In Vivo Flow Cytometry Research Articles

Real-time monitoring of small extracellular vesicles (sEVs) by in vivo flow cytometry.

Extracellular vesicles (EVs) are vesicular structures comprised of a bilayer lipid membrane, released by living cells. There is a growing body of evidence for their functionality, indicating that small EVs (sEVs) can mediate specific forms of intercellular communication. The future applications of sEVs hold great promise, not only as diagnostic markers but also as therapeutic agents. However, the greatest difficulty in the clinical translation of sEVs is that they are currently poorly understood, especially concerning their in vivo behaviour. In this study, we provide a novel method for monitoring sEVs in blood circulation based on in vivo flow cytometry (IVFC). We have demonstrated that the height of the IVFC signal baseline is proportional to the concentration of sEVs. Moreover, we have found out that the peaks in the IVFC signal are generated by the aggregation or cellular uptake of sEVs. In vivo monitoring of sEVs clearance from the blood indicates that PEGylated sEVs have a longer residence time and exhibit less aggregation in circulation compared to non-PEGylated sEVs. These studies reveal that IVFC enables real-time in vivo monitoring of circulating sEVs, which can provide valuable insights into the pharmacokinetics and cellular targeting capabilities of sEVs.

Monitoring Circulating Myeloid Cells in Peritonitis with an In Vivo Imaging Flow Cytometer.

Peritonitis is a common and life-threatening inflammatory disease. Myeloid cells are elevated in the peripheral blood and contribute to peritonitis, but their circulating dynamics are not clear. In vivo flow cytometry (IVFC) is a noninvasive technique for monitoring the dynamics of circulating cells in live animals. It has been extensively used to detect circulating tumor cells, but rarely for monitoring immune cells. Here, we describe a method adapting an intravital microscope for IVFC so that we can monitor LysM-EGFP-labeled circulating myeloid cells in a tumor necrosis factor (TNF) α-induced peritonitis mouse model. Using this IVFC method, we quantified the blood flow velocity and cell concentration in circulation. We observed a significant increase in LysM-EGFP+ cells in circulation after TNFα intraperitoneal (i.p.) injection, which reached a plateau in ~20 min. Conventional cytometry analysis showed that most LysM-EGFP+ cells were neutrophils. Increasing blood neutrophils were accompanied by neutrophil recruitment to the peritoneal cavity and neutrophil emigration from the bone marrow. We then monitored neutrophil CD64 expression in vivo and found a significant increase in TNFα-induced peritonitis. We also found that CD18 blockade doubled the circulating neutrophil number in TNFα-induced peritonitis, suggesting that CD18 is critical for neutrophil recruitment in peritonitis. Overall, we demonstrate that IVFC techniques are useful for studying the circulating dynamics of immune cells during inflammatory diseases.

Progress and challenges of in vivo flow cytometry and its applications in circulating cells of eyes.

Circulating inflammatory cells in eyes have emerged as early indicators of numerous major diseases, yet the monitoring of these cells remains an underdeveloped field. In vivo flow cytometry (IVFC), a noninvasive technique, offers the promise of real-time, dynamic quantification of circulating cells. However, IVFC has not seen extensive applications in the detection of circulating cells in eyes, possibly due to the eye's unique physiological structure and fundus imaging limitations. This study reviews the current research progress in retinal flow cytometry and other fundus examination techniques, such as adaptive optics, ultra-widefield retinal imaging, multispectral imaging, and optical coherence tomography, to propose novel ideas for circulating cell monitoring.

In vivo flow cytometry reveals an anti-metastatic effect of Rujifang in triple-negative breast cancer.

