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Chimeric antigen receptor cell therapy is a successful immunotherapy for the treatment of blood cancers. However, hurdles in their manufacturing remain including efficient isolation and purification of the T-cell starting material. Herein, we describe a one-step separation based on inertial spiral microfluidics for efficient enrichment of T-cells in B-cell acute lymphoblastic leukemia (ALL) and B-cell chronic lymphocytic leukemia patient's samples. In healthy donors used to optimize the process, the lymphocyte purity was enriched from 65% (SD ± 0.2) to 91% (SD ± 0.06) and T-cell purity was enriched from 45% (SD ± 0.1) to 73% (SD ± 0.02). Leukemic samples had higher starting B-cells compared to the healthy donor samples. Efficient enrichment and recovery of lymphocytes and T-cells were achieved in ALL samples with B-cells, monocytes and leukemic blasts depleted by 80% (SD ± 0.09), 89% (SD ± 0.1) and 74% (SD ± 0.09), respectively, and a 70% (SD ± 0.1) T-cell recovery. Chronic lymphocytic leukemia samples had lower T-cell numbers, and the separation process was less efficient compared to the ALL. This study demonstrates the use of inertial microfluidics for T-cell enrichment and depletion of B-cell blasts in ALL, suggesting its potential to address a key bottleneck of the chimeric antigen receptor-T manufacturing workflow., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Objectives: Non-small-cell lung carcinoma (NSCLC) is the most prevalent and lethal form of lung cancer. The need for biomarker-informed stratification of targeted therapies has underpinned the need to uncover the underlying properties of the tumor microenvironment (TME) through high-plex quantitative assays., Methods: In this study, we profiled resected NSCLC tissues from 102 patients by targeted spatial proteomics of 78 proteins across tumor, immune activation, immune cell typing, immune-oncology, drug targets, cell death and PI3K/AKT modules to identify the tumor and stromal signatures associated with overall survival (OS)., Results: Survival analysis revealed that stromal CD56 (HR = 0.384, P  = 0.06) and tumoral TIM3 (HR = 0.703, P  = 0.05) were associated with better survival in univariate Cox models. In contrast, after adjusting for stage, BCLXL (HR = 2.093, P  = 0.02) and cleaved caspase 9 (HR = 1.575, P  = 0.1) negatively influenced survival. Delta testing indicated the protective effect of TIM-3 (HR = 0.614, P  = 0.04) on OS. In multivariate analysis, CD56 (HR = 0.172, P  = 0.001) was associated with better survival in the stroma, while B7.H3 (HR = 1.72, P  = 0.008) was linked to poorer survival in the tumor., Conclusions: Deciphering the TME using high-plex spatially resolved methods is giving us new insights into compartmentalised tumor and stromal protein signatures associated with clinical endpoints in NSCLC., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s). Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

The purpose of this study was to evaluate the potential of zerumbone-loaded liquid crystalline nanoparticles (ZER-LCNs) in the protection of broncho-epithelial cells and alveolar macrophages against oxidative stress, inflammation and senescence induced by cigarette smoke extract in vitro. The effect of the treatment of ZER-LCNs on in vitro cell models of cigarette smoke extract (CSE)-treated mouse RAW264.7 and human BCi-NS1.1 basal epithelial cell lines was evaluated for their anti-inflammatory, antioxidant and anti-senescence activities using colorimetric and fluorescence-based assays, fluorescence imaging, RT-qPCR and proteome profiler kit. The ZER-LCNs successfully reduced the expression of pro-inflammatory markers including Il-6, Il-1β and Tnf-α, as well as the production of nitric oxide in RAW 264.7 cells. Additionally, ZER-LCNs successfully inhibited oxidative stress through reduction of reactive oxygen species (ROS) levels and regulation of genes, namely GPX2 and GCLC in BCi-NS1.1 cells. Anti-senescence activity of ZER-LCNs was also observed in BCi-NS1.1 cells, with significant reductions in the expression of SIRT1, CDKN1A and CDKN2A. This study demonstrates strong in vitro anti-inflammatory, antioxidative and anti-senescence activities of ZER-LCNs paving the path for this formulation to be translated into a promising therapeutic agent for chronic respiratory inflammatory conditions including COPD and asthma., (© 2023. The Author(s).)

Lung cancer is the second most prevalent type of cancer and is responsible for the highest number of cancer-related deaths worldwide. Non-small-cell lung cancer (NSCLC) makes up the majority of lung cancer cases. Zerumbone (ZER) is natural compound commonly found in the roots of Zingiber zerumbet which has recently demonstrated anti-cancer activity in both in vitro and in vivo studies. Despite their medical benefits, ZER has low aqueous solubility, poor GI absorption and oral bioavailability that hinders its effectiveness. Liquid crystalline nanoparticles (LCNs) are novel drug delivery carrier that have tuneable characteristics to enhance and ease the delivery of bioactive compounds. This study aimed to formulate ZER-loaded LCNs and investigate their effectiveness against NSCLC in vitro using A549 lung cancer cells. ZER-LCNs, prepared in the study, inhibited the proliferation and migration of A549 cells. These inhibitory effects were superior to the effects of ZER alone at a concentration 10 times lower than that of free ZER, demonstrating a potent anti-cancer activity of ZER-LCNs. The underlying mechanisms of the anti-cancer effects by ZER-LCNs were associated with the transcriptional regulation of tumor suppressor genes P53 and PTEN, and metastasis-associated gene KRT18. The protein array data showed downregulation of several proliferation associated proteins such as AXL, HER1, PGRN, and BIRC5 and metastasis-associated proteins such as DKK1, CAPG, CTSS, CTSB, CTSD, and PLAU. This study provides evidence of potential for increasing the potency and effectiveness of ZER with LCN formulation and developing ZER-LCNs as a treatment strategy for mitigation and treatment of NSCLC., (© 2023. The Author(s).)

Stem cells are specialised cells characterised by their unique ability to both self-renew and transform into a wide array of specialised cell types. The widespread interest in stem cells for regenerative medicine and cultivated meat has led to a significant demand for these cells in both research and practical applications. Despite the growing need for stem cell manufacturing, the industry faces significant obstacles, including high costs for equipment and maintenance, complicated operation, and low product quality and yield. Microfluidic technology presents a promising solution to the abovementioned challenges. As an innovative approach for manipulating liquids and cells within microchannels, microfluidics offers a plethora of advantages at an industrial scale. These benefits encompass low setup costs, ease of operation and multiplexing, minimal energy consumption, and the added advantage of being labour-free. This review presents a thorough examination of the prominent microfluidic technologies employed in stem cell research and explores their promising applications in the burgeoning stem cell industry. It thoroughly examines how microfluidics can enhance cell harvesting from tissue samples, facilitate mixing and cryopreservation, streamline microcarrier production, and efficiently conduct cell separation, purification, washing, and final cell formulation post-culture., Competing Interests: Declaration of Competing Interest Authors, L.D, P.R., M.E.W and S.O, own equity in the Smart MCs PTY LTD. Some schematics used in this review were designed with Biorender.com., (Copyright © 2023. Published by Elsevier Inc.)

