Anti-PD-L1 Antibody Enhances T Cell Immune Responses and Reduces Resistance of Breast Cancer Cells to Radiotherapy

Immune escape is a frequent occurrence, which limits the duration of antitumor immune responses to radiotherapy. Here, we aimed to ascertain the roles and underlying mechanisms of programmed death ligand 1 (PD-L1) in tolerance of breast cancer (BC) to radiotherapy. We first quantified microRNA-21 (miR-21) and PD-L1 expression in BC tissues and cells, followed by identification of the interactions between miR-21, PD-L1, and programmed cell death protein 4 (PDCD4). miR-21 knock-in mice were used to construct tumor-bearing models, which were then treated with anti-PD-L1 antibody and irradiation, followed by measurement of tumor growth and tumor immune escape. Finally, we evaluated the synergistic effects of radiotherapy and anti-PD-L1 antibody in vivo. The results showed increased miR-21 expression in BC tissues and cells, which was positively correlated with PD-L1 expression. The treatment with radiotherapy or anti-PD-L1 antibody in the miR-21 knock-in mice diminished tumor weight and volume, along with decreased CD3+CD8+ positive cells, serum IL-2 and IFN-γ levels, and lower PD-L1 expression, but augmented apoptosis of T and BC cells. Moreover, miR-21 significantly augmented PD-L1 expression via PI3K/Akt pathway activation by targeting PDCD4 in BC cells. Thus, radiotherapy and anti-PD-L1 antibody synergistically accelerated the therapeutic effect against BC in mice, thereby implicating a close interplay between radiotherapy, T cells, and the miR-21/PDCD4/PI3K/Akt/PD-L1 axis.


Introduction
Breast cancer (BC) is the most prevalent malignancy in female populations worldwide and accounts for a quarter of all cancers in women, causing around 500,000 deaths annually [1]. In spite of remarkable advancements in molecular targeted therapy [2], BC remains the predominant contributor to the cancer-related deaths in female populations [3]. Adjuvant whole breast radiotherapy is an important treatment modality in the early stage of BC, lowering the risk of local recurrence and death [4]. However, the application of radiotherapy in BC patients is often limited by the risk of its complications, in particular due to poor lung tissue tolerance, which often requires dose reductions or treatment breaks [5]. microRNAs (miRNAs) exert crucial roles in the development of human diseases, particularly in cancers, by acting as tumor suppressors or oncogenes; these properties have made miRNAs attractive tools and targets for novel therapeutic approaches [6]. Overexpression of miR-21 reduces ovarian cancer cell apoptosis and promotes cell proliferation by decreasing phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression and enhancing phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) activity [7]. In addition, miR-21 can potentiate hepatocellular carcinoma cell proliferation and migration while inhibiting apoptosis, by downregulating the TETs/PTENp1/PTEN pathway [8]. High miR-21 expression is linked to lower overall survival time among patients with BC [9]. Notably, miR-21 overexpression is linked to the onset of drug resistance in BC [10].
A recent study indicated that programmed death ligand 1 (PD-L1) protein expression was regulated by miR-21 in gastric cancer [11]. In a previous study of response biomarkers, radiotherapy at a low dose was found to trigger upregulated PD-L1 in tumor cells in various syngeneic models of cancer in mice [12]. BC tissues are frequently infiltrated by inflammatory cells, especially T lymphocytes and macrophages, and T lymphocytes have antitumor activity which can benefit patients' survival [13]. T cell immune responses have the potential to drive epithelial to mesenchymal transition in vivo, thereby producing BC stem cells [14]. PD-L1 has been documented as a critical immune checkpoint molecule that can enable escape of tumor cells from the host immune response via induction of T cell exhaustion and inhibition of effector T cell function [15]. Thus, prolonged treatment with anti-PD-L1 antibody results in persistent tumor regression in 6-17% of cases, together with prolonged stabilization of disease progression manifested in 12-41% cases of patients suffering from advanced BC [16]. In addition, Avelumab, an anti-PD-L1 antibody, presents with an acceptable safety profile and clinical activity in patients with locally advanced or metastatic BC; high PD-L1 expression in tumor-associated immune cells predicts an elevated possibility of clinical response to Avelumab in metastatic BC [17]. Furthermore, radiotherapy potentially provokes a local inflammatory response, thus simultaneously upregulating PD-L1 in the tumor microenvironment, which consequently represses sensitivity to antitumor immune responses [18]. Hence, if it were possible to avoid PD-L1-triggered immune escape, radiotherapy might induce longer lasting control of tumor cells. In this context, the current research intended to delineate the possible effects and elucidate the underlying mechanisms of anti-PD-L1 antibody involvement in T cell immune responses and subsequent radiotherapy tolerance of BC cells.

