Investigating the Effects of Chordoma Cell-Derived Exosomes on the Tumorigenicity of Nucleus Pulposus Cells

Abstract

Objective  Interaction of tumor cells with the surrounding environment is essential for tumor growth and progression that eventually leads to metastasis. Growing evidence shows that extracellular vesicles also known as exosomes play a crucial role in signaling between the tumor and its microenvironment. Tumor-derived exosomes have generally protumorigenic effects such as metastasis, hypoxia, angiogenesis, and epithelial-mesenchymal transition.

Methods  In this study, exosomes were isolated from a chordoma cell line, MUG-Chor1, and characterized subsequently. The number of exosomes was determined and introduced into the healthy nucleus pulposus (NP) cells for 140 days. The protumorigenic effects of a chordoma cell line-derived exosomes that initiate the tumorigenesis on NP cells were investigated. The impact of tumor-derived exosomes on various cellular events including cell cycle, migration, proliferation, apoptosis, and viability has been studied by treating NP cells with chordoma cell-line-derived exosomes cells.

Results  Upon treatment with exosomes, the NP cells not only gained a chordoma-like morphology but also molecular characteristics such as alterations in the levels of certain gene expressions. The migratory and angiogenic capabilities of NP cells increased after treatment with chordoma-derived exosomes.

Conclusion  Based on our findings, we can conclude that exosomes carry information from tumor cells and may exert tumorigenic effects on nontumorous cells.

Introduction

Malignancies might occur almost in every tissue in human body and start with a transformation of a single cell that undergoes several mutations. Although each cancer is unique with specific characteristics, the basic processes that cause the diversity of tumors are quite similar. ^{1 2} As proposed by Weinberg and Hanahan, ³ one of the hallmarks of cancer is the ability to migrate from the primary site, invade nearby tissues, and eventually reconstruct the tumors at the new residing sites. The interaction of tumor cells with their surrounding is also essential to understand the mechanism that drives the tumor initiation and progression. ⁴

Exosome-mediated signaling and molecule transport is a key feature for tumor progression and metastasis of cancer. ⁵ Exosomes are small, single membrane-enclosed secreted organelles ranging from 30 to 150 nm in size and are found in most of the body fluids such as breast milk, tears, urine, saliva, and plasma. ⁶ Exosomes carry proteins, RNAs, DNAs, enzymes, complex glycans, miRNAs, adhesion molecules, and scaffolds. ⁵ Exosomes have a wide range of biological functions along with being important components and remodelers of the extracellular matrix. ⁷ They are secreted from all types of cells including cancerous cells with higher levels than normal counterparts mediating the transmission of signals and molecules from cell to cell. ⁸ Tumor-derived exosomes have generally protumorigenic effects ⁹ triggered by Wnt and tumor growth factor-β signaling that favor the formation and further trigger angiogenesis, hypoxia, and metastasis for the progression of the tumor. ¹⁰

Chordoma, a rare type of malignant tumor affecting 1 in 10 ^{^} 6 people yearly, can occur anywhere along the spine, locating from the base of the skull to the tailbone. ¹¹ Albeit growing slowly, chordoma is considered as a malignant tumor owing to high resistance to conventional chemotherapies. ¹² Chordoma is thought to arise from the remnants of the notochord, what is known as nucleus pulposus (NP) in adults, that becomes malignant later in life. ¹³ It is very crucial to understand the mechanism that derives the prognosis in chordoma and tumor-derived exosomes may have a role in this process. Therefore, in our study, the effects of exosomes on triggering events in chordoma including metastasis, proliferation, viability, apoptosis, and changing the cell cycle profile were studied at the molecular level.

Materials and Methods

Cell Culture

MUG-Chor1 cell line was kindly obtained from Chordoma Foundation (North Carolina, United States) and grown in culture media containing Iscove's Modified Dulbecco's Medium (IMDM, # 31980030, Invitrogen, Gibco, UK) and RPMI 1640 in 4:1 ratio, respectively, supplemented with %1 Penicillin/Streptomycin/Amphotericin (Invitrogen, Gibco, UK) and %10 fetal bovine serum (FBS, #10500–064, Invitrogen, Gibco, UK). For exosome harvesting, the media in which the chordoma cells were grown were changed with serum-free IMDM/RPMI mix and the cells were incubated another day. Culturing media were harvested for exosome isolation later. Human nucleus pulposus cells (HNPC) (4800, ScienCell, United States) were cultured as recommended by the manufacturer. HNPCs will be referred as NP cells throughout the paper.

