Single Cell Population Research Articles

Circulating cellular biomarkers associated with delayed time to progression among bladder cancer patients treated with immune checkpoint inhibitors.

398 Background: Circulating tumor cells (CTCs) are an important emerging biomarker in bladder cancer that allow for a minimally invasive assessment of tumor activity and response to treatment. Characterization of CTC and other single cell populations associated with improved clinical outcomes can help guide treatment recommendations for patients with metastatic bladder cancer. Methods: Patients with metastatic bladder cancer who received treatment with anti-PD-1 or anti-PD-L1 agents were enrolled in this study. Patient response to treatment was assessed by treating physicians according to RECIST v1.1. Blood samples were prospectively collected from patients prior to the initiation of therapy and then while on treatment and shipped to Epic Sciences for processing. All nucleated cells were subjected to immunofluorescent (IF) staining and CTC and leukocyte identification by fluorescent scanners using algorithmic analysis. Kaplan-Meier analysis was utilized to compare time to progression of patients whose CTC and leukocyte values were above and below several pre-determined parameters. Results: A total of 27 patients (median age 74 years, 70% male) were enrolled in this study and were treated with anti-PD-1/PD-L1 agents pembrolizumab (n=15), atezolizumab (n=11), or nivolumab (n=1). For 20 patients who had evaluable responses, objective response rate (ORR) was 2/20 (10%), all partial responses; another 5/20 (35%) had stable disease. Increased CD4% (>8% of total leukocytes) was associated with delayed time to progression (TTP) (p=0.002) whereas increased baseline total CTCs (>2) had a statistically non-significant trend towards shorter TTP (p=0.09). Baseline CD8%, CD4/CD8 ratio and CTC PD-L1 status were not associated with TTP. Conclusions: In a preliminary analysis among metastatic bladder cancer patients treated with immune checkpoint inhibitors, patients with an increased baseline CTC count had a statistically non-significant trend towards shorter TTP whereas increased baseline CD4 cells had an association with delayed TTP. This prospective study is ongoing, and the results will be further validated in larger patient cohorts.

Simultaneous polychromatic flow cytometric detection of multiple forms of regulated cell death

Currently the study of Regulated Cell Death (RCD) processes is limited to the use of lysed cell populations for Western blot analysis of each separate RCD process. We have previously shown that intracellular antigen flow cytometric analysis of RIP3, Caspase-3 and cell viability dye allowed the determination of levels of apoptosis (Caspase-3+ ve/RIP3− ve), necroptosis (RIP3Hi + ve/Caspase-3− ve) and RIP1-dependent apoptosis (Caspase-3+ ve/RIP3+ ve) in a single Jurkat cell population. The addition of more intracellular markers allows the determination of the incidence of parthanatos (PARP), DNA Damage Response (DDR, H2AX), H2AX hyper-activation of PARP (H2AX/PARP) autophagy (LC3B) and ER stress (PERK), thus allowing the identification of 124 sub-populations both within live and dead cell populations. Shikonin simultaneously induced Jurkat cell apoptosis and necroptosis the degree of which can be shown flow cytometrically together with the effects of blockade of these forms of cell death by zVAD and necrostatin-1 have on specific RCD populations including necroptosis, early and late apoptosis and RIP1-dependent apoptosis phenotypes in live and dead cells. Necrostatin-1 and zVAD was shown to modulate levels of shikonin induced DDR, hyper-action of PARP and parthanatos in the four forms of RCD processes analysed. LC3B was up-regulated by combined treatment of zVAD with chloroquine which also revealed that DNA damage was reduced in live cells but enhanced in dead cells indicating the role of autophagy in maintaining cell health. This approach to RCD research should be a great advance to understanding the mechanisms of drugs and their effects upon RCD populations.

Recently Discovered Interstitial Cell Population of Telocytes: Distinguishing Facts from Fiction Regarding Their Role in the Pathogenesis of Diverse Diseases Called "Telocytopathies".

