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BioAssay: AID 463074

Dose Response HTS Screen to Identify Cytotoxic Compounds of HMLE_sh_eGFP

Assay Overview: The objective of the experiment is to isolate compounds that are toxic to the background control HMLE cell line. The HMLE cell line expressing shRNA against eGFP(HMLE_sh_eGFP) is used as a counterscreen line to identify grossly toxic compounds in HMLE_sh_ECad. Two thousand HMLE_sh_eGFP are plated in 50 uL of media in each well and cells are allowed to adhere overnight. The next more ..
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 Tested Compounds
 Tested Compounds
All(2242)
 
 
Active(2116)
 
 
Inactive(114)
 
 
Inconclusive(12)
 
 
 Tested Substances
 Tested Substances
All(2243)
 
 
Active(2117)
 
 
Inactive(114)
 
 
Inconclusive(12)
 
 
 Related BioAssays
 Related BioAssays
AID: 463074
Data Source: Broad Institute (2058-02_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK_TOXICITY)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-08-02
Modify Date: 2011-06-13

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 2116
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AIDNameTypeProbeComment
2721Summary of Broad Institute MLPCN Breast Cancer Stem Cell Toxicity ProjectSummary4 depositor-specified cross reference: Project Summary
2717Luminescence Cell-Based Primary HTS to Identify Inhibitors of Cancer Stem CellsScreening same project related to Summary assay
449748Luminescence Cell-Based Dose Retest to Confirm Inhibitors of Cancer Stem CellsConfirmatory same project related to Summary assay
493176Luminescence Cell-Based Primary HTS to Identify Inhibitors of Cancer Stem Cells Measured in Cell-Based System Using Plate Reader - 2058-01_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
493196HTS Screen to Identify Cytotoxic Compounds of HMLE_sh_eGFP Measured in Cell-Based System Using Plate Reader - 2058-02_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
493226Luminescence Cell-Based Primary HTS to Identify Inhibitors of Cancer Stem Cells Measured in Cell-Based System Using Plate Reader - 2058-01_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
504325HTS Screen to Identify Cytotoxic Compounds of HMLE_sh_eGFP Measured in Cell-Based System Using Plate Reader - 2058-02_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
504449Luminescence Cell-Based Primary HTS to Identify Inhibitors of Cancer Stem Cells Measured in Cell-Based System Using Plate Reader - 2058-01_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
504450HTS Screen to Identify Cytotoxic Compounds of HMLE_sh_eGFP Measured in Cell-Based System Using Plate Reader - 2058-02_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
504533HTS Screen to Identify Cytotoxic Compounds of HMLE_sh_eGFP Measured in Cell-Based System Using Plate Reader - 2058-02_Inhibitor_Dose_DryPowder_Activity_Set5Confirmatory same project related to Summary assay
504535Luminescence Cell-Based Primary HTS to Identify Inhibitors of Cancer Stem Cells Measured in Cell-Based System Using Plate Reader - 2058-01_Inhibitor_Dose_DryPowder_Activity_Set5Confirmatory same project related to Summary assay
504623Orthogonal assay for the inhibition of tumorsphere formation Measured in Cell-Based System Using Imaging - 2058-03_Inhibitor_SinglePoint_DryPowder_ActivityConfirmatory same project related to Summary assay
