Exosomes derived from mesenchymal stem cells curbs the progression of clear cell renal cell carcinoma through T-cell immune response

Abstract

Exosomes derived from mesenchymal stem cells (MSC-Exo) are effective in modulating immunity. However, the role of MSC-Exo in clear cell renal cell carcinoma (ccRCC) is unclear. Our study was performed to identify if exosomal microRNA (miRNA) can be used as potential noninvasive biomarkers for ccRCC therapy. An orthotopic ccRCC mouse model was established, followed by MSC-Exo injection (1 mL, 20 μg/mL). The metastases of tumors were observed using HE staining, while number of dendritic cells, natural killing (NK) T cells and CD8⁺ T cells was measured using flow cytometry. It was observed that MSC-Exo treatment significantly inhibited metastasis and growth of tumors, and improved immune response in vivo. As for in vitro assay, naive T cells were treated with MSC-Exo, followed by detection of T cell proliferation using EdU staining and CFSE assay. Results also showed that MSC-Exo facilitated sensitivity of ccRCC cells to NK T cells. Our experimental data further showed that miR-182 could be delivered by MSC-Exo in ccRCC, which targeted vascular endothelial growth factor A (VEGFA), as dual-luciferase reporter assays validated. In conclusion, miR-182 contained in MSC-Exo promoted immune response of T cells by suppressing VEGFA expression, thus alleviating ccRCC development.

Introduction

Mesenchymal stem cells (MSCs) are popular in regenerative medicine field for their immunomodulatory abilities (Maumus et al. 2013). Besides, MSCs play significant roles in tumor microenvironment, specifically, by exerting anti-tumor effects (Whiteside 2018). Exosomes (Exo) are vesicles with diameter of 30–150 nm, which function in cell–cell communication and can be derived from MSCs (Joo et al. 2020). MSC-derived Exo (MSC-Exo) treatment has been demonstrated to ameliorate a variety of diseases, such as spinal cord injury (Huang 2017), stroke (Zhang and Chopp 2016), renal fibrosis (Ji, 2020) and acute kidney injury (Cao 2020). MSC-Exo also decreases tumorigenicity in breast cancer by regulating the function of cancer stem cells and cancer cell proliferation (Naseri et al. 2018, 2020). However, to our best knowledge, the detailed mechanism of MSC-Exo in clear cell renal cell carcinoma (ccRCC) has been rarely explored. ccRCC is a common urological cancer with high mortality rate and metastatic potential which is caused by insufficient diagnosis of the early stage ccRCC, and the resistance to therapy, thus requiring the discovery of new therapeutic or diagnostic biomarkers (Braga et al. 2019). Recently, RCC cells-derived exosomes have been displayed to participate in different aspects of RCC progression, such as angiogenesis, immune escape and tumor growth, by transferring microRNAs (miRNAs) (Grange et al. 2019).

miRNAs are small RNAs that bind to target mRNAs to regulate their translation, which are known as noninvasive biomarkers for diagnosis or treatment of diseases (Duttagupta et al. 2011). miR-182 is associated with cell-cycle progression and shows anti-tumor properties in ccRCC progression (Kulkarni, 2018). A previous study also points out that long noncoding RNA UCA1 promotes the expression of delta-like 4 by competitively binding to miR-182, thereby accelerating the biological behavior of ccRCC cells (Wang et al. 2020). miR-182 can be encapsulated in human bone marrow MSCs to boost bone metastasis and osteoclastogenesis during chondrogenesis (Bai et al. 2019). Nevertheless, the role of miR-182 from MSC-Exo in ccRCC remains elusive. Additionally, miR-182 is detected to specifically bind to vascular endothelial growth factor (VEGF) in colon cancer (Yan, 2020). As one member of the VEGF family, VEGFA has been identified as an independent factor for prognosis of ccRCC, which links to shorter survival rate for ccRCC patients (Wierzbicki, 2019). Yet, the targeting relationship between miR-182 and VEGFA in ccRCC needs to be further explored. Based on the aforesaid references, we assumed that miR-182 may be contained in MSC-Exo and exert certain functions in ccRCC development by interacting with VEGFA. The goal of this study is to validate our hypothesis and to identify that if exosomal miRNA can be used as potential noninvasive biomarkers for ccRCC therapy.

