Impaired ARID1A expression attenuated the immune response in gastric cancer via histone acetylation.

Introduction
Gastric cancer ranks as the fifth most common malignant disease worldwide and stands as the fourth leading cause of cancer-related mortality [1]. Traditional treatments for advanced gastric cancer often yield limited therapeutic efficacy. In recent years, immune checkpoint blockade has emerged as a promising strategy for a variety of solid tumors. Notably, PD-1/PD-L1 blockade has shown significant therapeutic benefits in cases of DNA mismatch repair-deficient or Epstein-Barr virus (EBV) positive gastric cancer [2, 3]. However, majority of other stomach tumor failed to possess satisfactory response to the immune checkpoint inhibitors (ICI). A deeper understanding of the interplay between genomic characteristics and the tumor’s immune response is essential to develop more effective immunotherapeutic approaches for gastric cancer.
AT-rich interactive domain 1A (ARID1A) encodes a substantial 250-kDa subunit of the mammalian Switch/Sucrose Non-fermentable (SWI/SNF) chromatin remodeling complex. This complex plays a critical role in binding transcription factors, coactivator/corepressor complexes, and participating in various nuclear activities [4]. Notably, ARID1A mutations have been identified in a wide range of cancers [4–9] with the majority resulting in reduced or complete loss of ARID1A expression [9]. Recent research, such as the work of Shen et al., has revealed that ARID1A interacts with the mismatch repair (MMR) protein MSH2, leading to compromised MMR and influencing the response to immune checkpoint inhibitors (ICI) [10]. Additionally, ARID1A mutations are strongly associated with increased tumor mutation burden (TMB) and are linked to improved overall survival in patients receiving ICI treatment across various cancer types [2, 11, 12]. It’s important to note, however, that ARID1A is traditionally recognized as a tumor suppressor gene, and decreased ARID1A expression is associated with higher cancer-specific mortality and increased cancer recurrence rates [13]. Despite these findings, the direct role of ARID1A in regulating the tumor immune response remains an area that has received limited investigation.
In the present study, we observed that reduced ARID1A expression in gastric cancer resulted in diminished infiltration of CD8+ T cells within the tumor microenvironment. Moreover, ARID1A deficiency hindered the transcription of critical immune-modulatory chemokines, CXCL9 and CXCL10. Exploring the underlying molecular mechanisms, we found that ARID1A played a pivotal role in modulating the acetylation of histone marks, H3K27 and H3K9. This, in turn, influenced the accessibility of the promoters for CXCL9 and CXCL10. Notably, when we inhibited histonedeacetylase (HDAC), we were able to restore the expression of these Th1-type chemokines. This intervention significantly enhanced the efficacy of immunotherapy, particularly anti-PD-L1 treatment, in ARID1A-deficient tumors. As a result, our study suggests that the effectiveness of immunotherapy in ARID1A-deficient gastric cancer could be notably improved through the incorporation of histone deacetylase inhibitors.

Materials and methods

Cell lines, antibodies, and chemicals
All cell lines were obtained from Cell Bank of Shanghai, Institutes for Biological Sciences, China and negative for mycoplasma infection. Cells were cultured in DMEM medium or RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in a humidified atmosphere with 5% CO2. Antibodies against ARID1A, HP-1a, STAT1, H3, acetyl-H3K9 and acetyl-H3K27 were purchased from Cell Signaling Technology (Danvers, MA, USA), when used for western blotting, immunofluorescence or immunohistochemistry antibodies were diluted according to manufacturer’s instructions. The dilution rate for each antibody was given in Table 1. For staining immune cells in tumor tissues in flow cytometry analysis, PE-anti-CD45, APC Cy7-anti-CD3, FITC-anti-CD4, PerCP Cy5.5-anti-IFNg and PE Cy7-anti-TNFa antibodies were purchased from BioLegend (San Diego, CA, USA) or BD Biosciences (San Jose, CA, USA). HDAC inhibitors (HDACi: LBH589 and TSA) were purchased from Selleck chem (Selleck Chem, Houston, TX, United States). PD-L1 mAb was purchased from BioXcell (Lebanon, NH, United States). Concentration of LBH589 used in vitro was between 10 and 200 nmol/L, when used in vivo the concentration was 10 mg/kg.

