M1-like tumor-associated macrophages activated by exosome-transferred THBS1 promote malignant migration in oral squamous cell carcinoma
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Treatment strategies targeting tumor-associated macrophages (TAMs) have been proposed in cancer areas. The functional alterations of macrophages in the microenvironment during the tumorigenesis of human epithelial cancer remain poorly understood. Here, we explored phenotypic alteration of macrophages during the development of oral squamous cell carcinoma (OSCC).
Conditioned media (CM) and exosome supernatants were harvested from normal oral epithelium, oral leukoplakia cells and OSCC cells. We measured phenotypic alteration of macrophages using flow cytometry, luminex assays, and quantitative real-time PCR assay. Intracellular signaling pathway analysis, mass spectrometry proteomics, western blotting, enzyme-linked immunosorbent assay, immunohistochemical staining, and bioinformatics analysis were performed to uncover the underlying mechanisms.
THP-1-derived and PBMCs derived macrophages exhibited an M1-like phenotype but not M2-like phenotype, when treated with CM from OSCC cells but not with the CM from normal epithelium or leukoplakia cells. Further investigations revealed that macrophages were activated by taking up exosomes released from OSCC cells through p38, Akt, and SAPK/JNK signaling at the early phase. We further provided evidences that THBS1 derived from OSCC exosomes participated in the polarization of macrophages to an M1-like phenotype. Reciprocally, CM from exosomes induced M1-like TAMs and significantly promoted migration of OSCC cells.
We proposed a novel paracrine loop between cancer cells and macrophages based on exosomes from OSCC. Therefore, target management of M1-like TAMs polarized by exosomes shows great potential as a therapeutic target for the control of cancerous migration in OSCC.
KeywordsMacrophage Exosome THBS1 Oral squamous cell carcinoma Migration
American Type Culture Collection
Enzyme-linked Immunosorbent Assay
Human immortal oral epithelial cell line cells
Head and neck squamous cell carcinoma
Oral leukoplakia cell line cells
Magnetic activated cell sorting
Nanoparticle tracking analysis
Oral squamous cell carcinoma
Peripheral blood mononuclear cells
Quantitative real-time PCR
The immune system is an indispensable regulator in the crosstalk between cancer cells and tumor microenvironment [1, 2]. Among immunological effector cells associated with tumor microenvironment, macrophages have been widely recognized to participate in cancer-related inflammation, immune escape, matrix remodeling, and cancer metastases [3, 4, 5]. Over the years, it has been reported that macrophages account for 5–40% of malignant solid tumors [6, 7]. Macrophages display considerable functional plasticity in response to local microenvironment stimuli . Activated macrophages are functionally classified into two populations in vitro, M1 and M2 [9, 10, 11]. Tumor-associated macrophages (TAMs) are termed as a macrophage population recruited and educated by cancer cells, which exert important roles in tumor microenvironment [4, 12, 13]. Due to these findings, strategies targeting macrophages have been proposed in cancer therapy .
Canonically, TAMs are characterized by a molecular signature consistent with that of M2 macrophages [6, 15, 16]. Recently, increasing evidence suggests that TAMs are not composed of a homogeneous population but are a mixed population of macrophages harboring both M1 and M2 phenotypes that have been detected in several malignant solid tumors [17, 18, 19]. In hepatocellular carcinoma, CD68(+) HLA-DR(+) M1-like TAMs were shown to suppress anti-tumor immunity and promote cancer metastasis through expression of B7-H1 . In pancreatic ductal adenocarcinoma, TAMs were reported to exhibit M1 and M2 properties, both of which promoted the epithelial-mesenchymal transition . In addition, a mixed population of macrophages with M1 and M2 phenotypes was detected in vitro in several types of cancer cells [12, 18, 19]. However, no study has elucidated the underlying mechanism of these alterations for M1- or M2-like TAMs, especially with respect to the M1-like polarization. Thus, it is urgent to fully understand the education of TAMs when considering the heterogeneity among various tissue-derived cancers.
