
==== Front
Transl Oncol
Transl Oncol
Translational Oncology
1936-5233
Neoplasia Press

S1936-5233(24)00203-1
10.1016/j.tranon.2024.102076
102076
Original Research
Effect of tumor-derived extracellular vesicle-shuttled lncRNA MALAT1 on proliferation, invasion and metastasis of triple-negative breast cancer by regulating macrophage M2 polarization via the POSTN/Hippo/YAP axis
Wang Xuedong ab
Jian Qiwei a
Zhang Ziyun b
Gu Juan b
Wang Xinping a
Wang Yueping wy1005@126.com
bc⁎
a School of Medicine, Anhui University of Science & Technology, Huainan, Anhui, 232001, China
b Center for Precision Medicine, Anhui No.2 Provincial People's Hospital, Hefei, Anhui, 230041, China
c Department of Molecular and Cellular Biology, University of Connecticut, Storrs, CT, 06269, USA
⁎ Corresponding author at: 1868# Dangshan Road, North 2nd Ring, Center for Precision Medicine, Anhui No. 2 Provincial People's Hospital, Hefei, Anhui, 230041, China. wy1005@126.com
01 9 2024
11 2024
01 9 2024
49 10207629 1 2024
1 7 2024
1 8 2024
© 2024 The Authors. Published by Elsevier Inc.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• MALAT1/POSTN are boosted, and related with M2 infiltration/poor prognosis in TNBC.

• MALAT1 is up-regulated in TNBC-derived EVs and induces M2 polarization via EVs.

• TNBC-EV-derived MALAT1 induces macrophage M2 polarization by promoting POSTN.

• POSTN inhibition curbs M2 polarization by suppressing the Hippo/YAP pathway.

• TNBC-EV-derived MALAT1 induces M2 polarization and facilitates TNBC development.

Objectives

Triple-negative breast cancer (TNBC) is the deadliest subtype of breast cancer (BC). Tumor-derived extracellular vesicles (EVs) trigger tumor progression by promoting M2 polarization. Some lncRNAs can be encapsulated into EVs for intercellular communication. Herein, we investigated the mechanism of TNBC-derived EV-shuttled lncRNA MALAT1 on macrophage polarization/tumorigenesis.

Methods

BC-associated targeted EV-derived lncRNAs were screened. Tumor tissues/tissues adjacent to cancer of TNBC patients, and blood samples of all subjects were collected. MALAT1/POSTN mRNA levels in tumor tissues/tissues adjacent to cancer, and MALAT1 expression in EVs and its correlation with TNBC patient overall survival were assessed by RT-qPCR/Kaplan-Meier survival analysis/log-rank test. TNBC patient M2 infiltration was detected by flow cytometry. MALAT1/POSTN levels in EVs/macrophages were regulated by transfection. Hippo/YAP activation was determined by Western blot. Nude mouse xenograft model was established and metastasis was detected by H&E staining.

Results

MALAT1/POSTN were up-regulated and correlated with M2 infiltration/poor prognosis in TNBC patients. TNBC-derived EVs induced M2 polarization. MALAT1 was highly expressed in TNBC-derived EVs and could be transferred to macrophages via EVs to induce M2 polarization. POSTN overexpression diminished the inhibitory effect of MALAT1 knockdown on M2 markers. EVs activated the Hippo/YAP pathway in macrophages. The Hippo/YAP pathway inhibition abrogated the effect of POSTN overexpression on M2 marker expression. TNBC-EV-derived MALAT1 facilitated M2 polarization, and thus promoting occurrence and metastasis of TNBC in vitro and in vivo.

Conclusions

TNBC-EV-derived MALAT1 activated the Hippo/YAP axis by up-regulating POSTN, thereby inducing M2 polarization to promote TNBC occurrence and metastasis in vivo.

Keywords

Triple negative breast cancer
Extracellular vesicles
MALAT1
Hippo/YAP
M2 macrophage
==== Body
pmcIntroduction

Breast cancer (BC) is considered as the second leading cause of cancer-related death in women, only after lung cancer [1]. Triple-negative breast cancer (TNBC) refers to a subtype of BC that lacks progesterone receptor and estrogen receptor, or human epidermal growth factor receptor 2 (HER2) overexpression, accounting for approximately 15 % of BC [2,3]. Unfortunately, TNBC does not respond effectively to HER2-targeted therapy and hormone therapy and has a poorer prognosis compared with other types of BC [4]. Chemotherapy has been the primary first-line treatment option for patients with TNBC for decades [5]. Although a number of advances in recent years, including targeted agents and immunotherapy, have improved TNBC prognosis, however, many patients with TNBC continue to experience disease progression within 2–5 years of initial diagnosis [6]. Therefore, it is vital to better understand the potential progression mechanism and effective treatment for TNBC.

Macrophages play a crucial part in the development of TNBC [7]. Especially, tumor-associated macrophages (TAMs) are an essential element of the tumor microenvironment (TME) and drive aggressive cellular phenotypes in many cancers [[8], [9], [10], [11]]. TAMs participate in the paracrine signaling circulation by invading tumor cells, thereby facilitating the release of macrophage-derived EGF and tumor-derived CSF-1 in lung and breast cancers [[12], [13], [14]]. It is also noteworthy that macrophages are functionally malleable and can change their polarization state from M1 to M2 to fit in various physiological conditions, where activated M2 macrophages play a role in stimulating anti-inflammatory responses and tumor progression [8,15]. M2 polarization activation relies on the unique external stimuli and microenvironment [16,17]. The development of immunotherapies for cancers targeting these TAMs has attracted the attention of many scholars [15,18].

Extracellular vesicles (EVs), present in body fluids, exert a pivotal effect in intercellular signaling by transporting a variety of cargoes (including proteins, lipids, nucleic acids, and metabolites, among others) from parental cells to recipient cells [19,20]. Importantly, tumor-derived EVs are key mediators of cell communication between immune cells and cancer cells, which facilitate tumor progression by stimulating M2 macrophage polarization [[21], [22], [23]]. A study conducted by Jang et al. has demonstrated that epigallocatechin gallate up-regulates miR-16 in BC cells, which can be transferred to TAM via EVs and suppresses M2 polarization and TAM infiltration, ultimately leading to reduced tumor growth [24]. It has also been documented that TNBC-derived EVs trigger M2 macrophages polarization, thus creating a favorable ecological niche for lymph node metastasis [25]. However, a recent report has unveiled that TNBC-derived EVs facilitate an inflammatory TME bound up with better prognosis [26]. EVs originate from cancer are not sufficient on their own to produce a distinct and robust M1 or M2 macrophage phenotype, however, they contribute to the maintenance of TME to mediate pro-survival and proliferative phenotypes [27]. Nevertheless, the role and potential mechanism of TNBC-derived EVs mediating macrophage polarization remain poorly understood.

Long non-coding RNAs (lncRNAs), transcripts of more than 200 nucleotides with no apparent protein-coding function, are found in the nucleus or cytoplasm of cells and can interact with DNA, RNA, or proteins [28]. Among these lncRNAs, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is widely known to modulate disease progression in various cancers, which serves as a new oncogenic lncRNA in modulating the progression of BC [29]. LncRNAs can also be encapsulated into EVs as messengers for intercellular communication [30]. Notably, some lncRNAs are enriched in EVs, while others are almost absent, suggesting that some lncRNAs are selectively sorted into EVs and tumor-derived EV-shuttled lncRNAs are responsible for tumorigenesis by facilitating coagulation and angiogenesis, manipulating the immune system, and remodeling peripheral parenchymal tissues [31,32]. For instance, pancreatic cancer cells produce FGD5-AS1-rich EVs to polarize M2 macrophages to boost malignant behaviors of pancreatic cancer cells [33]. Tumor-derived EV-carried BCYRN1 distinctly enhances migration and tube formation of human lymphatic endothelial cells, as well as lymph node metastasis and lymphangiogenesis of bladder cancer [34]. Additionally, EVs derived from renal cell carcinoma induce macrophage polarization and facilitate tumor development by delivering lncARSR [35]. More importantly, BC-derived EV-shuttled lncRNAs are considered a new frontier for shaping malignant behaviors, such as the proliferation, angiogenesis, migration, and immunomodulation) of recipient BC cells [36]. Nevertheless, what is not yet clear is the effect and the underlying mechanism of TNBC-EV-MALAT1 mediating macrophage polarization on tumorigenesis. Therefore, this study highlights the importance of TNBC-EV-MALAT1 on macrophage polarization and tumorigenesis, thereby offering some important insights into the possible targets of TNBC.

Materials and methods

Ethics statement

All animal experimental procedures were ratified by the Research Ethics Committee of Anhui Medical University and conducted in strict accordance with the National Guide for Laboratory Animals (Approval number: LLSC20220982). We implemented various laboratory procedures to alleviate the discomfort of the mice, including the use of heating pads, disinfection, and fluid supplementation with saline.

