Reactive oxygen species reprogram macrophages to suppress antitumor immune response through the exosomal miR-155-5p/PD-L1 pathway

Human ovarian cancer cell line A2780, Ovcar-3, and SKOV-3, murine ovarian cancer cell line ID8, and human peripheral blood monocytes THP-1 were purchased from the American Type Culture Collection (ATCC). Peripheral blood mononuclear cells (PBMC) were purchased from Stemcell Technologies. All cell lines were cultured in RPMI 1640 or in Dulbecco’s modified Eagle’s (DMEM) media supplemented with 10% FBS. All cell lines did not have mycoplasma contamination determined by RT-PCR. N-acetyl-L-cysteine (NAC) was purchased from Selleckchem, and phorbol 12-myristate 13-acetate (PMA), catalase-PEG, and rotenone were from Sigma Aldrich. The sources of antibodies used in immunoblotting, flow cytometry, and IHC were shown in Suppl. Table 2.

Exosomes from cell culture medium were isolated by ultracentrifugation. Ovarian cancer cells were grown in T75 flasks until they reached to 70% confluency, and then were rinsed with PBS and then replaced with serum-free medium for 48 h. The supernatant was collected and centrifuged at 2000×g for 30 min to discard cellular debris. Next, the collected medium were centrifuged at 12000×g for 30 min at 4 °C followed by filtration through 0.22-μm pore filters (Steriflip, Millipore) to remove larger extracellular vesicles. The supernatant were then and ultracentrifuged at 110,000×g for 2 h at 4 °C (Beckman Coulter, Ti45 rotor). The exosomes pellets were re-suspended in 10 ml PBS, and loaded over 10 ml of 40% sucrose solution, and was ultracentrifuged at 110,000×g for 90 min. The lower sucrose layer, which contained the exosomes was diluted with PBS and ultracentrifuged at 110,000×g for 90 min. The pellet was resuspended in 100 μl PBS and stored in − 80 °C. Exosomes from ascites in mice were isolated using the same method except for erythrocytes lysis with ACK lysis buffer (Thermo Fisher Scientific) after the initial centrifuge at 1500×g for 15 min to remove debris. Exosomes from the serum in mice were isolated using Exosome Isolation Reagent (Invitrogen, Thermo Fisher Scientific). The serum exosomes were characterized by NTA.

NTA was performed using a NanoSight NS300 instrument according to the manufacturer’s instructions. Isolated exosome samples were diluted with PBS at 1:100 before analysis. The settings after NTA collection were optimized and remained constant between samples. Three videos of 60 s length were recorded for each sample and analyzed to give estimates of mean size and number of particles.

All samples were prepared and processed according to NanoString nCounter Expression CodeSet Design Manual. 100 ng of exosomal RNAs were used as input for Nanostring nCounter miRNA sample preparation. Raw data were normalized to the top 100 miRs using nSolver analysis 2.5 software (Nanostring). Expression heatmaps were generated with the R package pheatmap.

In situ hybridization was performed using double digoxigenin (DIG)-labeled miRCURY LNA miRNA detection probe hsa-miR-155-5p (Advanced Cell Diagnostics, CA, USA) in human ovarian carcinoma tissue microarray (US Biomax, MD, USA). The TMA sections contain 160 cases of ovarian cancer tissue sections and 40 cases of normal human ovary tissue sections. The experiments were performed according to the miRNAscope LS Automated Assay (ACD, CA, USA) protocol. The signal was detected with miRNAscope™ LS Reagent Kit - RED (ACD, CA, USA). Slow Fade Gold anti-fade reagent with DAPI (Life Technologies) was employed as the mounting medium. The positive and negative control of the method was verified employing miRNAscope LS Positive Control Probe (ACD, CA, USA) and miRNAscope LS Negative Control Probe (ACD, CA, USA), respectively. Bright field pictures were scanned by Pannoramic slide scanner (3DHISTECH) and converted to fluorescence signal with Caseviewer 2.2 software. Pictures were taken by Image-Pro Plus 6.0 software. Slides were analyzed with Quant Center 2.1 (Thermo Fisher Scientific) and Graphpad Prism software (La Jolla, CA, USA).

