Introduction
Cell death is strictly regulated by complex intracellular and extracellular signals, and is essential for various biological processes, including homeostasis, development and disease. The imbalance between proliferation and death of cancer cells is the important basis that leads to malignant biological characteristics. Cancer cells have shown complicated strategies of metabolic adaptation to survive under metabolic stress conditions and to allow tumor progression, including blocking of apoptosis, as well as non-apoptotic cell death pathways [
1,
2]. Ferroptosis, a novel form of regulated cell death, involves iron-dependent lipid peroxides (lipid-ROS) accumulation and leads to lethal damage of cells [
3]. Recent studies have identified the essential role of ferroptosis in mediating tumor development and drug-resistance in some types of cancer, but the detailed molecular mechanism remains poorly understood [
4‐
7].
Tumor microenvironment (TME) consists of extracellular matrix and mesenchymal cell types, including fibroblasts, inflammatory cells, pericytes and endothelial cells [
8]. Cancer-associated fibroblasts (CAFs) are the main type of stromal cells in tumor microenvironment and exhibit distinct tumorigenic properties [
9‐
11]. CAFs, as well as the other tumor stromal cells, surrounding the primary foci generate signals or factors to regulate tumor phenotypes, displaying the capacity to influence each stage of tumor development [
12,
13].
Exosomes belong to the family of extracellular vesicles (EVs) with a typical diameter of 30–100 nm, and are secreted by most cell types [
14]. In the past decade, exosomes have been treated as the novel message transmitters in intercellular communication by delivering proteins, lipids, lncRNAs, circRNAs and microRNAs [
15,
16]. Recent studies indicated that exosomes derived from CAFs promote tumor metastasis and increase chemo-resistance of cancer cells [
17,
18]. Exosome-miR-21 derived from CAFs was found to be involved in oxaliplatin resistance in colorectal cancer [
19]; and CAF-secreted exo-miR-146a are proved to regulate survival and proliferation of pancreatic cancer cells [
20]. However, the potential roles of exosomes derived from CAFs in regulating lipid metabolism and ferroptosis in cancer cells have not been defined yet.
In this study, ferroptosis is found to be significantly inhibited in gastric cancer (GC), contributing to tumor growth and decreased sensitivity to cisplatin and paclitaxel. And arachidonate lipoxygenase 15 (ALOX15), the main mediator of lipid-ROS production in GC cells, is observably down-regulated and is found to be closely linked with the suppression of ferroptosis. Moreover, exosomal miR-522 secreted from CAFs plays a dominant role in regulating ALOX15 expression in GC cells. Additionally, we demonstrated that ubiquitin-specific protease 7 (USP7) and recombinant human heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) are involved in the process of miR-522 sorting into exosomes. Therefore, this study identifies a novel network mediated by exosomes that regulates ferroptosis of cancer cells, and provides novel methods to enhance chemo-sensitivity of gastric cancer.
Methods
Human tissues
The tumor tissue samples and plasma samples of GC patients were obtained from Tianjin Medical University Cancer Institute and Hospital.
Animals
Male nude mice (BALB/c-nu, 6B8 weeks) were housed in a pathogen free animal facility with access to water and food, and allowed to eat and drink adlibitum.
Cell culture
Human gastric cancer cell lines, SGC7901 (human gastric adenocarcinoma cell), MGC803 cells and MKN45 cells were bought from cell bank of Chinese Academy of Sciences (Shanghai, China), and were cultured in DMEM medium (Gibco, USA) supplemented with 10% fetal bovine serum. Each cell line was tested for mycoplasma contamination before use.
Isolation of exosomes from medium and plasma
Exosomes in medium and plasma were isolated from cell by differential centrifugation, according to previous publications [
21]. After removing cells and other debris by centrifugation at 300 g and 3000 g respectively, the supernatant was centrifuged at 10,000 g for 30 min to remove shedding vesicles and the other vesicles with larger sizes. Finally, the supernatant was centrifuged at 110,000 g for 70 min, and exosomes were collected from the pellet and re-suspended in PBS (all steps were performed at 4 °C).
