Viruses are changing the biosensing and biomedicine landscape due to their multivalency, orthogonal reactivities, and responsiveness to genetic modifications. As the most extensively studied phage model for constructing a phage display library, M13 phage has received much research attention as building blocks or viral scaffolds for various applications including isolation/separation, sensing/probing, and in vivo imaging. Through genetic engineering and chemical modification, M13 phages can be functionalized into a multifunctional analysis platform with various functional regions conducting their functionality without mutual disturbance. Its unique filamentous morphology and flexibility also promoted the analytical performance in terms of target affinity and signal amplification. In this review, we mainly focused on the application of M13 phage in the analytical field and the benefit it brings. We also introduced several genetic engineering and chemical modification approaches for endowing M13 with various functionalities, and summarized some representative applications using M13 phages to construct isolation sorbents, biosensors, cell imaging probes, and immunoassays. Finally, current issues and challenges remaining in this field were discussed and future perspectives were also proposed. 

/pdf/m13-phage-a-versatile-building-block-for-a-highly-specific-2w1zkowy.pdf

M13 phage: a versatile building block for a highly specific analysis platform

Colonization of cancer cells at secondary sites, a decisive step in tumor metastasis, is strongly dependent on the formation of metastatic microenvironments regulated by intrinsic single-cell metabolism traits. Herein, we report a single-cell microfluidic platform for high-throughput dynamic monitoring of tumor cell metabolites to evaluate tumor malignancy. This microfluidic device empowers efficient isolation of single cells (>99%) in a squashed state similar to tumor extravasation, and employs enzyme-packaged metal-organic frameworks to catalyze tumor cell metabolites for visualization. The microfluidic evaluation was confirmed by in vivo assays, suggesting that the platform allowed predicting the tumorigenicity of captured tumor cells and screening metabolic inhibitors as anti-metastatic drugs. Furthermore, the platform efficiently detected various aggressive cancer cells in unprocessed whole blood samples with high sensitivity, showing potential for clinical application.

An Enzyme-Loaded Metal-Organic Framework-Assisted Microfluidic Platform Enables Single-Cell Metabolite Analysis.

Microfluidic systems for particle capture and release: A review.

The demand for the ultrasensitive and rapid quantitative analysis of trace target analytes has become increasingly urgent. However, the sensitivity of traditional immunoassay-based detection methods is limited due to the contradiction between molecular recognition and signal amplification caused by the size effect of nanoprobes. To address this dilemma, we describe versatile M13 phage-assisted immunorecognition and signal transduction spatiotemporal separation that enable ultrasensitive light-scattering immunoassay systems for the quantitative detection of low-abundance target analytes. The newly developed immunoassay strategy combines the M13 phage-assisted light scattering signal fluctuations of gold nanoparticles (AuNPs) with gold in situ growth (GISG) technology. Given the synergy of M13 phage-mediated leverage effect and GISG-amplified light scattering signal modulation, the practical detection capability of this strategy can achieve the ultrasensitive and rapid quantification of ochratoxin A and alpha-fetoprotein in real samples at the subfemtomolar level within 50 min, displaying about 4 orders of magnitude enhancement in sensitivity compared with traditional phage-based ELISA. To further improve the sensitivity of our immunoassay, the biotin-streptavidin amplification scheme is implemented to detect severe acute respiratory syndrome coronavirus 2 spike protein down to the attomolar range. Overall, this study offers a direction for ultrasensitive quantitative detection of target analytes by the synergistic combination of M13 phage-mediated leverage effect and GISG-amplified light scattering signal modulation.

M13 Bacteriophage-Assisted Recognition and Signal Spatiotemporal Separation Enabling Ultrasensitive Light Scattering Immunoassay.

Decoding the interplay between genetic and non-genetic drivers of metastasis.

Advances in single-cell level profiling of the proteome require quantitative and versatile platforms, especially for rare cell analyses such as circulating tumor cell (CTC) profiling. Here we demonstrate an integrated microfluidic chip that uses magnetic nanoparticles to capture single tumor cells with high efficiency, permits on-chip incubation, and facilitates in situ cell-surface protein expression analysis. Combined with phage-based barcoding and next-generation sequencing technology, we were able to monitor changes in the expression of multiple surface markers stimulated in response to CTC adherence. Interestingly, we found fluctuations in the expression of Frizzled2 (FZD2) that reflected the microenvironment of the single cells. This platform has a high potential for in-depth screening of multiple surface antigens simultaneously in rare cells with single-cell resolution, which will provide further insights regarding biological heterogeneity and human disease.

Phage-Based Profiling of Rare Single Cells Using Nanoparticle-Directed Capture.