The global burden of respiratory diseases is enormous, with many millions of people suffering and dying prematurely every year. The global COVID-19 pandemic witnessed recently, along with increased air pollution and wildfire events, increases the urgency of identifying the most effective therapeutic measures to combat these diseases even further. Despite increasing expenditure and extensive collaborative efforts to identify and develop the most effective and safe treatments, the failure rates of drugs evaluated in human clinical trials are high. To reverse these trends and minimize the cost of drug development, ineffective drug candidates must be eliminated as early as possible by employing new, efficient, and accurate preclinical screening approaches. Animal models have been the mainstay of pulmonary research as they recapitulate the complex physiological processes, Multiorgan interplay, disease phenotypes of disease, and the pharmacokinetic behavior of drugs. Recently, the use of advanced culture technologies such as organoids and lung-on-a-chip models has gained increasing attention because of their potential to reproduce human diseased states and physiology, with clinically relevant responses to drugs and toxins. This review provides an overview of different animal models for studying respiratory diseases and evaluating drugs. We also highlight recent progress in cell culture technologies to advance integrated models and discuss current challenges and present future perspectives., (© 2023 The Authors. Medicinal Research Reviews published by Wiley Periodicals LLC.)

Biological barriers are multicellular structures that precisely regulate the transport of ions, biomolecules, drugs, cells, and other organisms. Transendothelial/epithelial electrical resistance (TEER) is a label-free method for predicting the properties of biological barriers. Understanding the mechanisms that control TEER significantly enhances our knowledge of the physiopathology of different diseases and aids in the development of new drugs. Measuring TEER values within microphysiological systems called organ-on-a-chip devices that simulate the microenvironment, architecture, and physiology of biological barriers in the body provides valuable insight into the behavior of barriers in response to different drugs and pathogens. These integrated systems should increase the accuracy, reproducibility, sensitivity, resolution, high throughput, speed, cost-effectiveness, and reliable predictability of TEER measurements. Implementing advanced micro and nanoscale manufacturing techniques, surface modification methods, biomaterials, biosensors, electronics, and stem cell biology is necessary for integrating TEER measuring systems with organ-on-chip technology. This review focuses on the applications, advantages, and future perspectives of integrating organ-on-a-chip technology with TEER measurement methods for studying biological barriers. After briefly reviewing the role of TEER in the physiology and pathology of barriers, standard techniques for measuring TEER, including Ohm's law and impedance spectroscopy, and commercially available devices are described. Furthermore, advances in TEER measurement are discussed in multiple barrier-on-a-chip system models representing different organs. Finally, we outline future trends in implementing advanced technologies to design and fabricate nanostructured electrodes, complicated microfluidic chips, and membranes for more advanced and accurate TEER measurements., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Mucosal head and neck squamous cell carcinoma (HNSCC) are the seventh most common cancer, with approximately 50% of patients living beyond 5 years. Immune checkpoint inhibitors (ICIs) have shown promising results in patients with recurrent or metastatic (R/M) disease, however, only a subset of patients benefit from immunotherapy. Studies have implicated the tumor microenvironment (TME) of HNSCC as a major factor in therapy response, highlighting the need to better understand the TME, particularly by spatially resolved means to determine cellular and molecular components. Here, we employed targeted spatial profiling of proteins on a cohort of pre-treatment tissues from patients with R/M disease to identify novel biomarkers of response within the tumor and stromal margins. By grouping patient outcome categories into response or non-response, based on Response Evaluation Criteria in Solid Tumors (RECIST) we show that immune checkpoint molecules, including PD-L1, B7-H3, and VISTA, were differentially expressed. Patient responders possessed significantly higher tumor expression of PD-L1 and B7-H3, but lower expression of VISTA. Analysis of response subgroups indicated that tumor necrosis factor receptor (TNFR) superfamily members including OX40L, CD27, 4-1BB, CD40, and CD95/Fas, were associated with immunotherapy outcome. CD40 expression was higher in patient-responders than non responders, while CD95/Fas expression was lower in patients with partial response (PR) relative to those with stable disease (SD) and progressive disease (PD). Furthermore, we found that high 4-1BB expression in the tumor compartment, but not in the stroma, was associated with better overall survival (OS) (HR= 0.28, p-adjusted= 0.040). Moreover, high CD40 expression in tumor regions (HR= 0.27, p-adjusted= 0.035), and high CD27 expression in the stroma (HR= 0.2, p-adjusted=0.032) were associated with better survival outcomes. Taken together, this study supports the role of immune checkpoint molecules and implicates the TNFR superfamily as key players in immunotherapy response in our cohort of HNSCC. Validation of these findings in a prospective study is required to determine the robustness of these tissue signatures., Competing Interests: NM, BC, NJ, and OB are employees of Akoya Biosciences. BH, advisory board member of Merck Sharpe and Dohme, Bristol Myers Squibb, Astra Zeneca, Pfizer, Takeda, Sanofi, Eisai. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sadeghirad, Liu, Monkman, Ma, Cheikh, Jhaveri, Tan, Warkiani, Adams, Nguyen, Ladwa, Braubach, O’Byrne, Davis, Hughes and Kulasinghe.)

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.

Sperm selection is an essential component of all assisted reproductive treatments (ARTs) and is by far the most neglected step in the ART workflow in regard to technological innovation. Conventional sperm selection methodologies typically produce a higher total number of sperm with variable motilities, morphologies, and levels of DNA integrity. Gold-standard techniques, including density gradient centrifugation (DGC) and swim-up (SU), have been shown to induce DNA fragmentation through introducing reactive oxygen species (ROS) during centrifugation. Here, we demonstrate a 3D printed, biologically inspired microfluidic sperm selection device (MSSP) that utilizes multiple methods to simulate a sperms journey toward selection. Sperm are first selected based on their motility and boundary-following behavior and then on their expression of apoptotic markers, yielding over 68% more motile sperm than that of previously reported methods with a lower incidence of DNA fragmentation and apoptosis. Sperm from the MSSP also demonstrated higher motile sperm recovery after cryopreservation than that of SU or neat semen. Experiments were conducted side-by-side against conventional SU methods using human semen ( n  = 33) and showed over an 85% improvement in DNA integrity with an average 90% reduction in sperm apoptosis. These results that the platform is easy-to-use for sperm selection and mimics the biological function of the female reproductive tract during conception., Competing Interests: Conflict of interestThe authors declare no competing interests., (© The Author(s) 2023.)

Effective isolation and in-depth analysis of Circulating Tumour Cells (CTCs) are greatly needed in diagnosis, prognosis and monitoring of the therapeutic response of cancer patients but have not been completely fulfilled by conventional approaches. The rarity of CTCs and the lack of reliable biomarkers to distinguish them from peripheral blood cells have remained outstanding challenges for their clinical implementation. Herein, we developed a high throughput Static Droplet Microfluidic (SDM) device with 38,400 chambers, capable of isolating and classifying the number of metabolically active CTCs in peripheral blood at single-cell resolution. Owing to the miniaturisation and compartmentalisation capability of our device, we first demonstrated the ability to precisely measure the lactate production of different types of cancer cells inside 125 pL droplets at single-cell resolution. Furthermore, we compared the metabolomic activity of leukocytes from healthy donors to cancer cells and showed the ability to differentiate them. To further prove the clinical relevance, we spiked cancer cell lines in human healthy blood and showed the possibility to detect the cancer cells from leukocytes. Lastly, we tested the workflow on 8 preclinical mammary mouse models including syngeneic 67NR (non-metastatic) and 4T1.2 (metastatic) models with Triple-Negative Breast Cancer (TNBC) as well as transgenic mouses (12-week-old MMTV-PyMT). The results have shown the ability to precisely distinguish metabolically active CTCs from the blood using the proposed SDM device. The workflow is simple and robust which can eliminate the need for specialised equipment and expertise required for single-cell analysis of CTCs and facilitate on-site metabolic screening of cancer cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2022. Published by Elsevier B.V. All rights reserved.)

