Mesenchymal Stem/Stromal Cells under Stress Increase Osteosarcoma Migration and Apoptosis Resistance via Extracellular Vesicle Mediated Communication

Abstract

Studies have shown that mesenchymal stem/stromal cells (MSCs) from bone marrow are involved in the growth and metastasis of solid tumors but the mechanism remains unclear in osteosarcoma (OS). Previous studies have raised the possibility that OS cells may receive support from associated MSCs in the nutrient deprived core of the tumors through the release of supportive macromolecules and growth factors either in vesicular or non-vesicular forms. In the present study, we used stressed mesenchymal stem cells (SD-MSCs), control MSCs and OS cells to examine the hypothesis that tumor-associated MSCs in nutrient deprived core provide pro-proliferative, anti-apoptotic, and metastatic support to nearby tumor cells. Assays to study of the effects of SD-MSC conditioned media revealed that OS cells maintained proliferation when compared to OS cells grown under serum-starved conditions alone. Furthermore, OS cells in MSCs and SD-MSC conditioned media were significantly resistant to apoptosis and an increased wound healing rate was observed in cells exposed to either conditioned media or EVs from MSCs and SD-MSCs. RT-PCR assays of OS cells incubated with extracellular vesicles (EVs) from SD-MSCs revealed microRNAs that could potentially target metabolism and metastasis associated genes as predicted by in silico algorithms, including monocarboxylate transporters, bone morphogenic receptor type 2, fibroblast growth factor 7, matrix metalloproteinase-1, and focal adhesion kinase-1. Changes in the expression levels of focal adhesion kinase, STK11 were confirmed by quantitative PCR assays. Together, these data indicate a tumor supportive role of MSCs in osteosarcoma growth that is strongly associated with the miRNA content of the EVs released from MSCs under conditions that mimic the nutrient deprived core of solid tumors.

Fig 1. Comparative analysis of OS survival in the presence of mesenchymal stem cells (MSCs) or serum-deprived MSCs (SD-MSCs).

(A) KRSOS cells grown with either complete culture media (CCM) or media without serum (SDM) in the presence of transwell inserts with donor-matched MSCs or SD-MSCs. (B) KHOS grown in either CCM or serum-free media in the presence of inserts containing either MSC or serum-deprived MSC cells. Data presented as means ± SD, Columns, mean of three independent experiments; bars, standard deviation (SD).

Fig 2. Apoptosis analysis of OS cells incubated with conditioned media from serum-deprived MSCs.

(A and B) DNA quantification of KRSOS and KHOS osteosarcoma cells treated with 0.1 μM doxorubicin for 24 h in the presence of complete culture media (control), SD-MSC media (CM) or EVs from SD-MSCs (EVs). (C) OS cells treated with doxorubicin in the presence of either SDM, CCM. Caspase activity measured as an increase in the amount of fluorogenic DEVD₂, a caspase 3 substrate. The data presented as the means ± SD of 3 independent experiments, *P < 0.05. (D) Western blot of cleaved caspase-3 in KRSOS and KHOS treatment with doxorubicin in the presence of SD-MSC EVs.

Fig 3. Effects of EVs and conditioned media from SD-MSCs on OS wound healing.

(A) KHOS cell monolayer was scratched with a p200 micropipette tip, incubated with CCM or SD-MSC conditioned media, and imaged every 6 hrs, after replacing the media with serum free media, SD-MSC conditioned media or EV free media. (B) Scratched KHOS cell monolayer treated with EVs from SD-MSCs for either 4 days or 12 days and imaged every 6 hrs after replacing the media with SDM + EVs. Wound closure area was determined by ImageJ software analysis. Data presented as the means of three independent measurements. * P< 0.05, ** P< 0.01, *** P< 0.005 compared to untreated controls.

Fig 4

(A) Schematic procedure for determining microRNAs and genes associated with changes in OS cells after treating with to EVs from MSCs (4day EVs) and SD-MSCs (12day EVs). (B) Quantitative RT-PCR validation of changes in metastasis and metabolism associated genes after OS incubation with MSCs and SD-MSC-EVs. (C) Quantitative RT-PCR showing changes in expression of shortlisted miRNA after MSC-EVs treatment.

Fig 5. miRNA transferred by EVs regulate PTK2 and expression.

(A) Quantitative RT-PCR showing changes in expression of four miRNA after MSC-EVs treatment. (B) Quantitative RT-PCR expression of downstream target PTK2/FAK. (C) Quantitative RT-PCR showing downregulation of FAK1repressor LKB1.

Fig 6

(A) A simplified interaction schema between EV-derived miRNAs and changes in gene expression in osteosarcoma cells (B) Metabolic pathways involved in osteosarcoma metastasis highlighting the lactate importer MCT1 (SLC16A1) and oxidative phosphorylation pathways (ATP5B). (C) Cell migration pathway in osteosarcoma associated with exposure to EV miRNAs emphasizing the interaction between the focal adhesion (PTK2) and oxidative stress pathways (NFE2L2).