Single-cell Events Research Articles

Tracking and measuring local protein synthesis in vivo.

Detecting when and how much of a protein molecule is synthesized is important for understanding cell function, but current methods either cannot be performed in vivo or have poor temporal resolution. Here, we developed a technique to detect and quantify subcellular protein synthesis events in real time in vivo. This Protein Translation Reporting (PTR) technique uses a genetic tag that produces a stoichiometric ratio of a small peptide portion of a split fluorescent protein and the protein of interest during protein synthesis. We show that the split fluorescent protein peptide can generate fluorescence within milliseconds upon binding the larger portion of the fluorescent protein, and that the fluorescence intensity is directly proportional to the number of molecules of the protein of interest synthesized. Using PTR, we tracked and measured protein synthesis events in single cells over time in vivo. We use different color split fluorescent proteins to detect multiple genes or alleles in single cells simultaneously. We also split a photoswitchable fluorescent protein to photoconvert the reconstituted fluorescent protein to a different channel to continually reset the time of detection of synthesis events.

Single cell-inductively coupled plasma-mass spectrometry (SC-ICP-MS) reveals metallic heterogeneity in a macrophage model of infectious diseases.

Single cell-inductively coupled plasma-mass spectrometry (SC-ICP-MS) offers an attractive option for rapidly measuring trace metal heterogeneity at the single cell level. Chemical fixation has been previously applied to mammalian cells prior to sample introduction so that they can be resuspended in a solution suitable for SC-ICP-MS. However, the effect of fixation on the elemental composition of suspended cells is unknown, and robust methodologies are urgently needed so that the community can measure the effects of intracellular pathogens on elemental composition of their host cells. We demonstrate that different fixatives impact measured cell elemental composition. We have compared suspensions treated using different fixatives (methanol 60-100% in H2O and 4% paraformaldehyde in phosphate-buffered saline solution), and the number of distinguishable single cell events, keeping a constant particle number concentration. Significantly more single cell events (n = 3, P ≤ 0.05) were observed for Ca and Mg when cells were fixed in 4% paraformaldehyde than for the methanol-based fixatives, confirming the hypothesis that methanol fixatives cause leaching of these elements from the cells. The impact of fixation on Mn and Zn was less pronounced. Microbial and viral infection of eukaryotic cells can have profound effects on their elemental composition, but chemical fixation is necessary to render infected cells safe before analysis. We have successfully applied our methodology to a macrophage model of tuberculosis demonstrating utility in understanding metal homeostasis during microbial infection of mammalian cells.

Investigating the Near-Surface Wind Fields of Downbursts Using a Series of High-Resolution Idealized Simulations

Abstract Short-lived and poorly organized convective cells, often called weakly forced thunderstorms (WFTs), are a common phenomenon during the warm season across the eastern and southeastern United States. While typically benign, wet downbursts emanating from such convection can have substantial societal impacts, including tree, power line, and property damage from strong outflow winds. Observational studies have documented the occurrence of severe (25.7 m s−1 or higher) wind speeds from wet downbursts, but the frequency of severe downbursts, including the spatial extent and temporal duration of severe winds, remains unclear. The ability for modern observing networks to reliably observe such events is also unknown; however, answering these questions is important for improving forecast skill and verifying convective warnings accurately. This study attempts to answer these questions by drawing statistical inferences from 97 high-resolution idealized simulations of single-cell downburst events. It was found that while 35% of the simulations featured severe winds, the spatial and temporal extent of such winds is limited—O(10) km2 or less and persisting for around 5 min on average. Furthermore, through a series of simulated network experiments, it is postulated that the probability that a modern mesonet observes a severe wind gust given a severe downburst is around 1%. From these results, a statistical argument is made that most tree impacts associated with pulse convection are likely caused by subsevere winds. Several implications for forecasting, warning, and verifying WFT events fall out from these discussions.

Complementary Tumor Diagnosis by Single Cell-Based Cytogenetics Using Multi-marker Fluorescence In Situ Hybridization (mFISH).

