Live Cell Tracking Research Articles

Multi-Dimensional single particle tracking in live cells

Optical microscopy imaging has become an indispensable approach in understanding dynamic biological processes in complex cellular environments. Biological processes usually involve multiple species (incl. biomolecules and subcellular organelles) and their chemical and physical responses to each other and/or to the environment. It is highly desired, often quintessential, to harvest the rich properties of photons for visualizing not only the sample morphology (location, thickness, density, etc.) but also the dynamics, including motion, orientation, binding kinetics, and transient states, of target biological and chemical structures and single molecules.

Long-term 3D live cell tracking and morphological studies by telecentric digital holographic microscopy with digital focusing correction

A continuous long-term quantitative 3D imaging system for live cells is one of the essential requirements to study fast cellular dynamics. Confocal microscopy is a conventional volumetric imaging technique that enables morphological studies of cells and organisms, but the need to scan the sample to form a volumetric image limits the temporal resolution of the system. Phototoxicity and photobleaching are also inevitable problems for fluorescence microscopy, and faster dynamics require better temporal resolution.

A Cas6-based RNA tracking platform functioning in a fluorescence-activation mode.

Given the fact that the localization of RNAs is closely associated with their functions, techniques developed for tracking the distribution of RNAs in live cells have greatly advanced the study of RNA biology. Recently, innovative application of fluorescent protein-labelled Cas9 and Cas13 into live-cell RNA tracking further enriches the toolbox. However, the Cas9/Cas13 platform, as well as the widely-used MS2-MCP technique, failed to solve the problem of high background noise. It was recently reported that CRISPR/Cas6 would exhibit allosteric alteration after interacting with the Cas6 binding site (CBS) on RNAs. Here, we exploited this feature and designed a Cas6-based switch platform for detecting target RNAs in vivo. Conjugating split-Venus fragments to both ends of the endoribonuclease-mutated Escherichia coli Cas6(dEcCas6) allowed ligand (CBS)-activated split-Venus complementation. We name this platform as Cas6 based Fluorescence Complementation (Cas6FC). In living cells, Cas6FC could detect target RNAs with nearly free background noise. Moreover, as minimal as one copy of CBS (29nt) tagged in an RNA of interest was able to turn on Cas6FC fluorescence, which greatly reduced the odds of potential alteration of conformation and localization of target RNAs. Thus, we developed a new RNA tracking platform inherently with high sensitivity and specificity.

Opportunities for agent based modeling of retinal stem cell transplantation.

Opportunities for agent based modeling of retinal stem cell transplantation.

Multicolor Photoactivatable Raman Probes for Subcellular Imaging and Tracking by Cyclopropenone Caging.

Photoactivatable probes, with high-precision spatial and temporal control, have largely advanced bioimaging applications, particularly for fluorescence microscopy. While emerging Raman probes have recently pushed the frontiers of Raman microscopy for noninvasive small-molecule imaging and supermultiplex optical imaging with superb sensitivity and specificity, photoactivatable Raman probes remain less explored. Here, we report the first general design of multicolor photoactivatable alkyne Raman probes based on cyclopropenone caging for live-cell imaging and tracking. The fast photochemically generated alkynes from cyclopropenones enable background-free Raman imaging with desired photocontrollable features. We first synthesized and spectroscopically characterized a series of model cyclopropenones and identified the suitable light-activating scaffold. We further engineered the scaffold for enhanced chemical stability in a live-cell environment and improved Raman sensitivity. Organelle-targeting probes were then generated to achieve targeted imaging of mitochondria, lipid droplets, endoplasmic reticulum, and lysosomes. Multiplexed photoactivated imaging and tracking at both subcellular and single-cell levels was next demonstrated to monitor the dynamic migration and interactions of the cellular contents. We envision that this general design of multicolor photoactivatable Raman probes would open up new ways for spatial-temporal controlled profiling and interrogations in complex biological systems with high information throughput.

Notch signaling mediates olfactory multiciliated cell specification.

