Exosomes released by hepatocarcinoma cells endow adipocytes with tumor-promoting properties
The initiation and progression of hepatocellular carcinoma (HCC) are largely dependent on its local microenvironment. Adipocytes are an important component of hepatic microenvironment in nonalcoholic fatty liver disease (NAFLD), which is a significant risk factor for HCC. Given the global prevalence of NAFLD, a better understanding of the interplay between HCC cells and adipocytes is urgently needed. Exosomes, released by malignant cells, represent a novel way of cell-cell interaction and have been shown to play an important role in cancer cell communication with their microenvironment. Here, we explore the role of HCC-derived exosomes in the cellular and molecular conversion of adipocytes into tumor-promoting cells.
Exosomes were isolated from HCC cell line HepG2 and added to adipocytes. Transcriptomic alterations of exosome-stimulated adipocytes were analyzed using gene expression profiling, and secretion of inflammation-associated cytokines was detected by RT-PCR and ELISA. In vivo mouse xenograft model was used to evaluate the growth-promoting and angiogenesis-enhancing effects of exosome-treated adipocytes. Protein content of tumor exosomes was analyzed by mass spectrometry. Activated phospho-kinases involved in exosome-treated adipocytes were detected by phospho-kinase antibody array and Western blot.
Our results demonstrated that HCC cell HepG2-derived exosomes could be actively internalized by adipocytes and caused significant transcriptomic alterations and in particular induced an inflammatory phenotype in adipocytes. The tumor exosome-treated adipocytes, named exo-adipocytes, promoted tumor growth, enhanced angiogenesis, and recruited more macrophages in mouse xenograft model. In vitro, conditioned medium from exo-adipocytes promoted HepG2 cell migration and increased tube formation of human umbilical vein endothelial cells (HUVECs). Mechanistically, we found HepG2 exosomes activated several phopho-kinases and NF-κB signaling pathway in exo-adipocytes. Additionally, a total of 1428 proteins were identified in HepG2 exosomes by mass spectrometry.
Our results provide new insights into the concept that tumor cell-derived exosomes can educate surrounding adipocytes to create a favorable microenvironment for tumor progression.
KeywordsExosomes Adipocyte HCC MSCs NF-κB
Enzyme-linked immunosorbent assay
Fetal bovine serum
High glucose of Dulbecco’s modified Eagle’s medium
Monocyte chemotactic protein 1
Mesenchymal stem cells
Nuclear factor kappa-light-chain-enhancer of activated B cells
Quantitative real-time PCR
Hepatocellular carcinoma (HCC) now represents the fifth most common cancer worldwide and the third leading cause of cancer-related mortality [1, 2]. Although both diagnostic and therapeutic strategies for HCC have improved over the past decades, the 5-year survival rate only is around 10%, and HCC continues to be a global health issue, especially in Asian countries [3, 4]. Emerging evidence suggested that nonalcoholic fatty liver disease (NAFLD), a common disorder in obese people, is a significant risk factor for HCC [5, 6]. Given the global prevalence of obesity, there is the looming threat of a rapidly rising occurrence of NAFLD-related HCC. Therefore, it is urgent and paramount to understand the mechanisms by which NAFLD contributes to HCC development.
Tumor behavior is determined by not only the malignant potential of tumor cell itself but also the signals from its microenvironment. Thus, it is clear that the crosstalk between tumor cells and their surrounding microenvironment is crucial for HCC development. In NAFLD, the hepatic microenvironment comprises multiple cell lineages including endothelial cells, hepatic satellite cells, immune cells, and adipocytes [7, 8]. Previous studies have focused intensively on the interactions between HCC cells and a wide variety of immune cells such as Kupffer cells, NK cells, T cells, and several antigen-presenting cells. For example, necrotic debris of HCC cells can induce potent IL-1β release by macrophages which subsequently promote HCC metastasis in mouse models . The work done by Wolf et al. showed that hepatic NKT cells promoted NAFLD by secreting LIGHT and activated NF-κB signaling in hepatocytes to enhance malignant transformation . However, the interplay between the HCC cells and adjacent adipocytes remains poorly understood so far.