Screening of TNBC-associated EV-carried lncRNAs

Exosome-associated lncRNAs were screened from exoRBase 2.0 online database (http://www.exorbase.org/exoRBaseV2/toIndex) and deepBase v3.0 online database (https://rna.sysu.edu.cn/deepbase3/index.html). BC-associated lncRNAs were obtained via RNADisease V4.0 online database (http://www.rnadisease.org/search_result) (Score = 1) and the LncRNADisease 2.0 online database (http://www.rnanut.net/lncrnadisease/index.php/home/search) (Score > 0.95) screening. The intersection of the 4 databases and Venn diagram was taken to screen the target EV lncRNA of BC. The regulatory relationship between BC-related lncRNAs and M2 macrophage infiltration-related target genes of TNBC was retrieved using LncTarD 2.0 online database (https://lnctard.bio-database.com/).

Patient and tissue samples

Paired tumor tissues and tissues adjacent to cancer of 80 TNBC patients who underwent surgery in Anhui No. 2 Provincial People's Hospital from January 2015 to December 2017 were collected, as well as 5 mL blood samples at admission. Blood samples (5 mL) were also acquired from 80 healthy subjects who underwent physical examinations in Anhui No. 2 Provincial People's Hospital during the same period. All collected tissue samples were pathologically confirmed by 2 experienced pathologists and stored at −80 °C for EV extraction. Later, the blood samples were centrifuged at 3000 rpm for 15 min to separate serum samples, which were stored at −20 °C. The clinical data of all subjects are listed in Table S1. All TNBC patients did not receive chemotherapy or other related treatments preoperatively, and patients with metastatic diseases or other malignant tumors were also excluded from the study. TNBC patients were followed up for 5 years with their overall survival (OS) analyzed.

Cell culture

Human leukemia monocytic cell line (THP-1) and TNBC cell lines (MDA-MB-231 and HCC1937) obtained from ATCC (Manassas, VA, USA) were fostered in Dulbecco's modified Eagle medium (Biological Industries, Kibbutz Beit Haemek, Israel) containing 10 % fetal bovine serum (Biological Industries) and 1 % penicillin/streptomycin (Gibco, Carlsbad, CA, USA) at 37 °C in an incubator with 5 % CO2.

Based on the previous study [10], M2 macrophage utilized in the assay were obtained by induction of THP-1 cells. Briefly, THP-1 cells were induced by 100 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA; Merck KGaA, Darmstadt, Germany) at 37 °C for 24 h, followed by addition of interleukin (IL)−4 (20 ng/mL) and IL-13 (20 ng/mL) (PeproTech, Rocky Hill, NJ, USA) at 37 °C for 24 h.

The steps for non-contact co-culture of macrophages and TNBC cells in vitro were as follows [37]: MDA-MB-231 cells and HCC1937 cells were seeded onto the apical chamber of the polyester membrane (0.4 µM) of the transwell inserts (BD Biosciences, Franklin Lakes, NJ, USA) in the wells, and PMA-led THP-1 cells were seeded onto the basolateral chamber of the system, followed by 48-h co-culture, with the 2 types of cells collected for further experimentation.

Cell transfection

Lung adenocarcinoma metastasis-associated transcript 1 (MALAT1) in MDA-MB-231 cells was knocked out by synthesizing small interfering RNAs (siRNAs; si1-lnc, si2-lnc) and siRNA-negative control (si-NC; Geneseed Biotech, Guangzhou, Guangdong, China) targeting MALAT1 [38]. The most effective siRNA si1-MALAT1 and si2-MALAT1 subclones (50 nM each) detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) were co-transfected into the lentivirus vector pHBLV-U6-MCS-CMV-ZsGreen-PGKPuro to construct the si-MALAT1 vector, with si-NC as NC. Lentivirus vectors carrying si-MALAT1 or si-NC and two auxiliary vectors (pCMV-VSV-G and pCMV-dR8.2 dvpr) were transiently transfected into MDA-MB-231 cells. Subsequently, the stably transfected MDA-MB-231 cells were screened by puromycin for the subsequent experiments.

Periostin (POSTN) knockdown/overexpression was achieved using lentivirus vectors carrying POSTN shRNA (sh-POSTN) or its NC shRNA NC (sh-NC), pcDNA3.1-POSTN (oe-POSTN), or its NC pcDNA3.1-NC (oe-NC). The specific method was the same as previously mentioned. The dose for shRNA was 50 nM and for pcDNA3.1 was 10 nM. All transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) as per the manuals. The sequences of siRNA and shRNA are shown in Table S2.

Cell treatment and grouping

MDA-MB-231 cells were divided into the following 3 groups: 1) the MDA-MB-231 group (without any treatment); 2) the si-MALAT1 group (cells were transfected with lentivirus vectors containing si1-MALAT1 and si2-MALAT1); 3) the si-NC group (cells were transfected with lentivirus vectors containing si-NC).

Macrophages were arranged into the following 11 groups: 1) the Blank group (THP-1 cells were induced with 100 ng/mL PMA for 24 h); 2) the co-cul group (PMA-induced THP-1 cells were non-contactly co-cultured with MDA-MB-231 or HCC1937 cells for 48 h and collected); 3) the co-cul + dimethylacetamide (DMA) group [PMA-induced THP-1 cells were non-contactly co-cultured with MDA-MB-231 or HCC1937 cells treated with DMA (EV release inhibitor, Sigma-Aldrich, 15 nM for 24 h) for 48 h, and collected); 4) the phosphate buffer saline (PBS) group (PMA-induced THP-1 cells were treated with PBS); 5) the EVs group [PMA-induced THP-1 cells were treated with 30 μg/mL MDA-MB-231 or HCC1937 cell-derived EVs for 24 h, with the dosage of EVs referred to an existing study [39]]; 6) the EVs-si-NC group (PMA-induced THP-1 cells were treated with MDA-MB-231 cell-derived EVs in the si-NC group for 24 h); 7) the EVs-si-MALAT1 group (PMA-induced THP-1 cells were treated with MDA-MB-231 cell-derived EVs of the si-MALAT1 group for 24 h); 8) the EVs-si-MALAT1 + oe-POSTN group (PMA-induced THP-1 cells were transfected with pcDNA3.1-POSTN for 24 h, then treated with MDA-MB-231 cell-derived EVs of the si-MALAT1 group for 24 h); 9) the EVs-si-MALAT1 + oe-NC group (PMA-induced THP-1 cells were transfected with pcDNA3.1-NC for 24 h, followed by the treatment with MDA-MB-231 cell-derived EVs of the si-MALAT1 group for 24 h); 10) the EVs-si-MALAT1 + oe-POSTN + verteporfin (VP) group [PMA-induced THP-1 cells were transfected with pcDNA3.1-POSTN for 24 h, and then treated with MDA-MB-231 cell-derived EVs of the si-MALAT1 group and 2 μM of the Hippo/YAP pathway inhibitor VP (Sigma-Aldrich) for 24 h, with the dosage of VP referred to a previous study [40]]; 11) the EVs-si-MALAT1 + oe-POSTN + dimethyl sulfoxide (DMSO) group (NC for the EVs-si-MALAT1 + oe-POSTN + VP group).

M2 macrophages were divided into the following 4 groups: 1) the M2 group (PMA-treated THP-1 cells were treated with IL-4 and IL-13 for 24 h to differentiate into M2 macrophages); 2) the M2 + si-MALAT1 group (M2 macrophages were transfected with lentivirus vectors containing si1-MALAT1); 3) the M2 + sh-NC group (M2 macrophages were transfected with lentivirus vectors containing sh-NC); 4) the M2 + sh-POSTN group (M2 macrophages were transfected with lentivirus vectors carrying POSTN shRNA).

Isolation and identification of EVs

According to pervious study [41], serum, MDA-MB-231 or HCC1937 cell culture medium sample was centrifuged for 10 min at 300 g to remove cellular debris. After recovery of the supernatant, sequential centrifugation was performed on the samples at incremental speeds: first centrifugation for 20 min at 2000 g to diminish dead cells, followed by a subsequent centrifugation for 30 min at 10,000 g to discard cellular debris, and then a final centrifugation at 10,000 g for 70 min to pellet microvesicles. Prior to ultra centrifugation again at the same speed, the resulting pellet was washed with PBS. Then, the pellet was resuspended with PBS to 200 μg/mL. To inhibit EV release, MDA-MB-231 cells were incubated typically for 24 h with 15 nM of the pharmacological inhibitor DMA (Sigma-Aldrich) [37].

