Single cell analytics for nanobiology

Considerable evidence suggests that cancer stem-like cells (CSCs) are critical in tumor pathogenesis. Though functional CSCs can be identified using dish-based tumorsphere assays, it is difficult to handle and monitor single cells in a dish-based format. Tumor-stromal interaction is another key regulator of cancer micro-environments, affecting tumorigenesis and metastasis. To enable tumorsphere assays that incorporate interaction with stromal cells, we developed a microfluidic dual adherent/suspension co-culture device. This device combined a suspension environment for single-cell tumorsphere assay and an adherent environment for co-culture of stromal cells in close proximity by selectively patterning polyHEMA in indented microwells. By minimizing dead volume and improving cell capture efficiency, the presented platform allowed the use of small samples (<100 cells), affording the potential to study rare cells such as circulating tumor cells (CTCs). As a proof-of-concept, we co-cultured single T47D breast cancer cells and primary cancer associated fibroblasts (CAF) on-chip for 14 days to monitor sphere formation and growth. Compared to mono-cultured T47D cells, co-cultured T47D have significantly higher tumorigenic potential (sphere formation rate) by three times and proliferation rates (larger sphere size). In addition to the phenotypic observation, the platform allows the retrieval of mono-cultured and co-cultured spheres for downstream analyses. Multiplexed single-cell gene expression analyses using C1 and BioMark HD platforms were performed to compare the expression of 96 target genes in co-cultured and mono-cultured T47D cells. Co-cultured cells expressed significantly higher MKI67, which suggests a possible mechanism for the elevated proliferation rate. Additionally, lower BAX (pro-apoptotic marker) expression matches well with higher sphere formation rate in the co-cultured cells. Interestingly, we noticed the down-regulation of CDH1, KRT8, and KRT18 in the co-cultured cells, indicating possible epithelial-mesenchymal transition induced by CAF. Single cell resolution, while to comparing between groups, also allows examination of cellular heterogeneity within one population. While cells in the mono-cultured group were more homogeneous co-cultured cells displayed clear bimodal gene expression distribution including, ANXA3, MTOR, MKI67, BAX, and PGR. This is suggestive of variable responses to the same stimuli at the single cell level. Combining the dual adherent/suspension co-culture platform presented here with the single cell gene expression analysis, we may successfully identify functional CSCs, investigate the phenotypic and transcriptional effects induced by tumor-stromal interactions, and study cellular heterogeneity in greater detail. Citation Format: Yu-Chih Chen, Shamileh Fouladdel, Zhixiong Zhang, Ebrahim Azizi, Euisik Yoon, Max S. Wicha. Single cell dual adherent-suspension co-culture microenvironment for studying tumor-stromal interactions in breast cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4076.

The quantification of elemental concentration in cells is usually performed by analytical assays on large populations missing peculiar but important rare cells. The present article aims at comparing the elemental quantification in single cells and cell population in three different cell types using a new approach for single cells elemental analysis performed at sub-micrometer scale combining X-ray fluorescence microscopy and atomic force microscopy. The attention is focused on the light element Mg, exploiting the opportunity to compare the single cell quantification to the cell population analysis carried out by a highly Mg-selective fluorescent chemosensor. The results show that the single cell analysis reveals the same Mg differences found in large population of the different cell strains studied. However, in one of the cell strains, single cell analysis reveals two cells with an exceptionally high intracellular Mg content compared with the other cells of the same strain. The single cell analysis allows mapping Mg and other light elements in whole cells at sub-micrometer scale. A detailed intensity correlation analysis on the two cells with the highest Mg content reveals that Mg subcellular localization correlates with oxygen in a different fashion with respect the other sister cells of the same strain. Graphical abstract Single cells or large population analysis this is the question!

Quantifying silver nanoparticle association and elemental content in single cells using dual mass mode in quadrupole-based inductively coupled plasma-mass spectrometry

Development of a poly-dimethylsiloxane microfluidic device for single cell isolation and incubation

Microfluidic-based single cells analysis has been of great interest in recent years, promising disease diagnosis and personalized medicine. Current technologies are challenging in bioselectively isolating specific single cells from complex matrices. Herein, a novel microfluidic platform integrated with cell-recognizable aptamer-encoded microwells was specifically developed to isolate single tumor cells with satisfied single-cell occupancy and unique bioselectivity. In this work, the designed microwell-structures enable us to encourage strong 3D local topographic interactions of the target cell surface with biomolecules and regulate the single-cell resolution. Under the optimized size of microwells, the single-cell occupancy was significantly enhanced from 0.5% to 88.2% through the introduction of the aptamer. Analysis of the target cells was directly performed in short time periods (<5.0 min) with small volumes (4.5 μL). Importantly, such an aptamer-enabled microfluidic device shows an excellent selectivity for target single cells isolation compared with three control cells. Subsequently, targeted isolation and analysis of single tumor cells were demonstrated by using artificial complex cell samples at simulated conditions, and various cellular carboxylesterases were studied by time-course measurements of cellular fluorescence kinetics at individual-cell level. Thus, our technique will open up a new opportunity in single-cell level-based disease diagnosis and personalize medicine screening.