Multi-color (or multi-marker) fluorescence in situ hybridization (mFISH) is a well-established, valuable, complementary tool for prenatal and pathological (tumor) diagnosis. A variety of chromosomal abnormalities, such as partial or total chromosomal gains, losses, inversions, or translocations, which are considered to cause genetic syndromes, can relatively easily be detected on a cell-by-cell basis. Individual cells either in suspension (e.g., in the form of a cytological specimen derived from body fluids) or within a tissue (e.g., a solid tumor specimen or biopsy) can be quantitatively evaluated with respect to the chromosomal hybridization markers of interest (e.g., a gene or centromeric region) and with due consideration of cellular heterogeneity. FISH is helpful or even essential for the (sub-)classification, stratification, and unambiguous diagnosis of a number of malignant diseases and contributes to treatment decision in many cases. Here, the diagnostic power and limitations of typical FISH and mFISH approaches (except chromosome painting and RNA hybridization) are discussed, with special emphasis on tumor and single-cell diagnostics. Well-established and novel FISH protocols, the latter addressed to accelerate and flexibilize the preparation and hybridization of formalin-fixed and paraffin-embedded tissues, are provided. Moreover, guidelines and molecular aspects important for data interpretation are discussed. Finally, sophisticated multiplexed approaches and those that analyze very rare single-cell events, which are not yet implemented in diagnostic procedures, will be touched upon. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: (m)FISH applied to formaldehyde-fixed paraffin-embedded tissues Basic Protocol 2: (m)FISH applied to cytological specimens.

Species-dependent uptake of gadolinium in Chlamydomonas reinhardtii algae

Single cell-inductively coupled plasma-mass spectrometry (sc-ICP-MS) was used in this study as a valuable tool to assess the species-dependent uptake of metallopharmaceuticals into algal cells. Chlamydomonas reinhardtii algae were incubated for 24 h with four Gadolinium-based contrast agents (GBCAs) and GdCl3. A species dependency towards the uptake of the tested Gd species was observed. Using single cell-ICP-MS, a Gd signal corresponding to single cell events was detected for GdCl3 and the linear GBCAs Omniscan® (Gadodiamide, Gd-DTPA-BMA) and Magnevist® (Gadodiamide, Gd-DTPA). For the macrocyclic complexes Dotarem® (Gadoteric acid, Gd-DOTA) and Gadovist® (Gadobutrol, Gd-BT-DO3A), no such Gd signal was visible. Total Gd analysis via ICP-MS confirmed the presence of Gd in the cells only after incubation with GdCl3 and the linear GBCAs, while only small amounts of Gd were detected for the incubations with macrocyclic GBCAs. Furthermore, the results showed that more Gd is bound to cell structures or macromolecules, while smaller amounts are present in the lysate. Using hydrophilic interaction liquid chromatography (HILIC)-ICP-MS, the soluble Gd species in the lysate were analyzed to determine if the initial Gd complexes were still intact. Surprisingly, no intact GBCAs were detected in the lysates of any incubation solution, possibly due to a change in Gd speciation. Further research is needed to assess which Gd species are present in the lysate, while “free” Gd ions or adducts with cell constituents are the most likely explanation. This study highlights the need for species-dependent investigation of elements in aquatic organisms. Moreover, the uptake of linear GBCAs and their species alteration raises the question of a potential accumulation of Gd in the food chain.

COORDINATED CHANGES IN THE TUMOR MICROENVIRONMENT (TME) ARE ASSOCIATED WITH INCREASED RISK OF THERAPEUTIC FAILURE IN NEWLY DIAGNOSED DIFFUSE LARGE B‐CELL LYMPHOMA (DLBCL)

