Abstract

   Although oxidative stress is closely associated with tumor invasion and
   metastasis, its’ exact role and mechanism in the initial stage of oral
   cancer remain ambiguous. Glutamine uptake mediated by
   alanine-serine-cysteine transporter 2 (ASCT2) participates in
   glutathione synthesis to resolve oxidative stress. Currently, we
   firstly found that ASCT2 deletion caused oxidative stress in oral
   mucosa and promoted oral carcinogenesis induced by
   4-Nitroquinoline-1-oxide (4-NQO) using transgenic mice of ASCT2
   knockout in oral epithelium. Subsequently, we identified an upregulated
   gene Thbs1 linked to macrophage infiltration by mRNA sequencing and
   immunohistochemistry. Importantly, multiplex immunohistochemistry
   showed M1-like tumor-associated macrophages (TAMs) were enriched in
   cancerous area. Mechanically, targeted ASCT2 effectively curbed
   glutamine uptake and caused intracellular reactive oxygen species (ROS)
   accumulation, which upregulated Thbs1 in oral keratinocytes and then
   activated p38, Akt and SAPK/JNK signaling to polarize M1-like TAMs via
   exosome-transferred pathway. Moreover, we demonstrated M1-like TAMs
   promoted malignant progression of oral squamous cell carcinoma (OSCC)
   both in vitro and in vivo by a DOK transformed cell line induced by
   4-NQO. All these results establish that oxidative stress triggered by
   ASCT2 deletion promotes oral carcinogenesis through Thbs1-mediated M1
   polarization, and indicate that restore redox homeostasis is a new
   approach to prevent malignant progression of oral potentially malignant
   disorders.

   Keywords: ROS, ASCT2, Thbs1, Macrophages, Oral carcinogenesis

Graphical abstract

   [37]Image 1
   [38]Open in a new tab

Highlights

     * •
       ASCT2 knockout induced oxidative stress in oral epithelium and
       promoted oral carcinogenesis.
     * •
       ASCT2 knockout simultaneously promoted high expression of Thbs1 and
       enrichment of M1-like TAMs in the cancerous sites.
     * •
       Oxidative stress caused by ASCT2 knockdown promoted the
       polarization of M1-like TAMs through exosome-transferred Thbs1.
     * •
       M1-like TAMs accelerated oral carcinogenesis by enhancing cell
       proliferation, stemness, migration and invasion.

1. Introduction

   Oxidative stress is defined as deregulation of reactive oxygen species
   (ROS) production and ROS limitation, characterized by unspecific
   oxidation of biomolecules and alteration of normal physiological
   function. Aberrant redox homeostasis plays important roles in
   regulating diverse aspects of cell behavior (e.g., proliferation,
   differentiation, migration and angiogenesis) [[39]1]. It has been
   proven that oxidative stress is closely associated with tumor
   progression. On the one hand, oxidative stress triggers tumor
   development by causing DNA damage and genomic instability
   [[40]2,[41]3]; on the other hand, the rapid growth of tumor enhances
   ROS production [[42]4,[43]5]. However, it remains obscure on the role
   and mechanism of oxidative stress in the initiation of tumor
   carcinogenesis.

   Glutamine (Gln) is the most abundant and widely used non-essential
   amino acid in the human body, which involves in tricarboxylic acid
   (TCA) cycle and inter-organ nitrogen exchange [[44]6]. In addition, Gln
   can balance redox homeostasis by participating in de novo biosynthesis
   of glutathione (GSH) to cope with oxidative stress [[45][7], [46][8],
   [47][9]]. Alanine-serine-cysteine transporter 2 (ASCT2, encoded by gene
   Slc1a5) has been acknowledged as the major Gln transporter
   [[48]10,[49]11], although there have been numerous membrane
   transporters generally recognized as four Solute Carrier (SLC) families
   (SLC1, SLC6, SLC7 and SLC38) [[50]12,[51]13]. ASCT2 is an obligatory
   Na^+-dependent amino acid transporter which mediates the cotransport of
   Na^+ with Gln or other neutral amino acids (alanine, serine and
   cysteine) to support the biosynthetic pathways [[52]12,[53]14]. There
   have been numerous studies showed that targeted interference with
   ASCT2-mediated Gln uptake significantly suppressed Gln metabolism,
   especially GSH production, which contributed to redox imbalance and
   induced cellular oxidative stress [[54][15], [55][16], [56][17],
   [57][18]].

