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BioAssay: AID 686992

Identification of agents that induce E-selectin on human endothelial cells Measured in Cell-Based System Using Imaging - 2152-01_Activator_SinglePoint_HTS_Activity

Squamous cell carcinomas of the skin (SCC) are a leading cause of death in organ transplant recipients and treatment of non-melanoma skin cancers, of which SCC is the second most frequent type, account for 4.5% of all Medicare cancer costs. Immune evasion in human SCC primarily results from aberrant T cell homing. Vessels in SCC lack expression of the critical cutaneous T cell homing E-selectin more ..
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 Tested Compounds
 Tested Compounds
All(257982)
 
 
Active(963)
 
 
Inactive(255944)
 
 
Inconclusive(1107)
 
 
 Tested Substances
 Tested Substances
All(260364)
 
 
Active(963)
 
 
Inactive(258294)
 
 
Inconclusive(1107)
 
 
AID: 686992
Data Source: Broad Institute (2152-01_Activator_SinglePoint_HTS_Activity)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-05-21
Modify Date: 2013-10-21

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 963
Related Experiments
AIDNameTypeComment
687019Broad Institute Identification of agents that induce E-selectin on human endothelial cells Activator Probe ProjectSummarydepositor-specified cross reference
977605Identification of agents that induce E-selectin on human endothelial cells Measured in Cell-Based System Using Imaging - 2152-01_Activator_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
1053191On Hold
1053192On Hold
1053193On Hold
1117270On Hold
Description:
Keywords:
E-Selectin, HUVEC, Squamous Cell Carcinoma, T Cell, Addressin, Non-Melanoma Skin Cancer


Assay Overview:

Squamous cell carcinomas of the skin (SCC) are a leading cause of death in organ transplant recipients and treatment of non-melanoma skin cancers, of which SCC is the second most frequent type, account for 4.5% of all Medicare cancer costs. Immune evasion in human SCC primarily results from aberrant T cell homing. Vessels in SCC lack expression of the critical cutaneous T cell homing E-selectin and exclude the population of antigen-experienced skin homing T cells most capable of recognizing the tumor. There is therefore a need to identify novel agents that can induce endothelial activation and restore appropriate T cell homing without broad, nonspecific activation of the immune system. This project plan describes a high-throughput screening assay to identify small molecules that induce E-selectin expression on human endothelial cells. 3,000 human umbilical epithelial vein cells (HUVEC) are added to gelatin-coated imaging plates. Compounds or TNF-alpha (positive control) are added 24 hours before cells are stained for E-selectin and nuclear dyes. Cells are imaged with a 4x microIX system and the data is analyzed using MetaX software to identify the number of E-selectin positive cells. Compounds increasing the percent of E-selectin positive cells beyond 3 times the standard deviation of the neutral controls and are not greater than 20% toxic to the cells will be considered 'actives'.

Expected Outcome: Compounds that singnificantly cause an increase in E-Selectin expression and therefore have an increased number of E-selectin positive cells (gain of signal) will be considered active compounds. Compounds causing greater than 20% decrease in cell number will be excluded from the study.
Protocol
. Coat plates with 0.1% Gelatin in PBS (20ul per well) for 20 minutes at room temp.
Day 0
. Plate 3,000 HUVEC cells/well in 384 well plates, 50l per well, in EGM-2 medium, incubate at 37 degrees Celsius
Day 2
. Pin 100nl of compound, DMSO
. Add 10 ul Positive Control (TNFalpha at 0.3ng/ml)
. Incubate 16h
Day 3
. Aspirate media using plate washer
. Add 70 ul wash buffer using Thermo Multidrop Combi or plate washer (assay provider uses combi)
. Aspirate wash buffer with plate washer
. Add 20 ul blocking antibody (IgG) with combi
. Incubate at room temp for 15 minutes
. Aspirate
. Add 20 ul Biotin conjugated anti-e-selectin antibody with combi
. Incubate 30 minutes at room temp (or 15 minutes at 37 degrees Celsius)
. Aspirate
. Add 70 ul wash buffer using combi or plate washer (assay provider uses combi)
. Aspirate
. Add 20 ul streptavidin-PE using combi or plate washer (assay provider uses combi)
. Incubate 30 minutes at room temp (or 15 minutes at 37 degrees Celsius)
. Aspirate
. Add 70 ul of PBS (not wash buffer)
. Aspirate
. Add 20ul Hoechst/4% PF/PBS mix (fix cells and stain nuclei)
. Incubate 15 minutes at room temp
. Aspirate (PF is a hazardous waste and is collected in a seperate hazardous waste container)
. Add 70ul PBS
. Aspirate
. Add 70 ul PBS
Plates are sealed with foil seal and stored overnight at 4 degrees Celsius prior to imaging
Plates are imaged on an automated microscope (ImageXpress Micro, Molecular Devices Inc.) at 2x magnification using appropriate filter sets for Hoechst and PE fluorescence.
Cell counts and % of cells positive for PE fluorescence are quantified by using a modified Cell Scoring application module in the image analysis software (MetaXpress, Molecular Devices Inc.)
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in increasing readout signal.
NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of 100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.
PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized sample replicate activities, rounded to the nearest integer .
Compounds decreasing cell viability by more than 20 per cent were eliminated. The minimum PUBCHEM_ACTIVITY_SCORE of E-selectin positive cells required for a compound to be called a hit (the activity threshold, or AT) was set at 10.
PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 60.
PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T
Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T
Samples passing AT only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
Samples passing PAR_T only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: HUV-EC-C
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the 'replicate vector' (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was Invalid# Float
2PCT_ACTIVE_REPLICATESThe percentage of replicates which pass the activity threshold.Float
3REPLICATE_A_ACTIVITY_SCORE_7.58uM_(%) (7.58μM**)The calculated activity for the indicated sampleFloat%
4REPLICATE_B_ACTIVITY_SCORE_7.58uM_(%) (7.58μM**)The calculated activity for the indicated sampleFloat%

** Test Concentration.
Additional Information
Grant Number: 1R03MH095529-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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