Ethics Statement. The Ethics Committee of the Affiliated Cancer Hospital of Zhengzhou University (Henan Cancer
Hospital) approved the current study protocol, which was in accordance with the Declaration of Helsinki. Written informed consent was provided by each participant prior to sample collection. The animal experiment protocols were approved by the Animal Ethic Committee of Affiliated Cancer Hospital of Zhengzhou University (Henan Cancer Hospital) and were strictly aligned with the Guide for the Care and Use of Laboratory Animal by the US National Institutes of Health.

Bioinformatics
Analysis. The BC-related miRNA expression dataset GSE44124, which contained 50 BC tissue samples and 3 normal breast tissue samples, was obtained from Gene Expression Omnibus database. The two groups of data in the microarray were subjected to differential expression analysis with |logFoldChange | >1 and p < 0:05 as the screening criteria. The downstream regulatory genes of target miRNA were predicted using PicTar, miRDB, TargetScan, and starBase, and the intersection of the four prediction results was obtained. The STRING database was employed to perform the protein-protein interaction analysis, and Cytoscape software was utilized for network visualization.

Study Subjects.
We collected 50 clinical samples from BC patients at an early stage (aged 32-65 years with a median age of 50 years) who underwent treatment at the Affiliated Cancer Hospital of Zhengzhou University (Henan Cancer Hospital) between June 2015 and December 2016. Based on the international Tumor Node Metastasis clinical staging of BC [19], 5 tumor tissue samples were at stage I, 33 cases at stage II, and 12 cases at stage III. All patients received breastconserving surgery after diagnosis by imaging examination and biopsy and were confirmed as invasive ductal breast cancer by postoperative pathology. Subsequent to formalin fixing and paraffin embedding, BC tissues and adjacent normal tissues were preserved at -80°C for follow-up experiments. Eagle's Medium (DMEM) encompassing 20% fetal calf serum (Invitrogen, Carlsbad, CA, USA) and human insulin (10 μg/mL), followed by placement in an incubator at 37°C with 5% CO 2 . On the next day, cell growth was observed, and the medium was renewed every 1-2 days.
2.6. Gamma Irradiation Treatment. In a mouse model, 5 days following injection of BC cells into the abdominal breast fat pad, the tumorigenic area in mice was irradiated with Cs-137 radiator (HWM D-2000, Siemens Erlangen, Germany) at a single dose rate of 0.95 Gy/min, 3 Gy in total. In addition, the tumorigenic area of control mice was exposed to sham irradiation. The mice were fed normally and the tumors were collected at specified time points.