Materials and methods

Ethics statement

All animal experiments were performed with approval from the Ethics Committee of Hainan General Hospital.

Culture and identification of MSCs

Human umbilical cord-MSC (hucMSCs) were purchased from American Type Culture Collection (#PCS-500-010; ATCC, Manassas, VA, USA). MSC surface markers were identified by flow cytometry using antibodies against CD44, CD34, CD29, and CD45 (all from BD Biosciences, Franklin Lakes, NJ, USA). MSCs were seeded in 6-well plates and cultured in minimal essential medium plus dexamethasone (100 nM), ascorbic acid-2-phosphate (0.05 μM), β-glycerophosphate (10 mM), penicillin (100 U/mL)-streptomycin (100 μg/mL), and 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) for 3 weeks. Osteogenic differentiation was assessed by Alizarin Red (Sigma-Aldrich, St Louis, MO, USA) staining. MSC morphology was observed with inverted microscope (Life Technologies, Carlsbad, CA, USA).

Extraction and identification of Exo

MSCs were cultured in a 100-mm plate for 24 h, followed by cells debris removal. Collected medium was ultracentrifuged at 20,000×g for 90 min, and at 160,000×g for 3 h to collect precipitates. The precipitate was resuspended in phosphate buffered saline (PBS) for nanoparticle tracking analysis (NTA). Additionally, western blot analysis was used to examine Exo-specific markers (CD63 and TGS101), while morphology and size of Exo were viewed by transmission electronic microscope (TEM; JEM-1400, JEOL, Tokyo, Japan). After the identification was completed, the protein concentration of the extracted exosomes was measured using the bicinchoninic acid (BCA) Protein Concentration Assay Kit (Beyotime Biotechnology Co., Ltd., Shanghai, China) strictly according to the instructions. The protein concentration of Exo measured was 349.82 μg/mL, which was subsequently diluted to 20 μg/mL for further use.

Animal experiments

Tumorigenic capacity of ccRCC cells in mice was analyzed using an orthotopic mouse tumor model. BALB/c nu/nu male mice (5 weeks old) were purchased from Sankyo Labo Service (Tokyo, Japan; http://www.sankyolabo.co.jp/). Kidneys of mice were orthogonally injected with 2 × 10⁶ Caki-1 cells. Subsequently, extracted exosomes (1 mL) were injected through the tail vein of mice every three days (a total of 15 injections), with PBS served as a control. Mice were euthanized after 45 days using an intraperitoneal injection of pentobarbital (150 mg/kg). Half an hour after the injection, we confirmed that the mice had no heartbeat, no blink reflex and no neurological reflex, nor respiratory arrest.

Cell culture and treatment

Human ccRCC cell lines 786-O (#CRL-1932) and Caki-1 (#HTB-46, both from ATCC) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) with 10% FBS (Gibco, Carlsbad, CA, USA), penicillin (25 units/mL), streptomycin (25 g/mL), and 1% l-glutamine at 37 °C in 5% CO₂.

Peripheral blood mononuclear cells (PBMCs) were purified using Ficoll-Paque. Naive T cells were purified from PBMC of healthy donors by a EasySep enrichment kit (STEMCELL Technologies, Vancouver, BC, Canada). The purity of T cells (CD3⁺/CD28⁺/F4/80⁻) was > 97% by flow cytometry screening. Human T cells were maintained in T-cell medium containing 50 μg/mL recombinant human interleukin-2.

Flow cytometry

Tumor or spleen tissues were weighed, fragmented and detached in 10 mL detachment solution containing PBS, collagenase type I (200 U/mL), hyaluronidase, and DNase I (100 μg/mL) for 60 min. Single cell suspension was obtained through grinding and detachment of tissues and subsequent filtering using a 70-μm cell filter (BD Biosciences, San Jose, CA, USA). Immune cells were isolated using Ficoll density gradient centrifugation, and stained with monoclonal anti-mouse antibodies (eBioscience, San Diego, CA, USA) against CD45-PECy5.5, CD3-pecy7, CD8-APC, NK1.1-APC, F4/80-APC, CD11b-pecy7, CD11c-APC, and MHCII-PE at 4 °C for 30 min. FACS Canto II flow cytometer (BD Biosciences) and FlowJo software (TreeStar, Ashland, OR, USA) were utilized for analysis. For dendritic cells (DCs), the gating was: CD11b⁺/Ly6G⁻-F4/80⁻/CD11c⁺; for natural killer (NK) T cells, the gating was: CD3⁺/F4/80⁻/NK1.1⁺; CD4 T cells: CD3⁺/F4/80⁻/CD8⁻CD4⁺; CD8 T cells: CD3⁺F4/80⁻/CD8⁺CD4⁻.

