Vivo Labeling Research Articles

Visual and Quantitative Analysis of Dietary Fiber-Microbiota Interactions via Metabolic Labeling In Vivo.

Dietary fiber (DF)-based interventions are crucial in establishing a health-promoting gut microbiota. However, directly investigating DFs' in vivo interactions with intestinal bacteria remains challenging due to the lack of suitable tools. Here, we develop an in vivo metabolic labeling-based strategy, which enables not only imaging and identifying the bacteria that bind with specific DF in the intestines, but also quantifying DF's impact on their metabolic status. Four DFs, including galactan, rhamnogalacturonan and two inulins, are fluorescently derivatized and used for in vivo labeling to visually record DFs' interactions with gut bacteria. The subsequent cell-sorting, 16S rDNA sequencing, and fluorescence in situ hybridization identify the taxa that bind each DF. We then select a DF-binding species newly identified herein and verify its DF-catabolizing capability in vitro. Furthermore, we find that the indigenous metabolic status of Gram-positive bacteria, whether inulin-binders or not, is significantly enhanced by the inulin supplement. This trend isn't observed in Gram-negative microbiota, even for the inulin-binders, demonstrating the ability of our methods in differentiating the primary, secondary DF-degraders from cross-feeders, a question that is difficult to answer by using other methods. Our strategy provides a novel chemical biology tool for deciphering the complex DF-bacteria interactions in the gut.

In Vivo Labeling and Detection of Circulating Tumor Cells in Mice Using OTL38

PurposeWe recently developed an optical instrument to non-invasively detect fluorescently labeled circulating tumor cells (CTCs) in mice called ‘Diffuse in vivo Flow Cytometry’ (DiFC). OTL38 is a folate receptor (FR) targeted near-infrared (NIR) contrast agent that is FDA approved for use in fluorescence guided surgery of ovarian and lung cancer. In this work, we investigated the use OTL38 for in vivo labeling and detection of FR + CTCs with DiFC.ProceduresWe tested OTL38 labeling of FR + cancer cell lines (IGROV-1 and L1210A) as well as FR- MM.1S cells in suspensions of Human Peripheral Blood Mononuclear cells (PBMCs) in vitro. We also tested OTL38 labeling and NIR-DIFC detection of FR + L1210A cells in blood circulation in nude mice in vivo.Results62% of IGROV-1 and 83% of L1210A were labeled above non-specific background levels in suspensions of PBMCs in vitro compared to only 2% of FR- MM.1S cells. L1210A cells could be labeled with OTL38 directly in circulation in vivo and externally detected using NIR-DiFC in mice with low false positive detection rates.ConclusionsThis work shows the feasibility of labeling CTCs in vivo with OTL38 and detection with DiFC. Although further refinement of the DiFC instrument and signal processing algorithms and testing with other animal models is needed, this work may eventually pave the way for human use of DiFC.

Non-radioactive In Vivo Labeling of RNA with 4-Thiouracil.

RNA molecules and their expression dynamics play essential roles in the establishment of complex cellular phenotypes and/or in the rapid cellular adaption to environmental changes. Accordingly, analyzing RNA expression remains an important step to understand the molecular basis controlling the formation of cellular phenotypes, cellular homeostasis or disease progression. Steady-state RNA levels in the cells are controlled by the sum of highly dynamic molecular processes contributing to RNA expression and can be classified in transcription, maturation and degradation. The main goal of analyzing RNA dynamics is to disentangle the individual contribution of these molecular processes to the life cycle of a given RNA under different physiological conditions. In the recent years, the use of nonradioactive nucleotide/nucleoside analogs and improved chemistry, in combination with time-dependent and high-throughput analysis, have greatly expanded our understanding of RNA metabolism across various cell types, organisms, and growth conditions.In this chapter, we describe a step-by-step protocol allowing pulse labeling of RNA with the nonradioactive nucleotide analog, 4-thiouracil , in the eukaryotic model organism Saccharomyces cerevisiae and the model archaeon Haloferax volcanii .

In Vivo Labeling and Tracking of Proliferating Corneal Endothelial Cells by 5-Ethynyl-2'-Deoxyuridine in Rabbits.

