Protocol for A-549 VIM RFP (ATCC CCL-185EMT) TGFβ1 EMT Induction and Drug Screening Introduction: Vimentin (VIM) intermediate filament (IF) proteins are associated with EMT in lung cancer and its metastatic spread. When epithelial cells transition to the mesenchymal phenotype, VIM expression is generally increased. ATCC CCL-185EMT is a VIM red fluorescent protein (RFP) reporter cell line derived from the A549 non-small cell lung cancer (NSCLC) cell line via the creation of a C-terminal RFP fusion to the VIM gene using CRISPR/Cas9 gene editing. The expression of the fusion VIM RFP gene enables the end-point and real-time tracking of the EMT status of cells as they transition from epithelial to mesenchymal phenotype under defined conditions. The A549 Vim RFP reporter cell line serves as a suitable and sensitive model for basic science research on the mechanisms of metastasis. Furthermore, it is also uniquely designed to be used in high throughput screening (HTS) applications including the identification of new anti-emt drugs for metastatic NSCLC. The protocol below depicts how to set up such an in vitro 2D TGF-β1 EMT induction or drug testing experiment. Materials: Material Company Cat No. A549 VIM RFP ATCC ATCC CCL-185EMT DMEM: F-12 Medium ATCC ATCC 30-2006 Fetal Bovine Serum (FBS) ATCC ATCC 30-2020 Trypsin-EDTA Solution (1X) ATCC ATCC 30-2101 TGFβ1 R & D Systems 240-B-002 PP1 VWR 10191-714 A83-01 VWR 10188-672 Blasticidin S HCI Thermo Fisher A1113903 Black 96-well plates Thermo Fisher 165305 Dimethylsulfoxide (DMSO) ATCC ATCC 4-X - 1 -
Protocol: A. TGβ1 EMT Induction 1. Warm culture media to 37 C. 2. Harvest epithelial cells of interest using a dissociation solution (Trypsin-EDTA Solution (1X) ATCC 30-2101). 3. Resuspend the cells in warmed culture media. Centrifuge the cell suspension at approximately 250 x g for 5 minutes. Aspirate the liquid. 4. Gently resuspend the cell pellet in warmed culture media and count viable cells using Trypan blue or equivalent. 5. Day 0: i. Seed cells @ 10000 cells/well (31,579 cells/cm 2 ) in a black 96-Well Optical-Bottom Plates (or equivalent) in the presence of 160 µl of complete growth media supplemented with 2.5 ng/ml TGF-β1 (+ EMT) or and equivalent volume of 1X DPBS (- EMT). Run at least three experimental replicates for each condition being tested. ii. Incubate plate at 37 C with 5% CO2 for 5 days. During this incubation time, live cell imaging of cells in the red channel (λ = 555 nm) on a fluorescence microscope or a high content image analysis system (e.g. CellInsight CX7 High Content Analysis (HCA) System) could be used to track the EMT status of cells. 6. Day 5: i. Aspirate media and wash cells with 160 µl of 1x DPBS. ii. Fix cells by adding 30 µl 4% PFA/1X DPBS solution per well for 10 min at room temperature. iii. Wash with 160 µl 1X DPBS for 3 min at RT. Add another 160 µl of 1X DPBS to washed cells. Cells can subsequently be submitted to immunocytochemistry to check for additional EMT marker expression or stored in 1X DPBS at 4 ᵒC for up to about a week for analysis. 7. After Day 6: Analyze fold red fluorescence intensity change on day 5 for +/- EMT induced samples using a high content image analysis system (e.g. CellInsight CX7 High Content Analysis (HCA) System) and export images (Figure 1). Please note that the fold change in red fluorescence should be analyzed between the 1X DPBS supplemented samples and the 2.5 ng/ml TGF-β1 supplemented wells). - 2 -
B. General Protocol for Testing Anti-EMT Drugs 1. Warm culture media to 37 C. 2. Harvest epithelial cells of interest using a dissociation solution (Trypsin-EDTA Solution (1X) ATCC 30-2101). 3. Resuspend the cells in warmed culture media. Centrifuge the cell suspension at approximately 250 x g for 5 minutes. Aspirate the liquid. 4. Gently resuspend the cell pellet in warmed culture media and count viable cells using Trypan blue or equivalent. 5. Day 0: i. Seed cells @ 10000 cells/well (31,579 cells/cm 2 ) in a black 96-Well Optical-Bottom Plates in the presence of 160 µl of complete growth media supplemented with 2.5 ng/ml TGF-β1 (+ EMT) or and equivalent volume of 1X DPBS (- EMT) and increasing concentrations of anti-emt drug (e.g. see plate map for A83-01 & PP1 set up in Figure 2). Run at least three experimental replicates for each condition being tested. ii. Incubate plate at 37 C with 5% CO2 for 5 days. During this incubation time, live cell imaging of cells in the red channel (λ = 555 nm) on a fluorescence microscope or a high content image analysis system (e.g. CellInsight CX7 High Content Analysis (HCA) System) could be used to track the EMT status of cells. 6. Day 5: i. Aspirate media and wash cells with 160 µl of 1x DPBS. ii. Fix cells by adding 30 µl 4% PFA/1X DPBS solution per well and incubate at room temperature for 10 min. iii. Wash with 160 µl 1X DPBS for 3 min at RT. Add another 160 µl of 1X DPBS to washed cells. Cells can subsequently be submitted to immunocytochemistry to check for additional EMT markers or stored in 1X DPBS at 4 ᵒC for up to about a week for analysis. 7. After Day 6: Analyze fold red fluorescence intensity change for all drug dose conditions tested on day 5 for +/- EMT induced samples using a high content image analysis system (e.g. CellInsight CX7 High Content Analysis (HCA) System) and export images. Plot dose response curves using analyzed data as exemplified in figures 3 for A83-01 and PP1 respectively. - 3 -
Vimentin-RFP, Nuclei Vimentin-RFP, Nuclei Figure 1. A-549 VIM RFP shows increased mesenchymal marker protein expression in addition to decreased epithelial marker protein expression after EMT. A-549 VIM RFP cells were incubated in complete growth media supplemented with either 2.5 ng/ml TGF-β1 (right column) or an equivalent volume of 1X Dulbecco s phosphate buffered saline (as a no EMT control; left column) for 5 days. Treatment of A-549 Vim RFP with TGF-β1 induced EMT and resulted in increased vimentin-rfp expression (red; top left and right). The nuclei of cells were counterstained with NucBlue fixed cell ReadyProbes reagent (blue). Figure 2. Plate map for A83-01 and PP1 drug testing setup. Highlighted wells depict the following, Purple: Wells supplemented with 2.5 ng/ml TGF-β1 (+ EMT); Red: Wells supplemented with 1X DPBS (- EMT cells); Green: Cells to be analyzed to avoid edge effects; Yellow: Cells to be excluded from analysis to avoid edge effects. - 4 -
A. B. Figure 3. Small molecule EMT inhibitors block transition in A-549 Vim RFP cells. Two pathways associated with EMT were targeted: TGFβ and SRC using A83-01 (A) and PP1 (B), respectively. In both cases, TGF-β1-induced EMT was inhibited by the compound and gave. The calculated Z factor values for plates depict values between 0.5 and 1, indicating that the assay is suitable for HTS. Error bars indicate the standard deviation over 3 wells. - 5 -