Ethical Statement
LAM tissue samples were obtained from lung transplant donors for generation of cell lines, in accordance with the Declaration of Helsinki, approved by the Institutional Review Board at the University of Pennsylvania [22] and provided by the National Disease Research Interchange (NDRI, Philadelphia, PA). LAM patients had given written informed consent and all the collected samples were treated anonymously. Paraffin embedded tissue samples were obtained retrospectively from the Departments of Pathology at Semmelweis University, Budapest, and from the University of Pecs, Pecs, Hungary and the National Koranyi Institute of Pulmonology, Budapest, Hungary. The study was approved by the Medical Research Council of Hungary (54034-4/2018/EKU).
LAM cell lines, bronchial smooth muscle cells (BSMC), normal human lung fibroblast (NHLF) S102 and S103 cell lines and cell culture conditions
Primary cultures of human LAM cells where established in the Department of Medicine, University of Pennsylvania, Pennsylvania, USA [22]. Briefly, the primary cultures of LAM cells were dissociated from the LAM nodules of transplant patients. Each LAM nodule was used to establish individual cell lines (characterized based on alpha smooth muscle actin (α-SMA) expression, mTORC1 activation, HMB45 immunoreactivity, DNA synthesis, and cell migration) [23]. In the current study, four patient-derived individual LAM cell lines were used including LAM-100, LAM-111C, LAM-D9065 and LAM-HUP. As controls, primary cultures of normal, human bronchial smooth muscle cells (BSMC) and normal human lung fibroblasts (NHLF), were purchased from Lonza (Basel, Switzerland). Normal, BSMC and LAM cells were cultured at 37°C, 5% CO2 in SMC Growth Medium (insulin, hFGF, GA, FBS and hEGF) (Lonza, Basel, Switzerland). The 621-101 cells were derived from an angiomyolipoma and carried bi-allelic inactivation of the TSC2 gene [24, 25]. The 621-102 cell line was generated by introduction of E6/E7 (pLXSN 16E6E7-neo) and human telomerase (pLXSN hTERT-hyg) into a primary culture of TSC2 null human angiomyolipoma cells [26]. 621-103 was generated by stable transfection of TRI102 with wild-type TSC2 (pcDNA3.1 TSC2-zeo) into 621-101 cells [24].
Haematoxylin eosin staining
5 µm thick tissue sections were stained in Mayer’s haematoxylin solution (Sigma-Aldrich, St. Louis, USA) for 10 min, washed, then differentiated with 0.25% acetic acid and in eosin solution. Sections were mounted using Vectashield mounting medium (Vector Laboratories, Burlingame, USA). Images were taken using Nikon Eclipse Ti-U inverted microscope.
Immunofluorescent staining
Normal, BSMC, NHLF, LAM, S103 and S102 cells were cultured for 3 days using Falcon™ chambered cell culture slides (Thermo Fisher Scientific, Waltham, USA). Cell cultures were then fixed with 4% formaldehyde and permeabilized with PBS containing 0.1% Triton-X and 5% BSA. Slides were incubated with primary antibodies Anti-alpha -Smooth Muscle Actin (MAB1420), Anti-mTOR Antibody (ab25880), Anti-p70 S6 kinase Antibody (Ab32529) and Anti-RAR beta (ab25880) overnight at 4°C. Slides were washed with TBS for three times then incubated with corresponding secondary antibody: Anti-mouse Alexa 488 (A28175 1:200), Anti-rabbit Alexa 647 (A27040 1:200), Anti-rabbit Alexa 488 (A11034 1:200) or Anti-mouse Alexa 647 (A32728 1:200), for 960 min at RT. Nuclei were counter stained with DAPI. Images were acquired using an Olympus IX-81 (OLYMPUS Corporation, Tokyo, Japan) both light and fluorescence microscope.
Immunohistochemistry
5 µm thick tissue sections were stained using immunohistochemistry. First, the slides were rinsed in heated xylene and were washed with a descending series of alcohol to remove paraffin. After deparaffination the slides were rehydrated in distilled water and antigen retrieval was performed by heating the slides in Target Retrieval Solution (pH 6, DAKO, Produktionsvej, Denmark) at 97°C for 20–30 min. Subsequently slides were washed in dH2O and endogenous peroxidase activity was blocked with 3% H2O2 containing TBS (pH 7.4) for 15 min. Then slides were washed three times with TBS containing Tween (0.05%, pH 7.4). Pre-blocking was carried out with 3% BSA in TBS for 20 min before overnight incubation with anti- Melanoma gp100 antibody (HMB-45) (1:100, HMB-45 mouse monoclonal antibody clone: Ab787, ABcam) and anti-RARβ (1:100, anti-RARβ rabbit monoclonal antibody clone: Ab124701, ABcam) primary antibody at 4°C. Following incubation slides were washed with TBS for three times then incubated with peroxidase conjugated secondary antibody (1:100, Polyclonal Goat Anti-Rabbit IgG, DAKO) for 90 min. Antibody labelling was visualized with the help of liquid DAB Substrate Chromogen System (DAKO). For nuclear counterstaining, haematoxylin staining was performed. Finally, slides were mounted with Faramount Aqueous Mounting Medium (DAKO, Produktionsvej, Denmark). Histological evaluation was performed with the help of Panoramic MIDI digital slide scanner (3DHistech, Budapest, Hungary). Image analysis was performed using ImageJ software with IHC toolbox plug-in.