Reverse Transcription Quantitative Polymerase Chain
Reaction (RT-qPCR). Total RNA extraction was implemented utilizing TRIzol (15596026, Invitrogen) and the RNA was employed as a template to generate complementary DNA (cDNA) using a reverse transcription kit (RR047A, Takara, Tokyo, Japan). For miRNA detection, cDNA of PolyAsupplemented miRNA was obtained using a PolyA tailing detection kit (B532451, Sangon, Shanghai, China). With SYBR Premix EX Taq (RR420A, Takara) for loading, RT-qPCR was implemented on an ABI 7500 instrument (Applied Biosystems, Foster City, CA, USA). At least 3 replicate wells were used. The primers of miR-21, PDCD4, and PD-L1 were prepared by Sangon (Table S2). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene to determine the relative expression levels of PDCD4 and PD-L1, and U6 was used as the reference for miR-21 expression. Fold changes were computed with the 2 -△△Ct method.
2.10. Immunohistochemistry. Subsequent to 10% formaldehyde fixing, the specimens were sliced at 4 μm, dehydrated, and paraffin-embedded. Antigen retrieval was then implemented. The slices were blocked by the addition of normal goat serum working fluid at 37°C for 10 min before 2-h probing with primary antibodies of rabbit antimouse PD-L1 (1: 100, ab48187, Abcam) and rabbit antibody to Ki67 (ab15580, 1: 200, Abcam) at 37°C. Next, the slices were reprobed with HRP-tagged secondary goat antirabbit IgG (ab6721, 1: 1000, Abcam) in a wet box at 37°C for 30 min, followed by 4-min counterstaining with hematoxylin (Shanghai Fusheng Industrial Co., Ltd., Shanghai, China) at ambient temperature. The excess dye solution was flushed away under running water and the slices were blocked 10% glycerol/PBS for microscopic observation. The results were scored independently by two examiners in a double-blind manner [20].
2.11. Immunofluorescence Staining. The cover glasses in the culture plate were fixed in 4% paraformaldehyde for 15 min and reacted with 0.5% Triton X-100 (Sangon) for 20 min at ambient temperature. Thereafter, normal goat serum (Solarbio) was supplemented to the glass for 30-min blocking at ambient temperature, followed by removal of the blocking solution. The glass was then reacted with the following primary rabbit antimouse antibodies (Abcam): PD-L1 (ab48187, 1: 100), CD8 (ab91279, 1: 400), Ym1 (ab192029, 1: 200), and Fizz (ab11429, 1: 100) at 4°C. Next, the glass was incubated with Alexa Fluor 647-labeled secondary donkey antirabbit IgG (ab150075, 1: 400, Abcam) in a wet box at 37°C for 1 h. Subsequent to 5-min nuclei staining with 4',6-diamidino-2-phenylindole (BioDee Biotechnology Co., Ltd., Beijing, China) in subdued light, the 3 Oxidative Medicine and Cellular Longevity glass was covered with fluorescence decay-resistant medium and the images were acquired with a fluorescence microscope (Olympus, Tokyo, Japan) for examination.  (g) PD-L1 protein level in BC tissues measured by Western blot analysis (n = 50). * p < 0:05 compared with adjacent normal tissues. Data (mean ± standard deviation) from two groups were compared using paired t-test or independent sample t-test. Each sample was evaluated three times independently.   2.15. Enzyme-Linked Immunosorbent Assay (ELISA). A 50 mg portion of tissue was placed in a 2.0 mL centrifuge tube and washed with 0.05 mmol/L precooled PBS. Following this, the fluid was replaced with 0.5 mL precooled PBS and the sample was placed in an ice bath for homogenization using a high-speed tissue homogenizer for 2 min. The supernatant was harvested subsequent to 10-min centrifugation at 3000 r/min and 4°C, and interleukin (IL)-2 and IFN-γ levels in the supernatant were determined using an ELISA kit (69-50049, Wuhan Moshake Biotechnology Co., Ltd., Wuhan, Hubei, China). Within 3 min, the measurement of absorbance (A) of each well at 450 nm was recorded using a multimode microplate reader (Synergy 2, BioTek Instrument, Inc., Winooski, VT, USA). A standard curve was calculated by linear regression equation for quantitation of samples.
2.16. Statistical Analysis. All data were described as mean ± standard deviation and analyzed by SPSS 21.0 statistical software (IBM Corp., Armonk, New York, USA). The paired t-test was adopted to compare the data between BC tissues and adjacent normal tissues, whereas unpaired t test was employed to compare data between the two groups. Oneway analysis of variance (ANOVA) was applied for the comparison of data between multiple groups, followed by Tukey's post hoc tests. Statistical analysis in relation to time-based measurements for tumor volume was made with ANOVA of repeated measures. A value of p < 0:05 was considered statistically significant.