Total RNAs were extracted using Trizol (Thermo Fisher Scientific). The cDNA synthesis was performed using oligo (dT)18 primers and M-MLV reverse transcriptase. The 100 ng of RT product was used for PCR reaction using Power SYBR Green PCR Master Mix (Thermo Fisher Sci). The primer sequences are listed in Supplementary Table 1.

Two-step Taqman-qPCR analysis was performed to assess miRNA levels using Taqman miRNA reverse transcription kit and Taqman universal PCR master mix (Thermo Fisher Sci) in accordance with manufacturer’s instructions.

Exosomes were fluorescently labeled using lipophilic carbocyanine DiR dye (Thermo Fisher Scientific). In brief, 5 μl of DiR (200 μg/ml) in ethanol was mixed with 200 μg exosomes in 100 μl PBS for 1 h. The unincorportated DiR was removed by a Sepharose CL-4B column (Sigma-Aldrich).

THP-1 cells were seeded in 12-well plates at a number of 5 × 10⁵ and were co-transfected with PD-L1-WT 3′-UTR reporter plasmids (Addgene) and miR-cont, miR-155-5p mimics, miR-152-3p mimics, or miR-137 mimics, using jetPRIME® reagent. The transfected cells were harvested 48 h post-transfection using Passive Lysis Buffer (Promega, US), and firefly and Renilla luciferase activities were measured in cell lysates using Dual-Luciferase Reporter Assay System (Promega).

A2780-miR-con and A2780-miR-155 cells were seeded onto 1% agarose (Sigma-Aldrich) substrate in the cover glass bottom of 48-well plates (MatTek, MA, USA) at 1 × 10⁴ cells/well. After 4 days of formation of tumor spheroids, macrophages were added and allowed to co-culture with tumor spheroids for an additional 4 days. The diameters of spheroids were measured at 3 d, 7 d, 10 d and 12 d, respectively. For the 3D live cell imaging, the cells were stained with Cell tracker green (Thermo Fisher Scientific) before seeding onto 1% agarose concave surface for spheroid formation. The infiltrated macrophages were stained by eFluor670 (Thermo Fisher Scientific) before adding into tumor spheroids. The pictures of the 3D co-culture were taken by an A1R+ Nikon confocal microscope for 3D live cell imaging. The number of A2780 and infiltrated macrophages were presented as accumulative average intensity of fluorescence. Z stack-overlay video was taken by A1R+ Nikon confocal microscope.

For cultured cells or 3D co-culture cells, single-cell suspensions were incubated with 2% human Fc Receptor block (eBioscience, Frankfurt, Germany) in PBS for 20 min on ice. After washing with PBS, cells were stained with conjugated antibodies for 30 min at room temperature in the dark. For tumors, spleen, and ascites from mouse models, single-cell suspensions were prepared by tissue extraction, mashing, digestion with Liberase DL, TL (Roche, USA) and DNase І (Sigma-Aldrich, Germany). The erythrocytes were lysed with ACK lysis buffer (Gibco, Thermo Fisher Scientific), filtered by 40 μm pore size filter (Corning, NY, USA), incubated with 2% human Fc receptor blocking (eBioscience, Frankfurt, Germany), and stained with conjugated antibodies. Isotype IgG control was used in each experiment as reference. Flow cytometry was performed using BD Celesta, BD LSRII, or BD Fortessa (BD Bioscience, US). FACS was performed using BD Aria II or BD Melody (BD Bioscience, US). Data were analyzed using FlowJo software (Tree Star Inc., CA, USA).

The human PBMCs were purchased from Stemcell Technologies (Cambridge, MA). CD3⁺ T cells were separated using CD3 microbeads (Miltenyi Biotec, USA) together with MACS columns and separators according to the manufacturer’s instructions. For stimulation of T cells, 24-well plates were coated with 10 μg/ml CD3 purified antibody. After washing with PBS twice, isolated CD3⁺ T cell were plated in CD3-coated plate in culture medium, and stimulated with CD28 purified antibody in the presence of 30 U/ml recombinant human IL-2 (Peprotech, USA).

