Exosomes Secreted by Epidural Adipose-Derived Stem Cells Promote Malignancy and EMT in B Cell Non-Hodgkin Lymphoma by Increasing S100A4 Expression

Our previous study revealed that epidural adipose-derived stem cells (ADSCs) respond to lung cancer cells and are involved in forming premetastatic niches. The biological functions of epidural ADSCs in lymphoma are still unclear. In this study, we rst conrmed the B cell lymphoma-promoting effects of epidural ADSC exosomes and then compared differentially expressed genes in lymphoma cells before and after exosome treatment. In ADSC exosome-treated lymphoma cells, S100A4 expression was much higher than that in cells cultured alone. S100A4 levels had a direct relationship with EMT in cells and were also associated with the survival of lymphoma patients. Then, we tested the roles of ADSC exosomes and S100A4 in the lymphoma-promoting impacts of ADSCs. Our results showed that ADSC exosomes could foster the proliferation, EMT and invasion of lymphoma cells, as could the increase in S100A4 expression. S100A4 inhibition in two lymphoma cell lines suppressed the lymphoma cell proliferation’s ADSC exosome-mediated promotion, invasion and EMT in vitro. Moreover, S100A4 inhibition attenuated metastasis and tumour growth, and ADSC exosomes promoted tumour progression, as demonstrated in a nude mouse model of lymphoma. On the basis of these data, ADSC exosomes foster B cell lymphoma progression by increasing S100A4 expression in lymphoma cells.


Exosomes Secreted by Epidural Adipose-Derived Stem Cells Promote Malignancy and EMT in B Cell
Non-Hodgkin Lymphoma by Increasing S100A4 Expression which occurs in approximately 5% of patients with cancer 1 . Breast, prostate, and lung cancer each account for 15% to 20% of all cases of MESCC; non-Hodgkin lymphoma, myeloma, and renal cell carcinoma each account for 5% to 10% 2 . MESCC may arise from hematogenous metastasis to vertebral body initial, and then invasion of the epidural space in most tumors, or direct through the intervertebral foramina to spread into the epidural space, such as lymphoma and Pancoast tumors of the lung 3,4 .
Metastases involving the dural sac, root sleeves, and their contents are major causes of neurological dysfunction 5 .
Spinal lymphoma is more frequently caused by secondary dissemination from advanced disease, and which is more often associated with non-Hodgkin lymphoma, especially the diffuse large B-cell subtype 5 . Different from other neoplastic diseases of the spine, the epidural soft tissue mass of spinal lymphoma is more obvious, and reveals circumferential compression of the spinal cord that epidural soft tissue mass of tumor grows around the dura and spreads into the intervertebral foramen [3][4][5] .
Epidural fat is the last barrier in the progression of MESCC, and in which adipose-derived mesenchymal stem cells (ADSCs) has been proved to be activated and exhibit cancer promoting properties in MESCC from lung cancer 6 . The special invasion mode in spinal lymphoma demonstrate that spinal lymphoma may have a closer relationship with epidural fat or ADSCs than other spinal metastases.
Our previous work revealed that epidural adipose-derived stem cells (ADSCs) were involved in the formation of premetastatic niches in the epidural fat and responded to lung cancer cells to promote tumour metastasis and proliferation 6 . However, the role of epidural ADSCs in non-Hodgkin lymphoma spinal metastasis and the exact mechanisms underlying their effects remain poorly understood.
Exosomes are extracellular vesicles that transmit lipids, messenger RNA, DNA, microRNA and proteins and function as crucial players in intercellular communication 7 . Exosomes derived from stromal cells are integral in the formation of the tumour microenvironment and involved in all stages of cancer progression 8 . Understanding the effects of epidural ADSC exosomes in the spinal microenvironment may allow ADSCs to serve as preventative targets for cancer therapy.
The small protein S100A4 is a member of the S100 family of Ca 2+ -binding proteins 9 . Moreover, it takes charge of diverse intracellular pathways and by regulating the intracellular Ca 2+ concentration, it causes different effects 10 . S100A4 is a famous makers who characterize epithelial-mesenchymal transition (EMT) 11 . EMT is a composite biological process. In this process endothelial cells make use of a mesenchymal phenotype, together with the typical cell functions and morphology which contains the acquisition of contractile properties and cellular motility 12 . In some cancer cells, S100A4 takes the responsibility of the ability to form metastases, and promote cell invasiveness and motility 13 . However, at present, we know nothing about S100A4's role in the lymphoma spinal metastasis-promoting in uence of ADSCs.
In our research, we isolated exosomes from epidural ADSCs and assessed the in uence of ADSC exosomes on B cell non-Hodgkin lymphoma cells. Signi cant promotion of proliferation and invasion were observed in two lymphoma cell lines, and transcriptomic analyses were performed to understand genes with differential expression after ADSC exosome treatment. Signi cantly increased S100A4 expression was found in lymphoma cells treated with ADSC exosomes, and gene set enrichment analysis (GSEA) revealed an enrichment in EMT-associated pathways. We predicted that ADSC exosomes could increase S100A4 expression in lymphoma cells, which supported lymphoma cell invasion and proliferation. In the research, we examined the potential roles of S100A4 in the communication between ADSCs and lymphoma cells through exosomes.