Catalytic DNAzymes have been used for isothermal amplification and rapid detection of nucleic acids, holding the potential for point-of-care testing applications. However, when Subzymes (universal substrate and DNAzyme) are tethered to the polystyrene magnetic microparticles via biotin-streptavidin bonds, the residual free Subzymes are often detached from the microparticle surface, which causes a significant degree of false positives. Here, we attached dithiol-modified Subzyme to gold nanoparticle and improved the limit of detection (LoD) by 200 times compared to that using magnetic microparticles. As a proof of concept, we applied our new method for the detection of exosomal programed cell-death ligand 1 (PD-L1) RNA. As the classical immune checkpoint, molecule PD-L1, found in small extracellular vesicles (sEVs, traditionally called exosomes), can reflect the antitumor immune response for predicting immunotherapy response. We achieved the LoD as low as 50 fM in detecting both the RNA homologous to the PD-L1 gene and exosomal PD-L1 RNAs extracted from epithelioid and nonepithelioid subtypes of mesothelioma cell lines, which only takes 8 min of reaction time. As the first application of isothermal DNAzymes for detecting exosomal PD-L1 RNA, this work suggests new point-of-care testing potentials toward clinical translations.

The emergence of enabling technologies for the analysis of circulating tumor cells has been shedding new lights into cancer management in the recent years. However, majority of the technologies developed suffer from excessive cost, time-consuming workflows, and reliance on specialized equipment and operators. Herein, we propose a simple workflow for the isolation and characterization of single circulating tumor cells using microfluidic devices. The entire process can be operated by a laboratory technician without relying on any microfluidic expertise and can be completed within few hours of sample collection., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Nutraceuticals have emerged as potential compounds to attenuate the COVID-19 complications. Precisely, these food additives strengthen the overall COVID treatment and enhance the immunity of a person. Such compounds have been used at a large scale, in almost every household due to their better affordability and easy access. Therefore, current research is focused on developing newer advanced formulations from potential drug candidates including nutraceuticals with desirable properties viz, affordability, ease of availability, ease of administration, stability under room temperature, and potentially longer shelf-lives. As such, various nutraceutical-based products such as compounds could be promising agents for effectively managing COVID-19 symptoms and complications. Most importantly, regular consumption of such nutraceuticals has been shown to boost the immune system and prevent viral infections. Nutraceuticals such as vitamins, amino acids, flavonoids like curcumin, and probiotics have been studied for their role in the prevention of COVID-19 symptoms such as fever, pain, malaise, and dry cough. In this review, we have critically reviewed the potential of various nutraceutical-based therapeutics for the management of COVID-19. We searched the information relevant to our topic from search engines such as PubMed and Scopus using COVID-19, nutraceuticals, probiotics, and vitamins as a keyword. Any scientific literature published in a language other than English was excluded. PRACTICAL APPLICATIONS: Nutraceuticals possess both nutritional values and medicinal properties. They can aid in the prevention and treatment of diseases, as well as promote physical health and the immune system, normalizing body functions, and improving longevity. Recently, nutraceuticals such as probiotics, vitamins, polyunsaturated fatty acids, trace minerals, and medicinal plants have attracted considerable attention and are widely regarded as potential alternatives to current therapeutic options for the effective management of various diseases, including COVID-19., (© 2022 The Authors. Journal of Food Biochemistry published by Wiley Periodicals LLC.)

With the global burden of respiratory diseases, rapid identification of the best therapeutic measures to combat these diseases is essential. Animal models and 2D cell culture models do not replicate the findings observed in vivo. To gain deeper insight into lung pathology and physiology, 3D and advanced lung-on-a-chip models have been developed recently. Lung-on-a-chip models more accurately simulate the lung's microenvironment and functions in vivo, resulting in more-accurate assessments of drug safety and effectiveness. This review discusses the transition from 2D to 3D models and the recent advances in lung-on-a-chip platforms, their implementation and the numerous challenges faced. Finally, a general overview of this platform and its potential applications in respiratory disease research and drug discovery is highlighted., Competing Interests: Conflicts of Interest There are no conflicts of interest to declare., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Multimodal analysis of circulating tumour cells (CTCs) has the potential to provide remarkable insight for cancer development and metastasis. CTCs and CTC clusters investigation using single-cell analysis, enables researchers to gain crucial information on metastatic mechanisms and the genomic alterations responsible for drug resistance, empowering treatment, and management of cancer. Despite a plethora of CTC isolation technologies, careful attention to the strengths and weaknesses of each method should be considered in order to isolate these rare cells. Here, we provide an overview of cutting-edge technologies used for single-cell isolation and analysis of CTCs. Additionally, we highlight the biological features, clinical application, and the therapeutic potential of CTCs and CTC clusters using single-cell analysis platforms for cancer management., Competing Interests: Declaration of interests N.R. is an employee and stockholder of Fluidigm Corporation. E.S.Y. is an employee and stockholder of NomoCan Corporation. All other authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Local recurrence and metastasis remain the major causes of death in head and neck cancer (HNC) patients. Circulating tumour cells (CTCs) are shed from primary and metastatic sites into the circulation system and have been reported to play critical roles in the metastasis and recurrence of HNC. Here, we explored the use of CTCs to predict the response to treatment and disease progression in HNC patients., Methods: Blood samples were collected at diagnosis from HNC patients (n = 119). CTCs were isolated using a spiral microfluidic device and were identified using immunofluorescence staining. Correlation of baseline CTC numbers to 13-week PET-CT data and multidisciplinary team consensus data were conducted., Results: CTCs were detected in 60/119 (50.4%) of treatment naïve HNC patients at diagnosis. Baseline CTC numbers were higher in stage III vs. stage I-II p16-positive oropharyngeal cancers (OPCs) and other HNCs (p = 0.0143 and 0.032, respectively). In addition, we found that baseline CTC numbers may serve as independent predictors of treatment response, even after adjusting for other conventional prognostic factors. CTCs were detected in 10 out of 11 patients exhibiting incomplete treatment responses., Conclusions: We found that baseline CTC numbers are correlated with treatment response in patients with HNC. The expression level of cell-surface vimentin (CSV) on CTCs was significantly higher in patients with persistent or progressive disease, thus providing additional prognostic information for stratifying the risk at diagnosis in HNC patients. The ability to detect CTCs at diagnosis allows more accurate risk stratification, which in the future may be translated into better patient selection for treatment intensification and/or de-intensification strategies., (© 2022. The Author(s).)