Transmission electron microscopy assay (TEM)
For conventional TEM, the exosome pellet was placed in a droplet of 2.5% glutaraldehyde in PBS buffer at pH 7.2 and fixed overnight at 4 °C. Samples were rinsed in PBS buffer (3 times, 10 min each) and post-fixed in 1% osmium tetroxide for 60 min at room temperature. The samples were then embedded in 10% gelatin and fixed in glutaraldehyde at 4 °C and cut into several blocks (less than 1 mm3). The samples were dehydrated for 10 min each step in increasing concentrations of alcohol (30, 50, 70, 90, 95, and 100% × 3). Pure alcohol was then exchanged by propylene oxide, and specimens were infiltrated with increasing concentrations (25, 50, 75, and 100%) of Quetol-812 epoxy resin mixed with propylene oxide for a minimum of 3 h per step. Samples were embedded in pure, fresh Quetol-812 epoxy resin and polymerized at 35 °C for 12 h, 45 °C for 12 h, and 60 °C for 24 h. Ultrathin sections (100 nm) were cut using a Leica UC6 ultra-microtome and post-stained with uranyl acetate for 10 min and with lead citrate for 5 min at room temperature before observation in a FEI Tecnai T20 transmission electron microscope, operated at 120 kV.
Nanoparticle tracking analysis (NTA)
The size and density of exosomes were directly tracked using the Nanosight NS 300 system (NanoSight technology, Malvern, UK) [
22,
23]. Exosomes were re-suspended in PBS at a concentration of 5 μg/mL were further diluted 100- to 500-fold to achieve between 20 and 100 objects per frame. Samples were manually injected into the sample chamber at ambient temperature. Each sample was configured with a 488 nm laser and a high-sensitivity sCMOS camera, and was measured in triplicate at camera setting 13 with an acquisition time of 30s and a detection threshold setting of 7. At least 200 completed tracks were analyzed per video. Finally, data was analyzed using the NTA analytical software (version 2.3).
Mass Spectrum analysis
LC-MS/MS analysis was performed on a Q Exactive mass spectrometer (Thermo Scientific) that was coupled to Easy nLC (Proxeon Biosystems, now Thermo Fisher Scientific) for 60/120/240 min (determined by project proposal). The mass spectrometer was operated in positive ion mode. MS data was acquired using a data-dependent top10 method dynamically choosing the most abundant precursor ions from the survey scan (300–1800 m/z) for HCD fragmentation. Automatic gain control (AGC) target was set to 3e6, and maximum inject time to 10 ms. Dynamic exclusion duration was 40.0 s. Survey scans were acquired at a resolution of 70,000 at m/z 200 and resolution for HCD spectra was set to 17,500 at m/z 200, and isolation width was 2 m/z.. Normalized collision energy was 30 eV and the underfill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as 0.1%. The instrument was run with peptide recognition mode enabled. The MS data were analyzed using MaxQuant software version 1.5.3.17 (Max Planck Institute of Biochemistry in Martinsried, Germany) [
24].
Isolation of CAFs
CAFs and primary cancer cells were isolated from gastric tumor tissues by primary culture, while NFs were derived from the paired adjacent normal tissues [
25]. The paired NFs and CAFs were further identified by the presence of CAF-specific markers (α-SMA, FAP, and FSP1).
Determination of lipid ROS levels
To determine the levels of lipid-ROS, cells were seeded in 6-well plate, and the culture media was replaced by serum-free media containing 10 μmol/L 2′,7′-dichlorodihydrofluorescein diacetate (Sigma) and placed in dark for 30 min, gently shaken every 5 min. Cells were harvested by centrifuging at 1000 rpm for 5 min and washed 3 times with serum-free media followed by re-suspending in serum-free media, then incubated with 5 μl 7-aminoactinomycin D (KeyGEN Bio-TECH, Nanjing, China) in dark for 5 min. Fluorescence was determined at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The average intensity of fluorescence in each group indicated the amount of ROS within cells.
Immunofluorescence
Cells were cultured on four-well chamber slides. At the time of harvest, cells were fixed with 4% paraformaldehyde and then permeabilized with 0.01% Triton X-100 for 10 min. Then cells were treated with anti-α-SMA antibody (Abcam, ab124964), anti-FAP antibody (Abcam, ab53066), and anti-FSP1 (Abcam, ab124805). In addition, all samples were treated with 40, 6-diamidino-2-phenylindole dye for nuclear staining (358 nm). For confocal microscopy, a Nikon C2 Plus confocal microscope was used.
Determination of cell death
Cells death was determined by using PI (Roche) assay. Briefly, cells were seeded in a 12-well plate and treated with indicated drugs. Then cells were harvested and stained with 2 μg/ml PI. Dead cells (PI-positive cells) were analyzed using a BD Accuri C6 flow cytometer (BD Biosciences).
