M2 macrophage-derived exosomal miR-193b-3p promotes progression and glutamine uptake of pancreatic cancer by targeting TRIM62

Background Pancreatic cancer (PC) is a highly lethal malignancy that requires effective novel therapies. M2 macrophages are abundant in the PC microenvironment and promote cancer progression. Exosomes are emerging mediators of the crosstalk between cancer cells and the microenvironment. This study was conducted to explore the role of M2 macrophage-derived exosomes in PC. Methods Exosomes derived from M2 macrophages were extracted. miR-193b-3p and TRIM62 were overexpressed or silenced to examine their function in PC. Luminescence assays were used to investigate the interaction between miR-193b-3p and TRIM62. Cell proliferation was examined by EdU staining. Would healing and transwell assays were applied to evaluate cell migration and invasion. Co-immunoprecipitation was used to assess the interaction between TRIM62 and c-Myc. Gene and protein expressions were analyzed by quantitative RT-PCR and immunoblotting, respectively. Results M2 macrophage-derived exosomal miR-193b-3p promoted the proliferation, migration, invasion, and glutamine uptake of SW1990 cells. Mechanism study revealed that TRIM62 is a target of miR-193b-3p. TRIM62 inhibited the proliferation, migration, invasion, and glutamine uptake of SW1990 cells by promoting c-Myc ubiquitination. Our data also suggested that TRIM62 expression negatively correlated with miR-193b-3p and c-Myc expression. High-expression of miR-193b-3p and c-Myc predicts poor prognosis, whereas low-expression of TRIM62 predicts poor prognosis in patients with PC. Conclusion M2 macrophage-derived exosomal miR-193b-3p enhances the proliferation, migration, invasion, and glutamine uptake of PC cells by targeting TRIM62, resulting in the decrease of c-Myc ubiquitination. This study not only reveals the mechanism underlying the crosstalk between M2 macrophages and PC cells but also suggests a promising therapeutic target for PC. Supplementary Information The online version contains supplementary material available at 10.1186/s13062-023-00356-y.


Introduction
Pancreatic cancer (PC) is the seventh cause of cancerrelated deaths worldwide [1]. With a 5-year survival rate of 10%, PC represents an increasing cause of cancerrelated deaths [2]. Early diagnosis of PC is difficult. It often presents at an advanced stage with highly invasive tumor cells [3]. Risk factors include nonmodifiable (age, sex, genetic susceptibility, etc.) and modifiable (smoking, alcohol, obesity, etc.) [3]. Despite the development of cancer treatments, the incidence of PC is increasing, especially in the western world [1], which may be due to the extensive tumor microenvironment (TME) in PC. In this context, the dysregulation of metabolic reprogramming and immune regulation plays a vital role in the TME [4,5] and closely associates with progression and treatment efficacy in cancer. Therefore, it is important to elucidate the relationship between tumor cells and the tumor immune-metabolism microenvironment. Tumor-suppressive macrophages (M1 type) and tumor-supportive macrophages (M2 type) constitute the major cell types of the tumor microenvironment, and M2 macrophage-associated markers are primarily associated with a poor clinical outcome [6]. It is well known that M2 macrophages act as a driving factor in tumor-associated macrophages, which generally promote tumor growth, malignance, and metastasis [7].
Tripartite motif (TRIM)-containing proteins are defined by the presence of an N-terminal RING finger, one or two B-boxes, and a coiled-coil domain [22]. Due to the N-terminal RING finger domain, which predominantly contributes to E3 ubiquitin ligase activity, TRIM proteins have been implicated in ubiquitination and are proposed to be a subfamily of E3 ligases. TRIM-containing proteins participate in various cellular processes, such as oncogenesis. For example, TRIM15 promotes the invasion and metastasis of PC cells by mediating APOA1 ubiquitination and degradation [23]. TRIM50 suppresses PC progression and reverses the epithelial-mesenchymal transition by facilitating the ubiquitous degradation of Snail1 [24]. In addition, loss of TRIM29 suppresses the cancer stem cell-like characteristics of PC by accelerating the degradation of ISG15 [25]. Another TRIM member, TRIM62, has been demonstrated to regulate antifungal immunity and intestinal inflammation by facilitating CARD9 ubiquitination [26]. TRIM62 also promotes the proliferation and invasion and increases the chemosensitivity of hepatocellular carcinoma cells by affecting the NF-κB pathway [27] and suppresses tumor proliferation and metastasis in cervical cancer through c-Jun/ Slug signaling [28]. Despite the development of scientific research, the role of exosomal miR-193b-3p/TRIM62 in PC cells remains to be elucidated. In this study, we investigated the mechanism by which exosomal miR-193b-3p derived from M2 macrophages participates in the proliferation, migration, invasion, and glutamine uptake of PC cells.

