Targeting SIRT7 shape an immune-activated microenvironment by both promoting CCL5-induced TILs infiltration and decreasing PD-L1 expression

doi:10.21203/rs.3.rs-8421416/v1

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Targeting SIRT7 shape an immune-activated microenvironment by both promoting CCL5-induced TILs infiltration and decreasing PD-L1 expression

https://doi.org/10.21203/rs.3.rs-8421416/v1

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Background Tumors adapt and survival under immune surveillance by manipulating various adaptive immune-repressive microenvironments. Less than 20% of patients with triple-negative breast cancer benefit from anti-PD-1/PD-L1 therapy, and the absence of tumour-infiltrating lymphocytes (TIL) that primarily comprise macrophage and T cells is likely to be a key factor leading to the immunotherapy failure. These cold tumors are characterised by a reduced immune response and immune cell infiltration in the microenvironment. It has been reported that the anti-PD-L1 therapy is only effective in patients who express PD-L1 and whose tumors are infiltrated by CD8+ T cells. In contrast, the PD-L1+/TIL- type tumors rarely respond to anti-PD-L1 therapy. This study reports that the inhibition of the histone deacetylase SIRT7 not only reduces PD-L1 expression but also enhances M1 macrophage polarization and CD8+T cell infiltration. SIRT7 suppression may facilitate the transition from cold to hot tumors potentially increasing their responsiveness to immunotherapy.

Methods SIRT7, PD-L1, and CCL5 protein levels in breast cancer cells and tissues were quantified using western blot (WB) analysis, immunohistochemistry staining (IHC) and multi-immunofluorescence (mIF) assays. Transwell assays were carried out to evaluate cell migration. The mRNA level of SIRT7/CD274/CCL5, IL-10/CD206 (M1 macrophage markers) and iNOS/CD40 (M2 macrophage markers) were determined by q-PCR. Flow cytometry was performed to assess the type and number of immune T cells. CHIP assay was conducted to assess the interaction between the histone deacetylase SIRT7 and the associated DNA promoter of CCL5 regions. respectively. Subcutaneous implantation models were used to assess the in vivo tumor growth.

Results Here, we reported that SIRT7 knockdown in immunogenicity mice resulted in restrained growth and activation of infiltrated CD8⁺T cells. SIRT7 inhibition led to CCL5 upregulation and reduced PD-L1 expression. Besides, SIRT7-dependent histone H3K18ac deacetylation inhibited CCL5 transcription, and SIRT7 transcriptionally upregulates PD-L1 expression by directly deacetylating YB-1. Additionally, SIRT7 inhibition promoted CCL5-induced M1-like polarization and CD8+T cell infiltration that were attributed to the mutually reinforced activation. Furthermore, the combined treatment with SIRT7 inhibitor and PD-1 monoclonal antibody significantly restrained tumor growth in mouse model. In clinical breast cancer samples, SIRT7 expression was negatively correlated with CCL5 expression and the number of infiltrating CD8+ T cells. SIRT7 suppression may promote the transition from cold tumors to hot tumors. This suggest that SIRT7 may be a superior target to PD-1/PD-L1 blocking alone.

Conclusion SIRT7 inhibition promoted CCL5-induced M1-like polarization and CD8+T cell infiltration. SIRT7 simultaneously enhanced PD-L1 expression, thus weakening T cell-mediated anti-tumor immunity. The cancer progression can be suppressed by SIRT7 inhibition, which leads to decreased PD-L1 protein levels and an elevated immune-activated microenvironment. A combined therapy of SIRT7 inhibitor and anti-PD-1 antibody exerted a synergistic antitumor effect.

Cancer Biology

Breast cancer

SIRT7

PD-L1

CCL5

macrophage polarization

TIL

Breast cancer (BC) is the most prevalent cancer among women worldwide, resulting in to a high percentage of deaths[2]. Triple-negative breast cancer (TNBC) is characterized by an immunosuppressive microenvironment that contributes to tumor progression, metastasis, and immunotherapy resistance[3]. Identification of the intrinsic factors that control the immunosuppressive microenvironment is urgently needed. Immune checkpoint blockade (ICB) therapy have yielded significant benefits in terms of survival and clinical benefits[4]. However, the objective response rate to ICB monotherapy for BC is less than 20%[5], and only a subset of PD-L1 positive patients benefits from anti-PD-1/PD-L1 therapy[6]. The FDA-approved PD-L1 inhibitors are used in treatment of various malignancies, PD-L1 inhibitors prevent cancer development and progression by activating the immune response[7]. However, since the proportion of patients with BC who benefit from immunotherapy is small, it is particularly important to develop valuable biomarkers to distinguish ICB therapy responders. Defects in DNA mismatch repair proteins and subsequent high microsatellite instability lead to the accumulation of mutation loads in cancer-related genes and the generation of neoantigens that faciliate in predicting the efficacy of the immune response[8]. The unappealing outcomes with single-agent immune checkpoint inhibitors (ICIs) have necessitated the development of combined therapies. Dynamic interactions between tumor and immunosuppressive cells like tumor-associated macrophages (TAMs), tolerant dendritic cells, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs) contribute to development of an immunotolerant micro-environment resulting in tumor progression and immunotherapy resistance. These cells hinder the anti-tumor immune response by modulating the formation of immune checkpoint molecules, producing suppressive cytokines and chemokines, and competing for nutrients necessary for the function of CD8 + T cells. Targeting the immunosuppressive cellular components in the microenvironment may be a promising strategy[9–11]. Although patients with TNBC benefit from the immunotherapy, acquired resistance frequently occurs after a certain period of treatment[12]. Additionally, the combination of anti-PD-1 therapy with other targeted therapies is likely overcome acquired ICIs resistance, since an increasing number of potential targets for ICIs resistance have been discovered[13–16]. The bispecific antibody YM101 that simultaneously inhibits TGF-β and PD-L1 facilitates hot tumor formation and prevents immunotherapy resistance[17, 18]. However, the mechanisms governing the accumulation of immunosuppressive cells in the BC microenvironment remain unclear. This impedes the discovery of new techniques against immunotherapy resistance.

