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Assessing the molecular profiles of bladder cancer (BC) from patients with locally advanced or metastatic disease provides valuable insights, such as identification of invasive markers, to guide personalized treatment. Currently, most molecular profiling of BC is based on highly invasive biopsy or transurethral tumor resection. Liquid biopsy takes advantage of less-invasive procedures to longitudinally profile disease. Circulating tumor cells (CTCs) isolated from blood are one of the key analytes of liquid biopsy. In this study, we developed a protein and mRNA co-analysis workflow for BC CTCs utilizing the graphene oxide (GO) microfluidic chip. The GO chip was conjugated with antibodies against both EpCAM and EGFR to isolate CTCs from 1 mL of blood drawn from BC patients. Following CTC capture, protein and mRNA were analyzed using immunofluorescent staining and ion-torrent-based whole transcriptome sequencing, respectively. Elevated CTC counts were significantly associated with patient disease status at the time of blood draw. We found a count greater than 2.5 CTCs per mL was associated with shorter overall survival. The invasive markers EGFR, HER2, CD31, and ADAM15 were detected in CTC subpopulations. Whole transcriptome sequencing showed distinct RNA expression profiles from patients with or without tumor burden at the time of blood draw. In patients with advanced metastatic disease, we found significant upregulation of metastasis-related and chemotherapy-resistant genes. This methodology demonstrates the capability of GO chip-based assays to identify tumor-related RNA signatures, highlighting the prognostic potential of CTCs in metastatic BC patients., 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 © 2024. Published by Elsevier Inc.)

3D structures are crucial to biological function in the human body, driving interest in their in vitro fabrication. Advances in shape-morphing materials allow the assembly of 3D functional materials with the ability to modulate the architecture, flexibility, functionality, and other properties of the final product that suit the desired application. The principles of these techniques correspond to the principles of origami and kirigami, which enable the transformation of planar materials into 3D structures by folding, cutting, and twisting the 2D structure. In these approaches, materials responding to a certain stimulus will be used to manufacture a preliminary structure. Upon applying the stimuli, the architecture changes, which could be considered the fourth dimension in the manufacturing process. Here, we briefly summarize manufacturing techniques, such as lithography and 3D printing, that can be used in fabricating complex structures based on the aforementioned principles. We then discuss the common architectures that have been developed using these methods, which include but are not limited to gripping, rolling, and folding structures. Then, we describe the biomedical applications of these structures, such as sensors, scaffolds, and minimally invasive medical devices. Finally, we discuss challenges and future directions in using shape-morphing materials to develop biomimetic and bioinspired designs., 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 © 2024 Mirzababaei, Towery and Kozminsky.)

The COVID-19 pandemic has revealed how an emerging pathogen can cause a sudden and dramatic increase in demand for viral testing. Testing pooled samples could meet this demand; however, the sensitivity of reverse transcription quantitative polymerase chain reaction (RT-qPCR), the gold standard, significantly decreases with an increasing number of samples pooled. Here, we introduce detection of intact virus by exogenous-nucleotide reaction (DIVER), a method that quantifies intact virus and is robust to sample dilution. As demonstrated using two models of severe acute respiratory syndrome coronavirus 2, DIVER first tags membraned particles with exogenous oligonucleotides, then captures the tagged particles on beads functionalized with a virus-specific capture agent (in this instance, angiotensin-converting enzyme 2), and finally quantifies the oligonucleotide tags using qPCR. Using spike-presenting liposomes and spike-pseudotyped lentivirus, we show that DIVER can detect 1 × 10 5 liposomes and 100 plaque-forming units of lentivirus and can successfully identify positive samples in pooling experiments. Overall, DIVER is well positioned for efficient sample pooling and clinical validation.

