Multiparametric Profiling Research Articles

Multi-parametric atlas of the pre-metastatic liver for prediction of metastatic outcome in early-stage pancreatic cancer.

Metastasis occurs frequently after resection of pancreatic cancer (PaC). In this study, we hypothesized that multi-parametric analysis of pre-metastatic liver biopsies would classify patients according to their metastatic risk, timing and organ site. Liver biopsies obtained during pancreatectomy from 49 patients with localized PaC and 19 control patients with non-cancerous pancreatic lesions were analyzed, combining metabolomic, tissue and single-cell transcriptomics and multiplex imaging approaches. Patients were followed prospectively (median 3 years) and classified into four recurrence groups; early (<6 months after resection) or late (>6 months after resection) liver metastasis (LiM); extrahepatic metastasis (EHM); and disease-free survivors (no evidence of disease (NED)). Overall, PaC livers exhibited signs of augmented inflammation compared to controls. Enrichment of neutrophil extracellular traps (NETs), Ki-67 upregulation and decreased liver creatine significantly distinguished those with future metastasis from NED. Patients with future LiM were characterized by scant T cell lobular infiltration, less steatosis and higher levels of citrullinated H3 compared to patients who developed EHM, who had overexpression of interferon target genes (MX1 and NR1D1) and an increase of CD11B+ natural killer (NK) cells. Upregulation of sortilin-1 and prominent NETs, together with the lack of T cells and a reduction in CD11B+ NK cells, differentiated patients with early-onset LiM from those with late-onset LiM. Liver profiles of NED closely resembled those of controls. Using the above parameters, a machine-learning-based model was developed that successfully predicted the metastatic outcome at the time of surgery with 78% accuracy. Therefore, multi-parametric profiling of liver biopsies at the time of PaC diagnosis may determine metastatic risk and organotropism and guide clinical stratification for optimal treatment selection.

Amino acid profiling as a screening and prognostic biomarker in active tuberculosis patients

BackgroundTuberculosis (TB) is one of the world's most deadly chronic infectious diseases; early diagnosis contributes to reducing disease transmission among populations. However, discovering novel diagnostic and prognostic biomarkers is still an important topic in the field of TB. Amino acid is the basic unit of protein composition, and its structure and physicochemical characteristics are more stable. Therefore, it is a potential target for TB diagnosis and the prediction of TB development. MethodsIn this study, the blood of healthy people (HC), TB patients (TB), cured TB (RxTB), and other non-TB pneumonia patients (PN) were collected to detect the levels of amino acids in whole blood and plasma using ultra-high performance liquid chromatography coupled with mass spectrometry. ResultsWe detected that the amino acid levels correlated with participants status (TB, HC, RxTB, or PN) and the degree of lung damage. The results showed that phenylalanine had a good effect on the screening of TB (AUC = 0.924). We then built a TB prediction model. The model, which was based on the ratio of plasma amino acid content to whole blood amino acid content, showed good performance for the screening of TB, with 84% (95% CI = 60–97) sensitivity and 97% (95% CI = 83–100) specificity. The result of correlation between the HRCT score and amino acid level indicated that the glutamine content of plasma was significantly inversely associated with disease severity. Additionally, ornithine levels in the plasma of RxTB group reduced and four amino acids of which the ratio in plasma to whole blood showed significantly changed. ConclusionsTaken together, amino acid profiling can be used for TB screening, and a multiparameter profiling model is better. The profiling can also reflect the severity of lung damage. Moreover, the amino acid profile is useful for reflecting the efficacy of TB treatment.

Multifunctional profiling of triple-negative breast cancer patient-derived tumoroids for disease modeling.

3D cell models derived from patient tumors are highly translational tools that can recapitulate the complex genetic and molecular compositions of solid cancers and accelerate identification of drug targets and drug testing. However, the complexity of performing assays with such models remains a hurdle for their wider adoption. In the present study, we describe methods for processing and multi-functional profiling of tumoroid samples to test compound effects using a novel flowchip system in combination with high content imaging and metabolite analysis. Tumoroids were formed from primary cells isolated from a patient-derived tumor explant, TU-BcX-4IC, that represents metaplastic breast cancer with a triple-negative breast cancer subtype. Assays were performed in a microfluidics-based device (Pu⋅MA System) that allows automated exchange of media and treatments of tumoroids in a tissue culture incubator environment. Multi-functional assay profiling was performed on tumoroids treated with anti-cancer drugs. High-content imaging was used to evaluate drug effects on cell viability and expression of E-cadherin and CD44. Lactate secretion was used to measure tumoroid metabolism as a function of time and drug concentration. Observed responses included loss of cell viability, decrease in E-cadherin expression, and increase of lactate production. Importantly, the tumoroids were sensitive to romidepsin and trametinib, while showed significantly reduced sensitivity to paclitaxel and cytarabine, consistent with the primary tumor response. These methods for multi-parametric profiling of drug effects in patient-derived tumoroids provide an in depth understanding of drug sensitivity of individual tumor types, with important implications for the future development of personalized medicine.

