Orthogonal assay for the inhibition of tumorsphere formation Measured in Cell-Based System Using Imaging - 2058-03_Inhibitor_SinglePoint_DryPowder_Activity_Set2
Assay Overview: The objective of these experiments is to identify chemical compounds that can selectively kill breast cancer stem cells (CSCs). Compounds identified in previous assays that selectively killed breast cancer stem cell-like cells were tested for inhibition of in vitro tumor models, tumorspheres or mammospheres. One thousand SUM159 cells are plated in 200 uL of media with 0.5% more ..
BioActive Compounds: 3
Keywords: Tumorsphere, mammosphere, tumor, breast cancer stem cells, SUM159
Assay Overview: The objective of these experiments is to identify chemical compounds that can selectively kill breast cancer stem cells (CSCs). Compounds identified in previous assays that selectively killed breast cancer stem cell-like cells were tested for inhibition of in vitro tumor models, tumorspheres or mammospheres. One thousand SUM159 cells are plated in 200 uL of media with 0.5% methylcellulose containing 200nl previously added compounds. Tumorspheres are allowed to form for 9 days and imaged in transmitted light using a 2x objective. Images are subsequently analyzed using MetaXpress (Molecular Devices) to quantify numbers of tumorspheres formed. Compounds found to inhibit tumorsphere formation would be interesting for new drug candidates for anti-cancer therapy. Compounds identified would also serve as useful probes to study CSC biology, which are currently lacking.
Expected Outcome: Compounds significantly suppressing tumorsphere formation.
F12/DMEM 465 ml
25 ml FBS (5% final)
5 ml Pen/Strep (1%)
5 ml Glutamax (1%)
600 ul Insulin
500 ul Gentamicin
250 ul Plasmocin750 Hydrocortisone
SUM159 (primary tumor cell line) were provided collaborators Dr. Piyush Gupta & Dr. Eric Lander, as described in Breast Cancer Research 2008, 10:R25. Cells are propagated in SUM159 media at 37 degrees C, 5% CO2.
On the day of the experiment, 96 well, Costar Ultra Low Cluster (3474) plates are filled with 100ul of SUM159 Media using a Thermo Scientific Multidrop Combi with a standard sized cassette. Compounds are pinned (200 nL, 2 dips of 100 nl) to media alone. Confluent cells grown in T225 flasks are split, collected, and spun at 1200rpm for 5 min. Trypsin is aspirated and cells are resuspended in media. Cells are counted and diluted into media with final concentration of 1% MethylCellulose (1:2.6 dilution) to 20,000 cells/ml.
100 ul of cells plus methylcellulose mixture is added into each well of the 96-well plates pre-aliquoted with compounds, using a Thermo Scientific Multidrop Combi with a standard sized cassette. The final concentration is 0.5% Methylcellulose and 1000 cells/well.
The plates are incubated at 37 degrees C, 5% CO2 for 8 days. Imaging was performed using a 2x objective using an ImageXpress Micro (Molecular Devices) to collect 9 sites/well. Cell clusters greater than 400 square uM were identified as tumorspheres, using MetaXpress software.
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The raw signals of the plate wells were 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).
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
This was set as equal to the mean of the normalized sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at -15.
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).
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 1.
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 2 (active) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
tSamples passing PAR_T only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)