In recent years, the interstitial cells telocytes, formerly known as interstitial Cajal-like cells, have been described in almost all organs of the human body. Although telocytes were previously thought to be localized predominantly in the organs of the digestive system, as of 2018 they have also been described in the lymphoid tissue, skin, respiratory system, urinary system, meninges and the organs of the male and female genital tracts. Since the time of eminent German pathologist Rudolf Virchow, we have known that many pathological processes originate directly from cellular changes. Even though telocytes are not widely accepted by all scientists as an individual and morphologically and functionally distinct cell population, several articles regarding telocytes have already been published in such prestigious journals as Nature and Annals of the New York Academy of Sciences. The telocyte diversity extends beyond their morphology and functions, as they have a potential role in the etiopathogenesis of different diseases. The most commonly described telocyte-associated diseases (which may be best termed “telocytopathies” in the future) are summarized in this critical review. It is difficult to imagine that a single cell population could be involved in the pathogenesis of such a wide spectrum of pathological conditions as extragastrointestinal stromal tumors (“telocytomas”), liver fibrosis, preeclampsia during pregnancy, tubal infertility, heart failure and psoriasis. In any case, future functional studies of telocytes in vivo will help to understand the mechanism by which telocytes contribute to tissue homeostasis in health and disease.

Disrupted-in-schizophrenia 1 overexpression disrupts hippocampal coding and oscillatory synchronization.

Aberrant proteostasis of protein aggregation may lead to behavior disorders including chronic mental illnesses (CMI). Furthermore, the neuronal activity alterations that underlie CMI are not well understood. We recorded the local field potential and single‐unit activity of the hippocampal CA1 region in vivo in rats transgenically overexpressing the Disrupted‐in‐Schizophrenia 1 (DISC1) gene (tgDISC1), modeling sporadic CMI. These tgDISC1 rats have previously been shown to exhibit DISC1 protein aggregation, disturbances in the dopaminergic system and attention‐related deficits. Recordings were performed during exploration of familiar and novel open field environments and during sleep, allowing investigation of neuronal abnormalities in unconstrained behavior. Compared to controls, tgDISC1 place cells exhibited smaller place fields and decreased speed‐modulation of their firing rates, demonstrating altered spatial coding and deficits in encoding location‐independent sensory inputs. Oscillation analyses showed that tgDISC1 pyramidal neurons had higher theta phase locking strength during novelty, limiting their phase coding ability. However, their mean theta phases were more variable at the population level, reducing oscillatory network synchronization. Finally, tgDISC1 pyramidal neurons showed a lack of novelty‐induced shift in their preferred theta and gamma firing phases, indicating deficits in coding of novel environments with oscillatory firing. By combining single cell and neuronal population analyses, we link DISC1 protein pathology with abnormal hippocampal neural coding and network synchrony, and thereby gain a more comprehensive understanding of CMI mechanisms.

Exploiting single-cell RNA sequencing data to link alternative splicing and cancer heterogeneity: A computational approach.

Cell heterogeneity studies using single-cell sequencing are gaining great significance in the era of personalized medicine. In particular, characterization of tumor heterogeneity is an emergent issue to improve clinical oncology, since both inter- and intra-tumor level heterogeneity influence the utility and application of molecular classifications through specific biomarkers. Majority of studies have exploited gene expression to discriminate cell types. However, to provide a more nuanced view of the underlying differences, isoform expression and alternative splicing events have to be analyzed in detail. In this study, we utilize publicly available single cell and bulk RNA sequencing datasets of breast cancer cells from primary tumors and immortalized cell lines. Breast cancer is very heterogeneous with well defined molecular subtypes and was therefore chosen for this study. RNA-seq data were explored in terms of genes, isoforms abundance and splicing events. The study was conducted from an average based approach (gene level expression) to detailed and deeper ones (isoforms abundance/splicing events) to perform a comparative analysis, and, thus, highlight the importance of the splicing machinery in defining the tumor heterogeneity. Moreover, here we demonstrate how the investigation of gene isoforms expression can help to identify the appropriate in vitro models. We furthermore extracted marker isoforms, and alternatively spliced genes between and within the different single cell populations to improve the classification of the breast cancer subtypes.

A Simple and Flexible Computational Framework for Inferring Sources of Heterogeneity from Single-Cell Dynamics.

Single-cell time-lapse data provide the means for disentangling sources of cell-to-cell and intra-cellular variability, a key step for understanding heterogeneity in cell populations. However, single-cell analysis with dynamic models is a challenging open problem: current inference methods address only single-gene expression or neglect parameter correlations. We report on a simple, flexible, and scalable method for estimating cell-specific and population-average parameters of non-linear mixed-effects models of cellular networks, demonstrating its accuracy with a published model and dataset. We also propose sensitivity analysis for identifying which biological sub-processes quantitatively and dynamically contribute to cell-to-cell variability. Our application to endocytosis in yeast demonstrates that dynamic models of realistic size can be developed for the analysis of single-cell data and that shifting the focus from single reactions or parameters to nuanced and time-dependent contributions of sub-processes helps biological interpretation. Generality and simplicity of the approach will facilitate customized extensions for analyzing single-cell dynamics.