For transmission electron microscopy (TEM) analysis [42], EVs were fixed with 5 % glutaraldehyde (Sigma-Aldrich) and then observed under a TEM (Hitachi, Tokyo, Japan). EVs were identified by detecting the expression of EV marker proteins CD63, CD81, TSG101 and negative marker Calnexin by Western blot. The antibodies (Abcam, Cambridge, UK) used were as follows: CD63 (1/1000, ab217345), CD81 (1/500, ab79559), TSG101 (1/1000, ab125011) and Calnexin (1/1000, ab92573). The size distribution and concentration of EVs were analyzed by ZetaView (Particle Metrix, Ammersee, Germany).

EV tracing

EVs were labelled for EV tracing assay using the PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich) as per the manuals. PBS-diluted EVs were added to Diluent C (1 mL) within PKH67 dye (4 mL) for 4-min incubation. Subsequently, 2 mL 0.5% bovine serum albumin was added to bind excess dye. The labelled EVs were extracted using ExoQuickTC EVs Isolation Reagent. Thereafter, EVs pellet was diluted in PBS (100 Ml) and utilized for tracing assay. THP-1 cells were treated with PKH67-tagged EVs (50 mg) at 37 °C for 12 h. Finally, THP-1 cells were imaged using a fluorescence microscope (Leica,Weztlar, Germany).

RNA extraction and RT-qPCR

RNA was isolated from tissues or cells using Trizol (Vazyme, Nanjing, Jiangsu, China). RNA from the nucleus or cytoplasm was extracted using Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek, Thorold, Canada). EV-RNA was isolated and extracted with the help of EV purification and RNA isolation kit (AmyJet Scientific, Wuhan, Hubei, China). circRNA was purified by detaching linear RNA using RNase R (Geneseed Biotech). The Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) was performed to determine the concentration and purity of the extracted RNA. Next, complementary DNA was synthesized with 500 ng of the total RNA using PrimeScript reverse transcription kit (Takara, Shiga, Japan). Then, SYBR Premix Ex Taq II (Takara) was utilized to perform RT-qPCR on the Light Cycler 480 II Real-Time PCR System (Roche, Basel, Switzerland) to determine the required RNA levels. The reaction procedure was 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 58 °C for 30 s and 72 °C for 1 min. The primer sequences are shown in Table S3. With GAPDH serving as a normalized reference, 2-ΔΔCt method was for relative RNA level analysis. All primers were designed and synthesized by Sangon Biotechnology (Shanghai, China).

Flow cytometry

Monoplasts from human tumor tissues and tissues adjacent to cancer were extracted using the human tumor tissue dissociation kit (135–095–929, NovoBiotechnology, Beijing, China) as per the provided instructions. Mouse tumor tissue monoplasts were extracted using the mouse tumor tissue dissociation kit (135–096–730, NovoBiotechnology). The extracted tissue-derived monoplasts or differently-treated THP-1 cells were prepared into monocyte suspension at a concentration of 2 × 107 cells/mL. Macrophage M2 percentage content in the tissues and the expression of CD206 in THP-1 cells of different groups were assessed by flow cytometry. The gating methods were monoplasts (FSC-A and FSC—H), live cells (7-AAD and SSC-A), TAMs (CD45+CD11b+F4/80+) and M2 TAMs (CD11b+F4/80+CD206+). In short, fluorescent dye-labelled antibodies were added directly to the cell suspension, and the cells were incubated on ice for 1 h and washed twice with PBS. The stained cells were analyzed on a FACSCalibur system. The antibodies (eBioscience, San Diego, CA, USA) information was as below: PE-CD45 (25–0459–42), APC—CD11b (17–0118–42), FITC-F4/80 (11–4801–82) and APC—CD206 (17–2069–42).

Western blot

Radioimmunoprecipitation assay buffer (Thermo Fisher Scientific) was utilized to extract proteins, with the protein concentration measured using bicinchoninic acid kit (Beyotime, Shanghai, China). Prior to transferring to polyvinylidene difluoride membranes (Bio-Rad, Richmond, CA, USA), protein (20 μg) was separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membranes were blocked with 3 % bovine serum albumin (Solarbio, Beijing, China), followed by the incubation overnight with primary antibodies POSTN (Q15063, 2 μg/mL, CUSABIO,Wuhan, Hubei, China), LATS1 (1:5000, ab70561, Abcam), p-LATS1 (1:1000, Ser909, Yanjing Biotechnology, Shanghai, China), YAP (ab205270, 1 μg/mL, Abcam), p-YAP (1:1000, ab254343, Abcam) or β-actin (1:5000, ab179467, Abcam), and 2-h incubation with horseradish peroxidase-tagged secondary antibody immunoglobulin G (1:8000, ab97080, Abcam), with β-actin serving as a normalized control. The visualized blots were observed by Image J v1.8 after the membranes were incubated with enhanced chemiluminescence reagent (Beyotime). Relative protein expression was expressed as fold-change of the control group.

Cell counting kit-8 (CCK-8) assay

MDA-MB-231 cells were cultured with conditioned medium of macrophages in different treatment groups, with their viability assayed by CCK-8. Cells were seeded onto the 96-well plates (1 × 104/well) and incubated for 24, 48, and 72 h, with 10 μL CCK-8 added at each time point. Afterwards, cells were incubated for 3 h. The optical density value at 450 nm was measured using a microplate reader (Bio-Rad).

Transwell assay

The invasive and migratory properties of TNBC cells were separately tested using Transwell chambers (Corning, Corning, NY, USA) coated with and without Matrigel (Becton Dickinson, Franklin Lakes, NJ, USA). The treated 100 μL MDA-MB-231 cells were seeded onto the chambers of 24-well plates (2.5 × 105 cells/well), as well as the 600 μL of conditioned medium of macrophages of different groups underneath the chambers. After 18 h, the non-migrated and non-invaded cells on the lower surface of the chambers were wiped off using a cotton swab, whereas the invaded and migrated cells on the lower surface were fixed for 5 min with methanol, followed by 10-min staining with Giemsa. Ultimately, the stained cell number in 4 random fields of view was counted under a light microscope (Leica).

Nude mouse xenograft model

The 18 female BALB/c nude mice (4–6 weeks old, 16–20 g) from Vital River Laboratory Animal Technology (Beijing, China) were randomly arranged into 3 groups (n = 6). Each mouse was injected intravenously with clodronate liposomes (200 μL of 5 mg/mL stock in PBS, Yeasen, Shanghai, China) [43,44] for macrophage ablation. Subsequently, MDA-MB-231 cells (5 × 106 cells/mouse) were injected subcutaneously into the right area of the mice. At the same time, THP-1-induced macrophages (106 cells/50 μL/mouse) were injected into mice via tail vein at 3-day intervals. After 10 days, PBS, EVs, or EVs-si-MALAT1 (200 μg EVs in 200 μL PBS) were injected directly into the xenograft tumors (corresponding to the TNBC + PBS, TNBC + EVs and TNBC + EVs-si-MALAT1 groups, respectively). The mice were sacrificed 30 days after cell injection, and the tumor tissues were collected for RT-qPCR, Western blot and M2 macrophage infiltration detection.

To investigate the effect of macrophages on tumor metastasis in vivo, 18 BALB/c nude mice were randomly divided into 3 groups (n = 6). MDA-MB-231 cells co-cultured with conditioned macrophages (stimulated by PBS, EVs, or EVs-si-MALAT1) were injected into the mice via tail vein (corresponding to the TNBC + PBS-M group, TNBC + EVs-M group and TNBC + EVs-si-MALAT1-M groups, respectively). The health and behaviors of the mice were observed once the tumor diameter reached approximately 5 mm or the tumor volume reached about 50 mm3 (typically occurring 5–7 days post tumor cell inoculation). Monitoring was conducted at intervals of 2–3 days. During the monitoring period, the status of nude mice and tumor growth were observed. The tumor weight in mice should not surpass 10 % of their body weight, with the average tumor diameter not exceeding 15 mm. If ulceration, infection, or necrosis occurred, the experiment should be terminated, and the animals should be sacrificed. The tumor volume was measured once a week (tumor volume = 0.5 × length × width2). After 6 weeks, all mice were sacrificed by intraperitoneal injection of 200 mg/kg of 3 % pentobarbital sodium (the sacrifice of mice was confirmed by cessation of breathing, complete cessation of heartbeat, and dilation of the pupils). The tumors were collected and weighed. Next, H&E staining was performed on the lungs, bones, and livers of all mice to check for suspicious metastatic sites. No experimental mice sacrificed throughout the experimental period (prior to euthanasia).

Statistical analysis

Data were statistically analyzed and graphed using SPSS 21.0 (IBM, Armonk, NY, USA) and GraphPadPrism 8.01 (GraphPad, San Diego, CA, USA) software. Data were categorized into counting and measurement data, with counting data expressed as the number of cases and measurement data confirme to be normally distributed by the Shapiro-Wilk test expressed as mean ± standard deviation. Comparisons between 2 groups were made using paired/independnet t-test. Comparisons among multiple groups were analyzed by one-way analysis of variance (ANOVA), and post hoc analysis were performed by Tukey's multiple comparisons test. p < 0.05 was considered statistically significant.