Comprehensive molecular characterization of lung tumors implicates AKT and MYC signaling in adenocarcinoma to squamous cell transdifferentiation

BackgroundLineage plasticity, the ability to transdifferentiate among distinct phenotypic identities, facilitates therapeutic resistance in cancer. In lung adenocarcinomas (LUADs), this phenomenon includes small cell and squamous cell (LUSC) histologic transformation in the context of acquired resistance to targeted inhibition of driver mutations. LUAD-to-LUSC transdifferentiation, occurring in up to 9% of EGFR-mutant patients relapsed on osimertinib, is associated with notably poor prognosis. We hypothesized that multi-parameter profiling of the components of mixed histology (LUAD/LUSC) tumors could provide insight into factors licensing lineage plasticity between these histologies.MethodsWe performed genomic, epigenomics, transcriptomics and protein analyses of microdissected LUAD and LUSC components from mixed histology tumors, pre-/post-transformation tumors and reference non-transformed LUAD and LUSC samples. We validated our findings through genetic manipulation of preclinical models in vitro and in vivo and performed patient-derived xenograft (PDX) treatments to validate potential therapeutic targets in a LUAD PDX model acquiring LUSC features after osimertinib treatment.ResultsOur data suggest that LUSC transdifferentiation is primarily driven by transcriptional reprogramming rather than mutational events. We observed consistent relative upregulation of PI3K/AKT, MYC and PRC2 pathway genes. Concurrent activation of PI3K/AKT and MYC induced squamous features in EGFR-mutant LUAD preclinical models. Pharmacologic inhibition of EZH1/2 in combination with osimertinib prevented relapse with squamous-features in an EGFR-mutant patient-derived xenograft model, and inhibition of EZH1/2 or PI3K/AKT signaling re-sensitized resistant squamous-like tumors to osimertinib.ConclusionsOur findings provide the first comprehensive molecular characterization of LUSC transdifferentiation, suggesting putative drivers and potential therapeutic targets to constrain or prevent lineage plasticity.

MA11.06 Multi-Omic Characterization of Lung Tumors Implicates AKT and MYC Signaling in Adenocarcinoma to Squamous Cell Transdifferentiation

Lineage plasticity, the ability to transdifferentiate among distinct phenotypic identities, facilitates therapeutic resistance in multiple cancers. In lung adenocarcinomas (LUADs), this phenomenon includes small cell and squamous cell (LUSC) histologic transdifferentiation in the context of acquired resistance to targeted inhibition of driver mutations. The incidence of transdifferentiation into squamous carcinoma in EGFR mutant tumors, the setting where this histologic shift has been most extensively described, occurs in up to 9% of cases relapsed on osimertinib and has been associated to poor prognosis. The paucity of well annotated pre- and post-transdifferentiation clinical samples has precluded the performance of informative molecular analyses: little is known about the molecular mechanisms leading to this histological transition. We hypothesized that multi-parameter profiling of the components of mixed histology (LUAD/LUSC) tumors, together with pre- and post-transdifferntiation clinical samples, could provide insight into factors licensing lineage plasticity between these histologies and promoting squamous transdifferentiation of LUAD. We performed detailed genomic (whole exome sequencing), epigenomic (bisulfite sequencing), transcriptomic (RNAseq) and proteomic (antibody arrays) characterization. Clinical findings were validated in preclinical models including cell lines and patient-derived xenograft treatments. Our results suggest that LUSC transdifferentiation is primarily driven by transcriptional reprogramming rather than mutational events, and indicate that the resulting squamous tumors retain transcriptomic and methylation profiles of their previous LUAD state. We observed coordinated upregulation of PI3K/AKT, MYC and PRC2 pathway genes in the LUSC component of mixed histology tumors. Concurrent activation of PI3K/AKT and MYC induced squamous features in EGFR-mutant LUAD preclinical models, further augmented under selective pressure of osimertinib. Pharmacologic inhibition of EZH1/2 in combination with osimertinib prevented relapse and squamous transdifferentiation in an EGFR-mutant patient-derived xenograft model, and inhibition of EZH1/2 or PI3K/AKT signaling re-sensitized resistant transdifferentiated LUSC tumors to osimertinib. Our findings provide the first comprehensive molecular characterization of LUSC transdifferentiation, suggesting putative drivers and promising therapeutic targets to constrain or prevent lineage plasticity in this setting.