Currently, how cancer cells communicate with their local and distant microenvironment is undergoing a re-evaluation with the discovery of a novel way of cell-cell interaction exosomes [11, 12]. In addition to diffusible factors, such as growth factors, cytokines, and extracellular bioactive molecules, exosomes are small membrane vesicles that are released by many different cell types, including cancer cells. Increasing evidence suggests that tumor-derived exosomes support tumor development and progression by generating a favorable milieu through immune suppression, angiogenesis enhancement, extracellular matrix remodeling, and stromal cell conversion [13, 14, 15]. Exosome-mediated transfer of proteins, DNA, noncoding RNAs, and mRNAs could induce phenotypic changes in target cells . In melanoma, the tumor-derived exosomes educated bone marrow progenitors toward a pro-metastatic phenotype through the receptor tyrosine kinase MET . In pancreatic cancer, the secreted exosomes induced lipolysis in subcutaneous adipose tissue . In HCC, exosomes derived from metastatic HCC cell lines significantly enhanced the migratory and invasive abilities of nonmotile hepatocytes . However, to our knowledge, no study has reported on the effects of tumor-derived exosomes on adipocytes, which represent an abundant cell type within tumor microenvironment in overweight patients.
In this study, we explored the role of HCC-derived exosomes in the cellular and molecular conversion of adipocytes into tumor-promoting cells. Our results demonstrated that HCC cell line HepG2-derived exosomes could be actively internalized by adipocytes differentiated from mesenchymal stem cells (MSCs) and caused significant transcriptomic alterations, and in particular, induced an inflammatory phenotype in adipocytes. The tumor exosome-treated adipocytes, named exo-adipocytes, promoted tumor growth, enhanced angiogenesis, and recruited more macrophages in mouse xenograft model. In vitro, conditioned medium from exo-adipocytes promoted HepG2 cell migration and increased tube formation of human umbilical vein endothelial cells (HUVECs). Mechanistically, we found HepG2 exosomes activated several kinases and NF-κB signaling pathway in exo-adipocytes. Our findings showed for the first time that HCC-derived exosomes could convert adipocytes into tumor-promoting cells, which may provide new insights into understanding the interactions between tumor cells and surrounding microenvironment.
Human adipose tissues and umbilical cords were obtained according to the procedures approved by the Ethics Committee at the Chinese Academy of Medical Sciences and Peking Union Medical College. MSCs were isolated and culture-expanded from healthy volunteers as previously reported . Passage 3 MSCs were used for following experiments. To obtain adipocytes, MSCs were induced under adipogenic differentiation medium, which is high glucose of Dulbecco’s modified Eagle’s medium (H-DMEM) supplemented with 10% FBS, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 5 μg/mL 0.1 mM l-ascorbic acid. Adipocytes were characterized by Oil Red O staining according to the manufacture’s (Beyotime Biotechnology) instructions. HUVECs were isolated and cultured as routinely described . HCC cell line HepG2 was purchased from cell bank at the Chinese Academy of Medical Sciences and cultured in DF12 containing 10% FBS, penicillin (100 U/mL), and streptomycin (100lg/mL) at 37 °C with 5% CO2.
Exosome extraction was performed as previously described . Briefly, HepG2 cells were cultured in serum-free DF12 medium for 24 h. Then, the culture medium was collected and centrifuged at 800g for 5 min and additional 2000g for 10 min to remove lifted cells. The supernatant was subjected to filtration on a 0.1-mm-pore polyethersulfone membrane filter (Corning) to remove cell debris and large vesicles, followed by concentration by a 100,000-Mw cutoff membrane (CentriPlus-70, Millipore). The volume of supernatant was reduced from approximately 250–500 mL to less than 5 mL. The supernatant was then ultracentrifuged at 100,000g for 1 h at 4 °C using 70Ti Rotor (Beckman Coulter). The resulting pellets were resuspended in 6 mL PBS and ultracentrifuged at 100,000g for 1 h at 4 °C using 100Ti Rotor (Beckman Coulter). In the experiments involving HepG2 exosomes, we use PBS as a negative control.
Transmission electron microscopy
Purified exosomes were fixed with 1% glutaraldehyde in PBS (pH 7.4). After rinsing, a 20-uL drop of the suspension was loaded onto a formvar/carbon-coated grid, negatively stained with 3% (w/v) aqueous phosphotungstic acid for 1 min, and observed by transmission electron microscope.
Quantitative real-time polymerase chain reaction
Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer’s instruction, and cDNA was prepared. Real-time PCR amplification was performed in triplicates according to the procedures reported previously . Relative expression of mRNA was evaluated by the 2-ΔΔCt method and normalized to the expression of GAPDH.
