Isolation and culture of human ADSCs
Adipose tissues were obtained from healthy female donors, aged 18 to 30 years, undergoing an abdominal liposuction bariatric procedure with written informed consent. This study was approved by the Ethics Committee for Human and Animal Research of Wuhan University. All experiments were performed in accordance with Declaration of Helsinki and other recognized standards. After washing with phosphate buffered saline (PBS), these lipoaspirates were finely minced and enzymatically digested with 2 mg/mL type I collagenase at 37 °C for 40 min. The tissues were then centrifuged to remove buoyant adipocytes. The top layers were retrieved to obtain the stromal vascular fractions (SVFs) and cultured in Dulbecco's modified eagle medium (DMEM, Hyclone, USA) F12 supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic. ADSCs were obtained after 3-4 passages of SVFs cultured in the corresponding medium as reports.
Identification of ADSCs
The expression of stromal markers on cell surface of expanded ADSCs were confirmed using immunofluorescence analysis. ADSCs cultured on 14 mm slides in 24-well plates were fixed in 4% paraformaldehyde at room temperature for 30 minutes. After washing with PBS for 3 times, the cells were blocked with 10% goat serum and incubated with primary antibodies against CD31 (rat, Abcam, USA), CD45 (mouse, Santa Cruz, USA), CD90 (mouse, R&D, USA), and CD105 (rat, Santa Cruz, USA) at 4 °C overnight. After incubating with Alexa Fluor 488- and Cy3-conjugated secondary antibodies, 40,6-diamidino-2- phenylindole (DAPI, Sigma-Aldrich, USA) was used to stain the nuclei. The images were examined with a confocal microscope system (Nikon C2+ Confocal Microscope, Japan), and quantitatively evaluated using Image-Pro Plus 6.0 software to measure mean densities (IOD/area). For adipogenic differentiation, cells were seeded in 6-well plates. When full confluent, cells were cultured in DMEM/Ham’s F12 media supplemented with 10μg/ml transferrin, 0.85 μM insulin, 0.2nM triiodothyronine, 1μM dexamethasone, 500μM isobutylmethylxanthine for 2 weeks. For osteogenic differentiation, cells were seeded in 6-well plates and maintained in DMEM/Ham’s F12 media supplemented with 0.1mM dexamethasone, 50mM ascorbate-2-phosphate and 10mM b-glycerophosphate for 4 weeks.. Differentiated ADSCs were stained with oil red for 30 minutes or alizarin for 30 minutes.
Culture and transfection of human tumor cells
The human breast cancer cells (MDA-MB-468) and pancreatic cancer cells (ASPC1, Procell, China) were maintained in DMEM supplemented with 10% FBS. All cells were cultured at 37 °C in a humidified 5% CO2 incubator. FAM83H-AS1 shRNAs (sh-1: 5’-GGACAGAGTAGGAGCGTAACT-3’, sh-2: 5’-GCTGATTAGCAACCTTAGTTC-3’, sh-3: 5’-GCAAAGCACTCCTTCTATCAG-3’) were ligated into the Plko.1-Puro vector and packaged into lentiviruses (Viraltherapy, China). Lentiviral particles were used to infect the cancer cells to downregulate FAM83H-AS1 expression according to the manufacturer’s instruction. To investigate the effects of ADSCs on cancer cell behaviors, ADSCs were cultured on the transwell inserts (Corning, USA) with 0.4 μm pore polycarbonate membranes, and cancer cells were cultured on the bottom chamber of the plates.
Proliferation, cell cycle and colony formation assay
The breast and pancreatic cancer cells were cultured with or without ADSCs for 3 days and then trypsinized. And they were seeded (2×103 cells/well) in 96-well plates and cultured in complete medium. Cell viability was measured using Cell Counting Kit-8 (CCK8) every 12 hours. Cancer cells were cultured with or without ADSCs for 48 hours. After washing with PBS for 3 times, the cells were stained with propidium iodide (Multisciences, China) and analyzed by flow cytometer (Beckman, USA). For the colony formation assay, tumor cells were seeded (1-1.5×103 cells/well) in the bottom chamber of 6-well plates and maintained in complete medium for 2 weeks, ADSCs were cultured in the upper chamber. The cells were fixed with 4% paraformaldehyde for 2 hours, and stained with 1% crystal violet.
Xenograft tumor model
BALB/c nude mice aged 3 weeks were obtained from Beijing HFK Bioscience Co., Ltd. in Beijing, China. 1X106 MDA-MB-468/ASPC1 cells with or without 1X106 ADSCs were injected to each mouse. The mice were maintained in a temperature and humidity‐controlled and specific pathogen‐free environment in the laboratory animal facility of Zhongnan Hospital of Wuhan University. Tumor sizes were measured every 5 days until the end of the experiment. The experiments were performed under the protocols approved by ethnic committee of Zhongnan Hospital of Wuhan University.
Wound healing assay
The breast and pancreatic cancer cells were seeded in the bottom chamber of 6-well transwell plates with or without ADSCs cultured in the upper chamber. When full confluent, the cell layer was scratched with a 200 μl sterile pipette tip and washed with PBS. Images were acquired at different time points (0, 24, 48 h), and 3 independent experiments were conducted.
Modified Boyden chamber assay
The modified Boyden chamber assay was performed in 24-well transwell chamber systems (Corning, USA) with 8.0 µm pore size. The breast and pancreatic cancer cells were cultured with or without ADSCs for 3 days and then trypsinized. Cancer cells (5×104 cells/well) were seeded in the upper chamber insert and cultured in serum-free media. The lower chamber was filled with complete medium (600 μL, 10% FBS). After incubated at 37°C for 24 hours, the cells on the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with crystal violet. The membranes were placed under an inverted phase contrast microscope and imaged to count the migrated cells. Three independent experiments were conducted.
Quantitative real‐time polymerase chain reaction (qRT‐PCR)
The total RNA was extracted from the cancer cells using the RNeasy Mini Kit (Qiagen, Germany). Reverse transcription was performed using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, China). qRT-PCR was performed using the ChamQ SYBR qPCR Master Mix (Vazyme) with the CFX96TM Real-time PCR Detection System (Bio-Rad, USA). Relative expression was calculated using the 2 −ΔΔCt method, which was normalized to glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). All assays were performed in triplicates. The primer sequences used for PCR are listed as follows. FAM83H-AS1 forward: 5′-TAGGAAACGAGCGAGCCC-3′, and reverse: 5′-GCTTTGGGTCTCCCCTTCTT-3′; GAPDH forward: 5′-GGGAAACTGTGGCGTGAT-3′, and reverse: 5′-GAGTGGGTGTCGCTGTTGA-3′.
The breast and pancreatic cancer cells were lysed with RIPA extraction reagent (Beyotime, China) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich, USA). Total protein was separated using 10-12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to 0.45 μm PVDF membrane (Millipore, USA). Primary antibodies were E-cadherin, N-cadherin, Vimentin, cyclin D1, CDK4, CDK6, GAPDH (Proteintech, Chicago), p-IRE1, XBP1s (Abcam, UK), AKT, or p-AKT (Cell Signaling Technologies, USA) antibodies. Bands were visualized using an enhanced chemiluminescence (ECL) kit (Boster, China) and detected by ChemiDoc XRS+ Imaging System (Bio-Rad).
Student’s t test and one-way ANOVA were used to compare 2 and more groups respectively. Multiple comparison with Bonferroni correction was performed when appropriate. A P value < 0.05 was considered as statistically significant and all tests were two-tailed. All statistical tests were performed with Prism 7.0 (GraphPad, USA).