producing cells in whole blood samples.The use of PMA plus Ionomycin produced highest cytokine-producing T cells whereas LPS was a better stimulant for cytokine producing monocytes. Stimulation of whole blood for 5 h was optimal for cytokine detection in T cells whereas 4 h was optimal for monocytes. BFA was found to be a better Golgi blocker than Monensin and the use of 15 ml Falcon-type polypropylene tubes while stationary resulted in the detection of the highest proportion of cytokine-producing cells. T cells were found to be producers of mainly TNF-α, IFN-γ and IL-2 whereas Monocytes were mainly producing TNF-α and IL-6. Anti-CD3-PerCP (used at a ratio of 1:25), anti-CD14-APC (used at a ratio of 1:50) and anti-cytokine-PE (used at a ratio of 1:12.5) resulted in the best results. The highest cytokine production monocytes were detected when 1 X FACS Lysing solution was used at a volume of 40X that of the whole blood sample compared to the other volumes. These optimal conditions are essential in determination of proportion of cytokine-producing cells using ICS in whole blood.

Improved harvest and fixation methodology for isolated human peripheral blood mononuclear cells in cytokinesis-block micronucleus assay

Purpose In suspected radiation exposures, cytokinesis-block micronucleus (CBMN) assay is used for biodosimetry by detecting micronuclei (MN) in binucleated (BN) cells in whole blood and isolated peripheral blood mononuclear cell (PBMC) cultures. Standardized harvest protocols for whole blood were published by the International Atomic Energy Agency (IAEA) in 2001 (Technical report no. 405) and 2011 (EPR-Biodosimetry). For isolated PBMC harvest, cytocentrifugation of fresh cells is recommended to preserve cytoplasmic boundaries for MN scoring. However, cytocentrifugation utilizes specialized equipment and long-term cell suspension storage is difficult. In this study, an alternative CBMN harvest protocol is proposed for laboratories interested in culturing PBMCs and storing fixed cells with routine biodosimetry methods. Materials and methods Peripheral blood from 4 males (24, 34, 41, 51 y.o.) and females (26, 37, 44, 56 y.o.) was irradiated with 0 and 2 Gy X-rays. For cells harvested with IAEA 2001 and 2011 protocols, whole blood was used. For cells harvested with our protocol (CRG), isolated PBMCs were used. CRG protocol was validated in DAPI, acridine orange and Giemsa stain, and in three other laboratories. Cytoplasm status, nuclear division index (NDI) and induced MN frequency (MN frequency at 2 Gy – background MN frequency at 0 Gy) (MN/1000 BN) of Giemsa-stained BN cells were compared in IAEA 2001, IAEA 2011, IAEA 2011 + formaldehyde (FA) and CRG protocols. Effects of low and high humidity spreading were evaluated. Results >94% of 1000 BN cells were scorable with clear cytoplasmic boundaries in all donors harvested with CRG protocol. FA addition in IAEA 2011 protocol reduced cell rupture in whole blood cultures, but cell rupture was affected by age, sex and humidity. Almost all cells harvested with IAEA 2001 protocol had cytoplasm loss. PBMCs harvested with CRG protocol stained well in DAPI, acridine orange and Giemsa, and showed high scorable BN frequency in all laboratories. A higher NDI and a lower induced MN frequency were seen in 2 Gy isolated PBMC than whole blood cultures. Conclusion This quick CBMN harvest protocol for isolated PBMCs is a viable alternative to cytocentrifugation, as many scorable BN cells were obtained with routine biodosimetry reagents and equipment. IAEA 2011 + FA protocol should be used to improve CBMN harvest in whole blood cultures. Humidity during spreading should be optimized depending on the harvest protocol. NDI and MN frequency should be separately evaluated for whole blood and isolated PBMC cultures.