Materials And Methods
Cell culture As previously described, from healthy adult donors who underwent posterior lumbar discectomy, human epidural ADSCs were obtained so as to treat or lumbar disc herniation or lumbar fracture 6 . According to the ethical guidelines of the Qingdao University A liated Hospital in Qingdao, China, all the procedures were performed. In this study, the human B cell non-Hodgkin lymphoma cell lines SU-DHL-10 and SU-DHL-4 for Type Culture Collection from the China Centre were applied. Both lymphoma cell lines were cultured in culture medium (DMEM/F12, 10% foetal bovine serum (FBS)) under the condition of a humidi ed 5% CO 2 chamber at 37°C.

Exosome isolation
Exosomes were obtained from epidural ADSC supernatants by differential centrifugation. The medium was discarded when ADSCs reached 70% con uence. Then, in serum-free DMEM/F12, the cells were cultured for another 24 h. The supernatants were collected and then cleared by sequential centrifugation at 3,000 x g for 30 min or 15,000 x g for 30 min. After ltration through 0.22-mm lters (Millipore, Billerica, MA), the supernatants were ultracentrifuged at 120,000 x g for 2 h. With sterile PBS, the exosomes were washed and collected several times. Exosome concentrations were determined with a Pierce BCA protein assay kit (Thermo Fisher Scienti c).

Electron microscopy
Nearly 50 μl of prepared exosomes were adsorbed and put onto formvar carbon-coated 300-mesh copper grids in approximately 10 min. Then, at room temperature for 30 min, the adsorbed exosomes were dried and with 3% phosphotungstic acid, they were negatively dyed. Later, the exosomes were studied through taking advantage of a transmission electron microscope (Olympus Software Imaging Solutions) at 120.0 kV. Moreover, to capture images of the exosomes, a digital camera was used.

Immunohistochemistry
Tumour tissue samples were split into 4-μm sections and embedded in para n. With 100 μl of a solution, antigen retrieval was conducted at 4°C including antibodies against Ki67 and S100A4 (1:200 dilution, Abcam MA, USA) after dehydrating the tissue samples in a graded alcohol series. Moreover, with the sections for 20 min at 37°C, a diluted biotinylated secondary antibody was incubated. To visualize target proteins, haematoxylin was used as a tissue counterstain, and a fresh 3,3-diaminobenzidine (DAB) solution was also used. Two observers assessed the expression of target proteins independently by taking advantage of an Olympus FV500 optical microscope (Olympus, Tokyo, Japan) . In addition, to investigate the intensity and area of staining in ve random regions (200× magni cation) and evaluate the protein expression level, Image-Pro Plus 5.1was applied.

