A Method for Rapid, Quantitative Evaluation of Particle Sorting in Microfluidics Using Basic Cytometry Equipment.

This paper describes, in detail, a method that uses flow cytometry to quantitatively characterise the performance of continuous-flow microfluidic devices designed to separate particles. Whilst simple, this approach overcomes many of the issues with the current commonly utilised methods (high-speed fluorescent imaging, or cell counting via either a hemocytometer or a cell counter), as it can accurately assess device performance even in complex, high concentration mixtures in a way that was previously not possible. Uniquely, this approach takes advantage of pulse processing in flow cytometry to allow quantitation of cell separation efficiencies and resulting sample purities on both single cells as well as cell clusters (such as circulating tumour cell (CTC) clusters). Furthermore, it can readily be combined with cell surface phenotyping to measure separation efficiencies and purities in complex cell mixtures. This method will facilitate the rapid development of a raft of continuous flow microfluidic devices, will be helpful in testing novel separation devices for biologically relevant clusters of cells such as CTC clusters, and will provide a quantitative assessment of device performance in complex samples, which was previously impossible.

Isolation of Circulating Tumor Cells from Seminal Fluid of Patients with Prostate Cancer Using Inertial Microfluidics.

Prostate cancer (PCa) diagnosis is primarily based on prostate-specific antigen (PSA) testing and prostate tissue biopsies. However, PSA testing has relatively low specificity, while tissue biopsies are highly invasive and have relatively low sensitivity at early stages of PCa. As an alternative, we developed a technique of liquid biopsy, based on isolation of circulating tumor cells (CTCs) from seminal fluid (SF). The recovery of PCa cells from SF was demonstrated using PCa cell lines, achieving an efficiency and throughput as high as 89% (±3.8%) and 1.7 mL min-1, respectively, while 99% (±0.7%) of sperm cells were disposed of. The introduced approach was further tested in a clinical setting by collecting and processing SF samples of PCa patients. The yield of isolated CTCs measured as high as 613 cells per SF sample in comparison with that of 6 cells from SF of healthy donors, holding significant promise for PCa diagnosis. The correlation analysis of the isolated CTC numbers with the standard prognostic parameters such as Gleason score and PSA serum level showed correlation coefficient values at 0.40 and 0.73, respectively. Taken together, our results show promise in the developed liquid biopsy technique to augment the existing diagnosis and prognosis of PCa.

Affibody Functionalized Beads for the Highly Sensitive Detection of Cancer Cell-Derived Exosomes.

Exosomes belong to the class of extracellular vesicles of endocytic origin, which are regarded as a promising source of cancer biomarkers in liquid biopsy. As a result, an accurate, sensitive, and specific quantification of these nano-sized particles is of significant importance. Affinity-based approaches are recognized as the most valuable technique for exosome isolation and characterization. Indeed, Affibody biomolecules are a type of protein scaffold engineered with small size and enjoy the features of high thermal stability, affinity, and specificity. While the utilization of antibodies, aptamers, and other biologically active substances for exosome detection has been reported widely, there are no reports describing Affibody molecules' usage for exosome detection. In this study, for the first time, we have proposed a novel strategy of using Affibody functionalized microbeads (AffiBeads) for exosome detection with a high degree of efficiency. As a proof-of-concept, anti-EGFR-AffiBeads were fabricated and applied to capture and detect human lung A549 cancer cell-derived EGFR-positive exosomes using flow cytometry and fluorescent microscopy. Moreover, the capture efficiency of the AffiBeads were compared with its counterpart antibody. Our results showed that the Affibody probe had a detection limit of 15.6 ng exosomes per mL (~12 exosomes per AffiBead). The approach proposed in the current study can be used for sensitive detection of low expression level markers on tumor-derived exosomes, providing a basis for early-stage cancer diagnosis.

Circulating tumor cell clusters: Insights into tumour dissemination and metastasis.

Introduction: Metastasis results in more than 90% of cancer-related deaths globally. The process is thought to be facilitated by metastatic precursor cells, commonly termed circulating tumor cells (CTCs). CTCs can exist as single cells or cell clusters and travel through the lymphovasculature to distant organs where they can form overt metastasis. Areas covered: Studies have highlighted that CTC clusters, which may be homotypic or heterotypic in composition, have a higher metastatic potential compared to single CTCs. The characterization of CTC clusters is becoming important as heterotypic clusters can provide a mechanism for immune evasion. This review summarizes the latest advances in CTC cluster-mediated metastasis and clinical significance. Expert opinion: Comprehensive characterization of CTC clusters is needed to understand the cell types and interactions within clusters, in order to identify ways in which to reduce CTC cluster-mediated metastasis. The role of CTC clusters in prognosticating disease progression needs to be determined by documenting CTC clusters from the time of diagnosis over the course of therapy.