Hematoxylin–eosin (HE) staining

Lung and liver tissues were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and cut into 4-μm sections. After deparaffinization and rehydration, sections were stained with HE and observed under a microscope (Leica, Heidelberg, Germany).

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from cells or exosomes using Trizol reagent (Invitrogen, Grand Island, NY, USA), and cDNA was transcribed using the SuperScript II RT kit (Invitrogen). Expression of miR-182 in each sample was determined by RT-qPCR using specific primers and normalized to U6. Following primers were used: miR-182 forward primer: GGCAATGGTAGAACTCAC, miR-182 reverse primer: GAACATGTCTGCGTATCTC; U6 forward primer: CTCGCTTCGGCAGCACA, U6 reverse primer: TTTGCGTGTCATCCTTGCG, and gene quantification levels were calculated using the 2^−∆∆Ct method.

Western blotting

Lysate harvested using radio immunoprecipitation assay buffer and 1× protease inhibitor cocktail (at a ratio of 100:1) was centrifuged at 12,000 rpm for 20 min to remove the debris. Meanwhile, the supernatant was diluted with 5× loading buffer (ratio of 4:1), denatured in a boiling water bath (at 95 °C) for 5 min, and then stored at − 80 °C. The protein concentration was measured by the BCA assay. The protein extract (30 μg) was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes and treated with 5% skimmed milk powder for 1 h at room temperature. The membranes were probed with primary antibodies to VEGFA (1:500, #AF5131, Affinity Bioscience, Cincinnati, OH, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5000, #10,494–1-AP, Proteintech, Chicago, IL, USA) at 4 °C, followed by another incubation with horseradish peroxide-conjugated secondary anti-rabbit Immunoglobulin G (1:10,000, #SA00001-2, Proteintech) at room temperature for 1 h. With an enhanced chemiluminescence system (Millipore, Billerica, MA, USA), blots were developed. The band intensity was measured using ImageJ software (Rawak Software, Germany).

Immunohistochemistry

Mouse lung and liver tissues were fixed with 4% paraformaldehyde for 48 h. Next, paraffin sections (5 μm) were treated with HE staining to monitor lung and liver metastasis. For immunohistochemistry, tumor tissues were fixed, paraffin-embedded, sectioned into 5 μm, and immune-stained with antibodies to CD8 (1:100; ABclonal, Wuhan, Hubei, China), CD11c (1:100; ABclonal), CD49b (1:100; ABclonal), and F4/80 (1:200; BioLegend, San Diego, CA, USA) at 4 °C overnight. After the treatment with anti-rabbit antibody (1:200; Cell Signaling Technology, Danvers, MA, USA) and diaminobenzidine solution (OriGene, Beijing, China), the sections were observed under a microscope (Olympus, Tokyo, Japan).

5-ethynyl-2′-deoxyuridine (EdU) staining

The cells were labeled with 50 μmol/L EdU at 37 °C for 2 h, fixed with 4% paraformaldehyde for 30 min, incubated with 0.5% Triton X-100 for 20 min, and washed with PBS containing 3% bovine serum albumin. Subsequently, the cells in each well were stained in 100 μL staining solution for 1 h in the dark and counter-stained with 1× Hoechst 33,342 for 30 min. Images of EdU-positive cells, 4,6-diamino-2-phenyl indole-labeled nuclei, and merged images were obtained under a microscope (100×).

5,6-carboxyfluorescein diacetate succinimidy ester (CFSE)

By following instructions of CellTrace™ CFSE Cell Proliferation Kit (Invitrogen) and referring to a previous literature (Yi 2017), CFSE assay was performed to label the cells in suspension. Briefly, naive T cells were stained and then seeded in 6-well plates at a density of 1 × 10⁵ cells/well. After 3-day culture, naive T cells were harvested and analyzed using a flow cytometer (FACSCalibur, BD Biosciences) with 635 nm excitation and emission filters. CFSE-positive cell ratio was measured by Modfit LT (Verity Software House, Topsham, ME, USA).

