Exosomes impact survival to radiation exposure in cell line models of nervous system cancer

Abstract

Radiation is utilized in the therapy of more than 50% of cancer patients. Unfortunately, many malignancies become resistant to radiation over time. We investigated the hypothesis that one method of a cancer cell's ability to survive radiation occurs through cellular communication via exosomes. Exosomes are cell-derived vesicles containing DNA, RNA, and protein. Three properties were analyzed: 1) exosome function, 2) exosome profile and 3) exosome uptake/blockade. To analyze exosome function, we show radiation-derived exosomes increased proliferation and enabled recipient cancer cells to survive radiation in vitro. Furthermore, radiation-derived exosomes increased tumor burden and decreased survival in an in vivo model. To address the mechanism underlying the alterations by exosomes in recipient cells, we obtained a profile of radiation-derived exosomes that showed expression changes favoring a resistant/proliferative profile. Radiation-derived exosomes contain elevated oncogenic miR-889, oncogenic mRNAs, and proteins of the proteasome pathway, Notch, Jak-STAT, and cell cycle pathways. Radiation-derived exosomes contain decreased levels of tumor-suppressive miR-516, miR-365, and multiple tumor-suppressive mRNAs. Ingenuity pathway analysis revealed the most represented networks included cell cycle, growth/survival. Upregulation of DNM2 correlated with increased exosome uptake. To analyze the property of exosome blockade, heparin and simvastatin were used to inhibit uptake of exosomes in recipient cells resulting in inhibited induction of proliferation and cellular survival. Because these agents have shown some success as cancer therapies, our data suggest their mechanism of action could be limiting exosome communication between cells. The results of our study identify a novel exosome-based mechanism that may underlie a cancer cell's ability to survive radiation.

Keywords: exosomes; glioblastoma; glioma; radiation; resistance.

Figure 1. Exosome confirmation analysis in U87 glioma cells

Zetasizer analysis in Panels A-C demonstrate that size of the exosomes was not affected by radiation exposure but there is a dose dependent increase in release intensity. (A) At 0Gy radiation, exosomes had a release intensity of 8.5 and 7.1. (B) At 3Gy radiation, the release intensity was 7.98 and 9.1. (C) At 12Gy radiation, the release intensity was 11.3 and 12.2. Exosomes were then quantified with (D) BCA assay and (E) nanoparticle tracking analysis. Panels F and G demonstrate exosome release from U87 glioma cells before and after exposure of radiation at 3Gy and 12Gy with immunoblots of exosome confirmation markers (F) CD81 and (G) TSG101. (H) Electron microscopy visualization of exosomes (white arrows) from U87 cells. (^*p<.05, ^**p<.01, ^***p<.001)

Figure 2. Cellular proliferation and survival effects of exosomes

In panels A-F, U87 glioma cells are represented with a “U”, STS26T MPNST cells with an “S”, and SH-SY5Y neuroblastoma cells with a “B”. The number following the cell line letter is the dosage of radiation used; either 3R or 12R. The cells were either exposed to exosomes from not irradiated cells “NR” or to exosomes (Exos) from cells that received one of the two doses of radiation (“3R” or “12R”). The effect of the exosomes on cellular proliferation is shown in Panels A-F: (A,B) U87 glioma cells, (C,D) STS26T MPNST cells, (E,F) SH-SY5Y neuroblastoma cells. Increased recipient cancer cell survival after radiation due to the effect of exosomes in Panels G-L: (G,H) U87 glioma cells, (I,J) STS26T MPNST cells, (K,L) SH-SY5Y neuroblastoma cells) (^*p<.05, ^**p<.01). In Panels M and N are data showing there was no increase in reactive oxygen species of cells incubated with exosomes compared to control for either (M) 3Gy or (N) 12Gy radiated (represented with an “R”) or non-radiated (“NR”). The results show increased proliferation and increased survival to radiation when cells are exposed to exosomes from irradiated cells.