PurposeTo develop a method to label proliferating corneal endothelial cells (ECs) in rabbits in vivo and track their migration over time.MethodsWe compared intraperitoneal (IP) and intracameral (IC) administration of 5-ethynyl-2′-deoxyuridine (EdU) in two experiments: (1) six rabbits received IP or IC EdU. Blood and aqueous humor (AH) samples were incubated with HL-60 cells. Flow cytometry detected the EdU incorporation, representing the bioavailability of EdU. (2) In vivo EdU labeling was investigated in pulse-chase study: 48 rabbits received EdU IP or IC. The corneas were flat-mounted after 1, 2, 5, or 40 days and imaged using fluorescence microscopy. EdU+ and Ki67+ ECs were quantified and their distance from the peripheral endothelial edge was measured.ResultsEdU was bioavailable in the AH up to 4 hours after IC injection. No EdU was detected in the blood or the AH after IP injection. High quality EdU labeling of EC was obtained only after IC injection, achieving 2047 ± 702 labeled ECs. Proliferating ECs were located exclusively in the periphery within 1458 ± 146 µm from the endothelial edge. After 40 days, 1490 ± 397 label-retaining ECs (LRCs) were detected, reaching 2219 ± 141 µm from the edge, indicating that LRCs migrated centripetally.ConclusionsIC EdU injection enables the labeling and tracking of proliferating ECs. LRCs seem to be involved in endothelial homeostasis, yet it remains to be investigated whether they represent endothelial progenitor cells.Translational RelevanceEdU labeling in animal models can aid the search for progenitor cells and the development of cell therapy for corneal endothelial dysfunction.

Quantification of Bacterial Metabolic Activities in the Gut by d-Amino Acid-Based In Vivo Labeling.

Herein, we propose a metabolic d-amino acid-based labeling and in situ hybridization-facilitated (MeDabLISH) strategy for the quantitative analysis of the indigenous metabolic status of gut bacteria. The fluorescent d-amino acid (FDAA)-based labeling intensities of bacteria were found to highly correlate with their temporal and steady-state metabolic status. Then, after taxonomic identification of bacterial genera in the in vivo FDAA-labeled mouse gut microbiota, by corresponding fluorescence in situ hybridization (FISH) probes, the metabolic activities of different gut bacterial genera are quantified by flow cytometry, using FISH signals to differentiate genera and FDAA signals to indicate their basal metabolic levels. It was found that Gram-negative genera in the mouse microbiota have stronger metabolic activities during the daytime, and Gram-positive genera have higher activities at the night. Our strategy will be instrumental in deepening our understanding of the highly complex microbiota.

Quantification of Bacterial Metabolic Activities in the Gut by d‐Amino Acid‐Based In Vivo Labeling

AbstractHerein, we propose a metabolic d‐amino acid‐based labeling and in situ hybridization‐facilitated (MeDabLISH) strategy for the quantitative analysis of the indigenous metabolic status of gut bacteria. The fluorescent d‐amino acid (FDAA)‐based labeling intensities of bacteria were found to highly correlate with their temporal and steady‐state metabolic status. Then, after taxonomic identification of bacterial genera in the in vivo FDAA‐labeled mouse gut microbiota, by corresponding fluorescence in situ hybridization (FISH) probes, the metabolic activities of different gut bacterial genera are quantified by flow cytometry, using FISH signals to differentiate genera and FDAA signals to indicate their basal metabolic levels. It was found that Gram‐negative genera in the mouse microbiota have stronger metabolic activities during the daytime, and Gram‐positive genera have higher activities at the night. Our strategy will be instrumental in deepening our understanding of the highly complex microbiota.

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins.

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

In Vivo Labeling of Plasma Proteins for Imaging of Enhanced Vascular Permeability in the Lungs.

