Endoplasmic reticulum stress‐induced exosomal miR‐27a‐3p promotes immune escape in breast cancer via regulating PD‐L1 expression in macrophages

Abstract Immune escape of breast cancer cells contributes to breast cancer pathogenesis. Tumour microenvironment stresses that disrupt protein homeostasis can produce endoplasmic reticulum (ER) stress. The miRNA‐mediated translational repression of mRNAs has been extensively studied in regulating immune escape and ER stress in human cancers. In this study, we identified a novel microRNA (miR)‐27a‐3p and investigated its mechanistic role in promoting immune evasion. The binding affinity between miR‐27a‐3p and MAGI2 was predicted using bioinformatic analysis and verified by dual‐luciferase reporter assay. Ectopic expression and inhibition of miR‐27a‐3p in breast cancer cells were achieved by transduction with mimics and inhibitors. Besides, artificial modulation of MAGI2 and PTEN was done to explore their function in ER stress and immune escape of cancer cells. Of note, exosomes were derived from cancer cells and co‐cultured with macrophages for mechanistic studies. The experimental data suggested that ER stress biomarkers including GRP78, PERK, ATF6, IRE1α and PD‐L1 were overexpressed in breast cancer tissues relative to paracancerous tissues. Endoplasmic reticulum stress promoted exosome secretion and elevated exosomal miR‐27a‐3p expression. Elevation of miR‐27a‐3p and PD‐L1 levels in macrophages was observed in response to exosomes‐overexpressing miR‐27a‐3p in vivo and in vitro. miR‐27a‐3p could target and negatively regulate MAGI2, while MAGI2 down‐regulated PD‐L1 by up‐regulating PTEN to inactivate PI3K/AKT signalling pathway. Less CD4+, CD8+ T cells and IL‐2, and T cells apoptosis were observed in response to co‐culture of macrophages and CD3+ T cells. Conjointly, exosomal miR‐27a‐3p promotes immune evasion by up‐regulating PD‐L1 via MAGI2/PTEN/PI3K axis in breast cancer.


| INTRODUC TI ON
Breast cancer is the most prevalent malignancy among females and its incidence and mortality rate are predicted to increase in the coming years. 1 Advances in breast cancer screening have been updated internationally and the development in genomics helps to establish a new molecular categorization of breast cancer. 2 However, conservation surgery accompanied by radiotherapy was likely to relapse and it's largely dependent on patient age and other clinicopathological factors. 3 Recently, immunotherapy has emerged as a promising option for treating breast cancer, wherein a patient's own immune system is activated to combat breast cancer. 4 microRNAs (miRs) are important post-transcriptional regulators of gene expression and involved in cellular gene regulatory pathways. 5 In addition, miRNA-containing exosomes released from cancer cells are known to contribute to tumour growth and progression. 6 miR-27a-3p has been identified as an oncogenic RNA in multiple cancers including gastric and colorectal cancer, and is one of the highly-enriched miRNAs found in exosomes of breast cancer cells. [7][8][9] Bioinformatics analysis has predicted the possible binding sites between miR-27a-3p and the mRNA of membrane-associated guanylate kinase inverted 2 (MAGI2), a scaffold protein required for phosphatase and tensin homolog (PTEN) stability. MAGI2 has been previously implicated in regulating the migration and evasion of the breast cancer cells. 10,11 In particular, MAGI2 has been found to regulate PTEN stability. 12 The PTEN-PI3K/AKT axis plays an essential role in cell proliferation, migration and apoptosis in multiple myeloma as well as many other cancers. 13 In addition, programmed cell death-Ligand 1 (PD-L1), a protein exhibiting immune-inhibitory effect, is commonly found expressed on cancer cells and mediates immune escape. 14,15 Therefore, we examined if breast cancer cell-derived exosomal miR-27a-3p is involved in immune evasion by regulating PD-L1 expression.

| Ethics statement
The study was carried out under the approval of the Ethics Committee of Renmin Hospital of Wuhan University and all procedures followed the tenets of the Declaration of Helsinki. All patients signed written informed consent prior to participation in the study.