The molecular profiles associated with intrinsic and acquired resistance to anti-PD-1/PD-L1 therapy in BC were assessed. The investigation focused on the functions of the histone methyltransferase SIRT7 in previous studies. SIRT7 is a member of the SIRTs family of NAD+ -dependent protein deacetylases, contributes to tumorigenesis and progression through histone deacetylation[19, 20]. Recent evidence has revealed that aberrant expression of SIRT7 occurs in almost all cancer types via different mechanisms, including those involved in cancer metastasis, genome stability, as well as tumor microenvironment[21–23]. SIRT7 dysregulation contributes to BC progression and poor prognosis[24]. SIRT7 acts as an oncogene and independently predicts poor outcomes in patients with BC[25]. It is widely believed that SIRT7 can promote cancer proliferation, metastasis, and drug resistance[19]. SIRT7 inhibition reversed sorafenib-acquired resistance in hepatocellular carcinoma[26]. SIRT7 not only influences tumor-intrinsic phenotypes, but also impacts the tumor microenvironment. Further studies have indicated that SIRT7 can promote cancer progression by evading immune response via UPR activation[27]. This study explored the critical role of SIRT7 in immunotherapy resistance and how the immunosuppressive micro-environment evolves during the development of immunotolerance.

The CCL5/CCR5 axis has been shown to plays a role in promoting inflammatory responses. Recent studies have shown that the CCL5/CCR5 combination is vital in various pathological processes like: inflammation, chronic diseases, and cancers. Besides, the CCL5/CCR5 axis helps in the adhesion and migration of different T-cell subsets during immune responses[28]. Research has indicated that CCL5 facilitated the migratory and invasive ability of human glioma cells[29]. CCL5 has been identified as a vital mediator for tumor cells immune evasion, which is regulated by p38-MAX signaling[30]. It has been demonstrated that CCL5-deficiency blocked tumor growth and metastasis by promoting CD8 + T cell accumulation in CRC mouse models[31]. In contrast, CCL5 plays a role in tumor suppression. Dangaj et al. reported that CCL5 is normally epigenetically silenced and that tumor cells with high CCL5 expression enable the infiltration of T cells into tumors[32]. Tumors with low CCL5 levels are immunotolerant and rarely respond to ICIs therapy. Enrichment of tumor-infiltrating lymphocytes (TIL) can be triggered through tumor-derived CCL5 stimulation, the TILs like M1 macrophage polarization and T cell infiltration are critical in orchestrating the immunoreactive microenvironment. The findings indicate that CCL5 can promote M1 macrophage polarization and inhibit M2 polarization by the CCR5-mediated activation of MAPK pathways[33]. The induction of CCL5 can facilitate CD8 + T cells activation and subsequently attenuate tumor growth, and this represents a promising therapeutic strategy for inducing TIL recruitment[34]. In contrast, chemokines can promote tumor development, and CCL20 elevation triggered by taxane-containing chemotherapy mediates chemoresistance and serves as a novel predictive marker[35].

We observed that SIRT7 knockdown in immunogenic mice resulted in restrained tumor growth and increased the number and activation of infiltrating CD8 + T cells, and this correlated with the immune response in the microenvironment. Moreover, the results showed that SIRT7 inhibition led to CCL5 upregulation and reduced PD-L1 expression through transcriptional regulation. Additionally, SIRT7 inhibition results in CCL5-induced M1 macrophage polarization and CD8 + T cell infiltration. The transformation from cold tumors to hot tumors was completed with the increase in TIL levels, and this indicated that SIRT7 is likely a more effective biphase regulation factor in immune-repressed microenvironments compared with PD-L1. Combined treatment with an SIRT7 inhibitor and anti-PD-1 therapy significantly inhibited tumor growth in BC considerably. The tumor immune microenvironment (TIME) is classified based on the levels of TILs and PD-L1 expression. Generally, tumors expressing PD-L1 in the absence of TILs exhibit primary resistance to anti-PD-1/PD-L1 therapy. In addition, tumors with both PD-L1 expression and T-cell infiltration are expected to respond to ICB therapy. It is possible that the levels of TILs can be used as predictive biomarkers to select potential patients who can benefit from immunotherapy[36]. The non-small cell lung cancer (NSCLC) patients with both high PD-L1 expression and TIL infiltration may benefit from immunotherapy[37]. The increasing number of TILs in the immune microenvironment has become a promising immunotherapy strategy. CD8 is a TIL marker, and it has been reported that the CD8-positive subpopulation of TNBC exhibit a higher possibility of benefiting from immunotherapy[38]. By reducing the tumor immunosuppressive microenvironment, combination therapy targeting TNFR2 and anti-PD-L1 has yielded encouraging outcomes[39].

Cell Culture and Inhibitors

SK-BR-3, MCF7, MDA-MB-231, and EMT6 cell lines were purchased from ATCC and were routinely screened for mycoplasma and other pathogen contamination. These cell lines were screened in DMEM (Gibco) screened with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (Gibco) within a humidified incubator at 37°C and 5% CO₂. Peripheral blood mononuclear cells (PBMCs) isolated from healthy human donors were cultured in RPMI-1640 medium. The anti-PD-1 antibody were purchased from Invivo Crown(UK)，the SIRT7 antibody for WB were purchased from Bioswamp(China)，the YB-1 and Flag antibody for IP assay were purchased from Biodragen(China)，PD-L1 antibody for FITC assay and Human TGF-β ELISA Kit were purchased from Elabscience(China), the FITC antibody of human CD8/mouse CD8a and anti-human/mouse GZMB were purchased from Biolegend(China), the recombinant human IL-13 protein and human IFN-γ ELISA kit were purchased from SinoBiological(China)，the recombinant human CCL5/TGF-β/IFN-γ protein were purchased from SinoBiological(China), the human and mouse CCL5 ELISA Kits were purchased from BOSTER(China), recombinant Human IL-4 Protein was purchased from Absin(China)，the maraviroc and paclitaxel were purchased from TargetMol (USA), the SIRT7 inhibitor 97491 was obtained from Med Chem Express (USA).

Cell Transfection

For overexpression studies, plasmids were obtained from Tsingke Biotechnology (Beijing, China). Following the manufacturer’s transfection protocol, SK-BR-3 and EMT6 cells were transfected utilizing Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). Moreover, lentiviral shRNA vectors were purchased from Tsingke Biotechnology. Lentiviruses were generated by co-transfecting HEK293T cells with recombinant lentiviral vectors and the packaging plasmids pMD2.G and psPAX2. After 48 hours, the supernatant containing viral particles was harvested and concentrated. The concentrated viral particles were then used to infect tumor cells in the presence of 8 µg/mL Polybrene. Following a 48-hour infection period, tumor cells were selected with puromycin at a final concentration of approximately 2 µg/mL to establish stable transfectants. A list of primers utilized in this study can be found in Supplementary Table S2.

Human Tissue Specimens

A total of 101 paraffin-embedded breast cancer tissue specimens were collected from the First Affiliated Hospital of Shenzhen University. This study received approval from the Ethics Committee of the First Affiliated Hospital of Shenzhen University. All tissue samples were obtained with informed consent from the patients, adhering to ethical guidelines. Clinical and pathological information for the breast cancer patients is detailed in Supplementary Table S3.