Novel fluorescence techniques to quantitate renal cell biology.

Fluorescence microscopy techniques are powerful tools to study tissue dynamics, cellular function and biology both in vivo and in vitro. These tools allow for functional assessment and quantification along with qualitative analysis, thus providing a comprehensive understanding of various cellular processes under normal physiological and disease conditions. The main focus of this chapter is the recently developed method of serial intravital multiphoton microscopy that has helped shed light on the dynamic alterations of the spatial distribution and fate of single renal cells or cell populations and their migration patterns in the same tissue region over several days in response to various stimuli within the living kidney. This technique is very useful for studying in vivo the molecular and cellular mechanisms of tissue remodeling and repair after injury. In addition, complementary in vitro imaging tools are also described and discussed, like tissue clearing techniques and protein synthesis measurement in tissues in situ that provide an in depth assessment of changes at the cellular level. Thus, these novel fluorescence techniques can be effectively leveraged for different tissue types, experimental conditions as well as disease models to improve our understanding of renal cell biology.

Crossing constriction channel-based microfluidic cytometry capable of electrically phenotyping large populations of single cells.

This paper presents a crossing constriction channel-based microfluidic system for high-throughput characterization of specific membrane capacitance (Csm) and cytoplasm conductivity (σcy) of single cells. In operations, cells in suspension were forced through the major constriction channel and instead of invading the side constriction channel, they effectively sealed the side constriction channel, which led to variations in impedance data. Based on an equivalent circuit model, these raw impedance data were translated into Csm and σcy. As a demonstration, the developed microfluidic system quantified Csm (3.01 ± 0.92 μF cm-2) and σcy (0.36 ± 0.08 S m-1) of 100 000 A549 cells, which could generate reliable results by properly controlling cell positions during their traveling in the crossing constriction channels. Furthermore, the developed microfluidic impedance cytometry was used to distinguish paired low- and high-metastatic carcinoma cell types of SACC-83 (ncell = ∼100 000) and SACC-LM cells (ncell = ∼100 000), distinguishing significant differences in both Csm (3.16 ± 0.90 vs. 2.79 ± 0.67 μF cm-2) and σcy (0.36 ± 0.06 vs.0.41 ± 0.08 S m-1). As high-throughput microfluidic impedance cytometry, this technique may add a new marker-free dimension to flow cytometry in single-cell analysis.

Tooth bioengineering from single cell suspensions

Recent advances in bioengineering and biomaterials, along with knowledge deriving from the fields of developmental biology and stem cell research, have rendered feasible functional replacement of full organs. Here, we describe the methodology for bioengineering a tooth, starting from embryonic epithelial and mesenchymal single cell suspensions. In addition, we describe the subsequent steps of processing this minute structure for use in applications such as histological examination, immunofluorescence and in situ hybridisation. This methodology can be used for any minute structure that needs to be used in paraffin blocks.•Detailed methodology for reproducible and reliable results•Extra step to ensure single cell populations•Subsequent minute structure processing for histological analysis

Cell-Substrate Lift-off Lithography: Proof of Concept

In this study, we demonstrate the effectiveness of the lift-off lithography principle for mechanical only and thus chemical-free formation of HeLa cells controlled spatial distribution on the multielectrode array (MEA) surface. It allows creating the cells carrying MEA electrode and cells-free control MEA electrode in close proximity and thus in the same environment (temperature, chemical composition etc.). This scheme provides the additional sensitivity to the single cell and cells population bio-impedance measurements, which was ascertained by detecting the cells reaction on the trypsin-Versene solution addition to the medium.

Single-Cell Atlas of Driver Mutations in Acute Myeloid Leukemia (AML)

Single-Cell Atlas of Driver Mutations in Acute Myeloid Leukemia (AML)

Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become a powerful tool for human disease modeling and therapeutic testing. However, their use remains limited by their immaturity and heterogeneity. To characterize the source of this heterogeneity, we applied complementary single-cell RNA-seq and bulk RNA-seq technologies over time during hiPSC cardiac differentiation and in the adult heart. Using integrated transcriptomic and splicing analysis, more than half a dozen distinct single-cell populations were observed, several of which were coincident at a single time-point, day 30 of differentiation. To dissect the role of distinct cardiac transcriptional regulators associated with each cell population, we systematically tested the effect of a gain or loss of three transcription factors (NR2F2, TBX5, and HEY2), using CRISPR genome editing and ChIP-seq, in conjunction with patch clamp, calcium imaging, and CyTOF analysis. These targets, data, and integrative genomics analysis methods provide a powerful platform for understanding in vitro cellular heterogeneity.

