Investigating the Metabolic Heterogeneity of Cancer Cells Using Functional Single-Cell Selection and nLC Combined with Multinozzle Emitter Mass Spectrometry.

Tumor metastasis and cancer recurrence are often a result of cell heterogeneity, where specific subpopulations of tumor cells may be resistant to radio- or chemotherapy. To investigate this physiological and phenotypic diversity, single-cell metabolomics provides a powerful approach at the chemical level, where distinct lipid profiles can be found in different tumor cells. Here, we established a highly sensitive platform using nanoflow liquid chromatography (nLC) combined with multinozzle emitter electrospray ionization mass spectrometry for more in-depth metabolomics profiling. Our platform identified 15 and 17 lipids from individual osteosarcoma (U2OS) and glioblastoma (GBM) cells when analyzing single-cell samples. Additionally, we used the functional single-cell selection (fSCS) pipeline to analyze the subpopulations of cells with a DNA damage response (DDR) in U2OS cells and fast migration in GBM cells. Specifically, we observed a down-regulation of polyunsaturated fatty acids (PUFAs) in U2OS cells undergoing DDR, such as fatty acids FA 20:3; O2 and FA 17:4; O3. Furthermore, ceramides (Cer 38:0; O3) and triglycerides (TG 36:0) were found to be down-regulated in fast-migrating GBM cells compared to the slow-migrating subpopulation. These findings suggest the potential roles of these metabolites and/or lipids in the cellular behavior of the subpopulations.

Genomic Exploration of Distinct Molecular Phenotypes Steering Temozolomide Resistance Development in Patient-Derived Glioblastoma Cells.

Chemotherapy using temozolomide is the standard treatment for patients with glioblastoma. Despite treatment, prognosis is still poor largely due to the emergence of temozolomide resistance. This resistance is closely linked to the widely recognized inter- and intra-tumoral heterogeneity in glioblastoma, although the underlying mechanisms are not yet fully understood. To induce temozolomide resistance, we subjected 21 patient-derived glioblastoma cell cultures to Temozolomide treatment for a period of up to 90 days. Prior to treatment, the cells' molecular characteristics were analyzed using bulk RNA sequencing. Additionally, we performed single-cell RNA sequencing on four of the cell cultures to track the evolution of temozolomide resistance. The induced temozolomide resistance was associated with two distinct phenotypic behaviors, classified as "adaptive" (ADA) or "non-adaptive" (N-ADA) to temozolomide. The ADA phenotype displayed neurodevelopmental and metabolic gene signatures, whereas the N-ADA phenotype expressed genes related to cell cycle regulation, DNA repair, and protein synthesis. Single-cell RNA sequencing revealed that in ADA cell cultures, one or more subpopulations emerged as dominant in the resistant samples, whereas N-ADA cell cultures remained relatively stable. The adaptability and heterogeneity of glioblastoma cells play pivotal roles in temozolomide treatment and contribute to the tumor's ability to survive. Depending on the tumor's adaptability potential, subpopulations with acquired resistance mechanisms may arise.

Dynamics of soft nanomaterials captured by transmission electron microscopy in liquid water.

In this paper we present in situ transmission electron microscopy of synthetic polymeric nanoparticles with emphasis on capturing motion in a solvated, aqueous state. The nanoparticles studied were obtained from the direct polymerization of a Pt(II)-containing monomer. The resulting structures provided sufficient contrast for facile imaging in situ. We contend that this technique will quickly become essential in the characterization of analogous systems, especially where dynamics are of interest in the solvated state. We describe the preparation of the synthetic micellar nanoparticles together with their characterization and motion in liquid water with comparison to conventional electron microscopy analyses.

Enzyme-directed assembly of nanoparticles in tumors monitored by in vivo whole animal imaging and ex vivo super-resolution fluorescence imaging.