Immuno uorescence
For 24 h, B cell non-Hodgkin lymphoma cells were cultured with or without puri ed ADSC exosomes (10 μg/ml). Then, all the cells were gathered and separated, and later for 60 min, they were xed with 4% paraformaldehyde. The xed cells were cut into 4-μm sections and embedded in para n. Later, in PBS they were washed for three times and blocked with 10% goat serum for 1 h. After that in 0.2% Triton X-100 the sections were washed twice. Next, they were incubated with primary antibodies (anti-S100A4 and anti-vimentin were purchased from Abcam, MA, USA), secondary antibodies (Invitrogen) and DAPI (Guangzhou RiboBio, Guangzhou, China). Using a uorescence microscope, images were captured.

Proliferation and invasion assays
For the following tests, B cell non-Hodgkin lymphoma cells were applied after an incubation with puri ed exosomes (10 μg/ml) for 48 h. In previous studies, cell invasion assays and growth assays were conducted according to the manufacturer's instructions described.
EdU/PI assay According to the manufacturer's instructions (Guangzhou RiboBio, Guangzhou, China), 5-Ethynyl-2'deoxyuridine (EdU)/propidium iodide (PI) assays were conducted by taking advantage of a Cell-Light EdU in vitro klow cytometry kit. In brief, in a 6-well plate cells were cultured overnight, with a 20-min incubation with EdU followed. Then, they were xed with 70% ethanol at -20°C overnight and washed twice with PBS. Later, the cells were stained with a FITC-conjugated secondary antibody for 1 h at room temperature and Through putting the fold change data from differential expression analysis into GSEA software (Broad Institute) by taking advantage of the gene set database c2.cp.v5.1.symbols, GSEA of the RNA-seq data was conducted.
qRT-PCR assay and RNA isolation B cell non-Hodgkin lymphoma cells were gathered. Additionally, total RNA was isolated through taking advantage of TRIzol reagent (Invitrogen, Carlsbad, USA). Later, 2 μg of RNA was applied for rst-strand cDNA synthesis. By using a reverse transcription kit (Toyobo, Osaka, Japan), rst-strand cDNA synthesis was conducted. According to the instructions of manufacturer, quantitative real-time PCR was conducted with gene-speci c TaqMan probes (Applied Biosystems) and master mix (Thermo Fisher Scienti c). Gene expression was normalized to GAPDH expression. The used TaqMan probes included S100A4, Hs00656410_ce; and GAPDH, Hs02786624_g1.
In vivo metastasis assays and tumour growth The As described before, the luminescence of SU-DHL-10 cells was evaluated 14 .

Statistical analysis
In our study, the data are expressed as the mean ± standard deviation. We also calculated the means after conducting no less than three independent experiments. , The signi cance of differences was analysed with a two-tailed Student's t-test or one-way analysis of variance, and P<0.05 was regarded as signi cant.

ADSC exosomes enhance B cell non-Hodgkin lymphomacell invasion and proliferationin vitro
Exosomes were puri ed from ADSC supernatants. To measure the diameters of the puri ed exosomes, an electron microscope (EM) was used. With diameters of approximately 50-150 nm, all kinds of exosomes appeared to be round vesicles (Fig. S1A). Then, western blot analysis was used to detect exosomeassociated proteins, including CD63 and TSG101 (Fig. S1B). Representative images of exosomes derived from epidural ADSCs and internalized by the B cell lymphoma cell line SU-DHL-10 were acquired at 4 h.
To research the in uences of ADSC exosomes on lymphoma cell proliferation, we determined cell viability after adding 10 μg/ml exosomes to two lymphoma cell lines for 48 h. We found that treatment with exosomes derived from epidural ADSCs signi cantly promoted proliferation in the two lymphoma cell lines (Fig. 1A). The same as this result, an EdU/PI assay showed that compared with no treatment, epidural ADSC exosome treatment raised the amount of lymphoma cells that incorporated EdU (Fig. 1B), indicating that exosomes increased DNA synthesis. Later, we examined whether exosomes collected from ADSC supernatants were able to augment lymphoma cell invasion. ADSC exosomes signi cantly increased the invasive capabilities of lymphoma cells, as shown by Transwell Matrigel invasion assays (Fig. 1C). These results revealed that epidural ADSC exosomes had a great in uence on the lymphomapromoting effect of ADSCs.