Generation of stable cells by lentiviral infection
ARID1A knockdown lentivirus was purchased from Genechem (Genechem, Shanghai, PR China). To select cells that ARID1A were stably knockdown, cells were plated at subconfluent densities and infected with a cocktail of 1 ml of virus-containing medium, 1 ml of regular medium and 8 μg/ml polybrene, and then selected in 1 μg/ml of puromycin (Sigma-Aldrich, Louis, MO, USA) 48 h after lentivirus infection.

Small interfering RNA (siRNA)
siARID1A was purchased from GenePharma siRNA. Transfection was performed at a concentration of 20 nM siRNA. GenePharma siRNA sequence was in Table 2.

Bioinformatics analysis
The GEO: GSE15460 dataset was downloaded from the Gene Expression Omnibus (GEO). Spearman correlation analysis between the expression of CXCL9, CXCL10 and ARID1A in human gastric cancer tissues was conducted using RNA-seq data from GSE15460 dataset (n = 329).

Total RNA extraction and cDNA synthesis
Total RNA was extracted from cells using TRIzol Reagent (Takara, Kusatsu, Japan) following the manufacturer’s instructions. After assessing quality and quantity, samples were then stored at − 80 °C. The extracted RNA was reverse-transcribed into cDNA using PrimeScript™ RT Master Mix (Takara, Kusatsu, Japan). The resulting cDNA was stored at − 20 °C for further analysis.

Quantitative real-time PCR (qRT-PCR)
Gene expression levels were quantified using qRT-PCR with gene-specific primers and One Step SYBR PrimeScript RT-PCR Kit II (Takara, Kusatsu, Japan). The qRT-PCR reaction was following the manufacturer’s instructions. Expression levels were normalized to β-actin, and relative quantification was performed using the 2^-ΔΔCt method. Primers used was in Table 2.

Protein extraction
The cells were suspended in RIPA buffer (Sigma-Aldrich, Louis, MO, USA). Protease inhibitor cocktail (1%, Thermo Fisher Scientific, Waltham, MA, USA) was also added to the mixture. The lysate was collected by centrifugation at 12,000 rpm at 4 °C for 15 min. The supernatant was transferred to a new tube, after using BCA protein quantification assay to determine concentration, supernatant was then mixed with loading buffer (Sigma-Aldrich, Louis, MO, USA) and boiled at 95 °C for 10 min.
Histone extraction was performed differently, cells were resuspended in hypotonic lysis buffer with protease inhibitor cocktail (1%). Nuclei were then isolated by centrifugation, resuspended in strong lysis buffer to extract histone, and histones were collected by centrifugation. After histone extraction, quantification was performed by same way using BCA protein quantification assay.

Western blotting analysis
Protein samples (0.5–20 μg) were subjected to SDS-PAGE gel electrophoresis, and transferred to PVDF membranes. After blocking with 5% non-fat milk in Tris-buffered saline with Tween (TBST) the membranes were incubated with specific antibodies overnight at 4 °C with gentle agitation. After washing, membranes were incubated with HRP-conjugated secondary antibodies. Protein bands were visualized using chemiluminescent substrates.

Immunohistochemistry (IHC)
The sections of human gastric cancer tissues (n = 90) were dewaxed and rehydrated. Antigen retrieval was performed using citrate buffer, and endogenous peroxidase activity was blocked with 3% H2O2. The sections were incubated with blocking buffer (5% BSA) for 1 h at room temperature. Primary antibodies were incubated with the sections overnight at 4 °C. After washing three times with PBS for 5 min, the sections were incubated with secondary antibodies for 1 h. Hematoxylin was used to counterstain the tissue for visualization of the architecture, and the sections were mounted for microscopic examination. Spearman correlation between CD8-positive cells and ARID1A expression scores were analyzed using ImageJ (NIH, Bethesda, MD, USA).