Background: Non-small cell lung cancer (NSCLC) often presents at an incurable stage, and majority of patients will be considered for palliative treatment at some point in their disease. Despite recent advances, the prognosis remains poor, with a median overall survival of 12-18 months. Liquid biopsy-based biomarkers have emerged as potential candidates for predicting prognosis and response to therapy in NSCLC patients. This pilot study evaluated whether combining circulating tumour cells and clusters (CTCs) and cell-free DNA (cfDNA) can predict progression-free survival (PFS) in NSCLC patients., Methods: CTC and cfDNA/ctDNA from advanced stage NSCLC patients were measured at study entry (T 0 ) and 3-months post-treatment (T 1 ). CTCs were enriched using a spiral microfluidic chip and characterised by immunofluorescence. ctDNA was assessed using an UltraSEEK® Lung Panel. Kaplan-Meier plots were generated to investigate the contribution of the presence of CTC/CTC clusters and cfDNA for PFS. Cox proportional hazards analysis compared time to progression versus CTC/CTC cluster counts and cfDNA levels., Results: Single CTCs were found in 14 out of 25 patients, while CTC clusters were found in 8 out of the 25 patients at T 0 . At T 1 , CTCs were found in 7 out of 18 patients, and CTC clusters in 1 out of the 18 patients. At T 0 , CTC presence and the combination of CTC cluster counts with cfDNA levels were associated with shorter PFS, p = 0.0261, p = 0.0022, respectively., Conclusions: Combining CTC cluster counts and cfDNA levels could improve PFS assessment in NSCLC patients. Our results encourage further investigation on the combined effect of CTC/cfDNA as a prognostic biomarker in a large cohort of advanced stage NSCLC patients., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors.)

Head and neck squamous cell carcinoma (HNSCC) represents a heterogeneous group of tumors. While significant progress has been made using multimodal treatment, the 5-year survival remains at 50%. Developing effective therapies, such as immunotherapy, will likely lead to better treatment of primary and metastatic disease. However, not all HNSCC tumors respond to immune checkpoint blockade therapy. Understanding the complex cellular composition and interactions of the tumor microenvironment is likely to lead to new knowledge for effective therapies and treatment resistance. In this review, we discuss HNSCC characteristics, predictive biomarkers, factors influencing immunotherapy response, with a focus on the tumor microenvironment., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Cigarette smoke is considered a primary risk factor for chronic obstructive pulmonary disease. Numerous toxicants present in cigarette smoke are known to induce oxidative stress and airway inflammation that further exacerbate disease progression. Generally, the broncho-epithelial cells and alveolar macrophages exposed to cigarette smoke release massive amounts of oxidative stress and inflammation mediators. Chronic exposure of cigarette smoke leads to premature senescence of airway epithelial cells. This impairs cellular function and ultimately leads to the progression of chronic lung diseases. Therefore, an ideal therapeutic candidate should prevent disease progression by controlling oxidative stress, inflammation, and senescence during the initial stage of damage. In our study, we explored if berberine (an alkaloid)-loaded liquid crystalline nanoparticles (berberine-LCNs)-based treatment to human broncho-epithelial cells and macrophage inhibits oxidative stress, inflammation, and senescence induced by cigarette-smoke extract. The developed berberine-LCNs were found to have favourable physiochemical parameters, such as high entrapment efficiency and sustained in vitro release. The cellular-assay observations revealed that berberine-LCNs showed potent antioxidant activity by suppressing the generation of reactive oxygen species in both broncho-epithelial cells (16HBE) and macrophages (RAW264.7), and modulating the genes involved in inflammation and oxidative stress. Similarly, in 16HBE cells, berberine-LCNs inhibited the cigarette smoke-induced senescence as revealed by X-gal staining, gene expression of CDKN1A (p21), and immunofluorescent staining of p21. Further in-depth mechanistic investigations into antioxidative, anti-inflammatory, and antisenescence research will diversify the current findings of berberine as a promising therapeutic approach for inflammatory lung diseases caused by cigarette smoking.

In order to regenerate myocardial tissues with functional characteristics, we need to copy some properties of the myocardium, such as its extracellular matrix and electrical conductivity. In this study, we synthesized nanosheets of Molybdenum disulfide (MoS 2 ), and integrated them into polycaprolactone (PCL) and electrospun on the surface of decellularized human amniotic membrane (DHAM) with the purpose of improving the scaffolds mechanical properties and electrical conductivity. For in vitro studies, we seeded the mouse embryonic cardiac cells, mouse Embryonic Cardiac Cells (mECCs), on the scaffolds and then studied the MoS 2 nanocomposites by scanning electron microscopy and Raman spectroscopy. In addition, we characterized the DHAM/PCL and DHAM/PCL-MoS 2 by SEM, transmission electron microscopy, water contact angle measurement, electrical conductivity, and tensile test. Besides, we confirmed the scaffolds are biocompatible by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT assay. Furthermore, by means of SEM images, it was shown that mECCs attached to the DHAM/PCL-MoS 2 scaffold have more cell aggregations and elongated morphology. Furthermore, through the Real-Time PCR and immunostaining studies, we found out cardiac genes were maturated and upregulated, and they also included GATA-4, c-TnT, NKX 2.5, and alpha-myosin heavy chain in cells cultured on DHAM/PCL-MoS 2 scaffold in comparison to DHAM/PCL and DHAM. Therefore, in terms of cardiac tissue engineering, DHAM nanofibrous scaffolds reinforced by PCL-MoS 2 can be suggested as a proper candidate.

Antibiotic resistance has become a threat to microbial therapies nowadays. The conventional approaches possess several limitations to combat microbial infections. Therefore, to overcome such complications, novel drug delivery systems have gained pharmaceutical scientists' interest. Significant findings have validated the effectiveness of novel drug delivery systems such as polymeric nanoparticles, liposomes, metallic nanoparticles, dendrimers, and lipid-based nanoparticles against severe microbial infections and combating antimicrobial resistance. This review article comprises the specific mechanism of antibiotic resistance development in bacteria. In addition, the manuscript incorporated the advanced nanotechnological approaches with their mechanisms, including interaction with the bacterial cell wall, inhibition of biofilm formations, activation of innate and adaptive host immune response, generation of reactive oxygen species, and induction of intracellular effect to fight against antibiotic resistance. A section of this article demonstrated the findings related to the development of delivery systems. Lastly, the role of microfluidics in fighting antimicrobial resistance has been discussed. Overall, this review article is an amalgamation of various strategies to study the role of novel approaches and their mechanism to fight against the resistance developed to the antimicrobial therapies.

Breast cancer (BC) is a leading cause of cancer death among women worldwide. To better understand and predict therapeutic response in BC patient developing a fast, low-cost, and reliable preclinical tumor from patient's tumor specimen is needed. Here, we describe the development of a preclinical model of BC through the generation and ex vivo culture of patient-derived organotypic tumor spheroids (PDOTS) in a 3D microfluidic device. Moreover, the real-time screening of conventional chemotherapy agents on cultured PDOTS is also described., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Harvesting adherent cells from microcarriers has become one of the major challenges of the downstream bioprocessing at large scale the current method has high maintenance and operation cost, which are the results of frequent clogging, due to cell lysing effect and microcarrier cake formation on membrane-based technology. These problems hugely impede the adaptation of microcarriers technologies in large-scale cell culture and hampered the supply of cells to the clinical need. Here, we describe two 3D printing-based methods to fabricate inertial microfluidic devices for separating adherent cells from microcarriers which overcome the above-mentioned limitations. The spiral devices are employed to separate mesenchymal stem cells from the microcarriers with 99% microcarrier removal rate and 77% cell recovery rate in one round of separation., (© 2021. Springer Science+Business Media, LLC.)