A new analytical platform for potential point-of-care testing of circulating tumor cells

It is of significance to detect circulating tumor cells (CTCs) in whole blood using transportable instruments at the point of care to assist evaluating chemotherapeutic efficacy and recurrence risk of cancer patients. However, the current widely used detection methods either require expensive and complex equipments, need complicated enrichment steps, or produce high rates of false positive and/or negative results. Aiming for solving the two critical challenges involved in instrumentation miniaturization and simplification of sample preparation for POCT of CTCs without sacrificing the detection sensitivity and accuracy, this work reports a custom-built, automatic, large field-of-view microscopic CTC cytometer and a novel enrichment strategy based on a synthesized peptide ligand discovered from One-Bead One-Compound library screening. The custom-built microscope has compact size, low weight and efficient cost while still maintaining a detection limit of as low as 5 target objects. The simplified sample preparation utilized a novel peptide LXW7 functionalized to magnetic beads and allows for rapid, highly selective and sensitive detection of CTCs. This analytical platform may fulfill the unmet need for possible point-of-care CTC counting, and provide a new option for early diagnosis of cancers and convenient evaluation of chemotherapeutic efficacy and cancer recurrence.

Immunomagnetic Capture and Multiplexed Surface Marker Detection of Circulating Tumor Cells with Magnetic Multicolor Surface-Enhanced Raman Scattering Nanotags.

Circulating tumor cells (CTCs) have substantial clinical implications in cancer diagnosis and monitoring. Although significant progress has been made in developing technologies for CTC detection and counting, the ability to quantitatively detect multiple surface protein markers on individual tumor cells remains very limited. In this work, we report a multiplexed method that uses magnetic multicolor surface-enhanced Raman scattering (SERS) nanotags in conjunction with a chip-based immunomagnetic separation to quantitatively and simultaneously detect four surface protein markers on individual tumor cells in whole blood. Four-color SERS nanotags were prepared using magnetic-optical iron oxide-gold core-shell nanoparticles with different Raman reporters to recognize four different cancer markers with respective antibodies. A microfluidic device was fabricated to magnetically capture the nanoparticle-bound tumor cells and to perform online negative staining and single-cell optical detection. The level of each targeted protein was obtained by signal deconvolution of the mixed SERS signals from individual tumor cells using the classic least squares regression method. The method was tested with spiked tumor cells in human whole blood with three different breast cancer cell lines and compared with the results of purified cancer cells suspended in a phosphate buffer solution. The method, with either spiked cancer cells in blood or purified cancer cells, showed a strong correlation with purified cancer cells by enzyme-linked immunosorbent assay, suggesting the potential of our method for the reliable detection of multiple surface markers on CTCs. Combining immunomagnetic enrichment with high specificity, multiplexed targeting for the capture of CTC subpopulations, multicolor SERS detection with high sensitivity and specificity, microfluidics for handling rare cells and magnetic-plasmonic nanoparticles for dual enrichment and detection, our method provides an integrated, yet a simple and an efficient platform that has the potential to more sensitively detect and monitor cancer metastasis.

Genome-wide association study reveals a novel locus for asthma with severe exacerbations in diverse populations.

Severe asthma exacerbations are a major cause of asthma morbidity and increased healthcare costs. Several studies have shown racial and ethnic differences in asthma exacerbation rates. We aimed to identify genetic variants associated with severe exacerbations in two high-risk populations for asthma. A genome-wide association study of asthma in children and youth with severe exacerbations was performed in 1283 exacerbators and 2027 controls without asthma of Latino ancestry. Independent suggestive variants (P ≤ 5 × 10-6 ) were selected for replication in 448 African Americans exacerbators and 595 controls. Case-only analyses were performed comparing the exacerbators with additional 898 Latinos and 524 African Americans asthma patients without exacerbations, while adjusting by treatment category as a proxy of asthma severity. We analyzed the functionality of associated variants with in silico methods and by correlating genotypes with methylation levels in whole blood in a subset of 473 Latinos. We identified two genome-wide significant associations for susceptibility to asthma with severe exacerbations, including a novel locus located at chromosome 2p21 (rs4952375, odds ratio = 1.39, P = 3.8 × 10-8 ), which was also associated with asthma exacerbations in a case-only analysis (odds ratio = 1.25, P = 1.95 × 10-3 ). This polymorphism is an expression quantitative trait locus of the long intergenic non-protein coding RNA 1913 (LINC01913) in lung tissues (P = 1.3 × 10-7 ) and influences methylation levels of the protein kinase domain-containing cytoplasmic (PKDCC) gene in whole-blood cells (P = 9.8 × 10-5 ). We identified a novel susceptibility locus for severe asthma exacerbations in Hispanic/Latino and African American youths with functional effects in gene expression and methylation status of neighboring genes.