Tumor heterogeneity represents a major obstacle to effective cancer treatment and personalized medicine. Hepatocellular carcinoma (HCC) is clinically and biologically heterogeneous, which is partly attributed to the presence of hepatic cancer stem cells (CSCs). CSC surface makers can have overlapping expression or exclusive expression of different subpopulations of hepatic CSCs, and these cells may contain different oncogenic changes. The existence of various CSC surface-marked subpopulations might pose a problem for CSC targeted therapeutics. Little is known about whether there are shared or distinct pathways in different subpopulations. Moreover, it remains unknown whether heterogeneity exists within certain subpopulations. Thus, we propose to profile the global transcriptome of surface-marked CSCs (EpCAM+, CD133+ and CD24+) at the single-cell level using index flow cytometric sorting and single-cell RNA sequencing technology. We statistically compared mRNA transcriptomes of individual single cells of triple positive (EpCAM+/ CD133+ /CD24+) CSCs and triple negative (EpCAM-/ CD133- /CD24-) cells. Further, we compared the triple positive and the triple negative cells at the single cell level in terms of their self-renew capability in both normoxic and hypoxic conditions. Our results show that at the single cell level, the transcriptomic profiles show a high-degree of heterogeneity in HCC cell lines, meanwhile, there is a dramatic difference between triple positive and triple negative cells in their transcriptomic profiles. There is a high-degree of intra-tumor and inter-tumor heterogeneity observed in single cell transcriptomes of HCC patients’ samples. Triple positive CSC single cells show continuous rather than discrete stemness-related gene expression patterns. In parallel, the self-renewal capability of triple positive cells also exhibits heterogeneity, while triple negative cells show little self-renewal capability. Thus, our single cell analysis study reveals molecular and biological heterogeneity of cancer stem cells in HCC, which may provide insight into the underlying mechanisms of how CSCs contribute to tumor heterogeneity and underscore key pathways and novel targets for hepatic CSC therapy. Citation Format: Hongping Zheng, Yotsawat Pomyen, Maria Hernandez, Caiyi Li, Ferenc Livak, Tim Greten, Anuradha Budhu, Xin Wang. Single cell analysis reveals cancer stem cell heterogeneities in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 936. doi:10.1158/1538-7445.AM2017-936

BackgroundTherapeutic decisions in cancer are generally guided by molecular biomarkers or, for some newer therapeutics, primary tumor genotype. However, because biomarkers or genotypes may change as new metastases emerge, circulating tumor cells (CTCs) from blood are being investigated for a role in guiding real-time drug selection during disease progression, expecting that CTCs will comprehensively represent the full spectrum of genomic changes in metastases. However, information is limited regarding mutational heterogeneity among CTCs and metastases in breast cancer as discerned by single cell analysis. The presence of disseminated tumor cells (DTCs) in bone marrow also carry prognostic significance in breast cancer, but with variability between CTC and DTC detection. Here we analyze a series of single tumor cells, CTCs, and DTCs for PIK3CA mutations and report CTC and corresponding metastatic genotypes.MethodsWe used the MagSweeper, an immunomagnetic separation device, to capture live single tumor cells from breast cancer patients’ primary and metastatic tissues, blood, and bone marrow. Single cells were screened for mutations in exons 9 and 20 of the PIK3CA gene. Captured DTCs grown in cell culture were also sequenced for PIK3CA mutations.ResultsAmong 242 individual tumor cells isolated from 17 patients and tested for mutations, 48 mutated tumor cells were identified in three patients. Single cell analyses revealed mutational heterogeneity among CTCs and tumor cells in tissues. In a patient followed serially, there was mutational discordance between CTCs, DTCs, and metastases, and among CTCs isolated at different time points. DTCs from this patient propagated in vitro contained a PIK3CA mutation, which was maintained despite morphological changes during 21 days of cell culture.ConclusionsSingle cell analysis of CTCs can demonstrate genotypic heterogeneity, changes over time, and discordance from DTCs and distant metastases. We present a cautionary case showing that CTCs from any single blood draw do not always reflect metastatic genotype, and that CTC and DTC analyses may provide independent clinical information. Isolated DTCs remain viable and can be propagated in culture while maintaining their original mutational status, potentially serving as a future resource for investigating new drug therapies.