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.

In this study, the motion and distribution of droplets containing coronaviruses emitted by coughing of an infected person in front of a classroom (e.g., a teacher) were investigated using CFD. A 3D turbulence model was used to simulate the airflow in the classroom, and a Lagrangian particle trajectory analysis method was used to track the droplets. The numerical model was validated and was used to study the effects of ventilation airflow speeds of 3, 5, and 7 m/s on the dispersion of droplets of different sizes. In particular, the effect of installing transparent barriers in front of the seats on reducing the average droplet concentration was examined. The results showed that using the seat partitions for individuals can prevent the infection to a certain extent. An increase in the ventilation air velocity increased the droplets' velocities in the airflow direction, simultaneously reducing the trapping time of the droplets by solid barriers. As expected, in the absence of partitions, the closest seats to the infected person had the highest average droplet concentration (3.80 × 10 -8 kg/m 3 for the case of 3 m/s)., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The aim of this study was to assess the effects of pirfenidone (PFD) on promoting epithelial-mesenchymal-transition (EMT) and stemness features in breast carcinoma cells through targeting cancer-associated-fibroblasts (CAFs). Using The Cancer Genome Atlas (TCGA) database, we analyzed the association between stromal index, EMT, and stemness-related genes across 1084 breast cancer patients, identifying positive correlation between YAP1, EMT, and stemness genes in samples with a high-stromal index. We monitored carcinoma cell invasion and spheroid formation co-cultured with CAFs in a 3D microfluidic device, followed by exposing carcinoma cells, spheroids, and CAFs with PFD. We depicted a positive association between the high-stromal index and the expression of EMT and stemness genes. High YAP1 expression in samples correlated with more advanced EMT status and stromal index. Additionally, we found that CAFs promoted spheroid formation and induced the expression of YAP1, VIM, and CD44 in spheroids. Treatment with PFD reduced carcinoma cell migration and decreased the expression of these genes at the protein level. The cytokine profiling showed significant depletion of various EMT- and stemness-regulated cytokines, particularly IL8, CCL17, and TNF-beta. These data highlight the potential application of PFD on inhibiting EMT and stemness in carcinoma cells through the targeting of critical cytokines.

Non-small cell lung cancer (NSCLC) is one of the most common cancers globally and has a 5-year survival rate ~20%. Immunotherapies have demonstrated long-term and durable responses in NSCLC patients, although they appear to be effective in only a subset of patients. A more comprehensive understanding of the underlying tumour biology may contribute to identifying those patients likely to achieve optimal outcomes. Profiling the tumour microenvironment (TME) has shown to be beneficial in addressing fundamental tumour-immune cell interactions. Advances in multiplexing immunohistochemistry and molecular barcoding has led to recent advances in profiling genes and proteins in NSCLC. Here, we review the recent advancements in spatial profiling technologies for the analysis of NSCLC tissue samples to gain new insights and therapeutic options for NSCLC. The combination of spatial transcriptomics combined with advanced imaging is likely to lead to deep insights into NSCLC tissue biology, which can be a powerful tool to predict likelihood of response to therapy., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Epithelial-mesenchymal transition (EMT) and its reverse process, mesenchymal-epithelial transition (MET), are believed to play key roles in facilitating the metastatic cascade. Metastatic lesions often exhibit a similar epithelial-like state to that of the primary tumour, in particular, by forming carcinoma cell clusters via E-cadherin-mediated junctional complexes. However, the factors enabling mesenchymal-like micrometastatic cells to resume growth and reacquire an epithelial phenotype in the target organ microenvironment remain elusive. In this study, we developed a workflow using image-based cell profiling and machine learning to examine morphological, contextual and molecular states of individual breast carcinoma cells (MDA-MB-231). MDA-MB-231 heterogeneous response to the host organ microenvironment was modelled by substrates with controllable stiffness varying from 0.2kPa (soft tissues) to 64kPa (bone tissues). We identified 3 distinct morphological cell types (morphs) varying from compact round-shaped to flattened irregular-shaped cells with lamellipodia, predominantly populating 2-kPa and >16kPa substrates, respectively. These observations were accompanied by significant changes in E-cadherin and vimentin expression. Furthermore, we demonstrate that the bone-mimicking substrate (64kPa) induced multicellular cluster formation accompanied by E-cadherin cell surface localisation. MDA-MB-231 cells responded to different substrate stiffness by morphological adaptation, changes in proliferation rate and cytoskeleton markers, and cluster formation on bone-mimicking substrate. Our results suggest that the stiffest microenvironment can induce MET., Competing Interests: The authors have declared that no competing interests exist.

In-depth study of cellular heterogeneity of rare cells (e.g. circulating tumour cells (CTCs) and circulating foetal cells (CFCs)) is greatly needed in disease management but has never been completely explored due to the current technological limitations. We have developed a retrieval method for single-cell detection using a static droplet array (SDA) device through liquid segmentation with almost no sample loss. We explored the potential of using SDA for low sample input and retrieving the cells of interest using everyday laboratory equipment for downstream molecular analysis. This single-cell isolation and retrieval method is low-cost, rapid and provides a solution to the remaining challenge for single rare cell detection. The entire process takes less than 15 min, is easy to fabricate and allows for on-chip analysis of cells in nanolitre droplets and retrieval of desired droplets. To validate the applicability of our device and method, we mimicked detection of single CTCs by isolating and retrieving single cells and perform real-time PCR on their mRNA contents.

Glioblastoma (GBM) is the most common and aggressive type of tumour arising from the central nervous system. GBM remains an incurable disease despite advancement in therapies, with overall survival of approximately 15 months. Recent literature has highlighted that GBM releases tumoural content which crosses the blood-brain barrier (BBB) and is detected in patients' blood, such as circulating tumour cells (CTCs). CTCs carry tumour information and have shown promise as prognostic and predictive biomarkers in different cancer types. Currently, there is limited data for the clinical utility of CTCs in GBM. Here, we report the use of spiral microfluidic technology to isolate CTCs from whole blood of newly diagnosed GBM patients before and after surgery, followed by characterization for GFAP, cell-surface vimentin protein expression and EGFR amplification. CTCs were found in 13 out of 20 patients (9/20 before surgery and 11/19 after surgery). Patients with CTC counts equal to 0 after surgery had a significantly longer recurrence-free survival (p=0.0370). This is the first investigation using the spiral microfluidics technology for the enrichment of CTCs from GBM patients and these results support the use of this technology to better understand the clinical value of CTCs in the management of GBM in future studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Müller Bark, Kulasinghe, Hartel, Leo, Warkiani, Jeffree, Chua, Day and Punyadeera.)