Materials and methods
Cell lines and cell culture SW1990 cells were obtained from ATCC (USA). Cell lines were cultured in DMEM containing 1% penicillin/streptomycin and 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) at 37 °C with 5% CO 2 humidification.

Adenovirus construction
The control pShuttle-CMV adenovirus, TRIM62 overexpression adenovirus, and c-Myc overexpression adenovirus were obtained from Obio (Shanghai). Transfection was performed using Lipo2000 (Invitrogen, USA) according to the manufacturer's instructions. At 48 h after transfection, the cell culture medium containing the viral particles was collected to infect SW1990 cells.

Macrophage polarization and cell co-culture
THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% exosome-free FBS. The medium was changed every 48 h. THP-1 cells were induced to differentiate into M0 macrophages by treatment with 100 ng/ mL of phorbol myristic acetate (PMA, Sigma) for 24 h. To induce the M2 polarization of macrophages, 30 ng/ mL of IL-4 (R&D Systems Inc., USA) was used to treat M0 macrophages for 24 h.
The conditioned medium (CM) of M0 or M2 macrophages was used for co-culture. To exclude the interference of exosomes in FBS, 10% (v/v) exosome-free FBS (SunBio, China) was used to configure the medium for the culture of macrophages. After 48 h, the supernatant of macrophages was collected and centrifuged at 5000 rpm for 5 min to remove excess cells. Finally, it was mixed at 1:1 with a complete medium containing 10% (v/v) exosome-free FBS and added to SW1990 cells for 24 h as the CM. Moreover, GW4869 (Umibio, China) was used as an exosome inhibitor, and macrophages were pretreated with 20 µM GW4869 for 24 h to inhibit the generation and secretion of exosomes.

Exosome isolation
Briefly, the supernatant medium from the macrophage culture was centrifuged at 500g for 15 min, 3000g for 15 min, and 12,000g for 30 min at 4 h. Exosomes were centrifuged at 140,000g for 80 min. After resuspension, they were centrifuged at 140,000g for 80 min. Then, the exosomes were placed on the copper grid for examination by transmission electron microscopy (TEM). The exosomes were labeled with a PKH-67 kit (Sigma, St. Louis, MO, USA) to detect their phagocytosis by SW1990 cells. SW1990 cells were treated with 100 µg/mL of M0 or M2 macrophage-derived exosomes.

EdU assay
Cell proliferation was determined using EdU assays. After the abovementioned treatment, cells were seeded into 24-well plates, incubated with fresh media containing 50 nM EdU reagent (RiboBioInc, China) for 2 h at 37 °C, fixed with 4% paraformaldehyde solution, followed by DAPI staining, and then subjected to fluorescence microscopy (Olympus, Tokyo, Japan).

Wound healing assay
The migration abilities of SW1990 cells were evaluated using wound healing assays. After the abovementioned treatment, SW1990 cells were seeded in a 35-mm culture dish (8 × 10 5 cells/dish). Wounds were created using a sterile pipette tip when the cells were fully confluent. After washing with phosphate-buffered saline (PBS) to clean the exfoliated cells, the cells were cultured in a pure medium without FBS. The images of wounds at the same position were taken at 0, 24, and 48 h to calculate the distance.

Transwell invasion assay
After the abovementioned treatment, transwell chambers were used to examine cell invasion. A culture medium containing 10% FBS was added to the lower chamber. Then, 5 × 10 4 SW1990 cells were suspended in serum-free culture medium and inoculated in the matrigel-coated upper chamber (BD Biosciences, USA). After 24 h of incubation, the SW1990 cells were removed from the upper chamber with a cotton swab. The cells that penetrated and adhered to the bottom of the filter membrane were fixed with 4% paraformaldehyde in PBS for 10 min, stained with 0.5% crystal violet for 20 min, and then imaged under a microscope.