1MO Multi-omic characterization of lung tumors identify AKT and EZH2 as potential therapeutic targets in adenocarcinoma-to-squamous transdifferentiation

Lineage plasticity, the ability to transdifferentiate among distinct phenotypic identities, contributes to therapeutic resistance in cancer. In lung adenocarcinomas (LUADs), this phenomenon drives squamous cell (LUSC) histologic transdifferentiation in the context of acquired resistance to targeted inhibition of driver mutations, with an up to 9% incidence in EGFR-mutant tumors relapsed on osimertinib, and leads to poor prognosis. Limitations on well annotated pre- and post-transdifferentiation clinical samples has hindered the performance of molecular analyses, and thus little is known about the molecular mechanisms leading to this histological transition. We performed multi-parameter profiling of LUAD-to-LUSC transdifferentiating clinical samples, including detailed genomic (whole exome sequencing), epigenomic (bisulfite sequencing), transcriptomic (RNAseq) and proteomic (antibody arrays) characterization. Clinical findings were validated in preclinical models including cell lines and patient-derived xenograft treatments. Our data suggest that LUSC transdifferentiation is driven by epigenetic, rather than mutational events, and indicate that the transdifferentiated LUSC tumors retain transcriptomic and methylation profiles of their previous LUAD state. We observed coordinated upregulation of PI3K/AKT, MYC and PRC2 pathway genes upon transdifferentiation. Concurrent activation of PI3K/AKT and MYC overexpression induced squamous features in EGFR-mutant LUAD preclinical models, further accentuated by EGFR inhibition. Pharmacologic inhibition of EZH1/2 or PI3K/AKT in combination with osimertinib delayed relapse and squamous transdifferentiation in an EGFR-mutant patient-derived xenograft model, and re-sensitized resistant transdifferentiated LUSC tumors to osimertinib. Here, we provide the first comprehensive molecular characterization of LUSC transdifferentiation and nominate potential therapeutic targets to constrain or prevent lineage plasticity in this setting.

Monocytes and macrophages in severe COVID-19 - friend, foe or both?

Monocytes and macrophages in severe COVID-19 - friend, foe or both?

Multi-Parametric Portfolio to Assess the Fitness and Gonadal Maturation in Four Key Reproductive Phases of Brown Trout

Simple SummaryBrown trout is a freshwater fish with economic importance and with a great potential to be used as an environmental biosensor species. Despite being selected as a model species in distinct scientific contexts, in cultured specimens, there is a surprising lack of works investigating the morpho-physiological changes associated with the reproductive cycle; particularly concerning the gonads. In this study, a multi-parameter portfolio of biometric, biochemical, hormonal, and morphological analysis was established, which allowed a seasonal and sex characterization of the gonad status of adult brown trout males and females. Sampling included four reproductive phases: spawning capable (December), regressing (March), regenerating (July), and developing (November). Sex- and season-specific changes were described. The discriminative parameters characterized here stand now as normal baseline values against which abnormal patterns can be compared with. These parameters have the potential to be used as tools for the environmental monitoring of the reproductive status of wild populations and for the control of breeding stocks in aquaculture.Brown trout is an environmental freshwater sentinel species and is economically important for recreational fishing and aquaculture. Despite that, there is limited knowledge regarding morpho-physiological variations in adults throughout the reproductive cycle. Thus, this study aimed to analyze the fitness and gonadal maturation of cultured adult brown trout in four reproductive phases (spawning capable—December, regressing—March, regenerating—July, and developing—November). The systematic evaluation of males and females was based on biometric, biochemical, and hormonal parameters, along with a histomorphological grading of gonads and the immunophenotype location of key steroidogenic enzymes. The total weight and lengths reached the lowest levels in December. Gonad weights were higher in December and November, while the opposite pattern was found for liver weights. The lowest levels of cholesterol and total protein were also noted during those stages. The 11-ketotestosterone (11-KT) and testosterone (T) for males, and estradiol (E2) and T for females, mostly explained the hormonal variations. The immunohistochemistry of cytochrome P450c17 (CYP17-I), aromatase (CYP19), and 17β-hydroxysteroid dehydrogenase (17β-HSD) showed sex and site-specific patterns in the distinct reproductive phases. The sex- and season-specific changes generated discriminative multi-parameter profiles, serving as a tool for environmental and aquaculture surveys.