504666HTS Screen to Identify Cytotoxic Compounds of HMLE_sh_eGFP Measured in Cell-Based System Using Plate Reader - 2058-02_Inhibitor_Dose_DryPowder_Activity_Set6Confirmatory same project related to Summary assay
504667Luminescence Cell-Based Primary HTS to Identify Inhibitors of Cancer Stem Cells Measured in Cell-Based System Using Plate Reader - 2058-01_Inhibitor_Dose_DryPowder_Activity_Set6Confirmatory same project related to Summary assay
504788Luminescence Cell-Based Primary HTS to Identify Inhibitors of Cancer Stem Cells Measured in Cell-Based System Using Plate Reader - 2058-01_Inhibitor_Dose_DryPowder_Activity_Set7Confirmatory same project related to Summary assay
504789HTS Screen to Identify Cytotoxic Compounds of HMLE_sh_eGFP Measured in Cell-Based System Using Plate Reader - 2058-02_Inhibitor_Dose_DryPowder_Activity_Set7Confirmatory same project related to Summary assay
504859Orthogonal assay for the inhibition of tumorsphere formation Measured in Cell-Based System Using Imaging - 2058-03_Inhibitor_SinglePoint_DryPowder_Activity_Set2Other same project related to Summary assay
588702Luminescence-based toxicity of Human Mammary Epithelial (HMLE) cells_2058-08_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
588703Luminescence-based toxicity of HMLE_shTWIST Breast Cancer Stem Cell-like cells_2058-07_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
588704Luminescence-based toxicity of HMLE_shECAD Breast Cancer Stem Cell-like cells_2058-06_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
588705Luminescence-based toxicity of MDA231 Breast Cancer cells_2058-09_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
588840uminescence-based toxicity of MDA231 Breast Cancer cells Measured in Cell-Based System Using Plate Reader - 2058-09_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
588844Luminescence-based toxicity of HMLE_shECAD Breast Cancer Stem Cell-like cells Measured in Cell-Based System Using Plate Reader - 2058-06_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
588845Luminescence-based toxicity of HMLE_shTWIST Breast Cancer Stem Cell-like cells Measured in Cell-Based System Using Plate Reader - 2058-07_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
588846Luminescence-based toxicity of Human Mammary Epithelial (HMLE) cells Measured in Cell-Based System Using Plate Reader - 2058-08_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
Description:
Assay Overview: The objective of the experiment is to isolate compounds that are toxic to the background control HMLE cell line. The HMLE cell line expressing shRNA against eGFP(HMLE_sh_eGFP) is used as a counterscreen line to identify grossly toxic compounds in HMLE_sh_ECad. Two thousand HMLE_sh_eGFP are plated in 50 uL of media in each well and cells are allowed to adhere overnight. The next day, 100 nl compound are added to the wells and are incubated ~72 hours. Cell viability was measured using CellTiter-Glo and luminescence is measured by the EnVision. The chemical compounds that selectively kill CSCs would be promising new drug candidates for anti-cancer therapy and would also serve as useful probes to study CSC biology, which are currently lacking.