Oral squamous cell carcinoma (OSCC), belonging to the head and neck squamous cell carcinoma (HNSCC), remains one of the most lethal cancers worldwide, involving the mucosa epithelial cells from the oral cavity . Development of OSCC is evolutionary and characterized by specific transformation from normal epithelium to epithelial precancerous condition, and to cancerous lesion . In malignant solid tumors, emerging evidence supports the notion that many secretory products from cancer cells participate in the education and polarization of macrophages through paracrine loops [3, 23]. However, the functional alterations of macrophages during the developing of OSCC are poorly understood. Herein, we aimed to investigate the phenotypic alterations of macrophages in the tumor microenvironment during the developing of OSCC.
Cell line cultures and cancer-conditioned media preparation
Human immortal oral epithelial cell line cells (HIOEC) and oral leukoplakia cell line cells (Leuk1) were cultured in keratinocyte serum-free media [24, 25]. OSCC cell lines SCC25 and Cal27 were obtained from American Type Culture Collection and cultured in DMEM supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 μM/ml) at 37 °C in the presence of 5% carbon dioxide . To obtain conditioned media (CM) of various cell lines, cultured cells, up to 80% confluence, were washed and cultured for an additional 24 h with fresh RPMI 1640 media . The cell-free supernatants were collected and centrifuged at 3900 rpm (4 °C) for 15 min. The CM was finally harvested after successively filtration through 0.45 μm and 0.22 μm Filter Units.
Stable THBS1 knockdown cells for SCC25 and Cal25 were generated by transfection with THBS1-specific short hairpin RNA (5’-TTC TCC GAA CGT GTC ACG T -3′ for Scrambled, 5′-GTA GGT TAT GAT GAG TTT AAT -3′ for sh1, 5′-GGA CAA CTG TCC ATT CCA TTA -3′ for sh2) lentivirus, and positively selected with puromycin (10 μg/mL, Calbiochem, USA).
Macrophage differentiation and polarization
THP-1 cells were obtained from ATCC and maintained in RPMI 1640 media supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 μM), and 2-mercaptoethanol (2-ME, 50 μM, Sigma-Aldrich, USA). To obtain resting macrophages (M0), THP-1 cells were differentiated under phorbol-12-myristate-13-acetate (PMA, 100 ng/ml, Sigma-Aldrich, USA) treatment for 24 h and rested for another 24 h [19, 27]. The M0 cells were polarized into M1 using 50 ng/ml IFN-γ (PeproTech, USA) and 1 μg/ml LPS (Sigma-Aldrich, USA) for 24 h, and polarized into M2 using IL-4 and IL13 (20 ng/ml, PeproTech, USA) for 7 days [28, 29].
Peripheral blood mononuclear cells (PBMCs) were obtained from healthy controls using a Ficoll-Hypaque (GE Healthcare, USA) density gradient . Monocytes were isolated from PBMCs with Human CD14 MicroBeads (MiltenyiBiotec, USA) and positively selected by magnetic activated cell sorting (MACS) separation technique according to the manufacturer’s protocol. Purified monocytes were incubated for 7 days in RPMI 1640 medium supplemented with 10% FBS and 100 ng/mL of M-CSF to obtain M0 [31, 32].
Exosome isolation and identification
The collected CM were filtered through 0.10 μm Filter Units, then transferred to Amicon® Ultra-15 centrifugal filter device (100 K, Merck Millipore, USA) and concentrated by centrifugation at 3900 rpm at 4 °C for 15 min. Exosomes were then carefully washed with PBS, and resuspended in PBS or RPMI 1640 fresh medium for validation or subsequent experiments . Freshly isolated exosomes were diluted 1:1000 for nanoparticle tracking analysis (NTA) measurements (NanoSight NS500, NTA 3.2 Dev Build 3.2.16). Size distribution and quantification of exosome samples were analyzed with NanoSight LM10 system (NanoSight, Wiltshire, UK). Size distribution of the detected exosomes was determined and was represented as mean ± SD .
Macrophage morphology and imaging
Morphologies of treated macrophages were observed and photographed under an inverted microscope (ZEISS, German). For fluorescent observation, macrophages were fixed in paraformaldehyde and permeabilized beforehand. The cytoskeleton was labeled with Acti-stain™ 555 Fluorescent Phalloidin (Cytoskeleton, USA) and nuclei with DAPI (YEASEN, China). Fluorescently labelled cells were examined using a ZEISS fluorescent imaging microscope (ZEISS, German).