Multiparametric Profiling of Single Nanoscale Extracellular Vesicles by Combined Atomic Force and Fluorescence Microscopy: Correlation and Heterogeneity in Their Molecular and Biophysical Features.

Being a key player in intercellular communications, nanoscale extracellular vesicles (EVs) offer unique opportunities for both diagnostics and therapeutics. However, their cellular origin and functional identity remain elusive due to the high heterogeneity in their molecular and physical features. Here, for the first time, multiple EV parameters involving membrane protein composition, size and mechanical properties on single small EVs (sEVs) are simultaneously studied by combined fluorescence and atomic force microscopy. Furthermore, their correlation and heterogeneity in different cellular sources are investigated. The study, performed on sEVs derived from human embryonic kidney 293, cord blood mesenchymal stromal and human acute monocytic leukemia cell lines, identifies both common and cell line-specific sEV subpopulations bearing distinct distributions of the common tetraspanins (CD9, CD63, and CD81) and biophysical properties. Although the tetraspanin abundances of individual sEVs are independent of their sizes, the expression levels of CD9 and CD63 are strongly correlated. A sEV population co-expressing all the three tetraspanins in relatively high abundance, however, having average diameters of <100 nm and relatively low Young moduli, is also found in all cell lines. Such a multiparametric approach is expected to provide new insights regarding EV biology and functions, potentially deciphering unsolved questions in this field.

Deep learning-enabled analysis reveals distinct neuronal phenotypes induced by aging and cold-shock

BackgroundAccess to quantitative information is crucial to obtain a deeper understanding of biological systems. In addition to being low-throughput, traditional image-based analysis is mostly limited to error-prone qualitative or semi-quantitative assessment of phenotypes, particularly for complex subcellular morphologies. The PVD neuron in Caenorhabditis elegans, which is responsible for harsh touch and thermosensation, undergoes structural degeneration as nematodes age characterized by the appearance of dendritic protrusions. Analysis of these neurodegenerative patterns is labor-intensive and limited to qualitative assessment.ResultsIn this work, we apply deep learning to perform quantitative image-based analysis of complex neurodegeneration patterns exhibited by the PVD neuron in C. elegans. We apply a convolutional neural network algorithm (Mask R-CNN) to identify neurodegenerative subcellular protrusions that appear after cold-shock or as a result of aging. A multiparametric phenotypic profile captures the unique morphological changes induced by each perturbation. We identify that acute cold-shock-induced neurodegeneration is reversible and depends on rearing temperature and, importantly, that aging and cold-shock induce distinct neuronal beading patterns.ConclusionThe results of this work indicate that implementing deep learning for challenging image segmentation of PVD neurodegeneration enables quantitatively tracking subtle morphological changes in an unbiased manner. This analysis revealed that distinct patterns of morphological alteration are induced by aging and cold-shock, suggesting different mechanisms at play. This approach can be used to identify the molecular components involved in orchestrating neurodegeneration and to characterize the effect of other stressors on PVD degeneration.

The Effect of Early Diagenesis in Methanic Sediments on Sedimentary Magnetic Properties: Case Study From the SE Mediterranean Continental Shelf

Microbial respiration in marine sediment can affect the magnetic properties of the sediment through a complicated interplay between reductive dissolution and authigenic precipitation of iron-bearing magnetic minerals. However, a direct link between the main diagenetic zones in the upper sedimentary column and sedimentary magnetic properties using high resolution multi parameter profiles has been demonstrated only in few studies. Here, we directly correlate early diagenetic processes and sedimentary magnetism using a composite high-resolution sedimentary record of pore water chemistry, solid phase chemical measurements and mineral-magnetic parameters. Measurements along the profiles include the entire redox cascade, from the water-sediment interface, down through the deep methanic zone, on a six-meter sediment core collected from the Southern Eastern Mediterranean continental shelf. The uppermost part of the sediment core, associated with oxic, nitrous, manganous, ferruginous, and sulfate reduction zones, is characterized by high ferrous iron and sulfate concentrations and high values of the measured magnetic parameters (susceptibility, Isothermal remanent magnetization (IRM) and Anhysteretic remanent magnetization (ARM)). This layer is underlain by a sulfate-methane transition zone (SMTZ) that shows a significant decrease in magnetic parameters due to the dissolution of magnetic minerals. Below the SMTZ, the methanic zone has been assumed to be magnetically inactive under steady-state conditions. However, we observe in the upper methanic zone an increase in microbial iron reduction, coupled to an abrupt increase in magnetic parameters. Our data indicate that the observed increase in the magnetic signal is related to the precipitation of authigenic magnetic minerals. These diagenetic changes should be considered when interpreting paleomagnetic data, and highlight the potential to use high-resolution magnetic data as a proxy for identifying diagenetic processes.