Animal Experiments

BALB/c and nude mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. EMT6 cells were subcutaneously injected into 4-week-old BALB/c mice, with six mice per group, using a 100 μL mixture of serum-free DMEM. Each mouse received approximately 1 × 10^6 cells. Tumor volume, calculated using the formula volume = length × width² × 0.5, and the body weight of the mice were measured every three days. Treatment began once the tumor volume reached 100 mm³. The vehicle group received intraperitoneal saline injections, while the treatment groups were administered the respective drugs intraperitoneally three times a week. Paclitaxel and anti-PD-1 antibody were given at a dose of 10 mg/kg, and compound 97491 was administered at 5 mg/kg. Tumor tissue samples were fixed in 4% paraformaldehyde for immunohistochemistry, while the remaining tumors were processed into single-cell suspensions for flow cytometric analysis of tumor-infiltrating immune cells. In the SIRT7 knockout experiment, both BALB/c nude mice and BALB/c mice were subcutaneously implanted with 1 × 10^6 shSIRT7-SK-BR-3 and shSIRT7-EMT6 cells, respectively. Approximately 14 days after implantation, the mice were sacrificed, and tumor samples were fixed in 4% paraformaldehyde for immunohistochemistry. The remaining tumors were also processed into single-cell suspensions for flow cytometric analysis.

Co-immunoprecipitation (Co-IP) and Western Blot (WB)

Cells were lysed using Western and IP cell lysis buffer (Beyotime) and incubated at 4°C for 20 minutes. The lysate was then centrifuged at 12,000 rpm for 10 minutes to collect the cellular proteins. A protease inhibitor mixture (Beyotime) and 0.1 mM PMSF (Beyotime) were added, and protein concentration was assessed using the BCA protein assay kit (Beyotime). For the Co-IP experiment, 2 mg of whole-cell lysate protein was incubated overnight at 4°C with 1–5 μg of a specific antibody. Pre-treated Protein A/G beads (Thermo Fisher) were then added and incubated with the lysate at 4°C for 2-4 hours. The Protein A/G beads were washed 3–4 times with IP buffer, and immunocomplexes were eluted using 2× SDS loading buffer (Beyotime). For Western blotting, proteins were electrophoresed and transferred to membranes according to the manufacturer’s instructions. The membranes were subsequently blocked, washed, and incubated overnight with primary antibodies. After a 2-hour incubation with secondary antibodies, target proteins were detected using the Beyo ECL Star Kit (Beyotime) and quantified with Bio-Rad Quantity One software. Antibody details for Western blot and Co-IP are provided in Supplementary Table S1.

Immunohistochemistry (IHC)

Immunohistochemistry was utilized to assess the expression levels of SIRT7, CCL5, and CD8 in breast cancer tissues. Initially, tissue slides were immersed in xylene for 10 minutes, followed by a series of washes with decreasing concentrations of ethanol to facilitate deparaffinization. Antigen retrieval was then conducted by microwaving the sections in citrate buffer for 5 minutes. To block endogenous peroxidase activity, the sections were treated with 1% hydrogen peroxide and subsequently incubated with goat serum. Primary antibodies were applied and left overnight at 4°C in a humidified chamber. Signal amplification was achieved using biotinylated rabbit anti-mouse antibodies (Vector, Germany). The slides were stained with diaminobenzidine (DAB) (Sigma, USA) to visualize SIRT7, CCL5, and CD8, and counterstained with hematoxylin to highlight the nuclei. To ensure consistency, staining results were collected from identical locations across consecutive sections. Detailed information about the antibodies used for immunohistochemistry can be found in Supplementary Table S1.

Quantitative RT-PCR

Total RNA was extracted, and its concentration was determined following the guidelines of the HiScript II Q RT SuperMix for qPCR kit. cDNA synthesis was performed using a reverse transcription kit from Invitrogen. Quantitative PCR (qPCR) analysis was executed on an ABI 7500HT Real-Time PCR System, employing SYBR Green dye for detection. Each experiment was repeated independently three times, utilizing GAPDH as the reference gene. The primer sequences are listed in Supplementary Table S2.

Flow Cytometry

A single-cell suspension was generated from tumor tissues, and fluorochrome-conjugated antibodies (BioLegend) against CD8 and granzyme B (GZMB) were employed for staining. Surface staining was conducted using the CD8 antibody, while GZMB staining necessitated prior fixation and permeabilization with the Transcription Factor Buffer Set. All stained cells were analyzed with a BD flow cytometer (BD Biosciences, San Jose, CA, USA), and data analysis was performed using FlowJo software (Tree Star Inc.).

ChIP Assay

ChIP samples from SK-BR-3 and EMT6 cells were fixed using 37% formaldehyde at a final concentration of 1% for 10 minutes. To terminate the cross-linking, 10 × 1.25 M glycine was added at room temperature. DNA-protein complexes were then sheared through sonication and subsequently purified. ChIP assays were conducted in accordance with the protocol provided by Beyotime (P2080S). The target DNA was analyzed through real-time quantitative PCR. The antibodies and primer sequences utilized in this study are detailed in Supplementary Tables S1 and S2.

Co-culture Cytotoxicity Assay

Primary human T cells were isolated from the peripheral blood of healthy donors, while macrophages were differentiated from THP-1 cells through stimulation with PMA, IL-4, and IL-13. Both T cells and tumor cells were activated using CD3/CD28 antibodies (100 ng/mL) and IL-2 (10 ng/mL) before being co-cultured in 24-well plates. Macrophages were placed in transwell inserts (0.4 μm pore size) at a ratio of 2:2:1 (T cells:macrophages:tumor cells). The co-culture was maintained for 24 hours in conditioned medium. Following this period, T cells were collected, and the surviving tumor cells were fixed and stained with crystal violet for imaging and quantification.

Migration Assays and T Cell Activation

Migration assays were performed using a transwell system equipped with polycarbonate membranes that had 5 μm pores. Tumor cell-conditioned media were generated through various treatments. Activated CD8+ T cells or macrophages were washed twice with PBS, resuspended in serum-free medium, and added to the upper chamber, while the conditioned media were placed in the lower chamber. After a 24-hour incubation, cells in the upper chamber were collected and fixed with 4% paraformaldehyde. Migrated macrophages were fixed and stained with 0.1% crystal violet for 30 minutes, followed by three washes with phosphate-buffered saline, air drying, and images were taken for final cells counting. The quantities of CD8+ and GZMB+ T cells were quantified using a BD flow cytometer.