| Sample collection and cell culture
Tumour tissues of primary breast cancer and paracancerous tissues (≥5 cm from the tumour margin) were collected from 26 triple-negative breast cancer patients (mean age of 51.23 ± 3.18 years) who underwent tumour excision between 2013 and 2017 at the Renmin Hospital of Wuhan University. All enrolled patients were diagnosed with metastatic or locally advanced triple-negative breast cancer.
Validation of oestrogen receptor (ER) and epidermal growth factor receptor 2 (HER2) negativity (ER < 10% and HER2 0, 1 or 2 in the absence of amplification assessed by in situ hybridization) was performed with a biopsy of a metastatic lesion or recurrence in the breast. 16 Breast cancer tissues and paracancerous tissues were fixed using formaldehyde and embedded in paraffin for diagnosis.
The histopathology of breast cancer sections was evaluated using Edmondson-Steiner grading. Patients were classified into stage I (10), II (7) and III (9) based on the World Health Organization and tumour-node-metastasis staging system. MCF-7, BT474, MDA-MB-23, MCF-10A and THP-1 breast cancer cell lines were obtained from American Type Culture Collection (Manssas, VA, USA). Cells were cultured in the Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal bovine serum (FBS; Gibco, Carlsbad, CA), followed by culture in 5% CO 2 at 37°C with relative humidity of 95%.
Cells were passaged at confluency of approximately 90%.

| ER stress measurement
Endoplasmic reticulum stress-related proteins including GRP78 (ER master stress regulator) and IRE1α, PERK, ATF6 (ER stress sensors) were measured to quantify the degree of ER stress. The percentage of positive cells was graded (criteria: 0 indicates negative; 1 indicates ≤ 10%; 2 indicates 11%-50%; 3 indicates 51%-75%; 4 indicates > 75%). Staining intensity was measured in 5 random fields (0 stands for no staining; 1 for light yellow; 2 for pale brown; 3 for dark brown). Endoplasmic reticulum stress-related protein expression was determined as the percentage of positive cells multiplied by staining intensity. A score of 5 was used as the threshold to define low or high expression level for GRP78, while a score of 3 was used for IRE1α, PERK and ATF6.

| Immunohistochemistry
Paraffin-embedded sections were dewaxed using xylene, hydrated using gradient ethanol and incubated in PBS containing 0.5% Triton at room temperature for 20 minutes. The antigen was retrieved under low pressure for 2 minutes, boiled in 0.01 mol/L citrate buffer con- (1:50, ab125212), PD-L1 (1:50, ab238697) and GAPDH (1:5000, ab8245), followed by incubation for 2 hours at 37°C. Biotin-labelled goat anti-rabbit IgG was added (1:500) and incubated for 20 minutes at 37°C, followed by counterstaining using haematoxylin (Shanghai Fusheng Industrial Co., Ltd., Shanghai, China) for 4 minutes, dehydration, clearing and mounting. All antibodies were obtained from Abcam (Cambridge, UK). The sections were then viewed under a microscope and scored in a double-blind manner by two independent examiners.

| Immunofluorescence
Breast cancer cells were fixed in 4% polyformaldehyde for 30 minutes and embedded in paraffin, followed by permeabilization using 0.5% Triton X-100 (Sangon Biotech). Normal goat serum (Solarbio, (Beijing Biodee Biotechnology Co., Ltd.) was added to the cells and incubated without exposure to light for 5 minutes. The cells were then blocked using anti-fluorescence quenching blocking solution and a fluorescence microscope (TE2000, Nikon, Tokyo, Japan) was finally used for photography. Research Institute, La Jolla, CA) was employed to analyse at least 10 000 cells.

| Enzyme-linked immunosorbent assay (ELISA)
Tissues (50 mg) were immersed in 0.5 mL pre-cooled PBS, followed by an ice bath and then homogenized using a high-speed homogenizer for 2 minutes. The tissues were then centrifugated at 3000 r/min for 10

| Dual-luciferase reporter assay
The 3′-untranslated region (3′UTR) of MAGI2 was synthesized.  Transmission electron microscope was applied to evaluate exosomes. Exosomes (30 μL) were added to a copper net and 30 μL phosphotungstic acid (pH = 6.8) was added, followed by counterstaining for 5 minutes, and finally photographed using a transmission electron microscope.