   Oral leukoplakia (OLK) is one of the most common oral potentially
   malignant disorders (OPMDs) in oral mucosa and has the potential to
   develop into oral squamous cell carcinoma (OSCC) which affects nearly
   377713 people worldwide in 2020 [[58][19], [59][20], [60][21]].
   Although the etiology and anatomical locations are varied, a hallmark
   of OSCC is redox imbalance characterized by decreased antioxidant and
   increased oxidant [[61]22,[62]23]. 4-Nitroquinoline-1-oxide (4-NQO) is
   a synthetic quinolone-derived molecule which is used to mimic the
   carcinogenic effect of tobacco [[63]21,[64]24]. It can induce
   intracellular oxidative stress, leading to the production of ROS
   metabolic substances that bind to DNA and generating DNA adducts
   [[65]24,[66]25]. So far, the 4-NQO-induced mouse model remains the most
   ideal model that accurately reflects the process of oral
   carcinogenesis.

   In current study, we firstly induced oxidative stress in oral mucosa by
   utilizing ASCT2 conditional knockout mice, and subsequently found that
   oxidative stress promoted oral mucosa carcinogenesis. Mechanically, it
   was associated with the polarization of M1-like tumor-associated
   macrophages (TAMs) activated by exosome-transferred Thbs1. Altogether,
   the results established restoring redox homeostasis as a new approach
   targeting ROS-Thbs1-M1 pathway to prevent oral carcinogenesis.

2. Materials and method

2.1. Transformation of human dysplastic oral keratinocyte cells by 4-NQO and
RNA transfection

   The human dysplastic oral keratinocytes (DOK) were obtained from Dr.
   Juan Xia (Hospital of Stomatology, Guanghua School of Stomatology, Sun
   Yat-sen University). DOK cells were cultured in high-glucose DMEM
   (Gibco, NY, USA) supplemented with 10 % fetal bovine serum (FBS, Gibco,
   NY, USA) and 5 μg/mL hydrocortisone (Sigma-Aldrich, St. Louis, MO,
   USA).

   DOK cells were transformed by 4-NQO (Sigma-Aldrich, St. Louis, MO, USA)
   in our laboratory. Cultures were exposed to 2.6 μM 4-NQO for 3 h as
   described previously [[67]26]. After passed 30 generations of
   intermittently 4-NQO-treatment over 2 months, the DOK-4-NQO cells were
   expanded for tumorgenicity in vitro and in vivo.

   To generate stable silenced DOK cells, shRNA lentiviral vectors
   targeting ASCT2 were purchased from Genechem Co., Ltd (Shanghai, China)
   and transfected into DOK cells with corresponding control vectors
   according to the manufacturer's protocols. After transfection, DOK
   cells were incubated in conditioned medium (CM) with 2 μg/mL puromycin
   (Aladdin Co., Ltd, Shanghai, China) to select the stably transfected
   cells. The sequences of the shRNAs were presented in [68]Table S1
   (Supporting information).

   To determine the function of Thbs1 in ASCT2 knockdown DOK cells, small
   interfering RNA (siRNA) oligonucleotide (Hanbio Co., Ltd, Shanghai,
   China) was used. RNAFit Transfection Reagent (Hanbio Co., Ltd,
   Shanghai, China) was used to facilitate the transfection of siRNA
   oligonucleotides into the targeted cells in accordance with
   manufacturer's instruction. The Thbs1 siRNA sequences were listed in
   [69]Table S1 (Supporting information).