Proteins were extracted with radioimmunoprecipitation (RIPA) lysis buffer with PMSF, quantified by BCA Protein Assay Kit (Beyotime). Western blot was performed in triplicates according to the procedures reported previously . GAPDH was used as an internal control. We used the following antibodies: p-AKT (1:2000; rabbit IgG, CST, 4060T), p-ERK1/2 (1:5000; rabbit IgG, Abcam, ab76299), p-STAT5α (1:1000; rabbit IgG, Abcam, ab30648), p-GSK (1:5000; rabbit IgG, Abcam, ab75814), AKT (1:1000; mouse IgG, proteintech, 60203-2-Ig), ERK1/2 (1:1000; rabbit IgG, proteintech, 16443-1-AP), STAT5α (1:1000; rabbit IgG, Abcam, ab32043), GSK3β (1:1000, rabbit IgG, proteintech, 22104-1-AP), CD63 (1:500; rabbit IgG, proteintech, 25682-1-AP), TSG101 (1:500; rabbit IgG, Abcam, ab83), HSP70 (1:100; rabbit IgG, SBI, EXOAB-KIT-1), calnexin (1:2000; rabbit IgG, CST, 2433s), GAPDH (1:10000; rabbit IgG, proteintech, 10494-1-AP) (1:10000; mouse IgG, proteintech, 60004-1-Ig), HRP-conjugated anti-rabbit-IgG (NeoBioscience), HRP-conjugated anti-goat-IgG (NeoBioscience), and HRP-conjugated anti-mouse-IgG (NeoBioscience).
Culture medium was collected 24 h after the treatment with or without exosomes. The concentrations of all cell cytokines in supernatants were measured using ELISA kits (BD Technologies).
The cultured cells were fixed at 4 °C in ice-cold methanol for 10 min, washed three times in phosphate-buffered saline (PBS), and then permeabilized in 0.1% Triton X-100/PBS for 10 min at room temperature. Nonspecific binding was blocked with 0.5% Tween-20/PBS containing 1% bovine serum albumin (BSA) for 30 min. The primary antibodies were incubated at 4 °C overnight. The secondary antibodies were incubated for 1 h at room temperature. The incubated cells were washed in PBS, and Hoechst 33342 (Sigma-Aldrich) was used to visualize nuclei. p65 antibody (10745-1-AP) was purchased from Proteintech.
Mouse xenograft experiments
Nude mice were purchased from the Laboratory Animal Center of the Chinese Academy of Medical Sciences (Beijing, China). Animal use and experimental procedures were approved by the Animal Care and Use Committee of the Chinese Academy of Medical Sciences. Mice were randomly divided into three groups, one group received a subcutaneous injection of 2 × 105 exo-adipocytes and 2 × 106 HepG2 cells, one group received 2 × 105 adipocytes and 2 × 106 HepG2 cells, and the last one received 2 × 106 HepG2 cells. The tumor weight was measured after 4 weeks. The tumor tissues were fixed with 10% PFA. Each group was treated with HE, IL-6, Ki67, CD31, and F4/80 staining.
Tube formation assay in Matrigel
In vitro capillary network formation was determined by performing a tube formation assay in Matrigel (BD Biosciences). 1 × 104 HUVECs were plated on a growth factor-reduced Matrigel (BD)-coated 96-well plate in triplicates with 100 uL serum-free medium (control), exo-adipocyte-conditioned medium, or adipocyte-conditioned medium. After 8 h of incubation, tube formation was examined by microscopy (Olympus, Tokyo, Japan), and the branch density and tube length were quantified by randomly selecting three fields per well.
Data are presented as mean ± SD. Comparisons between groups were analyzed via Student’s t test. Differences were considered statistically significant at *P < 0.05, **P < 0.01, and ***P < 0.001.
HepG2 exosomes are actively internalized by adipocytes
HepG2 exosomes induce an inflammatory phenotype in adipocytes
exo-adipocytes promote tumor growth in vivo
exo-adipocyte-conditioned medium is chemotaxic and promotes HepG2 migration
exo-adipocyte-conditioned medium enhanced tube formation of HUVECs
HepG2 exosomes activate various kinases and NF-κB signaling pathway in adipocytes
Moreover, when NFκB inhibitor PDTC was added, the enhanced expression of IL-6, IL-8, and MCP-1 induced by HepG2 exosomes in adipocytes was reduced (Fig. 6d). Taken together, these results demonstrated that HepG2 exosomes are able to activate various kinases and NF-κB signaling pathway in adipocytes.
