In Situ Visualization of Dynamic Cellular Effects of Phospholipid Nanoparticles via High-Speed Scanning Ion Conductance Microscopy.

Phospholipid nanoparticles have been actively employed for numerous biomedical applications. A key factor in ensuring effective and safe applications of these nanomaterials is the regulation of their interactions with target cells, which is significantly dependent on an in-depth understanding of the nanoparticle-cell interactions. To date, most studies investigating these nano-bio interactions have been performed under static conditions and may lack crucial real-time information. It is, however, noteworthy that the nanoparticle-cell interactions are highly dynamic. Consequently, to gain a deeper insight into the cellular effects of phospholipid nanoparticles, real-time observation of cellular dynamics after nanoparticle introduction is necessary. Herein, a proof-of-concept in situ visualization of the dynamic cellular effects of sub-100 nm phospholipid nanoparticles using high-speed scanning ion conductance microscopy (HS-SICM) is reported. It is revealed that upon introduction into the cellular environment, within a short timescale of hundreds of seconds, phospholipid nanoparticles can selectively modulate the edge motility and surface roughness of healthy fibroblast and cancerous epithelial cells. Furthermore, the dynamic deformation profiles of these cells can be selectively altered in the presence of phospholipid nanoparticles. This work is anticipated to further shed light on the real-time nanoparticle-cell interactions for improved formulation of phospholipid nanoparticles for numerous bioapplications.

Addressing cellular heterogeneity in tumor and circulation for refined prognostication.

Despite pronounced genomic and transcriptomic heterogeneity in non-small-cell lung cancer (NSCLC) not only between tumors, but also within a tumor, validation of clinically relevant gene signatures for prognostication has relied upon single-tissue samples, including 2 commercially available multigene tests (MGTs). Here we report an unanticipated impact of intratumor heterogeneity (ITH) on risk prediction of recurrence in NSCLC, underscoring the need for a better genomic strategy to refine prognostication. By leveraging label-free, inertial-focusing microfluidic approaches in retrieving circulating tumor cells (CTCs) at single-cell resolution, we further identified specific gene signatures with distinct expression profiles in CTCs from patients with differing metastatic potential. Notably, a refined prognostic risk model that reconciles the level of ITH and CTC-derived gene expression data outperformed the initial classifier in predicting recurrence-free survival (RFS). We propose tailored approaches to providing reliable risk estimates while accounting for ITH-driven variance in NSCLC.

Personalized Treatment Through Detection and Monitoring of Genetic Aberrations in Single Circulating Tumor Cells.

Circulating tumor cells (CTCs) present a viable alternative to access tumor materials other than primary biopsies in cancer. This disease is among the most widespread in the world and is difficult to target because of its complex nature, challenges in getting quality samples and dynamic temporal changes in response to treatment. Conventional methods of detection and monitoring the disease profile do not suffice to be able to target the heterogeneity that exists at the cellular level. CTCs have been identified as a possible substitute for tumor tissue samples, and can be used to complement current disease management. Challenges in CTCs molecular analysis lie in the purity of the sample, which is masked by the presence of large quantities of white blood cells (WBCs) . In this chapter, we present a microfluidic biochip platform that performs secondary purification to isolate single CTCs efficiently. Studying single CTCs will allow for sensitive detection of critical mutations and addressing intercellular variances that will be otherwise missed easily due to low mutation frequencies when evaluating bulk cell retrieval. Using the biochip, we isolated single CTCs, and conducted personalized integrated EGFR mutational analysis using conventional polymerase chain reaction (PCR) and Sanger sequencing. We also demonstrated that high quality next generation sequencing (NGS) libraries can be readily generated from these samples. In our initial study, we revealed that the dominant EGFR mutations such as L858R and T790M could be detected in Non Small Cell Lung Cancer (NSCLC) patients with low CTC counts. We envision the biochip will enable efficient isolation of rare single cells from samples. This technology coupled with downstream molecular characterization of CTCs will aid in realizing the personalized medicine for cancer patients.

Exosomal mRNA Cargo are biomarkers of tumor and immune cell populations in pediatric osteosarcoma.