Abstract A22: Simultaneous multiparametric profiling of cell-free RNA (cfRNA) and cell-free tumor DNA (ctDNA) in advanced-stage colorectal cancer

Abstract Background: It has been recognized that intratumoral heterogeneity together with therapeutic intervention-associated multiple genomic alterations added complexity for precision therapy in late-stage colorectal cancer (CRC). The clinical utility/validity of ctDNA assays have been systematically analyzed and proved under clinical settings in select cancer types with demonstrated specificity and sensitivity. Notably, cfRNA sequencing confirmed and enhanced detection sensitivity for variants with low allele frequency. In this study, plasma-based ctDNA mutation and cfRNA gene expression in patients with advanced CRC were simultaneously tested using NGS liquid biopsy panels. Methods: ctDNA and cfRNA were extracted from plasma of 25 treatment-naive patients with advanced CRC (stage III/IV), then were tested using the 600-gene PredicineATLAS ctDNA NGS assay and 180-gene PredicinePLUS cfRNA NGS assay, respectively. Various genomic alterations including single-nucleotide variation (SNV), Indel, copy number variation (CNV) and genome rearrangement are detected, along with tumor mutation burdens (TMB) evaluation/measurement. RNA-based variants and gene expression are also analyzed. Results: At ctDNA level, 96% (24/25) ctDNA samples were detected with at least 1 genomic alteration and total 265 SNV/Indels and 27 CNVs were identified across samples, including 180 genes with reported SNVs/indels and 23 genes with CNV. 20% (5/25) of the patients with RAS somatic mutations, 32% (8/25) with TP53 mutations, 8% (2/25) with APC mutations, and 8% (2/25) with PIK3CA mutations. TPM3-NTRK1 fusion was also detected in one patient. A subset of these variants detected at ctDNA level were also confirmed by cfRNA sequencing. Conclusions: Our results demonstrated the clinical utility/feasibility of simultaneous multiparametric profiling of ctDNA and cfRNA for comprehensive molecular insight/characterization in advanced-stage cancer. cfRNA-derived variant detection confirms the variants originally detected in ctDNA test and further validates the high-specificity results for report interpretation. Further investigation of exploring concordance between temporally matched tissue and plasma samples is ongoing. The ctDNA and cfRNA-based combined liquid biopsy solution provides holistic molecular overview for detecting driver mutations, monitoring efficacy/disease burden, and identifying subclonal alterations that may related to drug resistance and tumor heterogeneity. Citation Format: Zheng Feng, Danyi Wang, Dennis Merkle, Jianjun Yu, Shidong Jia, Juergen Scheuenpflug. Simultaneous multiparametric profiling of cell-free RNA (cfRNA) and cell-free tumor DNA (ctDNA) in advanced-stage colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A22.

Design of a multi-parametric profile for assessing the acclimation period of juvenile brown trout after an acute transport to new housing environment

Fish batches are often transported from aquacultures to laboratory facilities, and this fact triggers a pre-stressed condition that can cause alterations in both physiological and behavioural status of animals. The acclimation before fish trials is highly recommended to ideally ensure basal levels for the species, but there is still limited information concerning the period of time and parameters to be included for the evaluation. Facing those facts, brown trout (Salmo trutta f. fario) juveniles subjected to an acute transport (around 2 h) were here tested during 42 days post-stress (at 12 h, 7, 14, 28 and 42 days), in order to: 1) define a multi-parameter approach, based on biochemical, physical, morphological, nutritional and behavioural parameters that best characterize the fish acclimation and welfare status; and (2) propose an adequate acclimation period for brown trout juveniles after transportation to new housing conditions.Behavioural changes were noted with an overall stabilization at the 17th day. Fish tended to occupy preferentially the bottom of the tank, a smaller number of tank sections, decreased the swimming activity and the number of abnormal movements. Reaction time to food decreased until it was instantaneous, and the reaction to food gradually changed over time, so that on the 1st day there was no reaction and, from the 30th day, most of the fish moved continuously to reach the food. Feed intake did not occur on the 1st day, and then increased gradually. Despite that, there were no differences in fish condition and hepatosomatic indices over time.In general, glucose was relatively stable along the assay, and cholesterol decreased at the 7th and 14th days, compared to 12 h, possibly as a consequence of the high metabolic demand induced by stress. Cortisol varied from 21,841.9 pg/mL (12 h) to 2162.2 pg/mL (42 day). As for cortisol, the volume-weighted nuclear volume of interrenal cells was not significantly distinct between sampling days, but a minimum of 96.33 μm3 was reached at 28 days. Higher haemoglobin values were obtained at 28th and 42th days, in relation to 12 h and the 14th day. Triglycerides, potassium and haematocrit did not change. The quantitative skin analyses (fragments and histological sections) showed a reduction in the pigmented area, with stabilization at 14th day.Acclimation of brown trout juveniles should be monitored using a multi-parameter evaluation including behavioural, biochemical, nutritional and morphological analyses. Overall, it is recommended an acclimation period of at least 28 days.