Matrix metalloproteinase enzymes, overexpressed in HT-1080 human fibrocarcinoma tumors, were used to guide the accumulation and retention of an enzyme-responsive nanoparticle in a xenograft mouse model. The nanoparticles were prepared as micelles from amphiphilic block copolymers bearing a simple hydrophobic block and a hydrophilic peptide brush. The polymers were end-labeled with Alexa Fluor 647 dyes leading to the formation of labeled micelles upon dialysis of the polymers from DMSO/DMF to aqueous buffer. This dye-labeling strategy allowed the presence of the retained material to be visualized via whole animal imaging in vivo and in ex vivo organ analysis following intratumoral injection into HT-1080 xenograft tumors. We propose that the material is retained by virtue of an enzyme-induced accumulation process whereby particles change morphology from 20 nm spherical micelles to micrometer-scale aggregates, kinetically trapping them within the tumor. This hypothesis is tested here via an unprecedented super-resolution fluorescence analysis of ex vivo tissue slices confirming a particle size increase occurs concomitantly with extended retention of responsive particles compared to unresponsive controls.

Protocol for profiling in vitro intratumor heterogeneity using spatially annotated single-cell sequencing.

Here, we present a protocol for spatially annotated single-cell sequencing, a technique for spatially profiling intratumor heterogeneity with deep single-cell RNA sequencing and single-cell resolution. By combining live-cell imaging and photopatterned illumination, we describe steps to identify regions of interest in an in vitro tumor model, label the selected cells with photoactivatable dyes, and isolate and subject them to scRNAseq. This protocol can be applied to a range of cell lines and could be expanded to tissue sections. For complete details on the use and execution of this protocol, please refer to Smit et al. (2022).1.

Spatial Transcriptomic Technologies.

Spatial transcriptomic technologies enable measurement of expression levels of genes systematically throughout tissue space, deepening our understanding of cellular organizations and interactions within tissues as well as illuminating biological insights in neuroscience, developmental biology and a range of diseases, including cancer. A variety of spatial technologies have been developed and/or commercialized, differing in spatial resolution, sensitivity, multiplexing capability, throughput and coverage. In this paper, we review key enabling spatial transcriptomic technologies and their applications as well as the perspective of the techniques and new emerging technologies that are developed to address current limitations of spatial methodologies. In addition, we describe how spatial transcriptomics data can be integrated with other omics modalities, complementing other methods in deciphering cellar interactions and phenotypes within tissues as well as providing novel insight into tissue organization.

Instant processing of large-scale image data with FACT, a real-time cell segmentation and tracking algorithm.

Quantifying cellular characteristics from a large heterogeneous population is essential to identify rare, disease-driving cells. A recent development in the combination of high-throughput screening microscopy with single-cell profiling provides an unprecedented opportunity to decipher disease-driving phenotypes. Accurately and instantly processing large amounts of image data, however, remains a technical challenge when an analysis output is required minutes after data acquisition. Here, we present fast and accurate real-time cell tracking (FACT). FACT can segment ∼20,000 cells in an average of 2.5 s (1.9-93.5 times faster than the state of the art). It can export quantifiable features minutes after data acquisition (independent of the number of acquired image frames) with an average of 90%-96% precision. We apply FACT to identify directionally migrating glioblastoma cells with 96% precision and irregular cell lineages from a 24 h movie with an average F1 score of 0.91.

Microscopy-based single-cell proteomic profiling reveals heterogeneity in DNA damage response dynamics.

Single-cell proteomics has the potential to decipher tumor heterogeneity, and a method like single-cell proteomics by mass spectrometry (SCoPE-MS) allows profiling several tens of single cells for >1,000 proteins per cell. This method, however, cannot link the proteome of individual cells with phenotypes of interest. Here, we developed a microscopy-based functional single-cell proteomic-profiling technology, called FUNpro, to address this. FUNpro enables screening, identification, and isolation of single cells of interest in a real-time fashion, even if the phenotypes are dynamic or the cells of interest are rare. We applied FUNpro to proteomically profile a newly identified small subpopulation of U2OS osteosarcoma cells displaying an abnormal, prolonged DNA damage response (DDR) after ionizing radiation (IR). With this, we identified the PDS5A protein contributing to the abnormal DDR dynamics and helping the cells survive after IR.

Spatially Annotated Single Cell Sequencing for Unraveling Intratumor Heterogeneity.