Figure 3. Exosome blockade analysis

Panels A-D show heparin (Hep) and simvastatin (SMV) were able to decrease the proliferation induced by the radiation derived exosomes (rad exos) in (A,B) U87 and (C,D) STS26T cells. Panels E-H show Hep and SMV were able to decrease the cell survival conferred by radiation-derived exosomes in (E,F) U87 and (G,H) STS26T cells. The addition of CD81 antibody (+Ab) was not as effective on either proliferation or survival. Panels I-M show microscopic examination of uptake of exosomes labeled with green PKH67 fluorescence under the various conditions in U87 glioma cells. (I) Exosomes from non-radiation cells show minimal uptake whereas (J) exosomes derived from irradiated cells are taken up robustly. (K) Radiation-derived exosomes plus anti-CD81 antibodies had minimal effect on exosome uptake similar to exosomes from non-radiated cells. (L) Radiation-derived exosomes plus heparin. (M) Radiation-derived exosomes plus simvastatin. Hep (L) and SMV (M) both decreased uptake of fluorescently labeled radiation-derived exosomes when compared to fluorescently labeled radiation-derived exosomes without treatment (J). (^*p<.05, ^**p<.01, ^***p<.001 significantly decreased compared to control, # previous increase due to radiation-derived exosomes is now not significant).

Figure 4. In vivo analysis of radiation derived exosome effect and therapeutic blockade

Representative IVIS images of (A) Control (B) Non-radiation exosomes (C) Radiation-derived exosomes, (D) Radiation-derived exosomes plus daily heparin (Hep), (E) Radiation-derived exosomes plus daily simvastatin (SMV) treatment. Mice treated with radiation-derived exosomes had visually larger tumors when compared to control. When co-treating mice with radiation-derived exosomes plus heparin or simvastatin, the tumor size decreased and was comparable to control levels. (F) Tumor progression over time was quantified with IVIS counts. Mice treated with radiation-derived exosomes (represented as “Rad Exos”) had an increase in tumor progression and when co-treating with Hep or SMV tumor progression was similar to baseline (p<0.05). (G) Mice treated with radiation-derived exosomes had a decrease in survival time but when co-treating with heparin or simvastatin the mouse survival increased.

Figure 5. Immunohistochemistry of glioblastoma tumor samples from each group

(A) H & E staining revealed increased necrotic tissue in the control saline treated tumors when compared to the radiation-derived exosome (Represented as “Rad Exos”) treated tumors. (B) Ki67 cellular proliferation marker analysis showed decreased proliferation in the control tumors when compared to the radiation-derived exosome treated tumors. (C) Cleaved caspase 3 marker for cell death increased in control tumors when compared to tumors treated with radiation derived exosomes. All of the effects associated with radiation-derived exosomes seen by immunohistochemical analysis were not present in tissue from tumors co-treated with heparin or simvastatin. The tumors from the heparin and simvastatin treated animals appeared similar to controls. The inserts are 40X images provided to show more cellular details within the tumors.

Figure 6. Analysis and comparison of miRNA contents within the non-radiation and radiation derived glioma exosomes

(A) Distinct heat map profiles were generated for exosomes derived from cells exposed to 0Gy (control glioma exosomes), 3Gy (low radiation), and 12Gy (high radiation). A total of 516 miRNA were identified in the exosomes following irradiation (B) Table showing the 4 statistically significant exosomal miRNAs following irradiation. The oncogenic miRNAs and tumor suppressive miRNAs were up and down regulated, respectively.

Figure 7. Analysis and comparison of mRNA contents within the non-radiation and radiation derived glioma exosomes

(A) The change in expression levels of 59 mRNA were identified following irradiation (p<0.05). Distinct heat map profiles were generated for exosomes derived from cells exposed to 0Gy (control glioma exosomes), 3Gy (low radiation), and 12Gy (high radiation). mRNA that have been demonstrated to have oncogenic or tumor suppressive functionality are highlighted with a red box. There is clearly a dose response to the patterns of expression. Panels B-C: Ingenuity Pathway Analysis and comparison of mRNA contents within the non-radiation and radiation derived glioma exosomes. (B) Molecular and cellular function pathways most highly represented in the radiation derived exosomes (C) The mRNA networks most represented in the radiation derived exosomes.

Figure 8. Analysis and comparison of protein contents within the non-radiation and radiation derived glioma exosomes

(A) 50 proteins were unique to the 3Gy derived exosomes, 92 were unique to the 12Gy derived exosomes, and 195 were overlapping in both radiation dose derived exosomes in comparison to non-radiation derived exosomes (B) Table showing the 4 most statistically significantly represented protein profiles in the exosomes following radiation were (C) Proteasome pathway proteins (D) Notch signaling pathway proteins (E) Jak-STAT pathways proteins (F) Cell cycle pathway proteins. All of the proteins upregulated in the exosomes are known to be associated with increased resistance to radiation and increased cellular proliferation.

Figure 9

Schematic representation of (A) Proposed model for the mechanism of exosomes enhancing the ability of recipient cancer cells to survive radiation therapy (B) Proposed model for the therapeutic blockade of exosome uptake.