2.2. Human macrophage differentiation in vitro

   The human monocyte cells THP-1 were provided as a gift from Dr.
   Zhengmei Lin (Hospital of Stomatology, Guanghua School of Stomatology,
   Sun Yat-sen University). THP-1 cells were maintained in RPMI 1640
   medium (Gibco, NY, USA) with 10 % FBS and 5 mM β-mercaptoethanol
   (Macklin Co., Ltd, Shanghai, China). M0 macrophages were differentiated
   from THP-1 cells using 100 ng/mL phorbol 12-myristate 13-acetate (PMA,
   Sigma-Aldrich, St. Louis, MO, USA) for 48 h. Then, the THP-1-derived
   macrophages were further differentiated into M1-like cells cultured in
   medium with 20 ng/mL IFN-γ (PeproTech Inc., NJ, USA) and 100 ng/mL LPS
   (Absin Bioscience Inc., Shanghai, China), or differentiated into
   M2-like cells cultured in medium with 20 ng/mL IL-4 (PeproTech Inc.,
   NJ, USA) and 20 ng/mL IL-13 (PeproTech Inc., NJ, USA) for 48 h. To
   obtain conditioned medium (CM) of M0 or M1 macrophages, cells, about
   70%–80 % confluence were washed and cultured for additional 24 h with
   fresh RPMI 1640 medium with 10 % FBS and 5 mM β-mercaptoethanol. Then
   the supernatants were centrifuged at 3000 g (4 °C) for 15 min and
   finally were filtrated through 0.22 μm Filter Units (Merk Millipore,
   MA, USA).

2.3. Animal models

   Slc1a5^tm1.1cyagen mice (CKOCMP’-02923-Slc1a5) were purchased from
   Cyagen Biosciences Inc (SF, USA). K14-CreER^tam mice (JAX-005107) were
   kindly given as a gift by Dr. Juan Xia (Hospital of Stomatology,
   Guanghua School of Stomatology, Sun Yat-sen University), which
   expressed Cre recombinase in squamous epithelium under the control of
   the K14 promoter. Slc1a5^f/f Cre ^± mice were generated by crossing the
   floxed Slc1a5 allele Slc1a5^tm1.1cyagen (Slc1a5^f/f) mice with
   K14-CreER^tam (K14-Cre^+/−) mice. Genotype of transgenic mice were
   identified by the One Step Geno Typing Kit (Vazyme Biotech Co, Nanjing,
   China) following the manufacturer's protocol.

   For acquiring Slc1a5 conditional knockout mice, Slc1a5^f/f Cre ^± mice
   were intraperitoneally injected with tamoxifen (TAM, Sigma Aldrich, St.
   Louis, MO, USA) five times every other day at a dose of 120 mg/kg. The
   PCR primers of genotyping were provided in [70]Table S2 (Supporting
   information).

   For mouse model of 4-NQO-induced oral carcinogenesis, 4-NQO was
   dissolved in propylene glycol (Macklin, Shanghai, China) to prepare
   stock solution (1 mg/mL) and further diluted to working solution
   (100 μg/mL) in drinking water every week. For inducing oral
   carcinogenesis, mice with the same gene background (Slc1a5^f/f Cre^+/−)
   were fed with working solution for 16 weeks and maintained with regular
   drinking water for a further 6 weeks. All animal experiments were
   approved by the Laboratory Animals Ethics Committee of Sun Yat-sen
   University (SYSU-IACUC-2019-000228).

   For xenograft model, male BALB/c nude mice (nu/nu, aged 3–5weeks) were
   purchased from the Laboratory Animal Center of Sun Yat-sen University
   (Guangzhou, China). In brief, 1*10^6 DOK or DOK-4-NQO cells/per site
   were subcutaneously injected under the skin of the axillary to
   establish the nude mouse xenograft tumor models (n ≥ 6 for each group).
   For assessing the effects of CM of macrophages on the tumorigenesis of
   DOK-4-NQO cells, mice with xenografts were intraperitoneally injected
   with 500 μL CM from M1-like TAMs, M0 or RPMI 1640 every other day.
   Tumor volumes (length * width^2/2) were monitored and compared. The
   above animal experiments were approved by the Laboratory Animals Ethics
   Committee of Sun Yat-sen University (SYSU-IACUC-2023-001462,
   SYSU-IACUC-2024-000773).