Animal experimental protocols were in consistent with the Care and Use of Laboratory Animals Guide and approved by the Institutional Animal Care & Use Committee of Thomas Jefferson University (No. 01159). For establishing human ovarian cancer xenograft model in nude mice, 1 × 10⁶ A2780 cells suspended in 100 μl PBS with 50% Phenol Red-free Matrigel (Corning, NY, USA) were injected into flanks of 6-week-old female NCr nude mice (Taconic, US). Tail vein injections of Exo-con or Exo-NAC (100 μg in 100 μl PBS) were performed every 3 days. Tumors were measured with calipers and tumor volume was calculated with formula (width)² × length/2. Mice were sacrificed 21 days after cell inoculation or the maximum tumor dimension reached 2.0 cm before 21 days.

For establishing immunocompetent mouse ovarian cancer model in C57BL/6 mice (Taconic), 8 × 10⁶ ID8-miR-con or ID8–155 mouse ovarian cancer tumor cells were inoculated intraperitoneally (i.p.) into C57/BL6 mice. Exosomes derived from ID8-miR-con (Exo-miR-con) or ID8-miR-155 (Exo-miR-155) tumor cells were prepared and injected via tail vein every 3 days. Starting at 60 days after tumor inoculation, mice were given IgG isotype control or anti-PDL1 antibodies at 200 μg/mouse every 3 days via intraperitoneal injection (IP). Mice were sacrificed when weight gain more than 30% and/or more than 40% waist circumference. Ascites, tumor nodules, spleens, and blood samples were collected and processed for further analysis.

We and others have shown that ROS alter the expression of miRNAs by regulating miRNA synthesis, transcription, and epigenetic modification [14, 15]. To determine whether ROS influence the TME through tumor exosomal miRNAs, we treated ovarian cancer A2780 cells with or without H₂O_2, and purified A2780-derived exosomes characterized by positive exosomal markers CD63 and CD81, and a negative exosomal marker calnexin (Fig. 1A). The exosome size distribution was determined by nanoparticle tracking analysis (NTA) (Fig. 1B). The transmission electron microscope image revealed intact vesicles around 100 nm (right panel) in diameter (Fig. S1A). The exosomal miRNA profile determined by a NanoString nCounter microarray analysis, showed a number of differentially expressed miRNAs in H₂O₂-treated cells compared with those in control cells (Fig. 1C-E). Notably, exosomal miR-155-5p emerged as the most strongly down-regulated miRNA in H₂O₂-treated A2780 cells. We validated the microarray result by RT-qPCR by showing that H₂O₂ decreased both cellular miR-155-5p and exo-miR-155-5p expression levels (Fig. 1F). N-acetyl-L-cysteine (NAC) is a non-selective antioxidant which can similarly inhibit intracellular ROS as other scavengers such as catalase and rotenone. We observed ROS reductions in A2780 cells treated with NAC, catalase-PEG, and mitochondria complex I inhibitor rotenone with flow cytometry and fluorescent microscope using different dyes based on the ROS species (Fig.S1B, C). We then treated three ovarian cancer cell lines with NAC. As expected, both cellular miR-155-5p and exosomal miR-155-5p levels were decreased in ovarian cancer cells (Fig. 1G, H). To confirm that miR-155-5p is a bona fide ROS-sensitive miRNA, we also treated A2780 cells with catalase-PEG and rotenone. The expression levels of miR-155-5p were significantly increased with the ROS inhibitors (Fig. 1I).

miR-155-5p has been considered as an oncomiR in lymphoma, pancreatic, and oral squamous cell carcinoma cancers, and an oncosuppressor-miR in melanoma and colon cancer, as well as ovarian cancer [16‐20]. To examine the abundance of miR-155-5p in ovarian cancer, we analyzed human ovarian carcinoma tissue microarray (TMA) for miR-155-5p expression by fluorescence in situ hybridization (FISH). Compared with normal ovarian tissues, the expression levels of miR-155-5p in ovarian cancer tissues were significantly lower (Fig. 1J), indicating a likely tumor suppressor role of miR-155-5p in ovarian cancer.