ELISA

The secretion levels of CCL5 in the supernatant were quantified using the Human RANTES ELISA Kit (EK0494, BOSTER) and the Mouse RANTES ELISA Kit (EK0495, BOSTER). Additionally, the levels of TGF-β and IFN-γ in the supernatant were measured using the ELISA Kit (E-EL-0162, Elabscience) and the ELISA Kit (KIT11725A, Sinobiological), respectively.

Dual Luciferase Reporter Gene Assay

The reporter plasmid pGL3-Basic, which includes a 2000 bp promoter region of CD274, was constructed by Tsingke in China. Cells were co-transfected with the Renilla luciferase reference plasmid pRL-TK, pGL3-Basic, and WT/K81Q-YB-1 plasmids, and incubated for 48 hours, using the empty pGL3-Basic plasmid as a control. Cells were lysed with PLB buffer, and 10 μL of the supernatant was transferred to 96-well plates. Subsequently, 100 μL of luciferase assay reagent was added, and luminescence intensity was measured in the dark. Subsequently, Renilla luciferase activity from the pRL-TK plasmid was assessed by adding 100 μL of Stop and Glo Reagent. To reduce experimental error, three replicate wells were used. RLU1 represented the intensity of firefly luciferase, while RLU2 indicated Renilla luciferase intensity. The ratio of RLU1 to RLU2 was then analyzed.

Immunofluorescence and multiple Immunofluorescence Assays

In the immunofluorescence assay conducted on Day 1, breast cancer cells were initially cultured on glass coverslips within 6-well plates. Following fixation, the cells were treated overnight with a PD-L1 antibody, while the nuclei were stained using DAPI. For the multiplex immunofluorescence (mIF), a five-color multiplex immunofluorescence kit (Shanghai Recordbio Technology Co. Ltd, RC0086-45RM) was utilized. The procedure began with the placement of tissue sections into a retrieval box containing EDTA (pH 9.0), which was then heated in water at 100°C for 15 minutes. After cooling the slides to room temperature, they were washed three times with PBS (pH 7.4), with each wash lasting 5 minutes. The slides underwent a 15-minute incubation with 3% H₂O₂, followed by washing and blocking with a solution of 3% BSA in PBS for 30 minutes. Primary antibodies were applied and incubated at room temperature for 1 hour. After washing, enzyme-labeled secondary antibodies were added and incubated at room temperature for 50 minutes. Following another wash, TSA dye (1:100) was applied for 10 minutes. This entire process was repeated three times for the PD-L1, SIRT7, and CD8 antibodies. Finally, nuclei were stainedThe final step involved staining the nuclei with DAPI. ImagesDAPI, and images were subsequently captured using fluorescence microscopy.

Single-Cell RNA Sequencing

After preparing the single-cell suspension, we conducted preliminary sequencing using the Chromium platform from 10x Genomics. The initial sequencing results were read and transformed into FASTQ files. Subsequently, we utilized CellRanger software (version 2.1.1, 10x Genomics) to convert unique molecular identifier (UMI) counts into a matrix. After de-duplicating the single-cell sequencing results from the 10x Genomics Chromium, we compared them to the human reference genome GRCh38.p12 using the CellRanger tool (version 3.1.0, 10x Genomics), which generated a UMI count matrix aligned with Ensembl gene annotations. Both the reference genome and gene annotations are accessible through the UCSC Genome Browser. The resulting count matrix was then analyzed using Scanpy software (version 1.9.3). Cells that did not meet quality criteria were filtered out, specifically those with fewer than 200 genes, over 20% mitochondrial content, or more than 50% ribosomal RNA. The integrated dataset was subsequently used for downstream analysis. To identify highly variable genes, we employed the sc.pp.highly_variable_genes function in Scanpy with parameters set to min_mean=0.0125, max_mean=3, and min_disp=0.5. Principal components were calculated using the sc.pp.pca function, and the number of principal components relevant for clustering was determined through sc.pl.pca_variance_ratio. We constructed a neighborhood graph using the sc.pp.neighbors function, followed by clustering with the sc.tl.leiden method, setting the resolution parameter to 0.4. Finally, UMAP projection was performed using sc.tl.umap to visualize the clustering results. We examined each cluster using known typical markers: epithelial cells (EPCAM, KRT19, KRT14, ERBB2, ESR1), endothelial cells (PECAM1, VWF), fibroblasts (DCN, COL1A1, COL1A2, COL3A1, CFD, PRGFRB), pericytes (ACTA2, TAGLN, MCAM), B cells (CD79B), macrophages (LYZ, IL1B, MSR1), plasma cells (JCHAIN, MZB1), and T cells (CD3G, CD3D, IL7R, NKG7, GNLY, CD8A).

Statistical Analysis

The experimental data were analyzed using SPSS version 23.0. Each experiment was replicated at least three times, and results are presented as mean ± standard error of the mean (SEM). We assessed differences between groups using Student’s t-test. Graphs and statistical analyses were created using GraphPad Prism version 5, with a p-value of < 0.05 considered statistically significant and a p-value of < 0.01 was considered highly significant.

SIRT7 inhibition restrained tumor growth by rescuing anti-tumor immune response.

The shSIRT7 cell lines were established in human-SK-BR-3 and mouse-EMT6 breast cancer cells, and these cells were then subcutaneously inoculated into T cell -deficient BALB/c nude mice. Besides, EMT6-shSIRT7 cells were implanted into BALB/c mice with normal immune function. The tumors were dissected and weighed after 3 weeks, and the results demonstrated that the SK-BR-3-shSIR7 group exhibited little difference in tumor volume compared to the control group in BALB/c nude mice(Fig. 1A-B), whereas the tumors in the EMT6-shSIRT7 group reduced greatly (Fig. 1C-D). Contrary, the restrained tumor growth was hardly observed in the nude mice. These results suggest that the demethyltransferase SIRT7 may promote tumor growth by impeding T-cell response. Hence, SIRT7 is likely to maintain tumor growth by suppressing the immune response. Furthermore, results on IHC staining for SIRT7 and CD8a demonstrated that the expression of CD8a was remarkably upregulated in EMT-6-shSIRT7 tumor tissues (Fig. 1E). T-cell activation act as an indicator of the immune response, and it was discovered that the population of GZMB+/CD8 + T cells was high in the tumor tissue (Fig. 1F-G). The expression of SIRT7 in most tumors was higher than that in normal tissues on the GEPIA and TIMER database (Fig. 1H-I). Additionally, it was also observed that SIRT7 was negatively correlated with the infiltration of CD8 + T cells in 1,100 patients with BC (Fig. 1J). Furthermore, 1,100 patients were classified into four subgroups that included Lumina A, Lumina B, HER2, and Basel type, and similar results indicated that SIRT7 was linked to an immunosuppressive environment (Fig. 1K).