2.4. Histological hematoxylin-eosin (HE) saining and immunohistochemistry
(IHC)

   For tissue HE staining, tissues were dissected and fixed overnight with
   4 % paraformaldehyde (Biosharp, Chongqing, China). Samples were then
   embedded in paraffin. For the HE staining of cells, cells were just
   fixed with 4 % paraformaldehyde for 15 min. Paraffin sections with 4 mm
   thick or cells were prepared and stained with hematoxylin and eosin
   (Servicebio Co, Ltd, Wuhan, China) for histology. IHC was carried out
   in accordance with the manufacturer's guidelines of the IHC Kit (Gene
   Tech, Shanghai, China) and quantified as our previous published methods
   [[71]17,[72]27]. The antibodies used in IHCassays were listed in
   [73]Table S3 (Supporting information).

2.5. Western blot

   The protocol was conducted according to the previous studies
   [[74]17,[75]28]. The primary antibodies used were listed in [76]Table
   S3 (Supporting information). The secondary antibodies were anti-rabbit
   or anti-mouse HRP-conjugated antibodies (1:5000, EMAR, Beijing, China).
   The bands were visualized by chemiluminescent HRP substrate (Millipore,
   MA, USA).

2.6. Tissue immunofluorescence (IF) staining and multiplex IHC (mIHC)

   Tissue IF was conducted as previously described [[77]27]. In short,
   sections were first incubated with primary antibodies against ASCT2,
   8-Hydroxyl-2-deoxyguanosine (8-OHdG) or γ-H2AX which was from DNA
   Damage Assay Kit (C2035S, Beyotime, Shanghai, China). Then these
   sections were incubated with corresponding fluorochrome-conjugated
   secondary antibodies for 1 h. Sections were then counterstained with
   DAPI (Solarbio, Beijing, China) and evaluated in an invert laser
   confocal microscope (Olympus FV3000, TKY, Japan). mIHC was performed
   using a Neon DendronFluor Kit (Histova Biotechnology Co, Ltd, Jilin,
   China) as previously described [[78]27]. The antibodies used in these
   sections were listed in [79]Table S3 (Supporting information).

2.7. Assessment of oxidative stress in tissue and cells

   Visualization of ROS in tongue epithelium samples was conducted by
   staining for oxidation of cytosolic dihydroethidium (DHE) (Applygen,
   Beijing, China) as described previously [[80]29]. In brief, 10 μm
   tongue cryosections were air-dried for 30 min and then incubated with
   0.5 μM DHE for 30 min at 37 °C. Then the sections were incubated with
   primary antibody against ASCT2 overnight at 4 °C, which were followed
   by incubation with fluorochrome-conjugated secondary antibody Alexa
   Flour488 for 1 h at room temperature. Sections were next counterstained
   with DAPI and evaluated in Olympus FV3000. The production of H[2]O[2]
   in tongue mucosa epithelium was assessed by H[2]O[2] Content Assay Kit
   (Solarbio, Beijing, China) according to the manufacture's guideline.
   GSH/GSSG ratio in tongue mucosa epithelium was determined by using GSH
   and GSSG Assay Kit (Beyotime, Shanghai, China) as the instruction
   described. For assessing ROS level in cells, intracellular ROS were
   detected using the DHE Kit (Applygen, Beijing, China) by flow cytometry
   as we previously described [[81]17,[82]27].