TAMs are major types of stromal cells within the ovarian cancer TME. To determine whether tumor exosomes can be taken up by macrophages, we first allowed differentiation of THP-1 cells into CD206⁺ and CD163⁺ macrophages with phorbol 12-myristate 13-acetate (PMA) treatment (Fig. 2A). We then treated macrophages with DiR-labeled exosomes for 24 h. Exosomes were detectable in the cytosol of macrophages as visualized by confocal microscopy indicating these exosomes are taken in by macrophages (Fig. 2B). To determine the impact of tumor exosomes on macrophages, we treated macrophages (PMA-treated THP-1) with exosomes derived from NAC-treated A2780 cells (Exo-NAC) and exosomes derived from A2780 cells (Exo-con). The result showed elevated expression levels of miR-155-5p in Exo-NAC treated macrophages compared to Exo-con treated macrophages (Fig. 2C). Moreover, to rule out the possibility that de novo expression of miR-155-5p in macrophages is the source for the increased miR-155-5p, we knocked down Dicer in macrophages with siRNAs in order to disrupt the formation of mature miRNAs. As seen in Fig. 2D, the expression levels of Dicer were markedly reduced in the knockdown cells. Dicer knockdown greatly reduced expression of miRNAs, as evidenced by examples of miR-9 and miR-145 (Fig. 2E). Moreover, neither tumor exosomes nor Dicer knockdown affected pre-miR-155-5p expression levels (Fig. 2F). However, regardless of Dicer knockdown, levels of mature miR-155-5p increased with Exo-NAC treatment (Fig. 2G), demonstrating that the increased miR-155-5p in macrophages comes from tumor exosomes. Collectively, the results showed that inhibiting ROS in ovarian cancer cells increases the release of exo-miR-155-5p that is taken up by macrophages, resulting in the elevation of miR-155-5p.

Next, we generated an A2780 cell line stably expressing miR-155-5p (A2780-miR-155) and miR-con cell line (A2780-miR-con). Expression levels of both cellular miR-155-5p and exosomal miR-155-5p were significantly higher in the A2780-miR-155 cells than in the control.

cells (Fig. S2A). To investigate if A2780-miR-155-derived exosomes (Exo-miR-155) affect macrophage migration, we performed a wound healing assay. The gap closure rates in macrophages treated with Exo-NAC or Exo-miR-155-5p for 24 h were markedly slower than their corresponding control cells. This indicates that Exo-NAC and Exo-miR-155-5p inhibit macrophage motility that is critical for macrophage recruitment into the TME (Fig. 3A). Furthermore, we examined a number of chemokines that are known to have chemotactic properties of various leukocyte subsets (Fig. S3A). We found CXCL10 and CCL2 were significantly decreased when treated with Exo-NAC or Exo-miR-155 in comparison to Exo-con or Exo-miR-con (Fig. 3B), suggesting these chemokines may contribute to macrophage mobilization.