Immunofluorescence
The sections were incubated with blocking buffer (5% BSA) for 1 h at room temperature, washed with blocking buffer. Diluted primary antibodies were incubated with the sections overnight at 4 °C. The primary antibodies were washed three times with PBS for 5 min, and the diluted secondary antibodies were dropped on the sections at room temperature for 2 h. The secondary antibodies were also washed three times with PBS for 5 min. After dropping the mounting medium with DAPI, the sections were covered with cover slides, and the edges were sealed. After observation under a fluorescence microscope, the sections were stored at 4 °C. The fluorescent intensity was analyzed using ImageJ (NIH, Bethesda, MD, USA).

Chromatin immunoprecipitation (ChIP)
Chromatin was cross-linked with 1% formaldehyde for 10 min and fragmented by sonication. Chromatin DNA was extracted using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, Danvers, MA, USA) following the protocol provided by the manufacturer. DNA was immunoprecipitated using specific antibodies. ChIP signals were quantified by qRT-PCR analysis with One Step SYBR PrimeScript RT-PCR Kit II (Takara, Kusatsu, Japan). Values obtained for immunoprecipitated samples (percent (%) input DNA) were normalized to values for respective normal IgG. The specific primer pairs for the CXCL9 promoter region and CXCL10 promoter region, respectively, are described in Table 1.
ChIP-sequencing was performed by BGI (BGI-Shenzhen, Shenzhen, PR China.). After DNA-end repair, 3′-dA overhang and ligation of methylated sequencing adaptor, samples were undergoing PCR amplification and size selection. Sequencing was performed after library qualification.

Chromatin accessibility analysis
Peaks were called using MACS2 and quantitated across samples using Seqmonk generating reads per kilobase per million mapped reads (RPKM). Peaks were annotated to genomic features and nearest promoter via the Homer annotate function. The reads from replicates for each peak were averaged and the peaks were ranked based on the difference between the average counts among conditions. The “pheatmap” R package was used to plot the top 1000 peaks heatmap.

DNA accessibility by MNase digestions
Chromatin DNA samples were prepared as method described above. Then samples were digested by MNase with different concentration. Transmission electron microscopy analysis was performed by Servicebio (Servicebio-Wuhan, Wuhan, PR China.). DNA accessibility was measured by agarose gel electrophoresis.

Transmission electron microscopy
Electron microscopy cells seeded at 1.105 per cm2 on the soft matrices were cultured for 24 h and fixed in 2% PFA-2% glutaraldehyde in 50 mM cacodylate buffer at pH 7.4 for 2 h and then fixed in 1% osmium tetroxide in 125 mM cacodylate for 30 min. After resin polymerization, resin blocks were cut to 60–80 nm thickness on the ultramicrotome, and the tissues were fished out onto the 150 meshes cuprum grids with formvar film. After 2% uranium acetate saturated alcohol solution stained for 8 min avoiding light, sections then rinsed in 70% ethanol and ultrapure water. Then sections were stained in 2.6% lead citrate solutions for 8 min avoiding CO2, then rinsed with ultrapure water. After dried by the filer paper, the cuprum grids were put into the grids board and dried overnight at room temperature. The cuprum grids are observed under HITACHI HT7800 (HITACHI, Tokyo, Japan) and take images.

Gastric cancer patient survival data analysis
RNA-seq and patient survival data of gastric cancer patients in GEO dataset GSE62254 was downloaded from website. ARID1A low and ARID1A high group were divided based on expression level of ARID1A. The “survival” R package was applied to plot Kaplan–Meier curves and to compute the associated log-rank p-value.