Breast cancer is the most common cancer, and triple-negative breast cancer (TNBC) has the highest metastasis and mortality rate among all breast cancer subtypes. Rujifang is a traditional Chinese medicine formula with many years of clinical application in breast cancer treatment. Here, we aim to investigate the effects of Rujifang on circulating tumor cell (CTC) dynamics and the tumor microenvironment in a ZsGreen/luciferase double-labeled TNBC orthotopic model. We report that the number of CTCs monitored by in vivo flow cytometry (IVFC) strongly correlates with disease progression. Rujifang treatment decreased the number of CTCs and suppressed the distant metastasis of TNBC. Moreover, immunofluorescence analysis revealed that Rujifang treatment could affect the tumor microenvironment by downregulating Kindlin-1, which has been reported to promote metastasis of TNBC. Our study provides evidence of the anti-metastatic effect of Rujifang against TNBC in an animal model using fluorescent cell lines. The results suggest the potential therapeutic value of Rujifang as an anti-metastatic drug, however, further clinical trials are needed to validate these findings in humans.

Detection of cells by flow cytometry: Counting, imaging, and cell classification

The study of circulating cells in the blood stream is critical, as it covers many fields of biomedicine, including immunology, cell biology, oncology, and reproductive medicine. In-vivo flow cytometry (IVFC) is a new tool to monitor and count cells in real time for long durations in their native biological environment. This review describes two main categories of IVFC, i.e., labeled and label-free IVFC. It focuses on label-free IVFC and introduces its technological development and related biological applications. Because cell recognition is the basis of flow cytometry counting, this review also describes various methods for the classification of unlabeled cells, including the latest machine learning-based technologies.

In vivo flow cytometry reveals a circadian rhythm of circulating tumor cells

Circulating tumor cells (CTCs) is an established biomarker of cancer metastasis. The circulation dynamics of CTCs are important for understanding the mechanisms underlying tumor cell dissemination. Although studies have revealed that the circadian rhythm may disrupt the growth of tumors, it is generally unclear whether the circadian rhythm controls the release of CTCs. In clinical examinations, the current in vitro methods for detecting CTCs in blood samples are based on a fundamental assumption that CTC counts in the peripheral blood do not change significantly over time, which is being challenged by recent studies. Since it is not practical to draw blood from patients repeatedly, a feasible strategy to investigate the circadian rhythm of CTCs is to monitor them by in vivo detection methods. Fluorescence in vivo flow cytometry (IVFC) is a powerful optical technique that is able to detect fluorescent circulating cells directly in living animals in a noninvasive manner over a long period of time. In this study, we applied fluorescence IVFC to monitor CTCs noninvasively in an orthotopic mouse model of human prostate cancer. We observed that CTCs exhibited stochastic bursts over cancer progression. The probability of the bursting activity was higher at early stages than at late stages. We longitudinally monitored CTCs over a 24-h period, and our results revealed striking daily oscillations in CTC counts that peaked at the onset of the night (active phase for rodents), suggesting that the release of CTCs might be regulated by the circadian rhythm.

Monitoring radiofrequency therapy-induced tumor cell dissemination by in vivo flow cytometry.

Clinical and experimental findings have disclosed a high recurrence rate after radiofrequency ablation (RFA), which might be due to the dissemination of malignant cells into the vasculature during ablation. Here, we apply in vivo flow cytometry (IVFC) to monitor circulating tumor cells (CTCs) while performing ablation in a real-time and noninvasive way in an orthotopic model of prostate cancer. We report that CTCs are dramatically increased during RFA. The CTCs induced by ablation eventually translate into enhanced distant metastasis and reduced survival as compared to resection. Immunofluorescence analysis reveals that RFA significantly increases the infiltration of tumor-associated macrophages (TAMs) in the lung. Our study thus suggests that the ablative procedure of prostate tumors causes immediate tumor cell dissemination and increases distant metastasis.

In Vivo Flow Cytometry.

In vivo flow cytometry (IVFC) was first designed to detect circulating cells in a mouse ear. It allows real-time monitoring of cells in peripheral blood with no need to draw blood. The IVFC field has made great progress during the last decade with the development of fluorescence, photoacoustic, and multiphoton microscopy. Moreover, the application of IVFC is no longer restricted to circulating cells. IVFC based on fluorescence and photoacoustic are most widely applied in biomedical research. Methods based on fluorescence are often used for object monitoring in superficial vessels, while methods based on photoacoustics have an advantage of label-free monitoring in deep vessels. In this chapter, we introduce technical points and key applications of IVFC. We focus on the principles, labeling strategies, sensitivity, and biomedical applications of the technology. In addition, we summarize this chapter and discuss important research directions of IVFC in the future.