Fluorescent labelling of exosomes and tracing exosome uptake by macrophages
Isolated exosomes were incubated with Exo-Red labelling reagent (Exo-Glow Exosome Labeling Kits, System Biosciences, USA) for 30 min at 37 °C. Labeled exosomes were washed with PBS using an ultra-filter device, and suspended in fresh RPMI 1640 media. Exosomes labeled with Exo-Red dyes were added to THP-1 derived or PBMCs derived M0 cells and incubated overnight. Subsequently, cells were washed out to remove free exosomes, and then fixed, permeabilized, and stained with Acti-stain™ 488-Phalloidin and DAPI as described above. Exosome uptake by macrophages was examined using Nikon’s A1 confocal laser microscope (Japan).
After treatment with CM or exosomes for 24 h, macrophages were washed, trypsinized, and resuspended in PBS containing 1% FBS. Next, cells were incubated with surface markers (FITC Mouse anti-Human CD14, Alexa Fluor Mouse anti-Human CD163, PE Mouse anti-Human CD86; FITC Mouse IgG2ακ, Alexa Fluor Mouse IgG1κ and PE Mouse IgG1κ used for Isotype Control; all from BD Biosciences, USA). For cell cycle analysis, cells were harvested and fixed in 70% ethanol at 4 °C and stained with propidium iodide (PI, BD Biosciences, USA). After staining, cells were analyzed by flow cytometry (Beckman CytoFLEX FCM, USA).
Enzyme-linked immunosorbent assay (ELISA)
Cytokine analyses were performed with Luminex™xMAP technology using High Sensitivity 9-Plex Human ProcartaPlex™ Panel (ThermoFisher Scientific, USA). Culture supernatants from treated macrophages were harvested, filtered, and stored at − 80 °C prior to analysis according to the protocols provided by the manufacturer. All samples were run in triplicate. THBS1 levels were quantified in cell culture supernatants and exosome suspensions using a Platinum ELISA assay according to the manufacturer’s instructions (eBioscience, USA).
Quantitative real-time PCR (qRT-PCR) assay
Total RNA was extracted and reversely transcribed using the PrimeScript RT reagent Kit (TaKaRa, Japan) according to the protocols recommended by the manufacturer. The cDNA was subjected to qRT-PCR detection using a SYBR Green Premix Kit (TaKaRa, Japan). The relative expression was calculated using the 2-ΔΔCT method for the following genes: TNFα, IL1β, IL6, IL10, CCL18, MRC1, CD80, HLA-DRα, PAI1, and THBS1.
Intracellular signaling pathway analysis
THP-1-derived macrophages were lysed after treatment with exosomes for 24 h. Cell lysates were assayed using the PathScan Immune Cell Signaling Antibody Array Kit according to the manufacturer’s protocol (Cell Signaling Technology, USA). Subsequently, THP-1-derived macrophages were lysed after treatment with exosomes for 2 h and 6 h, and cell lysates were assayed using the PathScan Intracellular Cell Signaling Antibody Array Kit (Cell Signaling Technology, USA) accordingly.
Mass spectrometry (MS)-based label-free quantitative proteomics
Exosomes were harvested and lysed for mass spectrometry-based label-free quantitative proteomics analysis by Beijing BangFei Bioscience Co., Ltd. . Eluted peptides from each sample underwent data acquisition in an Orbitrap Fusion mass spectrometer (Thermo Scientific, USA). MS RAW data files were uploaded into the Mascot 2.1 via Proteome Discoverer, and the referred database was uniprot-human_160701.fasta. Functions of the filtered proteins were analyzed through the UniProtKB database.