Introduction: Despite advances, 20%–40% of patients with DLBCL do not achieve remission or relapse after an initial response. We aimed to identify immunological factors associated with early clinical failure in DLBCL. Methods: Patients with DLBCL treated with rituximab and anthracycline-based chemotherapy were prospectively enrolled in the University of Iowa/Mayo Clinic Lymphoma Specialized Program of Research Excellence (SPORE). Tissue microarrays (TMAs) containing specimens from diagnosis were analyzed using the Digital Spatial Profiling (DSP) system quantifying expression of 58 proteins (Figure 1A). Event-free survival (EFS) was defined as time from diagnosis to relapse, progression, or death from any cause. Patients with EFS of at least 24 months were classified as achieving EFS24. Following normalization, logistic regression was performed on DSP data to adjust for IPI and cell of origin. Multiplex immunofluorescence (MxIF) using the CODEX system was performed on a biopsy specimen from a representative patient. Machine learning models were used for cell segmentation and classification (Figure 1B). Results: Specimens from 446 DLBCL patients were available on TMAs, of which 321 (72%) achieved EFS24. DSP data was acquired in 395 unique DLBCL patients and showed that increased expression of CD163 (Log2FC = 2.9; p = 0.0009), BCL2 (Log2FC = 1.4; p = 0.047), and PDL1 (Log2FC = 1.3; p = 0.05) were significantly associated with increased risk of EFS24 failure (Figure 1A, red arrows). Conversely, increased expression of CD11c (Log2FC = 0.5; p = 0.003), HLA-DR (Log2FC = 0.4; p = 0.01), CD357/GITR (Log2FC = 0.8; p = 0.01), CD137 (Log2FC = 0.7; p = 0.02), and B7H3 (Log2FC = 0.9; p = 0.02) were significantly associated with decreased risk of EFS24 failure (Figure 1A, green arrows). A total of 3004 single cell events were acquired using MxIF, of which 55% were classified as B cells, 17% as T cells, 8% as macrophages, and 20% as other cells. Within B cells, 81% were classified as HLA-DR+, 73% as CD11c+, 54% as BCL2+, while; 45% were positive for all 3 antigens (Figure C1–4). GITR expression was confirmed in 3% of T cells and showed a memory (CD45RO+), activated (CD38+) phenotype (Figure D1–4). The research was funded by: Department of Defense (DOD), Lymphoma Research Foundation (LRF), National Cancer Institute (NCI) Keywords: aggressive B-cell non-Hodgkin lymphoma, genomics, epigenomics, and other -omics, microenvironment No conflicts of interests pertinent to the abstract.

Shared regulation and functional relevance of local gene co-expression revealed by single cell analysis

Most human genes are co-expressed with a nearby gene. Previous studies have revealed this local gene co-expression to be widespread across chromosomes and across dozens of tissues. Yet, so far these studies used bulk RNA-seq, averaging gene expression measurements across millions of cells, thus being unclear if this co-expression stems from transcription events in single cells. Here, we leverage single cell datasets in >85 individuals to identify gene co-expression across cells, unbiased by cell-type heterogeneity and benefiting from the co-occurrence of transcription events in single cells. We discover >3800 co-expressed gene pairs in two human cell types, induced pluripotent stem cells (iPSCs) and lymphoblastoid cell lines (LCLs) and (i) compare single cell to bulk RNA-seq in identifying local gene co-expression, (ii) show that many co-expressed genes – but not the majority – are composed of functionally related genes and (iii) using proteomics data, provide evidence that their co-expression is maintained up to the protein level. Finally, using single cell RNA-sequencing (scRNA-seq) and single cell ATAC-sequencing (scATAC-seq) data for the same single cells, we identify gene-enhancer associations and reveal that >95% of co-expressed gene pairs share regulatory elements. These results elucidate the potential reasons for co-expression in single cell gene regulatory networks and warrant a deeper study of shared regulatory elements, in view of explaining disease comorbidity due to affecting several genes. Our in-depth view of local gene co-expression and regulatory element co-activity advances our understanding of the shared regulatory architecture between genes.

Histologically resolved multiomics enables precise molecular profiling of human intratumor heterogeneity.

Both the composition of cell types and their spatial distribution in a tissue play a critical role in cellular function, organ development, and disease progression. For example, intratumor heterogeneity and the distribution of transcriptional and genetic events in single cells drive the genesis and development of cancer. However, it can be challenging to fully characterize the molecular profile of cells in a tissue with high spatial resolution because microscopy has limited ability to extract comprehensive genomic information, and the spatial resolution of genomic techniques tends to be limited by dissection. There is a growing need for tools that can be used to explore the relationship between histological features, gene expression patterns, and spatially correlated genomic alterations in healthy and diseased tissue samples. Here, we present a technique that combines label-free histology with spatially resolved multiomics in unfixed and unstained tissue sections. This approach leverages stimulated Raman scattering microscopy to provide chemical contrast that reveals histological tissue architecture, allowing for high-resolution in situ laser microdissection of regions of interests. These microtissue samples are then processed for DNA and RNA sequencing to identify unique genetic profiles that correspond to distinct anatomical regions. We demonstrate the capabilities of this technique by mapping gene expression and copy number alterations to histologically defined regions in human oral squamous cell carcinoma (OSCC). Our approach provides complementary insights in tumorigenesis and offers an integrative tool for macroscale cancer tissues with spatial multiomics assessments.