In vivo flow cytometry combined with intravital microscopy to monitor kinetics of transplanted bone marrow mononuclear cells in peripheral blood and bone marrow.

Bone marrow mononuclear cells (BM-MNCs) transplantation has evolved as a promising experimental treatment in various regenerative therapy fields, especially in clinical hematopoietic stem cells transplantation (HSCT). In vitro methods have mainly been used to study the pre-clinical kinetics of BM-MNCs in mice after transplantation. And it is difficult to monitor the dynamic homing of BM-MNCs in living mice. The present study obtained the kinetics of transplanted BM-MNCs in the peripheral blood (PB) and the dynamic homing of BM-MNCs in the BM in living mice by a combination of in vivo flow cytometry (IVFC) and calvarium intravital microscopy. We found out that BM-MNCs were cleared rapidly from the PB and mainly localized to various hematopoietic tissues after transplantation. The number of BM-MNCs in the PB decreased over time accompanied by an increase in the BM indeed after transplantation. In addition, a lower number of BM-MNCs were found home to calvaria than long bone, probably indicating long bone marrow might also be an important hematopoietic organ. Clinical studies will benefit from non-invasive measurements to monitor the dynamic homing of transplanted cells. Our pre-clinical kinetics of BM-MNCs in living mice will have important clinical guiding significance in HSCT and other regenerative therapy fields.

Recent advances in fluorescence-based in vivo flow cytometry

The fluorescence-based in vivo flow cytometry (IVFC) is an emerging tool to monitor circulating cells in vivo. As a noninvasive and real-time diagnostic technology, the fluorescence-based IVFC allows long-term monitoring of circulating cells without changing their native biological environment. It has been applied for various biological applications (e.g., monitoring circulating tumor cells). In this work, we will review our recent works on fluorescence-based IVFC. The operation principle and typical biological applications will be introduced. In addition, the recent advances in IVFC flow cytometry based on photoacoustic effects and other label-free detection methods such as imaging-based methods, diffuse-light methods, hybrid multimodality methods and multispectral methods are also summarized.

Advances of In Vivo Flow Cytometry on Cancer Studies.

Cancer is a big threat to human life. Asia has about 60% of the global population and accounts for half of global cancer incidence and mortality. Circulating tumor cells (CTCs) have been a good biomarker for cancer diagnosis, staging, and prognosis. Conventional detection methods of CTCs require drawing blood. It may disturb the biological environment and limited real-time monitoring. in vivo flow cytometry (IVFC) is a burgeoning technique that allows noninvasive detection of CTCs in vivo. Here, we review the technical development of IVFC based on various contrast principles, including fluorescence IVFC, photoacoustic IVFC, imaging IVFC, and label-free IVFC. This powerful tool has been applied widely in many areas of cancer-related studies, especially the CTC studies. We review applications of IVFC in preclinical studies on prevalent cancers in Asia, including liver cancer, blood cancer, and so forth. Other cancer-related studies in breast cancer, prostate cancer, cancer-related stem cell research and drug studies are also reviewed. © 2019 International Society for Advancement of Cytometry.

Monitoring circulating tumor cells in vivo by a confocal microscopy system.

Circulating tumor cells (CTCs) play a key role in cancer metastasis but are very difficult to detect. in vivo monitoring CTCs has been recognized as an important technique for cancer research and clinical diagnosis. Recently, a noninvasive method, in vivo flow cytometry (IVFC) has been developed to enable continuous, real-time, and long-duration detection of CTCs in animal models by detecting CTC fluorescence in blood vessels excited by lasers. In this study, we present a simple optical scheme for direct noninvasive CTC detection using confocal microscopes. We demonstrate that line scanning of confocal microscopy can provide effective and quantitative CTC detection in live mice during cancer development. Rare CTC signals can be acquired at the early stage of the tumor development after implantation of subcutaneous tumor and monitored continuously to the end. Signals from CTC clusters can also be acquired and distinguished from single CTCs. Our results suggest confocal microscopy is a simple and reliable method for biologists and doctors to use for cancer research. © 2018 International Society for Advancement of Cytometry.