Animal model
All experiments were approved and performed according to the guidelines of the Ethical Committee of Union Hospital, Huazhong University of Science and Technology. 615 mice (6 weeks old) were purchased from Shulaibao (Shulaibao, Wuhan, PR China). The MFC or MFC/ ARID1A knockdown cells were inoculated s.c. (5 × 105 cells/100 μL in PBS/mice) into the flanks of anesthetized mice. Mice in each group intra-peritoneally received LBH589 in 10% DMSO solution (10 mg/kg/day) or vehicle and anti-PD-L1 antibody (100 μg/day) (BioXcell, Lebanon, NH, United States) twice a week. The tumor volume was assessed every two days starting from day 3 after tumor inoculation.

Flow cytometry
Subcutaneous tumors were entirely separated from tumor-bearing mice, then were all pestled through 40 μm mesh. Tumor cells were removed by centrifuging using FACS buffer. Single cell suspensions of each sample were then used in following flow cytometry analysis after activation. Flow cytometry was performed using fluorescence-conjugated antibodies and their controls followed by species-specific conjugate using a FACS CantoII flow cytometer (BD Biosciences, San Jose, CA, USA). Positive cells in percentage (%) were calculated as follows: Surface expression in percent obtained with the specific antibody—surface expression in percent obtained with isotype control. T cells were preselected by CD45+ and CD3+. All flow cytometry data were analyzed using FlowJo software (Treestar Software, Ashland, OR, United States).

Clinical data collection
A total of 90 eligible patients were finally enrolled in this study. Study collected data from locally advanced gastric cancer patients who underwent surgery at the Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, between April 2019 and January 2020. We included the patients on the basis of the following inclusion criteria: (1) gastric cancer confirmed in histological biopsy; (2) clinical TNM stages II, III, and IVa; (3) radical gastrectomy combined with D2 lymph node dissection; and (4) complete clinicopathological data.

Statistical analysis
Significance was determined using GraphPad Prism software (GraphPad Software, San Diego, CA, United States). All statistical analyses were based on data obtained from a minimum of three independent experiments. For comparisons involving more than three means, we utilized one- or two-way ANOVA with Bonferroni correction. Two means were compared using the unpaired Student’s t-test. A significance level of p < 0.05 was considered statistically significant.

