DOI: 10.5281/zenodo.11238551
title: Evaluation of transcription factor knockout impact on paclitaxel response for Triple Negative Breast Cancer

description: Data and code related to Zenodo repository: 10.5281/zenodo.11238552

 

Two experimental formats included:

'fixed' prefix: data from terminal time point of siRNA screen applied to HCC1143, HCC1806, and MDA-MB-468 Triple Negative Breast Cancer cell lines.

'live' prefix: data from live-cell imaging of cell cycle reporter (HDHB-mClover/NLS-mCherry) HCC1143 Triple Negative Breast Cancer cell line.

Note: 'live' level 1 data is available upon request (heiserl@ohsu.edu, calistri@ohsu.edu).

 

Experimental goal:

Evaluate whether siRNA knockdown of transcription factors elevated during paclitaxel response impact cell count, cell morphology or cycling dynamics.

 

Methods:

siRNA Knockdown: Cells were plated in 90ul of serum free media per well of a 96 well plate. 24 hours later, siRNA knockdown mixture was prepared using a cell-line optimized concentration of Lipofectamine RNAiMAX (cat 13778075-075, Invitrogen) and siRNA (Horizon Discovery ON-TARGETplus) following RNAiMAX recommended protocol. The final concentration of siRNA per well was 1pmol and the final volume of RNAiMAX per well was 75nL for HCC1143, and 37.5nL for HCC1806 or MDA-MB-468 in 100uL of cell containing volume. 24 hours after siRNA transfection cells were treated with an addition of 100uL complete media containing either DMSO vehicle control or paclitaxel. 

 

Fixed-cell assays: Cells were plated at 3000 cells in 100ul of complete media per well in a 96 well plate (#08-772-225, FisherScientific). After 24 hours, an additional 100ul of either vehicle (0.1% DMSO) or paclitaxel containing complete media was added. After 72 hours cells were fixed with 4% Formaldehyde (#28908, ThermoFisher Scientific) for 15 minutes at room temperature, then permeabilized with 0.3% Triton X-100 (#X100-100ML, Sigma Aldrich) for 10 minutes at room temperature, then washed twice with PBS. Fixed cells were then stained with 0.5ug/mL DAPI (4083S, Cell Signaling Technology) in PBS for 15 minutes at room temperature. Following DAPI staining, wells were washed once with PBS, then stained with 1:20,000 HCS CellMask Green in PBS (H32714, Invitrogen) for 15 minutes at room temperature. Wells were washed twice with room temperature PBS and then 4 fields of view per well imaged on an InCell 6000 (GE Healthcare). Images were segmented with two custom Cellpose models to segment the nucleus (using parameters: diameter = 50, chan = DAPI, chan2 = Cellmask Orange) and cytoplasm (using parameters: diameter = 90, chan = Cellmask Orange, chan2 = DAPI). Image quantification was performed in R (v4.3.1) using EBImage (v4.42.0), and cells were annotated based on the number of distinct nuclei segmented within each cytoplasmic mask. 

 

HDHB reporter live-cell assays: siRNA knockdown and drug treatment was performed as described above, and then the plate was loaded on an Incucyte S3 (Sartorious) and cells imaged every 15 minutes for 72 hours post drug treatment. At each timepoint 4 fields of view were captured at 20x magnificantion in each well using the phase, red and green channels. A cytoplasmic mask was computed from the mean of normalized red/green channel (cellpose parameters: diameter = 57, chan = mean(normalized(red), normalized(green)), and a nuclear mask was computed from the red channel (cellpose parameters: diameter = 30, chan = DAPI) using custom trained Cellpose models. Image quantification was performed in R (v4.3.1) using EBImage (v4.42.0). An additional perinuclear ring mask was computed as the 11 pixel dilation from the nuclear mask, but still bound by the cytoplasmic mask. To determine mClover localization thresholds for cell cycle assignment, 250 cell images were randomly selected and manually assigned to the G1, S/G2 or M cell cycle state based on mClover localization. The mClover intensity ratios were then used to determine thresholds for automated cell cycle phase calling which was applied to the rest of the data set (Supplemental Figure 5A). Mononuclear cells with a Perinuclear:Nuclear mean intensity ratio greater than 0.8 and Nuclear:Cytoplasmic total intensity less than 0.5 were assigned to the S/G2 phase. Mononuclear and Multinuclear cells with a Nuclear:Cytoplasmic total intensity ratio greater than 0.8 and Perinuclear:Nuclear mean intensity ratio less than 0.8 were assigned to the ‘M’ phase. The remainder of mononuclear cells were assigned ‘G1’, and the remainder of multinucleated cells were assigned ‘Multinucleated’. 

 

Included files:

fixed_level_1-plate_#.zip : Six .zip archives containing the raw images (DAPI/CellMask/Brightfield) from fixed-cell experiments.

plate 1: HCC1143 cells treated with plate A schema

plate 2: HCC1143 cells treated with plate B schema

plate 3: HCC1806 cells treated with plate A schema

plate 4: HCC1806 cells treated with plate B schema

plate 5: MDA-MB-468 cells treated with plate A schema

plate 6: MDA-MB-468 cells treated with plate B schema

fixed_level_2: Data quantified from cellpose masks at the single-nuclei level (redundant cytoplasm information)

fixed_level_3: Data from 'fixed_level_2.csv' collapsed to the single cell level, including staining intensity and aggregate nuclear information

fixed_incell_to_cellpose.rmd: R markdown code for converting original incell files (fixed_level_1) to RGB images for cellpose segmentation

fixed_image_quantification.rmd: R markdown code for quantifying images using cellpose segmentation masks and original images (fixed_level_1)

fixed_cellpose_models.zip: Archive including cellpose models used for fixed experiment

live_level_2: Data quantified from cellpose masks at the single-nuclei level (redundant cytoplasm information)

live_level_3: Data from 'live_level_2.csv' collapsed to the single cell level, including staining intensity and aggregate nuclear information

live_level_4: Data from 'live_level_3.csv' collapsed to the single condition level summarizing the number, multinucleation status and phase of cells at each time point.

live_image_quantification.rmd: R markdown code for quantifying images using cellpose segmentation masks and original images (live_level_1).

l ive_incu_archive2rgb.rmd: R markdown code for converting incucyte archive formatted data into RGB images, where the blue channel is the arithmetic mean of the min-max (0-1) normalized red and green channels.

live_cellpose_models.zip: Archive including cellpose models used for live experiment.