Keywords: Breast Cancer Stem Cells, HMLE, toxicity

Expected Outcome: Compounds significantly suppressing luminescence, and therefore toxic to HMLE_sh_eGFP will be identified as hits in the screen.
Protocol
CSC media complete media + serum= Propagation media
Using already filtered/sterile components, add:
440 ml DMEM (Cellgro 10-013-CM)
50 ml FBS (HyClone SH30071.03)
5 ml Pen/Strep
5 ml Glutamax-1 (Invitrogen 35050-061)
700ul 50 uM Hydrocortisone (Sigma H039K8402)
600 ul 10mg/ml Insulin (Sigma I9278)
500 ul 50 mg/ml Gentamicin (Sigma G1397)
250 ul 25 mg/ml Plasmocin (Invivogen ant-mpt)
50 ul 100 ug/ml EGF (Sigma E9644) resuspended in 2% serum/PBS
+
500 ml MEGM Complete Medium (Lonza CC-3051) or MEGM Bullet Kit with components CC-3150
1 ml BPE (Lonza CC-4009)
Makes 1 liter
CSC media complete media - serum= Screening media
Using already filtered/sterile components, add:
490 ml DMEM (Cellgro 10-013-CM)
5 ml Pen/Strep
5 ml Glutamax-1 (Invitrogen 35050-061)
700ul 50 uM Hydrocortisone (Sigma H039K8402)
600 ul 10mg/ml Insulin (Sigma I9278)
500 ul 50 mg/ml Gentamicin (Sigma G1397)
250 ul 25 mg/ml Plasmocin (Invivogen ant-mpt)
50 ul 100 u/ml EGF (Sigma E9644) resuspended in 2% serum/PBS
+
500 ml MEGM Complete Medium (Lonza CC-3051) or MEGM Bullet Kit with components CC-3150
1 ml BPE (Lonza CC-4009)
Makes 1 liter
Using HMLE_sh_eGFP provided by collaborators Dr. Piyush Gupta & Dr. Eric Lander (as described in Gupta PB, Onder TT, Jiang G, Tao K, Kuperwasser C, Weinberg RA, Lander ES.Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell. 2009 Aug 21;138(4):645-59. Epub 2009 Aug 13. PMID: 19682730), 10 million cells/vial were frozen down in CSC media complete media +Serum+10% DMSO and stored in liquid nitrogen.
Cell propagation
2-3 days prior to screening, vials were thawed and plated into T225 Falcon flasks with 40 ml CSC complete + Serum and are incubated at 37 deg C, 5% CO2 for ~16 hours.
Cells were washed with 1x PBS and 5 ml Trypsin + EDTA are added to cells for 2-5 minutes.
Cells are re-suspended in 25 ml CSC complete + serum, spun at 1400 rpm for 4 minutes.
Media is aspirated and cells are resupended in CSC complete + Serum and are plated in T225 flasks (as above) at 1:4 or 1:8 density and allowed to grow 1-2 days.
Cell Screening
Cells are trypsinized and spun as above.
Cell pellet is resuspended in CSC complete -serum.
50 ul Cell supension is added to 50 ul trypan blue and counted using Nexecelom Cellometer.
Cells are diluted to 40,000 cells/ml in CSC complete - serum.
Using standard sized cassette, 2000 cells/ 50 ul are dispensed into Tissue Culture treated 384-well plates (Corning 3850) using Thermo Scientific Multidrop Combi.
Cells are incubated at 37 deg C, 5% CO2 for at least 4 hours.
Cells are pinned with 100 nL compounds and incubated for ~70-74 hours. Neutral (DMSO only) and positive controls (160 uM Puromycin) were included on every plate.
Cell Titer Glo is prepared as Promega describes and diluted 1:3 with 1x PBS.
Assay plates are incubated at room temperature for 30 minutes.
30 ul CellTiter Glo dilution is dispensed using standard sized cassette by Thermo Scientific Multidrop Combi.
Plates are incubated 12 minutes and read using the PerkinElmer EnVision (Luminescence 0.1 sec/well).
Comment
Dose Data Analysis
Neutral control (NC) wells and inhibitor positive control (PC) wells were included on every plate.
Active inhibitor compounds result in decreased signal.
The raw luminescent signal was normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Therefore a negative activity value is expected when signal decreases relative to NC.
No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
AC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). AC50 values were extrapolated up to 1 log
over the highest tested concentration.
PubChem Activity Score and Outcome
PUBCHEM_ACTIVITY_SCORE:
Inactive compounds = 0
Active compounds = -10*Log(AC50)
Superactive compounds (full inhibition at all concentrations; constant curve fit) = 100
PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive)
AC50 > 1 log over the highest tested concentration
Activity_Outcome = 2 (active)
AC50 <= 1 log over the highest tested concentration
Activity_Outcome = 3 (inconclusive)
Curve fitting strategy resulted in a constant fit with activity >30% but <70%
or
The fit was not valid due to poor fit quality.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>', '=', or '<'String
2AC50_uM*the concentration at which activity reaches 50% of the maximumFloatμM
3Log_AC50_Mthe log of the EC50 (molar)Float
4Log_AC50_M_Standard_Errorthe standard error for the log of the EC50 valueFloat
5Hill_Slopethe slope at EC50Float
6S0the fitted activity level at zero concentrationFloat%
7SInfthe fitted activity level at infinite concentrationFloat%
8Num_Pointsthe number of data points included in the plotInteger
9Max_Activitythe maximum activity value observedFloat%
10Activity_at_0.16uM (0.16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity_at_0.3uM (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.6uM (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_1.2uM (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_2.6uM (2.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_5uM (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_10uM (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_19.5uM (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R03MH089663-01

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