Detection of caspase-3 activity

Caspase-3 activity was determined by a commercially obtained caspase-3 activity detection kit (Beyotime Biotechnology, Beijing, China). After a 3-day culture in 6-well plates with conditioned medium, ccRCC cells were lysed with the lysis buffer. Then, the lysate was incubated with caspase-3 substrate for 6 h in the dark at 37 °C. Caspase-3 activity was measured according to the manufacturer's instructions with the optical density (OD) value at 405 nm.

Lactate dehydrogenase (LDH)-based cytotoxicity assay

NK cells were lysed and determined as previously described (Ma, 2019). After 24-h transfection, Caki-1 and 768-O cells were seeded into 96-well plates at a density of 4–6 × 10³ cells/well. Adherent cells were co-cultured with NK cells at different effector-to-target (E:T) ratios for 4–6 h. Tumor cell lysate was measured with LDH cytotoxicity assay kit (Dojindo, Kumamoto, Japan).

Dual-luciferase reporter assay

Wild-type (WT) and mutant-type (MUT) of VEGFA 3'untranslated region (UTR) reporter (RiboBio, Guangzhou, Guangdong, China) were used. 293 T cells (#CRL-1573, ATCC) were co-transfected with miR-182 mimic or negative control (NC) and 500 ng luciferase reporter. Luciferase activities were evaluated using dual-luciferase reporter assay (Promega, Madison, WI, USA).

Statistical analysis

Every experiment was repeated at least thrice. Data were displayed as mean ± standard deviation. Paired t-test was used for comparison between two groups, while one-way ANOVA and Tukey’s post hoc test was for comparison among multiple groups. The p value of 0.05 was used as the threshold of significance.

Results

Exo are successfully extracted from hucMSCs

MSCs exhibited a characteristic fibroblast-like morphology (Fig. 1a) and showed the ability to differentiate into osteoblasts (Fig. 1b). MSC surface markers were identified using flow cytometry, which showed that CD34 and CD45 were negative, yet CD44 and CD29 were positive (Fig. 1c–f). NTA and TEM unraveled that size of MSC-Exo was around 100 ± 55 nm (Fig. 1g–h). Using Western blotting, we validated the expression of specific markers (CD63, CD81 and CD9) in isolated MSC-Exo (Fig. 1i).

Fig. 1.

Successful extraction of Exo is identified. a Morphology of hucMSCs observed under the light microscope. b Osteogenic differentiation ability of hucMSCs. c–f expression of MSC surface markers CD29 (c), CD44 (d), CD45 (e), and CD34 (f) detected by flow cytometry. g–h morphology and size distribution of Exo determined using TEM (g) and NTA (h). i Exo markers (CD63, CD81, and CD9) determined using Western blotting. Cell experiments are repeated 3 times

Growth and metastasis of ccRCC are suppressed by hucMSCs in vivo

To clarify the role of hucMSC-Exo in ccRCC, BALB/c-nu/nu mice were injected with 2 × 10⁶ cells/mL Caki-1 cells at the right kidney, and injected with 20 μg/mL Exo or PBS via the tail vein at an interval of 3 days. After 45 days, mice were euthanized (Fig. 2a). Subsequently, tumor nodules size in kidney tissues was examined by HE staining, which was significantly smaller in hucMSC-Exo-treated mice than that in PBS-treated mice (Fig. 2b). Furthermore, immunohistochemistry was conducted to detect the number of KI67-positive cells in tissues, showing that the number of KI67-positive cells was reduced after hucMSC-Exo treatment (Fig. 2c). However, TUNEL staining showed that the number of apoptotic cells was increased (Fig. 2d). HE staining showed that Caki-1 cells could develop metastases in liver and lung tissues, but hucMSC-Exo treatment could lessen the number of metastases (Fig. 2e–f).

Fig. 2.