2.8. RNA sequencing analysis

   For mouse model of 4-NQO-induced oral carcinogenesis, mouse tongues
   were treated with dispase Ⅱ enzyme (Roche, Basel, Switzerland) to
   obtain tongue mucosa epithelium for RNA-Seq analysis, which was
   conducted at OE biotech Co., Ltd. (Shanghai, China). The libraries were
   sequenced on an Illumina HiSeq X Ten system and 125/150bp paired-end
   reads were generated. Raw data (raw reads) of the fastq format were
   first processed using Trimmomatic. Then about 40–55 million clean reads
   for each sample were acquired by abolishing reads containing adapter or
   ploy-N and low-quality reads. The clean reads were next mapped to the
   human genome using HISAT2. The fragments per kb of transcript per
   million reads (FPKMs) value of each gene were calculated using
   Cufflinks, and the read counts of each gene were obtained by
   HTSeq-count. Differentially expressed genes (DEGs) analysis was
   performed using the DESeq (2012) R package. A P value of <0.05 and a
   fold change of >2 or <0.5 were set as the threshold for significantly
   differential expression. Hierarchical cluster analysis of
   differentially expressed genes was conducted to analyze transcript
   expression patterns. Gene ontology enrichment (GO) and Kyoto
   Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of
   DEGs were performed using R based on the hypergeometric distribution.

2.9. Bioinformatics analysis

   Bioinformatics analysis was conducted on the basis of the TCGA cohort
   of head and neck squamous cell carcinoma (HNSCC). Gene expression data
   of corresponding number pairs of adjacent and cancer samples was
   extracted to analyze the Thbs1 expression in HNSCC samples. The
   survival data was analyzed using the “survival” (v3.3.1) R package. K-M
   survival curve analysis was used to identify the survival outcomes of
   HNSCC patients based on the Thbs1 expression levels. The ssGSEA
   algorithm in the “GSVA” (v1.46.0) R package [[83]30] was used to assess
   the tumor infiltration status of 24 immune cell types [[84]31].
   Besides, we used the spearman's correlation analysis to evaluate the
   relationship between Thbs1 expression level and the immune cell
   infiltration status.

2.10. Flow cytometry

   For cell analysis, Zombie NIR™ Fixable Viability Kit (Biolegend, CA,
   USA, diluted at 1:500) was used to label dead cells at room temperature
   for 30 min firstly. Then, cells were incubated with specific antibodies
   for 30 min on ice. Data was acquired on LSRFortessa (BD Bioscience, NJ,
   USA) and was analyzed using Flowjo software (BD Bioscience, NJ, USA).
   The antibodies were listed in [85]Table S3 (Supporting information).

2.11. RNA extraction and quantitative real-time PCR (qPCR)

   Total RNA was isolated from cells with RNA-Quick Purification kit (ES
   Science, Shanghai, China). RNA concentration was measured by NanoDrop
   One (Thermo Fisher Scientific Inc., CA, USA). HiScript Ⅲ RT SuperMix
   for qPCR kit (Vazyme Biotech Co., Nanjing, China) was used to
   reversed-transcribe 1 μg of RNA to acquire cDNA. Quantitative RT-PCR
   was conducted to quantify gene expression level of β-actin, ASCT2 and
   Thbs1 by applying ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech
   Co., Nanjing, China) and quantified by 2^−ΔΔCT method. The primers used
   in this study, purchased from GeneRay biotechnology (Guangzhou, China)
   were listed in [86]Table S1 (Supporting information).

2.12. Exosome isolation, characterization and tracing

   Exosomes were isolated from the cell culture supernatants by
   ultracentrifugation. In brief, equal amounts of DOK cells treated
   differently were seeded on 15-cm dishes and cultured in high glucose
   DMEM medium with 10 % exosome-free FBS for 48 h. These supernatants
   were collected and centrifuged at 3000 g for 30 min at 4 °C to abolish
   dead cells and cell fragments, and then filtered through a 0.22 μm
   filter (Merk Millipore, MA, USA). Then the solution was
   ultracentrifuged at 110000 g for 2 h at 4 °C. The precipitate was
   collected and washed using sterilized PBS (Solarbio, Beijing, China),
   followed by next ultracentrifugation at 110000 g for 2 h at 4 °C. The
   pellets of exosome were resuspended with 200 μL PBS.

