Response Profiles Of Cells Research Articles

Peritoneal B1 and B2 cells respond differently to LPS and IL-21 stimulation

Peritoneal B cells can be divided into B1 cells (CD11b+CD19+) and B2 cells (CD11b-CD19+) based on CD11b expression. B1 cells play a crucial role in the innate immune response by producing natural antibodies and cytokines. B2 cells share similar traits with B1 cells, influenced by the peritoneal environment. However, the response of both B1 and B2 cells to the same stimuli in the peritoneum remains uncertain. We isolated peritoneal B1 and B2 cells from mice and assessed differences in Interleukin-10(IL-10) secretion, apoptosis, and surface molecule expression following exposure to LPS and Interleukin-21(IL-21). Our findings indicate that B1 cells are potent IL-10 producers, possessing surface molecules with an IgMhiCD43+CD21low profile, and exhibit a propensity for apoptosis in vitro. Conversely, B2 cells exhibit lower IL-10 production and surface markers characterized as IgMlowCD43-CD21hi, indicative of some resistance to apoptosis. LPS stimulates MAPK phosphorylation in B1 and B2 cells, causing IL-10 production. Furthermore, LPS inhibits peritoneal B2 cell apoptosis by enhancing Bcl-xL expression. Conversely, IL-21 has no impact on IL-10 production in these cells. Nevertheless, impeding STAT3 phosphorylation permits IL-21 to increase IL-10 production in peritoneal B cells. Moreover, IL-21 significantly raises apoptosis levels in these cells, a process independent of STAT3 phosphorylation and possibly linked to reduced Bcl-xL expression. This study elucidates the distinct functional and response profiles of B1 and B2 cells in the peritoneum to stimuli like LPS and IL-21, highlighting their differential roles in immunological responses and B cell diversity.

Abstract 4241: Development of stroma cells-rich oral cancer organoid for functional analysis of tumor microenvironment

Abstract Background: Tumor microenvironment (TME), composed of cancer cells, endothelial cells, fibroblast, immune cells, and etc, plays crucial roles in tumor progression and treatment resistance. Thus, it is extremely important to find its role involved in the interaction of TME. However, it is more difficult to examine the biological activities of those cells in vitro assay. The purpose of this study is to develop stroma cells-incorporating preclinical model, termed stroma cells-rich oral cancer organoid, for functional analysis of TME. Materials and Methods: We have first developed the stroma cells-rich oral cancer organoid by selecting the types of stroma cells such as endothelial cells, fibroblast, ratio of those cells, culture method, 3-D methods, culture media, and supplement of growth factors. Then, growth rate, morphology, radiosensitivity, and drug response were analyzed to investigate the effect of TME. Finally, the function of tumor-associated macrophage was determined using this organoid model. The growth rate of cancer cells and drug response were investigated using the organoid treated with supernatant of M1 and M2 macrophages polarized from THP-1 monocyte. Results: Oral cancer cells were co-cultured with endothelial cells (ECs) and mesenchymal stem cells (MSCs) in a particular ratio in 3D-culture, and the formed organoid became sphere. The vascular formation in the organoid was confirmed by immunofluorescent analysis. The organoid also showed higher tumorigenicity in nude mice when inoculated, compared to cancer cells alone or mixed cells with ECs and MSCs. Cell viability of luciferase-expressing tongue cancer cells (OSC-19-luc) in the organoid was assessed after treatment with cisplatin using the IVIS system showing that OSC-19-luc organoid was resistance to cisplatin compared to 3D-cultured OSC-19-luc alone or OSC-19-luc co-cultured with ECs/MSCs. The supernatant of M1 or M2 macrophage had no effect on cancer cell growth in the organoid. However, when the organoid was treated with cisplatin in the presence of M2 macrophage supernatant, the drug sensitivity was low compared to those of M1 suggesting that secretory factor from M2 macrophage may lead to drug resistance of oral cancer cells in the presence of TME. Conclusions: We have successfully established stroma cells-incorporating preclinical model that recreates the tumor microenvironment by co-culturing stromal cells. This will serve as a platform that can recapitulate the tumor biology and drug response profiles of cancer cells derived from individual oral cancer patients. This will also enable us to elucidate the role of other factors in TME and to identify novel targets for the treatment of chemo-resistant oral cancer. Citation Format: Ami Shimoda, Shizuka Shiiya, Haruka Yoshii, Kenji Mitsudo, Mitomu Kioi. Development of stroma cells-rich oral cancer organoid for functional analysis of tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4241.

