Exosomes Derived from Squamous Head and Neck Cancer Promote Cell Survival after Ionizing Radiation

Abstract

Exosomes are nanometer-sized extracellular vesicles that are believed to function as intercellular communicators. Here, we report that exosomes are able to modify the radiation response of the head and neck cancer cell lines BHY and FaDu. Exosomes were isolated from the conditioned medium of irradiated as well as non-irradiated head and neck cancer cells by serial centrifugation. Quantification using NanoSight technology indicated an increased exosome release from irradiated compared to non-irradiated cells 24 hours after treatment. To test whether the released exosomes influence the radiation response of other cells the exosomes were transferred to non-irradiated and irradiated recipient cells. We found an enhanced uptake of exosomes isolated from both irradiated and non-irradiated cells by irradiated recipient cells compared to non-irradiated recipient cells. Functional analyses by exosome transfer indicated that all exosomes (from non-irradiated and irradiated donor cells) increase the proliferation of non-irradiated recipient cells and the survival of irradiated recipient cells. The survival-promoting effects are more pronounced when exosomes isolated from irradiated compared to non-irradiated donor cells are transferred. A possible mechanism for the increased survival after irradiation could be the increase in DNA double-strand break repair monitored at 6, 8 and 10 h after the transfer of exosomes isolated from irradiated cells. This is abrogated by the destabilization of the exosomes. Our results demonstrate that radiation influences both the abundance and action of exosomes on recipient cells. Exosomes transmit prosurvival effects by promoting the proliferation and radioresistance of head and neck cancer cells. Taken together, this study indicates a functional role of exosomes in the response of tumor cells to radiation exposure within a therapeutic dose range and encourages that exosomes are useful objects of study for a better understanding of tumor radiation response.

Fig 1. Characterization of isolated exosomes.

(A) Scheme of exosome analysis. (B) Transmission electron micrograph showing exosomes isolated from the cell culture supernatant of 3 Gy-irradiated BHY cells [scale bar: 100 nm]. (C) Representative immunoblot of HSP70, actin, CD63 and calnexin performed with exosome lysates (EXO) and cell lysates (CP) harvested 24 hours after irradiation. DMEM medium, DMEM medium supplemented with exosome-depleted fetal calf serum as well as supernatant after ultracentrifugation were loaded as controls. (D) Size distribution of exosomes from non-irradiated BHY cells measured with NanoSight technology. (E) Relative exosome abundance of BHY exosomes isolated 24 hours after irradiation with 0, 3, 6 and 9 Gy [n = 6, p-value < 0.05].

Fig 2. Uptake of exosomes by recipient cells.

PKH67-labeled exosomes isolated from irradiated and non-irradiated BHY cells were co-cultivated with BHY cells. (A) Representative fluorescence microscopy images for exosome uptake after 3, 6 and 24 hours incubation. Exosomes were stained in green and nuclei were stained blue with Hoechst 33342. (B) Uptake of exosomes isolated from 6 Gy-irradiated (EXO 6 Gy) and non-irradiated BHY cells (EXO 0 Gy) after 3, 6, 8, 10 and 24 hours incubation. Mean fluorescence of untreated cells and cells after incubation with stained exosomes or an exosome-negative control (-EXO) is shown (n = 3). (C) Dependency of exosomal uptake was determined after 24 hours by using a serial dilution of an exosome preparation. (D) Uptake of labeled exosomes by 0, 2 and 4 Gy-irradiated recipient cells after 24 hours. In all experiments a minimum of 10,000 cells were analyzed for each sample [n ≥ 3, ± SD, p-value < 0.05].

Fig 3. Exosomes affect proliferation, colony formation and clonogenic survival.

(A) Proliferation of cells cultivated for 3 days in medium containing exosomes isolated from irradiated or non-irradiated cells. As a control an equal amount of PBS without exosomes was added to the recipient cells. (B) Plating efficiency of cells cultivated for 5 days in medium containing exosomes isolated from irradiated or non-irradiated cells. As a control an equal amount of PBS without exosomes was added to the recipient cells. (C) Clonogenic survival of BHY cells co-cultivated with exosomes isolated from irradiated or non-irradiated cells and control cells (BHY + PBS) were incubated for 5 days after irradiation with the indicated doses [n = 3, ± SD, p-value: * if p < 0.05, ** if p < 0.01 and **** if p < 0.0001].

Fig 4. Exosomes modulate the repair of DNA DSBs in irradiated recipient cells.

(A) Number of 53BP1 foci in BHY cells 1 hour after irradiation with 0 and 2 Gy and transfer of BHY exosomes isolated 24 hours after irradiation with 0 and 6 Gy [n = 5]. (B) Representative images of 53BP1 foci in BHY cells 6 hours after 2 Gy and transfer of BHY exosomes isolated 24 hours after irradiation with 0, 3, 6 or 9 Gy (53BP1 foci green, nuclei blue). (C) Number of 53BP1 foci in BHY cells 6 hours after 2 Gy and transfer of BHY exosomes isolated 24 and 48 hours after irradiation [n₁ (control; EXO 0 Gy 24 h; EXO 6 Gy 24 h) = 6, n₂ (EXO 0 Gy 48 h; EXO 3 Gy; EXO 6 Gy 48 h; EXO 9 Gy) = 3]. (D) Number of 53BP1 foci in FaDu cells 6 hours after 2 Gy and transfer of FaDu exosomes [n = 3]. (E) Number of 53BP1 foci in FaDu cells 6 hours after 2 Gy and transfer of BHY exosomes [n = 3]. (F) Number of 53BP1 in BHY cells after 2 Gy and transfer of destabilized BHY exosomes. Exosomes from BHY cells isolated 24 hours after irradiation with 0 and 6 Gy were treated with RNase A or a mixture of Triton and Trypsin [n₁ (control; intact) = 6; n₂ (RNase A 5 μg/μl) = 2; n₃ (RNase A 400 μg/μl; Triton + Trypsin) = 3]. For all experiments the ± SD was shown and p-values calculated on control were considered to be significant if * p < 0.05 and highly significant ** if p < 0.01, while ^▲ p < 0.05 and ^▲▲ p < 0.01 indicate significant differences to EXO 0 Gy.