Results

MALAT1 in serum EVs and postn in tumor tissues of tnbc patients were up-regulated and significantly correlated with M2 tam infiltration and poor prognosis

To probe for EV-derived lncRNAs involved in the regulation of M2 macrophage infiltration in TNBC, we obtained EV-associated lncRNAs and BC-related lncRNAs from databases, and the final 11 BC-associated target EV-derived lncRNAs (Supplementary Table 4 and Fig. 1A). Subsequently, we further screened for lncRNAs connected with M2 macrophage infiltration in TNBC. Wu H et al. have screened for 6 genes (OLFML2B, MS4A7, SPARC, POSTN, THY1 and CD300C) affecting the prognosis of TNBC patients [4]. We found that among the 11 BC EV-lncRNAs screened above, only H19 and MALAT1 were regulated by POSTN in the 6 genes associated with M2 macrophage infiltration in TNBC by LncTarD 2.0 online database (Supplementary Figure 1). MALAT1 has been reported to be promising in regulating the oncogenic immunomodulatory proteins MSLN and CD80 in TAMs of TNBC [45]. In addition, POSTN secreted by glioblastoma stem cells stimulates malignant growth by recruiting M2 tumor-associated macrophages [46]. Therefore, we speculated that TNBC-derived EVs might be involved in regulating M2 macrophage polarization and tumorigenesis via MALAT1/POSTN.Fig. 1 MALAT1 in serum EVs and POSTN expression in tumor tissues were up-regulated in TNBC patients and prominently linked with infiltration of M2 TAMs and poor prognosis. (A) 11 BC-associated target EV-derived lncRNAs were obtained by exoRBase 2.0, deepBase v3.0, RNADisease V4.0 (Score = 1), and LncRNADisease 2.0 (Score > 0.95) screening; (B) MALAT1 and (C) POSTN mRNA expression levels in tumors and tissues adjacent to cancer of TNBC patients were determined by RT-qPCR; Kaplan-Meier survival analysis and log-rank test were performed to determine the correlations between (D) MALAT1 and (E) POSTN mRNA in tumor tissues with OS; (F) EV morphology in serum of TNBC patients and healthy subjects was observed by TEM; (G) Western blot was implemented to measure the expression levels of CD63, CD81, TSG101 and Calnexin in serum EVs of TNBC patients and healthy subjects; (H) The concentration and size of EVs were analyze by NET; (I) RT-qPCR was carried out to determine the expression of MALAT1 in serum EVs of TNBC patients and healthy subjects; (J) Kaplan-Meier survival analysis and log-rank test were utilized to determine the correlation between serum EV-MALAT1 levels and OS in TNBC patients; (K) The percentages of TAMs (CD11b+F4/80+) and (L) M2 TAMs (CD11b+F4/80+CD206+) in tumors and tissues adjacent to cancer of TNBC patients were detected by flow cytometry; (M-N) Pearson was used to analyzed the correlations between MALAT1 level in serum EVs and POSTN mRNA level in tumor tissues with M2 macrophage infiltration level in TNBC patients. Data were expressed as mean ± standard deviation, and comparisons between 2 groups were made using paired/idependent t tests. ** p < 0.01.

Fig. 1

As reflected by RT-qPCR results, the expression levels of MALAT1 and POSTN mRNA were prominently elevated in tumor tissues compared to tissues adjacent to cancer (all P < 0.01, Fig. 1B-C). Kaplan-Meier survival analysis unveiled that highly-expressed MALAT1 and POSTN mRNA were distinctly correlated with OS reduction (all P < 0.01, Fig. 1D-E). MALAT1 expression in serum EVs of TNBC patients was subsequently examined. We obtained EVs from serum of TNBC patients and healthy subjects, respectively. TEM and Western blot were utilized to detect specific markers (positive markers CD63, CD81 and TSG101, and negative marker Calnexin) (Fig. 1F-G). NET analysis showed that both TNBC patients and healthy subjects had EVs in the diameter range of 90–150 nm, but TNBC patients (7.4 × 105 EVs/mL) had a higher concentration of EVs than healthy subjects (5.2 × 106 EVs/mL) (Fig. 1H). MALAT1 expression was notably boosted in an equal number of serum EVs in TNBC patients versus healthy subjects (P < 0.01, Fig. 1I), and observably correlated with reduced OS (P < 0.01, Fig. 1J). Detection of macrophage infiltration by flow cytometry suggested that the percentages of TAMs (CD11b+F4/80+) and M2 TAMs (CD11b+F4/80+CD206+) in tumor tissues of TNBC patients were dramatically elevated relative to tissues adjacent to cancer (P < 0.01, Fig. 1K-L). Additionally, MALAT1 level in serum EVs and POSTN mRNA level in tumor tissues were significantly favorably interrelated with the percentage of M2 macrophages (all P < 0.01, Fig. 1M-N). These results unraveled that EV-MALAT1 and POSTN might be involved in the regulation of M2 TAM infiltration in TNBC patients.

TNBC tumor-derived EVs induced macrophage M2 polarization

To investigate whether TNBC-derived EVs induced macrophage M2 polarization, human THP-1 cells were incubated with PMA to induce differentiation into M0 macrophages, which had the corresponding adherent morphology and highly-expressed macrophage marker CD68 (P < 0.01, Fig. 2A-B). M0 macrophages were non-contactly co-cultured with MDA-MB-231 and HCC1937 cells and collected. The results of flow cytometry and RT-qPCR assays unraveled that the expression patterns of M2 macrophage markers (CD206, Arg-1 and IL10) were distinctly elevated after co-culture with TNBC cells (all P < 0.01, Fig. 2C-D). Afterwards, DMA-treated TNBC cells were co-cultured with macrophages, and the promoting effect of TNBC cells on macrophage M2 polarization was partially averted after DMA treatment (all P < 0.01, Fig. 2C-D). To further confirm that the facilitating effect of TNBC cells on macrophage M2 polarization was mediated by the release of EVs, TNBC-derived EVs were extracted from TNBC cells and the specific markers (positive markers CD63, CD81 and TSG101, and the negative marker Calnexin) were identified by TEM and Western blot (Fig. 2E-F). The concentration and size of EVs were analyzed by NET (90–120 nm in diameter, with MDA-MB-231-derived EVs concentration of approximately 10.7 × 106 EVs/mL and HCC1937-derived EVs concentration of approximately 3.8 × 106 EVs/mL, Fig. 2G). Macrophages were treated with TNBC-derived EVs. The results demonstrated that treatment with EVs conspicuously stimulated M2 macrophage polarization versus the PBS group (all P < 0.01, Fig. 2H-I). Altogether, TNBC-derived EVs induced macrophage M2 polarization.Fig. 2 TNBC-derived EVs induced macrophage M2 polarization. (A) Representative graphs of macrophages obtained from THP-1 cells treated with PMA for 24 h; (B) RT-qPCR to determine CD68 expression; (C/H) Flow cytometry to determine CD206 expression; (D/I) RT-qPCR to assess Arg-1 and IL10 expression patterns; (E) TEM to observe EV morphology; (F) Western blot to measure the expression levels of CD63, CD81, TSG101 and Calnexin in EVs; (G) NET to analyze the concentration and size of EVs. The cellular experiments were repeated three times. Data were expressed as the mean ± standard deviation. T test was conducted for the comparisons between 2 groups. One-way ANOVA was utilized for the comparisons of data among multiple groups. Tukey's multiple comparisons test was used for post-hoc analysis. ** P < 0.01, *** P < 0.001.

