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BioAssay: AID 2717

Luminescence Cell-Based Primary HTS to Identify 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(300626)
 
 
Active(3187)
 
 
Inactive(296924)
 
 
Inconclusive(517)
 
 
 Tested Substances
 Tested Substances
All(300718)
 
 
Active(3190)
 
 
Inactive(297011)
 
 
Inconclusive(517)
 
 
 Related BioAssays
 Related BioAssays
AID: 2717
Data Source: Broad Institute (2058-01_INHIBITORS_SINGLE-POINT_MLPCN-HTS)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-03-29
Modify Date: 2011-03-01

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 3187
Related Experiments
Show more
AIDNameTypeProbeComment
2721Summary of Broad Institute MLPCN Breast Cancer Stem Cell Toxicity ProjectSummary4 depositor-specified cross reference
449748Luminescence Cell-Based Dose Retest to Confirm Inhibitors of Cancer Stem CellsConfirmatory depositor-specified cross reference
463074Dose Response HTS Screen to Identify Cytotoxic Compounds of HMLE_sh_eGFPConfirmatory 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:
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, 100 nL 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 40 ml CSC complete + Serum and were incubated at 37 deg C, 5% CO2 for ~16 hours.
Cells were washed with 1x PBS and 5 ml Trypsin + EDTA was added to cells for 2-5 minutes.
Cells were re-suspended in 25 ml 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.
50 ul Cell supension was added to 50 ul 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/ 50 ul were dispensed into Tissue Culture treated 384-well plates (Corning 3850) using a Thermo Scientific Multidrop Combi.
Cells were incubated at 37 deg C, 5% CO2 for at least 4 hours.
Cells were pinned with 100 nL 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
HTS Data Analysis

Negative control wells (NC) and positive controls wells (PC) were included on every plate.
Active compounds result in decreased readout signal.

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 NC wells was set to a normalized activity value of 0.
The median raw signal of the intraplate PC wells was set to a normalized activity value of -100.
Experimental wells were scaled to this range, giving an activity score as percent change in signal relative to the intraplate controls.
For one run, the postive control did not work, so the -100 full inhibition value was set to (0.33)(median of NC), and the compound wells scaled accordingly.

No plate pattern correction algorithm was applied to the data.

The replicate activity scores were multiplied by -1 to convert Genedata negative percent inhibition values to Pubchem positive percent activity values.
The final PUBCHEM_ACTIVITY_SCORE was set as equal to the integer portion of the mean of the valid replicates (i.e., no rounding up).

The PUBCHEM_ACTIVITY_OUTCOME class was assigned as described below, based on an inhibition threshold of 75% inhibition:

Activity_Outcome = 1 (inactive)
<50% of the replicates fell outside the threshold AND the pubchem_activity_score did not fall outside the threshold

Activity_Outcome = 2 (active)
More than 50% of replicates fell outside the mean
or
50% of the replicates fell outside the threshold AND the pubchem_activity_score fell outside the threshold

Activity_Outcome = 3 (inconclusive)
50% of the replicates fell outside the threshold AND the pubchem_activity_score did not fall outside the threshold
or
<50% of the replicates fell outside the threshold AND the pubchem_activity_score fell outside the threshold
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: HMLE
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 InvalidFloat
2BROAD_SCREENING_RUNIDSThis is a comma separated list of unique IDs given to each screening run at the Broad Institute.String
3REPLICATE_A_ACTIVITY_SCORE (7.5μM**)The calculated activity score for the indicated sample, a NULL here denotes data was not used or was not producedFloat%
4REPLICATE_B_ACTIVITY_SCORE (7.5μM**)The calculated activity score for the indicated sample, a NULL here denotes data was not used or was not producedFloat%
5REPLICATE_C_ACTIVITY_SCORE (7.5μM**)The calculated activity score for the indicated sample, a NULL here denotes data was not used or was not producedFloat%
6REPLICATE_D_ACTIVITY_SCORE (7.5μM**)The calculated activity score for the indicated sample, a NULL here denotes data was not used or was not producedFloat%
7DATE_REPORTEDThe date the data was internally reportedString

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

Data Table (Concise)
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