Intratumor heterogeneity is a major obstacle to effective cancer treatment. Current methods to study intratumor heterogeneity using single-cell RNA sequencing (scRNA-seq) lack information on the spatial organization of cells. While state-of-the art spatial transcriptomics methods capture the spatial distribution, they either lack single cell resolution or have relatively low transcript counts. Here, we introduce spatially annotated single cell sequencing, based on the previously developed functional single cell sequencing (FUNseq) technique, to spatially profile tumor cells with deep scRNA-seq and single cell resolution. Using our approach, we profiled cells located at different distances from the center of a 2D epithelial cell mass. By profiling the cell patch in concentric bands of varying width, we showed that cells at the outermost edge of the patch responded strongest to their local microenvironment, behaved most invasively, and activated the process of epithelial-to-mesenchymal transition (EMT) to migrate to low-confluence areas. We inferred cell-cell communication networks and demonstrated that cells in the outermost ∼10 cell wide band, which we termed the invasive edge, induced similar phenotypic plasticity in neighboring regions. Applying FUNseq to spatially annotate and profile tumor cells enables deep characterization of tumor subpopulations, thereby unraveling the mechanistic basis for intratumor heterogeneity.

Dynamic Visualization of TGF-ß/SMAD3 Transcriptional Responses in Single Living Cells.

Transforming growth factor-ß (TGF-ß) signaling is tightly controlled in duration and intensity during embryonic development and in the adult to maintain tissue homeostasis. To visualize the TGF-ß/SMAD3 signaling kinetics, we developed a dynamic TGF-ß/SMAD3 transcriptional fluorescent reporter using multimerized SMAD3/4 binding elements driving the expression of a quickly folded and highly unstable GFP protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-ß/SMAD3 transcriptional responses in both 2D and 3D systems in vitro, as well as in vivo, using live-cell and intravital imaging. Using this reporter in B16F10 cells, we observed single cell heterogeneity in response to TGF-ß challenge, which can be categorized into early, late, and non-responders. Because of its broad application potential, this reporter allows for new discoveries into how TGF-ß/SMAD3-dependent transcriptional dynamics are affected during multistep and reversible biological processes.

Linking the genotypes and phenotypes of cancer cells in heterogenous populations via real-time optical tagging and image analysis.

Linking single-cell genomic or transcriptomic profiles to functional cellular characteristics, in particular time-varying phenotypic changes, could help unravel molecular mechanisms driving the growth of tumour-cell subpopulations. Here we show that a custom-built optical microscope with an ultrawide field of view, fast automated image analysis and a dye activatable by visible light enables the screening and selective photolabelling of cells of interest in large heterogeneous cell populations on the basis of specific functional cellular dynamics, such as fast migration, morphological variation, small-molecule uptake or cell division. Combining such functional single-cell selection with single-cell RNA sequencing allowed us to (1) functionally annotate the transcriptomic profiles of fast-migrating and spindle-shaped MCF10A cells, of fast-migrating MDA-MB-231 cells and of patient-derived head-and-neck squamous carcinoma cells, and (2) identify critical genes and pathways driving aggressive migration and mesenchymal-like morphology in these cells. Functional single-cell selection upstream of single-cell sequencing does not depend on molecular biomarkers, allows for the enrichment of sparse subpopulations of cells, and can facilitate the identification and understanding of the molecular mechanisms underlying functional phenotypes.

Controlling and switching the morphology of micellar nanoparticles with enzymes.

Micelles were prepared from polymer-peptide block copolymer amphiphiles containing substrates for protein kinase A, protein phosphatase-1, and matrix metalloproteinases 2 and 9. We examine reversible switching of the morphology of these micelles through a phosphorylation-dephosphorylation cycle and study peptide-sequence directed changes in morphology in response to proteolysis. Furthermore, the exceptional uniformity of these polymer-peptide particles makes them amenable to cryo-TEM reconstruction techniques lending insight into their internal structure.

The fusion peptide domain is the primary membrane-inserted region and enhances membrane interaction of the ectodomain of HIV-1 gp41.

To execute the membrane fusion function, it is necessary for the fusion protein of the virus to penetrate into the hydrophobic milieu of membrane bilayer. Hence identification of the region(s) of the ectodomain of viral fusion proteins involved in the membrane insertion and their interaction with the rest of the fusion protein in the membrane would be important for the mechanistic study of membrane fusion. To this end, we examined membrane activity of the fusion peptide, and the ectodomain protein with or without the fusion peptide domain of HIV-1 gp41 by several biophysical measurements. The results revealed that the ectodomain protein containing the fusion peptide domain had higher membrane-perturbing activity and deeper membrane insertion, while the construct lacking the fusion peptide domain had much lower membrane activity. Strikingly, the N-terminal heptad repeat region was found to be induced deeper into the membrane by the fusion peptide, consistent with the role of the latter in the membrane penetration. We concluded that the fusion peptide is the only stretch of gp41 ectodomain that embeds deeply in the membrane interior in the prefusion stage. The function of fusion peptide in terms of membrane interaction and the implications of its interplay with other domains of gp41 on the membrane fusion cascade were discussed.

