Response Of Cell Populations Research Articles

Maximum Entropy Framework for Predictive Inference of Cell Population Heterogeneity and Responses in Signaling Networks.

Predictive models of signaling networks are essential for understanding cell population heterogeneity and designing rational interventions in disease. However, using computational models to predict heterogeneity of signaling dynamics is often challenging because of the extensive variability of biochemical parameters across cell populations. Here, we describe a maximum entropy-based framework for inference of heterogeneity in dynamics of signaling networks (MERIDIAN). MERIDIAN estimates the joint probability distribution over signaling network parameters that is consistent with experimentally measured cell-to-cell variability of biochemical species. We apply the developed approach to investigate the response heterogeneity in the EGFR/Akt signaling network. Our analysis demonstrates that a significant fraction of cells exhibits high phosphorylated Akt (pAkt) levels hours after EGF stimulation. Our findings also suggest that cells with high EGFR levels predominantly contribute to the subpopulation of cells with high pAkt activity. We also discuss how MERIDIAN can be extended to accommodate various experimental measurements.

Culture on Tissue-Specific Coatings Derived from α-Amylase-Digested Decellularized Adipose Tissue Enhances the Proliferation and Adipogenic Differentiation of Human Adipose-Derived Stromal Cells.

While extracellular matrix (ECM)-derived coatings have the potential to direct the response of cell populations in culture, there is a need to investigate the effects of ECM sourcing and processing on substrate bioactivity. To develop improved cell culture models for studying adipogenesis, the current study examines the proliferation and adipogenic differentiation of human adipose-derived stem/stromal cells (ASCs) on a range of ECM-derived coatings. Human decellularized adipose tissue (DAT) and commercially available bovine tendon collagen (COL) are digested with α-amylase or pepsin to prepare the coatings. Physical characterization demonstrates that α-amylase digestion generates softer, thicker, and more stable coatings, with a fibrous tissue-like ultrastructure that is lost in the pepsin-digested thin films. ASCs cultured on the α-amylase-digested ECM have a more spindle-shaped morphology, and proliferation is significantly enhanced on the α-amylase-digested DAT coatings. Further, the α-amylase-digested DAT provides a more pro-adipogenic microenvironment, based on higher levels of adipogenic gene expression, glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, and perilipin staining. Overall, this study supports α-amylase digestion as a new approach for generating bioactive ECM-derived coatings, and demonstrates tissue-specific bioactivity using adipose-derived ECM to enhance ASC proliferation and adipogenic differentiation.

Biomarkers of cytogenetic disorders under the external environmental isolated exposure of manganese and stable strontium from drinking water

In blood of children underwent to the long-term isolated exposure of manganese and stable strontium with drinking water exceeding the acceptable level by up to 1.4 times the elevated concentrations of mentioned metals in blood were shown to be formed in relation to reference level by 2,0-4,4 times. The developing at that mutagenic effect was proved to have the dependence on the level of manganese and stable strontium concentration in blood and is pronounced in the increased rate of cells with cytogenetic anomalies on the background of the enhancement of the proliferative activity, destructive changes in the cell population and oxidative responses at the cell DNA level. The activity of genetic instability and processes of DNA oxidation in the somatic cells in children under the exposure to manganese has more pronounced character in comparison to given indices under the exposure to the stable strontium.

DNA damage in aging, the stem cell perspective.

DNA damage is one of the most consistent cellular process proposed to contribute to aging. The maintenance of genomic and epigenomic integrity is critical for proper function of cells and tissues throughout life, and this homeostasis is under constant strain from both extrinsic and intrinsic insults. Considering the relationship between lifespan and genotoxic burden, it is plausible that the longest-lived cellular populations would face an accumulation of DNA damage over time. Tissue-specific stem cells are multipotent populations residing in localized niches and are responsible for maintaining all lineages of their resident tissue/system throughout life. However, many of these stem cells are impacted by genotoxic stress. Several factors may dictate the specific stem cell population response to DNA damage, including the niche location, life history, and fate decisions after damage accrual. This leads to differential handling of DNA damage in different stem cell compartments. Given the importance of adult stem cells in preserving normal tissue function during an individual's lifetime, DNA damage sensitivity and accumulation in these compartments could have crucial implications for aging. Despite this, more support for direct functional effects driven by accumulated DNA damage in adult stem cell compartments is needed. This review will present current evidence for the accumulation and potential influence of DNA damage in adult tissue-specific stem cells and propose inquiry directions that could benefit individual healthspan.