Lipid-based nanoparticles have been applied extensively in drug delivery and vaccine strategies and are finding diverse applications in the coronavirus disease 2019 (COVID-19) pandemic-from vaccine-component encapsulation to modeling the virus, itself. High-throughput, highly flexible methods for characterization are of great benefit to the development of liposomes featuring surface proteins. DNA-directed patterning is one such method that offers versatility in immobilizing and segregating lipid-based nanoparticles for subsequent analysis. Here, oligonucleotides are selectively conjugated onto a glass substrate and then hybridized to complementary oligonucleotides tagged to liposomes, patterning them with great control and precision. The power of DNA-directed patterning is demonstrated by characterizing a novel recapitulative lipid-based nanoparticle model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-S-liposomes-that presents the SARS-CoV-2 spike (S) protein on its surface. Patterning a mixture of S-liposomes and liposomes that display the tetraspanin CD63 to discrete regions of a substrate shows that angiotensin-converting enzyme 2 (ACE2) specifically binds to S-liposomes. Subsequent introduction of S-liposomes to ACE2-expressing cells tests the biological function of S-liposomes and shows agreement with DNA-directed patterning-based assays. Finally, multiplexed patterning of S-liposomes verifies the performance of commercially available neutralizing antibodies against the two S variants. Overall, DNA-directed patterning enables a wide variety of custom assays for the characterization of any lipid-based nanoparticle., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Antibodies provide the functional biospecificity that has enabled the development of sensors, diagnostic tools, and assays in both laboratory and clinical settings. However, as multimarker screening becomes increasingly necessary due to the heterogeneity and complexity of human pathology, new methods must be developed that are capable of coordinating the precise assembly of multiple, distinct antibodies. To address this technological challenge, we engineered a bottom-up, high-throughput method in which DNA patterns, comprising unique 20-base pair oligonucleotides, are patterned onto a substrate using photolithography. These microfabricated surface patterns are programmed to hybridize with, and instruct the multiplexed assembly of, antibodies conjugated with the complementary DNA strands. We demonstrate that this simple, yet robust, approach preserves the antibody-binding functionality in two common applications: antibody-based cell capture and label-free surface marker screening. Using a simple proof-of-concept capture device, we achieved high purity separation of a breast cancer cell line, MCF-7, from a blood cell line, Jurkat, with capture purities of 77.4% and 96.6% when using antibodies specific for the respective cell types. We also show that antigen-antibody interactions slow cell trajectories in flow in the next-generation microfluidic node-pore sensing (NPS) device, enabling the differentiation of MCF-7 and Jurkat cells based on EpCAM surface-marker expression. Finally, we use a next-generation NPS device patterned with antibodies against E-cadherin, N-cadherin, and β-integrin-three markers that are associated with epithelial-mesenchymal transitions-to perform label-free surface marker screening of MCF10A, MCF-7, and Hs 578T breast epithelial cells. Our high-throughput, highly versatile technique enables rapid development of customized, antibody-based assays across a host of diverse diseases and research thrusts.

Cancer is the second leading cause of death worldwide. Despite the immense research focused in this area, one is still not able to predict disease trajectory. To overcome shortcomings in cancer disease study and monitoring, we describe an exciting research direction: cellular mechanophenotyping. Cancer cells must overcome many challenges involving external forces from neighboring cells, the extracellular matrix, and the vasculature to survive and thrive. Identifying and understanding their mechanical behavior in response to these forces would advance our understanding of cancer. Moreover, used alongside traditional methods of immunostaining and genetic analysis, mechanophenotyping could provide a comprehensive view of a heterogeneous tumor. In this perspective, we focus on new technologies that enable single-cell mechanophenotyping. Single-cell analysis is vitally important, as mechanical stimuli from the environment may obscure the inherent mechanical properties of a cell that can change over time. Moreover, bulk studies mask the heterogeneity in mechanical properties of single cells, especially those rare subpopulations that aggressively lead to cancer progression or therapeutic resistance. The technologies on which we focus include atomic force microscopy, suspended microchannel resonators, hydrodynamic and optical stretching, and mechano-node pore sensing. These technologies are poised to contribute to our understanding of disease progression as well as present clinical opportunities., (Copyright © 2020 Author(s).)