Epithelial multiciliated cells (MCCs) use motile cilia to direct external fluid flow, the disruption of which is associated with human diseases in a broad array of organs such as those in the respiratory, reproductive, and renal systems. While many of the signaling pathways that regulate MCC formation in these organ systems have been identified, similar characterization of MCC differentiation in the developing olfactory system has been lacking. Here, using live cell tracking, targeted cell ablation, and temporally-specific inhibition of the Notch signaling pathway, we identify the earliest time window of zebrafish olfactory MCC (OMCC) differentiation and demonstrate these cells' derivation from peridermal cells. We also describe regionally segregated Notch signaling across time points of rapid OMCC differentiation and show that Notch signaling downregulation yields an increase in OMCCs, suggesting that OMCC fate is normally repressed in a region-specific manner during olfactory development. Finally, we describe Notch signaling's regulation of the differentiation/ciliogenesis-associated genes foxj1a and foxj1b. Taken together, these findings provide new insights into the origins and developmental programming of OMCCs in vivo.

Live Plant Cell Tracking: Fiji plugin to analyze cell proliferation dynamics and understand morphogenesis.

Arabidopsis (Arabidopsis thaliana) primary and lateral roots (LRs) are well suited for 3D and 4D microscopy, and their development provides an ideal system for studying morphogenesis and cell proliferation dynamics. With fast-advancing microscopy techniques used for live-imaging, whole tissue data are increasingly available, yet present the great challenge of analyzing complex interactions within cell populations. We developed a plugin "Live Plant Cell Tracking" (LiPlaCeT) coupled to the publicly available ImageJ image analysis program and generated a pipeline that allows, with the aid of LiPlaCeT, 4D cell tracking and lineage analysis of populations of dividing and growing cells. The LiPlaCeT plugin contains ad hoc ergonomic curating tools, making it very simple to use for manual cell tracking, especially when the signal-to-noise ratio of images is low or variable in time or 3D space and when automated methods may fail. Performing time-lapse experiments and using cell-tracking data extracted with the assistance of LiPlaCeT, we accomplished deep analyses of cell proliferation and clonal relations in the whole developing LR primordia and constructed genealogical trees. We also used cell-tracking data for endodermis cells of the root apical meristem (RAM) and performed automated analyses of cell population dynamics using ParaView software (also publicly available). Using the RAM as an example, we also showed how LiPlaCeT can be used to generate information at the whole-tissue level regarding cell length, cell position, cell growth rate, cell displacement rate, and proliferation activity. The pipeline will be useful in live-imaging studies of roots and other plant organs to understand complex interactions within proliferating and growing cell populations. The plugin includes a step-by-step user manual and a dataset example that are available at https://www.ibt.unam.mx/documentos/diversos/LiPlaCeT.zip.

Toward Understanding the Binding Synergy of Trastuzumab and Pertuzumab to Human Epidermal Growth Factor Receptor 2

Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast, gastric, esophageal, ovarian, and endometrial cancer. Combination therapy using trastuzumab and pertuzumab antibodies targeting HER2 has shown better survival outcomes in breast cancer patients. In the quest to understand the synergistic effect observed due to combination therapy, trastuzumab, pertuzumab, and their F(ab')2 fragments were labeled with radioisotope and fluorescent probes. Detailed in vitro studies to understand binding synergism in HER2 overexpressing cell lines were done. Antibodies and their F(ab')2 fragments prepared by enzyme digestion with pepsin were radiolabeled with iodine-125. In vitro binding studies to evaluate immunoreactivity, specificity, affinity, and binding synergism between radiolabeled trastuzumab, pertuzumab, and their F(ab')2 fragments were carried out. Synergism was observed by 20-30% enhanced uptake of radiolabeled pertuzumab and its F(ab')2 fragments in the presence of excess of unlabeled trastuzumab or F(ab')2-trastuzumab. However, uptake of radiolabeled trastuzumab was not enhanced in the presence of excess pertuzumab or its fragments; rather inhibition or competition in binding to HER2 was observed. Studies using fluorescent antibodies by flow cytometry confirmed enhanced binding of pertuzumab in the presence of trastuzumab. Live cell tracking was done to give insights into the binding synergy and fate of fluorescent antibodies . Colocalization of antibodies on HER2 followed by internalization in the cells was observed. The radiolabeled immunoconjugates served as an important tool for experimental characterization of interaction between pertuzumab and trastuzumab to HER2. Studies with fluorescent antibodies corroborated the binding data and provided evidence of colocalization and internalization of both the antibodies in HER2-positive cells.