Osteosarcoma is the commonest malignant bone tumor of children and adolescents and is characterized by a high risk of recurrence despite multimodal therapy, especially in metastatic disease. This suggests the presence of clinically undetected cancer cells that persist, leading to cancer recurrence. We sought to evaluate the utility of peripheral blood exosomes as a more sensitive yet minimally invasive blood test that could aid in evaluating treatment response and surveillance for potential disease recurrence. We extracted exosomes from the blood of pediatric osteosarcoma patients at diagnosis (n=7) and after neoadjuvant chemotherapy (n=5 subset), as well as from age-matched cancer-free controls (n=3). We also obtained matched tumor biopsy samples (n=7) from the cases. Exosome isolation was verified by CD9 immunoblot and characterized on electron microscopy. Profiles of 780 cancer-related transcripts were analysed in mRNA from exosomes of osteosarcoma patients at diagnosis and control patients, matched post-chemotherapy samples, and matched primary tumor samples. Peripheral blood exosomes of osteosarcoma patients at diagnosis were significantly smaller than those of controls and overexpressed extracellular matrix protein gene THBS1 and B cell markers MS4A1 and TCL1A. Immunohistochemical staining of corresponding tumor samples verified the expression of THBS1 on tumor cells and osteoid matrix, and its persistence in a treatment-refractory patient, as well as the B cell origin of the latter. These hold potential as liquid biopsy biomarkers of disease burden and host immune response in osteosarcoma. Our findings suggest that exosomes may provide novel and clinically-important insights into the pathophysiology of cancers such as osteosarcoma.

Application of Stand-Alone Oblique Lateral Interbody Fusion in L4-5 Lumbar Diseases.

Lumbar spine diseases often cause lower back pain, lower extremity pain, numbness, and paresthesia. In severe cases, intermittent claudication may occur, affecting the quality of life of patients. Surgery is often required when conservative treatment fails, or when patients' symptoms become unbearable. Surgical treatments include laminectomy and discectomy, as well as interbody fusion. The main purpose of laminectomy and discectomy is to relieve nerve compression; however, recurrence is common due to spinal instability. Interbody fusion improves stability while relieving nerve compression and significantly reduces the risk of recurrence compared to non-fusion surgery. Nonetheless, conventionally posterior intervertebral fusion requires separation of the muscles to expose the operated segment, which causes more trauma to the patient. In contrast, the oblique lateral interbody fusion (OLIF) technique achieves spinal fusion with minimal trauma to the patients and shortens the recovery time. This article introduces procedures of stand-alone OLIF surgery performed in the lumbar spine, providing a reference for other spine surgeons.

Pro-metastatic and mesenchymal gene expression signatures characterize circulating tumor cells of neuroblastoma patients with bone marrow metastases and relapse.

Existing marker-based methods of minimal residual disease (MRD) determination in neuroblastoma do not effectively enrich for the circulating disease cell population. Given the relative size differential of neuroblastoma tumor cells over normal hematogenous cells, we hypothesized that cell size-based separation could enrich circulating tumor cells (CTCs) from blood samples and disseminated tumor cells (DTCs) from bone marrow aspirates (BMA) of neuroblastoma patients, and that their gene expression profiles could vary dynamically with various disease states over the course of treatment. Using a spiral microfluidic chip, peripheral blood of 17 neuroblastoma patients at 3 serial treatment timepoints (diagnosis, n=17; post-chemotherapy, n=11; and relapse, n=3), and bone marrow samples at diagnosis were enriched for large intact circulating cells. Profiling the resulting enriched samples with immunohistochemistry and mRNA expression of 1490 cancer-related genes via NanoString, 13 of 17 samples contained CTCs displaying cytologic atypia, TH and PHOX2B expression and/or upregulation of cancer-associated genes. Gene signatures reflecting pro-metastatic processes and the neuroblastoma mesenchymal super-enhancer state were consistently upregulated in 7 of 13 samples, 6 of which also had metastatic high-risk disease. Expression of 8 genes associated with PI3K and GCPR signaling were significantly upregulated in CTCs of patients with bone marrow metastases versus patients without. Correspondingly, in patients with marrow metastases, differentially-expressed gene signatures reflected upregulation of immune regulation in bone marrow DTCs versus paired CTCs samples. In patients who later developed disease relapse, 5 genes involved in immune cell regulation, JAK/STAT signaling and the neuroblastoma mesenchymal super-enhancer state (OLFML2B, STAT1, ARHGDIB, STAB1, TLR2) were upregulated in serial CTC samples over their disease course, despite urinary catecholamines and bone marrow aspirates not indicating the disease recurrences. In summary, using a label-free cell size-based separation method, we enriched and characterized intact circulating cells in peripheral blood indicative of neuroblastoma CTCs, as well as their DTC counterparts in the bone marrow. Expression profiles of pro-metastatic genes in CTCs correlated with the presence of bone marrow metastases at diagnosis, while longitudinal profiling identified persistently elevated expression of genes in CTCs that may serve as novel predictive markers of hematogenous MRD in neuroblastoma patients that subsequently relapse.

Differential Collective Cell Migratory Behaviors Modulated by Phospholipid Nanocarriers.