High-content screen in human pluripotent cells identifies miRNA-regulated pathways controlling pluripotency and differentiation

BackgroundBy post-transcriptionally regulating multiple target transcripts, microRNAs (miRNAs or miR) play important biological functions. H1 embryonic stem cells (hESCs) and NTera-2 embryonal carcinoma cells (ECCs) are two of the most widely used human pluripotent model cell lines, sharing several characteristics, including the expression of miRNAs associated to the pluripotent state or with differentiation. However, how each of these miRNAs functionally impacts the biological properties of these cells has not been systematically evaluated.MethodsWe investigated the effects of 31 miRNAs on NTera-2 and H1 hESCs, by transfecting miRNA mimics. Following 3–4 days of culture, cells were stained for the pluripotency marker OCT4 and the G2 cell-cycle marker Cyclin B1, and nuclei and cytoplasm were co-stained with Hoechst and Cell Mask Blue, respectively. By using automated quantitative fluorescence microscopy (i.e., high-content screening (HCS)), we obtained several morphological and marker intensity measurements, in both cell compartments, allowing the generation of a multiparametric miR-induced phenotypic profile describing changes related to proliferation, cell cycle, pluripotency, and differentiation.ResultsDespite the overall similarities between both cell types, some miRNAs elicited cell-specific effects, while some related miRNAs induced contrasting effects in the same cell. By identifying transcripts predicted to be commonly targeted by miRNAs inducing similar effects (profiles grouped by hierarchical clustering), we were able to uncover potentially modulated signaling pathways and biological processes, likely mediating the effects of the microRNAs on the distinct groups identified. Specifically, we show that miR-363 contributes to pluripotency maintenance, at least in part, by targeting NOTCH1 and PSEN1 and inhibiting Notch-induced differentiation, a mechanism that could be implicated in naïve and primed pluripotent states.ConclusionsWe present the first multiparametric high-content microRNA functional screening in human pluripotent cells. Integration of this type of data with similar data obtained from siRNA screenings (using the same HCS assay) could provide a large-scale functional approach to identify and validate microRNA-mediated regulatory mechanisms controlling pluripotency and differentiation.

Long-Term Signs of T Cell and Myeloid Cell Activation After Intestinal Transplantation With Cellular Rejections Contributing to Further Increase of CD16+ Cell Subsets.

The intestine mediates a delicate balance between tolerogenic and inflammatory immune responses. The continuous pathogen encounter might also augment immune cell responses contributing to complications observed upon intestinal transplantation (ITx). We thus hypothesized that ITx patients show persistent signs of immune cell activation affecting both the adaptive and innate immune cell compartment. Information on the impact of intestinal grafts on immune cell composition, however, especially in the long-term is sparse. We here assessed activated and differentiated adaptive and innate immune subsets according to time, previous experience of cellular or antibody-mediated rejections or type of transplant after ITx applying multi-parametric flow cytometry, gene expression, serum cytokine and chemokine profiling. ITx patients showed an increase in CD16 expressing monocytes and myeloid dendritic cells (DCs) compared to healthy controls. This was even detectable in patients who were transplanted more than 10 years ago. Also, conventional CD4+ and CD8+ T cells showed persistent signs of activation counterbalanced by increased activated CCR4+ regulatory T cells. Patients with previous cellular rejections had even higher proportions of CD16+ monocytes and DCs, whereas transplanting higher donor mass with multi-visceral grafts was associated with increased T cell activation. The persistent inflammation and innate immune cell activation might contribute to unsatisfactory results after ITx.

