Single-cell Scale Research Articles

An Intra-Tissue Radiometry Microprobe for Measuring Radiance In Situ in Living Tissue.

Organisms appear opaque largely because their outer tissue layers are strongly scattering to incident light; strongly absorbing pigments, such as blood, typically have narrow absorbances, such that the mean free path of light outside the absorbance peaks can be quite long. As people cannot see through tissue, they generally imagine that tissues like the brain, fat, and bone contain little or no light. However, photoresponsive opsin proteins are expressed within many of these tissues, and their functions are poorly understood. Radiance internal to tissue is also important for understanding photosynthesis. For example, giant clams are strongly absorbing yet maintain a dense population of algae deep in the tissue. Light propagation through systems like sediments and biofilms can be complex, and these communities can be major contributors to ecosystem productivity. Therefore, a method for constructing optical micro-probes for measuring scalar irradiance (photon flux intersecting a point) and downwelling irradiance (photon flux crossing a plane perpendicularly) to better understand these phenomena inside living tissue has been developed. This technique is also tractable in field laboratories. These micro-probes are made from heat-pulled optical fibers that are then secured in pulled glass pipettes. To change the angular acceptance of the probe, a 10-100 µm sized sphere of UV-curable epoxy mixed with titanium dioxide is then secured to the end of a pulled, trimmed fiber. The probe is inserted into living tissue, and its position is controlled using a micromanipulator. These probes are capable of measuring in situ tissue radiance at spatial resolutions of 10-100 µm or on the scale of single cells. These probes were used to characterize the light reaching the adipose and brain cells 4 mm below the skin of a living mouse and to characterize the light reaching similar depths within living algae-rich giant clam tissue.

T cell functional phenotyping on a single cell scale using hydrogel microparticles.

Abstract Engineered cell therapies, especially chimeric antigen receptor (CAR) T cell therapies, have shown much promise in treating various cancers, most notably hematologic malignancies. However, the success of these therapies is highly variable between cancer types and across patient populations. A growing body of literature suggests that certain T cell functional properties are key drivers of response; thus, it is imperative to develop a more thorough understanding of the functional heterogeneity in T cell populations in order to inform the design of the next generation of cancer immunotherapies. Here, we used a new technology our team developed known as nanovials, which are microfluidically manufactured hydrogel microparticles, to enable the specific activation and capture of secreted products of T cells on a single-cell level. Leveraging this platform, we characterized the secretion of IFN-γ and TNF-α by individual CD8 +T cells harvested from OT-I mice. After initial expansion after isolation, T cells were loaded into functionalized nanovials and specifically activated with OVA peptide. The cells loaded in the nanovials were then separated based on cytokine secretion levels via fluorescence-activated cell sorting (FACS). Unlike traditional cytokine secretion assays which require fixation and permeabilization, cells remain viable throughout our process, allowing for sorted subpopulations to be dissociated from the microparticles, expanded, and further analyzed via cytotoxicity, co-culture, or phenotypic studies. Collectively, our efforts introduce a new paradigm for elucidating the functional properties of immune cells, providing critical insights that will guide the future design of engineered cell therapeutics. M.K. is a recipient of a National Science Foundation Graduate Research Fellowship Program award.

Quantitative comparison of EGFR expression levels of optically trapped individual cells using a capacitance biosensor

Cellular endocytosis is an essential phenomenon which induces cellular reactions, such as waste removal, nutrient absorption, and drug delivery, in the process of cell growth, division, and proliferation. To observe capacitance responses upon endocytosis on a single-cell scale, this study combined an optical tweezer that can optically place a single cell on a desired location with a capacitance sensor and a cell incubation chamber. Single HeLa cancer cell was captured and moved to a desired location through optical trapping, and the single-cell capacitance change generated during the epidermal growth factor (EGF) molecule endocytosis was measured in real time. It was found that single HeLa cells showed a larger increase in capacitance values compared to that of the single NIH3T3 cells when exposed to varying EGF concentrations. In addition, the capacitance change was in proportion to the cell's EGF receptor (EGFR) level when cells of different levels of EGFR expression were tested. An equation derived from these results was able to estimate the EGFR expression level of a blind-tested cell. The biosensor developed in this research can not only quickly move a single cell to a desired location in a non-invasive manner but also distinguish specific responses between cancer and normal cells by continuous measurement of real-time interactions of a single cell in culture to the external ligands.