Treatment of hucMSC-Exo suppresses metastasis of ccRCC in vivo. a Schematic diagram showing the treatment of mice. b Volume of tumors in hucMSC-Exo or PBS-treated mice. c KI67 expression in tumors detected by immunohistochemistry. d The proportion of apoptotic cells in tumor tissues examined using TUNEL staining. e–f Metastasis of lung (e) and liver (f) tissues determined by HE staining. Five mice in each treatment. Cell experiment is repeated 3 times. Data are shown as mean ± standard deviation. **p < 0.01 vs. PBS group

hucMSC-Exo promotes immune response in vivo

Subsequently, immune cells were isolated from spleen and tumor tissues in tumor-bearing mice by Ficoll density gradient centrifugation and sorted by flow cytometry. The proportion of DCs (CD11b⁺/CD11c⁺) in spleen and tumor tissues of mice was elevated after hucMSC-Exo treatment (Fig. 3a), along with increased number of NK T cells (CD3⁺/NK1.1⁺) and CD8⁺ T cells (Fig. 3b–c). However, the proportion of macrophages did not change significantly after hucMSC-Exo treatment (Fig. 3d). Immunohistochemistry was further performed to detect the infiltration of NK T and CD8⁺ T cells in lung and liver tissues, showing that the infiltration of NK T and CD8⁺ T cells was accumulated following hucMSC-Exo treatment (Fig. 3e–f).

Fig. 3.

Immune response is promoted by hucMSC-Exo treatment in mice. a Proportion of DCs (CD11b⁺/CD11c⁺) in spleen and tumor tissues of mice. b Proportion of CD8⁺ T cells in spleen and tumor tissues of mice determined by flow cytometry. c Proportion of NK T cells (CD3⁺/NK1.1⁺) in spleen and tumor tissues of mice determined by flow cytometry. d Proportion of macrophages in spleen and tumor tissues of mice determined by flow cytometry. e–f Number of positive-cells in CD8⁺ T (e) and NK T (f) cells measured by immunohistochemistry. Five mice in each treatment. Cell experiment is repeated 3 times. Data are shown as mean ± standard deviation. **p < 0.01 vs. PBS group

hucMSC-Exo facilitates growth and mature of T-cell in vitro

Since naive T cells proliferate, express CD8⁺ or CD4, and promote immune response after being stimulated by superior signals (e.g., signaling by mature DCs), naive T cells were treated with hucMSC-Exo. EdU staining and CFSE assay were conducted to detect the proliferation of T cells. After hucMSC-Exo treatment, the proliferation rate of T cells increased distinctly (Fig. 4a, b). Flow cytometry was applied to detect the phenotype of T cells. We found that hucMSC-Exo treatment promoted the maturation of T cells, in which proportion of CD8⁺ T and NK T cells increased (Fig. 4c, d). However, the number of CD4⁺ T cells increased, but was not significantly different (Fig. 4e).

Fig. 4.

hucMSC-Exo treatment promotes the maturation of T cells. a Proliferation of T cells detected by EdU staining. b Proportion of cells in proliferation stage determined by CFSE assay. c–e, Proportion of Naive T cells to CD8⁺ (c), NK T cells (d), and CD4⁺ T (e) cells measured by flow cytometry. Cell experiment is repeated 3 times. Data are shown as mean ± standard deviation. **p < 0.01 vs. PBS group

hucMSC-Exo boosts sensitivity of ccRCC cells to T-cell

To explore the immune response of hucMSC-Exo to ccRCC cells, ccRCC cell lines Caki-1 and 786-O were treated with hucMSC-Exo, which were then co-cultured with NK T cells at different ratios. An LDH kit was applied to detect cell lysis in ccRCC cells. Caki-1 and 786-O cells were more sensitive to NK T cells following hucMSC-Exo treatment (Fig. 5a, b). Moreover, the activity of Caspase-3 was strengthened in cells (Fig. 5c, d).

Fig. 5.

ccRCC cells are sensitive to T cells after hucMSC-Exo treatment. a and b Cell lysates in Caki-1 (a) and 786-O (b) detected using an LDH kit. c and d, Caspase-3 activity in Caki-1 (c) and 786-O (d) cells examined by Caspase-3 kit. Cell experiment is repeated 3 times. Data are shown as mean ± standard deviation. **p < 0.01 vs. PBS group