Pulpbow: A Method to Study the Vasculogenic Potential of Mesenchymal Stem Cells from the Dental Pulp.

Understanding how Mesenchymal Stem Cells (MSCs) form blood vessels is critical for creating mechanism-based approaches for the therapeutic use of these cells. In addition, understanding the determinants and factors involved in lineage hierarchy is fundamental to creating accurate and reliable techniques for the study of stem cells in tissue engineering and repair. Dental Pulp Stem Cells (DPSC) from permanent teeth and Stem cells from Human Exfoliated Deciduous teeth (SHED) are particularly interesting sources for tissue engineering as they are easily accessible and expandable. Previously, we have shown that DPSCs and SHEDs can differentiate into endothelial cells and form functional blood vessels through vasculogenesis. Here, we described how we created the “pulpbow” (pulp + rainbow), a multicolor tag experimental model that is stable, permanent, unique to each cell and passed through generations. We used the pulpbow to understand how dental pulp stem cells contributed to blood vessel formation in 3D models in in vitro and ex vivo live cell tracking, and in vivo transplantation assays. Simultaneous tracking of cells during sprout formation revealed that no single multicolor-tagged cell was more prone to vasculogenesis. During this process, there was intense cell motility with minimal proliferation in early time points. In later stages, when the availability of undifferentiated cells around the forming sprout decreased, there was local clonal proliferation mediated by proximity. These results unveiled that the vasculogenesis process mediated by dental pulp stem cells is dynamic and proximity to the sprouting area is critical for cell fate decisions.

The zebrafish presomitic mesoderm elongates through compaction-extension.

In vertebrate embryos the presomitic mesoderm becomes progressively segmented into somites at the anterior end while extending along the anterior-posterior axis. A commonly adopted model to explain how this tissue elongates is that of posterior growth, driven in part by the addition of new cells from uncommitted progenitor populations in the tailbud. However, in zebrafish, much of somitogenesis is associated with an absence of overall volume increase, and posterior progenitors do not contribute new cells until the final stages of somitogenesis. Here, we perform a comprehensive 3D morphometric analysis of the paraxial mesoderm and reveal that extension is linked to a volumetric decrease and an increase in cell density. We also find that individual cells decrease in volume over successive somite stages. Live cell tracking confirms that much of this tissue deformation occurs within the presomitic mesoderm progenitor zone and is associated with non-directional rearrangement. Taken together, we propose a compaction-extension mechanism of tissue elongation that highlights the need to better understand the role tissue intrinsic and extrinsic forces in regulating morphogenesis.

Human Cytomegalovirus RNA2.7 Is Required for Upregulating Multiple Cellular Genes To Promote Cell Motility and Viral Spread Late in Lytic Infection.