| Extraction and characterization of exosomes from breast cancer cells
Flow cytometry was adopted to detect surface markers of exosomes CD63 content. Specifically, breast cancer cells-transported exosomes were digested using pancreatin, centrifugated at 503.1 g for 5 minutes and were triturated into a single cell suspension.
The cells were aliquoted and stored in 1.5 mL EP tube. PBS (1 mL) containing 1% BSA was used to triturate cells, followed by incubation for 30 minutes to block non-specific antigens. PBS contained in 200 µL/EP tube was then used to resuspend cells and each group was added with CD63-PE antibody (ab234251, PE, Abcam), followed by culture for 30 minutes. Samples without antibody were used as controls, and PE-labelled anti-human IgG was used as a homotype control. Thereafter, PBS containing 1% BSA was used to suspend cells and Guava easyCyte™ system flow cytometry was used to determine surface marker CD63 expression. Western blot was used to measure exosome surface markers CD63 (ab216130), CD9 (ab223052), CD81 (ab59477) and Tsg101 (ab30871) and negative marker GRP94 (ab13509). All antibodies were obtained from Abcam. miR-27a-3p expression in exosomes was determined using RT-qPCR.

| Breast cancer cells induced by tunicamycin
To induce ER stress, MDA-MB-231 and MCF-7 breast cancer cells were seeded in 24-well plates and cultured in eagle's minimum essential medium (EMEM) containing 10% FBS. Tunicamycin (1.0 μg/ mL) was added into EMEM containing 2% FBS and cells were cultured for 7 days, with cell counting performed every 24 hours.

| Statistical analysis
All data were processed and analysed using SPSS 21.0 statistical software (IBM Corp., Armonk, New York, USA). Measurement data were expressed as mean ± standard deviation. Data conforming to normal distribution and homogeneity of variance in intergroup settings were compared using paired t test, while comparisons between two groups were conducted using unpaired t test and one-way analysis of variance (ANOVA) was utilized to compare data among multiple groups, followed by Tukey's post hoc test. P < 0.05 was considered statistically significant.

| ER stress-induced macrophage infiltration and up-related PD-L1 expression
To evaluate the role of ER stress on breast cancer, firstly, the expression of ER stress-related proteins: GRP78, PERK, ATF6 and IRE1α was measured in breast cancer tissues and paracancerous tissues.
Immunohistochemistry results ( Figure 1A) showed that tumour tissues displayed significantly elevated GRP78, PERK, ATF6 and IRE1α, relative to paracancerous tissues. Furthermore, an obvious increase in GRP78, PERK, ATF6 and IRE1α expression was noted in tumour tissues with high ER stress as compared to that with low ER stress. The same trend was observed in tumour tissues than that in paracancerous tissues determined using RT-qPCR and Western blot assay ( Figure 1B,C). These findings together indicated that ER stress was frequently activated in triple-negative breast cancer.
Endoplasmic reticulum stress was previously found to exert antitumour effects via its role in immune regulation. 17 We found ER stress-related proteins to be interrelated with macrophage infiltration. Immunohistochemistry results showed markedly increased CD68 expression in tumour tissues relative to paracancerous tissues, and elevated CD68 expression was also observed in response to high ER stress as compared to low ER stress ( Figure 1D). It has been reported that PD-L1 mediates immune escape by inhibiting activation of T cells. 18 We identified higher PD-L1 expression in tumour tissues than in paracancerous tissues. PD-L1 expression was also found appreciably increased in high ER stress when compared to low ER stress ( Figure 1E). RT-qPCR and Western blot assay ( Figure 1F,G) similarly showed higher PD-L1 expression in tumour tissues compared to paracancerous tissues. Immunofluorescence results demonstrated the co-localization of PD-L1 and CD68+ macrophage in tumour stroma of the high ER stress group ( Figure 1H).
Taken together, our findings showed ER stress-induced macrophage infiltration and elevated PD-L1 expression.