Expression of miR-21 Was Abundant in Patients with BC after Radiotherapy and Positively Correlated with PD-L1
Expression. As tumor suppressors or oncogenes, miRNAs assume a crucial role in the occurrence and development of human diseases, especially malignant tumors [6].
Through differential expression analysis of the BC-related expression dataset GSE44124, 25 differentially expressed miRNAs were obtained, with miR-21 showing the most significant difference (Figures 1(a) and 1(b)). Recent evidence has indicated that miR-21 overexpression inhibits apoptosis of tumor cells and promotes tumor cell proliferation and migration properties by activating the PI3K/Akt pathway [7,8]. In addition, miR-21 enhances the resistance of BC cells to paclitaxel [10]. Additionally, miR-21 was evident as highly expressed in BC tissues in The Cancer Genome Atlas (TCGA) database (Figure 1(c)). In agreement, RT-qPCR revealed an increased miR-21 expression in BC tissues (Figure 1(d)). Therefore, miR-21 was chosen as the research focus in this study. A recent study indicates that PD-L1 protein expression is regulated by miR-21 in gastric cancer [11]. Analysis of BC-related datasets in TCGA database illustrated a positive correlation between PD-L1 and miR-21 expression in BC (Figure 1(e)). Moreover, elevated PD-L1 expression was also noted in BC tissues (Figures 1(f) and 1(g), Figure S1A). This indicated that miR-21 was positively correlated with PD-L1, and both of these were highly expressed in BC tissues. (f) Level of IFN-γ in peripheral blood and tumor tissues of mice measured using ELISA. * p < 0:05 compared with WT mice; # p < 0:05 compared with WT mice treated with radiotherapy. Each sample was evaluated three times independently. Data (mean ± standard deviation) from two groups were compared using independent sample t-test while data for tumor volume were compared using repeated measures ANOVA. There were 10 mice in each group.   Oxidative Medicine and Cellular Longevity weight of the mice following a total radiotherapy of 3 Gy (0.95 Gy/min) were monitored for 20 days. The results depicted in Figures 2(a) and 2(b) showed that the tumor weight and volume decreased in the WT mice and miR-21 +/+ mice following radiotherapy as compared with the miR-21 +/+ mice without treatment. In comparison to the WT mice, the tumor weight and volume of miR-21 +/+ mice displayed a significant increase under exposure to radiotherapy. These results suggested that the tumors of miR-21 +/+ mice showed high radiotherapy tolerance. At the same time, flow cytometry and ELISA documented that the WT mice and miR-21 +/+ mice following radiotherapy exhibited increased numbers of CD3 + CD8 + positive cells and IL-2 and IFN-γ levels and decreased T lymphocyte apoptosis rate in peripheral blood and tumor tissues as compared with the miR-21 +/+ mice without treatment. The numbers of CD3 + CD8 + positive cells and levels of IL-2 and IFN-γ were slightly reduced and T lymphocyte apoptosis rate was slightly elevated in peripheral bloods and tumor tissues of the miR-21 +/+ mice following radiotherapy in contrast to the WT mice following radiotherapy (Figures 2(c)-2(f)). However, no significant differences were witnessed in these features between WT and miR-21 +/+ mice without radiotherapy. Collectively, the number of immunocompetent cells decreased in the miR-21 +/+ mice, which promoted the immune escape of BC cells. 10 Oxidative Medicine and Cellular Longevity

miR-21 Promoted PD-L1 Expression via the PI3K/Akt
Pathway Activation by Targeting PDCD4 in BC Cells. Aiming to dissect the mechanism of miR-21 regulating PD-L1 in BC cells, we initially predicted the target genes of miR-21 via the PicTar, miRDB, TargetScan, and starBase databases. Intersection of the predicted results showed 51 target genes (Figure 3(a)). The protein interaction of 51 candidate target genes was analyzed using STRING database, which displayed that PDCD4 was one of the core genes ( Figure 3(b)). In addition, an inverse correlation was noted between PDCD4 and miR-21 expression by analyzing the BC dataset of TCGA database (Figure 3(c)). Previously, miR-21 was found to enhance the progression and facilitate the resistance of BC cells to paclitaxel treatment by targeting PDCD4 [19]. Therefore, PDCD4 was selected as a focus of this research project. The TargetScan database revealed a binding site between miR-21 and PDCD4 (Figure 3(d)). In addition, luciferase assay manifested that miR-21 mimic diminished the luciferase activity of PmirGLO-PDCD4-WT, but there was no significant difference in the luciferase activity of PmirGLO-PDCD4-MUT (Figure 3(e)), highly suggestive of the targeting relation between miR-21 and PDCD4.
As depicted in Figure S2, compared with MCF-10A cells, miR-21 expression increased, while PDCD4 expression decreased in BC cell lines, and the highest expression of miR-21 and the lowest expression of PDCD4 were found in MCF-7 cells. Thus, MCF-7 cells were selected to further demonstrate the regulatory relationships between miR-21, PDCD4, and PD-L1. As described in Figure 3(f), miR-21 mimic treatment induced elevated expression of miR-21 and PD-L1 yet decreased PDCD4 expression, while miR-21 inhibitor treatment caused opposite effects. In addition, there was a downward trend regarding PD-L1 expression in cells treated with both miR-21 mimic and oe-PDCD4.
It has been found that disruption of the PI3K/Akt pathway can reduce PD-L1 expression and prevent tumor immune escape [20]. To identify whether PDCD4 regulates PD-L1 through the PI3K/Akt pathway, we first constructed siRNAs targeting PDCD4. The silencing efficiency of si-PDCD4-2 was significantly higher and was consequently used for the follow-up experiments, designated as si-PDCD4 (Figure 3(g)). MCF-7 cells were treated with oe-PDCD4, si-PDCD4, or both si-PDCD4 and the PI3K inhibitor AZD6482. There was increased PDCD4 expression and reduced PD-L1 expression and PI3K and Akt phosphorylation in cells upon oe-PDCD4 treatment. However, treatment with si-PDCD4 had the opposite effects. In addition, PI3K and PD-L1 expression together with PI3K and Akt phosphorylation was decreased in cells following both si-PDCD4 and AZD6482 (Figure 3(h), Figure S1B). We further chose an orthotopic xenograft tumor model established using the miR-21 +/+ mice after radiotherapy to detect differential changes. The results manifested that miR-21 expression and PI3K and Akt phosphorylation levels were augmented, and PDCD4 mRNA and protein expression were diminished in the miR-21 +/+ mice treated with radiotherapy in contrast to the WT mice treated with radiotherapy ( Figure S3). Therefore, miR-21 targets PDCD4 and then induces activation of the PI3K/ Akt pathway to upregulate PD-L1 in BC cells.