For immunoblotting, cellular extracts or exosomes extracts were acquired by using RIPA Lysis Buffer containing proteinase inhibitor cocktail (Innovation, USA). After subjecting the lysates to SDS-PAGE electrophoresis, proteins were transferred onto a polyvinylidene difluoride membrane by electroblotting. The membranes were then blocked and incubated with primary antibodies (anti-Alix antibody, from Thermo Fisher; anti-CD9 and anti-CD63 antibodies from System Biosciences, USA; anti-Rab5 antibody, from BioVision; anti-phospho-Akt (Ser473), anti-Akt (pan), anti-phosphor-SAPK/JNK (Thr183/Tyr185), anti-SAPK/JNK, anti-phosphor-p38 MAPK (Thr180/Tyr182), and anti-p38 MAPK antibodies, from Cell Signaling Technology, USA; anti-β-tubulin antibody, from BOSTER Biological Technology, China). Specific antibody-bound protein bands were detected with ECL Plus reagent (Millipore, USA) under Amersham Imager 600 (GE, USA).
Sections of 5 μm were prepared from paraffin-embedded samples. After deparaffinization, rehydration, and antigen retrieval, endogenous peroxidase activity was quenched. Immunohistochemistry (IHC) staining was performed with primary antibody (mouse anti-human THBS1 antibody and mouse anti-human CD68 antibody from Santa Cruz, USA; rabbit anti-human CD80 antibody from Abcam, USA). For THBS1 staining, the samples were incubated with a biotinylated secondary antibody followed by staining with a DAB kit (GTVision, China). For CD68 and CD80 double-staining, a multiplex mouse-HRP/rabbit-AP IHC kit (Enzo Life Sciences, Switzerland) was used according to the protocol provided by the manufacturer. The proportion of CD68+ CD80+ areas in CD68+ areas were measured in 20 OSCC cases by using the freeware Image J Version 1.51 t, downloaded from the National Institutes of Health (NIH) website (https://imagej.nih.gov/ij/).
Bioinformatics analysis and validation
Bioinformatics analysis was performed based on the TCGA HNSCC cohort using the UCSC Xena Browser [36, 37]. In total, 604 cases were searched for gene expression under RNAseq (polyA+IlluminaHiSeq). Only cases of primary HNSCC were filtered and included for further analysis of expression patterns of THBS1, TNFα, IL6, IL1B, CD68, CD80, and CD86. Expression heat-maps of defined gene sets were generated and clustered online, and detailed data were downloaded for subsequent statistical analysis. A validated cohort was constructed based on 30 primary OSCC cases. The patients involved in this study signed written informed consent, and the study was approved by the Medical Ethics Committee of the Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine.
Cell migration assays
Transwell assays were performed to examine the migration ability of OSCC cells in response to the CM of exosome-treated macrophages. Cancer cells were suspended in 200 μl (5 × 104 cells) of fresh medium and plated into Millicell chambers (8 μm, Millipore Corporation, USA) with 300 μl of culture media containing 10% FBS and 300 μl CM from exosome-treated macrophages in the bottom chamber. After 24 h, cells that migrated through the filter were fixed with paraformaldehyde, stained with 10% crystal violet. The migrated cells were photographed and counted.
All statistical analyses in this study were conducted with SPSS16.0 software (SPSS, Inc., Chicago, IL, USA), and data are presented as the mean ± SD. The significant difference between two groups was determined by Student’s t-test. Pearson Chi-squared tests were performed to assess the statistical significance for correlations between two variables. A p-value < 0.05 was considered statistically significant.
Conditioned media from OSCC cells polarizes macrophages to the M1-like phenotype
Exosomes released from OSCC cells activate macrophages
Exosomes from OSCC trigger M1-like macrophages through activation of p38, Akt, and SAPK/JNK signaling
Exosome-mediated THBS1 transfer polarizes macrophages to an M1-like phenotype
CMs from exosome-activated macrophages promote the migration of OSCC cells
Herein, CM from normal epithelium, precancerous lesions, and oral cancers were harvested to educate macrophages in vitro, respectively. Unexpectedly, an obvious M1-like polarization status was observed only in CM-OSCC treated macrophages, suggesting that characterized activation of TAMs occurred after malignant transformation. Our results were in agreement with studies that have identified the presence and biological roles for a mixed population of macrophages with M1 and M2 phenotypes in cancers [17, 19, 20]. In malignant solid tumors, the education of TAMs is achieved by both direct contact through membrane molecules and paracrine loops between macrophages and cancer cells [3, 15, 46, 47]. We observed an early-phase M1-like phenotypic alteration under the treatment with CM-OSCC. In OSCC samples, M1-like TAMs accounted for about 31% of all macrophages, indicating the simultaneous existence of M2-like TAMs in OSCC. A previous study reported that M1-M2 transition of TAMS occurred during tumor progression, but the underlying signals involved in the M1/M2 switch are poorly understood . Further studies are still required to determine the underlying mechanisms for the co-existence of M1 and M2-like TAMs in the tumor microenvironment of OSCC.