VEGFA is a putative target of miR-182 in ccRCC

To further determine the role of hucMSC-Exo in T cell immunity and the development of ccRCC, miR-182 expression was detected in hucMSC-Exo-treated T cells, Caki-1, and 786-O cells by RT-qPCR. miR-182 was upregulated by hucMSC-Exo treatment (Fig. 6a). Moreover, we found through StarBase website that miR-182 could target VEGFA (Fig. 6b). RT-qPCR and Western blotting were conducted to detect the mRNA and protein expression of VEGFA in hucMSC-Exo-treated T cells, Caki-1, and 786-O cells. It was found that VEGFA was downregulated by hucMSC-Exo treatment (Fig. 6c, d). The binding relationship between miR-182 and the 3'-UTR sequence of VEGFA was verified by dual-luciferase reporter assay, and we found that the luciferase activity was curtailed in 293 T cells transfected with miR-182 mimic and WT-VEGFA (Fig. 6e). Moreover, we found that miR-182 was highly conserved in mice and humans (http://www.mirbase.org/) (Fig. 6f) and that miR-182 is mainly distributed in exosomes (Fig. 6g) in MSCs through the RNA locate website (http://www.rna-society.org/rnalocate/index.html).

Fig. 6.

VEGFA is a putative target of miR-182 in ccRCC. a miR-182 expression in naive T cells, Caki-1, and 786-O cells treated with hucMSC-Exo detected using RT-qPCR. b Prediction result of StarBase website. c and d, VEGFA mRNA (c) and protein (d) expression in naive T cells, Caki-1, and 786-O cells treated with hucMSC-Exo detected using RT-qPCR and Western blotting. e Binding relation between miR-182 and VEGFA verified by dual-luciferase reporter assay. f sequence information of miR-182 in mice and humans predicted using miRBase website. g the distribution of miR-182 in MSCs predicted using RNAlocate website. Cell experiment is repeated 3 times. Data are shown as mean ± standard deviation. **p < 0.01 vs. PBS group

Loss of miR-182 in hucMSC-Exo partially reverses growth and mature of T-cell

As the above-mentioned, hucMSC-Exo promoted the immune function of T cells by delivering miR-182 which negatively regulated VEGFA expression. To further validate our results, hucMSCs were transfected with the miR-182 inhibitor or its control (Mock), followed by extraction of Exo as described in Fig. 1. RT-qPCR displayed that miR-182 expression was reduced in hucMSCs and the derived Exo (Fig. 7a, b). Subsequently, naive T cells were treated with Exo/Inhibitor or control Exo/Mock, and we found that the proliferation of T cells was retarded (Fig. 7c, d), accompanied with decreased number of CD8⁺ cells (Fig. 7e). VEGFA expression was further detected by RT-qPCR and Western blotting, which displayed that VEGFA expression was restored after miR-182 was depleted in Exo (Fig. 7f, g).

Fig. 7.

miR-182 inhibition in hucMSC-Exo partially reverses growth of T cells. a and b miR-182 expression in hucMSCs transfected with miR-182 inhibitor (a) and extracted Exo (b) determined by RT-qPCR. c Proliferation of T cells detected using EdU staining. d Proportion of proliferative cells detected by CFSE assay. e Proportion of naive T cells to CD8⁺ cells examined by flow cytometry. f and g VEGFA mRNA (f) and protein (g) expression in naive T cells detected by RT-qPCR and Western blotting. Cell experiment is repeated 3 times. Data are shown as mean ± standard deviation. **p < 0.01 vs. Mock or Exo/Mock group

Discussion

ccRCC represents one of the most frequent kidney tumors and is often accompanied by immune cell infiltration (Jiao et al. 2020). Moreover, infiltration of T cells is closely related to the clinical outcome of ccRCC, therefore targeting immune microenvironment might be a possible strategy for ccRCC (Senbabaoglu 2016). We paid particular attention to the role of exosomal miRNA contained in hucMSCs, as well as its effects on immune microenvironment in ccRCC, aiming to offer a novel therapeutic or diagnostic biomarker for ccRCC treatment.

Initially, we isolated Exo from hucMSCs. Through observation and identification, these membrane vesicles showed a diameter of 100 ± 55 nm, which conforms to the definition of exosomes as described in a previous literature (Wang, 2019). We further examined the surface markers of MSCs, showing that CD34 and CD45 were negative, while CD44 and CD29 were positive. As demonstrated, MSCs can be identified when the surface markers are CD34-, CD45-, and CD11b/c-negative and CD90-, CD29-, CD44-, CD54-, CD73-, and CD105-positive (Ise et al. 2019). Besides, we detected the protein expression of CD63, CD81, and CD9, which are known as tetraspanins (Salunkhe 2020). CD63, CD81, and CD9 are scaffolding membrane proteins localized at the exosome surface as markers (Andreu and Yanez-Mo 2014). By combining these references, it could be concluded that these membrane vesicles isolated from hucMSCs were Exo.

