Cell Lines and Reagents
The PC9 (Del19) and PC9GR (T790M&Del19 mutant) LUAD cell lines were generously granted from Dr. Changzhi Xu (Anhui University, Hefei, China). Both PC9 and PC9GR cells were cultured in DMEM medium supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin, and incubated in 5% CO2 at 37℃.
Lv-SHP2, Lv-SHP2-RNAI (RNA interference) and scrambled controls were recruited from Shanghai Genechem Co.,Ltd.. Recombinant human EGF (SRP3027) and FGF (GF003AF) were purchased from Sigma-Aldrich. SHP2 inhibitor (SHP099, Cat#: HY-100388), recombinant human CXCL8 (Cat#: HY-P7224), CXCR2 inhibitor (Danirixin, Cat#:HY-19768), CXCR1/2 inhibitor (Reparixin, CAT#: HY-15251) and Osimertinib (AZD-9291, Cat#: HY-15772) were bought from MCE LLC. Human CXCL8 Immunoassay Quantikine ELISA Kit (Cat#:D8000C) was acquired from R&D Systems.
Antibodies against SHP2(Clone:D50F2, Cat#:3397), CD133(Clone:D2V8Q, Cat#:64326), GSK3β(Clone:D5C5Z, Cat#:12456), p-GSK3β(ser9) (Clone:5B3, Cat#:9323), βCatenin(Clone:D10A8, Cat#:8480), ERK1/2(Clone:137F5, Cat#:4695), p-ERK1/2(Thr202/Tyr204) (Clone:20G11, Cat#:4376), AKT(Clone:11E7, Cat#:4685), p-AKT(Ser473) (Clone:D9E, Cat#:4060), p-RelA/p65(Ser536) (Clone:93H1, Cat#:3033), p-IkBβ(Thr19/Ser23) (Cat#:4921), Ki67(Clone:D2H10, Cat#:9027) and β-actin (Clone:8H10D10, Cat#:3700) were purchased from Cell Signaling Technology. For Flow assays, anti-CD133 monoclonal antibody conjugated with Super Bright 436 (Clone:TMP4, Cat#:62-1338-42), and mouse IgG1 kappa Isotype Control (Cat#:62-4714-42) were from eBioscience.
Lentiviral transduction was performed following user`s protocol using GV298 plasmids encoding over-expression (Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin) and RNAI (U6-MCS-Ubiquitin-Cherry-IRES-puromycin) against SHP2 and homologous control. Briefly, 1×105 cells were seeded in 6 well plate and incubated overnight. 20μL of 1×108 TU/mL lentivirus diluted in 1mL complete DMEM medium was added into each well, and medium changed after incubated for another 12 hours. Fluorescence expression was used for monitoring the efficacy of transfection. Infected PC9 and PC9GR cells were selected by puromycin (0.5 µg/ml) for 7 days to generate the stable clones and maintained in 0.3 µg/mL of puromycin condition over 1 month.
Transcriptome Analyses based on next-generation sequencing (NGS)
In order to analyze the relevant gene transcription of SHP2 modification in LUAD, the NGS of lentivirus modified and parental PC9GR cells were performed by the Beijing Genome Institute (BGI, Shenzhen, China). Total RNA was extracted, mRNA was enriched and then the cDNA libraries were prepared. Transcriptome expression were generated by equal quantities of RNA from lentivirus modified and parental PC9GR cells, 3 biological duplications were repeated in each group. Bowtie2 (v2.2.5) was applied to align the clean reads to the gene set, a database for this organism built by BGI, which known and novel, coding transcripts were included, then expression level of gene was calculated by RSEM (v1.2.12). The heatmap was drawn by pheatmap (v1.0.8) according to the gene expression in different samples. To take insight to the change of phenotype, KEGG (https://www.kegg.jp/) enrichment analysis of annotated different expression gene was performed by Phyper based on Hypergeometric test. The significant levels of terms and pathways were corrected by Q value with a rigorous threshold (Q value ≤ 0.05) by Bonferroni.
The online Kaplan–Meier analysis of the overall survival (OS) and progression free survival (PFS) of all 719 LUAD patients with different SHP2 and other EGFR downstream genes expression were performed by the Kaplan–Meier Plotter (http://kmplot.com/analysis/) with the auto select best cutoff of patients sectionalization for significance was set to p < 0.001.
Cell Viability testing by MTT assay
Cell Viability was tested by MTT assay. Briefly, 2×103 cells were seeded in each well of 96-well plates with 200μL medium and incubated for time that indicated in the text. Then, 50 μl MTT solution was added to the bottom of wells. Supernatant discarded after 4 hours incubation, MTT formazan crystal was then dissolved in DMSO, and the absorbance was measured by a Multi-Mode Microplate Reader (Varioskan Flash, Thermo Scientific) at a wavelength of 490nm.
For soft agar colony formation testing, 1.5ml of 0.6% agarose was filled into the wells of 6-well plate and solidified in fume hood for 30 mins. lentivirus transduced and parental PC9, PC9GR cells were washed with phosphate buffered saline (PBS) and 2000 cells were suspended in 0.35% top agarose in DMEM supplemented with 10% FBS and plated on top of the bottom agarose. Clones were scored after 2 weeks of incubation.