In Vivo flow Cytometry Combined with Intravital Microscopy to Monitor Synchronous Kinetics of Transplanted BM-MNCs in Peripheral Blood and Bone Marrow

Limited sources and finite numbers of hematopoietic stem cells (HSCs) have always been difficult problems to overcome in clinical HSC transplantation (HSCT). Improving the bone marrow (BM) homing efficiency of HSC has become an intensely studied topic. HSC homing involves the kinetics of transplanted cells in the peripheral blood (PB), the cell distribution in various organs and tissues, and the targeted homing of HSC to the BM. However, it is difficult to monitor the dynamic homing in living mice. The objective of this study is to establish a platform for monitoring the kinetics of transplanted BM-MNCs in the PB and the synchronous homing of BM-MNCs in the BM in living mice by a combination of in vivo flow cytometry (IVFC) and calvarium intravital microscopy.IVFC is a new in vivo technology that can quantitatively and continuously monitor fluorescence-labeled cells circulating in an anesthetized mouse. When a circulating fluorescent cell passes through the laser slit across the selected ear artery, fluorescence can be excited and recorded. Therefore, this method can provide continuous information on cellular behavior over time in the same living mouse without requiring extracting a blood sample at every time point. Calvarium intravital microscopy is another in vivo technology that allows a direct view of the dynamic homing of transplanted cells in the BM of a living mouse. Although the skull and femur are different bone types, the homing behavior and function of HSCs in the cross-road intersection region of coronal and sagittal sutures are comparable to those in the long bones in terms of immunophenotype and propagation. With calvarium intravital microscopy combined with in vivo flow cytometry, we here continually monitor the synchronous dynamic homing of transplanted BM-MNCs in the skull marrow in living mouse.By detecting the fluorescent cells in circulation, we obtained the kinetics of transplanted BM-MNCs in the PB over 24 h. Interestingly, the number of BM-MNCs decreased rapidly about 90% in the PB within the first 4 h after transplantation, further decreasing from 4 h to 6 h, and remaining at a low level from 6 h to 24 h. We further applied calvarium intravital microscopy to directly visualize the localization and the synchronous homing kinetics of BM-MNCs in the mouse skull marrow after transplantation. Interestingly, the BM-MNCs were mainly distributed over the large vessels in the cross-road intersection of coronal and sagittal sutures at 24 h, especially within the anterior half of this region (90-95% of total fluorescent cells). We then mainly measured the synchronous homing kinetics of BM-MNCs in the anterior half region at 2, 6, and 24 h in the mice, and found out that the homing of BM-MNCs in the skull marrow indeed increased gradually in the same mouse with the same pattern in different transplanted mice. Both the data obtained by IVFC and the calvarium intravital microscopy had a good linear correlation with their respective conventional in vitro flow cytometry data. By combining the conventional in vitro data, we also found a similar synchronous kinetics of BM-MNCs in the PB and the BM. All these data suggest that the BM-MNCs cleared from the PB might home to the BM over time after transplantation.In summary, as an important source of HSCs, BM-MNCs have been successfully applied to clinical HSCT. Understanding the early homing mechanism of transplanted BM-MNCs can enhance the marrow homing efficiency and engraftment of HSCs. Some other in vivo methods have been developed to study the homing of transplanted BM-MNCs to BM, including magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), endoscopic probing, and femur drilling to a laser-detectable depth. Compared to these methods, our method by calvarium intravital microscopy is mildly invasive, while the homing kinetics of BM-MNCs to the BM can be directly viewed and measured at the single-cell level. DisclosuresNo relevant conflicts of interest to declare.

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

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.

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).

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. © 2016 International Society for Advancement of Cytometry.