Mitochondrial membrane potential (MMP)
Cells were seeded in a 6-well plate, and 0.5 mM TMRE was added and incubated for 30 min. Excess TMRE was removed by washing the cells with PBS. Labeled cells were trypsinized and resuspended in PBS plus 2% FBS. Fluorescence at Ex/Em = 549/575 nm was analyzed using a flow cytometer.
RNA isolation and quantitative RT-PCR
Assays to quantify mature miRNAs were conducted as previously described with slight modifications [
26,
27]. Total RNA was extracted from the cultured cells and tissues using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. miR-522 determination was performed using Taqman microRNA probes (Catalog number: 4427975, ThermoFisher, US). All of the reactions were run in triplicate. After the reactions were complete, the cycle threshold (C
T) data were determined using fixed threshold settings, and the mean C
T was determined from triplicate PCRs. A comparative C
T method was used to compare each condition to the control reactions. U6 snRNA was used as an internal control of miRNAs, and mRNA levels were normalized to GAPDH. The relative amount of gene normalized to control was calculated with the eq. 2
-ΔCT, in which ΔC
T = C
T gene-C
T control.
The miRNA target prediction
Western blotting
The ALOX15, USP7 and hnRNPA1 expression were assessed by western blotting analysis and samples were normalized to GAPDH. Protein extraction was blocked with PBS-5% fat-free dried milk at room temperature for 1 h and incubated at 4 °C overnight with anti-ALOX15 (1:1000, Santa cruz), anti-hnRNPA1 (1:1000, Santa cruz), anti-CD63 (1:2000, Abcam), anti-TSG101 (1:1000, Santa Cruz), anti-Alix (1: 1000, Santa Cruz), anti-ubiquitin (1:1000, Santa Cruz), anti-α-SMA (1: 1000, Abcam), anti-FAP (1: 1000, Abcam), andti-FSP1 (1: 1000, Abcam), anti-CEA (1:1000, Abcam), anti-CK-18 (1:1000, Abcam) and anti-GAPDH (1:3000, Santa Cruz) antibodies respectively.
Immuno-precipitation
Immuno-precipitation using anti-hnRNPA1 antibody was performed at 48 h or 72 h after treatment. Cells were lysated by the lysis buffer containing 150 mM KCl, 25 mM Tris-HCl, pH 7.4, 5 mM EDTA, 0.5% Triton X-100, 5 mM dithiothreitol (DTT), PMSF and cocktail. The supernatant was mixed with hnRNPA1 antibody (Abcam) overnight at 4 °C, and then co-cultured with beads (santa cruz) for 2–4 h at RT. The beads were washed five times in lysis buffer followed by western blotting (WB) analysis.
Biotin miRNA pull-down assay
The cell lysates and exosomal lysates of CAFs were incubated overnight at 4 °C, with 100 pmol of synthetic single-stranded miR-522 or controlled miR-24 oligonucleotides containing a biotin modification. Agarose beads (Invitrogen, USA) were added to each binding reaction, which was further incubated at 4 °C for 4 h. Precipitates were washed five times and boiled in SDS buffer, followed by western blotting analysis or RT-qPCR analysis.
Luciferase assay
The reporter plasmid p-MIR-ALOX15 containing the predicted miR-522 targeting regions was designed by Genescript (Nanjing, China). Part of the wild-type and mutated 3′-UTR of ALOX15 was cloned immediately downstream of the firefly luciferase reporter. The 2 mg of beta-galactosidase expression vector (Ambion) was used as a transfection control. For the subsequent luciferase reporter assays, 2 mg of firefly luciferase reporter plasmid, 2 mg of beta-galactosidase vector and equal doses (200 pmol) of mimics, inhibitors or scrambled negative control RNA were transfected into the prepared cells. At 24 h after transfection, cells were analyzed using the Dual Luciferase Assay Kit (Promega) according to the manufacturer’s instructions. Each sample was prepared in triplicate and the entire experiment was repeated three times.
Immunohistochemistry (IHC)
The tumors were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned and then stained with corresponding antibodies (Abcam). Quantitative analysis was conducted by quantifying the fluorescence intensity from at least five sections.