Subsequently, an orthotopic mouse model was established utilizing the Caki-1 cell line. It was found that treatment of hucMSC-Exo suppressed metastasis of ccRCC in vivo. Similar to our results, MSC-Exo inhibit metastasis in colorectal cancer by targeting integrin alpha6 in vivo (Li et al. 2020). Also, Exo derived from bone marrow MSCs restrain tumor growth in vivo, thus abating non-small cell lung cancer (Liang et al. 2020). Furthermore, we found that the immune response was promoted in modeled mice following MSC-Exo treatment. MSC-Exo are considered as effective immunomodulators and are preferred by its immunogenicity properties (Bulut and I 2020). Specifically, the numbers DCs, NK T cells, and CD8⁺ T cells were observed to be increased after MSC-Exo treatment in the present study. DCs play a key role in antigen presentation to naive T cells, which can be increased when inflammation was alleviated (Salem and Thiemermann 2010). Besides, MSCs can interact with NK cells to contribute to the immunomodulatory effects, since NK cells are key effector molecules of innate immunity (Castro-Manrreza and Montesinos 2015). In addition, even though antigen cross-presentation by macrophages is less investigated than that for DCs, it is increasingly aware that proinflammatory macrophages are capable of cross-presentation (Muntjewerff et al. 2020). Based on these results, we concluded that miR-182 carried by MSC-Exo can promote T cell proliferation and activation. However, the mechanisms behind, whether it was direct activation, or indirect modulation through infiltration of DCs or macrophages, might be the direction of our further study.

As for in vitro assay, naive T cells were treated with hucMSC-Exo, and our experimental results displayed that T cells were activated by treatment of MSC-Exo. Besides, consistent with previous finding that DCs are able to activate naive T cells to promote immune response (Farag et al. 2019), our study also showed that maturation of T cells facilitated immune response. In our study, after ccRCC cells were treated with MSC-Exo, ccRCC cells were more sensitive to NK T cells, accompanied with activated caspase-3. Since NK T cells are mediators of killing tumor cells (Im 2020), sensitivity of ccRCC cells to NK T cells represented that the tumorigenicity was dampened.

The detailed molecular mechanism was further explored. Our study showed that hucMSC-Exo carried miR-182 to exert promoting function in immune response of ccRCC. miR-182 is located in a miR-183/-96/-182 cluster which is the highly conserved cluster to play an important part in cancer development and tumorigenesis (Fasihi-Ramandi et al. 2017). miR-182 is preferentially encapsulated in exosomes, which is detectable and can be transferred among cells (Mihelich et al. 2016). Moreover, loss of miR-182 in MSC-Exo partially weakened the effects on macrophage polarization (Zhao, 2019). These studies supported our statement that miR-182 could be delivered by MSC-Exo. Besides, it has been reported that miR-182 is downregulated in ccRCC samples, which suppresses migration of ccRCC cells (Wang et al. 2016). Our mechanistic investigation showed that VEGFA was the target of miR-182, which is similar to a previous study addressing that VEGFA is a direct target gene of miR-185 in ccRCC (Ma, 2015). Silencing of VEGFA could significantly promote the killing effect of T cells on colon cancer cells (Ragusa 2020). Coincidentally, it was also found that high expression of VEGFA could inhibit CD8⁺ differentiation of T cells, thereby promoting immune escape of hepatoma cells (Deng, 2020).

Conclusion

Our study showed that miR-182 contained in hucMSC-Exo may serve as a therapeutic biomarker for ccRCC by suppressing metastasis both in vitro and in vivo. This study replenished the MSC-Exo-based therapy for ccRCC, which may drive immune response of T cells. Future studies and prospective validations are still warranted to better understand the reported molecular mechanism.

Funding

This work was supported by Scientific Research Projects of Health Industry in Hainan Province (No. 20A200030).

Data availability

All the data generated or analyzed during this study are included in this published article.

Declarations

Conflict of interest

The authors declare that they have no competing interests.