Towards 'end-to-end' analysis and understanding of biological timecourse data.

Petabytes of increasingly complex and multidimensional live cell and tissue imaging data are generated every year. These videos hold large promise for understanding biology at a deep and fundamental level, as they capture single-cell and multicellular events occurring over time and space. However, the current modalities for analysis and mining of these data are scattered and user-specific, preventing more unified analyses from being performed over different datasets and obscuring possible scientific insights. Here, we propose a unified pipeline for storage, segmentation, analysis, and statistical parametrization of live cell imaging datasets.

Abstract 1597: Characterization of genomic diversity in high grade serous ovarian and triple negative breast cancer at single cell resolution

Abstract Copy number alterations and structural variants are associated with disease progression, therapeutic response, and metastasis in human cancers, yet the extent and mechanisms driving continued genomic instability remain poorly understood. We generated more than 20,000 single-cell whole genomes from 25 high-grade serous ovarian and triple-negative breast cancers across a range of mutational subtypes. Through the development of new computational methods, we resolved alterations into cancer haplotypes, providing fine-grained resolution of genomic events in single-cells and exposing extensive cell-to-cell genomic variation. We identified 3 key processes that contribute to this diversity. Firstly, we observed that parallel copy number events - whereby different cancer cells harbor chromosomal aberrations with identical total copy number but different combinations of maternal and paternal alleles - were present in > 80% of the tumors. This is consistent with extensive convergent evolution in these tumors. Secondly, we identified considerable cell-to-cell variability in the amplitude of high-level amplifications, often impacting oncogenes. Single-cell transcriptomes and immunohistochemistry from the same samples showed that this genomic diversity led to phenotypic differences between cells. Finally, we observed copy number break-point variability consistent with progressive genomic diversification, which we termed serriform structural variation (SSV). Both SSV’s and variable high-level amplifications were enriched in foldback inversion (FBI) bearing tumors relative to homologous recombination deficient tumors, highlighting that FBI tumors have greater levels of structural instability. Our study sheds new light on the evolution of genomically unstable tumors with implications for cancer evolution, therapeutic targeting, and patient stratification. Citation Format: Marc J. Williams, Tyler Funnell, Ciara H. O'Flanagan, Andrew McPherson, Samuel Aparicio, Sohrab P. Shah. Characterization of genomic diversity in high grade serous ovarian and triple negative breast cancer at single cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1597.

Abstract 5383: Dexter࣪ microfluidic platform spatially resolves antigen presentation and T cell functions in microdroplets and deciphers dynamic tumor-immune interaction network

Abstract Background: Immune checkpoint inhibitors are being used in clinics for a diverse set of cancers. However, the outcome is highly contextual and personalized with consistently low response rates. Understanding the mechanistic barriers at single cell levels is key for novel candidate selection. Here we describe Dexter࣪ microfluidic platform that can create highly parallel nanobioreactors on a chip to study cellular functions and interactions at the single cell level. Using microliter volumes of live tumor and human PBMC, we showed modulation of dynamic tumor-immune crosstalk at single cell level. Methods: The Dexter࣪ microfluidic platform was used for functional characterization at single cell resolution. The platform integrates an array of nanoliter sized droplets with cell encapsulation, multicolor fluorescence imaging and selective cell retrieval. PBMC, RBC and cancer cells were fluorescently (CFSE, PKH) labelled and used to interrogate effector cell functions following exposure to immune modulating milieu in nanoliter droplets. CD8+T cells were activated and labelled. Individual and clustered tumor cells were challenged with stimulated PBMC. Finally, using bioinformatic tools, we construct a Tumor immune Interactive Network (TiIN) and simulate perturbation. Results: Our data confirmed preservation of surface markers, T cells and tumor-immune specific intracellular and surface events including cell-cell contacts at the level observed before their encapsulation in microfluidic droplets. Both size and intensity of the signals were maintained without significant loss. Platform displayed critical events like antigen presentation, tumor-probiotic interface, T cell engagement with microbeads similar to APC dimensionalities and function. Both spiked tumor and immune cells showed high degree of recovery. Spatially resolved dynamic profiling in this platform further revealed immune phenotypic plasticity and vulnerabilities of tumor cells upon exposure to immune checkpoint agents. Finally, we, measured the killing of tumor cells by using different effector : target ratio that complement hot and cold niche. We observed differential killing of target when T cells were primed using immunomodulators. Conclusion: Our data highlights the versatility of Dexter࣪ platform in deciphering mechanistically critical tumor-immune interactions while parallelly resolving single cell events. This offers suitable means of profiling response to novel immunooncology candidates. Citation Format: Ashwin Lal, Ranjeet Singh, Abhay Sane, Rushil Manglik, Ravi R. Keshari, SM Mohanasundaram, Pradip K. Majumder, Biswanath Majumder. Dexter࣪ microfluidic platform spatially resolves antigen presentation and T cell functions in microdroplets and deciphers dynamic tumor-immune interaction network [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5383.