The Isolation of the Exosomes and Characterization Assays

MUG-Chor1 cells were incubated in serum-free chordoma culture media for 2 days. Upon incubation, conditioned serum-free media were harvested and exosomes were isolated by using polyethylene glycol/dexytran (PEG/DEX) (2X) solution (prepared as 30 g of DEX and 70 g of PEG powder and dissolved in 1 L distilled water) described by a previous method. ¹⁴ The exosomes were cleared off DEX by mixing with ice-cold methanol at a 1:1 volume ratio and centrifugated at 22.000 g for 20 minutes. Supernatants were collected and methanol was removed from the mixture by a using a concentrator. Exosomes were filtered and quantified by nanoparticle tracking assay (NTA). Video shootings were captured for 60-second intervals at camera level 16 and analyzed by NTA Software version 3.4.

Cell Viability Assay for Exosome Dosage Determination

NP cells were seeded onto a 48-well plate as 10,000 cells/well. Exosome particles were introduced to the cells at various concentrations as 0, 10 ² , 10 ³ ,10 ⁴ , and 5 × 10 ⁴ with three replicates. The viability of the cells incubated with exosomes was measured by a colorimetric 3-(4,5-di-methyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium assay (#G3582, CellTiter 96 Aqueous One Solution, Promega, Southampton, United States). Absorbance values were determined by a Plate Reader (ELx800, Biotek Instruments, United States) at 490 nm and according to IC50 value, 10.000 exosome particles per cell was found to be safe to introduce.

Treating the NP Cells with Exosome Particles

NP cells were seeded in T25 flasks as 100,000 cells/flask, cultured with DMEM with chordoma media-derived exosomes (10.000 particles per cell) with renewing the media twice a week for more than 140 days.

Gene Expression Assay

Cultured cells were collected for RNA isolation by silica column purification with NucleoSpin RNA kit (#740955.50, Macherey-Nagel, Germany) according to the manufacturer's protocol and cDNAs were synthesized by QuantiTect Reverse Transcription Kit (#205313, Qiagen, Netherland) as described in the manufacturer's instructions. The primers targeting chordoma and cancer-related genes including MMP2 (Hs01548727_m1), MYC (Hs00153408_m1), MET (Hs01565584_m1), BCL-2 (Hs04986394_s1), NANOG (Hs02387400_g1), CD24 (Hs02379687_s1), OCT4 (At02611156_m1), SMAD2 (Hs00998187_m1), KLF4 (Hs00358836_m1), BAX (Hs00180269_m1), BAK (Hs00832876_g1), MUC1 (Hs00159357_m1), ABCG2 (Hs01053790_m1), and GAPDH (Hs02786624_g1) were purchased from ThermoScientific. Expression values were normalized to GAPDH . VEGF primers ¹⁵ were as follows: F:5′-TTGCCTTGCTGCTCTACCTC-3′ R:5′-AGCTGCGCTGATAGACATCC-3′were. As for housekeeping gene 18S ¹⁶ F:5′-GTAACCCGTTGAACCCCATT-3′ and R:5′-CCATCCAATCGGTAGTAGCG-3′was used.

Cell Cycle Assay

The effect of exosome treatment on cell cycle was determined by the protocol described previously. ¹⁷ Shortly, NP cells—treated with exosomes—were harvested and suspended with ice-cold 70% ethanol for at least 2 hours prior to the analysis. The pellets were suspended with a solution containing 0.1% (v/v) Nonidet and Rnase A and incubated at 37°C for 30 minutes. Propidium iodide was added to the samples and measured with a Flow Cytometry (BD FACS Calibur, United States).

Cell Proliferation Assay

The changes in the number of proliferation cells treated with MUG-Chor1-derived exosomes and their negative counterparts were investigated. Shortly, cells were seeded as three replicates on 48-well plates with varying numbers between 5 × 10^3 and 40 ×10^3 on each well and following day a standard curve was created by measuring the absorbance of each well with the viability assay (as used in Exosome Dosage Determination method ) . In a separate setting, 10.000 cells/well were seeded on three 48-well plates and labeled for day 1, 4, and 7 and absorbance of each plate on its day was read. Viability readings were plotted and proliferation rate was determined.

Cell Migration (Wound Healing) Assay

Cells treated with MUG-Chor1-derived exosomes and their negative counterparts were seeded on 24-well plate with a 95% confluence on each plate. Cells were incubated for 24 hours and following day a linear scratch was made with a pipette tip. Floating cells were discarded and replaced with 2% FBS containing media. Images of the gaps were taken with an invert microscope at day 0, 24, 48, and 72 hours.

Statistical Analysis

Experiments were performed in triplicates to enable a proper statistical analysis. GraphPad Prism 7.0 program was used for multiple comparisons among cells treated with MUG-Chor1-derived exosomes and their negative counterparts. One-way analysis of variance with Tukey's post-hoc test was applied to data to determine the significance of differences.