Results
To evaluate the role of ARID1A in regulation of CD8+ T cell infiltration in gastric tumor microenvironment, we quantified the CD8A and ARID1A via immunohistochemistry in human stomach adenocarcinoma tissues by the H-score method and analyzed their clinical relevance. Based on the median values of ARID1A intensity, we divided 90 patients into “low” and “high” groups. CD8A intensity was dramatically decreased in ARID1A low groups (Fig. 1A, B), patients’ clinicopathological characteristics was shown in Table 3. ARID1A H-score was positively related with CD8A H-score (Fig. 1C) and patients with lower ARID1A expression had poor overall survival as compared with those with higher ARID1A expression (Fig. 1D).
In order to further value the relationship between ARID1A and CD8 + T cells, we performed implantation model of tumor with ARID1A knockdown. CD8+ T cell in tumor microenvironment was firstly detected by IHC staining. As expected, CD8+ T cell was absent in shARID1A tumor tissue (Fig. 1E and F). Flow cytometry dots demonstrated similar lack of CD8+ T cell in ARID1A depleted tumor microenvironment (Fig. 1G and H). ARID1A depletion favored tumor growth dramatically (Fig. 1I) and resulted in shorter survival time for the experimental mice (Fig. 1J). Effector T cell was typically attracted by the Th1-type chemokines such CXCL9 and CXCL10 [14]. We conducted a correlation analysis to examine the expressions of CXCL9 and CXCL10 in relation to ARID1A expression in gastric cancer, using RNA-seq data from the GSE15460 database. Our findings indicated that there was a significant positive correlation between the expressions of chemokines (CXCL9, CXCL10) and ARID1A. (Fig. 1K–L).
To explore the mechanism by which ARID1A control CD8 + T cell migration into tumor microenvironment, we first knocked down ARID1A in three human gastric cancer cell lines including AGS, SNU-1 and SNU-719 (Fig. S1A, B and C). ARID1A depletion caused a dramatic decrease in the transcription of CXCL9 (Fig. 2A, E, I) and CXCL10 (Fig. 2B, F, J). The same influence was also reproduced in murine gastric adenocarcinoma cell MFC when ARID1A was knocked down (Fig. 2M, N; Fig. S1D). To confirm the effects of ARID1A on the expression of CXCL9 and CXCL10, we evaluated the protein level of CXCL9 and CXCL10 in supernatant of aforementioned gastric tumor cells with ARID1A knockdown. Both human and murine gastric cancer cells showed a significant reduction of CXCL9 and CXCL10 in the culture supernatant when ARID1A was depleted (Fig. 2C, D, G, H, K, L, O, P). In vivo experiment further confirmed that knockdown of ARID1A reduced the expression of Th1 chemokines CXCL9 and CXCL10 (Fig. S1E and F).
SNF/SWI remodeling complex was revealed to recruit histone acetyltransferase (HAT) to bind the promoters of many genes [15] and acetylation of K9 and K27 in H3 was correlated with high transcriptional activity [16]. To further explore the molecular mechanism of ARID1A depletion affecting CXCL9 and CXCL10 expression, we assessed H3K9 and H3K27 acetylation status in ARID1A knockdown and control cells (Fig. 3A). The results demonstrated that H3K9Ac and H3K27Ac were significantly decreased in different cell lines with ARID1A depletion (Fig. 3B–C). To determine the role of histone acetylation in the expression of CXCL9 and CXCL10, we conducted experiments using various HDACi and found that the inhibition of histone deacetylases can upregulate the expression of CXCL9 and CXCL10 (Fig. 3D, Fig. S2A–D). More importantly, deacetylase inhibitor LBH589 completely recovered these acetylation alterations in the ARID1A defective cells (Fig. 3D). Also, deacetylase inhibition restored the expression of CXCL9 and CXCL10 that attenuated by ARID1A knockdown in both mRNA level and protein level (Fig. 3E–H).