Community-specific diffusion characteristics determine resistance of biofilms to oxidative stress

Biofilms are multicellular communities with a spatial structure. Different from single-cell scale diffusion in planktonic systems, the diffusion distance becomes the dimension of multicellular clusters in a biofilm. Such differences in diffusion behavior affect the tolerance and response to exogenous stress. Here, we found that at the same doses of exogenous hydrogen peroxide (H2O2), planktonic Escherichia coli were completely killed within twohours, whereas the biofilm resumed growth in sixhours by building a catalase barrier to block H2O2 penetration, despite the growth burden. Unexpectedly, when we changed the carbon source from glucose to glycerol, H2O2 instantly counterintuitively boosted biofilm growth due to supplemental oxygen, which was the growth-limiting factor. We further demonstrated that the energy metabolism modes determined the growth-limiting factor, which then determined the two patterns of biofilms resistances to H2O2.

Multiscale NIR-II Imaging-Guided Brain-Targeted Drug Delivery Using Engineered Cell Membrane Nanoformulation for Alzheimer's Disease Therapy.

Effective drug delivery in the central nervous system (CNS) needs to have long blood-circulation half-lives, to pass through the blood-brain barrier (BBB), and subsequently to be taken up by target cells. Herein, a traceable CNS delivery nanoformulation (RVG-NV-NPs) is developed by encapsulating bexarotene (Bex) and AgAuSe quantum dots (QDs) within Lamp2b-RVG-overexpressed neural stem cell (NSC) membranes. The high-fidelity near-infrared-II imaging by AgAuSe QDs offers a possibility of in vivo monitoring the multiscale delivery process of the nanoformulation from the whole-body to the single-cell scale. It was revealed the synergy of acetylcholine receptor-targeting of RVG and the natural brain-homing and low immunogenicity of NSC membranes prolong the blood circulation, facilitate BBB crossing and nerve cell targeting of RVG-NV-NPs. Thus, in Alzheimer's disease (AD) mice, the intravenous delivery of as low as 0.5% of oral dose Bex showed highly effective up-regulation of the apolipoprotein E expression, resulting rapid alleviation of ∼40% β-amyloid (Aβ) level in the brain interstitial fluid after a single dose administration. The pathological progression of Aβ in AD mice is completely suppressed during a 1 month treatment, thus effectively protecting neurons from Aβ-induced apoptosis and maintaining the cognitive abilities of AD mice.

Beyond Thresholds: Quorum‐Sensing as Quantitatively Varying Reaction Norms to Multiple Environmental Dimensions

AbstractMany types of bacteria engage in complex collective behaviors, often controlled by a form of cell‐cell communication mediated by diffusible signal molecules called quorum sensing (QS). Each individual produces and responds to these diffusible signaling molecules, inferring properties of their environment, and modulating the regulation of hundreds of downstream genes in response. A threshold concept is ingrained in the QS literature following the use of the legal ‘quorum’ analogy, where populations of bacteria are synchronously sub‐quorate below a threshold population size or synchronously quorate above a threshold population size. However, a growing body of literature highlights that QS can produce more nuanced, graded responses to environmental variation, implying there is no simple threshold ‘quorum’ on either the single cell or population scale. In this commentary, we discuss reaction norms as a tool to quantify and compare QS‐controlled behaviors in an environmental context.

Trends in single-impact electrochemistry for bacteria analysis.