Increased vascular permeability is an important hallmark of many diseases, including cancer, cerebral ischemia, and severe inflammatory disorders. In this regard, the noninvasive assessment of pathologically increased vascular permeability in vivo is of great interest. In this study, the potential of albumin- and transthyretin-binding radioligands was evaluated for imaging of vascular hyperpermeability. For this purpose, the bleomycin-induced lung injury model was used as a model of inflammation-associated vascular leakage. The plasma protein-binding ligands, which bind to albumin (DOTA-PPB-01) and transthyretin (DOTA-PPB-03), were radiolabeled and used for nuclear imaging and biodistribution studies. In this regard, 177Lu was employed as a surrogate nuclide for detailed preclinical investigations, including single-photon emission computed tomography (SPECT) studies, whereas 44Sc was proposed as a radionuclide for positron emission tomography (PET), which may be relevant for future clinical translation. Mice were administered with these radioligands 6-9 days after intratracheal instillation of bleomycin or saline. Bleomycin-treated mice developed pronounced lung inflammation with enhanced vascular permeability that was reflected in significantly increased lung size and weight due to edema and infiltration with inflammatory cells. Biodistribution studies revealed significantly higher accumulation of 177Lu-DOTA-PPB-01 in injured lungs as compared to lungs of control animals at all investigated time points (4-48 h p.i.). The best contrast was achieved at late time points (16.1 ± 2.91% IA/g vs 2.03 ± 1.22% IA/g, 48 h p.i.) when the blood activity levels were ∼7.5% IA/g. Injection of 177Lu-DOTA-PPB-03 also resulted in increased lung accumulation in bleomycin-treated mice at all investigated time points (2-8 h p.i.). The pharmacokinetics was significantly faster, however, resulting in good contrast already at 8 h p.i. (4.32 ± 0.85% IA/g vs 1.06 ± 0.10% IA/g) when blood activity levels were ∼2% IA/g. The absolute lung accumulation of 177Lu-DOTA-PPB-03 was significantly lower than that of 177Lu-DOTA-PPB-01. PET/CT scans performed with 44Sc-DOTA-PPB-01 distinguished injured from healthy lungs only at late time points (20 h p.i.), whereas 44Sc-DOTA-PPB-03 already allowed the differentiation at 4 h p.i. due to its faster clearance. The investigated radioligands, 44Sc/177Lu-DOTA-PPB-01 and 44Sc/177Lu-DOTA-PPB-03, hold promise for the visualization of vascular leakage in a variety of pathological conditions. 44Sc would be the radionuclide of choice for clinical application as it can be stably coordinated with a DOTA chelator and enables PET imaging over extended periods.

Correlating Anatomy and Function with Gene Expression in Individual Neurons by Combining in Vivo Labeling, Patch Clamp, and Single Cell RNA-seq.

The classification of neurons into distinct types is an ongoing effort aimed at revealing and understanding the diversity of the components of the nervous system. Recently available methods allow us to determine the gene expression pattern of individual neurons in the mammalian cerebral cortex to generate powerful categorization schemes. For a thorough understanding of neuronal diversity such genetic categorization schemes need to be combined with traditional classification parameters like position, axonal projection or response properties to sensory stimulation. Here we describe a method to link the gene expression of individual neurons with their position, axonal projection, or sensory response properties. Neurons are labeled in vivo based on their anatomical or functional properties and, using patch clamp pipettes, their RNA individually harvested in vitro for RNAseq. We validate the methodology using multiple established molecularly and anatomically distinct cell populations and explore molecular differences between uncharacterized neurons in mouse visual cortex. Gene expression patterns between L5 neurons projecting to frontal or contralateral cortex are distinct while L2 neurons differing in position, projection, or function are molecularly similar. With this method we can determine the genetic expression pattern of functionally and anatomically identified individual neurons.

Metabolic in Vivo Labeling Highlights Differences of Metabolically Active Microbes from the Mucosal Gastrointestinal Microbiome between High-Fat and Normal Chow Diet.