Glutamine uptake assay
After the abovementioned treatment, a glutamine assay kit (ab197011; Abcam, USA) was used to determine the concentration of glutamine according to the manufacturer's protocol. Based on the principle of glutamine conversion into glutamic acid and ammonia, the amount of glutamine was calculated by measuring the amount of ammonia.

Luciferase activity assay
SW1990 cells were transfected with an miR-193b-3p mimic/inhibitor. Then, the wild-type pGL3-promoter TRIM62 3ʹUTR (WT) or the mutant-type pGL3promoter TRIM62 3ʹUTR (MUT) luciferase plasmid was transfected into SW1990 cells. The pRL-TK vector was transfected into SW1990 cells, which served as an internal control reporter. A dual-luciferase assay was performed according to the manufacturer's protocol. The luciferase activity was evaluated using a Dual-Luciferase Reporter Assay system (Promega Biotech Co, Ltd, Beijing, China) at 48 h posttransfection and normalized to Renilla luciferase activity.

Quantitative RT-PCR (qRT-PCR)
RNAs were isolated using TRIzol and reverse-transcribed using Superscript II (Invitrogen, Shanghai). SYBR master mix (Bio-Rad, Philadelphia, PA) was used for qRT-PCR that was performed using the primers listed in Additional file 1: Table S1. The fold change for the relative gene expression was determined using the 2 −ΔΔCt method. GAPDH/U6 served as an internal reference gene.

Protein stability
Cycloheximide (CHX, 0.1 mg/ml; Sigma, Shanghai) was used to inhibit protein synthesis. SW1990 cells were treated with CHX for 0, 1, 4, and 8 h, cell lysates were obtained as described earlier, and then protein expression was assayed by western blotting. The CHX treatment time was plotted on the abscissa, and the amount of protein was plotted on the ordinate. The time required to degrade half the protein was the protein half-life.

Animal models
Experiments were performed according to the principles of the Committee on Ethics of Animal Experiments of Fudan University Shanghai Cancer Center. In a mice lung metastasis model, 5 × 10 6 SW1990 cells were inoculated through tail veins (n = 5). Exosomes (10 µg) derived from M0 macrophages (M0-exo) or M2 macrophages transfected with the miR-193b-3p inhibitor (M2/Inhibitorexo) or NC (M2/NC-exo) were inoculated every 3 days. Otherwise, 5 × 10 6 SW1990 cells with TRIM62 and c-Myc overexpression adenovirus infection were inoculated through tail veins (n = 5). Mice were euthanized 6 weeks post injection, and the lung was examined and collected for hematoxylin-eosin (HE) staining. The number of lung nodules was recorded.

Patient samples
Two cohorts of PC cases in Fudan University Shanghai Cancer Center recruited from October 2019 to March 2021 were included in this study. Cohort 1 consisted of patients with tumor and adjacent normal tissues (n = 20), and cohort 2 consisted of patients with tumor (n = 60) and adjacent normal (n = 10) tissues. This study was approved by the Ethics Committee of Fudan University Shanghai Cancer Center and was performed according to the Declaration of Helsinki. Written informed consents were obtained.

Statistical analysis
Data were analyzed using GraphPad8.4.3 (La Jolla, CA). Results are expressed as mean ± SD. Between-group differences were evaluated using Student's t-test or ANOVA. The Kaplan-Meier method and log-rank tests were used to analyze and compare overall survival. P values of < 0.05 were considered to indicate statistical significance.