Real - time monitoring of circulation tumor cells in a surgical orthotopic implantation model of human metastatic prostate cancer

Objective To study the relationship of circulation tumor cells (CTCs) and metastatic process, we use subcutaneous model and surgical orthotopic implantation model of human metastatic prostate cancer and detected by in vivo flow cytometry (IVFC). Methods The human prostate cancer cell line PC3 cells were transduced with pLVX-EGFP-3FLAG lentivirus vector containing the green fluorescent protein (GFP) and puromycin resistance gene. PC3 cells, high-expression of GFP, were obtained by screening of stably transfected clones. PC3-GFP cells or unlabeled tumor cells were implanted into the right armpit of 10 nude mice to build up subcutaneous xenograft prostate cancer mode1, which provided tumor fragments to implant the orthotopic prostate tumor mode1. Tumor tissue with high fluorescent from the subcutaneous tumor was harvested and cut into pieces of 1 mm3, and grafted into the anterior prostates of 10 nude mice. The number of CTCs monitored by IVFC real-time detecting fluorescent signal noninvasively at 5 d, 10 d and 15 d after tumor established. Results A PC3-GFP cell line was established which had the same growth pattern as well as tumorigenesis ability as mock-transfected PC3 cells. IVFC detected 2.37, 6.25, 99.75 CTCs/h at 5, 10, and 15 days. Conclusion The orthotopic prostate cancer models are better to assess the morphology, growth, metastatic pattern and development characteristics of clinical disease. They are also more representative of a primary tumor with respect to tumor site and metastasis, especially CTCs dynamics. The in vivo flow cytometry technique combined with orthotopic tumor models might provide insights into tumor hematogenous metastasis and guidance to cancer therapy. Key words: Prostate cancer; Metastasis; Circulating tumor cells; In vivo flow cytometry

Neovasculature and circulating tumor cells dual-targeting nanoparticles for the treatment of the highly-invasive breast cancer.

Antiangiogenesis therapy has been served as a potent cancer treatment strategy for decades, yet disrupting neovasculature would provoke tumor cells into invasive growth and result in distal metastasis. The basic cause of cancer metastasis can be traced down to the presence of circulating tumor cells (CTCs) which detach from primary tumor site and act as 'seeds'. Epithelial cell adhesion molecule (EpCAM) is a potential biomarker for selective capture of epithelium-derived CTCs. Here, we integrated tumor neovessles-targetable ligands K237 peptide with Ep23 aptamer against EpCAM into a single drug-loaded nanoplatform using paclitaxel (PTX) as the model drug, aiming at damaging the primary tumor and neutralizing CTCs simultaneously to achieve a synergistic anti-tumor therapeutic effect. Enhanced cellular uptake, cell apoptosis-induction and cell-viability inhibition efficiency of the peptide and aptamer dual-functionalized nanoparticles (dTNP) were observed in both human umbilical vein endothelial cells (HUVEC) and 4T1 cells invitro. Using cone-and-plate viscometer to create venous flow velocity, dTNP was also found to be able to capture CTCs under shear stress. The CTC-targeting and neutralization effect of dTNP in bloodstream and 4T1-GFP cell-derived lung metastasis mice model was confirmed via invivo flow cytometry (IVFC), intravital imaging and confocal microscopy analysis. As a result, the orthotropic breast tumor-bearing mice administrated with PTX-loaded dTNP exhibited the optimal therapeutic effect. Taken together, the findings here provided direct evidence that the tumor neovasculature and CTCs dual-targeting drug delivery system could provide a novel modality for the treatment of highly-invasive breast cancer.

Circulating tumor cells are correlated with disease progression and treatment response in an orthotopic hepatocellular carcinoma model.

Hepatocellular carcinoma (HCC) is a highly malignant tumor characterized by rapid progression, poor prognosis, and frequent hematogenous metastasis. A minimally invasive diagnostic biomarker that can predict disease progression and treatment response would be of extraordinary benefit. Therefore, we have investigated whether the number of circulating tumor cells (CTCs) is correlated with disease progression and treatment response in HCC. Here we report that the number of CTCs, monitored by in vivo flow cytometry (IVFC), is strongly correlated with disease progression and treatment response in a highly metastatic orthotopic nude mouse model of green fluorescent protein (GFP)-labeled HCC. Sorafenib treatment reduces the number of CTCs significantly. The decreased number of CTCs is consistent with low lung metastasis. This study has demonstrated a considerable clinical value of CTCs as a biomarker in predicting disease progression and monitoring therapeutic efficacy in patients with HCC.

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.