ABSTRACTLong noncoding RNAs (lncRNAs) are frequently associated with broad modulation of gene expression and thus provide the cell with the ability to synchronize entire metabolic processes. We used transcriptomic approaches to investigate whether the most abundant human cytomegalovirus-encoded lncRNA, RNA2.7, has this characteristic. By comparing cells infected with wild-type virus (WT) to cells infected with RNA2.7 deletion mutants, RNA2.7 was implicated in regulating a large number of cellular genes late in lytic infection. Pathway analysis indicated that >100 of these genes are associated with promoting cell movement, and the 10 most highly regulated of these were validated in further experiments. Morphological analysis and live cell tracking of WT- and RNA2.7 mutant-infected cells indicated that RNA2.7 is involved in promoting the movement and detachment of infected cells late in infection, and plaque assays using sparse cell monolayers indicated that RNA2.7 is also involved in promoting cell-to-cell spread of virus. Consistent with the observation that upregulated mRNAs are relatively A+U-rich, which is a trait associated with transcript instability, and that they are also enriched in motifs associated with mRNA instability, transcriptional inhibition experiments on WT- and RNA2.7 mutant-infected cells showed that four upregulated transcripts lived longer in the presence of RNA2.7. These findings demonstrate that RNA2.7 is required for promoting cell movement and viral spread late in infection and suggest that this may be due to general stabilization of A+U-rich transcripts.IMPORTANCE In addition to messenger RNAs (mRNAs), the human genome encodes a large number of long noncoding RNAs (lncRNAs). Many lncRNAs that have been studied in detail are associated with broad modulation of gene expression and have important biological roles. Human cytomegalovirus, which is a large, clinically important DNA virus, specifies four lncRNAs, one of which (RNA2.7) is expressed at remarkably high levels during lytic infection. Our studies show that RNA2.7 is required for upregulating a large number of human genes, about 100 of which are associated with cell movement, and for promoting the movement of infected cells and the spread of virus from one cell to another. Further bioinformatic and experimental analyses indicated that RNA2.7 may exert these effects by stabilizing mRNAs that are relatively rich in A and U nucleotides. These findings increase our knowledge of how human cytomegalovirus regulates the infected cell to promote its own success.

Basal epidermis collective migration and local Sonic hedgehog signaling promote skeletal branching morphogenesis in zebrafish fins

Teleost fish fins, like all vertebrate limbs, comprise a series of bones laid out in characteristic pattern. Each fin's distal bony rays typically branch to elaborate skeletal networks providing form and function. Zebrafish caudal fin regeneration studies suggest basal epidermal-expressed Sonic hedgehog (Shh) promotes ray branching by partitioning pools of adjacent pre-osteoblasts. This Shh role is distinct from its well-studied Zone of Polarizing Activity role establishing paired limb positional information. Therefore, we investigated if and how Shh signaling similarly functions during developmental ray branching of both paired and unpaired fins while resolving cellular dynamics of branching by live imaging. We found shha is expressed uniquely by basal epidermal cells overlying pre-osteoblast pools at the distal aspect of outgrowing juvenile fins. Lateral splitting of each shha-expressing epidermal domain followed by the pre-osteoblast pools precedes overt ray branching. We use ptch2:Kaede fish and Kaede photoconversion to identify short stretches of shha+basal epidermis and juxtaposed pre-osteoblasts as the Shh/Smoothened (Smo) active zone. Basal epidermal distal collective movements continuously replenish each shha+domain with individual cells transiently expressing and responding to Shh. In contrast, pre-osteoblasts maintain Shh/Smo activity until differentiating. The Smo inhibitor BMS-833923 prevents branching in all fins, paired and unpaired, with surprisingly minimal effects on caudal fin initial skeletal patterning, ray outgrowth or bone differentiation. Staggered BMS-833923 addition indicates Shh/Smo signaling acts throughout the branching process. We use live cell tracking to find Shh/Smo restrains the distal movement of basal epidermal cells by apparent ‘tethering’ to pre-osteoblasts. We propose short-range Shh/Smo signaling promotes these heterotypic associations to couple instructive basal epidermal collective movements to pre-osteoblast repositioning as a unique mode of branching morphogenesis.