CCL5 is positively associated with immuno-activated microenvironment

It was hypothesized that certain classes of cytokines secreted by tumor cells impact tumor cell function and inhibit the immune microenvironment. Based on information from the TIMER database, it was observed that the chemokine CCL5 was positively correlated with M1-like macrophage polarization and CD8 + T cell infiltration in 1100 BC patients (Fig. 2A). Similarly, the same results demonstrated that CCL5 is linked to an immune-activating environment in the Lumina A, Lumina B, HER2, and Basel type subgroups (Fig. 2B-E). Additionally, the relationship between the level of immune cell infiltration and CCL5 expression is was determined and illustrated (Fig. 2F). It was also realized that the CCL5 gene tended to be deleted at the arm level in patients with BC, suggesting that the CCL5 protein was typically expressed at low levels (Fig. 2G). The results revealed that patients showing high CCL5 expression exhibited a higher survival rate, and the prognosis of patients with high CCL5 expression and CD8 + T infiltration was favorable (Fig. 2H-I). Transcriptome sequencing results suggested that the upregulation of CCL5 levels is likely negatively correlated with the elevated activation of CD8 + T cells.

SIRT7 negatively regulates CCL5 expression through epigenetic transcription pathway

To determine whether CCL5 contributes to the construction of the SIRT7-mediated immunosuppressive microenvironment, the protein level of CCL5 when SIRT7 was overexpressed in MCF7 and HCC1937 BC cells was assessed. Western blot analysis demonstrated that SIRT7 overexpression lowered the expression of CCL5 and H3K18ac (Fig. 3A-B), as well as CCL5 mRNA levels (Fig. 3C-D). Furthermore, two shRNA sequences were designed to silence SIRT7 in SK-BR-3 and MDA-MB-231 BC cells and it was observed that SIRT7 knockdown enhanced CCL5 expression and H3K18ac levels (Fig. 3E-F). CCL5 and H3K18ac levels were also detected in the EMT6-shSIRT7 cells (Fig. 3G), and CCL5 mRNA levels increased when SIRT7 was knocked down (Fig. 3H-I). The compound 97491, a SIRT7 inhibitor, increased the CCL5 expression and H3K18ac levels(Fig. 3J-K). The CCL5 mRNA levels increased considerably as well (Fig. 3L-M). The mRNA level of CCL5 in shSIRT7 cell increased markedly as compared with other chemokines (Fig. 3N). The CCL5 protein levels in cell supernatants were assessed in the context of SIRT7 inhibition using ELISA assays, whereby the results demonstrated that the CCL5 level increased almost 20-fold when SIRT7 was inhibited in BC cells (Fig. 3O-P). Based on the current outcomes, it was concluded that SIRT7 negatively regulates CCL5 protein and mRNA levels, and considering that SIRT7 is a histone deacetylase, three sequence primers were designed for the CCL5 promoter region in human and mouse species. The ChIP assay results revealed that H3k18ac can bind to the P1 primers in the CCL5 promoter region in human SK-BR-3-shSIRT7 BC cells (Fig. 3Q-S). Similar results were obtained from the shSIRT7-EMT6 BC cells (Fig. 3T-V). SIRT7-mediated H3K18Ac reduction in the CCL5 promoter negatively regulates CCL5 expression via transcriptional epigenetic silencing pathways.

SIRT7 promote the transcriptional expression of PD-L1 by deacetylating YB-1

To determine the potential role of SIRT7 in regulating PD-L1, the stable knock-down cell lines were established to elucidate the regulatory effect of SIRT7 on PD-L1. Immunoblotting assays showed that SIRT7 knockdown decreased PD-L1 expression in MDA-MB-231,SK-BR-3 and EMT6 breast cancer cells (Fig. 4A-B,E). Additionally, the decreased expression of PD-L1 was observed when 97491 treatment (Fig. 4C-D). Overexpression of STRT7 increased the PD-L1 level (Fig. 4F). The patient-#2 who hardly responded to ICB treatment had high expression of SIRT7 and PD-L1, an opposite expression was observed in the patient-#4 who response to ICB treatment (Fig. 4G). Furtherly, the GEPIA database revealed that the mRNA of SIRT7 was positively correlated with CD274 (Fig. 4H). The fluorescence level of PD-L1 was decreased in shSIRT7-SK-BR-3 cells in comparison to the control group (Fig. 4I). Flow cytometry analyses demonstrated that the PD-L1 expression decreased due to SIRT7 inhibition (Fig. 4J). Data from mIF assays showed that patient-#4 who response to ICB treatment was expressed low SIRT7 and PD-L1(Fig. 4K). YB-1, as a previously studied transcription factor in our lab, it was discovered that YB-1 inhibition decreased the protein level of PD-L1 that YB-1 inhibition decreased the protein level of PD-L1(Fig. 4L). The fluorescence and flow cytometry analyses revealed that decreased PD-L1 expression when YB-1 knockdown (Fig. 4M-N). The acetylation on YB-1 protein was realized when perform IP assay using YB-1 antibody (Fig. 4O). However, SIRT7 inhibition barely affect the level of YB-1 protein, based on the deacetylation function of SIRT7[40], the acetylation at site of lysine 81 of YB-1 has been reported in the literature[41]. The IP assay revealed that the binding of SIRT7 and YB-1 protein decreased when SIRT7 inhibition (Fig. 4P). Moreover, the level of K-Ac on YB-1 protein increased when performing IP assay in shSIRT7 BC cells(Fig. 4Q), and decreased K-Ac on YB-1 when overexpression of SIRT7 (Fig. 4R). Moreover, the mutant plasmids YB-1-WT and YB-1-K81Q (Lysine at site 81 was replaced by glutamine to simulate hyperacetylation) were constructed to study the effects on transcription of PD-L1, and the results showed that the decreased PD-L1 in YB-1-K81Q group compared with YB-1-WT group (Fig. 4S). The acetylated enzyme activity assay showed that SIRT7 can directly remove the acetyl groups from YB-1 protein, the level of K-Ac on YB-1 protein decreasd when adding of SIRT7 protein(Fig. 4T). To further determine the effect of mutant YB-1-K81Q plasmid transfection on PD-L1 transcription, an upstream 2000bp fragment in the promoter of CD274 was constructed into a luciferase vector for the dual-luciferase reporter assay. The changes in luciferase activity can be used as the basis predicting the effect of PD-L1 transcription when transfected with YB-1-WT and YB-1-K81Q plasmids. The luciferase activity in the YB-1-K81Q groups was decreased considerably than that in the YB-1-WT group (Fig. 4U). To verify whether YB-1 can regulate the PD-L1 level by epigenetic ways, 3 primers for CD274 were designed respectively in ChIP experiment. The data demonstrated that YB-1 can bind to the P1 promoter region of CD274 in shSIRT7 BC cells(Fig. 4V). The PD-L1 transcription may be influenced by YB-1 acetylation at K81, how the transcriptional activity of YB-1 is affected by K81-acetylation is still under investigation. Generally, it can be concluded that SIRT7 positively regulated the protein level of PD-L1 based on YB-1 deacetylation.