Fig. 2

MALAT1 was highly expressed in TNBC-derived EVs and was transferred to macrophages via EVs to induce M2 polarization

RT-qPCR assay results displayed that the MALAT1 expression was dramatically boosted in TNBC-derived EV-treated macrophages relative to the PBS group (all P < 0.05, Fig. 3A). MDA-MB-231 cells with higher MALAT1 expression were selected for subsequent mechanistic investigations. After co-incubation of PKH67-labelled MDA-MB-231 cell-derived EVs with macrophages, PKH67 lipid staining spots were observed in macrophages (Fig. 3B), indicating that EVs released by MDA-MB-231 cells could be internalized by macrophages. MALAT1 expression in MDA-MB-231 cells and EVs was knocked down by cell transfection with lentivirus vectors containing si1-MALAT1 and si2-MALAT1 (all P < 0.01, Fig. 3C-D). We chose si1-MALAT1, which was more efficient in MALAT1 knockdown, for subsequent experiments. MALAT1 expression in macrophages was also significantly abated when macrophages were incubated with EVs with silenced MALAT1 (P < 0.01, Fig. 3B). Relative to the EV-treated group, there was no prominent change in MALAT1 expression in macrophages when macrophages were incubated with RNase-treated EVs, whereas the expression was markedly reduced when macrophages were incubated with EVs treated with RNase and Triton X-100. Additionally, RNA polymerase II inhibitor did not affect the upregulation of MALAT1 in recipient macrophages (all P < 0.01, Fig. 3E). These results suggested that MALAT1 was encapsulated in MDA-MB-231 cell-derived EVs and that the upregulation of MALAT1 in macrophages was caused by MALAT1 transfer mediated by TNBC-derived EVs rather than endogenous MALAT1 induction. Next, the effect of TNBC-derived EV-shuttled MALAT1 on macrophage M2 polarization was further explored. The results revealed that the stimulating effect of EVs on macrophage M2 polarization was partially abrogated after MALAT1 knockdown in EVs (all P < 0.01, Fig. 3F-G). In addition, THP-1 cells were incubated with PMA, IL-4 and IL-13 to differentiate into M2 macrophages, and the effect of MALAT1 on macrophage M2 polarization was further verified by MALAT1 knockdown in M2 macrophages by cell transfection (all P < 0.01, Fig. 3H). MALAT1 downregulation resulted in prominent reductions in the expression patterns of the M2 macrophage markers CD206, Arg-1 and IL10 (all P < 0.01, Fig. 3I-J). Collectively, these results suggested that MALAT1 was highly-expressed in TNBC-derived EVs and was transferred to macrophages by EVs to induce M2 polarization.Fig. 3 MALAT1 was highly expressed in TNBC-derived EVs and was transferred to macrophages via EVs to induce M2 polarization. (A/C/D/E/H) RT-qPCR to measure MALAT1 expression; (B) Representative immunofluorescence images showed that PKH67-labelled TNBC-derived EVs (green) were internalized by macrophages, and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI); (F/I) Flow cytometry to determine CD206 expression; (G/J) RT-qPCR to assess Arg-1 and IL10 expression levels. The cellular experiments were repeated three times. Data were expressed as mean ± standard deviation. One-way ANOVA was used to compare the data among multiple groups, and Tukey's multiple comparisons test was applied for post hoc analysis.** P < 0.01, *** P < 0.001.

Fig. 3

TNBC-derived EV-shuttled MALAT1 induced macrophage M2 polarization by facilitating postn expression

Western blot manifested that POSTN level was remarkably increased in EV-treated macrophages compared to the PBS group, while MALAT1 knockout in EVs led to a distinct decrease in POSTN level (all P < 0.01, Fig. 4A). Knockdown of POSTN in M2 macrophages induced by PMA, IL-4 and IL-13 was performed (all P < 0.01, Fig. 4B) to verify the effect of POSTN on macrophage M2 polarization. The results depicted that POSTN knockdown memorably reduced the expression levels of M2 macrophage markers CD206, Arg-1 and IL10 (all P < 0.01, Fig. 4C-D). Additionally, POSTN was overexpressed in macrophages upon treatment with EVs with silenced MALAT1 (all P < 0.01, Fig. 4A). Flow cytometry and RT-qPCR unveiled that overexpression of POSTN diminished the inhibitory effects of MALAT1 knockdown on the expression levels of M2 macrophage markers CD206, Arg-1 and IL10 (all P < 0.01, Fig. 4E-F). The aforesaid results unraveled that TNBC-derived EV-carried MALAT1 induced macrophage M2 polarization by promoting POSTN expression.Fig. 4 TNBC-EV-derived MALAT1 induced macrophage M2 polarization by promoting POSTN expression. (A-B) Western blot to assess POSTN expression; (C/E) Flow cytometry to determine CD206 expression; (D/F) RT-qPCR to measure Arg-1 and IL10 expression patterns. The cellular experiments were repeated three times, and the data were expressed as mean ± standard deviation. One-way ANOVA was used to compare the data among multiple groups, and Tukey's multiple comparisons test was used for post-hoc analysis. ** P < 0.01.

Fig. 4

POSTN induced macrophage M2 polarization via activating the Hippo/YAP pathway

The Hippo/YAP pathway has been reported to regulate TAM recruitment and M2 polarization in tumor tissues [[47], [48], [49]]. What's more, the Hippo-YAP pathway manipulates the tumor immune microenvironment via the TAZ/IL-34 axis to facilitate TNBC progression [50]. We thus speculated that POSTN might regulate macrophage M2 polarization through the Hippo/YAP pathway. Western blot results demonstrated up-regulated p-LATS1, p-YAP, and YAP levels and the activated Hippo/YAP pathway in EV-treated macrophages versus the PBS group. The activation of the Hippo/YAP pathway was partially suppressed by down-regulating the expression of MALAT1 in EVs, whereas partially restored after POSTN overexpression (all P < 0.05, Fig. 5A). Macrophages treated with EV-si-MALAT1 + oe-POSTN were co-treated with VP, an inhibitor of the YAP signaling, to inhibit the activation of the YAP signaling (all P < 0.05, Fig. 5B). Flow cytometry and RT-qPCR results displayed that the promoting effects of POSTN overexpression on the expression levels of M2 macrophage markers CD206, Arg-1 and IL10 were partially nullified by VP (all P < 0.01, Fig. 5C-D). The results revealed that TNBC-EV-derived MALAT1 activated the Hippo-YAP pathway by up-regulating POSTN expression, and thus inducing macrophage M2 polarization.Fig. 5 POSTN induced macrophage M2 polarization by activating the Hippo-YAP pathway. (A-B) Western blot to assess the expression levels of the Hippo/YAP pathway-related proteins (p-LATS1, LATS1, p-YAP and YAP); (C) Flow cytometry to assess the expression of CD206; (D) RT-qPCR to determine the expression levels of Arg-1 and IL10. The cellular experiments were repeated three times, and the data were expressed as mean ± standard deviation. One-way ANOVA was utilized to compare the data among multiple groups, and Tukey's multiple comparisons test was used for post hoc analysis.* P < 0.05, ** P < 0.01.

Fig. 5

Activated M2 macrophages stimulated proliferative, migratory and invasive properties of tnbc cells in vitro

MDA-MB-231 cells were cultured with conditioned medium of macrophages in different treatment groups. The proliferative, migratory and invasive abilities of MDA-MB-231 cells were distinctly enhanced after culture with the conditioned medium of TNBC-EVs-treated macrophages, which were prominently weakened after co-culture with the conditioned medium of TNBC-EV-treated macrophages with silenced MALAT1 (all P < 0.05, Fig. 6A-B). Taken together, M2 macrophages activated by TNBC-EV-derived MALAT1 contributed to promoting the proliferation, migration and invasion of TNBC cells.Fig. 6 M2 macrophages activated by TNBC-EV-derived MALAT1 faciltated the proliferation, migration and invasion of TNBC cells. MDA-MB-231 cells were collected after indirect co-culture with macrophages. (A) CCK-8 to detect changes of cell viability; (B) Transwell to detect changes of migration and invasion of cells. The cellular experiments were repeated three times. Data were expressed as mean ± standard deviation. One-way ANOVA was utilized to compare the data among multiple groups. Tukey's multiple comparisons test was applied for post-hoc analysis. ** P < 0.01.

Fig. 6

TNBC-EV-derived MALAT1 induced macrophage M2 polarization and promoted the onset and metastasis of tnbc in vivo

The role of TNBC-EV-derived MALAT1 in macrophage M2 polarization was explored by in vivo experiments. As reflected by RT-qPCR and Western blot results, EVs markedly elevated the expression patterns of MALAT1 and POSTN, and activated the Hippo/YAP pathway in tumor tissues, while MALAT1 knockdown in EVs suppressed the expression of POSTN and the activation of the Hippo/YAP pathway (all P < 0.01, Fig. 7A-B). Flow cytometry assay results manifested that TNBC-EV-derived MALAT1 induced macrophage M2 polarization (all P < 0.01, Fig. 7C-D).Fig. 7 TNBC-EV-derived MALAT1 facilitated the macrophage M2 polarization, thereby promoting the occurrence and metastasis of TNBC in vivo. (A) RT-qPCR to assess the expression of MALAT1 in tumor tissues; (B) Western blot to determine the expression patterns of POSTN and the Hippo/YAP pathway-related proteins (p-LATS1, LATS1, p-YAP and YAP); flow cytometry to assess the percentages of (C) TAMs (CD11b+ F4/80+) and (D) M2 TAMs (CD11b+F4/80+CD206+) in tumor tissues of mice; (E) The tumor volume and (F) weight of mice were recorded; (G) H&E staining of lungs, bones and livers. The blue arrow indicated metastatic tumor cells. n = 6. Data were expressed as mean ± standard deviation. One-way ANOVA was utilized to compare the data among multiple groups, and Tukey's multiple comparisons test was applied for post-hoc analysis. ** P < 0.01.