Smart lipids for programmable nanomaterials.

Novel, responsive liposomes are introduced, assembled from DNA-programmed lipids allowing sequence selective manipulation of nanoscale morphology. Short, single-stranded DNA sequences form polar head groups conjugated to hydrophobic tails. The morphology of the resulting lipid aggregates depends on sterics and electronics in the polar head groups and, therefore, is dependent on the DNA hybridization state. The programmability, specificity, and reversibility of the switchable system are demonstrated via dynamic light scattering, transmission electron microscopy, and fluorescence microscopy.

Photoactivated voltage imaging in tissue with an archaerhodopsin-derived reporter.

Photoactivated genetically encoded voltage indicators (GEVIs) have the potential to enable optically sectioned voltage imaging at the intersection of a photoactivation beam and an imaging beam. We developed a pooled high-throughput screen to identify archaerhodopsin mutants with enhanced photoactivation. After screening ~105 cells, we identified a novel GEVI, NovArch, whose one-photon near-infrared fluorescence is reversibly enhanced by weak one-photon blue or two-photon near-infrared excitation. Because the photoactivation leads to fluorescent signals catalytically rather than stoichiometrically, high fluorescence signals, optical sectioning, and high time resolution are achieved simultaneously at modest blue or two-photon laser power. We demonstrate applications of the combined molecular and optical tools to optical mapping of membrane voltage in distal dendrites in acute mouse brain slices and in spontaneously active neurons in vivo.

A comprehensive enhancer screen identifies TRAM2 as a key and novel mediator of YAP oncogenesis.

BACKGROUND: Frequent activation of the co-transcriptional factor YAP is observed in a large number of solid tumors. Activated YAP associates with enhancer loci via TEAD4-DNA-binding protein and stimulates cancer aggressiveness. Although thousands of YAP/TEAD4 binding-sites are annotated, their functional importance is unknown. Here, we aim at further identification of enhancer elements that are required for YAP functions. RESULTS: We first apply genome-wide ChIP profiling of YAP to systematically identify enhancers that are bound by YAP/TEAD4. Next, we implement a genetic approach to uncover functions of YAP/TEAD4-associated enhancers, demonstrate its robustness, and use it to reveal a network of enhancers required for YAP-mediated proliferation. We focus on EnhancerTRAM2, as its target gene TRAM2 shows the strongest expression-correlation with YAP activity in nearly all tumor types. Interestingly, TRAM2 phenocopies the YAP-induced cell proliferation, migration, and invasion phenotypes and correlates with poor patient survival. Mechanistically, we identify FSTL-1 as a major direct client of TRAM2 that is involved in these phenotypes. Thus, TRAM2 is a key novel mediator of YAP-induced oncogenic proliferation and cellular invasiveness. CONCLUSIONS: YAP is a transcription co-factor that binds to thousands of enhancer loci and stimulates tumor aggressiveness. Using unbiased functional approaches, we dissect YAP enhancer network and characterize TRAM2 as a novel mediator of cellular proliferation, migration, and invasion. Our findings elucidate how YAP induces cancer aggressiveness and may assist diagnosis of cancer metastasis.

Recruitment of HIV-1 envelope occurs subsequent to lipid mixing: a fluorescence microscopic evidence.

