Yuanzhen Suo

Near infrared in vivo flow cytometry for tracking fluorescent circulating cells

The in vivo flow cytometry (IVFC) is now a powerful technique in biomedical research, especially for tracking specific cells in circulatory system. The current fluorescence‐based IVFC is limited to visible spectrum, while near infrared (NIR) dyes have their advantages, such as deeper penetration, less absorption and less scattering for NIR fluorescence. Here, using an NIR in vivo flow cytometer with a 785 nm laser excitation, the measurement of fluorescent dye IR‐780 labeled circulating cells is demonstrated. Representative peaks corresponding to NIR fluorescent circulating cells are detected and quantified. In addition, blood flow information, including the blood flow velocity and flow volume per unit time, is obtained. By simultaneous detection of IR‐780 and enhanced green fluorescent protein (EGFP) signals from dual labeled cells, the IR‐780 is shown to be a suitable fluorescent dye for multicolor detection by IVFC, including NIR. Thus, the IVFC is extended to the NIR range and shows potential application in biomedical research.

Morphological change of CD4(+) T cell during contact with DC modulates T-cell activation by accumulation of F-actin in the immunology synapse.

BackgroundThe changes in T-cell morphology during immunological synapse (IS) formation are essential for T-cell activation. Previous researches have shown that T cell changed from spherical to elongated and/or flattened during in contact with B cell. As most powerful antigen presenting cell, dendritic cell (DC) has a strong ability to activate T cells. However, the morphological change of T cell which contacts DC and the relationship between morphological change and T-cell activation are not very clear. Thus, we studied the morphological change of CD4+ T cell during contact with DC.ResultsUsing live-cell imaging, we discovered diversity in the T-cell morphological changes during contact with DCs. The elongation-flattening of CD4+ T cells correlated with a low-level Ca2+ response and a loss of T-cell receptor (TCR) signalling molecules in the IS, including zeta-chain associated protein kinase 70 (ZAP-70), phospholipase C-γ (PLC-γ) and protein kinase C-θ (PKC-θ), whereas rounding-flattening correlated with sufficient CD4+ T-cell activation. Different morphological changes were correlated with the different amount of accumulated filamentous actin (F-actin) in the IS. Disruption of F-actin by cytochalasin D impaired the morphological change and the localisation of calcium microdomains in the IS and decreased the calcium response in CD4+ T cells.ConclusionOur study discovered the diversity in morphological change of T cells during contacted with DCs. During this process, the different morphological changes of T cells modulate T-cell activation by the different amount of F-actin accumulation in the IS, which controls the distribution of calcium microdomains to affect T-cell activation.

Proportion of circulating tumor cell clusters increases during cancer metastasis

Circulating tumor cell (CTC) clusters are found among CTCs and show significantly greater potential for an important role in cancer metastasis than single CTCs, which have been traditionally believed as the majority of CTCs. The accurate proportion and dynamics of CTC clusters remain unclear due to the fact that CTCs in blood flow are very difficult to detect in vivo and in vitro. CTC clusters are even more difficult to be distinguished from CTCs without perturbation by state‐of‐the‐art detection methods. Here, we demonstrate that by using in vivo flow cytometry (IVFC), we can reliably measure the proportion and dynamics of CTC clusters in two murine tumor models. CTC clusters are easily identified by their unique fluorescent pattern with multiple peaks and wider time duration. We find that the proportion of CTC clusters increases significantly during cancer metastasis in both mouse models, the orthotopic liver cancer and the subcutaneous prostate cancer models. Our results suggest that CTC clusters account for a much larger proportion of CTCs than previously anticipated. Hence this report might provide a new‐level of understanding of CTCs during cancer development and progression.