Abstract CT189: Improved progression-free and overall survival (PFS/OS) in patients (pts) with emergence of JTX-2011 associated biomarker (ICOS high CD4 T cells) on the ICONIC trial

Abstract Background: ICOS is a costimulatory molecule upregulated on activated T cells. JTX-2011 is an ICOS agonist antibody intended to stimulate primed CD4 T effector cells. JTX-2011 was assessed in pts with advanced solid tumors as monotherapy (mono) and combination (combo) with nivolumab (nivo) in the Phase I/II ICONIC trial (NCT02904226). Peripheral T cell phenotyping in ICONIC demonstrated emergence of an ICOS high (hi) subset of CD4 T cells associated with tumor reductions in mono and combo pts. In ex vivo studies, soluble JTX-2011 stimulated a polyfunctional cytokine response only in ICOS hi cells. Methods: Ad hoc flow cytometry phenotyping on PBMCs from a subset of pts with evaluable samples (n=50) was initiated retrospectively in early 2018 in ongoing pts, then prospectively on newly enrolled pts. Clinical characteristics and outcomes were analyzed, including unadjusted p-values for post-hoc statistical analyses. Phenotyping was also done on samples from pts treated with PD-1/L1 inhibitor (PD-1/L1i) mono collected outside of ICONIC. Results: Emergence of ICOS hi CD4 T effector cells (all FoxP3-, subset Tbet+ Ki67+) was observed in all pts with ≥30% target lesion tumor reduction by investigator assessment on mono and combo therapy (n=7). Emergence was seen in pts with stable target lesions (n=11/23) including loss of these cells with disease progression. ICOS hi cells were not seen in ICONIC pts with target lesion increase ≥20% (n=14), or in pts treated with PD-1/L1i mono, including responders. Emergence of ICOS hi CD4 T cells correlated with improved PFS and OS (Table). ICONIC Pt characteristicsICOS hi (N=18)ICOS low (N=32)≥3 prior therapies, n (%)13 (72.2)18 (56.3)Prior immunotherapy, n (%)6 (33.3)15 (46.9)Tumor type, n (%)Gastric n=9 (50), NSCLC n=3 (16.7), TNBC n=2 (11.1), Other n=4 (22.2)Gastric n=8 (25), NSCLC n=6 (18.8), TNBC n=4 (12.5), Other n=14 (43.8),Mono vs Combo, n (%)Mono n=2 (11.1), Combo n=16 (88.9)Mono n=11 (34.4), Combo n=21 (65.6)G3-4 treatment-related adverse events, n (%)1 (5.6)2 (6.3)Time on JTX-2011, median mths (range), p=0.00065.6 (1.45-18.4)1.41 (0.03-6.28)PFS, investigator and central imaging review, median mths, (investigator, p<0.0001; central p=0.0011)6.22OS, median mths, p=0.0183(not yet reached)9 Conclusion: Emergence of a distinct ICOS hi population of peripheral CD4 T cells is associated with improved PFS and OS with JTX-2011 mono and combo therapy. Two JTX-2011 development paths are planned: (1) combo with agents that induce ICOS hi CD4 T cells; (2) use of potential putative biomarkers predictive of emergence of this T cell population and JTX-2011 response. Citation Format: Timothy Anthony Yap, Justin F. Gainor, Margaret K. Callahan, Gerald S. Falchook, Russell Kent Pachynski, Patricia LoRusso, Shivaani Kummar, Geoffrey Thomas Gibney, Howard A. Burris, Scott S. Tykodi, Osama E. Rahma, Tanguy Seiwert, Kyriakos P. Papadopoulos, Ellen Hooper, Christopher J. Harvey, Amanda Hanson, Sean Lacey, Rachel McComb, Courtney Hart, Haley Laken, Ty McClure, Elizabeth Trehu. Improved progression-free and overall survival (PFS/OS) in patients (pts) with emergence of JTX-2011 associated biomarker (ICOS high CD4 T cells) on the ICONIC trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT189.

Abstract 4351: Single-cell RNA sequencing reveals transcriptomic heterogeneity in response to epigenetic inhibitors

