Cell culture
Human lung cancer cell lines (A549, NCI-H1299, NCI-H2170, NCI-H358, NCI-H460 and NCI-H446), normal human lung fibroblast cells (MRC-5) and human embryonic kidney HEK293T cell line were purchased from American Type Culture Collection (ATCC). Cell lines were tested and authenticated, and were not cultured continuously for more than 3 months. Each cell line was cultured in its standard medium as recommended by ATCC. A549 cells were cultured in F-12K Medium (Hyclone) supplemented with 10% FBS (Gibco). HEK293T cells were cultured in DMEM Medium (Life Technologies) supplemented with 10% FBS (Gibco). NCI-H1299, NCI-H2170, NCI-H358, NCI-H460 and NCI-H446 cells were cultured in RPMI1640 Medium supplemented with 10% FBS (Gibco). All cells were maintained at 37 °C in a humidified 5% CO2 atmosphere.
Plasmid construction
Full length of MACC1 fragment was cloned from human genome cDNA and ligated into pLVX-pTRE3G vector. The primers were as follows: 5’ NotI, 5’-CACGCGGCCGCTATGCTAATCACTGAAAGAAAACATT; 3’ MluI, 5-CACACGCGTCTATACTTCCTCAGAAGTGGAGAAT. The KLF4 promoter fragment was cloned from human genomic DNA and ligated into pGL3-basic vector (Promega) upstream of the luciferase reporter. Primers were as follows: 5’ SacI, 5’-CACGAGCTCAAGAATGCTTCTGTGGTCGG; 3’ BglII, 5’-CACAGATCTTTCTCTCTGGTCGGGAAACT. The full-length KLF4 plasmid was kindly provided by Dr. Chuan-Chun Han (Institute of Cancer Stem Cell, Dalian Medical University, Dalian, 116044, China). The shRNA targeting MACC1(1#, 2#, 3#) was ligated into pLKO-tet-on vector and pLKO.1 vector (Addgene) in following sequence: shMACC1-1#, TGCCTTGATTTGAATACA; shMACC1-2#, CTGCCACCATTTGGGATT. shMACC1-3#, GAGTTAGTCGCACGTCTC. The shRNA targeting KLF4 was ligated into pLKO.1 vector (Addgene) in following sequence: TTGGTGAGTCTTGGTTCTAA. All plasmids were verified by sequencing.
Lentivirus preparation and transfection
Lentivirus was produced in HEK293T cells using the 2nd generation packaging system plasmids psPAX2 (Addgene) and pMD2.G (Addgene). The cells were transfected using lipofectamine plus (Sagecreation) according to the manufacturer’s instructions. Supernatant was collected after 48 h transfection by centrifugation to remove any cellular debris. The resulting viral particles were used to infect the target cell line. The stably integrated cells were selected with 2 μg/ml of puromycin (Sigma-Aldrich).
To generate stable cell line expressing MACC1 on doxycycline (Dox) induction, we used the response vector pTRE3G with HA tagged, regulator vector pCMV-Tet3G and A549 cells. The full-length MACC1 was cloned into the response vector, packaged both vectors into lentivirus respectively, and infected A549 cells in 1:1 ratio. The stably integrated cells were selected with 5 μg/ml of puromycin and 800 μg/ml of G418 at 2 days after infection for 7 days to obtain individual colonies. To generate stable cell line knocking down MACC1 on doxycycline, we packaged pLkO tet-on-shMACC1#1, pLkO tet-on-shMACC1#2 and pLKO tet-on empty vector into lentivirus, and infected H1299 cell line, the stably integrated cells were selected with 2 μg/ml of puromycin for 7 days. The inductions were carried out by adding doxycycline to a final concentration of 2 μg/ml. The expression of transgenes was confirmed by western blots before further analysis.
Dual-luciferase reporter assay
The pGL3basic (Promega, USA) was used as control promoter and pRSV-Renilla luciferase expression vector (Promega, USA) was used to monitor transfection efficiency. H1299 or H2170 MACC1 knockdown stable cell lines were transfected with KLF4 promoter-driven luciferase vector pGL3-KLF4 or control pGL3-Basic luciferase vector using lipofectamine plus (Sagecreation) according to the manufacturer’s instruction. Cells Luciferase activity was measured using the Dual-Luciferase Reporter Assay system (Promega) according to the manufacturer’s instructions 24 h later. The Firefly luciferase activity was normalized to Renilla levels and is shown relative to control conditions.