The isolation of sperm cells from background cell populations and debris is an essential step in all assisted reproductive technologies. Conventional techniques for sperm recovery from testicular sperm extractions stagnate at the sample processing stage, where it can take several hours to identify viable sperm from a background of collateral cells such as white bloods cells (WBCs), red blood cells (RBCs), epithelial cells (ECs) and in some cases cancer cells. Manual identification of sperm from contaminating cells and debris is a tedious and time-consuming operation that can be suitably addressed through inertial microfluidics. Microfluidics has proven an effective technology for high-quality sperm selection based on motility. However, motility-based selection methods cannot cater for viable, non-motile sperm often present in testicular or epididymal sperm extractions and aspirations. This study demonstrates the use of a 3D printed inertial microfluidic device for the separation of sperm cells from a mixed suspension of WBCs, RBCs, ECs, and leukemic cancer cells. This technology presents a 36-fold time improvement for the recovery of sperm cells (> 96%) by separating sperm, RBCS, WBCs, ECs and cancer cells into tight bands in less than 5 min. Furthermore, microfluidic processing of sperm has no impact on sperm parameters; vitality, motility, morphology, or DNA fragmentation of sperm. Applying inertial microfluidics for non-motile sperm recovery can greatly improve the current processing procedure of testicular sperm extractions, simplifying the fertility outcomes for severe forms of male infertility that warrant the surgery.

Biphasic calcium phosphate ceramics (BCPs) have been extensively used as a bone graft in dental clinics to reconstruct lost bone in the jaw and peri-implant hard tissue due to their good bone conduction and similar chemical structure to the teeth and bone. However, BCPs are not inherently osteoinductive and need additional modification and treatment to enhance their osteoinductivity. The present study aims to develop an innovative strategy to improve the osteoinductivity of BCPs using unique features of zeolitic imidazolate framework-8 (ZIF8). In this method, commercial BCPs (Osteon II) were pre-coated with a zeolitic imidazolate framework-8/polydopamine/polyethyleneimine (ZIF8/PDA/PEI) layer to form a uniform and compact thin film of ZIF8 on the surface of BCPs. The surface morphology and chemical structure of ZIF8 modified Osteon II (ZIF8-Osteon) were confirmed using various analytical techniques such as XRD, FTIR, SEM, and EDX. We evaluated the effect of ZIF8 coating on cell attachment, growth, and osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADSCs). The results revealed that altering the surface chemistry and topography of Osteon II using ZIF8 can effectively promote cell attachment, proliferation, and bone regeneration in both in vitro and in vivo conditions. In conclusion, the method applied in this study is simple, low-cost, and time-efficient and can be used as a versatile approach for improving osteoinductivity and osteoconductivity of other types of alloplastic bone grafts., (Copyright © 2021. Published by Elsevier B.V.)

Microfluidics-based technologies for single-cell analysis are becoming increasingly important tools in biological studies. With the increasing sophistication of microfluidics, cellular barcoding techniques, and next-generation sequencing, a more detailed picture of cellular subtype is emerging. Unfortunately, the majority of the methods developed for single-cell analysis are high-throughput and not suitable for rare cell analysis as they require a high input cell number. Here, we report a low-cost and reproducible method for rare single-cell analysis using a highly hydrophobic surface and nanosized static droplets. Our method allows rapid and efficient on-chip single-cell lysis and subsequent collection of genetic materials in nanoliter droplets using a micromanipulator or a laboratory pipette before subsequent genetic analysis. We show precise isolation of single cancer cells with high purity using two different strategies (i- cytospin and ii- static droplet array) for subsequent RNA analysis using droplet digital polymerase chain reaction (PCR) and real-time PCR. Our highly controlled isolation method opens a new avenue for the study of subcellular functional mechanisms, enabling the identification of rare cells of potential functional or pathogenic consequence.

Cell co-culture serves as a standard method to study intercellular communication. However, random diffusion of signal molecules during co-culture may arouse crosstalk among different types of cells and hide directive signal-target responses. Here, a microfluidic chip is proposed to study unidirectional intercellular communication by spatially controlling the flow of the signal molecules. The chip contains two separated chambers connected by two channels where the culture media flows oppositely. A zigzag signal-blocking channel is designed to study the function of a specific signal. The chip is applied to study the unidirectional communication between tumor cells and stromal cells. It shows that the expression of α-smooth muscle actin (a marker of cancer-associated fibroblast (CAF)) of both MRC-5 fibroblasts and mesenchymal stem cells can be up-regulated only by the secreta from invasive MDA-MB-231 cells, but not from non-invasive MCF-7 cells. The proliferation of the tumor cells can be improved by the stromal cells. Moreover, transforming growth factor beta 1 is found as one of the main factors for CAF transformation via the signal-blocking function. The chip achieves unidirectional cell communication along X-axis, signal concentration gradient along Y-axis and 3D cell culture along Z-axis, which provides a useful tool for cell communication studies., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The recent years have been passed with significant progressions in the utilization of microfluidic technologies for cellular investigations. The aim of microfluidics is to mimic small-scale body environment with features like optical transparency. Microfluidics can screen and monitor different cell types during culture and study cell function in response to stimuli in a fully controlled environment. No matter how the microfluidic environment is similar to in vivo environment, it is not possible to fully investigate stem cells behavior in response to stimuli during cell proliferation and differentiation. Researchers have used stem cells in different fields from fundamental researches to clinical applications. Many cells in the body possess particular functions, but stem cells do not have a specific task and can turn into almost any type of cells. Stem cells are undifferentiated cells with the ability of changing into specific cells that can be essential for the body. Researchers and physicians are interested in stem cells to use them in testing the function of the body's systems and solving their complications. This review discusses the recent advances in utilizing microfluidic techniques for the analysis of stem cells, and mentions the advantages and disadvantages of using microfluidic technology for stem cell research., (Copyright © 2021 Aghlmandi et al.)

According to World Health Organization (WHO) reports about 70 million couples suffer from infertility all over the world. A lot of research groups are working on this issue and have made therapeutic approaches by integrating biology, medicine, genetics, chemistry, psychology, mechanic, and many other branches of science. However, these methods have their own pros and cons. Assisted Reproductive Technologies (ART) has appeared to solve infertility problems. In Vitro Fertilization (IVF), Intracytoplasmic Sperm Injection (ICSI), Intrauterine Insemination (IUI) are the most common and conventional technologies in this regard. There are at least two characteristics of microfluidics, mechanical and biochemical, which can be influential in the field of mammalian gamete and preimplantation embryo biology. These microfluidic characteristics can assist in basic biological studies on sperm, oocyte and preimplantation embryo structure, function and environment. Using microfluidics in sorting sperm, conducting different steps of oocyte selection and preparation, and transferring embryo by passing sub-microliter fluid through microchannels results in low cost and short time. The size and shape of microchannels and the volume of used fluid differs from non-human cells to human cells. The most progressions have been seen in animal models. Results suggest that microfluidic systems will lead to improved efficiencies in assisted reproduction., (Copyright © 2020. Published by Elsevier Inc.)