Shedding light on the dark genome: Insights into the genetic, CRISPR-based, and pharmacological dependencies of human cancers and disease aggressiveness.

Investigating the human genome is vital for identifying risk factors and devising effective therapies to combat genetic disorders and cancer. Despite the extensive knowledge of the "light genome", the poorly understood "dark genome" remains understudied. In this study, we integrated data from 20,412 protein-coding genes in Pharos and 8,395 patient-derived tumours from The Cancer Genome Atlas (TCGA) to examine the genetic and pharmacological dependencies in human cancers and their treatment implications. We discovered that dark genes exhibited high mutation rates in certain cancers, similar to light genes. By combining the drug response profiles of cancer cells with cell fitness post-CRISPR-mediated gene knockout, we identified the crucial vulnerabilities associated with both dark and light genes. Our analysis also revealed that tumours harbouring dark gene mutations displayed worse overall and disease-free survival rates than those without such mutations. Furthermore, dark gene expression levels significantly influenced patient survival outcomes. Our findings demonstrated a similar distribution of genetic and pharmacological dependencies across the light and dark genomes, suggesting that targeting the dark genome holds promise for cancer treatment. This study underscores the need for ongoing research on the dark genome to better comprehend the underlying mechanisms of cancer and develop more effective therapies.

Response Profiles of Retinal Ganglion Cells to Sinusoidal Electric Stimulation vary for Low vs. High Frequencies.

Microelectronic retinal prostheses electrically stimulate retinal neurons with the goal of restoring vision in patients blinded by outer retinal degeneration. Despite some success in clinical trials, the quality of vision elicited by these devices is still limited. To improve the performance of retinal prostheses, our group studied how retinal neurons respond to electric stimulation. Our previous work showed that responses of retinal ganglion cells (RGCs) are frequency-dependent and different types of RGCs can be preferentially activated with a specific frequency and current amplitude. In the present study, we systemically examined responses of RGCs to sinusoidal electric stimulation with varying frequencies and amplitudes. We found that ON sustained alpha RGCs show distinct stimulus-response relationships to low and high frequency stimulation. For example, RGCs showed monotonic response curves to 500 Hz sinusoidal stimulation, whereas they showed non-monotonic response curves to 2000 Hz stimulation. We also described how increasing stimulus frequency gradually changed the response curves of RGCs.

Clonal heterogeneity influences drug responsiveness in renal cancer assessed by ex vivo drug testing of multiple patient-derived cancer cells.

Renal cell cancer (RCC) has become a prototype example of the extensive intratumor heterogeneity and clonal evolution of human cancers. However, there is little direct evidence on how the genetic heterogeneity impacts on drug response profiles of the cancer cells. Our goal was to determine how genomic clonal evolution impacts drug responses. Finding from our study could help to define the challenge that clonal evolution poses on cancer therapy. We established multiple patient-derived cells (PDCs) from different tumor regions of four RCC patients, verified their clonal relationship to each other and to the uncultured tumor tissue by genome sequencing. Furthermore, comprehensive drug-sensitivity testing with 460 oncological drugs was performed on all PDC clones. The PDCs retained many cancer-specific copy number alterations and mutations in driver genes such as VHL, PBRM1, PIK3C2A, KMD5C and TSC2 genes. The drug testing highlighted vulnerability in the PDCs toward approved RCC drugs, such as the mTOR-inhibitor temsirolimus, but also novel sensitivities were uncovered. The individual PDC clones from different tumor regions in a patient showed distinct drug-response profiles, suggesting that genomic heterogeneity contributes to the variability in drug responses. Studies of multiple PDCs from a patient with cancer are informative for elucidating cancer heterogeneity and for the determination on how the genomic evolution is manifested in cancer drug responsiveness. This approach could facilitate tailoring of drugs and drug combinations to individual patients.

Differentiation status of primary chronic myeloid leukemia cells affects sensitivity to BCR-ABL1 inhibitors.