RT-PCR and quantitative RT-PCR validation
Total RNA was extracted from cells according to the manufacturer’s instructions. Genome DNAs were removed by 30 min DNase I (Takara) treatment at 37 °C and then heat inactivation using 50 nM EDTA. Total RNA (2 µg) was then reverse-transcribed with PrimeScript RT reagent kit (Takara) with random primer, and 1 µl of the cDNA was used as the template for PCR amplification. RT-PCR products were separated on 2% gels. The amount of each was measured by comparison of the integrated optical density of detected bands using the Image J. Real-time quantitative PCR was performed using Takara SYBR II kit on system according to the manufacturer’s instructions. The primer sequences see the supplementary table.
RNA sequencing analysis
We constructed H1299 shNC and shMACC1 stable cell lines, and then total RNAs were extracted using Trizol. We used the Illumina TruSeq Total RNA Sample Prep kits to purify poly-adenylated RNA (not exceeding 2 µg) after RNase free DNase digested as per manufactures instruction. Cytoplasmic rRNA was removed by the RiboZero Human. Prior to generation of cDNA library with bar coded ends the mRNA purified were further analyzed using Bio-analyzer (Agilent Technologies). The protocol employed to prepare RNA-seq libraries was based on the use of Illumina TruSeq Total RNA Sample Prep kits. The cluster generation and sequencing were carried out by standard procedures in HiSeq 2000 Illumina platform with pair-end 150 bp (PE 150) sequencing strategy.
RNA stability assay
H1299, H2170 MACC1 knockdown stable cells and control cells were treated with 5 µg/ml actinomycin D (ActD, Sigma, CA1201) and collected at the indicated time points. The total RNAs were extracted by Trizol reagent (Invitrogen) and analyzed by RT-PCR.
Immunoblotting
Cells were harvested with RIPA lysis buffer containing 1 mM Cocktail and 1 mM PMSF. Cell debris was removed by centrifugation. The protein samples (20 µg) were boiled at 100°C for 5 min in 1× SDS sample buffer and fractionated by 10% SDS-PAGE and transferred to PVDF membrane. The membranes were gently rinsed with TBST and then incubated for 1 hour at room temperature in a solution containing 5% fat-free milk. The following antibodies were used: MACC1 (Abcam, Ab106579, 1:1000), KLF4 (Proteintech, 17402-1-AP, 1:1000), Gapdh (Sigma-Aldrich, SAB1405848, 1:5000), P21 (Cell Signaling Technology, 12D1, 1:1000), P27 (Cell Signaling Technology, D69C12, 1:1000), Ki-67 (Cell Signaling Technology, 9449 s, 1:1000), HA-Tag (Convance, mms-101p-1000, 1:1000). Secondary antibody was used: Anti-mouse or Anti-rabbit IgG, 1:5000 dilution (Cell Signaling Technology). Bound antibodies were visualized with enhanced chemiluminescence (Tanon) by MiniChmei Chemiluminescence imager (SAGECREATION, Beijing).
ALDEFLUOR assay and flow cytometric analysis & sorting
The ALDEFLUORTM kit (Stem Cell Technologies, Catalog #01700) was used to identify the cells expressing ALDH activity for sorting and analysis. Cells (2 × 105) were incubated in assay buffer containing ALDH substrate BODIPY-aminoacetaldehyde (BAAA). As negative control, an aliquot of the ALDH substrated treated cells were immediately quenched with specific ALDH inhibitor, DEAB. Both tubes were incubated for 45 min at 37 °C. After incubation, cells were centrifuged and the pellets were resuspended with 500 μl PBS and store the cells at 4 °C. The result in fluorescence intensity of ALDHhigh cells and ALDHlow cells was analyzed by flow cytometer (Beckman Coulter, CytoFLEX). For flow cytometric sorting, cells were washed twice with PBS and resuspended in PBS and were performed on a FACS flow cytometer (Beckman).
Sphere formation assay
Sphere formation was performed in 96-wells ultra-low attachment plates (Corning) with DMEM/F-12 (Gibco) medium supplemented with 2% B27, 20 ng/ml EGF, and 20 ng/ml bFGF. Basal medium containing methylcellulose gel matrix (2%, M0512, Sigma-Aldrich, US) was used to prevent reaggregation of the cells, as described previously [56]. A549, NCI-H1299 and NCI-H2170 cells were seeded at a density of around 2 cells/μl and cultured at 37 °C with 5% CO2 After 9–14 days, spheres greater than 50 μm diameter were counted at ×40 magnification (Olympus) and analyzed using Image pro plus 6.0 software (Media Cybernetics).