Self-assembled fibrinogen nanofibers support fibroblast adhesion and prevent E. coli infiltration

Fibrinogen nanofibers hold great potential for wound healing applications since they mimic the native blood clot architecture and offer important binding sites to support fibroblast adhesion and migration. Recently, we introduced a new method of salt-induced self-assembly to prepare nanofibrous fibrinogen scaffolds. Here, we present our results on the mechanical properties of these scaffolds and their interaction with 3T3 fibroblasts and E. coli bacteria, which we used as model systems for wound healing. Hydrated, nanofibrous fibrinogen scaffolds showed a Young's modulus of 1.3 MPa, which is close to the range of native fibrin. 3T3 fibroblasts adhered and proliferated well on nanofibrous and planar fibrinogen up to 72 h with a less pronounced actin cytoskeleton on nanofibers in comparison to planar fibrinogen. Fibroblasts on nanofibers were smaller with many short filopodia while larger cells with few long filopodia were found on planar fibrinogen. Live cell tracking revealed higher migration velocities on nanofibers in comparison to planar fibrinogen. The growth of E. coli bacteria on nanofibrous fibrinogen was significantly reduced as compared to agar controls with no bacteria migrating through the nanofibers. In summary, we conclude that self-assembled fibrinogen nanofibers could become highly attractive as future scaffolds for wound healing applications.

Human naive epiblast cells possess unrestricted lineage potential.

SummaryClassic embryological experiments have established that the early mouse embryo develops via sequential lineage bifurcations. The first segregated lineage is the trophectoderm, essential for blastocyst formation. Mouse naive epiblast and derivative embryonic stem cells are restricted accordingly from producing trophectoderm. Here we show, in contrast, that human naive embryonic stem cells readily make blastocyst trophectoderm and descendant trophoblast cell types. Trophectoderm was induced rapidly and efficiently by inhibition of ERK/mitogen-activated protein kinase (MAPK) and Nodal signaling. Transcriptome comparison with the human embryo substantiated direct formation of trophectoderm with subsequent differentiation into syncytiotrophoblast, cytotrophoblast, and downstream trophoblast stem cells. During pluripotency progression lineage potential switches from trophectoderm to amnion. Live-cell tracking revealed that epiblast cells in the human blastocyst are also able to produce trophectoderm. Thus, the paradigm of developmental specification coupled to lineage restriction does not apply to humans. Instead, epiblast plasticity and the potential for blastocyst regeneration are retained until implantation.

Precise monitoring of mesenchymal stem cell homing to injured kidney with an activatable aptamer probe generated by cell-SELEX

Deficiency of precise tracking tools to monitor the migration and survival status of mesenchymal stem cells (MSCs) in the animal body has bottlenecked the clinical translation of stem cell therapies. In this study, we have for the first time successfully screened an aptamer, termed seq3, with high specificity and affinity for mouse bone marrow MSCs (mBMSCs) by MSC-based systematic evolution of ligands by exponential enrichment (MSC-SELEX), and ingeniously designed seq3-based activatable aptamer probes (AAP) for live-cell tracking. As proof of concept, real-time monitoring of transplanted mBMSCs in a mouse model of chronic kidney disease (CKD) was performed with the AAPs, and compared with Fe3O4-based magnetic resonance imaging (MRI), a widely utilized cell imaging module. It was confirmed that seq3 specifically binds to mBMSCs in the animal body and the binding activates the fluorescence of otherwise quenched dyes in AAPs, substantially minimizing the background signals originating from non-target tissues and avoiding the presence of false positive and/or false negative results occurred in MR imaging. Furthermore, the seq3-AAP-based fluorescence imaging was advantageous over Fe3O4-based MR imaging in terms of indicating survival status of the migrating and colonized mBMSCs, critical information required to evaluate the effectiveness and efficiency of cell therapy strategies. AAP fluorescence imaging results showed that the transplanted mBMSCs were well distributed in the bodies of normal mice and mainly aggregated in the livers, while they tended to migrate to injured kidneys of CKD mice and facilitated the repair of kidneys as a result. Besides cell tracking, the high specificity and affinity of seq3 to mBMSCs can be widely explored to understand the fate and biological roles of stem cells in regenerating tissues in the future.