A hydrocyclone is a macroscale separation device employed in various industries, with many advantages, including high-throughput and low operational costs. Translating these advantages to microscale has been a challenge due to the microscale fabrication limitations that can be surmounted using 3D printing technology. Additionally, it is difficult to simulate the performance of real 3D-printed micro-hydrocyclones because of turbulent eddies and the deviations from the design due to printing resolution. To address these issues, we propose a new experimental method for the direct observation of particle motion in 3D printed micro-hydrocyclones. To do so, wax 3D printing and soft lithography were used in combination to construct a transparent micro-hydrocyclone in a single block of polydimethylsiloxane. A high-speed camera and fluorescent particles were employed to obtain clear in situ images and to confirm the presence of the vortex core. To showcase the use of this method, we demonstrate that a well-designed device can achieve a 95% separation efficiency for a sample containing a mixture of (desired) stem cells and (undesired) microcarriers. Overall, we hope that the proposed method for the direct visualization of particle trajectories in micro-hydrocyclones will serve as a tool, which can be leveraged to accelerate the development of micro-hydrocyclones for biomedical applications., (© 2020 Author(s).)

Application of microfluidic systems for the study of cellular behaviors has been a flourishing area of research in the past decade. In the process of probing cell biomechanics the passage of a cell through a narrow microchannel or a small pore has attracted much attention during the recent years. And the study of cellular deformability and transportability using these systems with enhanced resolution and accuracy has opened a new paradigm for high-throughput characterization of both healthy and diseased cell populations.Here we use the level-set method to explore the relationship between the transit time and mechanical properties of normal white blood cells (WBCs) and breast cancer epithelial cells (MCF7) under different microenvironmental parameters (i.e., pressure difference, cell size, effective cell surface tension, constriction size and taper angle) in a 2-D computational domain by considering the cell as a viscous drop. The novel biomechanical relations are obtained for each cell type by the Response Surface Method (RSM), relating microenvironmental parameters to the dimensionless entry time of the normal and cancer cells. Our results revealed that MCF7 cells show asignificantly different behavior (a bifurcating behavior when the pressure difference of inlet/outlet increases) in regards to the dimensionless entry time as a function of microchannel taper angle in comparison with the WBC. These results suggest that the microenvironmental parameters have a significant effect on the transportability of the cells and different cells have different behaviors in response to a specific microenvironmental parameter. Finally, it can be claimed that this method can be also utilized to distinguish between benign and cancerous cells or even to probe tumor heterogeneity toward high throughput cell cytometry., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Tumour tissue-based information is limited. Liquid biopsy can provide valuable real-time information through circulating tumour cells (CTCs). Profiling and expanding CTCs may provide avenues to study transient metastatic disease., Methods: Seventy non-small cell lung cancer (NSCLC) patients were recruited. CTCs were enriched using the spiral microfluidic chip and a RosetteSep™ using bloods from NSCLC patients. CTC cultures were carried out using the Clevers media under hypoxic conditions. CTCs were characterized using immunofluorescence and mutation-specific antibodies for samples with known mutation profiles. Exome sequencing was used to characterized CTC cultures., Results: CTCs (>2 cells) were detected in 38/70 (54.3%) of patients ranging from 0 to 385 CTCs per 7.5 mL blood. In 4/5 patients where primary tumours harboured an EGFR exon 19 deletion, this EGFR mutation was also captured in CTCs. ALK translocation was confirmed on CTCs from a patient harbouring an ALK-rearrangement in the primary tumour. Short term CTC cultures were successfully generated in 9/70 NSCLC patients. Whole exome sequencing (WES) confirmed the presence of somatic mutations in the CTC cultures with mutational signatures consistent with NSCLC., Conclusions: We were able to detect CTCs in >50% of NSCLC patients. NSCLC patients with >2 CTCs had a poor prognosis. The short-term CTC culture success rate was 12.9%. Further optimization of this culture methodology may provide a means by which to expand CTCs derived from NSCLC patient's bloods. CTC cultures allow for expansion of cells to a critical mass, allowing for functional characterization of CTCs with the goal of drug sensitivity testing and the creation of CTC cell lines., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tlcr-20-521). KOB serves as an unpaid editorial board member of Translational Lung Cancer Research. The other authors have no conflicts of interest to declare., (2020 Translational Lung Cancer Research. All rights reserved.)

The programmed cell death ligand 1 (PD-L1) protein has emerged as a predictive cancer biomarker and sensitivity to immune checkpoint blockade-based cancer immunotherapies. Current technologies for the detection of protein-based biomarkers, including the enzyme-linked immunosorbent assay (ELISA), have limitations such as low sensitivity and limit of detection (LOD) in addition to degradation of antibodies in exposure to environmental changes such as temperature and pH. To address these issues, we have proposed a metal-organic framework (MOF)-based ELISA for the detection of the PD-L1. A protective coating based on Zeolitic Imidazolate Framework 8 (ZIF-8) MOF thin film and polydopamine-polyethylenimine (PDA-PEI) was introduced on an ELISA plate for the improvement of antibody immobilization. Sensitivity and LOD of the resulting platform were compared with a conventional ELISA kit, and the bioactivity of the antibody in the proposed immunoassay was investigated in response to various pH and temperature values. The LOD and sensitivity of the MOF-based PD-L1 ELISA were 225 and 15.12 times higher, respectively, compared with those of the commercial ELISA kit. The antibody@ZIF-8/PDA-PEI was stable up to 55 °C and the pH range 5-10. The proposed platform can provide sensitive detection for target proteins, in addition to being resistant to elevated temperature and pH. The proposed MOF-based ELISA has significant potential for the clinical and diagnostic studies.

Various factors in the tumor microenvironment (TME) regulate the expression of PD-L1 in carcinoma cells. The cancer-associated fibroblasts (CAFs) play a crucial role in regulating and rewiring TME to enhance their immune suppressive function and to favor the invasion of the malignant cells. Tumor progression may be retarded by targeting CAFs in the TME. Various studies highlighted the ability of targeting CAF with pirfenidone (PFD), leading to increased efficacy of chemotherapy. However, its potential for the reduction of immune-suppression capacity of CAFs remains to be elusive. Here, we assessed the effect of PFD on the expression of PD-L1 on CAF cells. Besides migration inhibitory effects of PFD on CAFs, the expression level of PD-L1 reduced in CAFs after treatment with PFD. The downstream analysis of released cytokines from CAFs showed that PFD significantly dropped the secretion of CCL17 and TNF-β, where a positive association between PFD-targeted proteins and PD-L1 was observed. These data suggest that the treatment of CAF within TME through the PFD may reduce the acquisition of CAF-mediated invasive and immune-suppressive capacity of breast carcinoma cells., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The development of stable and effective iodine removal systems would be highly desirable in addressing environmental issues relevant to water contamination. In the present research, a novel iodine adsorbent was synthesized by self-polymerization of dopamine (PDA) onto inert polypropylene (PP) membrane. This PP/PDA membrane was thoroughly characterized and its susrface propeties was analyzed by various analytical techniques indcluding field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH), contact angle, and surface free energy measurement. The PP/PDA membranes were subsequently used for batchwise removal of iodine at different temperatures (25-70 °C), pH (2-7), and surface areas (1-10 cm 2 ) to understand the underlying adsorption phenomena and to estimate the membrane capacity for iodine uptake. The increase in temperature and pH both led to higher adsorption of iodine. The present approach showed a removal efficiency of over 75% for iodine using 10 cm 2  PP/PDA membrane (18.87 m 2  g -1 ) within 2 h at moderate temperatures (∼50 °C) and pH > 4, about 15 fold compared to the PP control membrane. The adsorption kinetics and isotherms were well fitted to the pseudo-second-order kinetic and Langmuir isotherm models (R 2  > 0.99). This adsorbent can be recycled and reused at least six times with stable iodine adsorption. These findings were attributed to the homogenous monolayer adsorption of the iodide on the surface due to the presence of catechol and amine groups in the PP/PDA membrane. This study proposes an efficient adsorbent for iodine removal., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cancer cells continuously secrete inflammatory biomolecules which play significant roles in disease progression and tumor metastasis toward secondary sites. Despite recent efforts to capture cancer cells' intercellular secretion heterogeneity using microfluidics, the challenges in operation of these systems as well as the complexity of designing a biosensing assay for long-term and real-time measurement of single cell secretions have become grand research barriers. Here, we present a new capillary-based microfluidic biosensing approach to easily and reliably capture ~500 single cells inside isolated dead-end nanoliter compartments using simple pipette injection, and quantify their individual secretion dynamics at the single cell resolution over a long period of culture (~16 h). We first present a detailed investigation of the fluid mechanics underlying the formation of nanoliter compartments in the microfluidic system. Based on the measurement of single cell capture efficiency, we employ a one-step FRET-based biosensor which monitors the single cancer cells' protease activity. The sensor reports the fluorescent signal as a product of amino acid chain cleavage and reduction in its quenching capability. Using the single cell protease secretion data, we identified modes of cell secretion dynamics in our cell sample. While most of the cells had low secretion levels, two other smaller and more aggressive secretion dynamics were cells with secretion modes that include sharp spikes or slow but progressive trend. The method presented here overcomes the difficulties associated with performing single cell secretion assays, enabling a feasible and reliable technique for high throughput measurement of metabolic activities in cancer cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

