https://orcid.org/0000-0003-4280-3031
Figures & data
Figure 1. Osteosarcoma cell exosomes exhibit exosome morphology and the exosome markers CD9 and CD81. Exosomes were extracted from K7M3, K7, DLM8, and Dunn osteosarcoma cells via ultracentrifugation. (a) Exosome size and concentration were assessed via Nanosight analysis. (b) Transmission electron microscopy and immunogold staining was used to analyze CD9 expression. Arrows indicated positive CD9 expression. (c) Western blot analysis was used to evaluate CD81, Calreticulin and HSP90B1 expression in K7M3, K7, DLM8, Dunn, and 3T3 cell exosomes. MHS cells were used as a positive control
Figure 2. Alveolar macrophages can uptake osteosarcoma cell exosomes. Osteosarcoma cell exosomes were extracted from non-metastatic K7 and Dunn cells and from metastatic K7M3 and DLM8 cells via ultracentrifugation and then labeled with Cell Tracker CM-DiI red fluorescent dye. Alveolar macrophages were cultured overnight and then treated with labeled osteosarcoma cell exosomes for 24 h. Both fluorescent and bright field images were collected using the (a) IncuCyte Live-Cell System at 20× and the (b) Nikon Eclipse Ti de-convolution inverted bright field and fluorescent microscope at 60× under oil immersion. Osteosarcoma cell exosomes are shown in red and DAPI in blue. Bright field images are also included, as well as the merged images from all three channels
Figure 3. Metastatic osteosarcoma cell exosomes inhibit alveolar macrophage phagocytosis and efferocytosis and macrophage-mediated tumor cell killing. (a) Alveolar macrophages (MHS cells) were cultured overnight and then incubated with exosomes from osteosarcoma cells (non-metastatic K7 and Dunn; metastatic K7M3 and DLM8) for 24 h. Osteosarcoma cells, labeled with IncuCyte pHrodo labeling reagent, were then added to the MHS cell culture. Phagocytic activity was determined using the IncuCyte S3 Live-Cell Analysis system and the data analyzed by IncuCyte software. Significant inhibition (p < .05) was seen at twenty hours for K7M3 and DLM8. (b) MHS cells were cultured overnight and then incubated with exosomes from the same four osteosarcoma cell lines for 24 h. Effercytosis is the process of clearing dying apoptotic cells. Therefore, osteosarcoma cells, previously treated with gemcitabine for 24 h at a dose that induced apoptosis were labeled with IncuCyte pHrodo labeling reagent, were then added to the MHS cell culture. Efferocytosis was determined by using the IncuCyte S3 Live-Cell Analysis system, and the data were analyzed by the IncuCyte software. Significant inhibition (p < .05) was seen at twelve and thirty hours for K7M3 and DLM8 respectively. Significant enhancement (p < .05) was seen at twenty-four hours for K7. (c) To assess the effect of exosomes on macrophage-mediated cytotoxicity, osteosarcoma cells were cultured overnight and then labeled with the IncuCyte Caspase 3/7 green apoptosis reagent. MHS cells were cultured overnight with exosomes from the four osteosarcoma cell lines and were then added to the labeled osteosarcoma cells. Cytotoxicity was determined using the IncuCyte S3 Live-Cell Analysis system and the data were analyzed by the IncuCyte software. MHS cells treated with PBS was used as a control. Significant inhibition (p < .05) was seen at twenty-four hours for K7M3 and DLM8. Significant enhancement (p < .05) was seen at twenty-four hours for Dunn
Figure 4. Metastatic human osteosarcoma cell exosomes inhibit macrophage phagocytosis and macrophage-mediated tumor cell killing.. (a) Human monocytes (THP1 cells) were cultured and activated with PMA (150 ng/mL) overnight and then incubated with exosomes from human osteosarcoma cells (non-metastatic SAOS2; metastatic LM7) for 24 h. Osteosarcoma cells, labeled with IncuCyte pHrodo labeling reagent, were then added to the THP1 cell culture. Phagocytic activity was determined using the IncuCyte S3 Live-Cell Analysis system and the data analyzed by IncuCyte software. The exosomes from LM7 cells induced a significant inhibition (p < .05) in phagocytic activity at twenty hours. (b) Osteosarcoma cells were cultured overnight and then labeled with the IncuCyte Caspase 3/7 green apoptosis reagent. THP1 cells were activated overnight with PMA (150 ng/mL) and then exposed to exosomes from the two osteosarcoma cell lines and were then added to the labeled osteosarcoma cells. Cytotoxicity was determined using the IncuCyte S3 Live-Cell Analysis system and the data were analyzed by the IncuCyte software. THP1 cells treated with PBS was used as a control. Significant inhibition (p < .05) in cytotoxic activity was seen at twenty-four hours for the THP1 cells incubated with LM7 exosomes
Figure 5. Metastatic osteosarcoma cell exosomes significantly increase mRNA expression of M2 macrophage–related cytokines and chemokines. Alveolar macrophages (MHS cells) were cultured overnight and then incubated with exosomes from osteosarcoma cells (non-metastatic K7 and Dunn; metastatic K7M3 and DLM8) for 24 h. The cells were harvested, total RNA was collected, and cDNA conversion was performed. Hf3fa was used as a loading control. Expression of IL10, TGFB2, and CCL22 mRNAs was quantitated by qPCR. * indicates a p value of < 0.05. ** indicates a p value < .005. *** indicates a p vale < 0.0005
Figure 6. Induction of TGFB2 by exosomes from K7M3 and DLM8 metastatic cells plays a role in inhibiting macrophage function. Alveolar macrophages (MHS cells) were cultured overnight and then incubated with exosomes from osteosarcoma cells (non-metastatic K7 and Dunn; metastatic K7M3 and DLM8) for 48 h. IL10 (a) and TGFB2 (b) secretion in culture medium was measured by ELISA. *** indicates a p vale < 0.0005 (c,d) Alveolar macrophages (MHS cells) were cultured overnight and then incubated with exosomes from metastatic K7M3 (c) cells and DLM8 (d) cells with or without TGFB2 antibody (0.1ug/mL) for 24 h. Osteosarcoma cells, labeled with IncuCyte pHrodo labeling reagent, were then added to the MHS cell culture. Phagocytic activity was determined using the IncuCyte S3 Live-Cell Analysis system and the data analyzed by IncuCyte software. Significant inhibition (p < .05) in phagocytic activity was seen at twelve hours for the MHS cells incubated with both K7M3 and DLM8 exosomes as compared to control. There was no significant differences observed at twelve hours between control and K7M3 or DLM8 exosomes plus anti-TGFB2