A high degree of TAM infiltration in tumor tissues correlated with poor prognosis in many cancers. Next, we investigated whether tumor exo-miR-155-5p can reduce macrophage infiltration in vitro using a three-dimensional (3D) model to recapitulate the interaction between macrophages and ovarian cancer cells. First, we established 3D tumor spheroids using A2780-miR-con and A2780-miR-155 cells (Fig. 3C, S3B). A2780-miR-155 spheroids grew slower than A2780-miR-con as reflected by spheroid diameters 10 days after seeding, suggesting a tumor suppressive role of miR-155-5p. To build a co-culture model, A2780 cells were seeded onto non-adherent plates and were allowed to form spheroids for 4 days. Then, 20,000 CD14-positive THP-1 cells were added to the spheroids for an additional 4 days (Fig. 3D). Flow cytometry analysis showed the amounts of infiltrating macrophages in the A2780-miR-155 spheroids were significantly lower than those in the A2780-miR-con spheroids (Fig. 3E). To exclude the possibility that the decrease of macrophages was apoptotic-related, we conducted the apoptosis assay by Annexin V and 7-AAD staining, and found no significant change regarding the percentages of apoptotic tumor cells and macrophages (Fig. 3F). Then tumor cells and macrophages in the spheroids were sorted by FACS with the indicated gates followed by total RNA extraction (Fig. S3C). As expected, the miR-155-5p level increased in macrophages when co-cultured with A2780-miR-155 spheroids, indicating the transfer of exosomes from tumor cells (Fig. 3G). Then we analyzed the mRNA levels of chemokines associated with invasive phenotype in macrophages by RT-qPCR (Fig. 3H, S3D). Compared with macrophages in A2780-miR-con spheroids, the expression levels of CXCL10 and CCL2 significantly downregulated in macrophages in A2780-miR-155 spheroids (Fig. 3H). Among all the chemokines we detected in A2780 cells, CXCL10 and CCL2 levels were also lower in A2780-miR-155 cells sorted from spheroids (Fig. S3E). To observe the co-culture model in a spatiotemporal manner, we utilized cell tracker green-labeled A2780 spheroids and eFluor670-labeled infiltrating macrophages for 3D live cell imaging using confocal microscopy. In line with the results that we showed in Fig. 3E, fewer macrophages infiltrated into A2780-miR-155 spheroids compared with the control as reflected by lower fluorescent intensities (Fig. 3I, Movie. S1, S2). Taken together, these data suggest that ROS-related tumor exo-miR-155-5p is able to inhibit macrophage migration and infiltration.

PD-L1 is a putative target of miR-155-5p with two potential miR-155-5p-binding sites in its 3′-UTR regions that are conserved in humans and mice (Fig. 4A). It was reported that miR-155-5p negatively regulates PD-L1 protein expression by directly binding the 3′ UTR of PD-L1 mRNA in human dermal lymphatic endothelial cells [21]. Interestingly, another study showed that miR-155 positively regulates the transcriptional activity of the PD-L1 gene in HEK-293 T cells [22]. To investigate the effect of miR-155-5p on PD-L1 expression levels, we co-transfected THP-1 cells with miR-cont, or miR-155-5p, and a PD-L1 3′ UTR full-length luciferase reporter. miR-152 served as positive control as it has been validated a direct target of PD-L1, andmiR-137 was used as a negative control since it does not contain predictive binding sites on PD-L1 3’UTR regions [23, 24]. Co-transfection of miR-155-5p or miR-152 with the PD-L1 reporter decreased luciferase activities while co-transfection of miR-cont or miR-137 did not affect luciferase activities, suggesting miR-155-5p inhibits PD-L1 expression at the transcriptional level in THP-1 cells (Fig. 4B). Moreover, overexpression of miR-155-5p decreased PD-L1 protein levels with or without IFN-ɣ induction (Fig. 4C).

To determine whether tumor exosomes inhibit PD-L1 protein levels in macrophages, we treated macrophages with Exo-miR-155 and Exo-miR-con, and found Exo-miR-155 treatment decreased PD-L1 expression significantly in macrophages compared to Exo-miR-con treatment (Fig. 4D). Immunoblotting showed that Exo-miR-155 decreased IFN-γ-induced PD-L1 protein levels in macrophages (Fig. 4E). We had shown that Exo-NAC treatment increased miR-155-5p in macrophages (Fig. 2D). In line with this result, Exo-NAC effectively suppressed PD-L1 protein levels in macrophages (Fig. 4F, G). In 3D coculture models, the PD-L1 expression levels in macrophages that were separated from A2780-miR-155 tumor spheroids were lower than those from A2780-miR-con spheroids (Fig. 4H). We transiently transfected a PD-L1 expressing plasmid or a control vector in macrophages for 48 h, then added into the tumor spheroids. We observed that overexpression of PD-L1 reversed the inhibition of miR-155-5p on macrophage tumor infiltration (Fig. 4I). These results suggest the uptake of tumor exo-155-5p reduced macrophage infiltration through PD-L1.

