Human BEAS‑2B and HEK 293T cell lines was obtained from ATCC. Cells were maintained in high-glucose DMEM (Sigma Aldrich) supplemented with 10% FBS (Capricorn Scientific) and 1% Pen-Strep (Sigma Aldrich) 1% Glutamin (Sigma Aldrich) at 37°C in 5% CO2 on 10 cm dishes (Greiner Bioscience). For cell detachment Trypsin (Sigma Aldrich) was used. All the cells were maintained in culture for 15 passages as maximum to maintain cell identity. Cells were routinely tested for mycoplasma via PCR. The reagents were dissolved in Dimethyl sulfoxide (DMSO) in specified concentrations and added to the cells.
DNA transfection and infection
For DNA transfection, a mix of 2,5 μg plasmid DNA, 200 μl free medium and 5 μl PEI was added into the 6-well dish medium (60% confluence), after 6 h incubation at 37°C the medium was changed to full supplemented medium. For DNA infection retroviruses or lentiviruses (MOI=10) were added to the cell medium in the presence of polybrene (5μg/ml) and incubated at 37°C for 72 h. After incubation, infected cells were selected with 2 μg/ml puromycin for 72 h or 20 µg/ml blasticidin for 10 days.
Irradiation was performed at room temperature using a 6 MV Siemens linear accelerator (Siemens, Concord, CA) at a dose rate of 9,5 Gy/min.
Dependent on the experiment cells were treated with two different protocols. With the direct seeding protocol exponential growing cells were seeded to 10 cm dishes in adequate amount to be 50-80% confluent next day. Cells were trypsinized, counted and diluted. The dilution was dispensed into different vials and cells were irradiated in suspension. Cells were directly seeded in adequate amounts into 10 cm plates to obtain 100-400 colonies per dish. With the re-seeding protocol exponential growing cells were seeded to 10 cm dishes in adequate amount to be 25-30% confluent next day. The attached cells were treated with different substances or DMSO as a control. 3 h after treatment cells were irradiated with 0, 2, 3, 5, 7, 8 Gy and cultured for 24 h, then cells were trypsinized, counted and re-seeded in adequate amounts into 10 cm plates to obtain 100-400 colonies per dish. For both protocols KP and KPP cells formed colonies after 6 days, BEAS‑2B cells formed colonies after 10-11 days. Cells were fixed with ice cold 25% acidic acid in methanol and stained with 0,5% crystal violet. Colonies were count manually. Only colonies containing at least 50 cells were scored. Surviving fractions were calculated by dividing the plating efficiency for the specified dose divided by the plating efficiency of untreated cells. Radiation treatment survival curves were fitted to the linear-quadratic model formula S= exp[-αD-βD2] (S=survival fraction; D=radiation dose; α and β fitted parameters). Curves were fitted and blotted using a non-linear regression and analysed with OriginPro (OriginPro, 2020, OriginLab Corporation, Northampton, MA, USA). Mean survival fractions at 2 Gy (SF2) and 4 Gy (SF4) were also obtained for each cell model and each substance and used to calculate the radiation enhancement ratio at 2 Gy (RER2Gy) and 4 Gy (RER4Gy) RER greater than 1 indicates enhancement of radiosensitivity, RER below the value of 1 indicates a radio resistance effect. Similarly, the radiation dose with 25% (D25) and 50% (D50) survival under different conditions was calculated to obtain the dose enhancement ratio (DER25 and DER50) that is calculated by dividing D25 without substance treatment by D25 with substance treatment, respectively D50 without substance treatment by D50 with substance. DER greater than 1 indicates a radio sensitising effect, a DER below the value of 1 indicates a radio protecting effect. Plating efficiency was calculated by dividing the number of colonies by the number of seeded cells. All calculated parameters are listed in supplementary table 1 (Table S1)