The gastrointestinal microbiota in the gut interacts metabolically and immunologically with the host tissue in the contact zone of the mucus layer. For understanding the details of these interactions and especially their dynamics it is crucial to identify the metabolically active subset of the microbiome. This became possible by the development of stable isotope probing techniques, which have only sparsely been applied to microbiome research. We applied the in vivo stable isotope approach using 15N-labeled diet with subsequent identification of metabolically active bacterial species. Four-week old male Sprague-Dawley rats were randomly assigned to chow diet (CD, n =15) and high-fat diet (HFD, n =15). After 11 weeks, three animals from each group were sacrificed for baseline characterization of anthropometric and metabolic obesity. The remaining animals were exposed to either a 15N-labeled (n =9) or a 14N-unlabeled experimental diet (n =3). Three rats from each cohort (HFD and CD) were sacrificed at 12, 24, and 72 h. The remaining three animals from each cohort, which received the 14N-unlabeled diet, were sacrificed after 72 h. The colon was harvested and divided into three equal sections (proximal, medial, and distal), and the mucus layer of each specimen was sampled by scraping. We identified the active subset in an HFD model of obesity in comparison with lean controls rats using metaproteomics. In addition, all samples were investigated by 16S rRNA amplicon gene sequencing. The active microbiome of the HFD group showed an increase in bacterial taxa for Verrucomicrobia and Desulfovibrionaceae. In contrast with no significant changes in alpha diversity, time- and localization-dependent effects in beta-diversity were clearly observed. In terms of enzymatic functions the HFD group showed strong affected metabolic pathways such as energy production and carbohydrate metabolism. In vivo isotope labeling combined with metaproteomics provides a valuable method to distinguish the active from the non-active bacterial phylogenetic groups that are relevant for microbiota-host interaction. For morbid obesity such analysis may provide potentially new strategies for targeted pre- or probiotic treatments.

Epicardium-Derived Cells Formed After Myocardial Injury Display Phagocytic Activity Permitting In Vivo Labeling and Tracking.

Epicardium-derived cells (EPDCs) cover the heart surface and can function as a source of both progenitor cells and trophic factors for cardiac repair. Currently, EPDCs cannot be conveniently labeled in vivo to permit imaging and cell tracking. EPDCs formed after myocardial infarction (MI) preferentially take up a perfluorocarbon-containing nanoemulsion (PFC-NE; 130 ± 32 nm) injected 3 days after injury, as measured by (19)F-magnetic resonance imaging ((19)F-MRI). Flow cytometry, immune electron microscopy, and green fluorescent protein (GFP)-transgenic rats (only immune cells, but not epicardial cells, are GFP(+)) demonstrated that PFC-containing EPDCs are nonhematopoietic (CD45(-)/CD11b(-)) but stain positive for markers of mesenchymal stem cells such as platelet-derived growth factor receptor α (PDGFR-α) CD73, CD105, and CD90. When rhodamine-coupled PFC-NE was used, we found that ρ(+) vessel-like structures formed within the infarcted myocardium, comprising approximately 10% of all large vessels positive for smooth muscle actin (SM-actin). The epicardial cell layer, positive for Wilms' tumor 1 (WT-1), PDGFR-α, or KI-67, was shown to be well capillarized (293 ± 78 capillaries per mm(2)), including fenestrated endothelium. Freshly isolated EPDCs were positive for WT-1, GATA-4, KI-67, and FLK-1 (75%), PDGFR-α (50%), and SM-actin (28%) and also exhibited a high capacity for nanoparticle and cell debris uptake. This study demonstrates that EPDCs formed after MI display strong endocytic activity to take up i.v.-injected labeled nanoemulsions. This feature permitted in vivo labeling and tracking of EPDCs, demonstrating their role in myo- and vasculogenesis. The newly discovered endocytic activity permits in vivo imaging of EPDCs with (19)F-MRI and may be used for the liposomal delivery of substances to further study their reparative potential. The present study reports that epicardium-derived cells (EPDCs) formed after myocardial infarction can specifically endocytose nanoparticles in vivo and in vitro. This novel feature permitted in vivo targeting of EPDCs with either a perfluorocarbon-containing or rhodamine-conjugated nanoemulsion to track migration and fate decision of EPDC with (19)F-magnetic resonance imaging and fluorescence microscopy. The liposomal nanoemulsions used in the present study may be useful in the future as a nanomedical device for the delivery of substances to direct cell fate of EPDCs.