Acetylation of histone residues influence gene transcription by opening chromatin structure and recruiting different transcription factors [17]. Heterochromatin protein 1 alpha (HP1a) is a central factor in establishing and maintaining the heterochromatin state. To verify the alteration of chromatin structure by ARID1A depletion and acetylation of histone, we stained HP1a and H3K9ac in cell nucleus with immunofluorescence. ARID1A knockdown promoted heterochromatin nucleation and condensation and less H3K9ac combination on the DNA (Fig. 4A). Deacetylation inhibition drove HP1a dispelled from chromosomes and led to more H3K9ac retention in the nucleus. Furthermore, we performed micrococcal nuclease assay to evaluate the accessibility of DNA. ARID1A depletion protected DNA from MNase digestion which was obliterated by deacetylase inhibitor LBH589 (Fig. 4B). Transmission electron microscopy unveiled a strong increase of condensed heterochromatin in ARID1A knockdown cells and LBH589 impelled heterochromatin structure loose (Fig. 4B).
Due to the decreased expression of ARID1A leading to reduced histone acetylation levels, we conducted a ChIP-qPCR experiment to investigate the enrichment of H3K9Ac and H3K27Ac following the depletion of ARID1A. These findings indicated that ARID1A depletion led to decreased histone acetylation at the CXCL9 and CXCL10 gene promoter (Fig. 4C). Furthermore, STAT1 is the key transcription factor to regulate CXCL9 and CXCL10 expression [14]. To confirm the role of histone acetylation in the transcription regulation of type-Th1 chemokines, we performed ChIP-PCR assay to assess STAT1 binding on the promoter of CXCL9 and CXCL10. The results showed ARID1A knockdown sharply attenuated the precipitation of STAT1 on the promoter of CXCL9and CXCL10 (Fig. 4D, E) and this effect was completely reversed by deacetylation inhibition. ChIP-Seq further proved ARID1A deficiency hindered STAT1 combination in the promoters of a series of genes which could be restored by deacetylation inhibitor LBH589 (Fig. 4F).
ARID1A depletion was revealed to decrease H3K9ac and H3K27ac and control Th1-type chemokine expression. Deacetylase inhibitor LBH589 could completely restore CXCL9 and CXCL10 expression in ARID1A deficient cells. We hypothesized that deacetylase inhibitor might reverse the effect of ARID1A depletion on tumor progression and improved immunotherapy efficacy. In order to verify this hypothesis, we treated ARID1A depleted tumor bearing mice with LBH589 and anti-PD-L1 antibody. LBH589 dramatically extinguished the additional growth promoted by ARID1A depletion (Fig. 5A, B) and significantly enhanced the immunotherapy efficacy of anti-PD-L1 in shARID1A tumor (Fig. 5A, B). Also LBH589 favored the prognosis of mice bearing shARID1A tumor both treated and untreated with anti-PD-L1 antibody (Fig. 5C). The alteration of CD8+ T cell infiltration and function in microenvironment was evaluated by flow cytometry test. LBH589 restored CD8+ T cell infiltration and promoted CD8+ T cell function in shARID1A tumor tissue treated with or without anti-PD-L1 antibody (Fig. 5D and E).