Background Individual survival prediction is of vital importance to optimize the individualized treatment of metastatic cervical cancer (mCC) patients. The goal of this study was to identify the potential risk factors for the survival of mCC patients and construct a nomogram for their prognosis. Methods Medical records of patients with newly diagnosed mCC at the First Affiliated Hospital of Xi’an Jiaotong University were reviewed retrospectively. Risk factors were identified using Cox proportional hazards analysis and Kaplan–Meier curves. Random forest was used to identify factors associated with therapy strategy. Nomogram and dynamic nomogram were established using ‘rms’ and “DynNom” R package. Results A total of 98 patients with mCC were finally identified. In Cox analyses, multiple metastases and concurrent chemoradiotherapy (CCRT) were identified as independent predictors for overall survival (OS). We further explored the prognostic value of metastatic number and sites and therapy strategies for mCC patients by Kaplan–Meier curves. A dynamic nomogram including metastases number and sites (multiple metastases, liver and lymph node (LN) above diaphragm metastases) and chemoradiotherapy strategies (CCRT, postradiotherapy chemotherapy, and radiotherapy to metastatic sites) was constructed for predicting the prognosis of mCC patients. For newly diagnosed patients, we strongly recommended the combination of chemotherapy and definitive pelvic radiotherapy and, if possible, radiation to metastatic site, but CCRT should be implemented with caution. We constructed a dynamic nomogram indicating that patients with younger age, shorter symptom duration, and better laboratory test results are suitable for CCRT. Conclusion Survival analyses showed that the metastatic number and sites and therapy strategies are associated with the prognosis of mCC patients. The CCRT and prognostic nomograms may help clinicians to make better clinical decisions and effectively predict the prognosis for newly diagnosed mCC patients.

Dynamic Nomogram Based on the Metastatic Number and Sites and Therapy Strategies Predicting the Prognosis of Patients with Metastatic Cervical Cancer

ARTICLES DISCUSSED: Ma, Y., Li., T. Monitoring dynamic growth of retinal vessels in oxygen-induced retinopathy mouse model. Journal of Visualized Experiments. (170), e62410 (2021). Guan, Y., Xie, B. Zhong, X. Retinal organoid induction system for derivation of 3D retinal tissues from human pluripotent stem cells. Journal of Visualized Experiments. (170), e62435 (2021). Rose, K., Lokappa, S. Chen, J. Two peeling methods for the isolation of photoreceptor cell compartments in the mouse retina for protein analysis. Journal of Visualized Experiments. (178), e62977 (2021). Ye., Q. et al. In vivo methods to assess retinal ganglion cell and optic nerve function and structure in large animals. Journal of Visualized Experiments. (180), e62879 (2022). Abbas, F., Vinberg, F., Becker, S. Optimizing the setup and conditions for ex vivo electroretinogram to study retina function in small and large eyes. Journal of Visualized Experiments. (184), e62763 (2022). Okan, I., Ahmadian, M., Tutuncu, Y., Altay, H., Agca, C. Digital droplet PCR method for the quantification of AAV transduction efficiency in murine retina. Journal of Visualized Experiments. (178), e63038 (2021). 

/pdf/fundamental-and-state-of-the-art-technologies-in-retinal-ujlpwom2.pdf

Fundamental and State-of-the-Art Technologies In Retinal Studies and Disease Repair

Circulating tumor cells (CTCs) break free from primary tumors and travel through the bloodstream and lymphatic system to seed metastatic tumors, which are the major cause of death from cancer. The identification of the major genetic factors that enhance production and persistence of CTCs in the bloodstream at a whole genome level would enable more comprehensive molecular mechanisms of metastasis to be elucidated and the identification of novel therapeutic targets, but this remains a challenging task due to the heterogeneity and extreme rarity of CTCs. Here, we describe the first in vivo genome-wide CRISPR KO screen using CTCs directly isolated from a mouse xenograft. This screen elucidated SLIT2 - a gene encoding a secreted protein acting as a cellular migration cue - as the most significantly represented gene knockout in the CTC population. SLIT2 knockout cells are highly metastatic with hypermigratory and mesenchymal phenotype. Loss of SLIT2 is observed in human tumors, indicating its role as a negative modulator of tumor progression and metastasis.

https://www.biorxiv.org/content/biorxiv/early/2021/08/21/2021.08.20.457126.full.pdf

Genome-wide in vivo screen of circulating tumor cells identifies SLIT2 as a regulator of metastasis

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Phage-Based Profiling of Rare Single Cells Using Nanoparticle-Directed Capture.

Dynamic Nomogram Based on the Metastatic Number and Sites and Therapy Strategies Predicting the Prognosis of Patients with Metastatic Cervical Cancer

Fundamental and State-of-the-Art Technologies In Retinal Studies and Disease Repair

Genome-wide in vivo screen of circulating tumor cells identifies SLIT2 as a regulator of metastasis