Hematoxylin-eosin (HE) staining
Tissues were fixed in 10% formalin, processed and embedded in paraffin. Multiple sections (10 μm in thickness) were prepared and stained with haematoxylin and eosin for morphological observation.
Establishment of tumor in nude mice
Equal number of SGC7901 cells and primary CAFs isolated from GC tissues were injected into nude mice by orthotopic implantation. Briefly, 5 × 105 SGC7901cells and CAFs were injected subcutaneously for one mouse. These tumor-implanted mice were injected with either cisplatin (5 μg/g) or saline every 5 days since the day ten, and were sacrificed and tumors were removed at the 30th Day.
Statistics
All experiments were performed in triplicate, and the results are presented as the mean value ± standard deviation. The data were statistically analyzed using Student’s t-test in SPSS statistical software, with p < 0.05 considered statistically significant. * indicates p < 0.05; ** indicates p < 0.01 and *** indicates p < 0.001.
Discussion
Ferroptosis, the novel form of non-apoptotic regulated cell death, has been characterized by the accumulation of lipid peroxidation products in a cellular-iron dependent manner [
3,
39,
40]. Recent studies indicated that activation of ferroptosis related pathways effectively prevents tumor progression and enhances effects of chemo-therapy, targeted therapy and even immunotherapy [
4,
7,
41]. The benign cells in microenvironment of solid tumors generally contribute to tumor development and drug resistance [
42‐
44]; but CD8
+ T cells are found to promote tumor ferroptosis during cancer immunotherapy in recent study [
41]. However, the potential roles of the other stromal cells, especially CAFs, in regulating ferroptosis of tumor cells are still blank.
The family of arachidonate lipoxygenases is regarded as the key mediator of lipid peroxidation production and eventually lead to ferroptosis, though the other types of dioxygenases are also reported to participate in this process [
28,
45,
46]. Furthermore, both the inhibition of cysteine intake by and the inactivation of the phospholipid peroxidase glutathione peroxidase 4 (GPX4) result in overwhelming lipid peroxidation that causes cell death [
47,
48]. Therefore, lipid ROS maintains a dynamic balance in cancer cells to avoid ferroptosis. Previous studies have focused on the potential use of GPX4 as a therapeutic target [
48,
49], but the mechanism of how ALOX15 expression is suppressed remains poorly understood. The current study suggested that CAF-derived exosomes play a key role in the regulation of ALOX15 expression and lipid-ROS production in cancer cells, proving that exosomes in tumor associated microenvironment are linked with ferroptosis for the first time.
Chemo-therapy is the main method of treatment for advanced cancers, and cisplatin and paclitaxel are the first-line chemotherapeutic drugs in GC. But resistance to cisplatin and paclitaxel has become increasingly severe in GC treatment [
50‐
52]. Resistance to chemo-therapy is generally associated with DNA damage repair, mutations of the molecules regulating cell apoptosis and increased levels of glutathione (GSH) [
53,
54]. Here, we show that changes on ferroptosis-related signaling pathway may provide a new idea to reverse chemotherapy resistance. Exosomes can promote the development of chemo-resistance in tumor cells, and the in-depth understanding of the mechanisms involved in drug resistance would contribute to improve therapeutic effects as well as prognosis. In this study, we demonstrated that the block of CAF-exosomes mediated lipid-ROS inhibition leads to increased ferroptosis levels in cancer cells, which enhanced sensitivity of chemo-therapy. Further studies are needed to learn more about the effects of ferroptosis in the other strategies of GC treatment, especially targeted therapy.
Our study suggested that USP7 promotes miR-522 secretion from CAFs through regulating deubiquitination on hnRNPA1, which is specifically involved in miR-522 packaging into exosomes. Knockdown of USP7 or hnRNPA1 sharply decreases miR-522 levels in microenvironment, causing elevated cell death and improved chemo-sensitivity. Hence, inhibiting the secretion of specific miRNA from CAFs serves as a novel method for the clinical therapy of GC.
However, none of the ferroptosis-related genes described in this study are classical tumor-promoting factors. Since miR-522 plays a key role in mediating the down-regulation of ALOX15 and lipid-ROS, resulting in suppressed cell death, we believe that miR-522 serve as the tumor-driving factor. Although USP7, hnRNPA1 and miR-522 are proved to be up-regulated in gastric cancer, they are also widely expressed in normal tissues. Given that the expression and modification of these genes are more complicated in human body, there is still a long way before these results can be used for clinical treatment.
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