The bullseye synapse formed between CD4 + T-cell and staphylococcal enterotoxin B-pulsed dendritic cell is a suppressive synapse in T-cell response

The immunological synapse (IS) is a supermolecular activation cluster formed between T cells and antigen‐presenting cells. Although diverse IS structures have been reported, the function of the IS in T‐cell activation remains unclear. Here, we found that the bullseye IS, one of IS types at the interface of CD4+ T cells and staphylococcal enterotoxin B‐pulsed dendritic cells, suppressed CD4+ T‐cell activation, whereas multifocal IS, another synapse type, stimulated CD4+ T‐cell activation. Consistent with these results, bullseye IS formation was accompanied by a low‐level calcium response in T cells and a loss of T‐cell receptor signalling molecules from the synapse, whereas multifocal IS exhibited the opposite. Furthermore, we found that CD4+CD25+ regulatory T cells (Tregs) more efficiently formed bullseye IS and promoted bullseye IS formation in CD4+ CD25− T cells. Cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4), an inhibitory molecule expressed continuously on Tregs, was localised in bullseye IS. Moreover, blocking CTLA‐4 reduced the percentage of bullseye IS formation and promoted T‐cell activation. Our data thus indicate that bullseye IS formation is mediated by CTLA‐4, and may negatively control T‐cell activation as a suppressive synapse.

Systemically Infused Mesenchymal Stem Cells Show Different Homing Profiles in Healthy and Tumor Mouse Models

Bone marrow‐derived mesenchymal stem cells (MSCs) can localize in injured, inflamed, and cancerous tissues after systemic infusion. However, the dynamic homing profile of MSCs in the peripheral blood is not well characterized. Here, using in vivo flow cytometry to noninvasively monitor the dynamics of fluorescence‐labeled cells, we found different clearance kinetics of systemically infused MSCs between healthy and tumor mouse models. The circulation times of MSCs in healthy mice and mice with subcutaneous tumors, orthotopically transplanted liver tumors, or metastatic lung tumors were 30, 24, 18, and 12 hours, respectively, suggesting that MSCs actively home to tumor environments. MSCs infiltrated into hepatocellular carcinoma (HCC) sites and preferentially engrafted to micrometastatic regions both in vivo and in vitro. The expression of epidermal growth factor, CXCL9, CCL25, and matrix metalloproteinases‐9 by HCC cells differed between primary tumor sites and metastatic regions. By characterizing the homing profiles of systemically perfused MSCs under physiological and cancerous conditions, these findings increase our understanding of the migration of MSCs from the circulation to tumor sites and constitute a basis for developing MSC‐based anti‐cancer therapeutic strategies. Stem Cells Translational Medicine 2017;6:1120–1131

Toluidine blue O and porphyrin-mediated photodynamic therapy on three main pathogenic bacteria of periodontitis using portable LED phototherapy device

Photodynamic therapy (PDT) has been commonly used in treating many diseases, such as cancer and infectious diseases. We investigated the different effects of PDT on three main pathogenic bacteria of periodontitis — Prevotella melaninogenica (P.m.), Porphyromonas gingivalis (P.g.) and Aggregatibacter actinomycetemcomitans (A.a.). The portable red light-emitting diode (LED) phototherapy device was used to assess the exogenous PDT effects with different light doses and photosensitizer concentrations (Toluidine blue O, TBO). The portable blue LED phototherapy device was used to assess the endogenous PDT effects with the use of endogenous photosensitizers (porphyrin) under different light doses. We found out that both exogenous and endogenous PDT were able to restrict the growth of all the three bacteria significantly. Moreover, the optimal PDT conditions for these bacteria were obtained through this in vitro screening and could guide the clinical PDT on periodontitis.