Tyrosine kinase inhibitors (TKI) are the mainstay treatment of BCR-ABL1-positive leukemia and virtually all patients with chronic myeloid leukemia in chronic phase (CP CML) respond to TKI therapy. However, there is limited information on the cellular mechanisms of response and particularly on the effect of cell differentiation state to TKI sensitivity in vivo and ex vivo/in vitro. We used multiple, independent high-throughput drug sensitivity and resistance testing platforms that collectively evaluated 295 oncology compounds to characterize ex vivo drug response profiles of primary cells freshly collected from newly-diagnosed patients with BCR-ABL1-positive leukemia (n = 40) and healthy controls (n = 12). In contrast to the highly TKI-sensitive cells from blast phase CML and Philadelphia chromosome-positive acute lymphoblastic leukemia, primary CP CML cells were insensitive to TKI therapy ex vivo. Despite maintaining potent BCR-ABL1 inhibitory activity, ex vivo viability of cells was unaffected by TKIs. These findings were validated in two independent patient cohorts and analysis platforms. All CP CML patients under study responded to TKI therapy in vivo. When CP CML cells were sorted based on CD34 expression, the CD34-positive progenitor cells showed good sensitivity to TKIs, whereas the more mature CD34-negative cells were markedly less sensitive. Thus in CP CML, TKIs predominantly target the progenitor cell population while the differentiated leukemic cells (mostly cells from granulocytic series) are insensitive to BCR-ABL1 inhibition. These findings have implications for drug discovery in CP CML and indicate a fundamental biological difference between CP CML and advanced forms of BCR-ABL1-positive leukemia.

ZnO nanoparticles and organic chemical UV-filters are equally well tolerated by human immune cells

An important part of assessing the toxic potential of nanoparticles for specific applications should be the direct comparison of biological activities with those of alternative materials for the same application. Nanoparticulate inorganic ultraviolet (UV) filters, such as zinc oxide (ZnO), are commonly incorporated into transparent sunscreen and cosmetic formulations. However, concerns have been raised about potential unwanted effects, despite their negligible skin penetration and inherent advantages over organic chemical UV-filters. To provide useful application-relevant assessments of their potential hazard with/without UVA co-exposure, we directly compared cytotoxic and immune response profiles of human THP-1 monocytic cells to ZnO nanoparticles (30 nm) with bulk ZnO particulates (200 nm) and five conventional organic chemical UV-filters – butylmethoxydibenzoylmethane (avobenzone), octylmethoxycinnamate, octylsalicylate, homosalate and 4-methylbenzylidene camphor. High exposure concentrations of both organic and particulate UV-filters were required to cause cytotoxicity in monocyte and macrophage cultures after 24 h. Co-exposure with UVA (6.7 J/cm2) did not alter cytotoxicity profiles. Particle surface area-based dose responses showed that ZnO NPs were better tolerated than bulk ZnO. Organic and particulate UV-filters increased apoptosis at similar doses. Only particulates increased the generation of reactive oxygen species. Interleukin-8 (IL-8) release was increased by all particulates, avobenzone, homosalate and octylsalicylate. IL-1β release was only increased in macrophages by exposure to avobenzone and homosalate. In conclusion, direct effects were caused in monocytes and macrophages at similar concentrations of both organic UV-filters and ZnO nanoparticulates – indicating that their intrinsic cytotoxicity is similar. With their lower skin penetration, ZnO nanoparticles are expected to have lower bioactivity when used in sunscreens.

Detection of bitterness in vitro by a novel male mouse germ cell-based biosensor

Bitter detection has attracted extensive attention in industries and researches due to pharmacological and/or food safety issues. Common electronic tongues constructed with electrochemical, optical or mass sensors are often subjected to the drawback of low-specificity in detecting a wide range of bitter compounds with diverse chemical structures and functions. However, biological gustatory components present unique specificity to bitter compounds, which may provide a promising approach in bitter detection. The activation of G protein coupled receptors (GPCRs) by ligands will affect cell morphology in a specific manner, which can be monitored by cell-impedance sensor. Thus cells expressing bitter receptors (a subfamily of GPCRs) can be used as sensing material in specific bitter detection. In this study, we explored the feasibility of utilizing male mouse germ cells in testis and cell-based impedance sensors (CIS) to build a cell-based bitter biosensor. Male mouse germ cells express bitter receptor T2Rs, which can sensitively and specifically respond to bitter compounds. To verify bitter sensing capacity of the germ cells, calcium responses of germ cells to three bitter compounds were examined prior to cell impedance response measurement. Furthermore, a bitter receptor blocker, probenecid, was used to verify the specificity of T2R-mediated impedance responses in germ cells. Cell-impedance response profiles of germ cells to four bitter compounds were investigated by analyzing the response intensity under various concentrations. Finally, impedance responses to five basic tastes were examined to evaluate the performance of cell-based bitter biosensor in bitter detection. The results revealed that this hybrid bitter biosensor could respond to those bitter compounds in a dose-dependent manner. The detection threshold for quinine was 0.125mM, lower than our previous cell-based bitter biosensor. The calcium imaging tests and use of probenecid confirmed that T2R activation contributed to the cell impedance responses of this cell-based biosensor. Moreover, this germ cell-based cell could specifically detect bitter compounds among five taste stimuli, which may provide a promising and valuable approach for detection of various bitter compounds.