Proteomic analysis of HepG2 exosomes
Tumor initiation and progression rely on the dynamic interactions between malignant tumor cells and multiple normal cell types within its microenvironment such as fibroblasts, various immune cells, endothelial cells, and adipocytes. Of these cell types, adipocytes are probably the least well studied, although they represent a significant part of the tissue surrounding a tumor . Emerging evidence suggests that adipocytes should not be considered simply as an energy-storage depot. Instead, adipose tissue can play a central role in both endocrine and metabolic processes by producing a battery of factors including growth factors and adipokines . Thus, understanding how obesity and adipose tissue-related factors are connected to tumor development is paramount. In 2010, Dirat’s group coined the term “cancer-associated adipocytes (CAA)” to demonstrate the bidirectional crosstalk between breast cancer cells and tumor-surrounding adipocytes and that CAA are a key player in tumor progression . Subsequently, several studies also showed the existence of the putative CAA in the vicinity of cancer cells [31, 32]. Here, we chose adipocytes as a cellular model which are differentiated by culturing human MSCs under adipogenic conditions and are fully characterized by morphology, staining, and marker gene expression. We demonstrated that HCC cell line HepG2-derived exosomes could be actively incorporated by adipocytes and convert adipocytes into tumor-promoting cells (exo-adipocytes). In the mouse xenograft model, we found that exo-adipocytes promoted tumor growth and enhanced angiogenesis. Fujisaki et al. reported that in the presence of breast cancer cell lines MCF7 and MDA-MB-231, adipocytes reverted to an immature and proliferative phenotype of CAA that could promote cancer cell migration . Lee et al. found that when indirectly co-cultured with breast cancer cells, adipocytes would be transited into CAA, resulting in proliferation-enhancing effect in ER-positive breast cancer cells such as MCF7 and ZR-75-1 but not in ER-negative cells . Thus, we postulate that the exo-adipocytes in our study are a kind of CAA as they exhibit tumor-promoting capacity and higher expression of pro-inflammatory factors such as IL-6, IL-8, and MCP-1 whose higher expression in CAA has been reported [33, 34]. IL-6 plays diverse regulatory roles in cancer pathogenesis including remodeling the tumor microenvironment, activation of EMT process, and promoting drug resistance [35, 36]. IL-8 is known to be a stimulatory factor for tumor angiogenesis , and MCP-1 promotes the recruitment of macrophages into tumors . These cytokines may be at least partially responsible for the tumor-promoting and angiogenesis-enhancing effects of exo-adipocytes.
The regulatory mechanisms of the CAA transition are not clearly understood. In this study, we explored the role of HCC-derived exosomes on the cellular and molecular changes of exo-adipocytes, which further confirmed that tumor cells could use exosomes as a novel way of cell-cell communication. Our study is consistent with previous findings that tumor exosomes from various cancer types can “educate” neighboring cells such as MSCs , endothelial cells , monocytes , and dendritic cells . For example, exosomes from ovarian and breast cancer cells can convert adipose-derived MSCs (AMSC) into myofibroblast-like cells [43, 44] while prostate cancer cell-derived exosomes trigger bone marrow MSCs (BMSC) to differentiate into pro-angiogenic and pro-invasive myofibroblasts . Our results support the postulation that the elements of adipose tissue can also be modified by cancer cells and participate in a highly complex vicious cycle to form a tumor-favorable microenvironment.
How exosomes cause significant cellular and molecular changes in target cells remains an area of intensive research. Using microarray, Fang et al. found that HCC exosomes could deliver miR-1247-3p into fibroblasts and converted them into cancer-associated fibroblast to foster lung metastasis . Using proteomic analysis, He et al. revealed that exosomes derived from metastatic HCC cell lines carried a large number of protumorigenic proteins, such as MET protooncogene, S100 family members, and the caveolins . Here, we also detected common exosomal markers, structure or surface proteins, exosomal formation or secretion-related proteins, and oncogenic proteins in HepG2 exosomes. Upon taking up HCC exosomes, 725 upregulated and 648 downregulated genes were identified, and several cell signaling pathways were activated. In our previous study , we found that lung tumor exosomes could activate NFκB signaling pathway through HSP70/TLR2. Here, we also detected the activation of the NFκB signaling pathway. However, several questions remain for future investigation, including which receptors on the surface of adipocytes participated in HCC exosome internalization and how the internalized exosome cargos activated the downstream signaling pathways.
This study was supported by CAMS Innovation Fund for Medical Sciences (2017-I2M-3-007), The National Key Research and Development Program of China (2016YFA0101000, 2016YFA0101003), Beijing Key Laboratory of New Drug Development and Clinical Trial of Stem Cell Therapy (BZ0381), and National Natural Science Foundation of China (81473450).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
SHW designed and analyzed the experiments and wrote the manuscript. MQX performed and analyzed the experiments and prepared the figures. XXL, XDS, and XX performed the experiments. AK and RCHZ designed the experiment. All authors have read and approved the final manuscript.
All experiments were performed under the approval of the Ethics Committee at the Chinese Academy of Medical Sciences and Peking Union Medical College.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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