Global transcriptomic characterization of lymphoma cells regulated by epidural ADSC exosomes
To better understand the changes in lymphoma cells that occurred after ADSC exosome treatment, we compared the transcriptomes of SU-DHL-10 cells cultured for 48 h with or without ADSC exosomes. Through RNA-seq, we found 654 upregulated and 967 downregulated genes that had changes in expression of more than two-fold ( Fig. 2A). Moreover, GSEA was used to compare changed signalling pathways between lymphoma cells treated with ADSC exosomes and control SU-DHL-10 cells. The results revealed that cytokine signalling pathways associated with EMT and in ammatory responses showed the most signi cant upregulation in the lymphoma cells treated with ADSC exosomes (Fig. 2B). Then, we examined the top 10 elevated genes, and S100A4 was found to be related to B cell non-Hodgkin lymphoma patient survival in GEPIA (Fig. 2C). Additionally, we used an available online resource, STRING 15 , to visualize the protein interaction networks of S100A4 and found that S100A4 has close relationships with EMT and the cell cycle (Fig. 2D). In summary, our analyses predicted that ADSC exosomes upregulated proliferation, EMT and invasion in lymphoma cells, while S100A4 expression was upregulated signi cantly in this process.

ADSCexosomes induceS100A4 upregulation in lymphoma cells, which is accompanied by increased EMT and MMP2/9 expression
To have a clear idea of the function of S100A4 in epidural ADSC exosomes' lymphoma-promoting effect, S100A4, E-cadherin and vimentin expression in exosome-treated lymphoma cells was rstly examined through western blotting. Lymphoma cells which were cultured alone played the role of controls, and ADSC exosome-treated lymphoma cells expressed higher levels of S100A4 and vimentin and a lower level of E-cadherin than the control cells (Fig. 2E). Furthermore, S100A4 mRNA expression was detected in ADSC exosome-treated lymphoma cells through qRT-PCR (Fig. 2F), which revealed that ADSC exosome treatment markedly increased S100A4 mRNA expression in lymphoma cells.

ADSC exosomes promote lymphoma cell proliferation, EMT and invasion viaS100A4
To assess whether S100A4 inhibition leads to decreased proliferation, EMT and invasion in lymphoma cells, we transfected lymphoma cells with an S100A4-speci c shRNA. qRT-PCR and western blot results con rmed that shRNA transfection slackened the S100A4's protein and mRNA expression in lymphoma cells (Fig. 3A-B). After con rming the inhibitory e ciency of the S100A4-speci c shRNA in SU-DHL-4 and SU-DHL-10 cells, our team tested the impacts of S100A4 inhibition on the invasion and proliferation of the two lymphoma cell lines. Similar to the increased vimentin expression results, shRNA transfection decreased E-cadherin and matrix metalloproteinase (MMP) 2/9 protein expression in the two lymphoma cell lines (Fig. 3A, C). Then, we observed that S100A4 inhibition attenuated the signi cant increases in lymphoma cell proliferation and invasion in the two lymphoma cell lines caused by ADSC exosomes (Fig.  4A-C). All the results suggest that S100A4 may mediate the lymphoma-promoting effects of epidural ADSC exosomes.
ADSC exosomes trigger lymphoma progression by activating S100A4 in nude mouse models In nude mice with human SU-DHL-10 lymphoma cells, subcutaneous tumour models of lymphoma were constructed to determine how the lymphoma-promoting effects of ADSC exosomes were in uenced by S100A4. With an IVIS imaging system, luciferase activity was examined to monitor tumour volume. Compared with the control group, the shRNA-transfected group exhibited slower signal increases, while the ADSC exosome-injected group displayed faster signal increases (Fig. 5A-B). Moreover, more Ki67 was found in the ADSC exosome-injected group's tumour tissues, which was accompanied by higher S100A4, lower E-cadherin expression, and vimentin and MMP2/9 expression. The results suggested that S100A4 inhibition signi cantly reduced the growth, EMT and invasion of lymphoma (Fig. 5C). Thus, ADSC exosomes promoted lymphoma metastasis and growth. In addition, these effects were counteracted by S100A4 inhibition.