Stroma-Derived Factors Significantly Impact the Drug Response Profiles of Patient-Derived Primary AML Cells: Implications for Drug Sensitivity Testing

Background:Bone marrow (BM) microenvironment plays an important role in development of drug resistance in acute myeloid leukemia (AML) by supporting survival of therapy resistant leukemic cells that eventually may lead to disease relapse. Consideration of tumor stroma factors is therefore critically important when assessing the efficacy of drugs in the ex vivo drug testing of primary AML cells. To study the effects of tumor stroma factors on the response of AML patient cells ex vivo to a panel of cancer drugs, we evaluated the effects of stroma-derived conditioned medium (CM) on the sensitivity of genomically defined primary AML cells.Methods:Primary AML cells were isolated by Ficoll gradient separation from BM aspirates or peripheral blood of AML patients (n=13). For drug testing, 303 small molecule inhibitors were plated on 384-well plates in 5 different concentrations over a 10,000-fold concentration range. The stromal-cell conditioned medium (CM) was made by culturing human bone marrow (BM) stromal cell line HS-5 (American Type Culture Collection) in RPMI 1640 medium for 3 days. AML cells were added to the plates in either CM diluted with RPMI 1640 medium (25% CM) or in mononuclear cell medium (MCM, Promocell), which was used as the standard medium comparison. Cell viability was measured after 72 h and dose response curves generated for each drug. Drug sensitivity scores (DSS) were calculated as described previously (Yadav et al, 2014). Phosphorylation profiles of 43 proteins were analyzed with a human phospho-kinase array (R&D Systems). In addition, somatic mutations were identified by exome sequencing using DNA from the leukemia cells and matched skin biopsies, while expressed fusion genes were identified by transcriptome sequencing.Results:AML samples with activating mutations to kinases such as FLT3 or PDGFRB exhibited more sensitive ex vivo drug response profiles, particularly to broad-spectrum kinase inhibitors, compared to samples driven by other types of mutations. When the same AML samples were compared between the two conditions, CM or MCM, the drug sensitivities were different for many classes of drugs (Table 1). In CM, samples typically lost sensitivity to many of the tested drugs, such as topoisomerase II inhibitors, BCL2 inhibitors and several other classes of tyrosine kinase inhibitors (TKIs). The loss of TKI sensitivity in CM was particularly striking in the FLT3 and PDGFRB mutated cases. Cluster analysis of overall drug responses for AML samples tested in MCM resulted in a tight group of most TKIs, reflecting their overlapping target profiles. However, when the analysis was applied to responses from the same cells tested in CM, the TKI grouping was more dispersed. Thus, these results indicate that tyrosine kinase signaling is stringently regulated in standard medium, whereas CM helps to support cell survival resulting in lower responses to a range of TKIs. To test this hypothesis, phosphorylation of 43 different kinases was measured with AML samples incubated in either CM or MCM. CM induced phosphorylation of multiple proteins including p38α, HSP27, Src, Lyn, Hck and STAT6 proving the activation of other signaling pathways.Conclusions:Our dataindicate that stromal cell conditioned medium may have prominent effects on ex vivo drug responses of AML cells. BM factors likely provide survival cues that make primary patient-derived AML cells resistant to several targeted agents, such as topoisomerases and TKIs. This underscores the need to develop drug testing methods that take into account tumor-microenvironment interactions. [Display omitted] DisclosuresGjertsen:BerGenBio AS: Membership on an entity’s Board of Directors or advisory committees; Boehringer Ingelheim : Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Kinn Therapeutics AS: Equity Ownership. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Kallioniemi:Medisapiens: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Wennerberg:Pfizer: Research Funding. Heckman:Celgene: Research Funding.