Extended live-tracking and quantitative characterization of wound healing and cell migration with SiR-Hoechst

Cell migration is essential to many life processes, including immune response, tissue repair, and cancer progression. A reliable quantitative characterization of the cell migration can therefore aid in the high throughput screening of drug efficacy in wound healing and cancer treatments. In this work, we report what we believe is the first use of SiR-Hoechst for extended live tracking and automated analysis of cell migration and wound healing. We showed through rigorous statistical comparisons that this far-red label does not affect migratory behavior. We observed excellent automated tracking of random cell migration, in which the motility parameters (speed, displacement, path length, directionality ratio, persistence time, and direction autocorrelation) obtained closely match those obtained from manual tracking. We also present an analysis framework to characterize the healing of a scratch wound from the perspective of single cells. The use of SiR-Hoechst is advantageous for the crowded environments in wound healing assays because as long as cell nuclei do not overlap, continuous tracking can be maintained even if there is cell-cell contact. In this paper, we report wound recovery based on the number of cells migrating into the wound over time, normalized by the initial cell count prior to the infliction of the wound. This normalized cell count approach is impervious to operator bias during the arbitration of wound edges and is also robust against variability that arises due to differences in the cell density of different samples. Additional wound healing characteristics were also defined based on the evolution of cell speed and directionality during healing. Not unexpected, the wound healing cells exhibited much higher tendency to maintain the same migratory direction in comparison to the randomly migrating cells. The use of SiR-Hoechst thus greatly simplified the automation of single cell and whole population analysis with high spatial and temporal resolution over extended periods of time.

A Single-Cell Sequencing Guide for Immunologists.

In recent years there has been a rapid increase in the use of single-cell sequencing (scRNA-seq) approaches in the field of immunology. With the wide range of technologies available, it is becoming harder for users to select the best scRNA-seq protocol/platform to address their biological questions of interest. Here, we compared the advantages and limitations of four commonly used scRNA-seq platforms in order to clarify their suitability for different experimental applications. We also address how the datasets generated by different scRNA-seq platforms can be integrated, and how to identify unknown populations of single cells using unbiased bioinformatics methods.

The Single Cells and Cell Populations Viability Estimation in vitro by the Time-Domain Impedance Spectroscopy

In the present study, we investigate the sensitivity and applicability of the time-domain electrical impedance spectroscopy (EIS) techniques, namely Fourier-EIS and adaptive filtering based EIS (AF-EIS), for studying cells' populations and single living cells in natural physiological environment in vitro. Using ultra-violet radiation for decreasing cell life-span we demonstrate the possibility to distinguish the living cells' population from the dead cells by both Fourier-EIS and AF-EIS. However, determining the viability of the single cell requires significant sensitivity, which is inaccessible with the conventional Fourier-EIS contrary to AF-EIS. The latter result stems from the high noise immunity of the AF-EIS, which also makes it possible to provide the measurements using the safe for cells 15-mV excitation voltage and 10–100 nA current response. The developed single-cell AF-EIS approach opens a direct roadmap for creating accurate, robust, and easy to implement toxin and radiation hazard sensors with the living cell as an acting element and proposes a solution for actual ecological and healthcare problems.

Reproductive mode, stem cells and regeneration in a freshwater cnidarian with postreproductive senescence

Abstract In many basal metazoans, both somatic and reproductive functions are performed by cellular derivatives of a single multipotent stem cell population. Reproduction can drain these stem cell pools, imposing a physiological cost with subsequent negative effects on somatic maintenance functions. In the freshwater cnidarian Hydra oligactis, both asexual (budding) and sexual reproductive modes (production of resting eggs) are present, and both of these are dependent on a common pool of interstitial stem cells. Resting eggs tolerate harsh abiotic conditions which neither the parental animals, nor asexual offspring can survive (e.g., freezing). Therefore, when facing unfavourable conditions and increased mortality risk, hydra polyps are expected to show higher level of differentiation of interstitial stem cells into germ cells (i.e., sexual reproduction) than other cell types needed for self‐maintenance or asexual reproduction. Here, by comparing sexually and asexually reproducing individuals to nonreproductives, we studied the physiological costs of reproduction (size of interstitial stem cell pools, their somatic derivatives and regeneration rate, which is dependent on these cell types) in H. oligactis polyps from a free‐living Hungarian population prior to the onset of winter. Sexual individuals were characterized by significantly smaller interstitial stem cell pools, fewer nematoblasts involved in food capture and lower regeneration ability compared to nonreproductives, but asexuals did not differ from nonreproductive animals. We also found a negative correlation between germ cell counts and stem cell numbers in males (but not in females). We suggest that the lower numbers of these cell types and lower regenerative ability in sexual individuals reflect a somatic cost of sexual reproduction. Our results also suggest that increased differentiation of stem cells into gametes might limit investment into somatic functions in hydra polyps. Exhaustion of cellular resources (stem cells) could be a major mechanism behind the extreme postreproductive senescence observed in this species. A plain language summary is available for this article.