Anti-PD-L1 Antibody Combined with Radiotherapy Inhibited BC Cell Growth and Promoted Apoptosis In Vivo.
Radiotherapy is the conventional treatment of BC, but most patients with BC suffer radiation resistance in the process of treatment [21]. At present, anti-PD-L1 antibody can The positive rate of Ki67 protein expression in tumor tissues of miR-21 +/+ mice following different treatment protocols, detected by immunohistochemistry (10 mice in each group, 3 sections for each mouse). (e) Cell apoptosis in tumor tissues of miR-21 +/+ mice following different treatment protocols, measured using TUNEL staining. (f) Protein levels of apoptosis-related factors in tumor tissues of miR-21 +/+ mice following different treatment protocols, determined by Western blot analysis. * p < 0:05, compared with miR-21 +/+ mice without treatment; # p < 0:05, compared with miR-21 +/+ mice treated with radiotherapy or anti-PD-L1 antibody. Each sample was evaluated three times independently. Data (mean ± standard deviation) from two groups were compared using independent sample t-test. There were 10 mice in each group. 11 Oxidative Medicine and Cellular Longevity effectively improve the radiation resistance of tumor cells [22], but there are few studies describing the impact of anti-PD-L1 antibody on the radiation resistance of BC cells.
To further explore the role of anti-PD-L1 antibody in BC radiotherapy, miR-21 +/+ mouse tumor models in situ were established and the BC mice were treated with radiotherapy or anti-PD-L1 antibody. After 20 days of observation, it was found that there was a remarkable reduction in the tumor volume and weight of mice following radiotherapy or anti-PD-L1 antibody alone, which was further reduced by cotreatment of radiotherapy and anti-PD-L1 antibody in miR-21 +/+ mice (Figures 4(a) and 4(b)).
As depicted in Figure S4A, radiotherapy treatment caused the decline of PD-L1 positive cells in the WT and miR-21 +/+ mice, while PD-L1 positive cells were enhanced in miR-21 +/+ mice in contrast to WT mice under exposure to radiotherapy. In comparison to miR-21 +/+ mice, PD-L1 positive cells (Figure 4(c)) and the positive rate of Ki67 protein expression (Figure 4(d)) were decreased, and apoptosis of BC cells (Figure 4(e)) was increased in the miR-21 +/+ mice treated with radiotherapy or anti-PD-L1 antibody alone. Radiotherapy combined with anti-PD-L1 antibody further reduced PD-L1 positive cells and the positive rate of Ki67 protein expression and elevated apoptosis of BC cells in the miR-21 +/+ mice.
Western blot analysis for quantification of apoptosisrelated factors also revealed that B-cell lymphoma-2-(Bcl-2-) associated protein X (Bax) and cleaved caspase 3 expression were facilitated, while Bcl-2 and Cyclin D1 expression was restrained by radiotherapy in WT and miR-21 +/+ mice, while the opposite tendency was witnessed in miR-21 +/+ mice in comparison to WT mice, in the presence of radiotherapy ( Figure S4B). In addition, we also observed elevated protein levels of Bax and cleaved caspase 3 and decreased T lymphocyte apoptosis rate in peripheral blood of miR-21 +/+ mice following different treatment protocols measured using Annexin V-FITC/PI. (c) Levels of IL-2 in the peripheral blood of miR-21 +/+ mice following different treatment protocols measured using ELISA. (d) Levels of IFN-γ in peripheral blood of miR-21 +/+ mice following different treatment protocols measured using ELISA. * p < 0:05 compared with miR-21 +/+ mice without treatment; # p < 0:05 compared with miR-21 +/+ mice treated with radiotherapy or anti-PD-L1 antibody. Each sample was evaluated three times independently. Data (mean ± standard deviation) from two groups were compared using independent sample t-test. The experiments were repeated 3 times independently.