Spiky: An ImageJ Plugin for Data Analysis of Functional Cardiac and Cardiomyocyte Studies

Introduction and objective: Nowadays, investigations of heart physiology and pathophysiology rely more and more upon image analysis, whether for the detection and characterization of events in single cells or for the mapping of events and their characteristics across an entire tissue. These investigations require extensive skills in image analysis and/or expensive software, and their reproducibility may be a concern. Our objective was to build a robust, reliable and open-source software tool to quantify excitation–contraction related experimental data at multiple scales, from single isolated cells to the whole heart. Methods and results: A free and open-source ImageJ plugin, Spiky, was developed to detect and analyze peaks in experimental data streams. It allows rapid and easy analysis of action potentials, intracellular calcium transient and contraction data from cardiac research experiments. As shown in the provided examples, both classical bi-dimensional data (XT signals) and video data obtained from confocal microscopy and optical mapping experiments (XYT signals) can be analyzed. Spiky was written in ImageJ Macro Language and JAVA, and works under Windows, Mac and Linux operating systems. Conclusion: Spiky provides a complete working interface to process and analyze cardiac physiology research data.

Identifying Bacterial Lineages in Salmonella by Flow Cytometry.

Advances in technologies that permit high-resolution analysis of events in single cells have revealed that phenotypic heterogeneity is a widespread phenomenon in bacteria. Flow cytometry has the potential to describe the distribution of cellular properties within a population of bacterial cells and has yielded invaluable information about the ability of isogenic cells to diversify into phenotypic subpopulations. This review will discuss several single-cell approaches that have recently been applied to define phenotypic heterogeneity in populations of Salmonella enterica.

Zn2+ influx activates ERK and Akt signaling pathways

Zinc (Zn2+) is an essential metal in biology, and its bioavailability is highly regulated. Many cell types exhibit fluctuations in Zn2+ that appear to play an important role in cellular function. However, the detailed molecular mechanisms by which Zn2+ dynamics influence cell physiology remain enigmatic. Here, we use a combination of fluorescent biosensors and cell perturbations to define how changes in intracellular Zn2+ impact kinase signaling pathways. By simultaneously monitoring Zn2+ dynamics and kinase activity in individual cells, we quantify changes in labile Zn2+ and directly correlate changes in Zn2+ with ERK and Akt activity. Under our experimental conditions, Zn2+ fluctuations are not toxic and do not activate stress-dependent kinase signaling. We demonstrate that while Zn2+ can nonspecifically inhibit phosphatases leading to sustained kinase activation, ERK and Akt are predominantly activated via upstream signaling and through a common node via Ras. We provide a framework for quantification of Zn2+ fluctuations and correlate these fluctuations with signaling events in single cells to shed light on the role that Zn2+ dynamics play in healthy cell signaling.

Cancer stem cell generation during epithelial-mesenchymal transition is temporally gated by intrinsic circadian clocks.

Epithelial-mesenchymal transition (EMT) is a key event preceding tumor cell metastasis that increases cell invasiveness and cancer stem cell (CSC)populations. Studies suggest that genes used in generating circadian rhythms also serve in regulating EMT. To test the role of circadian clocks in cellular EMT events two cancer cell lines were compared, one that has a well-established circadian clock, C6 from rat glioma, and one that does not, MCF-7 from human breast tumor. MCF-7 tumorsphere cultures were tested for evidence of circadian rhythms because of previously reported circadian rhythm enhancement in C6 tumorspheres shown by elevated rhythm amplitude and increased expression of circadian clock gene Per2. Bioluminescence imaging of Per2 gene expression in MCF-7 tumorspheres revealed a previously unconfirmed circadian clock in this important cancer research model. Inducing CSCgeneration through EMT in C6 and MCF-7 monolayer cultures revealed circadian oscillations in the size of the post-EMT CSCpopulation, confirming that circadian rhythms are additional processes controlling this stage of cancer progression. EMT was verified by distinct cellularmorphological changes and expression of stem cell proteins OCT4, nestin, MSI1, and CD133 along with EMT-related proteins ZEB1, vimentin, and TWIST. Quantifying single-cell events and behaviors through time-lapse imaging indicated the post-EMT population size was determined largely by circadian rhythms in epithelial-like cancer cells undergoing EMT. We then identified a specific phase of the circadian rhythm in Per2 gene activation as a potential target for therapeutic treatments that may suppress EMT, minimize CSCs, and limit metastasis.