Integrated Multiparametric High-Content Profiling of Endothelial Cells

Endothelial cells (ECs) are widely heterogeneous at the cell level and serve different functions at the vessel and tissue levels. EC-forming colonies derived from induced pluripotent stem cells (iPSC-ECFCs) alongside models such as primary human umbilical vein ECs (HUVECs) are slowly becoming available for research with future applications in cell therapies, disease modeling, and drug discovery. We and others previously described high-content analysis approaches capturing unbiased morphology-based measurements coupled with immunofluorescence and used these for multidimensional reduction and population analysis. Here, we report a tailored workflow to characterize ECs. We acquire images at high resolution with high-magnification water-immersion objectives with Hoechst, vascular endothelial cadherin (VEC), and activated NOTCH staining. We hypothesize that via these key markers alone we would be able to distinguish and assess different EC populations. We used cell population software analysis to phenotype HUVECs and iPSC-ECFCs in the absence or presence of vascular endothelial growth factor (VEGF). To our knowledge, this study presents the first parallel quantitative high-content multiparametric profiling of EC models. Importantly, it highlights a simple strategy to benchmark ECs in different conditions and develop new approaches for biological research and translational applications for regenerative medicine.

Abstract B31: Multiparameter profiling of abscopal effect (AbE) in a pediatric fibrosarcoma with EML4-NTRK3 fusion

Abstract Background: Abscopal effect (AbE) is a rare phenomenon in pediatric oncology, defined as regression of tumor at distal sites following radiation therapy. AbE is widely believed to result from the local activation and expansion of tumor-specific T cells in the area radiated, followed by the migration of these T cells to distant sites. We present a case of AbE in a 1-year-old with congenital fibrosarcoma (forearm) bearing a novel EML4-NTRK3 fusion who developed bilateral lung metastasis 6 months post resection of the initial forearm tumor. The patient’s lung lesions were unresponsive to chemotherapy prompting palliative, hypofractionated radiation therapy (RT) to the right lung lesion only. One month following RT, patient showed significant decrease in size of bilateral lung lesions, with no evidence of disease at 3 months post RT. Using whole-exome sequencing (WES), high-throughput sequencing of T cell receptors (TCRseq), and quantitative multiplex immunofluorescence (qmIF), we investigated the relationship between tumor progression and metastasis, the pre- and post-RT microenvironment, and the co-evolution of the tumor-infiltrating lymphocyte (TIL) populations in this patient. We believe this unusual case offers key insights into mechanisms underlying AbE. Methods: Formalin-fixed, paraffin-embedded (FFPE) tissue specimens from the initial diagnostic lesion (forearm), pretreatment lung tumor, and bilateral post-RT lung lesions (right (RT treated) and left (outside RT field) were obtained. An FFPE slide was stained for each specimen using traditional immunohistochemistry (IHC) for CD3 and CD8 and qmIF for 7 biomarkers including DAPI, CD3, CD4, CD8, CD68, HLA-DR, and Ki67. QmIF images were taken using MANTRA and cell density analysis was performed using inForm software and R. DNA was isolated, from the tissue specimens and peripheral blood, for profiling using WES and TCRseq, generating a multiplex snapshot of each site. The TIL populations were described by the complementarity determining region 3 (CDR3) of each TCR (as identified by Adaptive), followed by analysis of the bias, diversity, and divergence of these TIL populations using entropy-based statistics. Results: IHC and qmIF of the primary fibrosarcoma and pre-RT lung tumor showed very low immune cell infiltration. In contrast, immune infiltration was markedly increased (both CD4+ and CD8+) post-RT, particularly in the nonirradiated lung lesion (left). TIL populations were analyzed using TCRseq, revealing little similarity between those of the primary fibrosarcoma and the recurrent lung lesions. However, the TCR repertoires comparing the post-RT lung lesions, irradiated (right) and non-irradiated (left), showed substantial similarity, sharing a larger percentage of CDR3 sequences with each other (6.7% of combined total) than with the primary tumor (≤0.6% combined total) or the pre-RT lung tumor (≤1.6% combined total). Additionally, the extent of the TIL repertoire and its binding-associated divergence from the peripheral blood was greatest in the irradiated (right) lung tumor post RT, suggesting a direct link between the evolution of trans-acting immunity and antigen-driven immunogenicity at that site. Of note, comparative WES of the initial fibrosarcoma and subsequent lung tumors identified the same EML4-NTRK3 fusion, validating their metastatic relationship, although the number of coding mutations had expanded upon recurrence. Conclusion: Using qmIF and TCRseq, we have extensively characterized immune infiltration in the tumor microenvironment prior to and following metastasis and RT, in the first reported case of AbE in a pediatric tumor. Our data show evidence of immune stimulation elicited by RT, and antitumor T-cell specificities are shared inside and outside of the radiation field. These data offer insight into AbE and how combination of radiation and immunotherapy may be useful to induce response in pediatric tumors. Citation Format: Robyn D. Gartrell, Jennifer S. Sims, Sunjay M. Barton, Angela Kadenhe-Chiweshe, Filemon Dela Cruz, Andrew T. Turk, Andrew L. Kung, Jeffrey N. Bruce, Yvonne M. Saenger, Darrell J. Yamashiro, Eileen P. Connolly. Multiparameter profiling of abscopal effect (AbE) in a pediatric fibrosarcoma with EML4-NTRK3 fusion [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B31.