Fig. 7

MDA-MB-231 cells co-cultured with macrophages (stimulated by PBS, EVs, or EVs-si-MALAT1) were injected into mice through tail vein. After 6 weeks, we observed that TNBC-EV-derived MALAT1 triggered tumor growth (all P < 0.01, Fig. 7E-F). Additionally, no conspicuous metastases were seen in bones and livers in all groups, while more lung metastatic nodules were found in the EV-stimulated macrophage group than in the control group, and the number of lung metastatic nodules was reduced in the EV-si-MALAT1-stimulated macrophage group (all P < 0.01, Fig. 7G). These results suggested that TNBC-EV-derived MALAT1 facilitated macrophage M2 polarization, thereby promoting the occurrence and metastasis of TNBC in vivo.

Discussion

TNBC accounts for approximately one quarter of newly diagnosed BC cases [51]. Although much progress has been made in the field of BC in recent years, the progression of TNBC metastases is still an unclear and complex process with suboptimal outcomes [52]. Evidence has shown that TNBC-released EVs can transfer cargo to target cells, facilitating cell proliferative capacity, tumor growth, cancer metastasis and invasive ability [53]. Herein, we highlighted the mechanism that TNBC-EV-derived MALAT1 could activate the Hippo/YAP pathway by up-regulating POSTN to induce macrophage M2 polarization, thereby facilitating the onset and metastasis of TNBC in vivo.

LncRNAs are sorted into EVs selectively and tumor-derived EV-carried lncRNAs contribute to tumorigenesis by stimulating coagulation and angiogenesis [31,32]. Meanwhile, M2 macrophage impacts many aspects of tumor cell biology, for instance, cell invasion, proliferation, metastasis, angiogenesis, and epithelial-mesenchymal transition [54]. We screened EV-linked and TNBC M2 macrophage-linked lncRNAs and obtained lncRNA MALAT1 and POSTN. Thus, we speculated that TNBC-EVs might modulate macrophage M2 polarization via the MALAT1/POSTN axis. Our findings disclosed highly expressed MALAT1 in tumor tissues and EVs, and POSTN in tumor tissues of the TNBC patients, with their high expression levels linked with reduced OS. As one of the most studied lncRNAs in cancer, MALAT1 is up-regulated in many cancer tissues [45]. For instance, MALAT1 is up-regulated in osteosarcoma and triggers osteosarcoma cell metastasis through activating the PI3K/Akt axis [55]. Yue and his colleagues have manifested that MALAT1 is highly expressed in both tissues and cells of BC, and silencing MALAT1 markedly repressed the proliferative, migrating and invasive properties of BC cells [29]. Also, POSTN is up-regulated in stromal fibroblasts of BC and contributes to maintaining BC stem cells via the Wnt signaling pathway, thereby playing a role in metastasis of BC [56], which are in line with our preliminary findings. Furthermore, macrophage infiltration was enhanced in tumor tissues of TNBC patients, and tumor tissue POSTN mRNA and serum EV-MALAT1 levels were positively linked to M2 macrophage percentage. Likewise, a study found that TAM density is favorably interrelated with POSTN protein levels of human glioblastoma, and silencing of POSTN prominently reduces M2 type of TAMs density, suppresses tumor growth, and enhances the survival of xenograft mice [46]. These evidence all suggested the possible involvement of POSTN and EV-MALAT1 in the modulation of M2 TAM infiltration in TNBC patients.

Tumor-derived EVs are a vital mediator for cellular communication between cancer cells and immune cells, which intensify tumor progression by amplifying macrophage M2 polarization [[21], [22], [23]]. Currently, there are few studies on tumor-derived EVs mediating macrophage polarization in BC, especially TNBC, and tumor-derived EVs show different contributions in driving TAM heterogeneity. Our findings confirmed for the first time that TNBC-EVs induced macrophage M2 polarization in TNBC. Next, we uncovered increased MALAT1 in TNBC-EVs and that MALAT1 was encapsulated in EVs derived from MDA-MB-231 cells, and the increase in MALAT1 expression in macrophages is caused by TNBC-derived EV-mediated MALAT1 transfer, rather than endogenous MALAT1 induction. What's more, MALAT1 silencing partly averted EV-mediated macrophage M2 polarization promotion. Consistently, knockdown of MALAT1 stimulates M1 macrophage polarization and suppresses M2 macrophage polarization [57]. Altogether, MALAT1 expression was facilitated in TNBC-EVs, and was transferred into macrophages to induced their M2 polarization via EVs. Subsequently, we uncovered that MALAT1 knockout in EVs led to reduction in POSTN expression. Furthermore, knockout of POSTN limited macrophage M2 polarization, while overexpression of POSTN partly abrogated MALAT1 silencing-repressed macrophage M2 polarization. Consistent with our study, a prior study reported that overexpression of POSTN enhances M2 macrophages and cancer-associated fibroblasts to facilitate ovarian cancer metastasis [58]. To conclude, the inductive role of TNBC-EV-derived MALAT1 in macrophage M2 polarization was achieved by stimulating POSTN expression.

Dysregulation of the Hippo pathway may facilitate tumor development due to its pivotal role in organ size control and cell proliferation [59]. YAP is boosted in TNBC, where it serves as a transcriptional cofactor and promote the signaling of multiple transcription factors [60]. The Hippo/YAP axis exerts an important effect on cell proliferative and invasive capacities and anti-apoptosis in TNBC [61]. Up-regulation of YAP was bound up with M2 TAM polarization and tumorigenesis in colorectal cancer, whereas downregulation of YAP curbs tumor metastasis and formation in colon cancer cells [47]. The Hippo pathway regulates XBP1-mediated NLRP3 activation through the interaction between YAP and β-catenin, thereby leading to M2 macrophage polarization [48]. In line with the prior study, our study found that POSTN exerted its effect to induce M2 macrophage polarization by activating the Hippo-YAP signaling pathway.

Moreover, in vivo and in vitro experiments by Jin et al. have demonstrated that knockout of MALAT1 hinders cell invasion, proliferation, and metastasis and facilitates apoptosis of epithelial ovarian cancer [62]. It has also been documented that tumor-derived EVs deliver the lncRNA PART1 into macrophages, thereby facilitating M2 macrophage polarization during the process of hepatocellular carcinoma development [63]. Our experiments revealed that activated M2 macrophages by TNBC-EV-MALAT1 promoted proliferative, migrating and invasive abilities of TNBC cells in vitro and TNBC-EV-derived MALAT1 induced M2 macrophage polarization and promotes onset and metastasis of TNBC in vivo. Additionally, in vitro experiments showed that POSTN expression, activation of the Hippo/YAP signaling pathway, and number of pulmonary metastatic nodules were remarkably decreased after TNBC-EVs-derived MALAT1 was knocked down, while were increased after MALAT1 was overexpressed. It is in line with a previous study that BC-derived EVs facilitate M2 macrophage polarization and create advantaged conditions for the lymph node metastasis in TNBC [25]. These data supported that TNBC-EV-derived MALAT1 contributed to M2 macrophage polarization, thus promoting the onset and metastasis of TNBC in vivo.

All in all, our study highlighted that TNBC-EV-derived MALAT1 activated the Hippo/YAP pathway by up-regulating POSTN, thereby inducing M2 polarization of macrophages to promote the occurrence and metastasis of TNBC in vivo. However, it is unfortunate that only MDA-MB-231 cell line was applied for in vitro and in vivo verification in this study, and the intermediate mechanism of MALAT regulating POSTN expression is still not elucidated. In the future, we will not only select multi-cell lines to carry out further in vitro and in vivo experimental validation, and will also explore the intermediate mechanism by which MALAT regulates POSTN expression.

Declarations

Funding

This study was partially supported by grants from the Major Project in Natural Science Research in Higher Education Institutions of Anhui Province, Grant/Award Numver: 2023AH040393.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

All animal experimental procedures were ratified by the Research Ethics Committee of Anhui Medical University and conducted in strict accordance with the National Guide for Laboratory Animals (Approval number: LLSC20220982). We implemented various laboratory procedures to alleviate the discomfort of the mice, including the use of heating pads, disinfection, and fluid supplementation with saline.

Patient consent for publication

Not applicable.

CRediT authorship contribution statement

Xuedong Wang: Writing – review & editing, Writing – original draft, Resources, Project administration, Data curation, Conceptualization. Qiwei Jian: Methodology, Investigation. Ziyun Zhang: Investigation, Formal analysis. Juan Gu: Data curation, Conceptualization. Xinping Wang: Formal analysis, Conceptualization. Yueping Wang: Visualization, Validation.

Declaration of competing interest

The authors declare that they have no competing interests.