Abstract Histone modification is a fundamental regulatory mechanism of gene transcription and is associated with multiple diseases including cancer and inflammatory diseases. The factors that place and read these modifications can be co-opted in pathological conditions to give rise to disease-promoting transcriptional states, and thus represent promising targets for therapeutic small molecule inhibition. A central challenge in the development of epigenetic cancer therapy is the identification of transcriptionally sensitive cells within a heterogeneous population as a way to maximize therapeutic efficacy. To explore this challenge, we used single cell RNA-sequencing to characterize the transcriptional response of intrinsically heterogeneous human cancer and primary cell populations to small molecule inhibitors of epigenetic factors including the BET bromodomains, EZH2 methyltransferase, and CBP/EP300 acetyltransferase. Through clustering and differential expression analysis, we show that bulk transcriptional response in a mixed population of primary or tumor cells can be driven by a profound response in a hypersensitive subpopulation. Further, through the use of trajectory analysis, we show that epigenetic inhibitors can skew cell fate or differentiation status depending on the heterogeneity of the starting population. Overall, our results demonstrate the value of single cell transcriptomics for understanding the response of cell populations to epigenetic small molecule inhibitors and for enhancing the therapeutic potential of targeting pathogenic transcriptional programs. Citation Format: Andrew R. Conery, Sungmi Park, Michael J. Steinbaugh, Barbara M. Bryant, Shruti Apte, Patricia J. Keller, C. C. Yuan, Bill Bradley, Archana Bommi-Reddy, Robert J. Sims. Single-cell RNA sequencing reveals transcriptomic heterogeneity in response to epigenetic inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4351.

Formal representation of immunology related data with ontologies

Abstract The Human Immunology Project Consortium (HIPC) is a multicenter collaboration between research centers performing large-scale human immunology studies that focus on profiling the human immune response to natural infection and vaccination. “Immune exposures” are events such as natural infection and vaccination whereby the immune system may or may not respond to the exposure. Many of the HIPC studies investigate the response of specific cell populations after a variety of immune exposures. In order to cross-compare results from the many different centers and projects, we established a standardized representation of immune exposures and cell descriptions that simplifies data collection. By standardizing how this data is collected and stored, the vast amount of data collected by these diverse projects is made significantly more useful and interoperable. The data collected by the HIPC projects is stored in the National Institute of Health, Division of Allergy, Immunology and Transplantation funded resource, the Immunology Database and Analysis Portal (ImmPort). ImmPort was modified to provide the necessary structured data fields to capture our standardized representation of immune exposures and studied cell populations with a set of data fields that primarily utilize formal ontology terms. We will discuss the process of modeling immune exposures and cell populations via ontology terms, including real life scenarios from HIPC projects, as well as collaborations with existing ontology projects in order to meet the specific needs of immunologists.

Quantitative cell-based model predicts mechanical stress response of growing tumor spheroids over various growth conditions and cell lines.

Model simulations indicate that the response of growing cell populations on mechanical stress follows the same functional relationship and is predictable over different cell lines and growth conditions despite experimental response curves look largely different. We develop a hybrid model strategy in which cells are represented by coarse-grained individual units calibrated with a high resolution cell model and parameterized by measurable biophysical and cell-biological parameters. Cell cycle progression in our model is controlled by volumetric strain, the latter being derived from a bio-mechanical relation between applied pressure and cell compressibility. After parameter calibration from experiments with mouse colon carcinoma cells growing against the resistance of an elastic alginate capsule, the model adequately predicts the growth curve in i) soft and rigid capsules, ii) in different experimental conditions where the mechanical stress is generated by osmosis via a high molecular weight dextran solution, and iii) for other cell types with different growth kinetics from the growth kinetics in absence of external stress. Our model simulation results suggest a generic, even quantitatively same, growth response of cell populations upon externally applied mechanical stress, as it can be quantitatively predicted using the same growth progression function.

Immunity Against Bacterial Infection of the Central Nervous System: An Astrocyte Perspective.

Bacterial infection of the central nervous system (CNS) is a severe and life-threatening condition with high mortality, and it may lead to permanent neurological deficits in survivors. Increasing evidence indicates that astrocytes, as the most abundant CNS glial cell population, regulate innate and adaptive immune responses in the CNS under pathological conditions in addition to their role in the maintenance of CNS homeostasis and neuronal function. Following antigen recognition, astrocytes participate in the initiation of innate immune responses, and prompt an adaptive immune response to recruit peripheral immune cells. Investigations have been conducted to understand the immunological role of astrocytes in CNS disease and injury, however, their part in bacterial infections of the CNS has not been fully evaluated. A better understanding will permit the identification of successful therapeutic targets for an improved prognosis and disease outcome.

Modeling the Encoding of Saccade Kinematic Metrics in the Purkinje Cell Layer of the Cerebellar Vermis.