Establishment of An Improved Rat Model of Sepsis Induced by Cecal Ligation and Puncture

Objective To establish an improved animal model of sepsis induced by cecal ligation and puncture(CLP). Methods Ninety-six male Sprague-Dawley rats were randomly divided into sham operation group(n=24),intubation group(n=24),CLP group(n=24),and CLP+intubation group(n=24).The mortality rate,abdominal cavity condition,pathological changes and pathological scores of heart,lungs,liver,and kidneys of rats in each group were observed after modeling.Blood samples were obtained from the inferior vena cava for measuring the whole blood cells(WBC)and platelets(PLT)counts and analyzing serum interleukin(IL)-6,tumor necrosis factor(TNF)-α,serum troponin T(cTnT),creatine kinase-MB(CK-MB),alanine aminotransferase(ALT),aspartate aminotransferase(AST),total bilirubin(TBIL),creatinine(CREA),and blood urea nitrogen(BUN)levels.Blood gas analysis of the aorta was also performed. Results The mortality rates 24 h after modeling were 0 in sham operation group and intubation group,20.8% in CLP group,and 54.2% in CLP+intubation group.Pathologically,swelling and inflammatory cell infiltration in the heart,lungs,liver,and kidneys were seen in the CLP+intubation group,inflammatory cell infiltration in a single organ was seen in most rats in the CLP group,and no obvious swelling and infiltration of inflammatory cells was observed in the sham-operation group and intubation group.The myocardial histopathology score,lung tissue injury pathology score,and kidney tissue injury pathology score in both the sham-operation group and the intubation group were significantly lower than those in the CLP group and the CLP+intubation group(all P=0.000).TNF-α,PaO2,CK-MB,cTnT,AST,TBIL,BUN,and CREA were significantly different between sham-operation group and intubation group/CLP group/CLP+intubation group and between intubation group and CLP group/CLP+intubation group(all P=0.000).The pH level was significantly different between sham operation group and intubation group/CLP group,between intubation group and CLP group/CLP+intubate group(all P=0.000). Conclusions Although both CLP and CLP+intubation can well mimic the pathophysiological mechanism of sepsis in rats,multiple organ dysfunction occurs in the latter.Thus,CLP+intubation can establish animal models of multiple organ dysfunction caused by sepsis induced by clinically effective abdominal infection.

Direct Analysis of Rare Circulating Tumor Cells in Whole Blood Based on Their Controlled Capture and Release on Electrode Surface.

The development of a simple, sensitive, and effective method for the analysis of circulating tumor cells (CTCs) is essential for cancer diagnosis and metastasis prediction. In this work, we have proposed an enzyme-free electrochemical method for specific capture, sensitive quantification, and efficient release of CTCs. To achieve this, the specific interaction between CTCs and the corresponding aptamer designed to be located in the identification probe (IP) will unfold the hairpin structure of IP. Consequently, IP will initiate a hybridization reaction to produce a duplex, which will further trigger the hybridization chain reaction (HCR) process to form a composite product of CTCs and double-stranded DNA polymers. Therefore, a significantly amplified signal readout can be obtained. Moreover, the composite product can be brought to the electrode surface by tetrahedral DNA nanostructures to achieve the purpose of capturing and quantifying CTCs. More significantly, these captured CTCs can be controlled released without compromising cell viability via a simple strand displacement reaction. Taking the breast cancer cell MCF-7 as a representative, the newly developed approach led to an ultralow detection limit of 3 cells mL-1, which is superior to several studies previously reported. The current method has also been demonstrated to analyze CTCs in human whole blood and hence revealed a great potential in the future.

Treatment of latent M. tuberculosis infection and use of antiretroviral therapy to prevent tuberculosis.

Even with treatment of HIV with antiretroviral therapy (ART), the risk of tuberculosis (TB) reactivation remains higher in HIV-infected than HIV-uninfected persons. In this issue of the JCI, Ganatra et al. explored TB reactivation in the context of ART using TB and simian immunodeficiency virus-coinfected (TB/SIV-coinfected) nonhuman primates. The authors found that treating rhesus macaques with ART restored CD4+ T cells in whole blood, spleen, and bronchoalveolar lavage (BAL) fluid, but not in the lung interstitium. TB risk was not decreased in the coinfected macaques treated with ART for 14-63 days, suggesting that ART does not decrease the short-term risk of reactivation. Reactivation occurred as CD4+ T cells were increasing, which is consistent with observations made in humans. This study provides a potential model for systematic evaluation of TB/SIV coinfection and different treatment regimens and strategies to prevent TB reactivation.