Single-impact electrochemistry for the analysis of bacteria is a powerful technique for biosensing applications at the single-cell scale. The sensitivity of this electro-analytical method has been widely demonstrated based on chronoamperometric measurements at an ultramicroelectrode polarized at the appropriate potential of redox species in solution. Furthermore, the most recent studies display a continuous improvement in the ability of this sensitive electrochemical method to identify different bacterial strains with better selectivity. To achieve this, several strategies, such as the presence of a redox mediator, have been investigated for detecting and identifying the bacterial cell through its own electrochemical behavior. Both the blocking electrochemical impacts method and electrochemical collisions of single bacteria with a redox mediator are reported in this review and discussed through relevant examples. An original sensing strategy for virulence factors originating from pathogenic bacteria is also presented, based on a recent proof of concept dealing with redox liposome single-impact electrochemistry. The limitations, applications, perspectives, and challenges of single-impact electrochemistry for bacteria analysis are briefly discussed, based on the most significant published data.

The Advance of the HSCT Evaluation at Single-Cell Scale --Review

Hematopoietic stem cell transplantation (HSCT) is one of the effective options for the treatment of irradiation-induced injury on hematopoiesis, malignant hematological diseases, and numerous benign severe hematopathy. However, the cellular composition of the graft for HSCT, as well as the significant events of transplanted HSCs in receipients including HSC homing, engraftment, differentiation, remains to be further elucidated. In recent years, with advances in single-cell techniques, the hematopoiesis has been decoding at single cell scale. In addition, single-cell RNA sequencing (scRNA-seq) has been used in the evaluation of hematopoietic dynamics post HSCT, which may be helpful to improve HSCT protocols and clinical outcomes. Hence, the recent advances of evaluating HSCT at single cell scale and the directions worthy paying attention to in the field have been reviewed briefly.

Immune checkpoint HLA-E:CD94-NKG2A mediates evasion of circulating tumor cells from NK cell surveillance.

Circulating tumor cells (CTCs), shed by primary malignancies, function as "seeds" for distant metastasis. However, it is still largely unknown how CTCs escape immune surveillance. Here, we characterize the transcriptomes of human pancreatic ductal adenocarcinoma CTCs, primary, and metastatic lesions at single-cell scale. Cell-interaction analysis and functional studies invitro and invivo reveal that CTCs and natural killer (NK) cells interact via the immune checkpoint molecule pair HLA-E:CD94-NKG2A. Disruption of this interaction by blockade of NKG2A or knockdown of HLA-E expression enhances NK-mediated tumor cell killing invitro and prevents tumor metastasis invivo. Mechanistic studies indicate that platelet-derived RGS18 promotes the expression of HLA-E through AKT-GSK3β-CREB signaling, and overexpression of RGS18 facilitates pancreatic tumor hepatic metastasis. In conclusion, platelet-derived RGS18 protects CTCs from NK-mediated immune surveillance by engaging the immune checkpoint HLA-E:CD94-NKG2A. Interruption of the suppressive signaling prevents tumor metastasis invivo by immune elimination of CTCs.

PIEZO1 regulates leader cell formation and cellular coordination during collective cell migration: An integrative multiscale modeling and experimental study.

The collective migration of keratinocytes during wound healing requires both the generation and transmission of mechanical forces for individual cellular locomotion as well as for the coordination of movement across cells. Leader cells initiated along the wound edge transmit mechanical and biochemical cues to ensuing follower cells, ensuring their uniform polarization and coordinated direction of migration, or directionality. Despite the observed importance of mechanical cues in leader cell formation and governing directionality, the underlying biophysical mechanisms remain elusive. The mechanically activated ion channel PIEZO1 was recently identified to play an inhibitory role during the reepithelialization of wounds through retraction of keratinocytes located along the wound edge. Here, through an integrative experimental and mathematical modeling approach, we elucidate PIEZO1's contributions to collective migration. Time-lapse microscopy reveals that PIEZO1 activity inhibits leader cell formation along the wound edge. To probe the relationship between PIEZO1 activity, leader cell formation and inhibition of reepithelialization, we developed an integrative 2D-multiscale model of wound closure that links observations at the single cell and collective cell migration scales. Through numerical simulations and subsequent experimental validation, we found that directionality plays a key role during wound closure and is inhibited by PIEZO1 activity. We propose that PIEZO1-mediated retraction suppresses leader cell formation which inhibits the coordination of directionality between cells during collective migration.