Specific In Vivo Labeling of Tyrosinated α-Tubulin and Measurement of Microtubule Dynamics Using a GFP Tagged, Cytoplasmically Expressed Recombinant Antibody

GFP-tagged proteins are used extensively as biosensors for protein localization and function, but the GFP moiety can interfere with protein properties. An alternative is to indirectly label proteins using intracellular recombinant antibodies (scFvs), but most antibody fragments are insoluble in the reducing environment of the cytosol. From a synthetic hyperstable human scFv library we isolated an anti-tubulin scFv, 2G4, which is soluble in mammalian cells when expressed as a GFP-fusion protein. Here we report the use of this GFP-tagged scFv to label microtubules in fixed and living cells. We found that 2G4-GFP localized uniformly along microtubules and did not disrupt binding of EB1, a protein that binds microtubule ends and serves as a platform for binding by a complex of proteins regulating MT polymerization. TOGp and CLIP-170 also bound microtubule ends in cells expressing 2G4-GFP. Microtubule dynamic instability, measured by tracking 2G4-GFP labeled microtubules, was nearly identical to that measured in cells expressing GFP-α-tubulin. Fluorescence recovery after photobleaching demonstrated that 2G4-GFP turns over rapidly on microtubules, similar to the turnover rates of fluorescently tagged microtubule-associated proteins. These data indicate that 2G4-GFP binds relatively weakly to microtubules, and this conclusion was confirmed in vitro. Purified 2G4 partially co-pelleted with microtubules, but a significant fraction remained in the soluble fraction, while a second anti-tubulin scFv, 2F12, was almost completely co-pelleted with microtubules. In cells, 2G4-GFP localized to most microtubules, but did not co-localize with those composed of detyrosinated α-tubulin, a post-translational modification associated with non-dynamic, more stable microtubules. Immunoblots probing bacterially expressed tubulins confirmed that 2G4 recognized α-tubulin and required tubulin’s C-terminal tyrosine residue for binding. Thus, a recombinant antibody with weak affinity for its substrate can be used as a specific intracellular biosensor that can differentiate between unmodified and post-translationally modified forms of a protein.

Spiked-in Pulsed in Vivo Labeling Identifies a New Member of the CCN Family in Regenerating Newt Hearts

The newt Notophthalmus viridescens , which belongs to the family of salamanders (Urodela), owns remarkable regenerative capacities allowing efficient scar-free repair of various organs including the heart. Salamanders can regrow large parts of the myocardium unlike mammals, which cannot replace lost cardiomyocytes efficiently. Unfortunately, very little is known about the molecules and the regulatory circuits facilitating efficient heart regeneration in newts or salamanders. To identify proteins that are involved in heart regeneration, we have developed a pulsed SILAC-based mass spectrometry method based on the detection of paired peptide peaks after ¹³C₆-lysine incorporation into proteins in vivo. Proteins were identified by matching mass spectrometry derived peptide sequences to a recently established normalized newt EST library. Our approach enabled us to identify more than 2200 nonredundant proteins in the regenerating newt heart. Because of the pulsed in vivo labeling approach, accurate quantification was achieved for 1353 proteins, of which 72 were up- and 31 down-regulated with a (|log 2 ratio| > 1) during heart regeneration. One deregulated member was identified as a new member of the CCN protein family, showing a wound specific activation. We reason that the detection of such deregulated newt-specific proteins in regenerating hearts supports the idea of a local evolution of tissue regeneration in salamanders. Our results significantly improve understanding of dynamic changes in the complex protein network that underlies heart regeneration and provides a basis for further mechanistic studies.

In Vivo Labeling of Cortical Astrocytes with Sulforhodamine 101 (SR101)

Fluorescent markers that stain particular cell types in the intact brain are essential tools for fluorescence microscopy because they enable studies of structure and function of cells identified in this way. Although cell type-specific fluorescence staining can be achieved through promoter-driven expression of fluorescent proteins, this genetic approach is generally labor- and cost-intensive. Alternative viral approaches for targeted fluorophore expression are relatively invasive. For astrocytes, there is a simple alternative. This protocol describes an easy and robust method for rapid (within minutes) and high-contrast staining of astrocytes in defined regions of the intact rodent cortex using the synthetic, water-soluble but non-fixable red fluorescent dye sulforhodamine 101 (SR101). Selective staining is achieved through local uptake and gap junction-mediated spread of SR101 following its topical application or injection into tissue. Applications, technical pitfalls, and limitations of the SR101-staining technique are discussed. Given its simplicity and reliability, SR101 staining is a valuable tool for the study of astrocyte function in the intact brain and for in vivo fluorescence microscopy in general.