Levels of alpha-toxin correlate with distinct phenotypic response profiles of blood mononuclear cells and with agr background of community-associated Staphylococcus aureus isolates.

Epidemiological studies of Staphylococcus aureus have shown a relation between certain clones and the presence of specific virulence genes, but how this translates into virulence-associated functional responses is not fully elucidated. Here we addressed this issue by analyses of community-acquired S. aureus strains characterized with respect to antibiotic resistance, ST types, agr types, and virulence gene profiles. Supernatants containing exotoxins were prepared from overnight bacterial cultures, and tested in proliferation assays using human peripheral blood mononuclear cells (PBMC). The strains displayed stable phenotypic response profiles, defined by either a proliferative or cytotoxic response. Although, virtually all strains elicited superantigen-mediated proliferative responses, the strains with a cytotoxic profile induced proliferation only in cultures with the most diluted supernatants. This indicated that the superantigen-response was masked by a cytotoxic effect which was also confirmed by flow cytometry analysis. The cytotoxic supernatants contained significantly higher levels of α-toxin than did the proliferative supernatants. Addition of α-toxin to supernatants characterized as proliferative switched the response into cytotoxic profiles. In contrast, no effect of Panton Valentine Leukocidin, δ-toxin or phenol soluble modulin α-3 was noted in the proliferative assay. Furthermore, a significant association between agr type and phenotypic profile was found, where agrII and agrIII strains had predominantly a proliferative profile whereas agrI and IV strains had a predominantly cytotoxic profile. The differential response profiles associated with specific S. aureus strains with varying toxin production could possibly have an impact on disease manifestations, and as such may reflect specific pathotypes.

The lateral intraparietal area codes the location of saccade targets and not the dimension of the saccades that will be made to acquire them

Activity in the lateral intraparietal area (LIP) represents a priority map that can be used to direct attention and guide eye movements. However, it is not known whether this activity represents the location of saccade targets or the actual eye movement made to acquire them. We recorded single neurons from rhesus macaques (Macaca mulatta) while they performed memory-guided delayed saccades to characterize the response profiles of LIP cells. We then separated the saccade target from the saccade end point by saccadic adaptation, a method that induces a change in the gain of the oculomotor system. We plotted LIP activity for all three epochs of the memory-guided delayed-response task (visual, delay period, and presaccadic responses) as a function of target location and saccade end point. We found that under saccadic adaptation the response profile for all three epochs was unchanged as a function of target location. We conclude that neurons in LIP reliably represent the locations of saccade targets, not the amplitude of the saccade required to acquire those targets. Although LIP transmits target information to the motor system, that information represents the location of the target and not the amplitude of the saccade that the monkey will make.

Comprehensive data-driven analysis of the impact of chemoinformatic structure on the genome-wide biological response profiles of cancer cells to 1159 drugs

BackgroundDetailed and systematic understanding of the biological effects of millions of available compounds on living cells is a significant challenge. As most compounds impact multiple targets and pathways, traditional methods for analyzing structure-function relationships are not comprehensive enough. Therefore more advanced integrative models are needed for predicting biological effects elicited by specific chemical features. As a step towards creating such computational links we developed a data-driven chemical systems biology approach to comprehensively study the relationship of 76 structural 3D-descriptors (VolSurf, chemical space) of 1159 drugs with the microarray gene expression responses (biological space) they elicited in three cancer cell lines. The analysis covering 11350 genes was based on data from the Connectivity Map. We decomposed the biological response profiles into components, each linked to a characteristic chemical descriptor profile.ResultsIntegrated analysis of both the chemical and biological space was more informative than either dataset alone in predicting drug similarity as measured by shared protein targets. We identified ten major components that link distinct VolSurf chemical features across multiple compounds to specific cellular responses. For example, component 2 (hydrophobic properties) strongly linked to DNA damage response, while component 3 (hydrogen bonding) was associated with metabolic stress. Individual structural and biological features were often linked to one cell line only, such as leukemia cells (HL-60) specifically responding to cardiac glycosides.ConclusionsIn summary, our approach identified several novel links between specific chemical structure properties and distinct biological responses in cells incubated with these drugs. Importantly, the analysis focused on chemical-biological properties that emerge across multiple drugs. The decoding of such systematic relationships is necessary to build better models of drug effects, including unanticipated types of molecular properties having strong biological effects.