Cells were lysed in RIPA lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40 and 1% sodium deoxycholate), containing proteinase inhibitor an phosphatase inhibitor (1/100; Bimake) by sonication using Branson Sonifier 150 with a duty cycle at 25%, output control set on level 2 and the timer set to 15 s. Protein concentration was quantified using Bradford assay (Biorad). After mixing of Bradford reagent with 2 μl of sample, the photometer was used to normalize the protein amounts with a previously performed bovine serum albumin (BSA) standard curve. The quantified protein (40-80 μg) was heated in 4x sample buffer (Thermo Fisher) and 10% sample reducing agent (Thermo Fisher) for 10 min at 70°C and separated on 4-12% Bis/Tris-gels or 3-8% Tris/Acetat-Gels (Thermo Fisher). After separation, protein was transferred to nitrocellulose membrane (Thermo Fisher) in transfer buffer (Thermo Fisher) and then, incubated with blocking buffer (5% low fat milk powder in TBS and 0.1% Tween20) for 60 min at RT. After blocking, membranes were incubated with indicated Primary antibodies (1/1000 dilution in a buffer composed 5% low fat milk powder or 5% BSA in TBS and 0.1% Tween20) over night at 4°C. Secondary HRP coupled antibody (Dako 1/1000 dilution in a buffer composed 5% low fat milk powder or 5% BSA in TBS and 0.1% Tween20) were incubated for 2 h at 4°C. Membranes were incubated for 5 min in luminol-solution (250 mg luminol in 100 mM Tris pH 8,6) with 10% v/v cumarinic acid solution (1,1 g cumarinic acid in DMS0 and 0,1% v/v H2O2)at RT, then membranes were recorded with my ECL Imaging System. Analysis and quantifications of protein expression was performed using Image Studio software (Licor Sciences, Lincoln, NE, USA). Antibodies used for this publication are listed in supplemantary table 2 (Table S2)
Cells growing as sub-confluent monolayers were pretreated with substance for 3 h before radiation with 0 Gy and 8 Gy. The cells were kept under standard conditions for normal cell growth. 24 h, 48 h, 72 h and 96 h after radiation cells were harvested with trysinization. Non irradiated cells, treated with camptothecin 5 µM (CPT) were harvested 48 h after treatment. Supernatant of cell culture dishes was pooled with trypsinized cells and pelleted by centrifugation. Further preparation for FACS measurement was following the protocol of the BioLegend APC Annexin V Apoptosis Detection Kit and DNA-staining with DAPI Reagent (25 μg/mL) (Biolegend, San Diego, CA, USA). 20 000 cells were assayed using a flow cytometer FACSCantoII (Becton Dickinson, San Jose, CA, USA). The output data presented as two-dimensional dot plot. Samples were analyzed using the Flowing software gating events to avoid debris, then dividing events in four quadrants. Flowing software was obtained from P. Terho (Turku Centre for Biotechnology, Turku, Finland). Column histograms and statistics were analyzed with Graphpad PRISM 8 (GraphPad Software, San Diego, California USA) and OriginPro. (OriginPro, 2020, OriginLab Corporation, Northampton, MA, USA).
sgRNAs were designed using the CRISPRtool ().
AAV and lentivirus production and purification
Virus was packaged and synthetized in HEK 293T cells seeded in 15 cm-dishes.
For AAV production, cells (70% confluence) were transfected with the plasmid of interest (10 μg), pHelper (15 μg) and pAAV-DJ or pAAV-2/8 (10 μg) using PEI (70 μg). After 96 h, the cells and medium of 3 dishes were transferred to a 50 ml Falcon tube together with 5 ml chloroform. Then, the mixture was shaken at 37°C for 60 min and NaCl (1 M) was added to the mixture. After NaCl is dissolved, the tubes were centrifuged at 20 000 x g at 4°C for 15 min and the chloroform layer was transferred to another Falcon tube together with 10% PEG8000. As soon as the PEG800 is dissolved, the mixture was incubated at 4°C overnight and pelleted at 20 000 x g at 4°C for 15 min. The pellet was resuspended in PBS with MgCl2 and 0.001% pluronic F68, then, the virus was purified using Chloroform and stored at -80C. AAV viruses were titrated using Coomassie staining and RT-PCR using AAV-ITR sequence specific primers.
For Lentivirus production, HEK 293T cells (70% confluence) were transfected with the plasmid of interest (15 μg), pPAX (10 μg) and pPMD2 (10 μg) using PEI (70 μg). After 96h, the medium containing lentivirus was filtered and stored at -80°C.