Abstract 243: Integration of transcriptome and metabolome provides unique insights to pathways associated with obese breast cancer patients

Abstract Information regarding transcriptome and metabolome has significantly contributed to the identification of potential therapeutic targets for the management of a variety of cancers. Obesity has been shown to have profound effects on both cancer cell transcriptome and metabolome and to affect the outcome of cancer therapy. The information regarding the potential effects of obesity on breast cancer (BC) transcriptome, metabolome, and its integration to identify novel pathways related to disease progression are still elusive. We assessed the whole blood transcriptome and serum metabolome as circulating metabolites, of obese BC patients and compared them with non-obese BC patients. In these patients, 186 differentially expressed genes (DEGs) were observed, with 156 upregulated and 30 downregulated, significantly. The DEGs were enriched in different cellular pathways: cell cycle, one carbon pathway, homologous recombination cellular senescence, and notch signaling pathway. Our results confirmed the altered expression of several DEGs by quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, 96 deregulated metabolites were identified when untargeted metabolomics was performed in obese BC patients when compared to non-obese BC patients, these were enriched in 71 pathways, most of them involved in ATP generation and cell proliferation. Finally, to provide a more comprehensive understanding of the association between obesity and BC, integration analysis between transcriptome and metabolomics data at the pathway level, revealed seven enriched pathways in obese BC vs. non-obese BC patients, that includes glutathione metabolism, glycine and serine metabolism, valine, leucine, and isoleucine degradation, purine metabolism, pyrimidine metabolism, thyroid hormone synthesis, and vitamin B6 metabolism; which may provide resistance for BC cell to dodge the circulating immune cells in whole blood. In conclusion, this study provides information on the unique pathways alteration at transcriptome and metabolome levels in obese BC patients, which may become an important tool for researchers and contribute to a rise in the knowledge on the molecular interaction between obesity and BC. Further studies are needed to confirm this and to elucidate the exact underlying mechanism for the effects of obesity on the BC initiation or/and progression. Citation Format: Mohammed Abdullah Hassan, Kaltoom Al-Sakkaf, Mohammed Razeeth Shait Mohammed, Ashraf Dallol, Jaudah Al-Maghrabi, Alia Aldahlawi, Sawsan Ashoor, Mabrouka Maamra, Jiannis Ragoussis, Wei Wu, Mohammad Imran Khan, Abdulrahman Al-Malki, Hani Choudhry. Integration of transcriptome and metabolome provides unique insights to pathways associated with obese breast cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 243.

Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment

Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRhiCD11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DRlo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.

Mapping systemic lupus erythematosus heterogeneity at the single-cell level.

Patients with Systemic Lupus Erythematosus (SLE) display a complex blood transcriptome whose cellular origin is poorly resolved. Using single-cell RNA-seq, we profiled ~276,000 PBMCs from 33 children with SLE (cSLE) with different degrees of disease activity (DA) and 11 matched controls. Increased expression of interferon-stimulated genes (ISGs) distinguished cSLE from healthy control cells. The high-ISG expression signature (ISGhi) derived from a small number of transcriptionally defined subpopulations within major cell types, including monocytes, CD4+ and CD8+ T cells, natural killer cells, conventional dendritic cells (cDCs), plasmacytoid DCs (pDCs), B cells and especially plasma cells. Expansion of unique subpopulations enriched in ISGs and/or in monogenic lupus-associated genes classified patients with the highest DA. Profiling of ~82,000 single peripheral blood mononuclear cells (PBMCs) from adult SLE patients confirmed the expansion of similar subpopulations in patients with the highest DA. This study lays the groundwork for resolving the origin of the SLE transcriptional signatures and the disease heterogeneity towards precision medicine applications.