SIRT7 suppression potentiate CCL5-induced M1-like macrophage polarization and CD8 + T cell activation

The results presented in Fig. 2 indicate a positive correlation between CCL5 and M1-like polarization. To understand the effect of CCL5 on macrophages, M2-like macrophages were obtained following the addition of PMA + IL-4 + IL-13 in THP-1 cells, and the expression of the M2 markers IL-10 and CD206 increased (Fig. 5A). Next, the stimulation with recombinant CCL5 protein facilitated the transformation from M2 to M1-like polarization (Fig. 5B), resulting in increase in the M1 markers CD40 and iNOS. Moreover, a co-culture system was used to link trafficking between BC cells and macrophages (Fig. 5C). Additionally, the supernatants from the coculture system containing CCL5 protein (induced by SIRT7 inhibition) enhanced the chemotactic migration of macrophages. The chemotactic effect could be blocked by treatment with maraviroc that suppressed CCL5-CCR5 axis activation (Fig. 5E-F). The chemotaxis assay revealed that the recombinant CCL5 protein promoted the chemotactic migration of macrophage. The variation in the chemotactic area of macrophages are presented in graph form (Fig. 5D). To understand the impact of CCL5 on the state of macrophages, the mRNA levels of M1-and M2-like phenotype markers were measured, and the results showed an increased M1-like mRNA level of iNOS and CD40 in response to CCL5 treatment or SIRT7 inhibition. In contrast, the M2-like mRNA levels of IL-10 and CD206 were decreased (Fig. 5G-L). The immune microenvironment consist of various immune cells, including macrophages and T cells, and the effects of M1-like phenotype on T cell activation are unclear. It has been previously reported that secreted TGF-β and IL-10 can lower T cell activation [42–44]. To assess the effect of M1-like macrophages on T cells activation, the level of T cell activation was weakened when TGF-β treatment (Fig. 5M). To verify the effect of co-cultured macrophages and tumor cells on the activation and killing ability of T cells, a two co-culture models containing three types of cells was designed. The model presented in Fig. 5N was used to measure T cell activation, and the model in Fig. 5U was used to assess the T cell killing ability (Fig. 5N,U). It was discovered that the lower TGF-β level was related to CCL5-induced M1-like polarization (Fig. 5O-P). The flow cytometry results indicated that T cell activation was augmented under CCL5-incduecd M1-like polarization (Fig. 5Q-T). It was also realized that the cytotoxic ability when macrophage and T cells were co-cultured was considerably stronger than that with macrophages alone (Fig. 5U-Y).

CCL5-induced CD8 + T cell infiltration promote M1-like polarization.

The previous results presented in Fig. 5W indicate that CCL5 can enhance the killing ability of T cells; and there is an association between CCL5 stimulation and T cell activation. The results demonstrated that the recombinant CCL5 protein promoted T cell activation, and CCL5-CCR5 blockade by maraviroc treatment attenuated T cell activation (Fig. 6A). A coculture system was used to link the trafficking between BC and T cells (Fig. 6B). It was confirmed that the elevated CCL5 secretion not only resulted in SIRT7 inhibition but also enhanced the activation of T cells (Fig. 6C-D). the results are presented in chart (Fig. 6E-F). It has been reported that response to immunotherapy is based on CD8 + T cells activation and M1-like macrophage polarization, and T cells can augment M1-like macrophage polarization[45]. It is unclear whether CCL5-induced activation of T cells can affect macrophage polarization. The effect of IFN-γ signaling, a known polarizer of macrophages towards anti-tumor M1-like phenotype has been reported [46, 47]. The expression level of IFN-γ was markedly increased in response to T cell activation by CCL5 stimulation, while treatment with Maraviroc attenuated this response (Fig. 6G). A comparable observation was made following 97491 treatment (Fig. 6H). Moreover, it was realized that IFN-γ can stimulate the polarization of M1 macrophages, with increased levels of iNOS and CD40 but decreased levels of IL-10 and CD206 in response to IFN-γ stimulation (Fig. 6I). The model presented was used to measure M1- and M2- like polarization of macrophages (Fig. 6J). The status of macrophage polarization was assessed when co-cultured with T cells and shSIRT7 BC cells, and it was observed that mRNA levels of iNOS and CD40 increased when SIRT7 inhibition (Fig. 6K-L). In contrast, the levels of IL-10 and CD206 markers of the M2-like phenotype decreased slightly (Fig. 6M-N). The model was used to assess the chemotactic effect (Fig. 6O). The chemotaxis assay revealed that activated T cells can promote the chemotactic migration of macrophages and that the chemotactic effect can be blocked by treatment with Maraviroc (Fig. 6P). Differences in the chemotactic area of macrophages are presented in graph form (Fig. 6Q). Based on these results, it is concluded that the CCL5 protein can trigger the activation of T cells, and activated T cells improve M1-like macrophage polarization. In summary, CCL5 not only promote the M1-like phenotype but also activate T cells, macrophages and T cells which mutually activate each other (Fig. 6R). This results in an immune-activated microenvironment that is characterized by a positive feedback activation loop.

SIRT7 inhibition enhance the anti-PD-1 immunotherapy effect

To investigate the potential effects of SIRT7 on breast cancer in vivo, a combination of the SIRT7 inhibitor 97491 and anti-PD-1 antibody was used to assess the immune-activated microenvironment in immunocompetent mouse models. The clinical application of anti-PD-1 therapy is often combined with paclitaxel, and this was also considered in our animal experiments. The results indicated that tumor size decreased significantly in the paclitaxel + 97491 + anti-PD-1 group compared to the control group (Fig. 7A). The representative images and statistical results of mouse tumors are as shown (Fig. 7B-C). Moreover, It was also observed that there was no significant fluctuation in body weight among the groups, suggesting that the integrated therapy exhibited limited dose toxicity in mice with breast cancer (Fig. 7D). Notably, according to the flow cytometric analyses, the combination treatment significantly induced tumor-infiltrating GZMB+/CD8 + T cells in the TME as compared to that of the control group (Fig. 7E-F). Likewise, multiple immunofluorescence (mIF) staining based on tyramide signal amplification (TSA) and quantitative analyses demonstrated a greater density of CCL5 and CD8a in the combination group than that in the control group, and a negative correlation between SIRT7 and CCL5/CD8a was observed(Fig. 7G). Data from mIF assays indicated that CCL5 and CD8 were coexpressed and colocalized among the groups, the codistribution of CCL5 and CD8a has obvious characteristics of regional consistency. Such negative relationship was confirmed by IHC results of the tumor tissue (Fig. 7H). Consistent with previous analyses, our results suggested that SIRT7 plays a pivotal role in the TME and that SIRT7 inhibitors may facilitate an antitumor response by enhancing tumor-infiltrating CD8 + T cells and downregulating PD-L1 in BC cells.