Recent electrophysiological observations related to saccadic eye movements in rhesus monkeys, suggest a prediction of the sensory consequences of movement in the Purkinje cell layer of the cerebellar oculomotor vermis (OMV). A definite encoding of real-time motion of the eye has been observed in simple-spike responses of the combined burst-pause Purkinje cell populations, organized based upon their complex-spike directional tuning. However, the underlying control mechanisms that could lead to such action encoding are still unclear. We propose a saccade control model, with emphasis on the structure of the OMV and its interaction with the extra-cerebellar components. In the simulated bilateral organization of the OMV, each caudal fastigial nucleus is arranged to receive incoming projections from combined burst-pause Purkinje cell populations. The OMV, through the caudal fastigial nuclei, interacts with the brainstem to provide adaptive saccade gain corrections that minimize the visual error in reaching a given target location. The simulation results corroborate the experimental Purkinje cell population activity patterns and their relation with saccade kinematic metrics. The Purkinje layer activity that emerges from the proposed organization, precisely predicted the speed of the eye at different target eccentricities. Simulated granular layer activity suggests no separate dynamics with respect to shaping the bilateral Purkine layer activity. We further examine the validity of the simulated OMV in maintaining the accuracy of saccadic eye movements in the presence of signal dependent variabilities, that can occur in extra-cerebellar pathways.

Single-Cell Analysis of the Transcriptional Response in AML Patients Treated with BET-Inhibitors

Acute Myeloid Leukemia (AML) is the most common myeloid leukemia in adults. Although substantial progress has been made in recent years, the long-term prognosis for patients remains poor which is mainly due to the dated treatments that consist of cytotoxic drugs with low specificity. AML is a clonal disease with multiple co-existing clones in each patient. Often, patients that initially respond to treatment may develop resistance due to lingering leukemic stem cells (LSC), or sub-clones that survive the treatment and cause a relapse. Therefore, novel therapeutic strategies are needed to fully eradicate all leukemic cells. AML has a strong epigenetic component meaning mutations in genes encoding epigenetic regulators are frequently acquired during early AML development, and are present in the initiating clones. Thus, targeting the epigenetic machinery may offer a new avenue for AML treatment. Among the newer epigenetic drugs are BET inhibitors, which bind reversibly to bromodomains of BRD proteins and prevent protein-protein interactions with acetylated histones and transcriptions factors. One of the most promising BET inhibitors is OTX015, which has already been in Phase II clinical trials for AML in the U.S. (Braun & Gardin, Expert Opinion on Investigational Drugs, 2017).We aim to analyze the heterogeneous response to OTX015 in AML, and normal stem/progenitor, cells in order to dissect the BET-inhibitor response. The main focus is the specific transcriptional signatures at promoters and enhancers as enhancers, and especially super-enhancers, have previously been shown to be sensitive to BET-inhibitors (Loven et al, Cell, 2013). To this effect, we have established a protocol that allowed for the transcriptional profiling of single cells from AML patients that were at different differentiation stages, using FACS- sorting. The patients were obtained from the Swedish Acute Leukemia Registry. To decrease population heterogeneity, the project focused on distinct subgroups of AML that previously has been shown to be sensitive for BET inhibitors. The different isolated AML, and normal progenitor populations, were exposed to OTX015 for 48hrs, and processed with both bulk transcriptional profiling of the general cell population response, and single cell profiling to analyze cell heterogeneity, and single cell response. For the transcriptional profiling, we utilized a unique technique called Cap Analysis of Gene Expression (CAGE), a powerful 5' start profiling technology, that allows for the identification of the transcription start site at base pair resolution, and determination of enhancer activity based on enhancer RNA expression. The single cell profiling was performed using C1 CAGE, which is a single-cell implementation CAGE (Kouno et al, bioRxiv 330845, 2018).We envision that the heterogenic transcriptional drug response, on the single-cell level, in AML and normal stem/progenitor cells will lead to the identification of key genes and pathways involved in the differential drug response. Additionally, the application of CAGE technology will lead to discovery of specific transcriptional signatures at promoters and enhancers that may be predictive of drug resistance.Clinical significance: Leukemic cell heterogeneity remains the main problem in AML, as chemotherapy often fails to completely eradicate all AML sub-clones including LSC, leading to relapses and high mortality of the disease. This study will shed light to the unique features of AML cell heterogeneity and how their drug response differs, not only between AML cells, but also between AML cells and their normal counterparts, on the single-cell level, based on the response to OTX015. The significance will be two-fold: the in-depth characterization of the features in AML populations and normal cells, and the potential this study will provide for novel, more targeted, combination treatments in AML. DisclosuresNo relevant conflicts of interest to declare.