Phospholipid nanocarriers have been widely explored for theranostic and nanomedicine applications. These amphiphilic nanocarriers possess outstanding cargo encapsulation efficiency, high water dispersibility, and excellent biocompatibility, which render them promising for drug delivery and bioimaging applications. While the biological applications of phospholipid nanocarriers have been well documented, the fundamental aspects of the phospholipid-cell interactions beyond cytotoxicity have been less investigated. In particular, the effect of phospholipid nanocarriers on collective cell behaviors has not been elucidated. Herein, we evaluate the interactions of phospholipid nanocarriers possessing different functional groups and sizes with normal and cancerous immortalized breast epithelial cell sheets with varying metastatic potential. Specifically, we examine the impact of nanocarrier treatments on the collective migratory dynamics of these cell sheets. We observe that phospholipid nanocarriers induce differential collective cell migratory behaviors, where the migration speed of normal and cancerous breast epithelial cell sheets is retarded and accelerated, respectively. To a certain extent, the nanocarriers are able to alter the migration trajectory of the cancerous breast epithelial cells. Furthermore, phospholipid nanocarriers could modulate the stiffness of the nuclei, cytoplasm, and cell-cell junctions of the breast epithelial cell sheets, remodel their actin filament arrangement, and regulate the expressions of the actin-related proteins. We anticipate that this work will further shed light on nanomaterial-cell interactions and provide guidelines for rational and safer designs and applications of phospholipid nanocarriers for cancer theranostics and nanomedicine.

A flexible multiplexed immunosensor for point-of-care in situ wound monitoring.

Chronic wounds arise from interruption of normal healing due to many potential pathophysiological factors. Monitoring these multivariate factors can provide personalized diagnostic information for wound management, but current sensing technologies use complex laboratory tests or track a limited number of wound parameters. We report a flexible biosensing platform for multiplexed profiling of the wound microenvironment, inflammation, and infection state at the point of care. This platform integrates a sensor array for measuring inflammatory mediators [tumor necrosis factor-α, interleukin-6 (IL-6), IL-8, and transforming growth factor-ß1], microbial burden (Staphylococcus aureus), and physicochemical parameters (temperature and pH) with a microfluidic wound exudate collector and flexible electronics for wireless, smartphone-based data readout. We demonstrate in situ multiplexed monitoring in a mouse wound model and also profile wound exudates from patients with venous leg ulcers. This technology may facilitate more timely and personalized wound management to improve chronic wound healing outcomes.

Complementary Sequential Circulating Tumor Cell (CTC) and Cell-Free Tumor DNA (ctDNA) Profiling Reveals Metastatic Heterogeneity and Genomic Changes in Lung Cancer and Breast Cancer.

INTRODUCTION: Circulating tumor cells (CTCs) and cell-free tumor DNA (ctDNA) are tumor components present in circulation. Due to the limited access to both CTC enrichment platforms and ctDNA sequencing in most laboratories, they are rarely analyzed together. METHODS: Concurrent isolation of ctDNA and single CTCs were isolated from lung cancer and breast cancer patients using the combination of size-based and CD45-negative selection method via DropCell platform. We performed targeted amplicon sequencing to evaluate the genomic heterogeneity of CTCs and ctDNA in lung cancer and breast cancer patients. RESULTS: Higher degrees of genomic heterogeneity were observed in CTCs as compared to ctDNA. Several shared alterations present in CTCs and ctDNA were undetected in the primary tumor, highlighting the intra-tumoral heterogeneity of tumor components that were shed into systemic circulation. Accordingly, CTCs and ctDNA displayed higher degree of concordance with the metastatic tumor than the primary tumor. The alterations detected in circulation correlated with worse survival outcome for both lung and breast cancer patients emphasizing the impact of the metastatic phenotype. Notably, evolving genetic signatures were detected in the CTCs and ctDNA samples during the course of treatment and disease progression. CONCLUSIONS: A standardized sample processing and data analysis workflow for concurrent analysis of CTCs and ctDNA successfully dissected the heterogeneity of metastatic tumor in circulation as well as the progressive genomic changes that may potentially guide the selection of appropriate therapy against evolving tumor clonality.

Mechanistic Understanding of the Biological Responses to Polymeric Nanoparticles.

Polymeric nanoparticles play important roles in the delivery of a multitude of therapeutic and imaging contrast agents. Although these nanomaterials have shown tremendous potential in disease diagnosis and therapy, there have been many reports on the failure of these nanoparticles in realizing their intended objectives due to an individual or a combination of factors, which have collectively challenged the merit of nanomedicine for disease theranostics. Herein, we investigate the interactions of polymeric nanoparticles with biological entities from molecular to organism levels. Specifically, the protein corona formation, in vitro endothelial uptake, and in vivo circulation time of these nanoparticles are systematically probed. We identify the crucial role of nanocarrier lipophilicity, zeta-potential, and size in controlling the interactions between nanoparticles and biological systems and propose a two-step framework in formulating a single nanoparticle system to regulate multiple biological effects. This study provides insight into the rational design and optimization of the performance of polymeric nanoparticles to advance their theranostic and nanomedicine applications.