M2 macrophage-derived exosomes enhance the proliferation, migration, invasion, and glutamine uptake of SW1990 cells
To explore the effect of macrophages on PC cells, THP-1 cells were treated with PMA and IL-4 to obtain M0 and M2 macrophages, respectively (Additional file 1: Figure S1A), and the expression levels of the macrophage marker CD68 and the M2 macrophage markers CD206 and ARG1 were measured to confirm the differentiation of macrophages (Additional file 1: Figure S1B). Next, we used a co-culture system to mimic the in vivo circumstance and examine the cell-cell communication of macrophages and SW1990 cells. Exosome-free condition medium (CM) was used in the co-culture system of macrophages and SW1990 cells. We found that the CM of M2 macrophages could substantially increase the proliferation (Fig. 1A, B), migration (Fig. 1C, D), invasion (Fig. 1E, F), and glutamine uptake (Fig. 1G) of SW1990 cells compared with the CM of M0 macrophages.
To elucidate how the CM of M2 macrophages affects SW1990 cells, the exosomes were successfully isolated, characterized by TEM (Additional file 1: Figure S1C), and confirmed by measuring the expression of exosome markers, including CD9, CD63, and TSG101 (Additional file 1: Figure S1D). The uptake of exosomes by SW1990 cells was confirmed by PHK-67 labeling (Additional file 1: Figure S1E). Then, the CM of M2 macrophages with or without treatment with the exosome inhibitor GW4869 was collected and used to treat SW1990 cells. Results showed that the administration of GW4869  Fig. 2A, B), migration (Fig. 2C, D), invasion (Fig. 2E, F), and glutamine uptake (Fig. 2G) of SW1990 cells. These findings suggest that the exosomes derived from M2 macrophages enhance the proliferation, migration, invasion, and glutamine uptake of SW1990 cells.

TRIM62 interacts with c-Myc and induces c-Myc ubiquitination
To further examine the mechanism by which TRIM62 is involved in migration and invasion, we performed a Co-IP assay using either anti-TRIM62 antibody or anti-c-Myc antibody, which confirmed the interaction between TRIM62 and c-Myc (Fig. 6A). The administration of MG132, a proteasome inhibitor, abolished the TRIM62 overexpression-induced decrease of c-Myc expression (Fig. 6B). TRIM62 overexpression also significantly increased the degradation of c-Myc (Fig. 6C). The IP results also demonstrated that TRIM62 overexpression promoted c-Myc ubiquitination (Fig. 6D). These results indicate that TRIM62 interacted with c-Myc and induced c-Myc ubiquitination.

C-Myc regulates the TRIM62-mediated proliferation, migration, invasion, and glutamine uptake of SW1990 cells in vitro and in vivo
We next examined the effects of c-Myc on proliferation, migration, invasion, and glutamine uptake. Our results showed that c-Myc overexpression significantly reversed the TRIM62-inhibited proliferation (Fig. 7A, B), migration (Fig. 7C, D), invasion (Fig. 7E, F), and glutamine uptake (Fig. 7G)  reversed the TRIM62 overexpression-induced decrease of c-Myc expression (Fig. 7H) and increased the numbers of lung metastatic nodules (Fig. 7I, J). Overall, these results indicate that c-Myc regulates the TRIM62-mediated proliferation, migration, invasion, and glutamine uptake of SW1990 cells.

MiR-193b-3p, TRIM62, and c-Myc levels are clinically associated with prognosis of patients with PC
To analyze the clinical relevance, we measured the levels of miR-193b-3p, TRIM62, and c-Myc in PC tissues obtained from cohort 2. Results showed that compared with than in adjacent normal tissues, miR-193b-3p levels were dramatically increased in PC tissues (Fig. 8A), whereas TRIM62 levels were significantly decreased (Fig. 8B). A correlation analysis suggested a strong negative correlation between TRIM62 and miR-193b-3p (Fig. 8C). We also evaluated the expression of c-Myc and TRIM62 at the protein level by IHC staining (Fig. 8D) and analyzed the correlation. TRIM62 expression negatively correlated with c-Myc expression (Fig. 8E). Analysis of the overall survival rate showed that patients with high-expression of miR-193b-3p had a low survival rate, patients with high-expression of TRIM62 had a high survival rate, and those with high-expression of c-Myc had a low survival rate (Fig. 8F-H). These results suggest that TRIM62 negatively correlates with miR-193b-3p and c-Myc, and high expressions of miR-193b-3p and c-Myc predict poor prognosis, whereas low-expression of TRIM62 predicts poor prognosis.