Effect of peripheral blood mononuclear cell cryopreservation on innate and adaptive immune responses

Cryopreservation of blood-derived immune cells is commonly used in clinical trials to examine immunological responses. However, studies elucidating the effects of cryopreservation on peripheral blood mononuclear cell (PBMC) responses have shown inconsistent results making it difficult to draw meaningful conclusions. Therefore we sought to address this issue by comparing key innate and adaptive immune parameters between freshly-isolated and cryopreserved PBMCs from healthy adults. We examined the effect of cryopreservation on the expression of key markers on innate and adaptive immune cell populations (i.e. CD4+ and CD8+ [T cells], CD14+ [monocytes], CD19+ [B cells], CD56+ [NK cells] or CD19 + CD27+ [memory B cells]), on cytokine secretion (TNF-α, INF-γ, IL-1β, IL-10, IL-6, MCP-1 and RANTES) in cultured PBMC supernatants following stimulation with a range of Toll-like receptor (TLR) agonists, as well as on antigen-specific memory B cell enumeration by ELISpot. We found that cryopreservation had no effect on the expression of immune markers on innate and adaptive immune cells as well on the number of antigen-specific memory B cells. However, the response to TLR ligands such as FLA-ST, CpG and LPS was variable with increased cytokine production by cryopreserved PBMCs observed compared to freshly-isolated PBMCs. Our results suggest that the effect of cryopreservation on the biological response of immune cell populations needs to be carefully considered, particularly in the context of clinical studies that rely on these immune outcomes.

Regulatory T Cells Suppress Effector T Cell Proliferation by Limiting Division Destiny.

Understanding how the strength of an effector T cell response is regulated is a fundamental problem in immunology with implications for immunity to pathogens, autoimmunity, and immunotherapy. The initial magnitude of the T cell response is determined by the sum of independent signals from antigen, co-stimulation and cytokines. By applying quantitative methods, the contribution of each signal to the number of divisions T cells undergo (division destiny) can be measured, and the resultant exponential increase in response magnitude accurately calculated. CD4+CD25+Foxp3+ regulatory T cells suppress self-reactive T cell responses and limit pathogen-directed immune responses before bystander damage occurs. Using a quantitative modeling framework to measure T cell signal integration and response, we show that Tregs modulate division destiny, rather than directly increasing the rate of death or delaying interdivision times. The quantitative effect of Tregs could be mimicked by modulating the availability of stimulatory co-stimuli and cytokines or through the addition of inhibitory signals. Thus, our analysis illustrates the primary effect of Tregs on the magnitude of effector T cell responses is mediated by modifying division destiny of responding cell populations.

Hypoxia-induced tRNA-derived fragments, novel regulatory factor for doxorubicin resistance in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of epithelial breast malignancy, and chemoresistance is the major obstacle for cancer therapy. TNBC is associated with a hypoxic phenotype, and hypoxia contributes to the chemoresistance in breast cancer. Transfer RNA-derived fragments (tDRs) represent a new class of small noncoding RNAs that can be induced specifically by hypoxia. Here, we conducted a comparative analysis of the aberrant expression of tDRs in hypoxia-treated TNBC cell lines through the use of high-throughput sequencing technique. Quantitative real-time polymerase chain reaction was used to validate the differently expressed tDRs between two samples. The results showed that tDR-0009 [derived from transfer RNA (tRNA)Gly-GCC-1-1 ] and tDR-7336 (derived from tRNA Gly-GCC-1-2 ) were significantly upregulated when the SUM-1315 cell lines were stimulated by hypoxia. Gene ontology (GO) and pathway analysis indicated that these two upregulated tDRs were mainly involved in maintenance of stem cell population and cellular response to interleukin (IL)-6, which may be the underlying mechanism of hypoxia-induced tDRs that facilitate the doxorubicin resistance in TNBC. The protein-protein interaction network for predicted target genes established by the STRING database manifested that tDR-0009 (tDR-7336) might be involved in the chemoresistance of TNBC via regulation of the activation of phosphorylation of STAT3. In summary, our study provided a comprehensive analysis of the deviant expression profiling of tDRs in hypoxia-treated TNBC cell lines. Specific tDRs may be a new class of regulatory factors involved in the hypoxia-induced chemoresistance in TNBC, and they could serve as potential biomarkers and intervention targets.

Separating intrinsic interactions from extrinsic correlations in a network of sensory neurons

Correlations in sensory neural networks have both extrinsic and intrinsic origins. Extrinsic or stimulus correlations arise from shared inputs to the network, and thus depend strongly on the stimulus ensemble. Intrinsic or noise correlations reflect biophysical mechanisms of interactions between neurons, which are expected to be robust to changes of the stimulus ensemble. Despite the importance of this distinction for understanding how sensory networks encode information collectively, no method exists to reliably separate intrinsic interactions from extrinsic correlations in neural activity data, limiting our ability to build predictive models of the network response. In this paper we introduce a general strategy to infer {population models of interacting neurons that collectively encode stimulus information}. The key to disentangling intrinsic from extrinsic correlations is to infer the {couplings between neurons} separately from the encoding model, and to combine the two using corrections calculated in a mean-field approximation. We demonstrate the effectiveness of this approach on retinal recordings. The same coupling network is inferred from responses to radically different stimulus ensembles, showing that these couplings indeed reflect stimulus-independent interactions between neurons. The inferred model predicts accurately the collective response of retinal ganglion cell populations as a function of the stimulus.