Discussion
ARID1A is a core component of the SWI/SNF complex, contributing to chromatin remodeling and DNA repair, and is a newly identified tumor suppressor across a wide range of cancer types. ARID1A was reported to have a high mutation frequency in ovarian clear cell carcinoma (45%), endometriod carcinoma (30%) [6], uterine carcinoma (20%) [18] and bladder cancer (18%) [19]. In gastric cancer, ARID1A mutation occured in around one third of all tumors and significantly associated with unfavorable overall survival [13]. Notwithstanding ARID1A was shown to promote proliferation [20], migration [21] and death [22] in gastric cancer cell lines, its role in vivo remains pronumeral. Study using a gastric tumor model showed ARID1A heterozygous deletion resulted in global loss of active enhancer marks and decreased activation of p53 and apoptosis-related genes [23]. Human gastric organoid model proved that loss of ARID1A driven hyperplastic and mucinous alteration in the initial stage of malignant transformation [24]. In the current study, we revealed the immunoregulator role of ARID1A in gastric tumor progression.
CXCL9 and CXCL10 are critical chemokines in the CXC subfamily that play an essential role in attracting CD8a + T cells to tumor microenvironments or sites of inflammation by binding to their receptor, CXCR3. Studies have demonstrated that CXCL9 and CXCL10 recruit activated Th1-type T cells and CD8 + T cells, thereby suppressing tumor growth [14]. Our results demonstrated that ARID1A controlled the expression of effector T cell chemokines CXCL9 and CXCL10 in gastric cancer cells. Loss of ARID1A led to dramatically reduction of CXCL9 and CXCL10 release and absence of CD8 + T cell infiltration in tumor microenvironment. Consistently, Jing et al. have reported similar results in ovarian cancer that ARID1A deficiency led to absence of Th1-type chemokines [25]. However, we uncovered a novel molecule mechanism of ARID1A controlling CXCL9 and CXCL10 expression. In this study, we found that ARID1A influences H3K9 and H3K27 acetylation as well as chromatin accessibility, which in turn facilitates STAT1 binding to the promoters of CXCL9 and CXCL10.
Our study, using electron microscopy and immunofluorescence, confirmed that ARID1A regulates chromatin decompaction with HP-1α in gastric cancer cells. Chromatin accessibility, closely linked to histone modifications, governs gene expression epigenetically, thereby epigenetic drugs like EZH2 inhibitors have been applied to ARID1A-mutated tumors. For instance, Patricia J. Keller et al. showed that EZH2 inhibitors reduce histone methylation in ARID1A-mutated tumors, improving therapy response [26]. Unlike their focus on histone methylation, our study highlights histone acetylation changes caused by ARID1A downregulation, finding a positive correlation between ARID1A expression and acetylation levels in gastric cancer cell lines, with LBH589, an HDAC inhibitor, reversing the reduction in acetylation induced by ARID1A knockdown. This suggests histone acetylation regulated by ARID1A may be the key mechanism in this process. The SNF/SWI remodeling complex has been shown to recruit histone acetyltransferases (HATs) to the promoters of various genes, which increases histone acetylation and changes gene expressions [15]. Also, study shows ARID1A directly suppresses HDAC in ovarian cancer cells, HDAC inhibition could help improve therapy response in ARID1A-deficient cells [27, 28].
In addition to altering the immune response by ARID1A through attracting effector T cell to chemokines, Young-Bae et al. found loss of ARID1A could activate AKT signaling to up-regulated PD-L1 expression in gastric cancer [29] which is a co-inhibitory molecule that provides secondary signals to inhibit effector T cell activity. All these studies indicated that ARID1A could positively regulated tumor immune response via various mechanisms which contribute to the poor predictive prognosis of ARID1A deficiency for tumors.
Recently, immune checkpoint blockade (ICB) has become as one of the most promising therapeutic options for a significant percentage of cancer patients. Effector T cells infiltrating in microenvironment and co-inhibitory molecule expressing on the tumor cells or antigen presenting cells play essential role in influencing therapeutic efficacy of immune checkpoint inhibitor [30]. Later on, tumor mutation burden (TMB), which indicated potential neo-antigen produced, was considered as a strongest predictor of ICB sensitivity [31]. ARID1A was found to interact with MutS Homolog 2 (MSH2) and promote mismatch repair (MMR) and ARID1A deficiency compromised MMR and increased tumor mutation burden across various types of human cancers [10]. Consequently, Shen et al. showed loss of ARID1A sensitized ovarian cancer to immune checkpoint blockade therapy [10]. However, our study demonstrated that ARID1A inactivation dramatically restricted the expression of Th1-type chemokines in gastric cancer and led to low infiltration of CD8 + T cell into tumor microenvironment, which gave a comprehensive view on its role in tumor immune regulation.
In view of fact that CD8 + T cell is the primary effector cell for anti-tumor response in most of cancers, we suspected the effect of ARID1A deficiency on the immune therapeutic efficacy for gastric cancer. When we treated shARID1A MFC-bearing mice with PD-L1 blockade, we did not found more sensitivity of therapy response than control group. Intriguingly, deacetylase inhibitor LBH589, which relieved the hindering effect of AIRID1A deficiency on the expression of CXCL9 and CXCL10, significantly reversed the un-response of gastric cancer to the anti-PD-L1 antibody. This inconsistency might attribute to the different tumor models used and context-dependent function of ARID1A [23, 32, 33]. Moreover, Li et al. did show ARID1A deficiency had negative effect on the expression of Th1-type chemokines in ovarian cancer cells [25]. In this case, restoration of CXCL9 and CXCL10 expression combining with PD-L1 blockade might yield more largely therapeutic efficacy in ARID1A loss ovarian tumor than anti-PD-L1 antibody alone.

Conclusion
In conclusion, we have demonstrated the immune regulation of ARID1A loss in gastric cancer both in vitro and in vivo and influence of ARID1A deficiency on the immune therapy response to gastric tumor. Our study indicated that restoration effector T cell infiltration with small molecular inhibitor such as LBH589 to improve tumor immune microenvironment and sensitize immunotherapy response would be a novel strategy for gastric cancer with ARID1A deficiency.

Supplementary Information