In Vivo Labeling of Brain Capillary Endothelial Cells after Intravenous Injection of Monoclonal Antibodies Targeting the Transferrin Receptor

The development of vectors for drug delivery to the central nervous system remains a major pharmaceutical challenge. Here, we have characterized the brain distribution of two monoclonal antibodies (MAbs) targeting the mouse transferrin receptor (TfR) (clones Ri7 and 8D3) compared with control IgGs after intravenous injection into mice. MAbs were fluorolabeled with either Alexa Fluor (AF) dyes 647 or 750. Intravenous injection of Ri7 or 8D3 MAb coupled with AF750 led to higher fluorescence emission in brain homogenates compared with control IgGs, indicating retention in the brain. Fluorescence microscopy analysis revealed that AF647-Ri7 signal was confined to brain cerebrovasculature, colocalizing with an antibody against collagen IV, a marker of basal lamina. Confocal microscopy analysis confirmed the delivery of injected Ri7 MAb into brain endothelial cells using the pericyte marker anti-α-smooth muscle actin, the endothelial marker CD31, and the collagen IV antibody. No evidence of colocalization was detected with neurons or astrocytes identified using antibodies specific for neuronal nuclei or glial fibrillary acidic protein, respectively. Our data show that anti-TfR vectors injected intravenously readily accumulate into brain capillary endothelial cells, thus displaying strong drug-targeting potential.

In Vivo Labeling of Mammalian Cone Photoreceptors by Intravitreal Injection of Fluorescently Tagged Peanut Agglutinin - A Potential Tool for Assessing Photoreceptor Degeneration in Humans

In Vivo Labeling of Mammalian Cone Photoreceptors by Intravitreal Injection of Fluorescently Tagged Peanut Agglutinin - A Potential Tool for Assessing Photoreceptor Degeneration in Humans

In Vivo Labeling Method Using a Genetic Construct for Nanoscale Resolution Microscopy

We demonstrate beam scanning-stimulated emission depletion microscopy with in vivo labeled cells. A red emitting fluorescent dye is introduced into membrane protein fused to a multifunctional reporter protein (HaloTag, Promega, Madison, WI) in live cells. This approach allows superresolution stimulated emission depletion imaging without the limitations of immunofluorescence-based staining.

The Stability and Transactivation Potential of the Mammalian MafA Transcription Factor Are Regulated by Serine 65 Phosphorylation

The level of the MafA transcription factor is regulated by a variety of effectors of beta cell function, including glucose, fatty acids, and insulin. Here, we show that phosphorylation at Ser(65) of mammalian MafA influences both protein stability and transactivation potential. Replacement of Ser(65) with Glu to mimic phosphorylation produced a protein that was as unstable as the wild type, whereas Asp or Ala mutation blocked degradation. Analysis of MafA chimeric and deletion constructs suggests that protein phosphorylation at Ser(65) alone represents the initial degradation signal, with ubiquitinylation occurring within the C terminus (amino acids 234-359). Although only wild type MafA and S65E were polyubiquitinylated, both S65D and S65E potently stimulated transactivation compared with S65A. Phosphorylation at Ser(14) also enhanced activation, although it had no impact on protein turnover. The mobility of MafA S65A was profoundly affected upon SDS-PAGE, with the S65E and S65D mutants influenced less due to their ability to serve as substrates for glycogen synthase kinase 3, which acts at neighboring N-terminal residues after Ser(65) phosphorylation. Our observations not only illustrate the sensitivity of the cellular transcriptional and degradation machinery to phosphomimetic mutants at Ser(65), but also demonstrate the singular importance of phosphorylation at this amino acid in regulating MafA activity.