Abstract 3188: Development of a cancer pharmacopeia-wide ex-vivo drug sensitivity and resistance testing (DSRT) platform for AML: Towards individually optimized therapy and improved understanding of drug resistance patterns

Abstract In order to discover unexpected anti-cancer efficacies of approved and emerging drugs, we established a diagnostic ex vivo drug sensitivity and resistance testing (DSRT) platform covering the entire cancer pharmacopeia as well as emerging anti-cancer compounds. Here, the platform was applied to analyze bone marrow (BM) mononuclear cells from 17 adult acute myeloid leukemia (AML) patients, 3 healthy donors as well as 7 AML cell lines. The DSRT panel covered FDA-approved small molecule oncology drugs (n=120), as well as emerging, investigational and pre-clinical oncology compounds (n=120), such as kinase (e.g. RTKs, checkpoint and mitotic kinases, Raf, MEK, JAKs, mTOR, PI3K), and non-kinase inhibitors (e.g. HSP, Bcl, activin, HDAC, PARP, Hh). To generate dose-response curves, each of the drugs was applied over a 10,000-fold concentration range. In addition, the samples underwent deep molecular profiling including exome- and transcriptome sequencing, as well as phosphoproteomic analysis. DSRT provided consistent and reliable data from ex vivo samples with a high correlation between data from individual healthy BM samples (r=0.93). Bioinformatic processing of the data from AML resulted in several key observations. First, overall drug response profiles of AML blast cells were distinctly different from healthy BM controls suggesting several potential leukemia-selective effects, such as multi-kinase (dasatinib), MEK, and mTOR inhibitors. Second, the overall drug responses from AML cell lines and the patient ex vivo samples showed differences, suggesting that ex vivo testing may reveal cancer-selective effects not previously seen in established cancer cell line panels. Third, the response data from patient samples clustered many drugs consistently into the expected functional classes (such as topoisomerase II inhibitors, MEK inhibitors and rapalogs), whereas other drug classes were more dispersed (such as FLT3 inhibitors with quizartinib clustering away from all other tyrosine kinase inhibitors), suggesting secondary targets playing a key role in drug efficacy. Fourth, analysis of serial samples from patients developing clinical resistance to targeted agents showed striking agreement between the ex-vivo DSRT profiles and clinical responses. In conclusion, comprehensive DSRT platform generated powerful novel insights on AML drug response and may enable individual optimization of therapies, particularly for recurrent leukemias. DSRT will also serve as a powerful hypothesis-generator for clinical trials, particularly for emerging drugs. The ability to correlate ex vivo response profiles for hundreds of drugs in clinical samples with deep molecular profiling data will yield exciting new translational and pharmacogenomic opportunities for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3188. doi:1538-7445.AM2012-3188

Abstract 895: Quantitative drug sensitivity and resistance testing (DSRT) of primary ex vivo AML blasts highlights mTOR and MEK as potential key molecular driver signals of therapeutic significance

Abstract Identification of signaling pathways that are required for the growth and differentiation block of cells from adult acute myeloid leukemia (AML) is urgently required to facilitate development of novel therapies. Here, we describe an approach to functionally determine molecular drivers of AML by quantitative drug sensitivity and resistance testing (DSRT) of AML blast cells in primary culture ex vivo. The selection of drugs covered the entire cancer pharmacopeia and much of the pipeline of drugs under development in the industry: 120 FDA approved small molecular cancer drugs and 120 emerging drugs, investigational compounds and signal transduction inhibitors. All compounds were tested over a 10,000-fold concentration range to generate quantitative and reliable dose-response data. In addition, whole exome and transcriptome sequencing and phophoproteomic profiling were also performed to derive a comprehensive understanding of the molecular AML-related aberrations on an individual basis. Comparison of 17 AML patient samples and 3 healthy bone marrow control samples based on ex vivo drug responses identified several classes of approved and investigational drugs that showed selective anti-AML activities: mTOR inhibitors (e.g. temsirolomus, everolimus, sirolimus), MEK inhibitors (e.g. AS703026, GSK1120212, RDEA119, selumetinib), tyrosine kinase inhibitors (e.g. dasatinib, ponatinib, sunitinib), Bcl-2 inhibitors (navitoclax) and HSP90 inhibitors (e.g. BIIB021, NVP-AUY922, tanespimycin). In particular, the rapamycin class of mTOR inhibitors and allosteric MEK inhibitors stood out as effective and selective inhibitors in 8/17 (47%) and 9/17 (52%) of the patients, respectively. Simultaneous data from other targeted inhibitors made it possible to dissect the critical steps in signaling and therapeutic efficacy. For example, PI3K and Akt inhibitors were not effective in these patients, suggesting that the mTOR dependency is mediated through a PI3K-Akt-independent pathway. Similarly, the dependency of MEK signaling appears to be through a Ras-Raf-independent pathway since Raf inhibitors were not effective. In conclusion, the DSRT platform allows us to derive quantitative data on the ex vivo drug response profiles of AML cells from individual patients. This information could be used as a diagnostic tool to optimize personalized therapies in the future. Our data demonstrate that mTOR and MEK signaling and the associated inhibitors are the most promising leads for improved AML therapeutics. This analysis also demonstrates gaps in our current understanding of the redundancy of key cancer cell signaling pathways and proves the significant value of data from experimental drug response testing ex vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 895. doi:1538-7445.AM2012-895