High resolution single cell microscopy analyses identifies the cellular redox state that supports conversion to a neoplastic stem cell state Brian Spurlock and Kasturi Mitra University of Alabama at Birmingham Introduction: Single cell analyses lie at the core of teasing out tumor cell heterogeneity at the level of their self-renewal and differentiation ability, gene mutation and expression, energetics, metabolism, redox status etc. Cellular redox states are modulated by Reactive Oxygen Species (ROS) that are primarily generated by mitochondria. The neoplastic stem cells, which lie at the base of tumor cell hierarchy, are supported by optimally elevated mitochondrial ROS levels. However, induction of higher levels of mitochondrial ROS can eliminate the neoplastic stem cells. Therefore, there appears to be a sweet spot of mitochondrial ROS levels for maintenance of neoplastic stem cells. Given our longstanding interest in mitochondrial biology, we aimed at probing mitochondrial redox modulation at a single cell level towards understanding cellular susceptibility for neoplastic transformation. Experimental procedures: For this study we have used our high resolution confocal microscopy based single cell assay for mitochondrial properties, cell based Seahorse respirometry assay, tumorsphere assay, xenograft assays and shRNA mediated gene knockdown. Summary of data: We have used carcinogen induced cell transformation model to study neoplastic transformation. We found that carcinogen induced enrichment of neoplastic stem cells was associated with a certain level of mitochondrial ROS beyond which the number of neoplastic stem cell frequency decreases dramatically. The increase in the lineage specific stem cell marker, Sox2, happens only in cells with repression of a mitochondrial property called mitochondrial fission. Knockdown of the master regulator of mitochondrial fission, Drp1 (Dynamin Regulated Protein 1), alters mitochondrial ROS levels and causes increase in Sox2 levels and accelerates carcinogen induced cell transformation. Therefore, our data suggested that repression of mitochondrial fission sustains mitochondrial ROS at a certain level to allow enrichment of neoplastic stem cells. Most importantly, our single cell microscopy analyses of mitochondrial ROS and mitochondrial fission in the same cells identifies the intermediate mitochondrial ROS levels that associates with a minimum mito-fission state that supports neoplastic stem cells. Conclusion: We conclude that the cells with a certain quantifiable level of mitochondrial-ROS status are maximally susceptible to (carcinogen induced) conversion to neoplastic stem cells and can be identified by our novel high resolution microscopy based single cell analyses. Currently, we are performing single cell RNAseq experiments with the goal of integrated understanding of gene expression and mitochondrial redox regulation in neoplastic stem cells. Citation Format: Brian Spurlock, Kasturi Mitra. High resolution single cell microscopy analyses identifies the cellular redox state that supports conversion to a neoplastic stem cell state [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-116.

Our understanding of the diversity of cells that escape the primary tumor and seed micrometastases remains rudimentary, and approaches for studying circulating and disseminated tumor cells have been limited by low throughput and sensitivity, reliance on single parameter sorting, and a focus on enumeration rather than phenotypic and genetic characterization. Here, we utilize a highly sensitive microfluidic and dielectrophoretic approach for the isolation and genetic analysis of individual tumor cells. We employed fluorescence labeling to isolate 208 single cells from spiking experiments conducted with 11 cell lines, including 8 neuroblastoma cell lines, and achieved a capture sensitivity of 1 tumor cell per 106 white blood cells (WBCs). Sample fixation or freezing had no detectable effect on cell capture. Point mutations were accurately detected in the whole genome amplification product of captured single tumor cells but not in negative control WBCs. We applied this approach to capture 144 single tumor cells from 10 bone marrow samples of patients suffering from neuroblastoma. In this pediatric malignancy, high-risk patients often exhibit wide-spread hematogenous metastasis, but access to primary tumor can be difficult or impossible. Here, we used flow-based sorting to pre-enrich samples with tumor involvement below 0.02%. For all patients for whom a mutation in the Anaplastic Lymphoma Kinase gene had already been detected in their primary tumor, the same mutation was detected in single cells from their marrow. These findings demonstrate a novel, non-invasive, and adaptable method for the capture and genetic analysis of single tumor cells from cancer patients.