Stable and Non-Disruptive In Vitro/In Vivo Labeling of Mesenchymal Stem Cells by Internalizing Quantum Dots

Stable and Non-Disruptive In Vitro/In Vivo Labeling of Mesenchymal Stem Cells by Internalizing Quantum Dots

In Vivo Labeling of Dividing Chronic Lymphocytic Leukemia B Cells Suggests That CXCR4 and CD5 Define the Clonal Subfraction That Recently Proliferatead and Emigrated from a Solid Tissue

B-cell chronic lymphocytic leukemia (B-CLL) is due, in most of the cases, to the slow progressive invasion of phenotypically homogeneous monoclonal B cells. Several studies are clarifying that B-CLL cells preferentially proliferate in the context of lymphoid solid tissues where they receive accessory signals (chemokines, cytokines, cell to cell contact). Deuterium (2H) incorporation into newly synthesized DNA can be used as a direct in vivo measure of cell division. Using this technique in B-CLL, it has been found that although the fraction of cells dividing each day is small (0.1 – ~2% of the clones), the absolute number of proliferating cells is sizeable (~109 – 1012/day). The study also elucidated a relationship between proliferation and disease activity. By using this same approach, we have tried to refine our understanding of this small percentage of proliferating cells. Thirteen patients drank deuterated water (2H2O) for 6–12 weeks (“labeling period”) and were followed for 24 weeks (“washout period”). Blood was drawn during and at the end of labeling (1st, 2nd time points) and at the end of washout (3rd time point). Based upon proliferation kinetics patients could be divided into 2 groups (Group A and Group B). Four/13 patients (Group A) showed unusual kinetic curves with a strikingly delayed appearance of labeled cells in the blood as compared to Group B patients. This suggested that B-CLL cells of these patients were retained longer at the site of proliferation. Therefore, the two groups were compared for expression of a panel of chemokine receptors (CCR1, CCR2, CCR4, CCR5, CCR7, CXCR1, CXCR3, CXCR4, and CXCR5) by flow cytometry. Only CXCR4 was found at significant higher densities in Group A compared to Group B (P = 0.002). CXCR4 expression was then used to identify B-CLL sub-populations enriched in cells displaying Ki67 and MCM6, two cell cycle related markers. We observed a relationship between CXCR4 and CD5 where CXCR4dim cells tended to be CD5bright and vice-versa with the majority of the cells falling in an intermediate category (CXCR4intCD5int). The CXCR4dimCD5bright subpopulation contained significantly more Ki67+ and MCM6+ cells. Based on this observation, B-CLL cells of 9 patients were sorted into CXCR4brightCD5dim, CXCR4intCD5int, CXCR4dimCD5bright and 2H was measured for each fraction. A hierarchy of 2H enrichment in the fractions was found: CXCR4dimCD5bright > CXCR4intCD5int >CXCR4brightCD5dim. These differences were statistically significant during labelling period (1st and 2nd time point) but completely disappeared at the end of washout (3rd time point). The 2H enrichment measured in the unfractionated clones at the 1st, 2nd and 3rd time point was similar (0.45±0.15 vs. 0.53±0.16 vs. 0.45±0.07, P = ns), suggesting a redistribution of label among the fractions, presumably due to a change of phenotype over time. In vitro studies have suggested that CXCR4 has a crucial role in trafficking and survival of B-CLL cells, and it is emerging as a powerful predictor for bone marrow infiltration and clinical evolution. CXCR4 surface expression is downregulated after encountering its ligand SDF-1. Our data indicate a relationship between CXCR4 levels and B-CLL cells kinetics. Moreover, since CLL cell subpopulations defined by relative CXCR4 and CD5 levels differ in the number of proliferating cells, we suggest that these markers ca be used to define cells that have emigrated recently (CXCR4dimCD5bright) or much earlier (CXCR4brightCD5dim) from the solid tissue in which they divided. The results strongly suggest, in vivo, the important role of the stroma and SDF-1-producing cells for B-CLL cell proliferation providing indications for disease control strategies.