Membrane potential response profiles of CA1 pyramidal cells probed with voltage-sensitive dye optical imaging in rat hippocampal slices reveal the impact of GABA A-mediated feed-forward inhibition in signal propagation

The spatial and temporal distribution of excitatory and inhibitory membrane potential responses on a cell plays an important role in neuronal calculations in local neuronal circuits in the brain. The electrical dynamics of excitatory and inhibitory inputs along the somatodendritic extent of CA1 pyramidal cells during circuit activation were examined by stimulating strata radiatum (SR), oriens (SO), and lacunosum-moleculare (SLM) and measuring laminar responses with voltage-sensitive dye (VSD) optical recording methods. We first confirmed the linearity of the optical signal by comparing fluorescence changes in CA1 to global membrane potential changes when slices were bathed in high-potassium ([K +] O = 25 mM) solution. Except for a TTX-sensitive component in stratum pyramidale, fluorescence changes were equal in all strata, indicating that VSD sensitivity had reasonable linearity across layers. We then compared membrane potential profiles in slices exposed to picrotoxin, a GABA A receptor antagonist. We attributed the picrotoxin-induced changes in the first peak of the excitatory membrane potential to feed-forward inhibition and the later response (appearing 30 ms after stimulation) to feedback inhibition. A difference in feed-forward components was observed in perisomatic and distal apical dendritic regions after SR stimulation. SLM stimulation produced large differences in perisomatic and apical dendritic regions. SO stimulation, however, produced no feed-forward inhibition at the perisomatic region, but produces feed-forward inhibition in distal dendritic regions. These results suggest that actual inhibition of membrane potential response by feed-forward inhibition is greater at perisomatic regions after SR or SLM stimulation but is smaller at distal dendritic regions after SR, SO, and SLM stimulation.

Simultaneous determination of depth and motion in early vision

We investigated computationally the role of V1 complex cells in the simultaneous determination of depth and motion, in conjunction with spatiotemporal response characteristics of the cells. First, we examined whether a pooling of simple-cell units with monocular, spatiotemporal response profiles evokes binocular, spatiotemporal response profiles of complex cells. Second, we examined whether a population of the cascade complex-cell models yields the responses being consistent with the perception of depth and motion. We tested various stimuli including ambiguous cases that consist of interocular time delay without spatial disparity. Our simulation study showed that a pooling of simple cells leads to the generation of binocular response profiles of complex cells, and that a population of the complex cells provides a basis for the simultaneous perception of depth and motion.

The role of spatial phase in texture segmentation and contour integration

It has been recently argued that the visual system possesses just two phase "detector" mechanisms, namely, +cosine and -cosine (P. C. Huang, F. A. Kingdom, & R. F. Hess, 2006). This suggests rather limited access to the rich distribution of receptive field phase that neurophysiologists tell us are represented in the different response profiles of striate simple cells. Whereas that study has suggested that striate receptive field phase is not directly available to perception for the detection/discrimination of localized stimuli, here we investigate whether such information might be used in more integrative striate or extrastriate functions such as texture segregation or contour integration. Specifically, given that simple cells have different local absolute phase response profiles, we ask whether a network of simple cells with similar phase preferences interact in such a way as to extract textures, contours, or both based on phase alone. Two novel texture segmentation experiments and one contour integration experiment were carried out with the intention of providing an answer to the question of how useful is local absolute spatial phase for texture segmentation and contour integration. The results support the possibility of two phase mechanisms (+/-cosine) for global texture segmentation, as well as for contour integration, when the elements that make up a given contour are orthogonal to contour paths.