Effects of pH manipulation, CatSper stimulation and Ca2+-store mobilization on [Ca2+]i and behaviour of human sperm

How do the alkaline pH, progesterone and 4-aminopyridine interact in their effects on human sperm? Behaviour of human sperm (proportion of hyperactivated cells and motility kinematics) were related directly to [Ca2+]i irrespective of pH or the agonist applied. CatSper channels of human sperm, which are central to generation of sperm [Ca2+]i signals and induction of hyperactivated motility, are activated by intracellular alkalinization and progesterone. Progesterone (P4) is much less effective than 4-aminopyridine (4-AP) (which mobilizes stored Ca2+ but also raises pHi) as an inducer of hyperactivation. This was a laboratory study, spanning ~18 months that used 15 sperm donors and involved more than 100 separate experiments. Semen donors and patients were recruited in accordance with local ethics approval (ERN_12-0570R). [Ca2+]i responses of suspended cell populations were examined by fluorimetric recording and motility parameters assessed by computer-assisted sperm analysis. Increasing pHo from 7.4 to 8.5 raised pHi (from 6.9 to 7.2) and significantly increased both [Ca2+]i and the proportion of hyperactivated cells. Stimulation of cells with P4 (1 nM-20 μM) induced a biphasic (transient and plateau) increase in [Ca2+]i. The [Ca2+]i increase was of similar amplitude and dose-dependency at pHo = 7.4 and pHo = 8.5. 4-aminopyridine (0.2-5 mM) induced a biphasic [Ca2+]i increase that was dose-dependent across the entire range tested and was strongly enhanced at pH 8.5. Motility was assessed 300 s post-stimulation, during the plateau phase of the progesterone and 4-AP-induced [Ca2+]i responses. Progesterone had only a small effect on hyperactivated motility even at the highest dose used (20 μM; < 5% increase in the proportion of cells classified as hyperactivated) which was insensitive to pHo. 4-Aminopyridine potently stimulated hyperactivated motility, this effect being dose-dependent and greatly enhanced at pHo = 8.5. The relationship between [Ca2+]i (fluorescence of fluo4) and proportion of hyperactivated cells, irrespective of pHo, agonist or dose, was fitted by a single curve (second order polynomial; R2 = 0.96). Similar analysis of curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) showed a linear relationship to [Ca2+]i (R2 > 0.9). This was an in-vitro study and caution must be taken when extrapolating these results to in vivo regulation of sperm. Though controls indicate that saturation of fluo4 did not affect the findings, at the highest doses of progesterone the true amplitude of the [Ca2+]i transient may not have been reported by the dye. These findings indicate that (i) activation of human sperm CatSper by progesterone (and presumably other ligands that act similarly) and consequent acquisition of hyperactivated motility is not significantly enhanced by intracellular alkalinization; (ii) VCL, ALH and hyperactivation are directly related to [Ca2+]i, irrespective of the mechanism by which Ca2+ is mobilized, and the ability of stimuli to induce prolonged [Ca2+]i elevation (as occurs upon mobilization of stored Ca2+) determines the observed effect on cell behaviour. CA was supported by the Nigerian government (Tertiary Education Trust (TET) Fund). The authors have no conflicts of interest.

Abstract 3791: Triggering receptor expressed in myeloid cells 1 (TREM1) is a predictive marker for breast cancer therapeutics

Abstract Triggering receptor expressed on myeloid cells-1 (TREM1) is a cell surface immunoglobulin that amplifies pro-inflammatory responses in myeloid cell populations and has been implicated in carcinogen-induced liver cancer. However, its role in breast cancer is unknown. In a recent screen to identify gene expression patterns associated with breast cancer outcomes, we identified a significant and reproducible positive association between TREM1 expression and breast cancer recurrence in two large breast cancer cohorts comprising 1,984 and 616 patients (p&amp;lt;0.001 and p=0.003, respectively). TREM1 expression was significantly associated with recurrence in both basal-like (p&amp;lt;0.001) and luminal tumor subtypes (Luminal A, p=0.047; Luminal B, p=0.022) and remained significant (p&amp;lt;0.001) in a multivariate Cox regression model, independent of histologic grade, tumor size, nodal status, patient age, treatment and molecular subtype. In pre-treatment biopsies of patients treated with neoadjuvant chemotherapy, high TREM1 expression was associated with a significant decrease in pathologic complete response (pCR) rate (p&amp;lt;0.001), especially in those tumors estimated to have high levels of lymphocytic infiltrate that otherwise associated with an increased pCR rate, consistent with an immune regulatory function for TREM1. Bioinformatics analysis of genes reproducibly correlated with TREM1 expression in breast tumors revealed significant enrichment in myeloid immune cell populations (p&amp;lt;0.001) in general, and neutrophils (p&amp;lt;0.001), M0 macrophages (p&amp;lt;0.001) and M2 macrophages (p&amp;lt;0.001), in particular. Immunohistochemical analysis of breast tumor tissues confirmed colocalization of TREM1 protein expression with the pan-macrophage marker, CD68. These findings, together with the established role for TREM1 in promoting inflammation, fit a model where tumor-infiltrating TREM1-positive myeloid cells impact breast cancer outcomes by promoting immune evasion. Citation Format: Ashok K. Pullikuth, Julia Chifman, Eric D. Routh, Jeff Chou, Guangxu Jin, Michael A. Black, Lance D. Miller. Triggering receptor expressed in myeloid cells 1 (TREM1) is a predictive marker for breast cancer therapeutics [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 3791.