Early Prediction of Single-Cell Derived Sphere Formation Rate Using Convolutional Neural Network Image Analysis.

Functional identification of cancer stem-like cells (CSCs) is an established method to identify and study this cancer subpopulation critical for cancer progression and metastasis. The method is based on the unique capability of single CSCs to survive and grow to tumorspheres in harsh suspension culture environment. Recent advances in microfluidic technology have enabled isolating and culturing thousands of single cells on a chip. However, tumorsphere assay takes a relatively long period of time, limiting the throughput of this assay. In this work, we incorporated machine learning with single-cell analysis to expedite tumorsphere assay. We collected 1,710 single-cell events as the database and trained a convolutional neural network model that predicts whether a single cell could grow to a tumorsphere on Day 14 based on its Day 4 image. With this future-telling model, we precisely estimated the sphere formation rate of SUM159 breast cancer cells to be 17.8% based on Day 4 images. The estimation was close to the ground truth of 17.6% on Day 14. The preliminary work demonstrates not only the feasibility to significantly accelerate tumorsphere assay but also a synergistic combination between single-cell analysis with machine learning, which can be applied to many other biomedical applications.

Abstract P5-04-09: Deep phenotyping using CyTOF identifies peripheral blood immune signatures associated with clinical outcomes and molecular subtypes in patients with early-stage triple negative breast cancer (TNBC)

Abstract Background: Local antitumor immunity—as assessed by quantifying tumor-infiltrating immune cells—is increasingly recognized as a critical factor influencing prognosis and therapy response in TNBC. However, an understanding of systemic antitumor immune responses occurring in peripheral blood, and their influence on prognosis and chemotherapy response has not been rigorously studied. Methods: Cytometry by time-of-flight (CyTOFTM, Fluidigm) was used to examine viably cryopreserved peripheral blood mononuclear cell (PBMC) suspensions prospectively collected from patients with early-stage TNBC prior to initiation of standard neoadjuvant paclitaxel followed by doxorubicin and cyclophosphamide (NACT) as part of the BEAUTY study [1]. Samples were stained using a panel of metal-tagged antibodies, recognizing 30 surface proteins optimized for immune monitoring of human peripheral blood. Differential abundance analysis of immune cell subsets was carried out to evaluate differences between patients who achieved pCR versus those with residual disease after NACT, and between patients with known luminal androgen receptor (LAR) versus basal TNBC subtypes defined by bulk tumor RNA sequencing. Results: Viably cryopreserved PBMC samples from 40 treatment-naive TNBC patients were available for analysis. The median age was 52 years (range 32 - 73), with 6 (15%) patients having tumors classified as LAR TNBC, and the remaining 34 (85%) as basal TNBC. Overall, 21 (53%) patients achieved pCR after NACT. After acquisition on the mass cytometer, the median yield per sample was 626,815 single-cell events (range 42,786 - 1,035,575), with a median percent debris of 13.7% (range 14 - 58). Across the 40 PBMC samples, the total yield was 23,507,094 single-cell events. The median frequencies of major circulating immune cell subsets across the 40 TNBC patients were: T cells 53.9% (range 25.4 - 71.3), with 33.4% CD4+ T cells (range 11.4 - 46.7) and 10.3% CD8+ T cells (range 5.8 - 19.9); B cells 10.8% (3.3 - 32.6), NK cells 8.6% (1.7 - 17.0) and monocytes 10.6% (2.7 - 29.8). Examining pre-treatment blood samples, patients with residual disease after NACT exhibited a higher median frequency of baseline CD14+CD16- classical monocytes (7.5% vs. 4.1%, p=0.025) and a lower frequency of terminally-differentiated effector memory cytotoxic (CD8+) T cells (0.6% vs. 1.7%, p=0.038) compared to patients who achieved pCR. Patients with LAR TNBC also exhibited a higher frequency of CD14+CD16- classical monocytes (11.5% vs 4.3%, p=0.058), and in addition exhibited a lower frequency of central memory CD4+ T cells (10.4% vs 15.2%, p=0.048). No difference in CD8+ T cells was seen by LAR status. Additional associations of peripheral blood immune cell subsets and classic tumor pathological features will be presented at the meeting. Conclusion: To our knowledge, this is the first study focused on TNBC to demonstrate variation in peripheral blood immune cell populations by molecular TNBC subtype (LAR vs. basal), and by chemotherapy response. A higher frequency of circulating classical monocytes—which can infiltrate into tissues and give rise to macrophages—appears to be detrimental; whereas a higher frequency of circulating antigen-experienced memory CD8+ T cells seems to be protective, suggesting a putative role of this cell subset in TNBC anti-tumoral immunity. Reference: [1] Goetz MP et al. JNCI 2017, PMID:28376176 Citation Format: Roberto A Leon-Ferre, Kaitlyn McGrath, Jodi M Carter, Krishna R Kalari, Vera J Suman, Richard Weinshilboum, Liewei Wang, Keith L Knutson, Stephen M Ansell, Judy C Boughey, J C Villasboas, Matthew P Goetz. Deep phenotyping using CyTOF identifies peripheral blood immune signatures associated with clinical outcomes and molecular subtypes in patients with early-stage triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-09.

