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BioAssay: AID 449748

Luminescence Cell-Based Dose Retest to Confirm Inhibitors of Cancer Stem Cells

Assay Overview: The objective of the experiments in this proposal is to identify compounds that can selectively kill breast cancer stem cells (CSCs). The HMLE cell line suppressing E-Cadherin expression (HMLE_sh_ECad) is a stable cell line that has been induced into epithelial-to-mesenchymal transdifferentiation (EMT). This cell line was used to represent a uniform population of CSCs. Two more ..
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 Tested Compounds
 Tested Compounds
All(2242)
 
 
Active(2180)
 
 
Inactive(53)
 
 
Inconclusive(9)
 
 
 Tested Substances
 Tested Substances
All(2243)
 
 
Active(2181)
 
 
Inactive(53)
 
 
Inconclusive(9)
 
 
 Related BioAssays
 Related BioAssays
AID: 449748
Data Source: Broad Institute (2058-01_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-07-13
Modify Date: 2011-03-01

Data Table ( Complete ):           Active    All
BioActive Compounds: 2180
Depositor Specified Assays
AIDNameTypeComment
2717Luminescence Cell-Based Primary HTS to Identify Inhibitors of Cancer Stem CellsscreeningPrimary HTS
2721Summary of Broad Institute MLPCN Breast Cancer Stem Cell Toxicity ProjectsummarySummary
Description:
Keywords: Breast Cancer Stem Cells

Assay Overview: The objective of the experiments in this proposal is to identify compounds that can selectively kill breast cancer stem cells (CSCs). The HMLE cell line suppressing E-Cadherin expression (HMLE_sh_ECad) is a stable cell line that has been induced into epithelial-to-mesenchymal transdifferentiation (EMT). This cell line was used to represent a uniform population of CSCs. Two thousand HMLE_sh_ECAD were plated in 50 uL of media in each well and cells were allowed to adhere overnight. The next day, 100nL compound were added to the wells and incubated ~72 hours. Cell viability was measured using CellTiter-Glo and luminescence was quantitated using an EnVision reader.

Expected Outcome: Compounds significantly suppressing luminescence, and therefore toxic to HMLE_sh_ECad, will be identified as active 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 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


HMLE_sh_ECad was 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). Ten 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 40ml CSC complete + Serum and were incubated at 37 degrees C, 5% CO2 for ~16 hours.
Cells were washed with 1x PBS and 5ml Trypsin + EDTA was added to cells for 2-5 minutes.
Cells were re-suspended in 25ml CSC complete + serum and spun at 1400 rpm for 4 minutes.
Media was aspirated. Cells were resupended in CSC complete + Serum, plated in T225 flasks (as above) at 1:4 or 1:8 density and allowed to grow 1-2 days.

Cell Screening
Cells were trypsinized and spun as above.
Cell pellet was resuspended in CSC complete -serum.
50ul Cell supension was added to 50ul trypan blue and counted using Nexecelom Cellometer.
Cells were diluted to 40,000 cells/ml in CSC complete - serum.

Using standard sized cassette, 2000 cells/50ul were dispensed into Tissue Culture treated 384-well plates (Corning 3850) using a Thermo Scientific Multidrop Combi.
Cells were incubated at 37 degrees C, 5% CO2 for at least 4 hours.
Cells were pinned with 100nL compounds and incubated for ~70-74 hours.

Cell Titer Glo was prepared as Promega describes and diluted 1:3 with 1x PBS.
Assay plates were incubated at room temperature for 30 minutes.
30 ul CellTiter Glo dilution was dispensed using a standard sized cassette by Thermo Scientific Multidrop Combi.
Plates were 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.
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)
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