Results

Exosome Particles Were Detected and Images of NP Cells Were Analyzed upon Treatment with the Tumor-Derived Exosomes

MUG-Chor1-derived exosome concentration was counted as 4.43 × 10 ¹⁰ particles per milliliter and average size of particles were found to be 200 nm ( Fig. 1A ). Untreated NP cells expanded homogenously in two-dimensional cell culture environment with a fibroblast-like appearance whereas the Mug-CHOR1 cells form clusters in which each cell looks epithelial, small, and circular ( Fig. 1B ). Upon treatment with tumor-derived exosomes, the morphology the NP cells was altered to become more small and circular as opposed to the untreated NP cells ( Fig. 1C ). At the end of 140 days of exosomes treatment, NP cells seemed to gather as clusters rather than a monolayer expansion in both low and high confluent cases ( Fig. 1D ).

Fig. 1.

Characterization of MUG-Chor1-derived exosomes: Concentration and size graph of ( A ) images of nucleus pulposus (NP) cells before the treatment with exosomes ( B ), MUG-Chor1cells ( C ), and NP cells treated with MUGChor1-derived exosomes ( D ). Images were taken under 5X objective under light microscope.

The Alterations in the Gene Expressions

The changes in the gene expression profile of exosome-treated NP cells were analyzed by quantitative polymerase chain reaction. According to the data, the expression of MUC1 gene was increased approximately 1.33, while expression of C-MYC and ABCG2 genes decreased approximately 0.91 and 0.28, respectively ( Fig. 2A ) upon exosome treatment. The expression of stem cell markers including OCT3/4 , NANOG , CD44, and KLF4 was increased by 4.18-, 2.03-, 2.25-, 1.83-fold, respectively ( Fig. 2B ). While the expression of BAK increased 1.76-fold, the expression of BCL2 remained unchanged ( Fig. 1C ).

Fig. 2.

Gene expression levels of drug resistance markers ( A ), stem cell markers ( B ), and apoptotic markers ( C ). C-MYC, cellular myelocytomatosis; NP, nucleus pulposus.

Tumor-Derived Cells Affect the Rate of Proliferation as well as the Cell Cycle Profile of NP Cells

The number of untreated NP cells ( Fig. 3A ) in the untreated NP cells at day 1, day 4, and day 7 were 11,600, 21,090 and 19,972, respectively, while in the exosome-treated control, the numbers were 12,526, 22,574, and 20,080, respectively.

Fig. 3.

Changes in the rates of proliferation ( A ) and cell cycle stages ( B ) of nucleus pulposus (NP) cells treated with and without exosomes.

According to the cell cycle data, there is only slight difference in the numbers of NP cells that were exposed to the tumor-derived exosomes. Total cell percentage of MUG-Chor1 exosome-treated NP cells ( Fig. 3B ) in G0/G1, S, and G2/M phases are found to be 85.82, 3.79, and 10.72, respectively, whereas the number of untreated NP cells in G0/G1, S and G2/M phases are 90.23, 4.99, and 4.97, respectively. Based on this data, the mitotic index of exosome-treated cells increased distinctly when compared with the untreated cells.

Chordoma Exosomes Exert Migratory Effects on NP Cells

To understand the effect of MUG-Chor1-derived exosomes on the ability of migration of NP cells, a wound healing assay was performed as described previously. ¹⁸ Results have shown that exosome treatment increased the migration capacity of cells. In untreated NP cells, 50% of the gap was closed in 48 hours, whereas in MUG-Chor1-derived exosomes-treated cells, 70% of gap was closed as shown in Fig. 4A . With the effect of MUG-Chor1 cell-derived exosomes, expression of C-MET , VEGF, and MMP2 genes was upregulated by 1.64-, 3.05-, and 1.47-fold, respectively. However, the expression of CD24 was decreased to 0.76-fold ( Fig. 4B )

Fig. 4.

The effects of MUG-Chor1-derived exosomes on migration. The changes in the gap distance ( A ) and relative expressions of genes associated with migration and invasion ( B ). C-MET, cellular mesenchymal epithelial transition factor; C-MYC, cellular myelocytomatosis; MMP2, matrix metalloprotease 2; NP, nucleus pulposus; VEGF, vascular endothelial growth factor.