| ER stress elevated breast cancer cell-derived exosomal miR-27a-3p expression
Exosomal miRs have been reported to be involved in macrophage infiltration and immune evasion. 19 To investigate how ER stress mod- Flow cytometry ( Figure 2E) was adopted to measure the level of exosome surface marker CD63, which was significantly increased. In addition, high expression of CD63, CD9, CD81 and Tsg101 were confirmed, along with poor expression of negative marker GRP94, in isolated exosomes using Western blot assay ( Figure 2F, P < 0.05). These findings suggested exosomes were isolated successfully. miR-27a-3p expression in breast cancer cells MCF-7 and MDA-MB-231 and in exosomes was determined using RT-qPCR ( Figure 2G,H, P < 0.05).
Up-regulated miR-27a-3p expression was found in both cancer cells and exosomes. miR-27a-3p up-regulation was also noted in cells treated with tunicamycin as compared to the controls. Meanwhile, RT-qPCR assay showed that miR-27a-3p was significantly up-regulated in tumour tissues and was negatively correlated with patient survival ( Figure 2I,J). Collectively, our results showed ER stress elevated breast cancer cell-derived exosomal miR-27a-3p expression.

| Breast cancer cell-derived exosomal miR-27a-3p up-regulated PD-L1 expression in macrophages in vitro
To further elucidate the role of breast cancer cell-derived exosomes in macrophages, we co-cultured PKH-67 (green)-traced exosomes and PMA-differentiated THP-1 macrophages ( Figure 3A,B). We found markedly increased red fluorescence signal within the macrophages at 12 hours after co-culture, suggesting a substantial uptake of exosomes by macrophages.
After co-culture for 12 hours, the uptake of PKH-67-labelled exosomes by macrophages was very obvious, indicating that exosomes can be transferred from breast cancer cells to macrophages.
To further validate that breast cancer cell-derived exosomes could deliver miR-27a-3p to macrophages, we overexpressed or knocked down miR-27a-3p in breast cancer cells MCF-7 and MDA-MB-231.
No significant differences were observed in exosomal miR-27a-3p expression between the mimic NC group and the inhibitor NC group. As expected, miR-27a-3p expression was markedly F I G U R E 1 ER stress-induced macrophage infiltration and contributed to PD-L1 overexpression. A, GRP78, PERK, ATF6 and IRE1α protein expression in breast cancer tissues in response to low/high ER stress was determined using immunohistochemistry (×400). B, GRP78, PERK, ATF6 and IRE1α protein expression in tumour tissues and paracancerous tissues was determined using Western blot assay. *P < 0.05 vs paracancerous tissues. C, GRP78, PERK, ATF6 and IRE1α mRNA expression in tumour tissues and paracancerous tissues was determined using RT-qPCR. *P < 0.05 vs paracancerous tissues. D, CD68 expression in response to low/high ER stress was assessed using immunohistochemistry (×400). E, PD-L1 expression in response to low/high ER stress was measured using immunohistochemistry (×400). F, PD-L1 mRNA expression in tumour tissues and paracancerous tissues was determined using RT-qPCR. *P < 0.05 vs paracancerous tissues. G, PD-L1 protein expression in tumour tissues and paracancerous tissues was determined using Western blot assay. *P < 0.05 vs paracancerous tissues. H, The co-localization of PD-L1, CD68 and macrophages in response to low/high ER stress was determined using immunofluorescence assay (×400). Measurement data were presented as mean ± standard deviation. n = 26. The unpaired t test was used to analyse the differences between two experimental groups F I G U R E 2 ER stress up-regulated breast cancer cell-derived exosomal miR-27a-3p expression. A, GRP78, PERK, ATF6 and IRE1α mRNA expression in breast cancer cells was determined using RT-qPCR, *P < 0.05 vs normal cells. B, GRP78, PERK, ATF6 and IRE1α protein expression in breast cancer cells was determined using Western blot assay, *, vs normal tissues, P < 0.05. C, Transmission electron microscope was used to evaluate exosomes (scale bar, 100 nm). D, Diameter distribution detected using Image-Pro Plus software. E, Flow cytometry was adopted to measure the content of exosome surface marker CD63. F, Contents of CD63, CD9, CD81, Tsg101 and GRP94 were observed using Western blot assay. G, miR-27a-3p expression was determined in MCF-7 and MDA-MB-231 breast cancer cells, *, vs control (MCF-7), P < 0.05; #, vs control (MDA-MB-231), P < 0.05. H, miR-27a-3p expression in MCF-7 and MDA-MB-231 breast cancer cell-derived exosomes was assessed using RT-qPCR, *P < 0.05, vs control. I, miR-27a-3p expression in tumour tissues and paracancerous tissues. J, correlation analysis of miR-27a-3p expression and survival of breast cancer patients. Measurement data were presented as mean ± standard deviation. The unpaired t test was used to analyse the differences between two experimental groups, and ANOVA was utilized to analyse data among multiple groups, followed by Tukey's post hoc test To test whether transferred miR-27a-3p may regulate PD-L1 expression in macrophages, we co-cultured MCF-7-derived exosomes with macrophages. miR-27a-3p and PD-L1 expression levels measured by RT-qPCR were significantly increased in response to Exo-miR-27a-3p mimic as compared to Exo-mimic NC, but no obvious change was observed in response to Exo-miR-27a-3p inhibitor or Exo-inhibitor NC ( Figure 3E, P < 0.05). Western blot assay ( Figure 3F) revealed a marked rise in PD-L1 expression in response to Exo-miR-27a-3p mimic as compared to Exo-mimic NC, while no significant change in PD-L1 was observed in response to Exo-miR-27a-3p inhibitor or Exo-inhibitor NC. These data indicated that breast cancer cell-derived exosomal miR-27a-3p up-regulated PD-L1 expression in macrophages in vitro.