Colony formation assay
MACC1 stable knockdown H1299 or H2170 cells and MACC1 stable over-expressing cells were seeded in 60 mm dishes (500 cells per dish) and incubated at 37 °C, 5% CO2 in humidified incubator for 10 days or two weeks (updated with fresh medium every 3 days). Each treatment was carried out in triplicate. Colonies were fixed with 4% paraformaldehyde and stained with crystal violet solution.
Cell viability assay
We determined cell viability by cell counting kit-8 assay. (CCK-8, APExBIO) Cells were added to a 96-well plate (1000 cells/well) and cultured overnight until cells were adherent, CCK-8 was added to the cells according to the manual and was incubated for 2 h. Then, the absorbance value at OD450 was measured.
Xenograft mouse model
Experimental procedures regarding the animal study have been subject to rigorous evaluation and received ethical clearance from the Animal Experimental Ethics Committee of Dalian Medical University (Approval No. AEE21015). 4-week-old BALB/c nude mice were purchased from Vital River Laboratories (VRL, Beijing) for in vivo tumorigenicity study. Nude mice were randomly allocated into each group, and were injected subcutaneously with 1 × 106 A549 cells stably Dox-induced overexpressing MACC1 (n = 6), NCI-H1299 cells stably knocking down MACC1 (n = 6). Doxycycline was administered in drinking water (dilute to 2 mg/ml in 5% sucrose) and the bottles were covered with aluminum foil to prevent degradation by light, and change the water every three days. Six mice were used for each group. The size of the tumor was determined by vernier caliper measurement of the subcutaneous tumor mass every 3 days. Mice were raised in the following 8 weeks. Nude mice died unexpectedly or tumor weight exceeded 10% of their body weight were excluded. At the end of measurement, all mice were euthanized, and tumors were excised for further analysis. Tumor volume was calculated as V = 1/2 (long diameter) × (short diameter) [2]. For all data points, five independent measurements were performed and means were used for calculation.
For limiting dilution assays, cells were serially diluted from 1 × 106 to 1 × 103 cells/100 μl in PBS containing 50% Matrigel (BD Biosciences), followed by subcutaneous injection into nude mice. Four mice were randomly allocated in each group. Tumor formation was examined once every 3 days, and the total observation period was 4 weeks. Solid tumor size measurement method as stated above.
Primary cell isolation and culture
The mouse tumor samples were chopped into small pieces, and incubated in fresh complete medium with 5 mg/ml collagenase I solution with 0.2 mg/ml hyaluronic acid in shaker at 37 °C for 3 h with 220 r/min. After incubation, the suspensions were passed through 70 μm (FALCON, REF352350) pore filter and centrifuge at 1000 rpm for 5 min. The pellet was resuspended in the fresh DMEM containing 10% FBS and plated into 60 mm cell culture plates.
Immunohistochemistry
Resected xenograft tumor tissues were fixed with 4% paraformaldehyde before embedded in paraffin. Prepared tissue sections (4 μm) were deparaffinized with xylene and rehydrated with graded alcohol. Tissue sections were then treated with blocking solution (ZSGB-Bio) for 30 min at room temperature, before primary antibody incubation overnight at 4 °C, followed by secondary antibody incubation for 1 h at room temperature. Tissue samples were stained using a peroxidase IHC assay kit (ZSGB-Bio, Beijing) according to the manufacturer’s instructions. Mounted sections were examined by light microscopy (Leica) and acquired images were analyzed with Image-Pro Plus software (version 6.0).
Clinical tissues samples collection
Fresh lung cancer tissues and adjacent normal tissues were continuous enrolled from patients with pathologically and clinically confirmed lung carcinomas. All of tissue specimens were kept in liquid nitrogen and sectioned for protein extraction. The use of the clinical samples in this study was approved by the Ethics Committee of the first affiliated hospital of Dalian Medical University (PJ-KS-KY-2021-94). All human tumor tissue samples were acquired with the written informed consent of patients or their guardians before involvement in the research.
Statistics
Statistical analyses were performed by prim 9.5.1 (GraphPad Software, San Diego, CA, USA). Biologically repeated experiments (n = 3) were carried out to allow statistical analysis. Results were illustrated by mean ± standard error of the mean (SE). Two-tailed Student’s t-test was performed to evaluate statistical differences between groups, one-way ANOVA was employed to compare data among multiple groups, post-hoc comparisons among groups were made using the Tukey’s HSD procedure. with calculated p-values of less than 0.05 deemed as significant differences. Overall survival and First Progression were assessed using the Kaplan-Meier method, and differences between survival curves were evaluated using the log-rank test. No blinding was used. Sample sizes were selected without predetermined effect size.
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- Source: https://www.nature.com/articles/s41420-024-02256-0