Vivo Flow Cytometry Research Articles

Lipid nanoparticle encapsulated large peritoneal macrophages migrate to the lungs via the systemic circulation in a model of clodronate-mediated lung-resident macrophage depletion.

Rationale: A mature tissue resident macrophage (TRM) population residing in the peritoneal cavity has been known for its unique ability to migrate to peritoneally located injured tissues and impart wound healing properties. Here, we sought to expand on this unique ability of large peritoneal macrophages (LPMs) by investigating whether these GATA6+ LPMs could also intravasate into systemic circulation and migrate to extra-peritoneally located lungs upon ablating lung-resident alveolar macrophages (AMs) by intranasally administered clodronate liposomes in mice. Methods: C12-200 cationic lipidoid-based nanoparticles were employed to selectively deliver a small interfering RNA (siRNA)-targeting CD-45 labeled with a cyanine 5.5 (Cy5.5) dye to LPMs in vivo via intraperitoneal injection. We utilized a non-invasive optical technique called Diffuse In Vivo Flow Cytometry (DiFC) to then systemically track these LPMs in real time and paired it with more conventional techniques like flow cytometry and immunocytochemistry to initially confirm uptake of C12-200 encapsulated siRNA-Cy5.5 (siRNA-Cy5.5 (C12-200)) into LPMs, and further track them from the peritoneal cavity to the lungs in a mouse model of AM depletion incited by intranasally administered clodronate liposomes. Also, we stained for LPM-specific marker zinc-finger transcription factor GATA6 in harvested cells from biofluids like broncho-alveolar lavage as well as whole blood to probe for Cy5.5-labeled LPMs in the lungs as well as in systemic circulation. Results: siRNA-Cy5.5 (C12-200) was robustly taken up by LPMs. Upon depletion of lung-resident AMs, these siRNA-Cy5.5 (C12-200) labeled LPMs rapidly migrated to the lungs via systemic circulation within 12-24 h. DiFC results showed that these LPMs intravasated from the peritoneal cavity and utilized a systemic route of migration. Moreover, immunocytochemical staining of zinc-finger transcription factor GATA6 further confirmed results from DiFC and flow cytometry, confirming the presence of siRNA-Cy5.5 (C12-200)-labeled LPMs in the peritoneum, whole blood and BALF only upon clodronate-administration. Conclusion: Our results indicate for the very first time that selective tropism, migration, and infiltration of LPMs into extra-peritoneally located lungs was dependent on clodronate-mediated AM depletion. These results further open the possibility of therapeutically utilizing LPMs as delivery vehicles to carry nanoparticle-encapsulated oligonucleotide modalities to potentially address inflammatory diseases, infectious diseases and even cancer.

Applications of Optical in Vivo Flow Cytometry in Tumor Metastasis Research (Invited)

Applications of Optical in Vivo Flow Cytometry in Tumor Metastasis Research (Invited)

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.

Fluorescence Labeling of Circulating Tumor Cells with a Folate Receptor-Targeted Molecular Probe for Diffuse In Vivo Flow Cytometry.

We recently developed a new instrument called "diffuse in vivo flow cytometry" (DiFC) for enumeration of rare fluorescently labeled circulating tumor cells (CTCs) in small animals without drawing blood samples. Until now, we have used cell lines that express fluorescent proteins or were pre-labeled with a fluorescent dye ex vivo. In this work, we investigated the use of a folate receptor (FR)-targeted fluorescence molecular probe for in vivo labeling of FR+ CTCs for DiFC. We used EC-17, a FITC-folic acid conjugate that has been used in clinical trials for fluorescence-guided surgery. We studied the affinity of EC-17 for FR+ L1210A and KB cancer cells. We also tested FR- MM.1S cells. We tested the labeling specificity in cells in culture in vitro and in whole blood. We also studied the detectability of labeled cells in mice in vivo with DiFC. EC-17 showed a high affinity for FR+ L1210A and KB cells in vitro. In whole blood, 85.4% of L1210A and 80.9% of KB cells were labeled above non-specific background with EC-17, and negligible binding to FR- MM.1S cells was observed. In addition, EC-17-labeled CTCs were readily detectable in circulation in mice with DiFC. This work demonstrates the feasibility of labeling CTCs with a cell-surface receptor-targeted probe for DiFC, greatly expanding the potential utility of the method for pre-clinical animal models. Because DiFC uses diffuse light, this method could be also used to enumerate CTCs in larger animal models and potentially even in humans.

In Vivo Flow Cytometry of Extremely Rare Circulating Cells

Circulating tumor cells (CTCs) are of great interest in cancer research, but methods for their enumeration remain far from optimal. We developed a new small animal research tool called “Diffuse in vivo Flow Cytometry” (DiFC) for detecting extremely rare fluorescently-labeled circulating cells directly in the bloodstream. The technique exploits near-infrared diffuse photons to detect and count cells flowing in large superficial arteries and veins without drawing blood samples. DiFC uses custom-designed, dual fiber optic probes that are placed in contact with the skin surface approximately above a major vascular bundle. In combination with a novel signal processing algorithm, DiFC allows counting of individual cells moving in arterial or venous directions, as well as measurement of their speed and depth. We show that DiFC allows sampling of the entire circulating blood volume of a mouse in under 10 minutes, while maintaining a false alarm rate of 0.014 per minute. In practice, this means that DiFC allows reliable detection of circulating cells below 1 cell per mL. Hence, the unique capabilities of DiFC are highly suited to biological applications involving very rare cell types such as the study of hematogenous cancer metastasis.