   The morphology and size of exosomes were determined by transmission
   electron microscopy (TEM, Hitachi, Tokyo, Japan). The size distribution
   and concentration were examined by nano flow cytometry (nanoFCM,
   Agilent, CA, USA). The expression of exosome markers (CD63, CD81,
   TSG101 and Histone 3) was analyzed by WB. Exosome-tracing experiments
   were conducted by EvLINK 505 Exosome-labeling Kit (Tingo Regenerative
   Medicine, Tianjin, China) according to the manufacturer's instruction.
   Exosomes labeled with fluorescent were incubated with THP-1 cells for
   24 h. Then, cells were fixed in 4 % paraformaldehyde and permeabilized
   in 0.25 % Triton X-100 (Solarbio, Beijing, China) for 30 min
   respectively. Cells were next incubated with primary antibody against
   β-Tubulin overnight at 4 °C, followed by incubated with Alex Fluor 594
   fluorescent-conjugated secondary antibody for 1 h and counterstained
   with DAPI. The cells were finally observed by Olympus FV3000. The
   antibodies were listed in [87]Table S3 (Supporting information).
   Exosomes were incubated with recipient cells at a concentration of
   50 μg exosome protein/10^5 cells in following experiments.

2.13. Glutamine uptake assay

   ^3H-glutamine (Perikin Elmer, MA, USA, NET55120UC) was used to examine
   glutamine uptake as we previously described [[88]16].

2.14. Metabolite determination

   Whole cell lysates of DOK cells were prepared for determined
   a-ketoglutarate (α-KG), citrate, malate, fumarate and succinate levels
   using the following ELISA kits: Human a-KG ELISA Kit (Jianglaibio, Co.,
   Ltd, Shanghai, China), Human Citrate ELISA Kit (Jianglaibio, Co., Ltd,
   Shanghai, China), Human Malate ELISA Kit (Jianglaibio, Co., Ltd,
   Shanghai, China), Human Fumarate ELISA Kit (Jianglaibio, Co., Ltd,
   Shanghai, China), and Human Succinate ELISA Kit (Jianglaibio, Co., Ltd,
   Shanghai, China). The optical density (OD) at 450 nm was examined by a
   microplate reader and the concentrations were calculated according to
   respective standard curves. Cellular glutamate, GSH and ATP levels were
   identified by using Glutamate Assay Kit (Jiancheng Engineering Research
   Institute, Nanjing, China), Reduced Glutathione Assay Kit (Solarbio,
   Beijing, China) and ATP Assay Kit (Beyotime, Shanghai, China)
   respectively according to the manufacturer's instructions.

2.15. Papanicolaou staining assay and cytoskeleton staining assay

   The morphology of cells was observed by using Papanicolaou Staining Kit
   (LBP Medicine Science and Technology Co., Ltd) in accordance with the
   manufacturer's guideline. For cytoskeleton staining assay, Cells with
   different treatment were fixed in 4 % paraformaldehyde for 15 min and
   then permeabilized in 0.25 % Triton X-100 for 30 min at room
   temperature. The cells were subsequently stained with Actin-Tracker
   Green-555 (Beyotime, Shanghai, China) for 30 min and counterstained
   with DAPI. Olympus FV3000 was used for observing.

2.16. Cell cycle assay and cell proliferation assay

   For cell cycle assay, DOK cells were seeded on 6-well plates at a
   density of 2*10^5 per well. Cell Cycle Staining Kit (Multi Sciences,
   Hangzhou, China) was applied to examine the DNA content of cells in
   different stages of cell cycle in accordance with the manufacture's
   instruction. For cell proliferation assay, DOK cells were plated on a
   96-well plate at a density of 1*10^3 cell per well and cultured in the
   corresponding conditions. The cell counting kit-8 solution (CCK-8,
   Dojindo, Kumanmoto, Japan) was added into wells and incubated for 1 h
   in a 37 °C incubator in accord with the instruction. the optical
   density (OD) value was measured at 450 nm and 600 nm.

2.17. Colony formation assay and tumor sphere formation assay

   For colony formation assay, 1000 cells/well were seeded in 6-well
   plates and cultured under the indicated conditions. After incubated for
   7 days, cells were fixed with 4 % paraformaldehyde for 15 min, and
   stained with the crystal violet staining solution (Beyotime, Shanghai,
   China) for 15 min. The number of colonies (>50 cells) were counted
   using Image J analysis software. For tumor sphere formation assay, the
   cells were cultured in ultra-low attachment 96-well plates (Corning
   Inc., NY, USA) at a density 1000 cells/well in serum-free tumor stem
   cell medium (TSCM, QIDABIO, Shanghai, China) in a humidified 5 % CO2
   incubator at 37 °C. Fresh medium was replenished every 3 days. The
   number of the valid spheres (>75 μm) was assessed under microscopy 1
   week later.