To date, a myriad of strategies has been suggested for targeting the chemical signaling of cancer cells. Also, biomechanical features are gaining much more attention. These features can be used as biomarkers which influence cancer progression. Current approaches on cancer treatment are mainly focused on changing the biochemical signaling of cancer cells, whereas less attention was devoted to their biomechanical properties. Herein, we propose targeting of cancer cell mechanics through the microenvironmental mechanical and chemical cues. As such, we examined the role of substrate stiffness as well as the effect of epidermal growth factor receptor (EGFR) blockade in the cell mechanics. As a mechanical stimulus, stiff and soft polydimethylsiloxane substrates were utilized, while as a chemical stimulus, EGFR blockade was considered. Thus, breast cancer cell lines, MCF7 and MDA-MB-231, were cultured among chemical and mechanical groups. The local elasticity of cancer cells was assessed by atomic force microscopy nanoindentation method. Furthermore, we evaluated the effect of mentioned mechanical and chemical treatments on the morphology, actin cytoskeleton structures, and cancer cell migration abilities. The stiffness and migration ability of cancer cells increased by substrate stiffening while Cetuximab treatment demonstrated an elevation in the elastic modulus of cells followed by a reduction in the migration ability. These findings indicate that cancer cell mechanics is modulated not only by the mechanical cues but also by the chemical ones through EGFR signaling pathway. Overall, our results illustrate that manipulation of cell mechanics allows for the possible modulation of tumor cell migration. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1569-1581, 2019., (© 2019 Wiley Periodicals, Inc.)

Curved microchannels allow controllable microparticle focusing, but a full understanding of particle behavior has been limited-even for simple rectangular and trapezoidal shapes. At present, most microfluidic particle separation literature is dedicated to adding "internal" complexity (via sheath flow or obstructions) to relatively simple cross-sectional channel shapes. We propose that, with sufficient understanding of particle behavior, an equally viable pathway for microparticle focusing could utilize complex "external" cross-sectional shapes. By investigating three novel, complex spiral microchannels, we have found that it is possible to passively focus (6, 10, and 13  μ m) microparticles in the middle of a convex channel. Also, we found that in concave and jagged channel designs, it is possible to create multiple, tight focusing bands. In addition to these performance benefits, we report an "additive rule" herein, which states that complex channels can be considered as multiple, independent, simple cross-sectional shapes. We show with experimental and numerical analysis that this new additive rule can accurately predict particle behavior in complex cross-sectional shaped channels and that it can help to extract general inertial focusing tendencies for suspended particles in curved channels. Overall, this work provides simple, yet reliable, guidelines for the design of advanced curved microchannel cross sections.

Inertial microfluidics represents a powerful new tool for accurately positioning cells and microparticles within fluids for a variety of biomedical, clinical, and industrial applications. In spite of enormous advancements in the science and design of these devices, particularly in curved microfluidic channels, contradictory experimental results have confounded researchers and limited progress. Thus, at present, a complete theory which describes the underlying physics is lacking. We propose that this bottleneck is due to one simple mistaken assumption-the locations of inflection points of the Dean velocity profile in curved microchannels are not fixed, but can actually shift with the flow rate. Herein, we propose that the dynamic distance ( δ ) between the real equilibrium positions and their nearest inflection points can clearly explain several (previously) unexplained phenomena in inertial microfluidic systems. More interestingly, we found that this parameter, δ , is a function of several geometric and operational parameters, all of which are investigated (in detail) here with a series of experiments and simulations of different spiral microchannels. This key piece of understanding is expected to open the door for researchers to develop new and more effective inertial microfluidic designs.

New tools for higher-resolution fetal genome analysis including microarray and next-generation sequencing have revolutionized prenatal screening. This article provides commentary on this rapidly advancing field and a future perspective emphasizing circulating fetal cell (CFC) utility. Despite the tremendous technological challenges associated with their reliable and cost-effective isolation from maternal blood, CFCs have a strong potential to bridge the gap between the diagnostic sensitivity of invasive procedures and the desirable noninvasive nature of cell-free fetal DNA (cffDNA). Considering the rapid advances in both rare cell isolation and low-input DNA analysis, we argue here that CFC-based noninvasive prenatal testing is poised to be implemented clinically in the near future., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Elasto-inertial microfluidics has drawn significant attention in recent years due to its enhanced capabilities compared to pure inertial systems in control of small microparticles. Previous investigations have focused mainly on the applications of elasto-inertial sorting, rather than studying its fundamentals. This is because of the complexity of simulation and analysis, due to the presence of viscoelastic force. There have been some investigative efforts on the mechanisms of elasto-inertial focusing in straight channels; however, these studies were limited to simple rectangular channels and neglected the effects of geometry and flow rates on focusing positions. Herein, for the first time, we experimentally and numerically explore the effects of elasticity accompanying channel cross-sectional geometry and sample flow rates on the focusing phenomenon in elasto-inertial systems. The results reveal that increasing the aspect ratio weakens the elastic force more than inertial force, causing a transition from one focusing position to two. In addition, they show that increasing the angle of a channel corner causes the elastic force to push the particles more efficiently toward the center over a larger area of the channel cross section. Following on from this, we proposed a new complex straight channel which demonstrates a tighter focusing band compared to other channel geometries. Finally, we focused Saccharomyces cerevisiae cells (3-5  μ m) in the complex channel to showcase its capability in focusing small-size particles. We believe that this research work improves the understanding of focusing mechanisms in viscoelastic solutions and provides useful insights into the design of elasto-inertial microfluidic devices.