This paper describes the use of nanosensor and pH-dependent fluorescent dye to monitor intracellular pH in single CA46 cell. The pH-dependent fluorescent dye 5 (6)-Carboxyeosin diacetate (CFDA) was used to measure intracellular pH from the surface fluorescence of the single CA46 Cell. Argon ion laser was focused on single cell with a fiber optic nanoprobe which is parked outside on the single cell, also the emitted light was collected with this fiber optic nanoprobe. To account for system instabilities, we have explored a ratiometric approach to determine the intracellular pH on the single-cell level after staining with CFDA. Calibration of the intracellular fluorescence spectra is obtained by equilibration of internal and external pH with Nigericin in k <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">+</sup> buffers at different external pH. Besides accurate estimated of intracellular pH (7.21 plusmn 0.05). This technique permitted monitor transient changes in intracellular pH caused by addition and removal of NH <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">4</sub> <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">+</sup> and allow to study pH-regulatory in Single CA46 cell.

Leveraging microfluidics and nano-plasmonics, we present in this paper a new method employing a micro-nano-device that is capable of monitoring the dynamic cell-substrate attachment process at single cell level in real time without labeling. The micro-nano-device essentially has a gold thin film as the substrate perforated with periodic, near-cm2-area, template-stripped nano-holes, which generate plasmonic extraordinary optical transmission (EOT) with a high sensitivity to refractive index changes at the metal-dielectric interface. Using this device, we successfully demonstrated label-free and real-time monitoring of the dynamic cell attachment process for single mouse embryonic stem cell (C3H10) and human tumor cell (HeLa) by collecting EOT spectrum data during 3-hour on-chip culture. We further collected the EOT spectral shift data at the start and end points of measurement during 3-hour on-chip culture for 50 C3H10 and 50 HeLa cells, respectively. The experiment results show that the single cell attachment process of both HeLa and C3H10 cells follow the logistic retarded growth model, but with different kinetic parameters. Variations in spectral shift during the same culture period across single cells present new evidence for cell heterogeneity. The micro-nano-device provides a new, label-free, real-time, and sensitive, platform to investigate the cell adhesion kinetics at single cell level.

Multielement analysis of single red blood cells by single cell - inductively coupled plasma tandem mass spectrometry

Optical tweezers assisted analyzing and sorting of tumor cells tagged with fluorescence nanospheres in a microfluidic chip

Choice of temporal and spatial scale for querying biological systems is key to opening up nature’s mysteries for investigation. For example, temporal resolution at which sampling is conducted is critical to answering granular details about a biological phenomenon, where a coarse sampling interval could not reveal fine level control on RNA transcription or protein translation. On the other hand, the spatial scale at which a biological question is posed concerns the validity of the conclusions drawn from the data obtained. Specifically, techniques and methods chosen for population level cellular assays would not be able to address questions at the single cell level, while the intricacies and caveats of single cell methodologies in understanding biological processes at the single cell level needs to be appreciated. More importantly, how single cell phenomena is aggregated to population level effects need to be factored into experiment design and data interpretation both for single cell and population level studies. Specifically, as biology transcends multiple levels of organization ranging from single cell to clusters of cells and cell population, it is critical to gain understanding of how different biological effects could manifest at different population sizes. Hence, understanding the nuances of how temporal and spatial concepts could be deployed in experiment design in biology would help yield experiments that would more likely help address specific questions posed at the interface of subpopulations and subcellular level.

Depending on age, phase in the cell cycle, nutrition, and environmental factors, individual cells exhibit large metabolic diversity. To explore metabolic variations in cell populations, laser ablation electrospray ionization (LAESI) mass spectrometry (MS) was used for the in situ analysis of individual cells at atmospheric pressure. Single cell ablation was achieved by delivering mid-IR laser pulses through the etched tip of a GeO(2)-based glass fiber. Metabolic analysis was performed from single cells and small cell populations of Allium cepa and Narcissus pseudonarcissus bulb epidermis, as well as single eggs of Lytechinus pictus. Of the 332 peaks detected for A. cepa, 35 were assigned to metabolites with the help of accurate ion masses and tandem MS. The metabolic profiles from single cells of the two plant species included a large variety of oligosaccharides including possibly fructans in A. cepa, and alkaloids, e.g., lycorine in N. pseudonarcissus. Analysis of adjacent individual cells with a difference in pigmentation showed that, in addition to essential metabolites found in both variants, the pigmented cells contained anthocyanidins, other flavonoids, and their glucosides. Analysis of single epidermal cells from different scale leaves in an A. cepa bulb showed metabolic differences corresponding to their age. Our results indicate the feasibility of using LAESI-MS for the in situ analysis of metabolites in single cells with potential applications in studying cell differentiation, changes due to disease states, and response to xenobiotics.