Discussion
In the current study, we used RNA-seq to explore the mechanisms underlying the malignancy-promoting effects of epidural ADSC exosomes on B cell lymphomas. According to the RNA-seq results for lymphoma cells treated with ADSC exosomes, GSEA revealed that the activation of EMT-associated pathways in exosome-treated lymphoma cells was accompanied by increased S100A4 expression.
Enhanced EMT plays important roles in the metastasis of aggressive B cell lymphomas, which may explain the special spinal metastatic a nity. Our data link S100A4 upregulation to the metastasispromoting effect of epidural ADSC exosomes, further implicating S100A4 as an important therapeutic target. Our ndings support the concept that the microenvironment in parts of cancer cells enhanced cancer cell metastasis and maintained cancer cell growth and survival through secretion, particularly exosome secretion.
In speci c organs, a premetastatic niche is a microenvironment prepared for colonizing circulating tumour cells 16 . Our previous studies showed that conditioned medium from epidural ADSCs promoted primary lung cancer forming premetastatic niches in the spine 6 . Increasing evidence suggests that premetastatic niche formation may determine metastatic organotropism in cancer patients 17 . In addition, several studies have revealed that ADSCs from subcutaneous adipose tissue promote the growth of acute lymphoblastic leukaemia cells and T cell lymphoma cells 18,19 . It is still unknown that whether B cell lymphoma which is the most common type non-Hodgkin's lymphoma, has a premetastatic niche in the spine that involves epidural ADSCs. Exosomes are extracellular vesicles with various biological functions.
Moreover, exosomes have great in uences on premetastatic niches, determine organotropic metastasis and cause extracellular matrix remodelling 20,21 . In this research, we isolated exosomes from ADSCs, and lymphoma cell EMT was induced by these exosomes. Although this nding still requires further investigation, RNA-seq results indicated that elevated S100A4 expression in lymphoma cells might be the cause of enhanced EMT, which was accompanied by the downstream activation of MMP2/9.
Metastasis is directly associated with cancer progression and is the main cause of cancer-related death.
Moreover, EMT enables cells to migrate and invade in cancer 22 . EMT results in the upregulation of mesenchymal markers' expression, containing vimentin, and downregulation of key adherence proteins' expression, containing E-cadherin, which enhances the invasive and migratory properties of tumour cells 12,23 . S100 is a family of low-molecular weight proteins consisting of 25 closely related members in humans 24 . It has been revealed that S100 proteins are related to EMT regulation and numerous biological processes that possibly contain cell survival, cell cycle regulation, apoptosis and cell growth and motility 25,26 . S100A4 is a signi cant member in the S100 family, and it plays a role in increasing tumour metastasis and progression 9 . In the early prediction of cancer metastasis and diagnosis of cancer, S100A4 has become a promising candidate biomarker; thus, S100A4 is likely to be a therapeutic target 27 .
Altogether, in this study, we focused on the special metastatic pattern of non-Hodgkin lymphoma in the vertebral body, and epidural ADSCs were found to be involved in the formation of the premetastatic niche in B cell non-Hodgkin lymphoma. Our results support the conclusion that exosomes secreted by ADSCs can activate the expression of S100A4 in lymphoma cells, which fosters lymphoma metastasis and growth. Furthermore, ADSC exosomes have been displayed so as to induce EMT and the expression of growth factors, including MMPs and vimentin, in tumour cells, which could partly explain the increased proliferation and invasion observed. However, our knowledge on S100A4's exact mechanism that in uences the organotropic metastasis of lymphoma is limited. In the tumour microenvironment, our ndings could result in the design of lymphoma therapies targeting protein modi cations to change ADSCs' tumour-promoting effects.

Declarations
Ethics approval and consent to participate The study was approved by the Ethics Committee of the Qingdao University A liated Hospital. All participants provided a written informed consent.

Consent for publication
Not applicable.
Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.  Figure 4