Cell and Nucleus Shape as an Indicator of Tissue Fluidity in Carcinoma

Tissue, cell, and nucleus morphology change during tumor progression. In 2D confluent cell cultures, different tissue states, such as fluid (unjammed) and solid (jammed), are correlated with cell shapes. These results do not have to apply a priori to three dimensions. Cancer cell motility requires and corresponds to a fluidization of the tumor tissue on the bulk level. Here, we investigate bulk tissue fluidity in 3D and determine how it correlates with cell and nucleus shape. In patient samples of mamma and cervix carcinoma, we find areas where cells can move or are immobile. We compare 3D cell spheroids composed of cells from a cancerous and a noncancerous cell line. Through bulk mechanical spheroid-fusion experiments and single live-cell tracking, we show that the cancerous sample is fluidized by active cells moving through the tissue. The healthy, epithelial sample with immobile cells behaves more solidlike. 3D segmentations of the samples show that the degree of tissue fluidity correlates with elongated cell and nucleus shapes. This correlation links cell shapes to cell motility and bulk mechanical behavior. We find two active states of matter in solid tumors: an amorphous glasslike state with characteristics of 3D cell jamming and a disordered fluid state. Individual cell and nucleus shape may serve as a marker for metastatic potential to foster personalized cancer treatment.

Folic Acid-Functionalized Nanomedicine: Folic Acid Conjugated Copolymer and Folate Receptor Interactions Disrupt Receptor Functionality Resulting in Dual Therapeutic Anti-Cancer Potential in Breast and Prostate Cancer

We have previously reported on a functionalized folic acid (FA) conjugated poly(styrene-alt-maleic anhydride) (SMA) via biological linker 2,4-diaminobutyric acid (DABA) (FA-DABA-SMA) copolymer. This biocompatible nanocopolymer self-assembles in a pH-dependent manner, providing stimuli responsiveness, active targeting, and extended release of hydrophobic chemotherapeutic agents and effectively penetrates the inner core of 3-dimensional cancer spheroid models. The empty FA-DABA-SMA decreased tumor spheroid volume, revealing a previously unknown mechanism of action. Here, we investigated the potential mechanism of the small (20 kDa) and large (350 kDa) FA-DABA-SMA empty copolymers affecting the folic acid receptor alpha (FRα) signaling properties in breast and prostate cancer cell lines. Microscopic imaging, immunocytochemistry, flow cytometry, Caspase 3/7 apoptosis assays, Incucyte live cell tracking, the scratch wound assay, the water-soluble tetrazolium salt-1 (WST-1) cell viability assay, morphologic changes, and Western blot for the expression levels of FRα on the cell surface were used on MDA MB-231 and MCF-7 breast and DU-145 prostate cancer cell lines. The findings indicate that FA-DABA-SMA increases FRα expression levels in breast MDA MB-231 cancer cells and then disrupts FR signaling by reducing HES1 and NOTCH1 protein expression levels. Also, FA-DABA-SMA induces apoptosis and further causes a change in the MDA MB-231 cells' morphology and significantly reduces their ability to migrate in a scratch wound assay. Collectively, these findings provide a novel insight into the functionalized FA-DABA-SMA copolymer. The 350 and 20 kDa copolymers actively target FRα to initialize internationalization. However, only the large size and sheet-shaped 350 kDa copolymers disrupt FRα signaling. The significance of these novel findings reveals that the copolymer's intracellular activity is critically dependent on the size and structural shape. This report offers novel therapeutic insight into a dual mechanism of the FA-DABA-SMA copolymer for its therapeutic potential to treat cancer.

