Spatial proteomic profiling of tumor and stromal compartments in non-small-cell lung cancer identifies signatures associated with overall survival.

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.

Enrichment of T-lymphocytes from leukemic blood using inertial microfluidics toward improved chimeric antigen receptor-T cell manufacturing.

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.

Zerumbone-incorporated liquid crystalline nanoparticles inhibit proliferation and migration of non-small-cell lung cancer in vitro.

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.

Zerumbone liquid crystalline nanoparticles protect against oxidative stress, inflammation and senescence induced by cigarette smoke extract in vitro.

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-1ß 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.

Scaling up stem cell production: harnessing the potential of microfluidic devices.

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.

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.

A Microfluidic Approach for Enrichment and Single-Cell Characterization of Circulating Tumor Cells from Peripheral Blood.

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.

Compartmentalized spatial profiling of the tumor microenvironment in head and neck squamous cell carcinoma identifies immune checkpoint molecules and tumor necrosis factor receptor superfamily members as biomarkers of response to immunotherapy.

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.

Advances in TEER measurements of biological barriers in microphysiological systems.

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.

Advanced models for respiratory disease and drug studies.

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.

Sperm quality metrics were improved by a biomimetic microfluidic selection platform compared to swim-up methods.

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.

Thiolate DNAzymes on Gold Nanoparticles for Isothermal Amplification and Detection of Mesothelioma-derived Exosomal PD-L1 mRNA.

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.

Rapid metabolomic screening of cancer cells via high-throughput static droplet microfluidics.

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.

Nutraceuticals and COVID-19: A mechanistic approach toward attenuating the disease complications.

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.

Prognostic value of integrating circulating tumour cells and cell-free DNA in non-small cell lung cancer.

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 (T0) and 3-months post-treatment (T1). 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 T0. At T1, CTCs were found in 7 out of 18 patients, and CTC clusters in 1 out of the 18 patients. At T0, 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.

Generation and Culture of Organotypic Breast Carcinoma Spheroids for the Study of Drug Response in a 3D Microfluidic Device.

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.

Understanding the tumor microenvironment in head and neck squamous cell carcinoma.

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.

Application of circulating tumour cells to predict response to treatment in head and neck cancer.

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.

Recent advances in lung-on-a-chip models.

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.