Diagnostic value of serum HER2 levels in breast cancer: a systematic review and meta-analysis.

BACKGROUND: Measurement of serum human epidermal growth factor receptor-2 (HER-2/neu) levels might play an essential role as a diagnostic/screening marker for the early selection of therapeutic approaches and predict prognosis in breast cancer patients. We aimed to undertake a systematic review and meta-analysis focusing on the diagnostic/screening value of serum HER-2 levels in comparison to routine methods. METHODS: We performed a systematic search via PubMed, Scopus, Cochrane-Library, and Web of Science databases for human diagnostic studies reporting the levels of serum HER-2 in breast cancer patients, which was confirmed using the histopathological examination. Meta-analyses were carried out for sensitivity, specificity, accuracy, area under the ROC curve (AUC), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), and negative likelihood ratio (NLR). RESULTS: Fourteen studies entered into this investigation. The meta-analysis indicated the low sensitivity for serum HER2 levels (Sensitivity: 53.05, 95%CI 40.82-65.28), but reasonable specificity of 79.27 (95%CI 73.02-85.51), accuracy of 72.06 (95%CI 67.04-77.08) and AUC of 0.79 (95%CI 0.66-0.92). We also found a significant differences for PPV (PPV: 56.18, 95%CI 44.16-68.20), NPV (NPV: 76.93, 95%CI 69.56-84.31), PLR (PLR: 2.10, 95%CI 1.69-2.50) and NLR (NLR: 0.58, 95%CI 0.44-0.71). CONCLUSION: Our findings revealed that although serum HER-2 levels showed low se nsitivity for breast cancer diagnosis, its specificity, accuracy and AUC were reasonable. Hence, it seems that the measurement of serum HER-2 levels can play a significant role as a verification test for initial negative screening test results, especially in low-income regions due to its cost-effectiveness and ease of implementation.

Fabrication of unconventional inertial microfluidic channels using wax 3D printing.

Inertial microfluidics has emerged over the past decade as a powerful tool to accurately control cells and microparticles for diverse biological and medical applications. Many approaches have been proposed to date in order to increase the efficiency and accuracy of inertial microfluidic systems. However, the effects of channel cross-section and solution properties (Newtonian or non-Newtonian) have not been fully explored, primarily due to limitations in current microfabrication methods. In this study, we overcome many of these limitations using wax 3D printing technology and soft lithography through a novel workflow, which eliminates the need for the use of silicon lithography and polydimethylsiloxane (PDMS) bonding. We have shown that by adding dummy structures to reinforce the main channels, optimizing the gap between the dummy and main structures, and dissolving the support wax on a PDMS slab to minimize the additional handling steps, one can make various non-conventional microchannels. These substantially improve upon previous wax printed microfluidic devices where the working area falls into the realm of macrofluidics rather than microfluidics. Results revealed a surface roughness of 1.75 µm for the printed channels, which does not affect the performance of inertial microfluidic devices used in this study. Channels with complex cross-sections were fabricated and then analyzed to investigate the effects of viscoelasticity and superposition on the lateral migration of the particles. Finally, as a proof of concept, microcarriers were separated from human mesenchymal stem cells using an optimized channel with maximum cell-holding capacity, demonstrating the suitability of these microchannels in the bioprocessing industry.

Promoted chondrogenesis of hMCSs with controlled release of TGF-ß3 via microfluidics synthesized alginate nanogels.

The field of cartilage tissue engineering has been evolved in the last decade and a myriad of scaffolding biomaterials and bioactive agents have been proposed. Controlled release of growth factors encapsulated in the polymeric nanomaterials has been of interest notably for the repair of damaged articular cartilage. Here, we proposed an on-chip hydrodynamic flow focusing microfluidic approach for synthesis of alginate nanogels loaded with the transforming growth factor beta 3 (TGF-ß3) through an ionic gelation method in order to achieve precise release profile of these bioactive agents during chondrogenic differentiation of mesenchymal stem cells (MSCs). Alginate nanogels with adjustable sizes were synthesized by fine-tuning the flow rate ratio (FRR) in the microfluidic device consisting of cross-junction microchannels. The result of present study showed that the proposed approach can be a promising tool to synthesize bioactive -loaded polymeric nanogels for applications in drug delivery and tissue engineering.

Upregulation of PD-L1 expression in breast cancer cells through the formation of 3D multicellular cancer aggregates under different chemical and mechanical conditions.