Elucidating how the spatial organization of extrinsic signals modulates cell behavior and drives biological processes remains largely unexplored because of challenges in controlling spatial patterning of multiple microenvironmental cues in vitro. Here, we describe a high-throughput method that directs simultaneous assembly of multiple cell types and solid-phase ligands across length scales within minutes. Our method involves lithographically defining hierarchical patterns of unique DNA oligonucleotides to which complementary strands, attached to cells and ligands-of-interest, hybridize. Highlighting our method's power, we investigated how the spatial presentation of self-renewal ligand fibroblast growth factor-2 (FGF-2) and differentiation signal ephrin-B2 instruct single adult neural stem cell (NSC) fate. We found that NSCs have a strong spatial bias toward FGF-2 and identified an unexpected subpopulation exhibiting high neuronal differentiation despite spatially occupying patterned FGF-2 regions. Overall, our broadly applicable, DNA-directed approach enables mechanistic insight into how tissues encode regulatory information through the spatial presentation of heterogeneous signals., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

In metastatic cancer, the secondary microenvironment consists of numerous cell types, each signaling with, and potentially supporting, the disseminated tumor cell. However, in vitro models have thus far been limited in their complexity, ultimately hindering study. To overcome this, we report the optimization and application of a high-throughput method, DNA-directed patterning, to pattern different cell types from the bone marrow microenvironment for the study of prostate cancer proliferation within this environment. We show that cells in our patterned microenvironment maintain their phenotype and behavior. Moreover, we demonstrate the successful introduction of prostate cancer cells in our microenvironment to investigate dormancy.

The enumeration of circulating tumor cells (CTCs) has shown prognostic importance in patients with breast cancer. However, CTCs are highly heterogeneous with diverse functional properties, which may also be clinically relevant. To provide a comprehensive landscape of the patient's disease, further CTC analysis is required. Here, a highly sensitive and reproducible graphene oxide based CTC assay is utilized to isolate and characterize CTCs from 47 metastatic breast cancer patients. The CTCs are captured with high purity, requiring only a few milliliters of blood and enabling efficient enumeration and subsequent analysis at both the protein and the transcription level. The results show that patient clinical outcomes correlate with the associated CTC profile and clearly demonstrate the potential use of the assay in the clinical setting. Collectively, these findings suggest that beyond simple enumeration, CTC characterization may provide further information that improves the diagnosis of the patients' disease status for proper treatment decisions. Moreover, this thorough validation study will facilitate the translation of the CTC assay into future clinical applications to broaden the utility of liquid biopsy., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rates of progression and treatment response in advanced prostate cancer are highly variable, necessitating non-invasive methods to assess the molecular characteristics of these tumors in real time. The unique potential of circulating tumor cells (CTCs) to serve as a clinically useful liquid biomarker is due to their ability to inform via both enumeration and RNA expression. A microfluidic graphene oxide-based device (GO Chip) is used to isolate CTCs and CTC clusters from the whole blood of 41 men with metastatic castration-resistant prostate cancer. Additionally, the expression of 96 genes of interest is determined by RT-qPCR. Multivariate analyses are conducted to determine the genes most closely associated with overall survival, PSA progression, and radioclinical progression. A preliminary signature, comprising high expression of stemness genes and low expression of epithelial and mesenchymal genes, potentially implicates an undifferentiated CTC phenotype as a marker of poor prognosis in this setting.

Activation of the EGF receptors EGFR (ErbB1) and HER2 (ErbB2) drives the progression of multiple cancer types through complex mechanisms that are still not fully understood. In this study, we report that HER2 expression is elevated in bone metastases of prostate cancer independently of gene amplification. An examination of HER2 and NF-κB receptor (RANK) coexpression revealed increased levels of both proteins in aggressive prostate tumors and metastatic deposits. Inhibiting HER2 expression in bone tumor xenografts reduced proliferation and RANK expression while maintaining EGFR expression. In examining the role of EGFR in tumor-initiating cells (TIC), we found that EGFR expression was required for primary and secondary sphere formation of prostate cancer cells. EGFR expression was also observed in circulating tumor cells (CTC) during prostate cancer metastasis. Dual inhibition of HER2 and EGFR resulted in significant inhibition of tumor xenograft growth, further supporting the significance of these receptors in prostate cancer progression. Overall, our results indicate that EGFR promotes survival of prostate TIC and CTC that metastasize to bone, whereas HER2 supports the growth of prostate cancer cells once they are established at metastatic sites. Cancer Res; 77(1); 74-85. ©2016 AACR., Competing Interests: The authors disclose no potential conflicts of interest., (©2016 American Association for Cancer Research.)