Towards prediction of acute and chronic drug effects using multi-parameter profiling of hiPSC-derived cardiomyocytes

Towards prediction of acute and chronic drug effects using multi-parameter profiling of hiPSC-derived cardiomyocytes

Multi-parametric liquid biopsy analysis of circulating tumor cells (CTCs), cell-free DNA (cfDNA), and cell-free RNA (cfRNA) in metastatic castrate resistant prostate cancer (mCRPC).

274 Background: Molecular profiling of prostate cancer using liquid biopsies such as CTC capture and cell-free nucleic acid analysis yield informative yet distinct datasets. Additional insights may be gained by simultaneously interrogating multiple liquid biopsy components to construct a more comprehensive molecular disease profile. We have conducted an initial proof of principle study aimed at piloting this multi-parametric approach. Methods: Blood was drawn under an IRB-approved protocol from 20 mCRPC patients. Samples were analyzed simultaneously for the following: CTC enumeration and single CTC and matched white blood cell capture for whole genome amplification and low-pass copy number variation; CTC DNA and matched cfDNA for somatic single nucleotide variant analysis; plasma cfRNA extraction and qRT-PCR for AR, AR-V7, and PCA3. When available, liquid biopsies were compared with matched tumor profiles. Results: Fifteen of 20 patients (75%) had detectable CTCs by CellSearch (range: 1-692/7.5mL, median: 16.5/7.5mL). Thirteen of 20 patients (65%) had detectable SSNVs in CTC DNA and/or matched cfDNA, including mutations in TP53, PIK3CA, HRAS, and EGFR. Matched CTC DNA and cfDNA demonstrated both shared and distinct SSNVs. Copy number analysis of single CTCs was performed in 2 patients, and both had CNVs in multiple cancer relevant genes. A majority of CNVs were present in matched solid tumor profiles, but some were exclusive to the liquid biopsies. Plasma PCA3 and AR transcripts were detected in 18/20 (90%) and AR-V7 in 4/20 (20%) cfRNA samples. Unique SSNVs were detected at second time points at disease progression (more are being collected). Conclusions: In this pilot cohort, simultaneous multi-parametric profiling was feasible for CTC DNA mutations and CNVs, and matched plasma cfDNA mutations and cfRNA gene expression. These disease-specific molecular profiles were often concordant with tumor tissues but also contained new, potentially actionable alterations unique to CTC DNA or cfDNA. Expanded studies will build upon this approach to optimally leverage liquid biopsies for molecularly directed patient management.

Multiparametric Profiling for Identification of Chemosensitizers against Gram-Negative Bacteria.

Antibiotic resistance is now a worldwide therapeutic problem. Since the beginning of anti-infectious treatment bacteria have rapidly shown an incredible ability to develop and transfer resistance mechanisms. In the last decades, the design variation of pioneer bioactive molecules has strongly improved their activity and the pharmaceutical companies partly won the race against the clock. Since the 1980s, the new classes of antibiotics that emerged were mainly directed to Gram-positive bacteria. Thus, we are now facing to multidrug-resistant Gram-negative bacteria, with no therapeutic options to deal with them. These bacteria are mainly resistant because of their double membrane that conjointly impairs antibiotic accumulation and extrudes these molecules when entered. The main challenge is to allow antibiotics to cross the impermeable envelope and reach their targets. One promising solution would be to associate, in a combination therapy, a usual antibiotic with a non-antibiotic chemosensitizer. Nevertheless, for effective drug discovery, there is a prominent lack of tools required to understand the rules of permeation and accumulation into Gram-negative bacteria. By the use of a multidrug-resistant enterobacteria, we introduce a high-content screening procedure for chemosensitizers discovery by quantitative assessment of drug accumulation, alteration of barriers, and deduction of their activity profile. We assembled and analyzed a control chemicals library to perform the proof of concept. The analysis was based on real-time monitoring of the efflux alteration and measure of the influx increase in the presence of studied compounds in an automatized bio-assay. Then, synergistic activity of compounds with an antibiotic was studied and kinetic data reduction was performed which led to the calculation of a score for each barrier to be altered.