Appendix Supplementary materials

Image, application 1

Image, application 2

Image, application 3

Supplementary Figure 1 LncTarD 2.0 online database showed regulatory relationship between H19 and MALAT1 with POSTN.

Image, image 4

Image, application 5

Acknowledgements

Not applicable.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.tranon.2024.102076.
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References

1 DeSantis C.E. Fedewa S.A. Goding Sauer A. Kramer J.L. Smith R.A. Breast cancer statistics, 2015: convergence of incidence rates between black and white women CA Cancer J. Clin. 66 2016 31 42 10.3322/caac.21320 26513636
2 Chodosh L.A. Breast cancer: current state and future promise Breast. Cancer Res. 13 113 2011 10.1186/bcr3045
3 Cronin K.A. Lake A.J. Scott S. Sherman R.L. Noone A.M. Annual report to the nation on the status of cancer, part I: national cancer statistics Cancer 124 2018 2785 2800 10.1002/cncr.31551 29786848
4 Wu H. Feng J. Zhong W. Zouxu X. Xiong Z. Model for predicting immunotherapy based on M2 macrophage infiltration in TNBC Front. Immunol. 14 2023 1151800 10.3389/fimmu.2023.1151800
5 So J.Y. Ohm J. Lipkowitz S. Yang L. Triple negative breast cancer (TNBC): non-genetic tumor heterogeneity and immune microenvironment: emerging treatment options Pharmacol. Ther. 237 2022 108253 10.1016/j.pharmthera.2022.108253
6 Tan D.S. Marchio C. Jones R.L. Savage K. Smith I.E. Triple negative breast cancer: molecular profiling and prognostic impact in adjuvant anthracycline-treated patients Breast. Cancer Res. Treat. 111 2008 27 44 10.1007/s10549-007-9756-8 17922188
7 Santoni M. Romagnoli E. Saladino T. Foghini L. Guarino S. Triple negative breast cancer: key role of Tumor-Associated Macrophages in regulating the activity of anti-PD-1/PD-L1 agents Biochim. Biophys. Acta Rev. Cancer 1869 2018 78 84 10.1016/j.bbcan.2017.10.007 29126881
8 Wu K. Lin K. Li X. Yuan X. Xu P. Redefining Tumor-Associated Macrophage Subpopulations and Functions in the Tumor Microenvironment Front. Immunol. 11 2020 1731 10.3389/fimmu.2020.01731 32849616
9 Cheng Y.Q. Wang S.B. Liu J.H. Jin L. Liu Y. Modifying the tumour microenvironment and reverting tumour cells: new strategies for treating malignant tumours Cell Prolif. 53 2020 e12865 10.1111/cpr.12865 32588948
10 Chen M. Lai R. Lin X. Chen W. Wu H. Downregulation of triggering receptor expressed on myeloid cells 1 inhibits invasion and migration of liver cancer cells by mediating macrophage polarization Oncol. Rep. 45 2021 10.3892/or.2021.7988
11 Chen D. Zhang X. Li Z. Zhu B. Metabolic regulatory crosstalk between tumor microenvironment and tumor-associated macrophages Theranostics. 11 2021 1016 1030 10.7150/thno.51777 33391518
12 Wolfsberger J. Sakil H.A.M. Zhou L. van Bree N. Baldisseri E. TAp73 represses NF-kappaB-mediated recruitment of tumor-associated macrophages in breast cancer Proc. Natl. Acad. Sci. U. S. A 118 2021 10.1073/pnas.2017089118
13 Pyonteck S.M. Akkari L. Schuhmacher A.J. Bowman R.L. Sevenich L. CSF-1R inhibition alters macrophage polarization and blocks glioma progression Nat. Med. 19 2013 1264 1272 10.1038/nm.3337 24056773
14 Liang Z.W. Ge X.X. Xu M.D. Qin H. Wu M.Y. Tumor-associated macrophages promote the metastasis and growth of non-small-cell lung cancer cells through NF-kappaB/PP2Ac-positive feedback loop Cancer Sci. 112 2021 2140 2157 10.1111/cas.14863 33609307
15 Xiang X. Wang J. Lu D. Xu X. Targeting tumor-associated macrophages to synergize tumor immunotherapy Signal. Transduct. Target. Ther. 6 2021 75 10.1038/s41392-021-00484-9 33619259
16 Xiao H. Guo Y. Li B. Li X. Wang Y. M2-like tumor-associated macrophage-targeted codelivery of STAT6 inhibitor and IKKbeta siRNA Induces M2-to-M1 repolarization for cancer immunotherapy with low immune side effects ACS. Cent. Sci. 6 2020 1208 1222 10.1021/acscentsci.9b01235 32724855
17 Bian X. Xiao Y.T. Wu T. Yao M. Du L. Microvesicles and chemokines in tumor microenvironment: mediators of intercellular communications in tumor progression Mol. Cancer 18 2019 50 10.1186/s12943-019-0973-7 30925930
18 Ngambenjawong C. Gustafson H.H. Pun S.H. Progress in tumor-associated macrophage (TAM)-targeted therapeutics Adv. Drug Deliv. Rev. 114 2017 206 221 10.1016/j.addr.2017.04.010 28449873
19 Crescitelli R. Lasser C. Szabo T.G. Kittel A. Eldh M. Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes J. ExtraCell Vesicles. 2 2013 10.3402/jev.v2i0.20677
20 Akers J.C. Gonda D. Kim R. Carter B.S. Chen C.C. Biogenesis of extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies J. Neurooncol. 113 2013 1 11 10.1007/s11060-013-1084-8 23456661
21 Tian X. Shen H. Li Z. Wang T. Wang S. Tumor-derived exosomes, myeloid-derived suppressor cells, and tumor microenvironment J. Hematol. Oncol. 12 2019 84 10.1186/s13045-019-0772-z 31438991
22 Jiang Z. Zhang Y. Zhang Y. Jia Z. Zhang Z. Cancer derived exosomes induce macrophages immunosuppressive polarization to promote bladder cancer progression Cell Commun. Signal. 19 2021 93 10.1186/s12964-021-00768-1 34521440
23 Baig M.S. Roy A. Rajpoot S. Liu D. Savai R. Tumor-derived exosomes in the regulation of macrophage polarization Inflamm. Res. 69 2020 435 451 10.1007/s00011-020-01318-0 32162012
24 Jang J.Y. Lee J.K. Jeon Y.K. Kim C.W. Exosome derived from epigallocatechin gallate treated breast cancer cells suppresses tumor growth by inhibiting tumor-associated macrophage infiltration and M2 polarization BMC. Cancer 13 2013 421 10.1186/1471-2407-13-421 24044575
25 Piao Y.J. Kim H.S. Hwang E.H. Woo J. Zhang M. Breast cancer cell-derived exosomes and macrophage polarization are associated with lymph node metastasis Oncotarget. 9 2018 7398 7410 10.18632/oncotarget.23238 29484119
26 Tkach M. Thalmensi J. Timperi E. Gueguen P. Nevo N. Extracellular vesicles from triple negative breast cancer promote pro-inflammatory macrophages associated with better clinical outcome Proc. Natl. Acad. Sci. U S. A 119 2022 e2107394119 10.1073/pnas.2107394119
27 Ham S. Lima L.G. Chai E.P.Z. Muller A. Lobb R.J. Breast Cancer-Derived Exosomes Alter Macrophage Polarization via gp130/STAT3 Signaling Front. Immunol. 9 2018 871 10.3389/fimmu.2018.00871 29867925
28 Rinn J.L. Chang H.Y. Genome regulation by long noncoding RNAs Annu. Rev. Biochem. 81 2012 145 166 10.1146/annurev-biochem-051410-092902 22663078
29 Yue X. Wu W.Y. Dong M. Guo M. LncRNA MALAT1 promotes breast cancer progression and doxorubicin resistance via regulating miR-570-3p Biomed. J. 44 2021 S296 S304 10.1016/j.bj.2020.11.002 35410813
30 Li C. Ni Y.Q. Xu H. Xiang Q.Y. Zhao Y. Roles and mechanisms of exosomal non-coding RNAs in human health and diseases Signal. Transduct. Target. Ther. 6 2021 383 10.1038/s41392-021-00779-x 34753929
31 Xu Z. Chen Y. Ma L. Chen Y. Liu J. Role of exosomal non-coding RNAs from tumor cells and tumor-associated macrophages in the tumor microenvironment Mol. Ther. 30 2022 3133 3154 10.1016/j.ymthe.2022.01.046 35405312
32 Sun Z. Yang S. Zhou Q. Wang G. Song J. Emerging role of exosome-derived long non-coding RNAs in tumor microenvironment Mol. Cancer 17 2018 82 10.1186/s12943-018-0831-z 29678180