A highly sensitive microfluidic system to capture circulating tumor cells from whole blood of cancer patients is presented. The device incorporates graphene oxide into a thermoresponsive polymer film to serve as the first step of an antibody functionalization chemistry. By decreasing the temperature, captured cells may be released for subsequent analysis., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The second leading cause of death in the United States, cancer is at its most dangerous as it spreads to secondary locations. Cancer cells in the blood stream, or circulating tumor cells (CTCs), present an opportunity to study metastasis provided they may be extracted successfully from blood. Engineers have accelerated the development of technologies that achieve this goal based on exploiting differences between tumor cells and surrounding blood cells such as varying expression patterns of membrane proteins or physical characteristics. Collaboration with biologists and clinicians has allowed additional analysis and will lead to the use of these rare cells to their full potential in the fight against cancer.

A high-throughput approach which automates the synthesis of polyelectrolyte-based layer-by-layer films (HT-LbL) to facilitate rapid film generation, systematic film characterization, and rational investigations into their interactions with cells is described. Key parameters, such as polyelectrolyte adsorption time and polyelectrolyte deposition pH, were used to modulate LbL film growth to create LbL films of distinct thicknesses using the widely utilized polyelectrolytes poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA). We highlight how HT-LbL can be used to rapidly characterize film-forming parameters and robustly create linearly growing films of various molecular architectures. Film thickness and growth rates of HT-LbL films were shown to increase as a function of adsorption time. Subsequently, we investigated the role that polyelectrolyte solution pH (ranging from 2.5 to 9) has in forming molecularly distinct films of weak polyelectrolytes and report the effect this has on modulating cell attachment and spreading. Films synthesized at PAA-pH of 5.5 and PAH-pH 2.5-5.5 exhibited the highest cellular attachment. These results indicate that HT-LbL is a robust method that can shift the paradigm regarding the use of LbL in biomedical applications as it provides a rapid method to synthesize, characterize, and screen the interactions between molecularly distinct LbL films and cells.

Circulating tumor cells (CTCs) are low frequency cells found in the bloodstream after having been shed from a primary tumor. These cells are research targets because of the information they may potentially provide about both an individual cancer as well as the mechanisms through which cancer spreads in the process of metastasis. Established technologies exist for CTC isolation, but the recent progress and future of this field lie in nanomaterials. In this review, we provide perspective into historical CTC capture as well as current research being conducted, emphasizing the significance of the materials being used to fabricate these devices. The modern investigation into CTCs initially featured techniques that have since been commercialized. A major innovation in the field was the development of a microfluidic capture device, first fabricated in silicon and followed up with glass and thermopolymer devices. We then specifically highlight the technologies incorporating magnetic nanoparticles, carbon nanotubes, nanowires, nanopillars, nanofibers, and nanoroughened surfaces, graphene oxide and their fabrication methods. The nanoscale provides a new set of tools that has the potential to overcome current limitations associated with CTC capture and analysis. We believe the current trajectory of the field is in the direction of nanomaterials, allowing the improvements necessary to further CTC research.

Background: Esotropia during opiate withdrawal is a new clinical syndrome that has only recently been reported in the literature., Methods: Clinical case series., Results: Five patients with acute esotropia during opiate withdrawal are presented. In four there was evidence of underlying hyperopia and/or other strabismogenic features., Conclusions: The precise cause of esotropia with heroin withdrawal is uncertain. Most (and possibly all) of these patients have one or more objective strabismogenic features, and these are probably a large factor in the causation.

Zosteriform connective-tissue nevus, because of its distribution and histopathologic characteristics, is considered to be a separate entity. The only previous case of zosteriform connective-tissue nevus to be reported in the American literature was in 1944. We report and discuss a similar case.