In vivo experiments and histology
All in vivo experiments were approved by the Regierung Unterfranken and the ethics committee under the license numbers 2532-2-362, 2532-2-367, 2532-2-374 and 2532-2-1003. The mouse strains used for this publication are listed. All animals are housed in standard cages in pathogen‐free facilities on a 12 h light/dark cycle with ad libitum access to food and water. FELASA2014 guidelines were followed for animal maintenance.
Adult mice were anesthetized with Isoflurane and intratracheally intubated with 50 μl AAV virus (3 × 107 PFU) as previously described (Prieto-Garcia et al. 2019). Viruses were quantified using Coomassie staining protocol62. Animals were sacrificed by cervical dislocation and lungs were fixed using 10% NBF. H&E, slides were de-paraffinized and rehydrated following the protocol: 2x 5 min. Xylene, 2x 3 min. EtOH (100%), 2x 3 min. EtOH (95%), 2x 3 min. EtOH (70%), 3 min. EtOH (50%) and 3 min. H2O. For all staining variants, slides were mounted with 200 μl of Mowiol® 40-88 covered up by a glass coverslip. IHC slides were recorded using Panoramic DESK scanner or using FSX100 microscopy system (Olympus) and analysed using Case Viewer software (3DHISTECH) and ImageJ.
Primary murine lung cancer cell lines
In brief, at endpoint of experiment, tumor bearing mice were sacrificed and lung lobes excised. The tissue was briefly rinsed in PBS and transferred to PBS containing Petri dishes. By using a binocular, macroscopically detectable tumor lesions on the lung lobes were excised with a scissor and transferred to a test tube containing Collagenase I (100 U/ml in PBS). The tumor containing tissue was digested for 30 min at 37°C, and the reaction was stopped by addition of 10% FCS. The tissue/collagenase/FCS mixture was briefly spun in a benchtop centrifuge and the supernatant discarded. Digested tissue was re-suspended in 10% FCS (Capricorn) DMEM (Sigma Aldrich), Pen/Strep (Sigma Aldrich) and washed 3 times in 1 ml solution prior to plating in a 6 well tissue culture plate. During subsequent re-plating fibroblasts were counter-selected, by selective trypsinisation, and cell clusters with a homogenous morphology were clonally expanded. These clones were then subjected to further biochemical analysis and characterisation, including genotyping PCR, RNA-sequencing.
FFPE fixed tissue sections from animals were de-parafinized and stained with haematoxylin and eosin (H&E). Each slide was scanned using a Roche Ventana DP200 slide scanner. To assess tumor area per animal, total lung area was measured by using the QuPath image analsyis tool. Subsequently, all tumor nodules were measured and the tumor surface calculated. Graph was generated using GraphPad Prism 8.
Survival curves mouse
Upon intratracheal administration of AAV, animals were monitored on a daily basis. Whenever experimentally defined termination points were reached, such as 20% weight loss, animals were sacrificed by cervical dislocation and tissue samples collected. Graphs were generated using Prism Graphpad 8.
RNA sequencing was performed with Illumina NextSeq 500 as described previously63. RNA was isolated using ReliaPrep™ RNA Cell Miniprep System Promega kit, following the manufacturer’s instruction manual. mRNA was purified with NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB) and the library was generated using the NEBNext® UltraTM RNA Library Prep Kit for Illumina, following the manufacturer’s instructions).