Nanomechanical Microfluidic Mixing and Rapid Labeling of Silica Nanoparticles using Allenamide-Thiol Covalent Linkage for Bioimaging.

Rapid surface functionalization of nanomaterials using covalent linkage following "green chemistry" remains challenging, and the quest for developing simple protocols is persisting. We report a nanomechanical microfluidic approach for the coupling of allenamide functionalized organic derivatives on the surface of thiol-modified silica nanoparticles using allenamide-thiol chemistry. The coupling principle involves the use of a microfluidic surface acoustic wave device that generates acoustic streaming-based chaotic fluid micromixing that enables mixing of laterally flowing fluids containing active components. This approach was used to demonstrate the direct surface labeling of thiol-modified silica nanoparticles using a selected group of modified fluorescent tags containing allenamide handles and achieved a total labeling efficiency of 83-90%. This green approach enabled a highly efficient surface functionalization under aqueous conditions, with tunable control over the conjugation process via the applied field. The dye-labeled silica particles were characterized using various analytical techniques and found to be biocompatible with potential in live cell bioimaging. It is envisaged that this bioconjugation strategy will find numerous applications in the field of bioimaging and drug delivery.

Microfluidics for cell sorting and single cell analysis from whole blood.

The complexity and dynamic evolution of cancer often result in tumor subpopulations containing distinctly heterogeneous cells. During metastasis, these also give rise to heterogeneous circulating tumor cells (CTCs) which are considered to be a hematogenous dissemination from the primary tumor. CTCs represent a viable less-invasive sampling opportunity, also known as liquid biopsy. However, current technological platforms that analyze entire CTC population are not effective due to cell-to-cell variability within the same population and this can manifest differences in genomic expression, cell cycle stages and eventually cellular responses to drug treatments. Here, we present a novel microfluidic approach that involves combination of two microfluidic chips operating under inertial fluid forces and hydrodynamic focusing to rapidly isolate and selectively retrieve bulk as well as single CTCs from whole blood for downstream single cell analysis. It is envisioned that this combinational approach to retrieve single CTCs can cater to several applications including more accurate disease diagnosis as well as formulation of personalized therapeutic strategies.

Highly Stretchable, Weavable, and Washable Piezoresistive Microfiber Sensors.

A key challenge in electronic textiles is to develop an intrinsically conductive thread of sufficient robustness and sensitivity. Here, we demonstrate an elastomeric functionalized microfiber sensor suitable for smart textile and wearable electronics. Unlike conventional conductive threads, our microfiber is highly flexible and stretchable up to 120% strain and possesses excellent piezoresistive characteristics. The microfiber is functionalized by enclosing a conductive liquid metallic alloy within the elastomeric microtube. This embodiment allows shape reconfigurability and robustness, while maintaining an excellent electrical conductivity of 3.27 ± 0.08 MS/m. By producing microfibers the size of cotton threads (160 µm in diameter), a plurality of stretchable tubular elastic piezoresistive microfibers may be woven seamlessly into a fabric to determine the force location and directionality. As a proof of concept, the conductive microfibers woven into a fabric glove were used to obtain physiological measurements from the wrist, elbow pit, and less accessible body parts, such as the neck and foot instep. Importantly, the elastomeric layer protects the sensing element from degradation. Experiments showed that our microfibers suffered minimal electrical drift even after repeated stretching and machine washing. These advantages highlight the unique propositions of our wearable electronics for flexible display, electronic textile, soft robotics, and consumer healthcare applications.

Microfluidic enrichment for the single cell analysis of circulating tumor cells.

Resistance to drug therapy is a major concern in cancer treatment. To probe clones resistant to chemotherapy, the current approach is to conduct pooled cell analysis. However, this can yield false negative outcomes, especially when we are analyzing a rare number of circulating tumor cells (CTCs) among an abundance of other cell types. Here, we develop a microfluidic device that is able to perform high throughput, selective picking and isolation of single CTC to 100% purity from a larger population of other cells. This microfluidic device can effectively separate the very rare CTCs from blood samples from as few as 1 in 20,000 white blood cells. We first demonstrate isolation of pure tumor cells from a mixed population and track variations of acquired T790M mutations before and after drug treatment using a model PC9 cell line. With clinical CTC samples, we then show that the isolated single CTCs are representative of dominant EGFR mutations such as T790M and L858R found in the primary tumor. With this single cell recovery device, we can potentially implement personalized treatment not only through detecting genetic aberrations at the single cell level, but also through tracking such changes during an anticancer therapy.