33 He Z. Wang J. Zhu C. Xu J. Chen P. Exosome-derived FGD5-AS1 promotes tumor-associated macrophage M2 polarization-mediated pancreatic cancer cell proliferation and metastasis Cancer Lett. 548 2022 215751 10.1016/j.canlet.2022.215751
34 Zheng H. Chen C. Luo Y. Yu M. He W. Tumor-derived exosomal BCYRN1 activates WNT5A/VEGF-C/VEGFR3 feedforward loop to drive lymphatic metastasis of bladder cancer Clin. Transl. Med. 11 2021 e497 10.1002/ctm2.497 34323412
35 Zhang W. Zheng X. Yu Y. Zheng L. Lan J. Renal cell carcinoma-derived exosomes deliver lncARSR to induce macrophage polarization and promote tumor progression via STAT3 pathway Int. J. Biol. Sci. 18 2022 3209 3222 10.7150/ijbs.70289 35637970
36 Yi Y. Wu M. Zeng H. Hu W. Zhao C. Tumor-derived exosomal non-coding RNAs: the emerging mechanisms and potential clinical applications in breast cancer Front. Oncol. 11 2021 738945 10.3389/fonc.2021.738945
37 Rao X. Zhou X. Wang G. Jie X. Xing B. NLRP6 is required for cancer-derived exosome-modified macrophage M2 polarization and promotes metastasis in small cell lung cancer Cell Death. Dis. 13 2022 891 10.1038/s41419-022-05336-0 36270983
38 Zheng X. Huang M. Xing L. Yang R. Wang X. The circRNA circSEPT9 mediated by E2F1 and EIF4A3 facilitates the carcinogenesis and development of triple-negative breast cancer Mol. Cancer 19 2020 73 10.1186/s12943-020-01183-9 32264877
39 Chen W.X. Wang D.D. Zhu B. Zhu Y.Z. Zheng L. Exosomal miR-222 from adriamycin-resistant MCF-7 breast cancer cells promote macrophages M2 polarization via PTEN/Akt to induce tumor progression Aging (Albany. NY) 13 2021 10415 10430 10.18632/aging.202802 33752173
40 Wang M. Dong Y. Gao S. Zhong Z. Cheng C. Hippo/YAP signaling pathway protects against neomycin-induced hair cell damage in the mouse cochlea Cell Mol. Life Sci. 79 79 2022 10.1007/s00018-021-04029-9
41 Pin F. Beltra M. Garcia-Castillo L. Pardini B. Birolo G. Extracellular vesicles derived from tumour cells as a trigger of energy crisis in the skeletal muscle J. Cachexia Sarcopenia Muscle 13 2022 481 494 10.1002/jcsm.12844 34931471
42 Zheng R. Du M. Wang X. Xu W. Liang J. Exosome-transmitted long non-coding RNA PTENP1 suppresses bladder cancer progression Mol. Cancer 17 143 2018 10.1186/s12943-018-0880-3
43 Tan R.Z. Zhong X. Han R.Y. Xie K.H. Jia J. Macrophages mediate psoriasis via Mincle-dependent mechanism in mice Cell Death. Discov. 9 2023 140 10.1038/s41420-023-01444-8 37117184
44 Grunz E. Jones B. Lateef O. Sen S. Wilkenson K. Adventitial macrophage accumulation impairs perivascular nerve function in mesenteric arteries with inflammatory bowel disease bioRxiv. 2023 10.1101/2023.04.04.535591
45 Amer H.T. Eissa R.A. El Tayebi HM: a cutting-edge immunomodulatory interlinkage between HOTAIR and MALAT1 in tumor-associated macrophages in breast cancer: a personalized immunotherapeutic approach Front. Mol. Biosci. 9 2022 1032517 10.3389/fmolb.2022.1032517
46 Zhou W. Ke S.Q. Huang Z. Flavahan W. Fang X. Periostin secreted by glioblastoma stem cells recruits M2 tumour-associated macrophages and promotes malignant growth Nat. Cell Biol. 17 2015 170 182 10.1038/ncb3090 25580734
47 Yang W. Yang S. Zhang F. Cheng F. Wang X. Influence of the Hippo-YAP signalling pathway on tumor associated macrophages (TAMs) and its implications on cancer immunosuppressive microenvironment Ann. Transl. Med. 8 2020 399 10.21037/atm.2020.02.11 32355843
48 Li C. Jin Y. Wei S. Sun Y. Jiang L. Hippo signaling controls NLR family pyrin domain containing 3 activation and governs immunoregulation of mesenchymal stem cells in mouse liver injury Hepatology 70 2019 1714 1731 10.1002/hep.30700 31063235
49 Ji D. Jia J. Cui X. Li Z. Wu A. FAP promotes metastasis and chemoresistance via regulating YAP1 and macrophages in mucinous colorectal adenocarcinoma iScience 26 2023 106600 10.1016/j.isci.2023.106600
50 Wang Z. Wang F. Ding X.Y. Li T.E. Wang H.Y. Hippo/YAP signaling choreographs the tumor immune microenvironment to promote triple negative breast cancer progression via TAZ/IL-34 axis Cancer Lett. 527 2022 174 190 10.1016/j.canlet.2021.12.016 34929335
51 Borri F. Granaglia A. Pathology of triple negative breast cancer Semin. Cancer Biol. 72 2021 136 145 10.1016/j.semcancer.2020.06.005 32544511
52 Al-Mahmood S. Sapiezynski J. Garbuzenko O.B. Minko T. Metastatic and triple-negative breast cancer: challenges and treatment options Drug Deliv. Transl. Res. 8 2018 1483 1507 10.1007/s13346-018-0551-3 29978332
53 Ozawa P.M.M. Alkhilaiwi F. Cavalli I.J. Malheiros D. de Souza Fonseca Ribeiro E.M. Extracellular vesicles from triple-negative breast cancer cells promote proliferation and drug resistance in non-tumorigenic breast cells Breast. Cancer Res. Treat. 172 2018 713 723 10.1007/s10549-018-4925-5 30173296
54 Zhang Y. Fan Y. Jing X. Zhao L. Liu T. OTUD5-mediated deubiquitination of YAP in macrophage promotes M2 phenotype polarization and favors triple-negative breast cancer progression Cancer Lett. 504 2021 104 115 10.1016/j.canlet.2021.02.003 33587979
55 Chen Y. Huang W. Sun W. Zheng B. Wang C. LncRNA MALAT1 promotes cancer metastasis in osteosarcoma via activation of the PI3K-Akt signaling pathway Cell Physiol. Biochem. 51 2018 1313 1326 10.1159/000495550 30481748
56 Malanchi I. Santamaria-Martinez A. Susanto E. Peng H. Lehr H.A. Interactions between cancer stem cells and their niche govern metastatic colonization Nature 481 2011 85 89 10.1038/nature10694 22158103
57 Hou Z.H. Xu X.W. Fu X.Y. Zhou L.D. Liu S.P. Long non-coding RNA MALAT1 promotes angiogenesis and immunosuppressive properties of HCC cells by sponging miR-140 Am. J. Physiol. Cell Physiol. 318 2020 C649 C663 10.1152/ajpcell.00510.2018 31693399
58 Lin S.C. Liao Y.C. Chen P.M. Yang Y.Y. Wang Y.H. Correction: periostin promotes ovarian cancer metastasis by enhancing M2 macrophages and cancer-associated fibroblasts via integrin-mediated NF-kappaB and TGF-beta2 signaling J. Biomed. Sci. 30 2023 54 10.1186/s12929-023-00948-w 37438746
59 Yu F.X. Zhao B. Guan K.L. Hippo Pathway in Organ Size Control, Tissue Homeostasis, and Cancer Cell 163 2015 811 828 10.1016/j.cell.2015.10.044 26544935
60 Liu-Chittenden Y. Huang B. Shim J.S. Chen Q. Lee S.J. Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP Genes Dev. 26 2012 1300 1305 10.1101/gad.192856.112 22677547
61 Davies C.C. Chakraborty A. Diefenbacher M.E. Skehel M. Behrens A. Arginine methylation of the c-Jun coactivator RACO-1 is required for c-Jun/AP-1 activation EMBO J. 32 2013 1556 1567 10.1038/emboj.2013.98 23624934
62 Jin Y. Feng S.J. Qiu S. Shao N. Zheng J.H. LncRNA MALAT1 promotes proliferation and metastasis in epithelial ovarian cancer via the PI3K-AKT pathway Eur. Rev. Med. Pharmacol. Sci. 21 2017 3176 3184 28770968
63 Zhou J. Che J. Xu L. Yang W. Zhou W. Tumor-derived extracellular vesicles containing long noncoding RNA PART1 exert oncogenic effect in hepatocellular carcinoma by polarizing macrophages into M2 Dig. Liver. Dis. 54 2022 543 553 10.1016/j.dld.2021.07.005 34497040