Tumor xenografts were generated by A2780 cells by injection onto the flanks of female NCr nude mice. At 3 days after the implantation, Exo-con or Exo-NAC (100 μg in 100 μl PBS) was intravenously administered via the tail vein every 3 days, and tumor growth was monitored 3 times a week for 21 days (Fig. 5A). The mice treated with Exo-NAC grew smaller tumors compared to the mice treated with Exo-con (Fig. 5B, C). FACS was used to isolate macrophages from tumors (Fig. S4A) and spleens (Fig. S4B) in single cell suspensions with the indicated gates. The amount of tumor infiltrating macrophages in mice treated with Exo-NAC was significantly lower than in mice treated with Exo-con (Fig. 5D). The mouse macrophage marker F4/80 was also stained by IHC in tumor tissues (Fig. 5E), which showed similar results as illustrated in flow cytometry (Fig. 5D). The miR-155-5p expression level was significantly higher in macrophages isolated from tumors and spleens in mice treated with Exo-NAC (Fig. 5F), indicating miR-155-5p might be a major functional molecule in NAC-treated exosome cargo to downregulate PD-L1. As expected, the expression levels of PD-L1 in tumor tissues and tumor macrophages were significantly lower in mice treated with Exo-NAC (Fig. 5G, H). Collectively, these findings suggest that NAC-derived tumor exosomes are able to inhibit xenograft tumor growth and macrophage infiltration, in which the Exo-miR-155-5p/PD-L1 pathway might play a pivotal role.

Accumulating evidence suggest that tumor-associated macrophage infiltration can promote tumor progression by impairing the immune responses of cytotoxic CD8⁺ T cells in the tumor microenvironment [25]. We asked whether macrophages treated with the exosomes impact T cell functions. To address this question, we isolated CD3⁺ T cells from human PMBC and activated T.

cells with CD3, CD28, and IL-2. Next, we co-cultured activated T cells with macrophages pre-treated with Exo-NAC, Exo-miR-155, or their respective controls for 24 h. The T cell function was evaluated by the percentage of cytotoxic CD8⁺ T cells (Fig. S5A) as well as the apoptosis of T cells using flow cytometry (Fig. S5B). Compared to the controls, Exo-NAC and Exo-miR-155 treated-macrophages significantly increased the CD8⁺ T cell proportion (Fig. 6A). The apoptosis rate of T cells after co-culture with exosome-treated macrophages was also examined by Annexin V/7-AAD staining. The results showed the percentage of apoptotic CD3⁺ T cells was significantly reduced after co-culturing with Exo-NAC and Exo-miR-155 treated macrophages compared with their respective controls (Fig. 6B).

Next, we isolated CD8⁺ T cells from the co-culture system to analyze the intracellular production of tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ) and interleukin-2 (IL-2). The mRNA levels of these cytokines in CD8⁺ T cells were significantly increased after being co-cultured with Exo-NAC- and Exo-miR-155-treated macrophages (Fig. 6C). To determine whether CD3⁺ T cell activation is mediated by PD-L1, we transfected PD-L1 in macrophages before co-culturing with the T cells. We observed that PD-L1 overexpression effectively reversed an exo-miR-155-5p-mediated increase of CD8⁺ T cell (Fig. 6D), suggesting that Exo-miR-155-5p-treated macrophages can activate T cell functions by increasing CD8⁺ T cell ratio and decreasing T-cell apoptosis mainly through PD-L1 downregulation.