For colony formation assays, lentivirus transduced PC9, PC9GR and parental cells were washed with PBS and plated at a cell density of 200 cells per well in DMEM supplemented with or without treatment, and clones were fixed with crystal violet and scored after 2 weeks of incubation.
For tumorsphere formation assays, lentivirus transduced and parental PC9, PC9GR cells were seeded in 1.2% agarose pre-coated 6-well plates at a density of 2000/ml in serum free DMEM/F12 medium (Gibco) with 10 ng/mL bFGF, 20 ng/mL EGF. The culture medium was changed every 2 days until the sphere generated. After 7 to 10 days culturing, the spheres were collected for further experiments.
Flow Cytometry Testing
1 x 106 cells/100μL was aliquot into FACS tubes. Added 5μL anti-CD133 antibody into each tube and vortex. And then incubated for 30 minutes at room temperature in the dark on ice. After centrifuging the suspended cells at 300 x g for 5 minutes, decant the buffer and re-suspend the cells with 2 mL of flow cytometry staining buffer. Repeat the wash for two times. Re-suspend the cells in 400μL of buffer for analysis (BD Bioscience, San Jose, CA, USA). Data was analyzed in FlowJo Software version 7.6 (Tree Star, Inc., Ashland, OR).
For ELISA analysis, 5×104 lentivirus transduced PC9, PC9GR and parental cells were seeded in 6-well plates for 48 hours. Culture supernate was collected to quantify the secreted CXCL8 using pre-coated ELISA kit according to the manufacturer’s protocol. Briefly, prepared CXCL8 dilute standard solution, sample or control (100 μL) was added to each well, and incubated at room temperature for 2 hours. After aspirated and washed each well four times with wash buffer, 100μL of CXCL8 conjugate was added into each well and incubated for 1 hour at room temperature. After four washes, 200μL of substrate solution was added to each well and the plate was incubate for 30 minutes at room temperature in dark. 50μL of stop solution was added to each well, and then determined the optical density of each well using a microplate reader set to 450nm. A standard curve was created for figuring the concentration of CXCL8.
Cell were lysed in protein lysis buffer containing protease and phosphatase inhibitors. Protein concentration was determined by the BCA Protein Assay Kit. Equal proteins were separated using electrophoresis by SDS-PAGE gels , and transferred to PVDF membrane. After blocking in 5% milk, PVDF membranes were incubated with specific antibodies against to targeted molecule,respectively. The bands were detected with enhanced chemiluminescence and quantified by Image J software.
For IHC examination, the tumors from homologous group were harvested, fixed, embedded in paraffin and examined for the further expression of the indicated proteins. Briefly, paraffin-embedded slides were dehydrated and antigen retrieved. Endogenous peroxidase was quenched by treatment with 3% hydrogen peroxide for 5 minutes. CD133, Ki67 were stained following the manufacturer`s protocol.
The animal protocols were approved by the Anhui Medical University Animal Care & Use Committee and conducted in accordance with the Guideline for laboratory animals of Anhui Medical University. Balb/c Nude mice were obtained from GemPharmtech Co., Ltd. (Nanjing, Jiangsu) and bred in SPF animal room with a positive pressure containment rack.
Tumorigenicity of SHP2 modified LUAD tumor cells in gradient concentration
Tumor cells were suspended into indicated solution (5 × 104, 5 × 103 and 5 × 102), and mixed with Matrigel (BD Biosciences) at the ratio of 1:1, injected into the lower flanks of 5 weeks Balb/c-Nude mice subcutaneously. The mice were monitored 2-3 times per week.
For testing the sensitivity of PC9 and PC9GR cells to Osimertinib. 1 × 107 cells were injected subcutaneously into the lower flanks of healthy 5 weeks old nude mice. Mice that bearing same tumor were randomly treated with DMSO and Osimertinib (5.0 mg/kg daily by oral gavage, S.C., 2 times/week for 3 week), treatment was began in the second week. The mice were monitored 2-3 times per week, and tumor curve was evaluated by formula: [length × (width)2] / 2.
For testing the sensitivity of SHP2 high and low expressed PC9GR cells to Osimertinib. 1 × 107 cells were injected subcutaneously into the lower flanks of healthy 5 weeks old nude mice. Mice that bearing same tumor were randomly treated with DMSO and Osimertinib (5.0 mg/kg daily by oral gavage, S.C., 2 times/week for 3 week), treatment was began in the second week. The mice were monitored 2-3 times per week, and tumor curve was evaluated by formula: [length × (width)2] / 2.
To identify the tumor formation ability of PC9GR Lv-SHP2 and Lv-SHP2RNAI cells in the condition of wild type and blockade of CXCL8-CXCR1/2. 1 × 107 cells were collected in PBS and injected S.C. into the lower flanks of healthy 5 weeks old nude mice. Mice that bearing same tumor were randomly separated in three groups, DMSO group, Danirixin (25mg/Kg, S.C., 3 times per week for 3 week) and Reparixin group (30mg/Kg, S.C., 3 times per week for 3 week), the treatment was began in the second week. The mice were monitored 2-3 times per week, and tumor curve was evaluated by formula: [length × (width)2] / 2.
Data in current experiment were represented by means ± SEM. For comparison between two groups, statistical significance was performed by Student's t-test, For comparison of more than two groups, statistical significance was performed by ANOVA. Statistical analyses were performed with GraphPad Prism 7 software. p values of <0.05 are considered statistically significant.