Expression of programmed death-ligand 1 (PD-L1) in cancer cells plays an important role in cancer-immune cell interaction. The emerging evidence suggests regulation of PD-L1 expression by several tumor microenvironmental cues. However, the association of PD-L1 expression with chemical and mechanical features of the tumor microenvironment, specifically epidermal growth factor receptor (EGFR) signaling and matrix stiffness, remains elusive. Herein, we determine whether EGFR targeting and substrate stiffness affect the regulation of PD-L1 expression. Breast carcinoma cell lines, MCF7 and MDA-MB-231, were cultured under different conditions targeting EGFR and exposing cells to distinct substrate stiffness to evaluate PD-L1 expression. Furthermore, the ability to form aggregates in short-term culture of breast carcinoma cells and its effect on expression level of PD-L1 was probed. Our results indicated that PD-L1 expression was altered in response to both EGFR inhibition and substrate stiffness. Additionally, a positive association between the formation of multicellular aggregates and PD-L1 expression was observed. MDA-MB-231 cells expressed the highest PD-L1 level on a stiff substrate, while inhibition of EGFR reduced expression of PD-L1. The results suggested that both physical and chemical features of tumor microenvironment regulate PD-L1 expression through alteration of tumor aggregate formation potential. In line with these results, the in-silico study highlighted a positive correlation between PD-L1 expression, EGFR signaling, epithelial to mesenchymal transition related transcription factors (EMT-TFs) and stemness markers in metastatic breast cancer. These findings improve our understanding of regulation of PD-L1 expression by tumor microenvironment leading to evasion of tumor cells from the immune system.

A simple coating method of PDMS microchip with PTFE for synthesis of dexamethasone-encapsulated PLGA nanoparticles.

Dexamethasone is a widely used drug in medical and biological applications. Since the systematic and controllable release of this drug is of significant importance, encapsulation of this anti-inflammatory drug in poly(lactic-co-glycolic acid) (PLGA) nanoparticles can minimize uncontrolled issues. As dexamethasone-encapsulated PLGA nanoparticles are synthesized in the presence of organic solvents, poly(dimethylsiloxane) (PDMS)-based microchannels collapse due to the swelling problem. In present study, PTFE nanoparticles were used for the surface modification of the microchannels to prevent absorption and adhesion of solvents into the microchannels' wall. The contact angle analysis of microchips after coating showed that the surface of microchannels bear the superhydrophobicity feature (140.30°) and SEM images revealed that PTFE covered the surface of PDMS, favorably. Then, the prepared microchip was tested for the synthesis of dexamethasone-loaded nanoparticles. SEM and atomic force microscopy (AFM) images of the synthesized nanoparticles represented that there was not any evidence of adhesion or absorption of nanoparticles. Furthermore, the monodispersity of nanoparticles was discernible. As AFM results revealed, the average diameters of 47, 63, and 82 nm were achieved for flow ratios of 0.01, 0.05, and 0.1, respectively. To evaluate the drug efficiency, cumulative release and encapsulation efficiency were analyzed which showed much more efficiency than the synthesized nanoparticles in the bulk mode. In addition, MTT test revealed that nanoparticles could be considered as a non-toxic material. Since the synthesis of drug-loaded nanoparticles is ubiquitous in laboratory experiments, the approach presented in this study can render more versatility in this regard.

Rapid and Label-Free Isolation of Tumour Cells from the Urine of Patients with Localised Prostate Cancer Using Inertial Microfluidics.

During the last decade, isolation of circulating tumour cells via blood liquid biopsy of prostate cancer (PCa) has attracted significant attention as an alternative, or substitute, to conventional diagnostic tests. However, it was previously determined that localised forms of PCa shed a small number of cancer cells into the bloodstream, and a large volume of blood is required just for a single test, which is impractical. To address this issue, urine has been used as an alternative to blood for liquid biopsy as a truly non-invasive, patient-friendly test. To this end, we developed a spiral microfluidic chip capable of isolating PCa cells from the urine of PCa patients. Potential clinical utility of the chip was demonstrated using anti-Glypican-1 (GPC-1) antibody as a model of the primary antibody in immunofluorescent assay for identification and detection of the collected tumour cells. The microchannel device was first evaluated using DU-145 cells in a diluted Dulbecco's phosphate-buffered saline sample, where it demonstrated >85 (±6) % efficiency. The microchannel proved to be functional in at least 79% of cases for capturing GPC1+ putative tumour cells from the urine of patients with localised PCa. More importantly, a correlation was found between the amount of the captured GPC1+ cells and crucial diagnostic and prognostic parameter of localised PCa-Gleason score. Thus, the technique demonstrated promise for further assessment of its diagnostic value in PCa detection, diagnosis, and prognosis.