2.18. Cell migration and invasion assay

   The in vitro cell migration and invasion were measured with the Corning
   Biocoat Matrigel Invasion Chamber (Corning Inc., NY, USA) according to
   the manufacturer's instruction. Briefly, 1*10^5 cells resuspended in
   200 μL of serum-free medium were seeded in the upper chambers, and
   500 μL culture medium with 20 % FBS was added into the lower chambers.
   After incubated with 24 h, cells of the reverse side of inserts were
   stained with the crystal violet staining solution and quantified with
   Image J analysis software.

2.19. Statistical analysis

   The results were presented as the mean ± SD from replicated
   experiments. Two-tailed Student's t-test or one-way ANOVA was performed
   to compare the means of different groups based on the raw data when the
   data conformed to normal distribution or homogeneity of variance.
   P-values <0.05 were considered statistically significant with Prism 9
   (GraphPad Software, San Diego, CA, USA), and the levels of significance
   were further defined at level of *P < 0.05, **P < 0.01, ***P < 0.001,
   ****P < 0.0001.

3. Results

3.1. ASCT2 conditional knockout triggered oxidative stress in oral mucosa

   It has been reported in vitro that intracellular Gln is mainly
   converted to glutamate which participates in the synthesis of GSH to
   protect cells from oxidative stress [[89][15], [90][16],
   [91][17],[92]32,[93]33]. However, it remains unknown whether in vivo
   targeted interference with ASCT2-mediated Gln uptake causes redox
   homeostasis imbalance. We therefore selectively deleted ASCT2 specially
   in oral mucosa epithelium, as briefly described in Methods ([94]Fig.
   S1a and b, Supporting information). As shown in [95]Fig. S1c
   (Supporting information),the tissue-specific gene deletion band (470
   bq) was confirmed by the genotyping. The expression of ASCT2 gradually
   decreased and stabilized 2 weeks after intraperitoneal injection of
   tamoxifen according to Western blot and IHC experiments ([96]Fig. 1a
   and b).

Fig. 1.

   [97]Fig. 1
   [98]Open in a new tab

   The identification of oxidative stress in oral mucosa epithelium of
   ASCT2 knockout mice

   Western blot (a) and IHC (b) were used to detect the efficiency of
   ASCT2 knockout in day 1,3,7,14,21 after tamoxifen injection
   (120 mg/kg). ([99]Fig. 1–b, magnification × 100 for the left,
   magnification × 100 for the right, scale bars, 400 μm for 100*
   magnification and 100 μm for 400* magnification. the red box showed the
   area of interest). (c) The GSH/GSSG ratio was measured in the 3 groups.
   (d) H[2]O[2] levels were evaluated in oral epithelium using the
   H[2]O[2]Content Assay Kit. (e) ROS levels in oral epithelium of the
   same gene background (Slc1a5^f/f Cre^+/−) were assessed by DHE staining
   in cryosections. The markers were ASCT2 (green), ROS (red) and nucleus
   (blue) respectively. Left: Representative captures from 3 mice for each
   group. (Scale bar 100 μm). Right: Quantification of ASCT2 and DHE
   staining using image J. (f) Staining of γ-H2AX of the 3 groups in
   paraffin sections. The markers were ASCT2 (green), γ-H2AX (red) and
   nucleus (blue) respectively. Left: Representative captures for each
   group (Scale bar 100 μm). Right: Quantification of ASCT2 and γ-H2AX
   staining using image J. (g) Staining of 8-OHdG of the 3 groups in
   paraffin sections. The markers were ASCT2 (green), 8-OHdG (red) and
   nucleus (blue) respectively. The above mice used to detect oxidative
   stress were all male. (For interpretation of the references to colour