Abstract 5913: ECO nanoparticle delivery of siRNA targeting DNA repair to enhance the radiation response of glioblastoma

Abstract Background: Glioblastoma (GBM) is among the most lethal of human cancers with a median survival of ~15 months. Novel treatment strategies targeting DNA damage response proteins such as ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNApk) are being investigated for the radiosensitization of GBM. Previously, (1-aminoethyl)iminobis[N-(oleicylcysteinyl-1-amino-ethyl)-propionamide (ECO) nanoparticles (NPs) were shown to effectively deliver siRNA targeting tumor growth and metastasis. Here we investigate the ability of these NPs to deliver siRNA targeting ATM and DNApk to enhance the radiation response of glioma and glioma stem cell populations. Methods: Normal astrocytes, glioma (U251), and glioma stem cells (NSC11, GBMJ1) were used. Protein analysis revealed the degree of silencing using ECO NPs with siRNA targeting ATM or DNApk. The effects of each siRNA alone (40nM) and in combination (20nM each) on radiation response was evaluated using clonogenic cell survival and γH2AX foci assays. A cranial guide screw model was used to assess the effectiveness of NPs in vivo. U251 tumor cells expressing luciferase were orthotopically implanted in nude mice and NPs targeting luciferase or DNApk were injected intratumorally. The efficacy of siRNA delivery was assessed via bioluminescence signaling and immunohistochemical staining. Results: ECO NPs effectively silenced both target genes in all tumor cells. The effect of this silencing on double strand break (DSB) repair varied with each cell line. γH2AX studies showed significantly impaired DSB repair in U251 cells with DNApk and ATM/DNApk combination treatment and in GBMJ1 stem cells with combination treatment. Treatment of NSC11 stem cells resulted in trends towards impaired DSB repair however the results were not statistically significant. Importantly, each treatment had no significant effect on DSB repair in normal astrocytes. Evaluation of radiation response in the long term using clonogenic assays resulted in dose enhancement factors for ATM, DNApkcs, and combination treatments of 1.24, 1.43, and 1.34 for U251, 1.34, 1.35, and 1.48 for NSC11, and 1.35, 1.36, and 1.51 for GBMJ1. In vivo studies showed evidence that ECO NPs were able to deliver siRNA targeting luciferase and reduce the bioluminescence signal output in vivo. Conclusions: The use of ECO NPs in normal astrocytes resulted in no significant effect on DSB repair demonstrating a potential therapeutic advantage to ECO NP/siRNA therapies. In contrast, NPs effectively delivered siRNA targeting ATM and DNApk to glioma and glioma stem cells showing the ability to target and treat a heterogeneous GBM cell population. The subsequent enhancement of radiation response in vitro coupled with in vivo observations of bioluminescence silencing provide the basis for future investigation into the use of ECO/siRNA complexes to enhance the radiation response of GBM in vivo. Citation Format: Jennifer A. Lee, Nadia Ayat, Zhanhu Sun, Tatsuya Kawai, Zheng-Rong Lu, Kevin Camphausen. ECO nanoparticle delivery of siRNA targeting DNA repair to enhance the radiation response of glioblastoma [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 5913.