Stereo Coverage and Overall Stiffness of Biomaterial Arrays Underly Parts of Topography Effects on Cell Adhesion.

Surface topography is a biophysical factor affecting cell behaviors, yet the underlying cues are still not clear. Herein, we hypothesized that stereo coverage and overall stiffness of biomaterial arrays on the scale of single cells underly parts of topography effects on cell adhesion. We fabricated a series of microarrays (micropillar, micropit, and microtube) of poly(l-lactic acid) (PLLA) using mold casting based on pre-designed templates. The characteristic sizes of array units were less than that of a single cell, and thus, each cell could sense the micropatterns with varied roughness. With human umbilical vein endothelial cells (HUVECs) as the model cell type, we examined spreading areas and cell viabilities on different surfaces. "Stereo coverage" was defined to quantify the actual cell spreading fraction on a topographic surface. Particularly in the case of high micropillars, cells were confirmed not able to touch the bottom and had to partially hang among the micropillars. Then, in our opinion, a cell sensed the overall stiffness combining the bulk stiffness of the raw material and the stiffness of the culture medium. Spreading area and single cell viability were correlated to coverage and topographic feature of the prepared microarrays in particular with the significantly protruded geometry feather. Cell traction forces exerted on micropillars were also discussed. These findings provide new insights into the surface modifications toward biomedical implants.

Enhanced mechanical heterogeneity of cell collectives due to temporal fluctuations in cell elasticity.

Cells are dynamic systems characterized by temporal variations in biophysical properties such as stiffness and contractility. Recent studies show that the recruitment and release of actin filaments into and out of the cell cortex-a network of proteins underneath the cell membrane-leads to cell stiffening prior to division and softening immediately afterward. In three-dimensional (3D) cell collectives, it is unclear whether the stiffness change during division at the single-cell scale controls the spatial structure and dynamics at the multicellular scale. This is an important question to understand because cell stiffness variations impact cell spatial organization and cancer progression. Using a minimal 3D model incorporating cell birth, death, and cell-to-cell elastic and adhesive interactions, we investigate the effect of mechanical heterogeneity-variations in individual cell stiffnesses that make up the cell collective-on tumor spatial organization and cell dynamics. We discover that spatial mechanical heterogeneity characterized by a spheroid core composed of stiffer cells and softer cells in the periphery emerges within dense 3D cell collectives, which may be a general feature of multicellular tumor growth. We show that heightened spatial mechanical heterogeneity enhances single-cell dynamics and volumetric tumor growth driven by fluctuations in cell elasticity. Our results could have important implications in understanding how spatiotemporal variations in single-cell stiffness determine tumor growth and spread.

215 Spatial transcriptomics on human epidermal equivalents

Single-cell RNA seq analyses on human epidermis have given new insights into cell fates of the tissue by characterizing the precise transcriptomic profile at single-cell scale, but information of spatial localizations of transcripts in each single cells is lacking. 3D transcriptomic analyses are therefore of great importance to characterize cell fates of the epidermis with spatial resolution. To study human epidermal biology and diseases, organotypic models can be grown using keratinocytes, which reproduce the architecture of the epidermis.

Trophic strategies of intertidal foraminifera explored with single-cell microbiome metabarcoding and morphological methods: What is on the menu?