PO-391 High-throughput single-cell T-cell receptor profiling by SMART technology

IntroductionSingle-cell T-cell receptor (scTCR) clonotype analysis permits the determination of the specific TCR alpha-beta (α/β) chain pairing expressed on each cell. This pairing information allows researchers to gain insight into Tcell heterogeneity and plasticity, determine the contribution of the pairing to antigen specificity of the individual TCR, and design therapeutic antibodies. Here we employ a 5’-RACE-like approach and SMART technology, in conjunction with two novel next-generation sequencing (NGS) library preparation kits, using the same primer pairs, to capture full-length variable regions of TCR-α and -β chains.Material and methodsMethod 1, using the SMARTer Human scTCRa/b Profiling Kit, permits NGS library preparation of FACS-sorted cells in 96-well plates. Method 2 is an adaptation of the process above that scales to ~1200 single cells using the SMARTer ICELL8 Single-Cell System, which enables single-cell isolation and nanoliter PCR in a nanowell chip.Results and discussionsWe present data showing α/β pairing from Jurkat, CCRF, PBMCs, and CD4 +T cells. In addition to the sensitivity of this method, the ability to pool the cDNA from 96wells into 12 sequencing libraries adds to the ease of use. Consistent with immunology reports, unstimulated CCRF-CEM cells examined with this kit expressed a TCR-β but not a TCR-α chain.scTCR pofiling with SMARTer ICELL8 Single-Cell System as a proof of principle of scTCRα/β was performed on Jurkat cells and CCRF-CEM cells.Jurkat cells and CCRF-CEM cells were processed using an ICELL8 chip preprinted with barcoded oligos. Paired TCRα/β Jurkat clonotypes were detected in 77% and 87% cells in mixed and single cell populations, respectively.ConclusionThe ability of the core biochemistry and PCR components of these kits to be used with either FACS-sorted cells in 96-well plates or >1,000 cells in novel ICELL8 chips (in development) points to the general utility and scalability of this approach in understanding paired scTCR clonotype diversity.

PO-207 Ferritin-engineered nanoparticles as targeted drug delivery system for cancer treatment

IntroductionT cell acute lymphoblastic leukaemia (T-ALL) is an aggressive haematological malignancy that originates from malignant transformation of T cell progenitors. It accounts for 15% of paediatric and 25% of adult ALLs. Most frequent genetic alterations associated with this neoplasia are activating mutations in NOTCH1 and loss of function mutation for FBXW7 and PTEN. Current chemotherapeutic approaches demonstrate good cure and survival rates in patients. However prognosis of relapsed patients is still dismal. Therefore there is an urgent need to identify new targets for the development of more effective therapeutic compounds.Material and methodsCRISPR/Cas9 editing technology was used to generate mosaic mutant animals in Xenopus tropicalis, a true diploid amphibian. sgRNAs were designed to induce activating (i.e. truncating) mutations in notch1* and loss-of-function mutations in fbxw7 and pten. Cas9 recombinant protein and different sgRNA combinations were injected in 4 cell stage embryos. Animals were raised for 6 weeks. Liver, spleen, kidneys and thymus were dissected and further processed.Results and discussionsCombined injections of sgRNAs that targeted notch1 and pten resulted in mosaic mutant animals that exhibited pale appearance, enlarged spleen and haemorrhagic hindlimbs. Furthermore, sudden unexplained full penetrant mortality was observed around the time of metamorphosis. From diseased animals, genomic DNA was extracted from dissected thymi and the targeted gene loci were PCR-amplified and deep sequenced. Interestingly, several thymi showed clonal enrichment of a single cell population with a unique INDEL mutation pattern in one notch1 allele and two pten alleles, indicative of a leukemic cell population in the mosaic mutant animals. Exploiting a concept of clonal enrichment and negative selection for mutations in genes essential for T-ALL formation, we are using multiplexed gene inactivation to identify T-ALL dependency factors and hence explore novel routes for targeted therapy. We are currently verifying the feasibility of this experimental concept by multiplexed injections of pten and notch1 together with myc. Furthermore, we are validating our model by conducting flow cytometric and immunohistochemical analysis of the blood.ConclusionWe generated a genetic T–ALL model in X. tropicalis by co-targeting of notch1* and pten. We expect this model to provide new opportunities for identification and validation of novel driver genes and dependency factors for T-ALL.