Disparity sensitivity in the superior colliculus of the cat

The present study aims at evaluating the spatial disparity response profiles of binocular cells in the superficial layers of the superior colliculus of the cat using drifting light bars and phase-shifted spatial frequency gratings. Results show that a total of 64% of the cells were sensitive to phase disparities and had large tuning profiles. Similarly, a large proportion (75%) of those tested with position offsets showed one of the four classic disparity profiles, those of the tuned cells being rather coarse. When tested with both position and phase disparities, 54% of the cells showed sensitivity profiles to the two types of stimuli. The overall results suggest that the superior colliculus is involved in the analysis of coarse stereopsis and/or the planning and initiation of saccades during vergence eye movements and/or the control of fine adjustments to maintain fixation as the stimulus moves in depth.

Activity in the Hypothalamus, Amygdala, and Cortex Generates Bilateral and Convergent Modulation of Pontine Gustatory Neurons

Evidence suggests that centrifugal modulation of brain stem gustatory cells might play a role in the elaboration of complex taste-guided behaviors like conditioned taste aversion and sodium appetite. We previously showed that activity in one forebrain area, the central nucleus of the amygdala (CeA), increased the chemical selectivity of taste cells in the parabrachial nucleus (PBN). The present study investigates how activity in 2 other similarly interconnected forebrain sites, the lateral hypothalamus (LH) and gustatory cortex (GC), might influence PBN gustatory processing in rats. The potential convergence of descending inputs from these sites, as well as the CeA, was also evaluated. After anesthesia (35 mg/kg Nembutal ip), 70 PBN gustatory neurons were tested before, during, and after electrical stimulation of these forebrain sites, while responding to 0.3 M sucrose, 0.1 M NaCl, 0.01 M citric acid, and 0.003 M QHCl. Although each forebrain site modulated taste-evoked responses, more PBN neurons were influenced by stimulation of the GC (67%) and CeA (73%) than of the LH (48%). Activation of cortex (71%) and amygdala (85%) most often produced inhibition, whereas inhibition and excitation occurred equally often during hypothalamic stimulation. Of the neurons tested for convergence (n = 60), 88% were influenced by > or =1 of the 3 sites. Twenty were modulated by stimulation at all 3 sites and another 17 by 2 of the 3 sites. The net effect of centrifugal modulation was to sharpen the across-stimulus response profiles of PBN cells, particular with regard to the NaCl- and citric acid-best cells.

Dynamic Coding of Taste Stimuli in the Brainstem: Effects of Brief Pulses of Taste Stimuli on Subsequent Taste Responses

Recent studies have suggested that the response profiles of taste-responsive cells in the brainstem may be modulated by inhibitory interactions, potentially originating from activity in peripheral taste nerves. This idea was explored by testing the hypothesis that brief (100 msec) pulses of taste stimuli would alter the responses to subsequently presented tastants in the nucleus of the solitary tract (NTS) of urethane-anesthetized rats. Pulses of taste stimuli, called prepulses, were followed by a 3 sec presentation of the same or different taste stimulus. The prepulse-stimulus interval was either 1 or 5 sec, during which the tongue was rinsed with distilled water. Taste stimuli consisted of 0.1 m NaCl, 0.5 m sucrose, 0.01 m quinine HCl, and 0.01 m HCl. Taste prepulses suppressed (or enhanced) subsequent taste responses in 30 of 49 (61%) units when the prepulse-stimulus interval was 1 sec but were ineffective when this interval was 5 sec. Most commonly, NaCl or HCl prepulses attenuated the response to quinine. Control experiments showed that these effects were not attributable to adaptation, mixture effects, or response variability. In 19 (39%) of the units tested, effects of prepulses were large enough to change the order of effectiveness of the taste stimuli. Taste responses in these cells were "dynamically tuned" in that the magnitude of response was a function of the taste stimulus that immediately preceded it. Dynamic tuning may be the result of inhibitory interactions within the NTS; cells that show dynamic tuning may have a unique function in taste coding.