In mudflats, interactions and transfers of nutrients and secondary metabolites may drive ecosystems and biodiversity. Foraminifera have complex trophic strategies as they often rely on bacteria and eukaryotes or on potential symbionts for carbon and nitrogen resources. The capacity of these protists to use a wide range of adaptive mechanisms requires clarifying the relationships between them and their microbial associates. Here, we investigate the interactions of three foraminiferal species with nearby organisms in situ, by coupling molecular (cloning/Sanger and high-throughput sequencing) and direct counting and morphological identification with microscopy. This coupling allows the identification of the organisms found in or around three foraminiferal species through molecular tools combined with a direct counting of foraminifera and diatoms present in situ through microscopy methods. Depending on foraminiferal species, and in addition to diatom biomass, diatom frustule shape, size and species are key factors driving the abundance and diversity of foraminifera in mudflat habitats. Three different trophic strategies were deduced for the foraminifera investigated in this study: Ammonia sp. T6 has an opportunistic strategy and is feeding on bacteria, nematoda, fungi, and diatoms when abundant; Elphidium oceanense is feeding mainly on diatoms, mixed with other preys when they are less abundant; and Haynesina germanica is feeding almost solely on medium-large pennate diatoms. Although there are limitations due to the lack of species coverage in DNA sequence databases and to the difficulty to compare morphological and molecular data, this study highlights the relevance of combining molecular with morphological tools to study trophic interactions and microbiome communities of protists at the single-cell scale.

Filamented Light (FLight) Biofabrication of Highly Aligned Tissue-Engineered Constructs.

Cell-laden hydrogels used in tissue engineering generally lack sufficient 3Dtopographical guidance for cells to mature into aligned tissues. A new strategy called filamented light (FLight) biofabrication rapidly creates hydrogels composed of unidirectional microfilament networks, with diameters on the length scale of single cells. Due to optical modulation instability, a light beam is divided optically into FLight beams. Local polymerization of a photoactive resin is triggered, leading to local increase in refractive index, which itself creates self-focusing waveguides and further polymerization of photoresin into long hydrogel microfilaments. Diameter and spacing of the microfilaments can be tuned from 2 to 30µm by changing the coherence length of the light beam. Microfilaments show outstanding cell instructive properties with fibroblasts, tenocytes, endothelial cells, and myoblasts, influencing cell alignment, nuclear deformation, and extracellular matrix deposition. FLight is compatible with multiple types of photoresins and allows for biofabrication of centimeter-scale hydrogel constructs with excellent cell viability within seconds (<10 s per construct). Multidirectional microfilaments are achievable within a single hydrogel construct by changing the direction of FLight projection, and complex multimaterial/multicellular tissue-engineered constructs are possible by sequentially exchanging the cell-laden photoresin. FLight offers a transformational approach to developing anisotropic tissues using photo-crosslinkable biomaterials.

Challenges of intracellular visualization using virtual and augmented reality.

Microscopy image observation is commonly performed on 2D screens, which limits human capacities to grasp volumetric, complex, and discrete biological dynamics. With the massive production of multidimensional images (3D + time, multi-channels) and derived images (e.g., restored images, segmentation maps, and object tracks), scientists need appropriate visualization and navigation methods to better apprehend the amount of information in their content. New modes of visualization have emerged, including virtual reality (VR)/augmented reality (AR) approaches which should allow more accurate analysis and exploration of large time series of volumetric images, such as those produced by the latest 3D + time fluorescence microscopy. They include integrated algorithms that allow researchers to interactively explore complex spatiotemporal objects at the scale of single cells or multicellular systems, almost in a real time manner. In practice, however, immersion of the user within 3D + time microscopy data represents both a paradigm shift in human-image interaction and an acculturation challenge, for the concerned community. To promote a broader adoption of these approaches by biologists, further dialogue is needed between the bioimaging community and the VR&AR developers.

Measuring Photonics in Photosynthesis: Combined Micro-Fourier Image Spectroscopy and Pulse Amplitude Modulated Chlorophyll Fluorimetry at the Micrometre-Scale.