Near-infrared-driven fluorescent nanomotors for detection of circulating tumor cells in whole blood

It is of great significance to develop micro/nanomotors with good biosafety and high efficiency for biomedical detection. In order to enhance the contact chances between the biosensors and the analytes in the complex blood sample, we report a near-infrared light (NIR)-driven fluorescent nanomotor, which can safely and efficiently capture and detect circulating tumor cells (CTCs) in the whole blood environment without pretreating procedures. Firstly, the structure of the fluorescent nanomotor was characterized and its motion behavior in different environments was analyzed. Subsequently, CTCs detection conditions were optimized to explore the effect of nanoscale autonomous mixing provided by nanomotor’s motion behavior on the efficiency of CTCs capture. The results showed that the NIR-driven fluorescent nanomotor can keep moving in the complex biological medium so as to increase their contact chances with CTCs and improve the binding efficiency with the target analyte (the capture efficiency increased from 67.5% – 85% (no NIR irradiation) to 93.75–98.75% (under NIR irradiation)). Finally, due to the excellent fluorescence properties of the nanomotors, they can be applied to the visual detection of the whole blood samples obtained from clinical cancer patients. The NIR-driven nanomotor designed in this paper can detect CTCs in whole blood environment, which is a beneficial extension of the existing cell detection system of most micro/nanomotors in water phase environment.

Discrimination between Breast Cancer Cells and White Blood Cells by Non-Invasive Measurements: Implications for a Novel In Vitro-Based Circulating Tumor Cell Model Using Digital Holographic Cytometry

Breast cancer is the second most common cancer worldwide. Metastasis is the main reason for death in breast cancer, and today, there is a lack of methods to detect and isolate circulating tumor cells (CTCs), mainly due to their heterogeneity and rarity. There are some systems that are designed to detect rare epithelial cancer cells in whole blood based on the most common marker used today, the epithelial cell adhesion molecule (EpCAM). It has been shown that aggressive breast cancer metastases are of non-epithelial origin and are therefore not always detected using EpCAM as a marker. In the present study, we used an in vitro-based circulating tumor cell model comprising a collection of six breast cancer cell lines and white blood cell lines. We used digital holographic cytometry (DHC) to characterize and distinguish between the different cell types by area, volume and thickness. Here, we present significant differences in cell size-related parameters observed when comparing white blood cells and breast cancer cells by using DHC. In conclusion, DHC can be a powerful diagnostic tool for the characterization of CTCs in the blood.

Complement Inhibitors Block Complement C3 Opsonization and Improve Targeting Selectivity of Nanoparticles in Blood.

Complement is one of the critical branches of innate immunity that determines the recognition of engineered nanoparticles by immune cells. Antibody-targeted iron oxide nanoparticles are a popular platform for magnetic separations, in vitro diagnostics, and molecular imaging. We used 60 nm cross-linked iron oxide nanoworms (CLIO NWs) modified with antibodies against Her2/neu and EpCAM, which are common markers of blood-borne cancer cells, to understand the role of complement in the selectivity of targeting of tumor cells in whole blood. CLIO NWs showed highly efficient targeting and magnetic isolation of tumor cells spiked in lepirudin-anticoagulated blood, but specificity was low due to high uptake by neutrophils, monocytes, and lymphocytes. Complement C3 opsonization in plasma was predominantly via the alternative pathway regardless of the presence of antibody, PEG, or fluorescent tag, but was higher for antibody-conjugated CLIO NWs. Addition of various soluble inhibitors of complement convertase (compstatin, soluble CD35, and soluble CD55) to whole human blood blocked up to 99% of the uptake of targeted CLIO NWs by leukocytes, which resulted in a more selective magnetic isolation of tumor cells. Using well-characterized nanomaterials, we demonstrate here that complement therapeutics can be used to improve targeting selectivity.

Electrochemical assay for detection of circulating tumor cells based on LiFePO4 as electrochemical probe

In this study, we reported an electrochemical assay for detecting circulating tumor cells (CTCs) in whole blood. Breast cancer cell MCF-7 was chosen as a model for study. Fe3O4 magnetic nanospheres (MNs) modified with anti-epithelial cell adhesion molecule (EpCAM) antibody were utilized to capture MCF-7 from samples and gold nanoparticles modified LiFePO4 (LiFePO4/Au) particles were chosen as electrochemical label. Aptamer can be linked onto LiFePO4/Au via Au-S bond, and then the LiFePO4/Au-aptamer complex can specifically bind to the MUC1 protein that is over-expressed on the cell surface of MCF-7. Electrochemical signals are generated by the reaction of phosphate in LiFePO4 with molybdate to form redox molybdophosphate. The utilization of LiFePO4 for signal probe may expand the application of LiFePO4 in biosensing fields.