Narrow Stimulated Resonance Raman Scattering and WGM Lasing in Small Conjugated Polymer Particles for Live Cell Tagging and Tracking

AbstractConjugated polymer particles are brightly fluorescing and stable materials for live cell imaging. Combination of conjugated polymers with a whispering gallery mode (WGM) resonator allows laser emission from microscale particles. Once internalized by cells, the mode pattern of the laser emission can be used for tagging and tracking, as each laser spectrum represents a bar code to identify individual cells. However, currently these particle systems are limited by their large size, which might interfere with cellular functions. Here, stimulated resonance Raman scattering (SRRS) in small conjugated polymer microparticles is presented as a new method for generating narrow emission as an alternative to WGM‐based laser emission. This opens up spectral range for multiplexing optical readout and multicolor imaging of live cells. The synthesis of monodisperse micrometer‐sized poly(fluorene‐co‐divinylbenzene) particles is discussed and their WGM and SRRS emission are characterized. Finally, how these particles and their emission can be employed in live cell imaging and tagging is showcased.

Spatial patterning of nanofibrous collagen scaffolds modulates fibroblast morphology

Current knowledge about cell-biomaterial interactions is often based on two-dimensional (2D) cell culture systems like protein-coated glass slides. However, such smooth surfaces cannot mimic the nanofibrous environment of the native extracellular matrix (ECM). It is therefore a major challenge to transfer the results from 2D surfaces to 3D protein scaffolds with biomimetic nanofiber architecture. To understand the influence of different protein topographies on the cell response we introduce a new process to fabricate binary collagen scaffolds of variable thickness with spatially controlled regions of nanofibrous and smooth topography. We used pH-induced self-assembly to prepare collagen nanofibers with diameters between 130 and 150 nm on glass surfaces, which were partly covered with a polymer mask. After cross-linking with glutaraldehyde, smooth collagen films were prepared on the remaining glass regions. Atomic force microscopy revealed a much lower surface roughness of smooth collagen compared to nanofibers. Subsequently, we studied the viability, morphology and migration of 3T3 fibroblasts on both collagen topographies. We found small, elongated fibroblasts with few, long filopodia on collagen nanofibers whereas large, flat fibroblasts with many short filopodia were observed on smooth collagen. Actin stress fibers on collagen nanofibers were substantially reduced in comparison to smooth collagen. Live cell tracking revealed that fibroblasts on thin nanofibrous collagen migrated faster than on smooth collagen. In summary, binary collagen scaffolds enabled us for the first time to study cell responses to topographical cues on a single protein scaffold. In future, it will be intriguing to transfer our patterning process to other proteins to study fundamental principles of topography-dependent cell recognition processes.

Regional Hyperthermia Enhances Mesenchymal Stem Cell Recruitment to Tumor Stroma: Implications for Mesenchymal Stem Cell-Based Tumor Therapy

The tropism of mesenchymal stem cells (MSCs) for tumors forms the basis for their use as delivery vehicles for the tumor-specific transport of therapeutic genes, such as the theranostic sodium iodide symporter (NIS). Hyperthermia is used as an adjuvant for various tumor therapies and has been proposed to enhance leukocyte recruitment. Here, we describe the enhanced recruitment of adoptively applied NIS-expressing MSCs to tumors in response to regional hyperthermia. Hyperthermia (41°C, 1 h) of human hepatocellular carcinoma cells (HuH7) led to transiently increased production of immunomodulatory factors. MSCs showed enhanced chemotaxis to supernatants derived from heat-treated cells in a 3D live-cell tracking assay and was validated invivo in subcutaneous HuH7 mousexenografts. Cytomegalovirus (CMV)-NIS-MSCs were applied 6-48h after or 24-48h before hyperthermia treatment. Using 123I-scintigraphy, thermo-stimulation (41°C, 1 h) 24h after CMV-NIS-MSC injection resulted in a significantly increased uptake of 123I in heat-treated tumors compared with controls. Immunohistochemical staining and real-time PCR confirmed tumor-selective, temperature-dependent MSC migration. Therapeutic efficacy was significantly enhanced bycombining CMV-NIS-MSC-mediated 131I therapy with regional hyperthermia. We demonstrate here for the first time that hyperthermia can significantly boost tumoral MSC recruitment, thereby significantly enhancing therapeutic efficacy of MSC-mediated NIS gene therapy.