Targeted therapy is effective in many tumor types including lung cancer, the leading cause of cancer mortality. Paradigm defining examples are targeted therapies directed against non-small cell lung cancer (NSCLC) subtypes with oncogenic alterations in EGFR, ALK and KRAS. The success of targeted therapy is limited by drug-tolerant tumor cells which withstand and adapt to treatment and comprise the residual disease state that is typical during treatment with clinical targeted therapies. Here, we integrate studies in patient-derived and immunocompetent lung cancer models and clinical specimens obtained from patients on targeted therapy to uncover a focal adhesion kinase (FAK)-YAP signaling axis that promotes residual disease during oncogenic EGFR-, ALK-, and KRAS-targeted therapies. FAK-YAP signaling inhibition combined with the primary targeted therapy suppressed residual drug-tolerant cells and enhanced tumor responses. This study unveils a FAK-YAP signaling module that promotes residual disease in lung cancer and mechanism-based therapeutic strategies to improve tumor response.

A focal adhesion kinase-YAP signaling axis drives drug tolerant persister cells and residual disease in lung cancer

Cells were seeded in 35mm glass-bottom dishes (MatTek Corporation) or µClear 96-well imaging plates (Greiner Bio-One). At the time of harvest, cells were washed carefully and fixed in 4% paraformaldehyde. Cells were then permeabilized in 0.1% Triton-X/PBS and blocked in 5% bovine serum albumin (BSA) / 0.1% Triton-X/PBS. Primary antibody was diluted in 1% BSA / 0.1% Triton-X/PBS and incubated overnight at 4 °C. After washing, secondary antibodies were diluted in 1% BSA / 0.1% Triton-X/PBS and added for 1 hour at room temperature. Where indicated, actin filaments were stained with rhodamine-phalloidin (Thermo Fisher Scientific) as described by the manufacturer. After secondary antibody staining, cells were washed and then stained with DAPI solution (1:1000 in PBS, stock 1 mg/mL, Thermo Fisher Scientific). For endogenously tagged YAP-mNeonGreen2 cells, permeabilization, blocking as well as primary and secondary antibody staining were omitted. Cells were imaged at a Yokogawa CSU22 spinning disk confocal microscope using a Plan Apo VC 60X/ 1.4 Oil objective (Nikon Imaging Center, UCSF). Image analysis was done via Fiji ImageJ software58. Quantification of relative integrated density for nuclear levels was performed by automated analysis quantifying the intensity for the protein of interest per nuclei.

Confocal analysis

Whole-cell lysates were prepared by using radio-immunoprecipitation assay buffer (RIPA) [10 mM Tris·Cl (pH 8.0), 1 mM EDTA, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl] supplemented with protease inhibitor and phosphatase inhibitor (Roche). Nuclear-cytoplasmic extracts were prepared using 0.1% NP-40 in PBS supplemented with protease inhibitor and phosphatase inhibitor (Roche) as previously described56. Whole-cell and nuclear lysates were clarified by centrifugation at 17,000 x g for 15 minutes at 4 °C. Lysates were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal masses of protein (5 - 20 ug) were separated by 4–15% of SDS/ PAGE and were transferred onto nitrocellulose membranes (Bio-Rad) for protein blot analysis. After blocking in 5 % milk/ Tris-buffered saline, 0.1% Tween-20 (TBS-T), membranes were incubated with primary antibody overnight at 4 °C, then washed and incubated with secondary antibody for 1 hour at room temperature. Protein bands were visualized using either a fluorescence system (LI-COR) or Amersham ECL chemiluminescent reagent (GE Life Sciences); chemiluminescent signals were visualized with an ImageQuant LAS 4000 instrument (GE Healthcare).

Western blot analysis

Apoptotic cell death was detected by flow cytometry using Annexin V and 7-amino-actinomycin (7-AAD) staining. Cells were harvested and resuspended in Annexin V-binding buffer containing 10% Annexin V-FITC and 10% 7-AAD staining solution (Thermo Fisher Scientific). After an incubation time of 15 min at 4°C, stained cells were analyzed by flow cytometry.

Apoptosis analysis

Cell lines were seeded in 96-well assay clear-bottom microplates at a density of 2,500-5,000 cells per well in a total volume of 90 μL per well and incubated at 37 °C, 5% CO2 overnight. Following drug exposure, cell confluency was measured by staining with Hoechst 33342 (Thermo Fisher Scientific) nuclear dye; apoptosis was measured using YO-PRO-1 early apoptosis dye (Thermo Fisher Scientific) and analyzed using a CellInsight High Content Microscope (Thermo Fisher Scientific) at the indicated time points.

High-content microscopy screening

Pilot experiment to determine general translation effects and optimal conditions for electroporation of TVA cell line.