Natural photonic structures are common across the biological kingdoms, serving a diversity of functionalities. The study of implications of photonic structures in plants and other phototrophic organisms is still hampered by missing methodologies for determining in situ photonic properties, particularly in the context of constantly adapting photosynthetic systems controlled by acclimation mechanisms on the cellular scale. We describe an innovative approach to determining spatial and spectral photonic properties and photosynthesis activity, employing micro-Fourier Image Spectroscopy and Pulse Amplitude Modulated Chlorophyll Fluorimetry in a combined microscope setup. Using two examples from the photosynthetic realm, the dynamic Bragg-stack-like thylakoid structures of Begonia sp. and complex 2.5 D photonic crystal slabs from the diatom Coscinodiscus granii, we demonstrate how the setup can be used for measuring self-adapting photonic-photosynthetic systems and photonic properties on single-cell scales. We suggest that the setup is well-suited for the determination of photonic–photosynthetic systems in a diversity of organisms, facilitating the cellular, temporal, spectral and angular resolution of both light distribution and combined chlorophyll fluorescence determination. As the catalogue of photonic structure from photosynthetic organisms is rich and diverse in examples, a deepened study could inspire the design of novel optical- and light-harvesting technologies.

BASS: multi-scale and multi-sample analysis enables accurate cell type clustering and spatial domain detection in spatial transcriptomic studies

Spatial transcriptomic studies are reaching single-cell spatial resolution, with data often collected from multiple tissue sections. Here, we present a computational method, BASS, that enables multi-scale and multi-sample analysis for single-cell resolution spatial transcriptomics. BASS performs cell type clustering at the single-cell scale and spatial domain detection at the tissue regional scale, with the two tasks carried out simultaneously within a Bayesian hierarchical modeling framework. We illustrate the benefits of BASS through comprehensive simulations and applications to three datasets. The substantial power gain brought by BASS allows us to reveal accurate transcriptomic and cellular landscape in both cortex and hypothalamus.

Stochastic simulation of enzymatic kinetics for 13C isotope labeling at the single-cell scale

Stochastic simulation of enzymatic kinetics for 13C isotope labeling at the single-cell scale

Multimodal approach to capture spatially resolved single-cell tumor heterogeneity in breast cancer.

e12597 Background: Tumor heterogeneity is known to play a key role in drug response and resistance. However, knowledge of tumor heterogeneity is largely limited to genetics and biopsy samples representing one region of the tumor. The study of spatial tumor heterogeneity is gaining more interest as researchers and clinicians realize that biopsies might miss biomarker information. Although several approaches like single-cell RNA seq and multiplex IHC/IF are used to study spatial heterogeneity, these methods tend to be costly and time consuming. Additionally, there is no consensus on how to interpret these results for optimizing therapy, making it harder to implement these approaches in the clinic. Methods: Here we present a state-of-the-art technique to characterize tumor heterogeneity within individual patient tumors by integrating single cell RNA-seq data with biophysical modeling, using SimBioSys’ TumorScope (TS) – a biophysical modeling platform. As a proof of concept, we compared intra-tumoral heterogeneity from experimental single cell RNA-seq studies from TNBC patients (n=8) to to simulated intra-tumoral heterogeneity within TNBC patients from the ISPY-1 trial (n = 10). We also compared simulated vs. experimentally measured bulk transcriptional profiles within individual patients. Results: We found that the correlation between individual single-cell transcriptomes to biopsy transcriptomes was low (median Spearman correlation ̃ 0.5). However, using TS to predict single-cell scale transcriptomics within a tumor based on biophysical characteristics of the tumor resulted in correlations of ̃ 0.86, between simulated and experimentally measured transcriptomes (range = 0.85 - 0.87). TS allowed the simulated single-cell RNA-seq data to create spatially resolved single-cell maps that quantify the spatial expression of key tumor biomarkers across the whole tumor including Ki67, HER2 and PDL1. The spatial distribution of these biomarkers can have important implications for therapy and patient prognosis. A more comprehensive understanding of the distribution of these molecular markers across the tumor could inform if some tumors deserved a different Ki67 score, if some patients are not receiving HER2-targeted therapy because of HER2 uneven expression across the tumor, and if PD-L1 could improve as a biomarker by considering its spatial expression. Conclusions: In summary, we present a novel approach to characterize spatial tumor heterogeneity directly from patient MRIs using the TS biophysical modeling platform. This technology is less time consuming and less costly than other approaches, making it accessible for use in hospitals at a larger scale.