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Oxidative Medicine and Cellular Longevity protein levels of Bcl-2 and Cyclin D1 in the miR-21 +/+ mice treated with radiotherapy and/or anti-PD-L1 antibody, while more significant changes were detected in miR-21 +/+ mice after cotreatment of radiotherapy and anti-PD-L1 antibody (Figure 4(f), Figure S1C). Thus, anti-PD-L1 antibody combined with radiotherapy inhibited BC cell growth and promoted apoptosis in vivo to increase the sensitivity of miR-21 +/+ mice to radiotherapy.

Anti-PD-L1 Antibody Combined with Radiotherapy
Suppressed Immune Escape of BC Cells In Vivo. We then determined whether anti-PD-L1 antibody and radiotherapy effectively curtailed T cell immune response in tumor tissues in vivo. Flow cytometry and ELISA exhibited that compared with the miR-21 +/+ mice without treatment, numbers of CD3 + CD8 + positive cells and levels of IL-2 and IFN-γ were elevated, and T lymphocyte apoptosis rate decreased in peripheral blood of miR-21 +/+ mice following radiotherapy or anti-PD-L1 antibody alone, and the most significant difference was found after cotreatment of radiotherapy and anti-PD-L1 antibody in miR-21 +/+ mice (Figures 5(a)-5(d)). These results support the notion that cotreatment of anti-PD-L1 antibody with radiotherapy greatly suppressed immune escape of tumor cells in miR-21 +/+ mice in comparison to anti-PD-L1 antibody treatment or radiotherapy alone, so as to ameliorate radiotherapy resistance of BC cells.