Abstract 2017: Phenotypic plasticity and class switching in ovarian cancer

Abstract The multifaceted evolution of cancer involves differential responses of tumor cell populations to intrinsic and extrinsic stimuli. Numerous reports have examined the role of epithelial-mesenchymal transition (EMT) in the progression of high-grade serous ovarian carcinoma (HGSC). An earlier study from our group successfully resolved molecular subclasses in HGSC wherein metastasis was mediated by cooperative cell migration in the epithelial subclass and EMT in the mesenchymal subclass. We now explore the transcriptional circuitry in expression datasets and cell lines regulating these states. This led to the identification that Tcf21 and Slug expression correlated with the epithelial and mesenchymal phenotypes, respectively, and inversely correlated with each other. Probing the differential altered localization and expression of TCF21 and Slug further resolved a spectrum of intermediary phenotypic states along the epithelial to mesenchymal spectrum, each of which is associated with the expression of specific markers. Further, functional assays reveal the inherent plasticity of each phenotype with migratory mechanisms emerging as a differential parameter. Rigidity and high degree of differentiation of the epithelial and mesenchymal state is evident from a lack of responsiveness to specific microenvironmental conditions. Importantly, such cellular plasticity induced shifts from a steady-state phenotype following modulation of the microenvironment only in the intermediate phenotypes to exhibit flexibility to transit towards either the two ends of the spectrum. Specifically, a tendency to acquire an epithelial phenotype on growth factor deprivation was noted. The effects of BMP7 and TGFβ to induce epithelial or mesenchymal phenotypes, respectively, were also noted exclusively within the intermediary states. The stress generated by chemotherapeutic agents like paclitaxel led to enrichment of and dominant effects of the epithelial state. We further queried if this plasticity is also a feature of HGSC tumors. Marker expression (Tcf21, E-cadherin, Parp1, Slug, Anxa2 and Hyaluronan) was scored based on their frequency, intensity and location in tumors to derive a definitive scoring system comprising biomarker and tumor class indices. Application of such scoring could effectively stratifiy 95 human HGSC cases comprising primary and metastatic tumors into epithelial and mesenchymal classes, and further resolve a third class likely to represent the intermediate states. A salient feature noted was that metastases and chemotherapy could lead to class switching. Our study thus provides a comprehensive view of the intrinsic and extrinsic molecular events governing cellular plasticity in HGSC. Citation Format: Sharmila A. Bapat, Sagar Varankar, Swapnil Kamble. Phenotypic plasticity and class switching in ovarian cancer [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 2017.

Abstract 708: Increase of senescent mesenchymal stromal/stem cells is predictive of unresponsiveness to the treatment with immune checkpoint inhibitors

Abstract Purposes: The therapeutic efficacy induced by immune inhibitory checkpoint inhibitors is still limited to a part pf the treated patients, and the methods effective in overcoming the treatment failures have not been established yet. A main issue under discussion is immune exhaustion as well as immune senescence with age, which is caused by persistent and chronic inflammation due to both internal and external stimuli ultimately leading to immune dysfunction in cancer patients. In literature, aging has been reported as a strong risk factor not only for causing cancers, but also for inducing hyper progression after treatment with immune checkpoint inhibitors in clinical settings. This suggests that a distinct strategy from releasing the brake on immune suppression might be needed for properly organizing anti-tumor immunity in aged hosts. In this study, we attempted to elucidate the molecular mechanisms underlying the immune exhaustion with age. Results: We used young (1 to 4-month-old) and aged (18 to 24-month-old) C57BL/6 mice, and comprehensively analyzed cell population and immune responses of spleen cells and bone marrow cells obtained from the mice. With age, CD45-CD166+ mesenchymal stromal/stem cells (MSCs) increased correlatively with increase of immunosuppressive CD4+CTLA4+Foxp3+ Tregs and CD11b+Gr1+ MDSCs, and dysfunctional CD3+PD1+Tim3+ T cells in mice. These cells were further expanded by implanting with tumor cells such as melanoma B16-F10 and lung cancer 3LL, and immune checkpoint inhibitors were ineffective in suppressing tumor progression in aged mice. As compared to the young MSCs, the aged MSCs generated much more dysfunctional T cells in vitro and in vivo, and differentiated into hypertrophic adipocytes with a large lipid droplet containing a huge amount of inflammatory adipokines. These suggest that the increasing MSCs play a key role in disturbing anti-tumor immunity both before and after differentiation in aged hosts. Utilizing cDNA microarray analysis, we identified the specific molecules those are significantly upregulated in the aged MSCs, but not young MSCs. Knockdown of the gene expressions in the aged MSCs using the specific siRNAs deprived of the morbid properties, and generated potent tumor-specific CTLs through balancing immunity in the aged mice. Conclusions: These results demonstrate that increase of the senescent and inflammatory MSCs are, at least in part, responsible for the unresponsiveness to the treatment with immune checkpoint inhibitors. Elimination of these MSCs may be a promising strategy for successfully controlling immunity against cancer. Citation Format: Chie Kudo-Saito, Takahiro Miyamoto, Mami Kawamura, Yamato Ogiwara, Kazunori Aoki. Increase of senescent mesenchymal stromal/stem cells is predictive of unresponsiveness to the treatment with immune checkpoint inhibitors [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 708.