| Breast cancer cell-derived exosomal miR-27a-3p up-regulated PD-L1 expression in macrophages in vivo
To confirm the role of breast cancer cell-derived exosomal miR-27a-3p in macrophages in vivo, we transfected different plasmids into breast cancer MCF-7 cells and inoculated the transfected cells into nude mice. Next, we injected PKH-67 (green)-labelled exosomes into the nude mice every other day for 20 days. At 12 hours after the last injection, we separated peritoneal macrophages and examined them using flow cytometry ( Figure 4A). Using confocal microscopy and flow cytometry, we found that PKH-67-labelled exosomes were enriched in the isolated peritoneal macrophages ( Figure 4B,C), suggesting in vivo uptake of labelled exosomes by macrophages. Figure 4D) demonstrated that miR-27a-3p and PD-L1 expression were significantly increased in response to Exo-miR-27a-3p mimic as compared to Exo-mimic NC, but no obvious F I G U R E 3 Breast cancer cell-derived exosome-encapsulated miR-27a-3p up-regulated PD-L1 expression in macrophages in vitro. A, Flow cytometry was used to validate PMA-induced differentiated THP-1 macrophages. B, Confocal fluorescence microscopy was used to observe the uptake of exosomes by macrophages (×400). C, miR-27a-3p expression in breast cancer cells was determined using RT-qPCR, *P < 0.05 vs mimic NC; #, P < 0.05 vs inhibitor NC. D, miR-27a-3p expression in exosomes secreted by breast cancer cells was determined using RT-qPCR, *P < 0.05 vs mimic NC. E, miR-27a-3p and PD-L1 expression in co-culture of exosomes and macrophages was determined using RT-qPCR, *P < 0.05 vs Exo-mimic NC. #P < 0.05 vs Exo-inhibitor NC. F, PD-L1 protein expression in co-culture of exosomes and macrophages was assessed using Western blot assay. *P < 0.05 vs Exo-mimic NC. #P < 0.05 vs Exo-inhibitor NC. Measurement data were presented as mean ± standard deviation, and ANOVA was utilized to analyse data among multiple groups, followed by Tukey's post hoc test. Experiments were conducted in triplicates A, Flow cytometry was used to evaluate macrophages. B, Confocal microscopy was used to observe exosomes uptake by macrophages (×400). C, Flow cytometry was used to determine exosomes uptake by macrophages; D, miR-27a-3p and PD-L1 expression in co-cultures of exosomes and macrophages determined using RT-qPCR, *P < 0.05, vs Exo-mimic NC. #P < 0.05 vs Exo-inhibitor NC. E, PD-L1 protein expression in macrophages co-cultured with exosomes was determined using Western blot assay. *P < 0.05 vs Exo-mimic NC. #P < 0.05 vs Exo-inhibitor NC. Measurement data were presented as mean ± standard deviation, and ANOVA was utilized to compare data among multiple groups, followed by Tukey's post hoc test. Experiments were conducted in triplicates F I G U R E 5 miR-27a-3p targeted MAGI2 in macrophages. A, TargetScan was used to predict the existence of putative binding sites between miR-27a-3p and MAGI2. B, Mutation of miR-27a-3p and MAGI2 binding sites and dual-luciferase reporter assay of luciferase activity. C, MAGI2 expression in macrophages in response to miR-27a-3p mimic, mimic NC, miR-27a-3p inhibitor, and inhibitor NC was determined using RT-qPCR, *P < 0.05 vs mimic NC. #P < 0.05 vs inhibitor NC. Measurement data were presented as mean ± standard deviation. Unpaired t test was used to analyse the differences between two experimental groups, while ANOVA was utilized to analyse differences among multiple groups, followed by Tukey's post hoc test. Experiments were conducted 3 times independently