SIRT7 was negatively correlated with the “CCL5-CD8” anti-tumor axis

Although anti-PD-1/PD-L1 therapy has been applied in breast cancer patients, Less than 20% of patients benefited from the immunotherapy and disease progression[5]. We collected paraffin sections from breast cancer patients in the First Afliated Hospital of Shengzhen University. Previous research has revealed the relationship between SIRT7 and CCL5/CD8a in cell line and animal experiments, but the expression of SIRT7/CCL5/CD8a in breast cancer tissues are unknown. The IHC staining results revealed that the SIRT7 expression was negatively correlated with CCL5/CD8. The 3 patients who hardly response to ICB treatment had high expression of SIRT7 and low expression of CCL5, an opposite expression was presented on the patinet who response to ICB treatment (Fig. 8A). Data from mIF assays indicated that patients who response to ICB treatment expressed both low SIRT7 and high CCL5(Fig. 8B). According to the expression of SIRT7 and CCL5, we divided samples into four groups: SIRT7^L + CCL5^L, SIRT7^L + CCL5^H, SIRT7^H + CCL5^L, and SIRT7^H + CCL5^H. Surprisingly, the percentage of the low SIRT7 group accounted for 100% of the high CCL5 group, which indicated that CCL5 was almost highly expressed while SIRT7 was low-expressed (Fig. 8C-D). Furthermore, the scatter plots clearly indicated that CCL5 was negatively correlated with SIRT7 in the BC (n = 101, r=-0.6315, p < 0.0001***), CD8 and SIRT7 (n = 101, r=-0.5709, p < 0.0001***) and CD8 and CCL5 (n = 101, r = 0.8434, p < 0.0001***) groups of patients (Fig. 8E-G). Furthermore, the expression of CD8 in the SIRT7^L + CCL5^H group was much higher than in the SIRT7^H + CCL5^L group(Fig. 8H). The results suggested that CD8 was negatively correlated with SIRT7 and positively related to CCL5 regardless of SIRT7 status(Fig. 8H). Based on the different histological grades of tumor patients, we found that almost 50% of patients in the SIRT7^H + CCL5^L group were classified into the high grade. Contrary, the SIRT7^L + CCL5^H group were wholely classified into the low and moderate grade (Fig. 8I). The single cell sequencing was used to detect the proportion and distribution of SIRT7/CCL5/CD8 positive cells in 7 breast cancer patients, the result indicated that the abundance of SIRT7 was negatively correlated with CCL5/CD8(Fig. 8J). It was concluded that the low CCL5/CD8 was linked to high SIRT7 expression in BC patients.

Working model of SIRT7 bidirectionally regulates TILs/PD-L1 levels to reshape the breast cancer immunosuppressive microenvironment.

To better understand the molecular mechanisms discussed in this paper, we have drawn a schematic model summarizing how the transition from cold tumors to hot tumors. SIRT7 inhibition may sensitive the cancer cells to the anti-PD-1 therapy by increasing the TILs level and decreasing the PD-L1. SIRT7 inhibition enhance the CCL5 level by H3K18ac deacetylation, and CCL5-induced M1-like polarization and CD8 + T cell infiltration that can activate and reinforce each other. SIRT7 suppresion may facilitate YB-1 acetylation, leading to the binding of YB-1 to the promoter region of CD274, resulting in transcriptional activation of PD-L1. Elevated PD-L1 weakens T cell-mediated anti-tumor immunity. Cancer progression can be blocked through SIRT7 inhibition, leading to the immune-activated microenvironment. Combination therapy with a SIRT7 inhibitor and anti-PD-1 antibody exerted a synergistic antitumor effect. transition from cold tumors to hot tumors. SIRT7 inhibition may sensitive the cancer cells to anti-PD-1 therapy by increasing the TILs level and decreasing PD-L1. SIRT7 inhibition enhance the CCL5 level by H3K18ac deacetylation. CCL5-induced M1-like polarization and CD8 + T cell infiltration that can activate and reinforce each other. SIRT7 suppresion may promote YB-1 acetylation, which leads to the binding of YB-1 to the promoter region of CD274, and results in transcriptional activation of PD-L1. Combination therapy with a SIRT7 inhibitor and anti-PD-1 antibody exerted a synergistic antitumor effect.

Herein, it was observed that SIRT7 is highly expressed and contributes to the immunosuppressive microenvironment of BC. It was demonstrated that SIRT7 inhibition facilitated CCL5-induced M1-like polarization and CD8 + T cell infiltration. SIRT7-mediated histone H3K18 deacetylation lowered the level of CCL5 protein transcription, which was attributed to the binding of SIRT7 to the promoter of the CCL5 gene. Besides, as to its direct effect on tumor cell survival, SIRT7 facilitates immune evasion in a biphasic manner by promoting PD-L1 expression and CCL5-mediated TILs infiltration. Moreover, SIRT7 inhibition can prominently increase the efficacy of ICI therapy by potentiating TIL infiltration and reducing PD-L1 levels. Taken together, the data indicate that SIRT7 orchestrates breast cancer progression by simultaneously decreasing CD8 + T cell infiltration and increasing PD-L1 levels.Targeting SIRT7 may be a promising strategy for restraining tumor growth and increasing the efficacy of immunotherapy in BC.

Dysregulation of SIRT7 expression has been realized in various cancers and is believed to contribute to tumor pathogenesis[21, 23, 24]. It has demonstrated that SIRT7 facilitates HCC cell proliferation and tumorigenesis by deacetylating USP39 protein and enhancing its stability[48]. Additionally, the high SIRT7 expression is characterised with oncogenic properties and is associated with poor prognosis in colorectal cancer[25]. SIRT7 also facilitates the activation of AKT and S6K tumor-promoting pathway[49]. In contrast, some studies have shown that SIRT7 can also act as a tumor suppressor. Representatively, SIRT7 can prevent tumor metastasis by antagonizing TGF-β signaling, and its protein expression is significantly down-regulated in BC[22]. Additionally, SIRT7 can inhibit tumor cell growth by directly promoting WDR77 deacetylation to interfere with the WDR77-PRMT5 interaction[50]. Moreover, SIRT7 also prevents oral squamous cell carcinoma metastasis by promoting SMAD4 deacetylation and restraining epithelial-to-mesenchymal transition[27]. Therefore, the pathogenic role of SIRT7 in cancer is tumor type-specific and context-dependent.