Discussion
Ionizing irradiation in high doses induces direct tumor cell death and augments tumor-specific immunity, thus enhanc-ing tumor regulation locally and distantly. However, local recurrence frequently occurs following irradiation treatment, which suggests that the irradiation-provoked response is not sufficient to sustain antitumor immunity [18]. Blockade of the T cell negative regulator PD-L1, also known as B7-H1, has the effect of activating T cell effectors in the context of elevated PD-L1 expression occurring in chronically inflamed tissues and tumors [23,24]. Here, we demonstrated that administration of anti-PD-L1 antibody enhanced the efficacy of radiotherapy through a T cell-dependent mechanism. We initially found a preferential expression of miR-21 in BC patients following radiotherapy. miR-21 is an oncogene that has been identified to aid in potentiating tumor cell growth and migration abilities in breast, bladder, and colon cancers [25]. Consistent with our present observations, BC tumor samples showed elevated miR-21 expression, and miR-21 expression was transiently upregulated 8 h upon irradiation treatment in radio-resistant T47D cells, and its inhibition attenuated radiation resistance at cellular level [26]. miR-21 has been identified as an independent biomarker of the efficacy of radiotherapy and chemotherapy and thus can be adopted as an effective predictor of response to chemotherapy and radiotherapy in esophageal squamous cell carcinoma [27]. In agreement, the experimental data in our study indicated that miR-21 knock-in enhanced tolerance of BC cells to radiotherapy and promoted tumor immune escape. In partially agreement with our findings, loss of miR-21 was shown to potentiate the polarization of macrophages to an inflammatory M1-like phenotype in tumor cells and thereby confer the host mice with enhanced antitumor immunity [28]. In addition, miR-21 expression has also been observed to be upregulated in colorectal cancer, which results in PD-L1 overexpression through inhibition of PTEN expression [29], suggesting a possible association between miR-21 and PD-L1. Here, we demonstrated that miR-21 expression was positively correlated with PD-L1 expression in BC patients after radiotherapy. miR-21 is implicated in maintenance of the balance between Th17 and Treg cells in patients following gastric cancer resection via the PD-1/PD-L1 pathway [11]. Moreover, PTEN induction has been found to reduce miR-21 expression, thus resulting in downregulation of PD-L1 via the PI3K/Akt/ p53 signaling pathway in embryonic cancer stem cells [30]. Accordingly, our experimental results suggested that miR-21 induced PD-L1 expression via the PI3K/Akt pathway activation by targeting PDCD4 in BC cells. Previously, computational analysis indicated that miR-21 can directly target PDCD4 and that the overexpression of miR-21 provokes a reduction in the transcription and protein expression of PDCD4 in cells presenting with the 3 ′ UTR of PDCD4 [31]. miR-21-5p has been demonstrated to augment the progression and paclitaxel resistance in drug-resistant BC cell lines by targeting PDCD4 [19]. In concurrence, the targeting relationship between miR-21 and PDCD4 was detected in our study, while PDCD4 was found poorly expressed in BC cells. PDCD4 overexpression has the capacity to restrict cell growth through its inhibitory role in the PI3K/Akt pathway in nonsmall-cell lung cancer [32], supporting our findings.
Another central observation in our study was that administration of anti-PD-L1 antibody increased the sensitivity of miR-21 +/+ mice to radiotherapy. A functional study has demonstrated that activation of the Akt/mammalian target of rapamycin (mTOR) pathway accelerates immune escape by elevating PD-L1 expression, where an mTOR inhibitor in combination with antibody PD-1 reduces tumor growth and regulatory T cells, while increasing the abundance of tumor-infiltrating T cells [33]. Avelumab, an anti-PD-L1 antibody, possesses antitumor property in treating metastatic BC and it has already shown clinical activity and safety in patients suffering from metastatic BC [17]. Notably, upregulated PD-L1 in bladder cancer cells correlates to the irradiation dose, whereas the blockade of PD-L1 delays tumor growth following radiotherapy [34]. Importantly, we verified that anti-PD-L1 combined with radiotherapy improved the therapeutic effect in BC by weakening radiation resistance. Concurrent inhibition of miR-21 in macrophages and anti-PD-1 antibody treatment presented with superior antitumor activity than either agent alone in an earlier study [28]. Of note, radiotherapy combined with immunotherapy is much more effective than monotherapy and has shown promising outcomes in various animal models [35]. Patients with non-small-cell lung cancer receiving combined therapeutic administration of anti-PD-1/PD-L1 antibody and radiotherapy have a significantly better prognosis than those who have not undergone radiotherapy [36]. In the context of invasive BC, radiotherapy combined with anti-PD1/PD-L1 therapy leads to more favorable clinical outcomes among patients [37]. These aforementioned findings provide a rationale for the combination of anti-PD-L1 antibody and radiotherapy in BC.

Conclusion
Overall, we have demonstrated that anti-PD-L1 antibody treatment can drive T cell immune responses and override the radiotherapy tolerance of BC cells by modulating the miR-21/PDCD4/PI3K/Akt/PD-L1 axis ( Figure 6). Our study provides evidence clarifying the effect of anti-PD-L1 antibody as a radiosensitizer. In addition, future studies should be conducted to verify whether miR-21 +/+ mice might become immunized against the tumor after radiotherapy and anti-PD-L1 treatment and how PI3K is phosphorylated by PDCD4.

Data Availability
The data and materials of the study can be obtained from the corresponding author upon request. and miR-21 +/+ mice after treatment with radiotherapy. B, PDCD4, PI3K, and Akt expression and PI3K and Akt phosphorylation levels in WT mice and miR-21 +/+ mice after treatment with radiotherapy. * p < 0:05 compared with WT mice treated with radiotherapy. There were 10 mice in each group. Each sample was evaluated three times independently. Figure S4: expression of PD-L1 and apoptosisrelated factors in WT mice and miR-21 +/+ mice treated with radiotherapy. A, PD-L1 positive cells in tumor tissues of WT mice and miR-21 +/+ mice treated with radiotherapy measured by immunofluorescence. B, Protein levels of apoptosis-related factors in tumor tissues of WT mice and miR-21 +/+ mice treated with radiotherapy determined by Western blot analysis. * p < 0:05, compared with WT mice treated with radiotherapy. There were 10 mice in each group. Table S1: intervention measures in each group.