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.

Influenza A Virus Infection in Pigs Attracts Multifunctional and Cross-Reactive T Cells to the Lung.

ABSTRACTPigs are natural hosts for influenza A viruses and play a critical role in influenza epidemiology. However, little is known about their influenza-evoked T-cell response. We performed a thorough analysis of both the local and systemic T-cell response in influenza virus-infected pigs, addressing kinetics and phenotype as well as multifunctionality (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukin-2 [IL-2]) and cross-reactivity. A total of 31 pigs were intratracheally infected with an H1N2 swine influenza A virus (FLUAVsw) and consecutively euthanized. Lungs, tracheobronchial lymph nodes, and blood were sampled during the first 15 days postinfection (p.i.) and at 6 weeks p.i. Ex vivo flow cytometry of lung lymphocytes revealed an increase in proliferating (Ki-67+) CD8+ T cells with an early effector phenotype (perforin+ CD27+) at day 6 p.i. Low frequencies of influenza virus-specific IFN-γ-producing CD4+ and CD8+ T cells could be detected in the lung as early as 4 days p.i. On consecutive days, influenza virus-specific CD4+ and CD8+ T cells produced mainly IFN-γ and/or TNF-α, reaching peak frequencies around day 9 p.i., which were up to 30-fold higher in the lung than in tracheobronchial lymph nodes or blood. At 6 weeks p.i., CD4+ and CD8+ memory T cells had accumulated in lung tissue. These cells showed diverse cytokine profiles and in vitro reactivity against heterologous influenza virus strains, all of which supports their potential to combat heterologous influenza virus infections in pigs.IMPORTANCE Pigs not only are a suitable large-animal model for human influenza virus infection and vaccine development but also play a central role in the emergence of new pandemic strains. Although promising candidate universal vaccines are tested in pigs and local T cells are the major correlate of heterologous control, detailed and targeted analyses of T-cell responses at the site of infection are scarce. With the present study, we provide the first detailed characterization of magnitude, kinetics, and phenotype of specific T cells recruited to the lungs of influenza virus-infected pigs, and we could demonstrate multifunctionality, cross-reactivity, and memory formation of these cells. This, and ensuing work in the pig, will strengthen the position of this species as a large-animal model for human influenza virus infection and will immediately benefit vaccine development for improved control of influenza virus infections in pigs.

In Vivo Myeloperoxidase Imaging and Flow Cytometry Analysis of Intestinal Myeloid Cells.

Myeloperoxidase (MPO) imaging is a non-invasive method to detect cells that produce the enzyme MPO that is most abundant in neutrophils, macrophages, and inflammatory monocytes. While lacking specificity for any of these three cell types, MPO imaging can provide guidance for further flow cytometry-based analysis of tissues where these cell types reside. Isolation of leukocytes from the intestinal tract is an error-prone procedure. Here, we describe a protocol for intestinal leukocyte isolation that works reliable in our hands and allows for flow cytometry-based analysis, in particular of neutrophils.

Real-time Detection of Circulating Apoptotic Cells by in Vivo Flow Cytometry

Survival of cancer cells in the circulation is an important step in metastasis. However, the fate of circulating tumor cells is difficult to assess with conventional methods that require blood sampling. We report the first in situ measurement of circulating apoptotic cells in live animals using in vivo flow cytometry, a novel method [1–3] that enables real-time detection and quantification of circulating cells without blood extraction. Injected cancer cells undergo cell death within 1–2 hr after entering the mouse circulation. Apoptotic cells are rapidly cleared from the circulation with a half-life of ~10 min. Real-time monitoring of circulating apoptotic cells can be useful for detecting early changes in disease processes, as well as for monitoring response to therapeutic intervention. To detect circulating apoptotic cells in vivo, annexin-V conjugated to Alexa Fluor 647 (AF647) was used to label exposed phosphatidylserine on the cell surface. The long wavelength of AF647 fluorescence (Molecular Probes, OR) allows its detection through blood with minimum attenuation by red blood cells. In initial studies conducted to demonstrate that our instrument had sufficient sensitivity to detect individual annexin-Vlabeled apoptotic cells in vivo, we used MatLyLu prostate cancer cells pretreated with camptothecin [4] as a positive control. We verified that >80% of the camptothecin-treated cells undergo apoptosis by conventional flow cytometry (i.e., >80% of the treated cells were FITC–annexin V positive and propidium iodide negative). The camptothecin-treated cells were then labeled with the AF647-conjugated annexin-V and injected into the mouse intravenously. The circulating annexin-V + cells were measured by focusing a He–Ne laser beam onto an ear vessel and detecting the fluorescent bursts as individual cells flowed