| MAGI2 inhibited PD-L1 expression in macrophages by up-regulating PTEN to inhibit PI3K/ AKT signalling pathway
It has been previously shown that MAGI2 regulates PTEN expression, while PTEN inhibits PI3K/AKT signalling pathway. 10 Western blot assay, *P < 0.05 vs OE-MAGI2-si-NC. Measurement data were presented as mean ± standard deviation. Unpaired t test was adopted to analyse the differences between two experimental groups, while ANOVA was utilized to analyse differences among multiple groups, followed by Tukey's post hoc test. Experiments were conducted 3 times independently  Figure 6C).
Using Western blot analysis, we did not find significant differences in MAGI2 or AKT expression between OE-PTEN or OE-NC groups, and between si-PTEN and si-NC groups (P > 0.05, Figure 6D).
Similarly, decreased expression of PI3K, p-AKT and PD-L1 was ob-

| Macrophages activated by exosomal miR-27a-3p promoted the immune escape of breast cancer cells
Macrophages have been suggested to promote tumour progression by stimulating angiogenesis, enhancing tumour cell migration and invasion, and suppressing antitumour immunity. 20 Therefore, we wondered whether breast cancer cells with ER stress could promote the M2-polarization of macrophages, which may in turn impair cytotoxic CD8 + T cell responses. We isolated exosomes from MCF-7 breast cancer cells and co-cultured them with THP-1-differentiated macrophages. Flow cytometry revealed significantly increased CD206 + cells in response to Exo-miR-27a-3p mimic treatment as compared to treatment with Exo-mimic NC, but there was no apparent change in the number of CD206 + cells in response to Exo-miR-27a-3p mimic vs Exo-inhibitor NC ( Figure 7A). These findings suggested that exosomal miR-27a-3p promoted macrophage M2-polarization.
To describe the functional role of exosome-treated macrophages, we co-cultured macrophages with CD3 + T cells. We found a reduction of CD8 + T cells and decreased IL-2 expression in T cells upon co-culture with macrophages treated with Exo-miR-27a-3p mimic as compared to Exo-mimic NC. The proportion of CD8 + T cells and IL-2 expression in T cells exhibited no obvious changes between macrophages treated with Exo-miR-27a-3p inhibitor and Exo-inhibitor NC ( Figure 7B,C). Annexin V/PI assay showed more apoptotic T cells in macrophages treated with Exo-miR-27a-3p mimic than in those treated with Exo-mimic NC ( Figure 7D). Taken together, we demonstrated that macrophage treatment with exosomal miR-27a-3p could lead to immune escape of breast cancer cells via inhibiting CD8 + T cells.