Entry of the human immunodeficiency virus (HIV) into the target cell is initiated by fusion with the cell membrane, mediated through the envelope glycoproteins gp120 and gp41, following engagement to CD4 and the co-receptor. Previous fusion kinetics studies on the HXB2 envelope protein (Env) revealed that Env recruitment occurred at about 13 min concurrent with the lipid mixing. To resolve the temporal sequence of lipid mixing and recruitment, we employed an inhibitory assay monitored by fluorescence microscopy using a gp41 ectodomain (gp41e) fragment, which blocked Env recruitment in stark contrast to the lack of gp41e effect on the lipid mixing. In addition, to demonstrate the mode of action for the inhibition of gp41e, our results strongly suggested that lipid mixing precedes the Env recruitment because lipid mixing can proceed with Env recruitment inhibited by exogeneous gp41e molecules. Importantly, it was found that the random clustering of Env molecules on the membrane surface occurred at approximately 1 minute whereas the Env recruitment was observed at 13 minutes after the attachment of Env-expressing cell to the target cell. This > 10-fold temporal discrepancy highlights that the productive assembly of Env molecules leading to fusion requires spatio-temporal coordination of several adjacent Env trimers aggregated via directed movement.

The function of coreceptor as a basis for the kinetic dissection of HIV type 1 envelope protein-mediated cell fusion.

The function of HIV-1 HXB2 envelope (Env) glycoprotein (gp) was investigated by surface plasmon resonance and fluorescence imaging techniques. Strikingly, it was found that gp120 shedding requires the presence of the X4 coreceptor. A similar coreceptor requirement was observed for the membrane mixing and the Env recruitment on the cell surface. However, exposure and membrane penetration of the fusion peptide do not require X4 and occur within the first minute after incubation of Env with CD4 and/or X4. Analogously X4 was not required but enhanced binding of the fusion inhibitor. In contrast, bundle formation of the gp41 ectodomain, as monitored by NC-1, was accelerated by the presence of X4. The kinetics of these key post-Env binding events as determined in real time by fluorescence microscopic imaging, coupled with the differential coreceptor requirement, led to the proposition that gp120 shedding, which takes place from 1 to 10 min after engagement of receptor and coreceptor to Env, is a primary function of the coreceptor. The shedding of the surface subunits is needed for the subsequent processes including hemifusion, full fusion, and Env recruitment. The temporal order of these fusogenic steps allows construction of a refined model on the Env-mediated cell fusion event.

Ultrawidefield microscope for high-speed fluorescence imaging and targeted optogenetic stimulation.

The rapid increase in the number and quality of fluorescent reporters and optogenetic actuators has yielded a powerful set of tools for recording and controlling cellular state and function. To achieve the full benefit of these tools requires improved optical systems with high light collection efficiency, high spatial and temporal resolution, and patterned optical stimulation, in a wide field of view (FOV). Here we describe our 'Firefly' microscope, which achieves these goals in a Ø6 mm FOV. The Firefly optical system is optimized for simultaneous photostimulation and fluorescence imaging in cultured cells. All but one of the optical elements are commercially available, yet the microscope achieves 10-fold higher light collection efficiency at its design magnification than the comparable commercially available microscope using the same objective. The Firefly microscope enables all-optical electrophysiology ('Optopatch') in cultured neurons with a throughput and information content unmatched by other neuronal phenotyping systems. This capability opens possibilities in disease modeling and phenotypic drug screening. We also demonstrate applications of the system to voltage and calcium recordings in human induced pluripotent stem cell derived cardiomyocytes.

Geometry-dependent functional changes in iPSC-derived cardiomyocytes probed by functional imaging and RNA sequencing.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising platform for cardiac studies in vitro, and possibly for tissue repair in humans. However, hiPSC-CM cells tend to retain morphology, metabolism, patterns of gene expression, and electrophysiology similar to that of embryonic cardiomyocytes. We grew hiPSC-CM in patterned islands of different sizes and shapes, and measured the effect of island geometry on action potential waveform and calcium dynamics using optical recordings of voltage and calcium from 970 islands of different sizes. hiPSC-CM in larger islands showed electrical and calcium dynamics indicative of greater functional maturity. We then compared transcriptional signatures of the small and large islands against a developmental time course of cardiac differentiation. Although island size had little effect on expression of most genes whose levels differed between hiPSC-CM and adult primary CM, we identified a subset of genes for which island size drove the majority (58%) of the changes associated with functional maturation. Finally, we patterned hiPSC-CM on islands with a variety of shapes to probe the relative contributions of soluble factors, electrical coupling, and direct cell-cell contacts to the functional maturation. Collectively, our data show that optical electrophysiology is a powerful tool for assaying hiPSC-CM maturation, and that island size powerfully drives activation of a subset of genes involved in cardiac maturation.