ICAM-1 depletion in the center of immunological synapses is important for calcium releasing in T-cells

T-cell activation requires the formation of the immunological synapse (IS) between a T-cell and an antigen-presenting cell (APC) to control the development of the adaptive immune response. However, calcium release, an initial signal of T-cell activation, has been found to occur before IS formation. The mechanism for triggering the calcium signaling and relationship between calcium release and IS formation remains unclear. Herein, using live-cell imaging, we found that intercellular adhesion molecule 1 (ICAM-1), an essential molecule for IS formation, accumulated and then was depleted at the center of the synapse before complete IS formation. During the process of ICAM-1 depletion, calcium was released. If ICAM-1 failed to be depleted from the center of the synapse, the sustained calcium signaling could not be induced. Moreover, depletion of ICAM-1 in ISs preferentially occurred with the contact of antigen-specific T-cells and dendritic cells (DCs). Blocking the binding of ICAM-1 and lymphocyte function-as...

Cell counting for in vivo flow cytometry signals with baseline drift

In biomedical research fields, the in vivo flow cytometry (IVFC) is a widely used technology which is able to monitor target cells dynamically in living animals. Although the setup of IVFC system has been well established, baseline drift is still a challenge in the process of quantifying circulating cells. Previous methods, i.e., the dynamic peak picking method, counted cells by setting a static threshold without considering the baseline drift, leading to an inaccurate cell quantification. Here, we developed a method of cell counting for IVFC data with baseline drift by interpolation fitting, automatic segmentation and wavelet-based denoising. We demonstrated its performance for IVFC signals with three types of representative baseline drift. Compared with non-baseline-correction methods, this method showed a higher sensitivity and specificity, as well as a better result in the Pearson’s correlation coefficient and the mean-squared error (MSE).

Noninvasive monitoring of nanoparticle clearance and aggregation in blood circulation by in vivo flow cytometry

ABSTRACT Nanoparticles have been widely used in biomedical research as drug carriers or imaging agents for living animals. Blood circulation is crucial for the delivery of nanoparticles, which enter the bloodstream through injection, inhalation, or dermal exposure. However, the clearance kinetics of nanoparticles in blood circulation has been poorly studied, mainly because of the limitations of conventional detection methods, such as insufficient blood sample volumes or low spatial‐temporal resolution. In addition, formation of nanoparticle aggregates is a key determinant for biocompatibility and drug delivery efficiency. Aggregation behavior of nanoparticles in blood is studied using dynamic light scattering in serum or serum protein solutions, which is still very different from in vivo condition. In this work, we monitored the dynamics of nanoparticle concentration and formation of nanoparticle aggregates in the bloodstream in live animals using in vivo flow cytometry (IVFC). The results indicated that nanoparticles in smaller size could stay longer in the bloodstream. Polyethylene glycol (PEG)‐modification could prolong circulating time and reduce the formation of aggregates in the blood circulation. Our work shows that IVFC can be a powerful tool for pharmacokinetic studies of nanoparticles and other drug carriers, assessing cell‐targeting efficiency, as well as potentially measuring cardiac output and hepatic function in vivo.

Monitoring circulating prostate cancer cells by in vivo flow cytometry assesses androgen deprivation therapy on metastasis: Monitoring Circulating Prostate Cancer Cells

It remains controversial whether surgical castration prolongs survival rate and improves therapy prospects in patients suffering from prostate cancer. We used PC3 cell line to establish prostate tumor models. In vivo flow cytometry and ultrasonic imaging were used to monitor the process of prostate cancer growth, development and metastasis. We found out that the number of circulating tumor cells (CTCs) in orthotopic tumor model was higher than that in subcutaneous tumor model. The CTC number in orthotopic tumor model was due to burst growth, while CTC number in subcutaneous tumor model showed a gradual increase with tumor size. After androgen deprivation therapy (ADT) through testicular extraction, we constructed GFP‐PC3 subcutaneous tumor models and orthotopic tumor models. We found dramatically decreased CTC number, relieved symptoms caused by the tumor, and significantly prolonged survival time after testicular extraction in orthotopically transplanted prostate tumor model, while the carcinogenesis process and metastases were little influenced by ADT in subcutaneous tumor model. ADT treatment can restrict tumor growth, decrease the CTC number significantly and inhibit distant invasion through inhibition of tumor proliferation and tumor angiogenesis in orthotopical prostate tumor model.