Sample preparation for mass spectrometry
Lysates of cells, solved from cell culture plates with cell scrapers pelleted and frozen at -80°C, were precipitated by methanol/chloroform and proteins resuspended in 8 M Urea/10 mM EPPS pH 8.2. Concentration of proteins was determined by Bradford assay and 300 µg of protein per samples was used for digestion. For digestion, the samples were diluted to 1 M Urea with 10 mM EPPS pH 8.2 and incubated overnight with 1:50 LysC (Wako Chemicals) and 1:100 Sequencing grade trypsin (Promega). Digests were acidified using TFA and tryptic peptides were purified by Oasis Prime HLB columns (30 mg, Waters). 80 µg peptides per sample were TMTpro labeled, and the mixing was normalized after a single injection measurement by LC-MS/MS to equimolar ratios for each channel. 100 µg of pooled peptides were dried for offline High pH Reverse phase fractionation by HPLC (whole cell proteome) and remaining ~1.1 mg of multiplexed peptides were used for phospho-peptide enrichment by High-Select Fe-NTA Phosphopeptide enrichment kit (Thermo Fisher) after manufacturer`s instructions. After enrichment, peptides were dried and resuspended in 70% acetonitrile/0.1% TFA and filtered through a C8 stage tip to remove contaminating Fe-NTA particles. Dried phospho-peptides then were fractionated on C18 (Empore) stage-tip. For fractionation C18 stagetips were washed with 100% acetonitrile twice, followed by equilibration with 0.1% TFA solution. Peptides were loaded in 0.1% TFA solution and washed with water. Elution was performed stepwise with different acetonitrile concentrations in 0.1% Triethylamine solution (5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 50%). The resulting 12 fractions were concatenated into six fractions and dried for LC-MS.
Peptides were fractionated using a Dionex Ultimate 3000 analytical HPLC. 250 µg of pooled and purified TMT-labeled samples were resuspended in 10 mM ammonium-bicarbonate (ABC), 5% ACN, and separated on a 250 mm long C18 column (X-Bridge, 4.6 mm ID, 3.5 µm particle size; Waters) using a multistep gradient from 100% Solvent A (5% ACN, 10 mM ABC in water) to 60% Solvent B (90% ACN, 10 mM ABC in water) over 70 min. Eluting peptides were collected every 45 s into a total of 96 fractions, which were cross-concatenated into 24 fractions and dried for further processing.
Liquid chromatography mass spectrometry
All mass spectrometry data was acquired in centroid mode on an Orbitrap Fusion Lumos mass spectrometer hyphenated to an easy-nLC 1200 nano HPLC system using a nanoFlex ion source (ThermoFisher Scientific) applying a spray voltage of 2.6 kV with the transfer tube heated to 300°C and a funnel RF of 30%. Internal mass calibration was enabled (lock mass 445.12003 m/z). Peptides were separated on a self-made, 32 cm long, 75 µm ID fused-silica column, packed in house with 1.9 µm C18 particles (ReproSil-Pur, Dr. Maisch) and heated to 50°C using an integrated column oven (Sonation). HPLC solvents consisted of 0.1% Formic acid in water (Buffer A) and 0.1% Formic acid, 80% acetonitrile in water (Buffer B).
For total proteome analysis, a synchronous precursor selection (SPS) multi-notch MS3 method was used in order to minimize ratio compression as previously described. Individual peptide fractions were eluted by a non-linear gradient from 7 to 40% B over 90 min followed by a step-wise increase to 95% B in 6 min which was held for another 9 min. Full scan MS spectra (350-1400 m/z) were acquired with a resolution of 120,000 at m/z 200, maximum injection time of 100 ms and AGC target value of 4 x 105. The most intense precursors with a charge state between 2 and 6 per full scan were selected for fragmentation and isolated with a quadrupole isolation window of 0.7 Th and a cycle time of 1.5 s. MS2 scans were performed in the Ion trap (Turbo) using a maximum injection time of 50 ms, AGC target value of 1.5 x 104 and fragmented using CID with a normalized collision energy (NCE) of 35%. SPS-MS3 scans for quantification were performed on the 10 most intense MS2 fragment ions with an isolation window of 0.7 Th (MS) and 2 m/z (MS2). Ions were fragmented using HCD with an NCE of 65% and analyzed in the Orbitrap with a resolution of 50,000 at m/z 200, scan range of 110-500 m/z, AGC target value of 1.5 x 105 and a maximum injection time of 120 ms. Repeated sequencing of already acquired precursors was limited by setting a dynamic exclusion of 45 seconds and 7 ppm and advanced peak determination was deactivated.
