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BioAssay: AID 493226

Luminescence 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_Set2

Assay Overview: The objective of the experiments in this proposal is to identify chemical compounds that can selectively kill breast cancer stem cells (CSCs). The HMLE cell line suppressing E-Cadherin expression (HMLE_shECad), which 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 more ..
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Active(31)
 
 
Inactive(24)
 
 
Inconclusive(8)
 
 
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 Tested Substances
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Active(33)
 
 
Inactive(24)
 
 
Inconclusive(8)
 
 
 Related BioAssays
 Related BioAssays
AID: 493226
Data Source: Broad Institute (2058-01_Inhibitor_Dose_DryPowder_Activity_Set2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-02-15
Hold-until Date: 2012-02-15
Modify Date: 2012-02-15

Data Table ( Complete ):           Active    All
BioActive Compounds: 31
Depositor Specified Assays
AIDNameTypeComment
2721Summary of Broad Institute MLPCN Breast Cancer Stem Cell Toxicity ProjectsummarySummary assay
Description:
Keywords: Breast Cancer Stem Cells

Assay Overview: The objective of the experiments in this proposal is to identify chemical compounds that can selectively kill breast cancer stem cells (CSCs). The HMLE cell line suppressing E-Cadherin expression (HMLE_shECad), which 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_shECAD 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.

Expected Outcome: Compounds significantly suppressing luminescence, and therefore toxic to HMLE_shECad will be identified as hits in the screen.
Protocol
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


Using HMLE_sh_ECad 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.

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
PRESENCE OF CONTROLS: Neutral control wells (NC; n=132) and positive control wells (PC; n=18) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:

ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (10)(Max_Concentration).

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
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.7.0.3) was applied.

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)

PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.

DATA AGGREGATION:
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:

1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.

2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.

3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.

Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
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
3pAC50_MEqual to -1*log10(AC50)String
4AC50_Qualifier_TEST1'>','=', or '<'String
5AC50_uM_TEST1The concentration at which activity reaches 50% of the maximumFloatμM
6pAC50_M_TEST1Equal to -1*log10(AC50)String
7Hill_Slope_TEST1The slope at AC50Float
8S0_(%)_TEST1The fitted activity value at zero concentrationFloat%
9Sinf_(%)_TEST1The fitted activity value at infinite concentrationFloat%
10Num_Points_TEST1The number of data points used to generate the plotInteger
11Max_Activity_(%)_TEST1The maximum activity value observed, based on mean of replicates per concentrationFloat%
12Max_Activity_Conc_uM_TEST1The concentration at which the maximum activity is observedFloatμM
13Max_Concentration_uM_TEST1Maximum valid test concentrationFloatμM
14Activity_at_0.0046uM_(%)_TEST1 (0.0046μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.0135uM_(%)_TEST1 (0.0135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_0.042uM_(%)_TEST1 (0.042μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_0.12uM_(%)_TEST1 (0.12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.38uM_(%)_TEST1 (0.38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_1.1uM_(%)_TEST1 (1.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_3.5uM_(%)_TEST1 (3.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_10uM_(%)_TEST1 (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_30uM_(%)_TEST1 (30μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_160uM_(%)_TEST1 (160μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24AC50_Qualifier_TEST2'>','=', or '<'String
25AC50_uM_TEST2The concentration at which activity reaches 50% of the maximumFloatμM
26pAC50_M_TEST2Equal to -1*log10(AC50)String
27Hill_Slope_TEST2The slope at AC50Float
28S0_(%)_TEST2The fitted activity value at zero concentrationFloat%
29Sinf_(%)_TEST2The fitted activity value at infinite concentrationFloat%
30Num_Points_TEST2The number of data points used to generate the plotInteger
31Max_Activity_(%)_TEST2The maximum activity value observed, based on mean of replicates per concentrationFloat%
32Max_Activity_Conc_uM_TEST2The concentration at which the maximum activity is observedFloatμM
33Max_Concentration_uM_TEST2Maximum valid test concentrationFloatμM
34Activity_at_0.0046uM_(%)_TEST2 (0.0046μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
35Activity_at_0.0135uM_(%)_TEST2 (0.0135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
36Activity_at_0.042uM_(%)_TEST2 (0.042μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
37Activity_at_0.12uM_(%)_TEST2 (0.12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
38Activity_at_0.38uM_(%)_TEST2 (0.38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
39Activity_at_1.1uM_(%)_TEST2 (1.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
40Activity_at_3.5uM_(%)_TEST2 (3.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
41Activity_at_10uM_(%)_TEST2 (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
42Activity_at_30uM_(%)_TEST2 (30μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
43Activity_at_160uM_(%)_TEST2 (160μM**)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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