Rapamycin and Retinoic acid (RA) treatments
BSMC, NHLF, LAM, S103 and S102 cell cultures were treated with mono rapamycin or RA treatments and in combined treatments using the following concentrations: 10 or 20 nM Rapamycin catalogue: tlrl-rap (InvivoGen, San Diego, USA) and 1 or 2 µM retinoic acid (Sigma-Aldrich, St. Louis, USA) for 24h at 37°C, 5% CO2.
Western blot
Cells were lysed in ice-cold RIPA buffer (Sigma-Aldrich, St. Louis, USA) supplemented with protease inhibitors (Roche Diagnostics, Mannheim, Germany) for 30 min on ice and centrifuged at 16,000 × g for 20 min at 4° C. The supernatant was then used as the cell lysate. The protein content of each cell lysate was assessed using a Qubit protein assay kit (Thermo Scientific, Waltham, MA). 30 µg of total protein was loaded onto Mini Protean gel (Bio-Rad, California, USA), then electrophoresis was followed by overnight blotting onto a nitrocellulose membrane using 10 mA current. The blots then were blocked in 5% non-fat skimmed milk blocking solution (Bio-Rad, California, USA) in TBS-T for 1 h and incubated with primary antibodies diluted 1:1000 in 2.5% non-fat skimmed milk powder in TBS-T overnight at 4° C. After washing with TBS-T, the blots were incubated with rabbit anti-goat/HRP diluted in 2.5% non-fat skimmed milk powder in TBS-T for 1 h at room temperature. The immunoreaction was developed with a chemiluminescence HRP substrate and recorded with ImageQuant LAS-4000 imager (GE Healthcare Life Sciences, USA).
RNA isolation
Total RNA was extracted from normal BSMC, NHLF and LAM cell cultures with MN NucleoSpin RNA isolation kit according to the manufacturer’s protocol (Macherey-Nagel, Düren, Germany). The concentration of RNA samples was measured using NanoDrop (Thermo Fisher Scientific, Waltham, USA). Total RNA from human lung tissues were obtained using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Waltham, USA). RNA (1 µg) was digested with DNase (Sigma-Aldrich, St. Louis, USA) to eliminate any DNA contamination. cDNA was synthesized with high capacity RNA to cDNA kit (Thermo Fisher Scientific, Waltham, USA). Reverse transcription was performed with random hexamer primers.
Quantitative qRT-PCR
qRT-PCR was performed using SensiFAST SYBR Green reagent (BioLine, London, UK) in an ABI StepOnePlus system. Gene expressions using sequence specific primers (S. Table 1) were analysed with StepOne software and normalized to beta-actin. Changes in gene expression were calculated according to the 2-ddCt method.
Quantstudio 12k flex
cDNA was prepared using TaqMan miRNA reverse transcriptase kit and Megaplex RT primers Pool A and B (Thermo Fisher Scientific, Waltham, USA) according to manufacturers’ protocol using 350ng-1000ng of total RNA as starting material. miRNA expression levels were performed using open array miRNA card Pool A and B and Quantstudio 12k flex (Thermo Fisher Scientific, Waltham, USA).
Metabolic enzymes RT2 array
cDNA was prepared using RT2 First Strand Kit (Qiagen, Hilden, Germany) according to manufacturers’ protocol using 350ng-1000ng of total RNA as starting material. Metabolic enzymes mRNA expression levels were performed using Human Drug Metabolism: Phase I Enzymes arrays (Qiagen, Hilden, Germany), RT2 SYBR® Green qPCR Mastermix (Qiagen, Hilden, Germany) and results were acquired by Quantstudio 12k flex (Thermo Fisher Scientific, Waltham, USA).
ALDH and ADH activity assay
ALDH Activity Assay Kit (Abcam, MA, USA, ab155893) and Alcohol Dehydrogenase Assay Kit (Abcam, MA, USA, ab102533) were used to test ALDH and ADH activity of LAM and S102 compared to their controls before and after treatments. Activity of cell lysates was assessed using a detection kit and following the manufacturer’s instructions. Enzyme activity induced colour changes were measured at OD450 nm with EnSpire® Multimode Plate Reader (PerkinElmer, Waltham, Massachusetts, USA). Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, USA) was used to measure protein content and results are presented as the fold change vs. control.
Wound healing assay
Cells were grown to 90% confluence in 24 well plates and wound gap was made by scratching the cell with rapamycin (10 nM), RA (2 μM) and rapamycin (10 nM) + RA (2 μM) was after inducing the wound gap. The healing of the wound gap by cell migration and the centre of the gap was monitored with images taking with EVOS light microscopy (Thermo Fisher Scientific, Waltham, USA) and the gap area was quantified using ImageJ software.
3D co-cultures
Normal human lung fibroblasts (NHLF) and bronchial smooth muscle cells (BSMC) were isolated from anonymous donors of different ages and sexes and were purchased from Lonza (Basel, Switzerland). All cells were cultured at 37°C and 5% CO2 in primary cell culture media. NHLF, BSMC and LAM cell types were sub-cultured and mixed at 1:1 ratio then dispensed 3*105 cells/well onto a low-attachment 96-well U-bottom plates (Corning, New York, USA). 3D aggregates were formed as described previously (Kovacs et al., 2014). The 3D aggregate co-cultures were incubated in the presence or absence of 10 nM rapamycin and/or 2 μM RA for 24 h, then collected into cryomold and sectioned for staining.
Statistical analysis
Unless otherwise noted, statistical analysis was performed with SPSS version 20 software. S102 and S103 data are presented as mean ± technical error of three replicates and statistical analysis was performed using the t-test. LAM primary samples and their controls (BSMC n=4 and NHLF n=4) data are presented as mean ± standard error of mean (SEM), and statistical analysis was performed using the one-way ANOVA. p<0.05 was considered as significant.