Abstract 4685: Phenotypic difference of CD103+ tissue-resident memory T cells in various cancers

Abstract Background: The presence and clinical importance of tissue-resident memory T cells (TRM) have been recently described in human lung, liver, and bladder cancer. However, the frequency and phenotypic characteristics of TRM in other tumors are largely unknown. Methods: We analyzed single-cell populations of renal cell carcinoma (n=20), breast (n=16), colorectal (n=20), and stomach cancer (n=15) by dissociation of tumor tissue with collagenase/hyaluronidase. We investigated population of memory T cells and TRM using anti-CD45RO or anti-CD103 antibodies by flow cytometry. Results: Colorectal cancer had higher level of CD4+ T cells (70.1%) among CD3+ T cells than other tumors (stomach cancer, 59.6%; renal cell carcinoma, 49.4%; breast cancer, 49.0%; p=0.001). In CD4+ T cells, the percentage of CD45RO+ memory T cells was higher in renal cell carcinoma (75.7%) and breast cancer (85.4%) than colorectal (36.9%) and stomach cancer (38.1%, p<0.001). About 4% of CD4+ T cells in renal cell carcinoma, colorectal and stomach cancer were TRM, while 14.3% of CD4+ T cells were TRM in breast cancer (p<0.001). The percentage of CD45RO+ memory T cells among CD4+ TRM was about 60% in all tumors (p=0.164). In CD8+ T cells, the percentage of CD45RO+ memory T cells was higher in renal cell carcinoma (73.5%), breast (66.4%) and stomach cancer (54.4%) than colorectal cancer (33.0%, p<0.001). Stomach cancer had higher level of TRM (74.3%) among CD8+ T cells than other tumors (colorectal cancer, 19.6%; renal cell carcinoma, 20.1%; breast cancer, 30.6%; p<0.001). The percentage of CD45RO+ TRM in renal cell carcinoma (89.3%) and breast cancer (89.5%) was higher than colorectal (43.0%) and stomach cancer (51.5%, p<0.001). Conclusions: The frequency and phenotypic characteristics of TRM in renal cell carcinoma, colorectal, stomach, and breast cancer were different. Further studies regarding functional difference and clinical significance of TRM in various tumors are warranted.Acknowledgement: This study was supported by Basic Science Research Programs through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning, Republic of Korea (NRF-2017R1D1A1B03033104). Citation Format: Hye Seon Park, Young-Ae Kim, Won Seon Bang, Heejae Lee, Miseon Lee, In Ah Park, In Hye Song, Sun-Hee Heo, Hyeonjin Lee, Hee Jin Lee, Gyungyub Gong. Phenotypic difference of CD103+ tissue-resident memory T cells in various cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4685.

Staphylococcus aureus biofilms release leukocidins to elicit extracellular trap formation and evade neutrophil-mediated killing

Bacterial biofilms efficiently evade immune defenses, greatly complicating the prognosis of chronic infections. How methicillin-resistant Staphylococcus aureus (MRSA) biofilms evade host immune defenses is largely unknown. This study describes some of the major mechanisms required for S. aureus biofilms to evade the innate immune response and provides evidence of key virulence factors required for survival and persistence of bacteria during chronic infections. Neutrophils are the most abundant white blood cells in circulation, playing crucial roles in the control and elimination of bacterial pathogens. Specifically, here we show that, unlike single-celled populations, S. aureus biofilms rapidly skew neutrophils toward neutrophil extracellular trap (NET) formation through the combined activity of leukocidins Panton-Valentine leukocidin and γ-hemolysin AB. By eliciting this response, S. aureus was able to persist, as the antimicrobial activity of released NETs was ineffective at clearing biofilm bacteria. Indeed, these studies suggest that NETs could inadvertently potentiate biofilm infections. Last, chronic infection in a porcine burn wound model clearly demonstrated that leukocidins are required for "NETosis" and facilitate bacterial survival in vivo.