- Renilla luciferase shows cap dependent translation 
- Firefly luciferase shows cap independent translation 
- Used Promega Dual-Luciferase Reporter Assay System and FB12 Luminometer from Berthold

 Protocol 

1. Warm 5 ml media in 6 well plate 

2. Resuspend cells in 400 ul serum free media. Use 3 million cells per condition. 

3. Add 1 ug pRSTF-CVB3 plasmid per 1 million cells. 

4. Electroporate cells in 0.4 cm cuvette electroporator 

5. Slowly add the 400 ul of electroporated cells to the warm 5 ml of media. 

6. Incubate the cells in media for 2 hours to let them recover. 

7. Aliquot 1ml of cell suspension per well and add the drug treatment. 

8. Incubate for 16 hours. 

9. Spin down cells (500 G, 5 min).  

10. Wash with PBS and spin down again. 

11. Resuspend in 25 ul passive lysis buffer (samples at this point can be stored in the -80 for a long period of time). 

12. Lyse by freezing cells in -80 and thawing, 5 minutes each. Do 2 freeze/thaw cycles. 

13. Combine 10 ul lysate and 50 ul LAR2 substrate and measure in luminometer to read Firefly luciferase (cap independent translation). 

14. Immediately add 50 ul Stop and Glow solution and measure in luminometer to read Renilla luciferase (cap dependent translation). 

15. Compare ratio of Renilla to Firefly luciferase for readouts. 

 

TVA Electroporation Optimization  

8 conditions, 3 million cells each (24 million total) 

Each condition is with or without pRSTF-CVB3 in separate 0.4 cm cuvettes 

250 uF 250 V 
250 uF 300 V 
975 uF 250 V 
975 uF 300 V 

Results 

All luciferase values too low, likely no transformation 

TVA Cell Line Electroporation Optimization

DNA stains enable a commonly used FACS method of monitoring a compound’s effects on cell cycle progression. 


1. Transfer cells from culture dishes to FACS tubes. Make sure you pipet vigorously to get all the lightly adherent cells. The protocol assume 0.5 million cells per sample, so scale up or down with the volumes for your samples. 

2. If needed, stain for surface antigens.

3. Spin cells for 5 minutes, 500 g in a microcentrifuge. Discard supernatants afterwards.

4. Resuspend the cell pellet in 250 uL wash buffer (1x PBS with 5mM EDTA). 

5. Add 125uL 100% COLD ethanol and vortex immediately. Repeat once. 

6. Incubate at room temperature for at least 30 minutes. This cell suspension can be left at 4 C overnight instead if necessary.

7. Spin cells at 5 min, 500 g. Discard supernatants afterwards.

8. Add 250 uL propidium iodide solution (0.1 mg/ml propidium iodide in wash buffer). Place samples on ice.

9. The exact parameters and protocol at this point are dependent on your flow cytometer, but in general start with gating out cell debris using SSC/FSC and doublets using FL2-H/FL2-W. PI has an excitation wavelength of 488nm and maximal emission at 617nm.

10. Collect at around 10,000 events at low speed settings using cell count vs FL2-H as the primary indicator of cell cycle. There will be 2 primary peaks representing G0/G1 and G2 phase respectively, with the G2 phase having double the FL2 height due to having twice the amount of DNA. Cells with less FL2 height than the G1 population are sub-G1 (dying) and cells in between the two main peaks are in the S phase.

Cell Cycle Progression Analysis

A commonly used quantifiable metric for protein phosphorylation is achieved through the use of fluorescence tagged antibodies specific for phosphorylated protein sites. Post treatment cell samples incubated with these antibodies can be analyzed via flow cytometry to measure their respective levels of phosphorylation. Be sure to include unstained controls and secondary antibody only controls if necessary.


1. Treat 0.5-2x106 cells per condition for 2-3 hours.

2. Collect cells to conical tubes / eppitubes and spin down cells (500g, 5 minutes).

3. Prechill 810µl Methanol in -20 C freezer.

4. Resuspend pellet in 100µl PBS (does not have to be sterile).

5. Add 100µl of 4% paraformaldehyde (PFA)  -or-  25µl of 16% PFA to each sample.

6. Incubate 10 min at room temperature.

7. Add 800µl of ice cold methanol 100% methanol. At this stage, you can store at -20oC indefinitely or incubate samples for 30 minutes before proceeding to the next step.

8. Spin down cells 12,000rpm, 5min. This spin should be as fast as possible to ensure maximum sample collection.

9. Add 1 ml PBS to each sample.

10. Spin down cells 12,000rpm, 5min.

11. Prepare 100 uL of primary antibody master mix per sample. Concentrations may need to be 2-5 times higher than an equivalent western blot procedure.

12. Discard sample supernatant and add 100µl of diluted antibody in PBS. 

13. Incubate 30 minutes in the dark on ice (or a fridge).

14. Wash by adding 1 mL PBS and incubate for 5 minutes at room temperature. Spin down cells at 8000 rpm, 5 minutes. Prepare secondary antibody solution in the meantime if necessary (100 uL solution per sample).

15. Remove supernatant. If using secondary antibody repeat resuspension, incubation, and wash steps with secondary antibody solution. If proceeding to FACS analysis, resuspend in 250 uL PBS.

16. Analyze via FACS.

Phosphoflow Protocol

Hi I'm Kris. I received my PhD in Medical Biology studying genome engineering with CRISPR/Cas, ZFNs, and TALENs. I have multiple peer-reviewed scientific publications and awards for my research. Over the last 8 years I've worked to develop gene therapies from HIV/AIDS to hemoglobinopathies. Please feel free to direct any gene editing or related workflow questions to me, I'm happy to help! 
www.arkitekt.bio                         
www.crisprclassroom.org

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