Discussion
This study demonstrated that M2 macrophage-secreted exosomal miR-193b-3p enhances the proliferation, migration, invasion, and glutamine uptake of SW1990 cells. The mechanism study indicated that exosomal miR-193b-3p targets TRIM62 that interacts with and induces c-Myc ubiquitination, resulting in the promotion of the proliferation, migration, invasion, and glutamine uptake of SW1990 cells. Our data also indicate that TRIM62 negatively correlates with miR-193b-3p and c-Myc, and high expressions of miR-193b-3p and c-Myc predict poor prognosis, whereas low-expression of TRIM62 predicts poor prognosis. To our knowledge, our study is the first to indicate that miR-193b-3p/TRIM62/c-Myc signaling promotes the proliferation, migration, invasion, and glutamine uptake of PC cells.
The TME consists of different types of cells, including cancer cells and macrophages. Tumor-associated macrophages counteract the cytotoxic effects of NK/T cells, promoting cancer proliferation and migration [29]. As the major immune cells in the TME, M2 macrophages exhibit immunosuppressive properties. Increasing evidence suggests that exosomes can transfer cargoes to target cells to promote cancer progression and metastasis [30]. It has been demonstrated that the transmission of HISLA through macrophage-secreted exosomes increases the apoptotic resistance of breast cancer cells [31]. M2-exos enhance cell migration in colon cancer through miRNAs [11]. In this study, we found that M2-exos enhanced the proliferation, migration, invasion, and glutamine uptake of SW1990 cells. Moreover, this effect was mediated by miR-193b-3p/TRIM62/c-Myc signaling. These findings improve our understanding of the crosstalk between macrophage-derived exosomes and the TME and also broaden our understanding of PC progression.
miR-193b-3p is involved in different biological and pathological processes. For instance, Li et al. reported that miRNA-193b suppresses proliferation and induces apoptosis in ovarian cancer [32]. miR-193b-3p also inhibits neuroblastoma cell growth [33]. Furthermore, decreasing the expression of miR-193b was found to impair PC cell growth [34]. The present study demonstrated that miR-193b-3p expression was dramatically upregulated in M2-exo, and exosomal miR-193b-3p enhanced the proliferation, migration, invasion, and glutamine uptake of SW1990 cells through TRIM62/c-Myc signaling. These findings indicate a novel role for miR-193b-3p in the progression of PC. TRIM62 has been implicated in different types of diseases. For example, Cao et al. indicated that TRIM62 promoted CARD9 ubiquitination to promote antifungal immunity and decrease susceptibility to fungal infection [26]. Moreover, TRIM62 was found to be activated in the muscles of critically ill patients [35]. Quintas-Cardama demonstrated that NSCLC lesions lose TRIM62 in a stepwise manner during disease progression [36]. In the present study, we demonstrated that TRIM62 overexpression significantly ameliorated the exosomal miR-193b-3p-promoted proliferation, migration, invasion, and glutamine uptake of SW1990 cells. These data widen our understanding of TRIM62 and PC progression. Glutamine is a major nutrient that participates in different aspects of cancer metabolism. It supports both biosynthesis and the tricarboxylic acid cycle [37,38]. A hallmark of metabolism reprogramming in cancer cells is the increased utilization of glutamine [39]. Demas et al. reported that glutamine metabolism drives the growth of mammary cancer [40]. Blockade of glutamine metabolism was found to suppress cancer growth by reprogramming myeloid cells and metabolically reshaping the TME [41]. In the present study, we confirmed that miR-193b-3p derived from M2-exos enhanced the glutamine uptake of SW1990 cells through TRIM62/c-Myc ubiquitination. These results elucidated a new role for miR-193b-3p/TRIM62/c-Myc in the regulation of glutamine uptake by PC cells. However, there are some limitations in our study. For instance, we used only SW1990 cells for these experiments. We intend to use other PC cells for further experiments. A PDX mouse model will provide more relevant data and will be used in a future study. Despite these limitations, we report a novel mechanism underlying the progression of PC induced by M2 macrophage-derived exosomal miR-193b-3p.
In conclusion, M2 macrophage-secreted exosomal miR-193b-3p enhances the proliferation, migration, invasion, and glutamine uptake of SW1990 cells by targeting TRIM62 and inhibiting c-Myc ubiquitination. These findings emphasize the importance of miR-193b-3p/TRIM62/c-Myc signaling in the progression of PC and provide novel insights into therapeutic strategies.