Previous studies have focused on soluble factors, i.e., cytokines, chemokines, and growth factors, produced by cancer cells. Until now, it has been suggested that exosomes derived from cancer cells have a wide range of biological functions [39, 48, 49, 50]. Exosomes are recognized as important signaling mediators, transferring lipids, proteins, mRNAs, microRNAs and lncRNAs to recipient cells, allowing the transfer of cancer-associated signaling molecules to surrounding cells, such as endothelial cells, immune cells, mesenchymal stromal cells, and etc. [39, 50, 51, 52]. Exosome-based communication in the microenvironments of cancer cells are some of the key events in cancer developing. In this study, we demonstrated that exosomes in CM from OSCC activated macrophages to M1-like phenotype.
We next applied intracellular signaling pathway analysis and found that exosomes from OSCC triggered macrophage polarization primarily through activation of p38, Akt, and SAPK/JNK signaling at an early-phase. The combined activation of Akt, p38 and JNK kinases participates in expression of pro-inflammatory mediators in macrophages . Above all, these results provided evidences for the M1-like activation of macrophages in response to exosomes from OSCC. Furthermore, early-phase activation of macrophages indicated a direct and quick response to an effector protein carried by exosomes. THBS1 was identified via mass spectrometry based quantitative proteomics analysis. THBS1 is a multi-functional protein with potent pro-inflammatory and pro-migratory signaling effects on macrophages [43, 45, 54]. In addition, THBS1 has been identified as the most abundant protein secreted by OSCC and was reported to be significantly up-regulated in OSCC compared to normal epithelium . Our study also identified higher mRNA expression and secreted levels of THBS1 in OSCC cells compared to pre-cancerous and normal epithelial cells. Additionally, bioinformatics analysis based on two cohorts and THBS1 knockdown assays indicated that THBS1 expression levels correlated well to the M1 activation in OSCC.
Previous studies have focused more on the roles of M2-like TAMs rather than M1-like TAMs in cancer pathology. We observed a centered distribution of M1-like macrophages in TAMs from OSCC samples. Therefore, we speculated that the biological effects of M1-like TAMs on cancer cells should be achieved through paracrine signaling. We discovered that exosome-activated macrophages could significantly promote the malignant migration of OSCC cells. M1 macrophages are always considered as key effector cells for the elimination of cancer cells either directly or indirectly through attraction and activation of NK and Th1 cells . Hence, the M1-like TAMs activated by exosomes in OSCC may present therapeutic targets to motivate the tumoricidal potentials of these cells. In addition, this study highlighted the need for a more profound understanding of the roles and mechanisms of M1-like TAMs in OSCC.
In conclusion, our findings demonstrated that exosomal transfer of THBS1 from oral cancer could polarize macrophages into M1-like TAMs. Targeted management of M1-like TAMs shows great potentials for the control of tumor cell migration in OSCCs.
This work was supported by the National Program on Key Research Project of China (2016YFC0902700); the National Natural Science Foundation of China (81472515 and 91229103); and the Science and Technology Commission of Shanghai Municipality (18DZ2291500). Wanjun Chen is supported by the Intramural Research Program of NIDCR, NIH.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
WtC and WjC conceived and designed the experiments. MX performed the experiments. MX and JjZ summarized and analyzed the data. MX, JjZ and WtC contributed to writing and revising the paper. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This study was approved by the Medical Ethics Committee of the Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine.
Consent for publication
The authors declare that they have no competing interests.
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