Next, we asked whether tumor exo-miR-155-5p could suppress tumor progression and activate the anti-tumor immune response in immune-competent mice. First, we examined the exosome distribution after in vivo delivery in C57BL/6 mice. The exosomes were detectable in the liver, spleen, and lung after 24 h (Fig. 7A). Next, we generated a murine ovarian cancer cell line ID8 stably expressing miR-155-5p (ID8-miR-155) and miR-con cell line (ID8-miR-con). The expression levels of both cellular miR-155-5p and exosomal miR-155-5p in ID8-miR-155 cells were 3-fold higher than those in control cells (Fig. S2B). We then established tumor models by intraperitoneal injection of ID8-miR-con or ID8-miR-155 cells into C57BL/6 mice. Exosomes from ID8-miR-con cells (Exo-miR-con) or exosomes from ID8-miR-155 cells (Exo-miR-155) were administered every 3 days, with anti-PD-L1 or anti-IgG antibodies administered starting at 60 days after tumor inoculation (Fig. 7B). Because ascites accumulation causes waist gain that can be used as an index of tumor progression in ID8 model [26], we monitored the tumor progression by measuring weight and waist circumference. Mice were euthanized when they gained more than 30% body weight and/or more than 40% waist circumference or showed any signs of cachexia or distress [26]. Mice in the control group developed visible ascites starting around 70 days after tumor implantation (Fig. 7C, left), and multiple tumor nodules were disseminated in the peritoneal cavity, including parietal and visceral surfaces (Fig. 7C, right). Delivery of Exo-miR-155 repressed tumor growth as evidenced by prolonged survival and delayed waist gain (Fig. 7D, E, F). Notably, Exo-miR-155 was more potent than anti-PD-L1 antibody treatment for inhibiting tumor growth. In addition, the combination of exo-miR-155-5p with anti-PD-L1 did not further improve the survival of the mice compared with exo-miR-155-5p treatment alone. These results indicated that additional factors are involved in miR-155-5p-mediated tumor suppression, and that the effects of anti-PD-L1 antibody and Exo-miR-155-5p on tumor growth may be repetitive instead of additive. Having demonstrated that tumor exo-miR-155-5p inhibited macrophage infiltration in vitro, we further investigated its effects on macrophage infiltration in vivo. Higher levels of miR-155-5p were found in exosomes isolated from ascites and serum in mice treated with Exo-miR-155, indicating a successful in vivo delivery of exo-miR-155-5p (Fig. 8A). The expression of macrophages marker F4/80 was lower in tumors in the Exo-miR-155-treated group by IHC (Fig. 8B). The proportion of macrophages in tumor samples was analyzed by FACS. The mice treated with either anti-PD-L1 antibody or exo-miR-155-5p had far fewer TAMs compared with the control mice (Fig. 8C). Moreover, the expression levels of miR-155-5p in ascites macrophages and spleen macrophages were elevated in the Exo-miR-155 group, suggesting tumor exo-miR-155-5p was uptaken by macrophages (Fig. 8D). Next, we examined PD-L1 expression in macrophages from ascites and tumors. Consistent with the findings obtained in nude mice, the expression of PD-L1 in ascites, and tumor macrophages were significantly lower in mice treated with Exo-miR-155 than in mice treated with Exo-miR-con (Fig. 8E). Similarly, the expression of PD-L1 in tumor tissue samples in the Exo-miR-155 group were generally downregulated determined by immunoblotting and IHC (Fig. 8F, G).

Lastly, we examined the effects of tumor exo-155-5p on T cell function in vivo. The percentages of CD8⁺ T cells from ascites and spleen in mice from Exo-miR-155 group were significantly increased compared with those in control groups (Fig. 8H). As expected, the anti-PD-L1 antibody increased the percentage of CD8⁺ T cells. Notably, adding anti-PD-L1 to Exo-miR-155 had no synergistic or additive effect on CD8⁺ T cells compared to Exo-miR-155 alone. Collectively, these in vivo findings indicate that exo-miR-155-5p inhibits tumor progression and prevent formation of the suppressive tumor microenvironment by reducing macrophage infiltration and subsequently activating T cell anti-tumor immune responses in ovarian cancer.