Single-Cell Biodetection by Upconverting Microspinners.

Near-infrared-light-mediated optical tweezing of individual upconverting particles has enabled all-optical single-cell studies, such as intracellular thermal sensing and minimally invasive cytoplasm investigations. Furthermore, the intrinsic optical birefringence of upconverting particles renders them light-driven luminescent spinners with a yet unexplored potential in biomedicine. In this work, the use of upconverting spinners is showcased for the accurate and specific detection of single-cell and single-bacteria attachment events, through real-time monitoring of the spinners rotation velocity of the spinner. The physical mechanisms linking single-attachment to the angular deceleration of upconverting spinners are discussed in detail. Concomitantly, the upconversion emission generated by the spinner is harnessed for simultaneous thermal sensing and thermal control during the attachment event. Results here included demonstrate the potential of upconverting particles for the development of fast, high-sensitivity, and cost-effective systems for single-cell biodetection.

IMAGING HOW THE PREIMPLANTATION EMBRYO FORMS IN REAL TIME

Preimplantation development has typically been studied using fixed specimens. To reveal the dynamics that form the embryo, we established advanced imaging technologies to visualize single-cell dynamics and molecular events in real time within the mouse embryo. With this approach, we discovered how transcription factors bind to DNA in single cells of the embryo, to regulate the first cell fate decisions during embryonic development. We also unveiled new forms of actin and microtubule cytoskeletal organization, which control how cells become polarized during compaction and how they interact with each other to establish the first forms of tissue architecture, the pluripotent and trophectoderm lineages, and the blastocyst cavity. Together, our findings uncover dynamic mechanisms controlling how the mammalian embryo forms.

High resolution monitoring of chemotherapeutic agent potency in cancer cells using a CMOS capacitance biosensor

Monitoring cell viability and proliferation in real-time provides a more comprehensive picture of the changes cells undergo during their lifecycle than can be achieved using traditional end-point assays. Particularly for drug screening applications, high-temporal resolution cell viability data could inform decisions on drug application protocols that might lead to better treatment outcomes. We describe a CMOS biosensor that monitors cell viability through high-resolution capacitance measurements of cell adhesion quality. The system consists of a 3 × 3 mm2 chip with an array of 16 sensors, on-chip digitization, and serial data output that can be interfaced with inexpensive off-the-shelf components. An imaging system was developed to provide ground-truth data of cell coverage concurrently with data recordings. Results showed the sensor's ability to detect single-cell binding events, track cell morphology changes, and monitor cell motility. A chemotherapeutic assay was conducted to examine dose-dependent cytotoxic effects on drug-resistant and drug-sensitive cancer cell lines. Concentrations higher than 5 μM elicited cytotoxic effects on both cell lines, while a dose of 1 μM allowed discrimination of the two cell types. The system demonstrates the use of real-time capacitance measurements as a proof-of-concept tool that has potential to hasten the drug development process.