Discussion

Exosomes are small, single membrane enclosed secreted organelles, and used for communication of cells in organisms' body. ¹⁹ The content of exosomes is RNA, DNA, miRNA, proteins, lipids, enzymes, and this content carries information about host cell and a part of the host cell. ²⁰ Tumor cells released more exosomes than healthy cells and use these exosomes for the growth and progression of tumor. Tumor cells form or find a premetastatic niche via exosomes, which is a well fitted place to form new tumor and metastasize to this site of the body. ²¹ Previous studies support the role of exosomes in various cellular mechanisms including metastasis, ²² invasion, and immune escape. ²³ To our knowledge, this is the first study that demonstrates that exosomes released from chordoma cells might initiate tumorigenicity on nontumorigenic NP cells.

Upon treatment with tumor-derived exosomes, normally fibroblast-like looking NP cells ²⁴ acquired more like clustering epithelial appearance as MUG-Chor1 cells. ²⁵ This was not surprising to see homogenously growing NP cells losing their long and linear cell structure and turning into smaller and round-shaped cells akin MUG-Chor1 cells that normally form colonies expand on flask surface heterogeneously. Study done by Hood et al ²⁶ shows that the melanoma exosomes affect the endothelial tubule morphology which in turn supports our findings that influence of chordoma-derived exosomes alters the shape of NP cells.

Migratory ability of tumor cells might be mediated through exosomes as shown in many studies. Wu et al showed that the inhibition of exosome secretion may prevent the metastasis of colorectal cancer cells. ²¹ Considering the fact that mesenchymal stem cells play a role in the migration process ²⁷ and chordoma has a mesenchymal ²⁸ and invasive origin, ²⁹ we may conclude that chordoma-derived exosomes may have enhanced the migratory capacity of NP cells. MUG-Chor1cell-derived exosomes increased the migration capacity of NP cells and ultimately exerted tumorigenic effects on NP cells. Based on our data, there is an increase in the expression levels of genes associated with metastasis that confirm the exosome-mediated migration of NP cells demonstrated with the enhanced expression of vascular endothelial growth factor (VEGF), matrix metalloprotease 2 (MMP2), ³⁰ and cellular mesenchymal epithelial transition factor (C-MET). ³¹ In the absence of sufficient O2, cancer cells form the blood vessels through a process called angiogenesis, a hallmark of cancer. ³ Previous study demonstrated that tumor-derived exosomes play a role in stimulating angiogenesis. ³² The present study shows the angiogenesis might be accomplished with enforced expression of C-MET, VEGF, and MMP2 genes. Due to a previous study, NP cells along with chordoma cells express CD24 gene. ³³ Therefore, an exposure to chordoma-derived exosome did not cause a significant increase in NP cells.

Exposure of NP cells to chordoma-derived exosomes caused the acquiring not only stem cell but also chordoma-related markers including OCT3/4, NANOG, CD44, and KLF4 that are coherent with our as well as others' data. ^{34 35} Evading apoptosis is one of the six hallmarks of cancer and by decreased expression of BAK and increased expression of BCL-2 may explain the effects of cancer-derived exosomes rendering NP cells to gain evasion in apoptosis. ³⁶

One of the significant diagnostics markers of chordoma is the elevated levels in the expression of MUCIN 1 (MUC1) gene. MUC1 and cellular myelocytomatosis (C-MYC) are multifaceted oncogenes that become mutated and consequently overexpressed that lead to ³⁷ mutation in oncogenes and their overexpression leads to formation of tumor. ³⁸ Though our previous data demonstrated the presence of C-MYC in the several chordoma tissue samples and hence considered to be a stem cell marker, ³⁴ after the exposure of tumor-derived exosomes, the C-MYC levels did not rise in NP cells. Considering the slow growth nature of chordoma cells, it is expected to see that the chordoma cell-derived exosomes would not have any increasing effect on the proliferation rate of NP cells. This was also demonstrated in the cell cycle in which both cells (exosome treated and untreated) presented a similar count in S phase. Both of these data also confirm with others ³⁹ that C-MYC expression affects the cell cycle progression. ABCG2 gene is responsible for drug resistance against chemotherapy treatments and known to be expressed in chordoma cells although at low levels. ⁴⁰ It was not surprising to see that upon treatment with tumor-derived exosomes, the gene expression levels of ABCG2 did not elevate. However, MUC1—one of the main diagnostic markers of chordoma ⁴¹ —was found to be amplified at the gene expression level suggesting that exosomes might have a role in mediating this increase.

Conclusion

Exosomes are known to be the important mediators of cancer. The transfer of exosomes from tumor cells may enable the nontumorigenic cells gain tumor-like properties. Our molecular as well as morphological data show that chordoma cell-derived exosomes exert tumorigenic features on primary noncancerous NP cells and become an evidence that exosomes convey substantial information among cells.

Funding Statement

Funding This study was supported by Yeditepe University research funding.