| D ISCUSS I ON
Chemotherapeutic resistance in certain subtypes of breast cancer is known as a cause of poor patient survival, and countering such resistance remains one of the major challenges in breast cancer management. 21 At the same time, immune-modulating treatments have shown efficacy for some breast cancers, given their immunogenic features. 22 In this study, we identified a novel regulatory mechanism promoting immune escape of breast cancer cells. We found that under ER stress, breast cancer cells produce exosomes containing miR-27a-3p. Exosomal miR-27a-3p up-regulates PD-L1 in macrophages and promotes immune evasion of breast cancer cells by activating the PTEN-AKT/PI3K pathway.
The first finding of this study was that ER stress was activated in breast cancer tissues, as evident by increased expression of GRP78, PERK, ATF6 and IRE1α in tumour tissues. In addition, ER stress-induced macrophage infiltration and up-regulated PD-L1 expression.
Endoplasmic reticulum stress was previously reported to be transmitted to myeloid cells, macrophages and dendritic cells, which are pivotal regulators of tumour immunity. 23 Higher expression of GRP78, PERK, ATF6 and IRE1α 24 has been noted in breast cancer tissues, indicative of stimulation of ER stress in breast cancer cells. It was previously reported that macrophages could stimulate angiogenesis, enhance tumour cell migration and invasion and thereby suppress antitumour immunity. 20 Notably, the activation of macrophages is found to be enhanced by ER stress. 25 PD-L1 expression on tumour cells inhibits CD8 + T cell cytotoxicity and is shown as a prerequisite for immune evasion in immunogenic tumours. 26 Marked elevation of PD-L1 expression has been found to promote tumour growth and escape from antitumour immune mechanisms. 27 In agreement with this finding, we found macrophage infiltration and PD-L1 elevation in response to high ER stress in breast cancer cells, suggesting ER stress could induce immune evasion of breast cancer cells.
Exosomes are cell-released, phospholipid-enclosed vesicles, which act as biological cargos to carry and exchange biological materials or signals. 28 Exosomes contain specific repertoires of miRNAs that can be functionally transferred to recipient cells. 28 Notably, miR-27a-3p was up-regulated in breast cancer cells and ER stress elevated miR-27a-3p expression shuttled by exosomes.
Besides, breast cancer cells can deliver miR-27a-3p to macrophages through exosomes. Concordant with our study, miR-27a was previously found up-regulated in breast cancer cells and its mimics at- We validated that MAGI2 is a target gene of miR-27a-3p.
MAGI2 has been reported as an independent predictor of recurrence in prostate cancer. 32 MAGI2, a scaffold protein required for PTEN, has also been identified as a target of miRNAs that are up-regulated in tumours. 33 The results of this study illustrated that MAGI2 up-regulated PTEN to inactivate the PI3K/AKT signalling pathway. Consistently, PTEN enhancement has been found to underscore the MAGI-2-induced inhibition of cell migration and proliferation in hepatocarcinoma cells. 34 A more recent study reported that MAGI2 binds to un-phosphorylated PTEN and this interaction contributes to PTEN stability. 35 Importantly, PTEN tumour suppressor is a major inhibitor of the PI3K/AKT pathway inactivation and a common target for suppressing multiple cancers. 36 Inhibition of PI3K has been found to down-regulate PD-L1 expression and potentiate an anti-proliferative effect, indicating that disruption of PI3K signalling may enhance the antitumour effect. 37 We found increased phosphorylation of AKT and increased expression of PTEN in OE-MAGI2-treated macrophages, which down-regulated PD-L1. Thus, our results indicated MAGI2 inhibited PD-L1 expression by up-regulating PTEN and inactivating PI3K/AKT.
In conclusion, the present study provides new insights into the mechanism of miR-27a-3p on immune evasion of breast cancer cells. Notably, miR-27a-3p was up-regulated in breast cancer cells and exosomal miR-27a-3p derived from breast cancer cells induced by ER stress up-regulated PD-L1 expression, thereby F I G U R E 7 Immune evasion of breast cancer cells was enhanced by macrophages treated with exosomal miR-27a-3p. A, CD206 in macrophages was determined using flow cytometry. B, CD4 and CD8 in T cells were detected using flow cytometry. C, IL-2 secreted by T cells was detected using ELISA assay. *P < 0.05 vs Exo-mimic NC. D, Annexin V/PI assay was used to assess macrophages treated with exosomal miR-27a-3p on T cells apoptosis. Measurement data were presented as mean ± standard deviation. ANOVA was utilized to analyse differences among multiple groups, followed by Tukey's post hoc test. Experiments were conducted 3 times independently   The discovery of this mechanism may direct the future development novel therapeutic targets for breast cancer. However, further studies warranted to explore the role of this mechanistic pathway in clinical settings.

ACK N OWLED G EM ENTS
We thank the patients who participated in this study, all investigators and research support staff, past and present, at the participating centres. We also acknowledge all trial unit staff who contributed to the central coordination of the study.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and/or analysed in the current study are available from the corresponding author upon reasonable request.