For phosphopeptide analysis, each peptide fraction was eluted by a linear gradient from 5 to 32% B over 120 min followed by a step-wise increase to 95% B in 8 min which was held for another 7 min. Full scan MS spectra (350-1400 m/z) were acquired with a resolution of 120,000 at m/z 200, maximum injection time of 100 ms and AGC target value of 4 x 105. The most intense precursors per full scan with a charge state between 2 and 5 were selected for fragmentation, isolated with a quadrupole isolation window of 0.7 Th and fragmented via HCD applying an NCE of 38% with an overall cycle time of 1.5 s. MS2 scans were performed in the Orbitrap using a resolution of 50,000 at m/z 200, maximum injection time of 86ms and AGC target value of 1 x 105. Repeated sequencing of already acquired precursors was limited by setting a dynamic exclusion of 60 s and 7 ppm and advanced peak determination was deactivated.
QUANTIFICATION AND STATISTICAL ANALYSIS
Fastq files were generated using Illuminas base calling software GenerateFASTQ v184.108.40.206 and overall sequencing quality was analyzed using the FastQC script. Reads were aligned to the human genome (hg19) using Tophat v2.1.164 and Bowtie2 v2.3.265 and samples were normalised to the number of mapped reads in the smallest sample. For differential gene expression analysis, reads per gene (Ensembl gene database) were counted with the “summarizeOverlaps” function from the R package “GenomicAlignments” using the “union”-mode and non- or weakly expressed genes were removed (mean read count over all samples <1). Differentially expressed genes were called using edgeR66 and resulting p-values were corrected for multiple testing by false discovery rate (FDR) calculations. GSEA analyses were done with signal2Noise metric and 1000 permutations. Reactome analysis were performed with PANTHER67 using the “Statistical overrepresentation test” tool with default settings. Genes were considered significantly downregulated for Reactome analysis when: Log2FC>0.75 and FDR p-value<0.05.
Analysis of publicly available data
All publicly available data and software used for this publication are listed (please see Star Methods). Oncoprints were generated using cBioportal68, 69. Briefly, Oncoprints generates graphical representations of genomic alterations, somatic mutations, copy number alterations and mRNA expression changes. TCGA data was used for the different analysis. Data were obtained using UCSC Xena. Data was downloaded as log2 (norm_count+1)
Kaplan-Meier curves were estimated with the KM-plotter70, cBioportal68 and R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl). The KM-plotter was used to analyse overall survival of lung cancer patients (Figure 1 and S1) based on gene expression data from microarrays obtained from GEO, caBIG and TCGA
Mass spectrometry data analysis
Raw files were analyzed using Proteome Discoverer (PD) 2.4 software (ThermoFisher Scientific). Spectra were selected using default settings and database searches performed using SequestHT node in PD. Database searches were performed against trypsin digested Mus musculus SwissProt database containing one sequence per gene without isoforms. Static modifications were set as TMTpro at the N-terminus and lysines and carbamidomethyl at cysteine residues. Search was performed using Sequest HT taking the following dynamic modifications into account: Oxidation (M), Phospho (S,T,Y), Met-loss (Protein N-terminus), Acetyl (Protein N-terminus) and Met-loss acetyl (Protein N-terminus). For whole cell proteomics, the same settings were used except phosphorylation was not allowed as dynamic modification. For phospho-proteomics all peptide groups were normalized by summed intensity normalization and then analyzed on peptide level71. For whole cell proteomics normalized PSMs were summed for each accession and data exported for further use. For proteomics analysis, significance was assessed via a two-sided unpaired students t-test with equal variance assumed. For pathway analysis, Protein/Peptide lists were filtered as indicated and a STRING network created in Cytoscape. For the resulting network a pathway enrichment analysis was performed using the STRING App Cytoscape plugin. For network views of enrichments, the Reactome pathways were filtered for a FDR < 0.001 and loaded into the Enrichment Map 3 plugin for Cytoscape to create visualization. Gene sets for visualization purposes were downloaded from the molecular signature gene set database (https://www.gsea-msigdb.org/) on 02-21-2021. Result files were filtered for the included genes to create pathway specific visualizations.
DATA AND SOFTWARE AVAILABILITY
Raw data is available via Mendeley Data: doi: RNA-sequencing data is available at the Gene Expression Omnibus under the accession number GEO:
Contact for reagent and resource sharing
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Markus E. Diefenbacher ([email protected]).