In this study, we respectively introduced the two different acetylation modifications of SIRT7 on histone H3K18 and non-histone protein YB-1 were introduced. SIRT7-mediated H3K18 deacetylation is important in gene transcription repression, and the results demonstrated that CCL5 transcription was suppressed by SIRT7-mediated H3K18 deacetylation. SIRT7 orchestrates BC progression by transcriptionally restraining the CCL5-induced immune-activated microenvironment. Additionally, SIRT7 increases the protein level of PD-L1 by directly deacetylating YB-1, thereby reducing its transcriptional activity. its transcriptional activity. It has been reported that YB-1 acetylation inhibit the gene transcription[41]. Studies have also shown that SIRT7 up-regulation eradicated anti-tumor immunity by promoting PD-L1 expression[27]. Targeting SIRTs-mediated deacetylation has been regarded as a viable target for therapeutic intervention[51, 52]. This study reports that SIRT7 inhibition reduces PD-L1 levels and promotes H3K18ac-mediated CCL5 transcription, inducing M1-like polarization and CD8 + T cell infiltration. Tumor cells are likely to be thought of as smart and cunning, and SIRT7 can shape the tumor immune microenvironment by regulating the PD-L1 levels in cancer cells and restricting TIL cell infiltration due to reduced CCL5 secretion. It has been confirmed that CCL5 can activate both M1-type macrophages and CD8 + T cells. Besides, the activation of CD8 + T cells by CCL5 stimulation has also been discovered[53, 54]. It was discovered that M1-macrophages and CD8 + T cells can also reinforce each other, forming an more effective immune-activated microenvironment through cascading expansion. It has been revealed that immune tolerance can be moderated by gene transcription and chemokine secretion simultaneously.

Immunotherapy utilizing T cell antigen immune responses has encountered challenges related to limited efficacy and significant toxicity[55, 56]. The induction of immune reactions through the blockade of the PD1/PD-L1 pathway has shown promising results in clinical immunotherapy[57–59]. Generally, immune responses are responsible for recognizing and eliminating cancer cells. However, as cancer progresses, tumor cells have developed mechanisms to evade immune surveillance, allowing them to adapt to the immune environment and survive attacks. This phenomenon, collectively called adaptive immune resistance(AIR), classifies the tumor immune microenvironment (TIME) into four categories: PD-L1−/TIL− (type I), PD-L1+/TIL+ (type II), PD-L1−/TIL+ (type III), and PD-L1+/TIL− (type IV). Notably, only the type II subpopulation, characterized by high immune scores and elevated PD-L1 mRNA expression, demonstrated a significant association with improved progression-free survival (PFS) when compared to combination therapies and chemotherapy[36]. Immunotherapy efficacy has been verified by large-scale randomized controlled clinical trials in patients with advanced non-small cell lung cancer (NSCLC), based on the TIME classification model[37]. Following a prolonged period of adaptation and resistance to immune surveillance, a notable heterogeneity arises among patients, even those with the same pathological cancer type. Consequently, assessing a patient's immune status and formulating an appropriate treatment plan becomes critically important. Our findings suggest that SIRT7 plays a role akin to type IV-AIR in fostering an immunosuppressive microenvironment. Additionally, we will evaluate the potential of SIRT7 as a biomarker for immunotherapy in a larger cohort of breast cancer (BC) patients.

Our results indicate that targeting SIRT7 may offer advantages over solely targeting PD-L1, as it fosters the development of a type III-AIR immune-activated microenvironment. This suggests SIRT7 could serve as a novel prognostic index for tumor immunotherapy and a promising target for synergistic combinations with anti-PD-1 therapies. The introduction of new SIRT7 inhibitors could significantly enhance the landscape of immunotherapy for breast cancer.

Ethical approval and consent to participate

All in vivo experiments were approved by the Institutional Animal Care and Use Committee of Shenzhen University (SZU-IACUC-2022–0087) and followed the Guide for the Care and Use of Laboratory Animals. All human breast cancer sample acquisitions were approved by the Committee on Ethics of Shenzhen Second People’s Hospital, Shenzhen University. Written informed consent was obtained from all the participants.

Consent for publication

The authors have declared that no competing interest exists.

Availability of data and material

The mentioned datebase website in this paper were listed as http://gepia.cancer-pku.cn/ and http://timer.cistrome.org/.

Competing interests

The authors have declared that no competing interest exists.

Funding

This project was supported by the National Natural Science Foundation of China, China (No. 82172356, No.82203519, No.82572665), the Natural Science Foundation of Guangdong, China (No.2023A1515220238), the Natural Science Foundation of Shenzhen, China (JCYJ20230807115112024), and the Shenzhen High-level Hospital Construction Fund and Medical-Engineering Interdisciplinary Research Foundation of ShenZhen University.

Author contributions

The experiments for Fig. 1, 2, 3, 4, 5 and 6 were mostly completed by Sohail Khan. The animal experiment was cooperatively done by Sohail Khan. The rest of experiments for Fig. 8 were completed by Iltaf Khan. The working mechanism diagram in Fig. 9 and manuscript were fnished independently by Sumreen Sohail. The assistant of experimental data analysis was obtained from Sumreen Sohail. The main manuscript was completed by Sohail Khan, and the assistant of English polishing work was obtained from Sohail Khan. The design, research scheme and paper writing of whole project were under the guidance of Sumreen Sohail.

Acknowledgements

Not applicable.

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The authors declare no competing interests.

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Targeting SIRT7 shape an immune-activated microenvironment by both promoting CCL5-induced TILs infiltration and decreasing PD-L1 expression

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Abstract

Figures

Introduction

Materials and Methods

Results

CCL5 is positively associated with immuno-activated microenvironment

SIRT7 negatively regulates CCL5 expression through epigenetic transcription pathway

SIRT7 promote the transcriptional expression of PD-L1 by deacetylating YB-1

SIRT7 suppression potentiate CCL5-induced M1-like macrophage polarization and CD8 + T cell activation

SIRT7 inhibition enhance the anti-PD-1 immunotherapy effect

SIRT7 was negatively correlated with the “CCL5-CD8” anti-tumor axis

Working model of SIRT7 bidirectionally regulates TILs/PD-L1 levels to reshape the breast cancer immunosuppressive microenvironment.

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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