Focused Ultrasound Hyperthermia Augments Release of Glioma-derived Extracellular Vesicles with Differential Immunomodulatory Capacity

Abstract

Background: Increasing evidence points to the critical role of extracellular vesicles (EVs) as molecular parcels that carry a diverse array of bioactive payloads for coordination of complex intracellular signaling. Focused ultrasound (FUS) hyperthermia is a technique for non-invasive, non-ionizing sublethal heating of cells in a near-instantaneous manner; while it has been shown to improve drug delivery and immunological recognition of tumors, its impact on EVs has not been explored to date. The goal of this study was to determine whether FUS impacts the release, proteomic profile, and immune-activating properties of tumor-derived EVs. Methods: Monolayered murine glioma cells were seeded within acoustically transparent cell culture chambers, and FUS hyperthermia was applied to achieve complete coverage of the chamber. Glioma-derived EVs (GEVs) were isolated for characterization by Nanoparticle Tracking Analysis, cryo-electron microscopy and mass spectrometry. An in vitro experimental setup was designed to further dissect the impact of GEVs on innate inflammation; immortalized murine dendritic cells (DCs) were pulsed with GEVs (either naïve or FUS hyperthermia-exposed) and assayed for production of IL-12p70, an important regulator of DC maturation and T helper cell polarization toward the interferon-γ-producing type 1 phenotype. Results: We confirmed that FUS hyperthermia significantly augments GEV release (by ~46%) as well as shifts the proteomic profile of these GEVs. Such shifts included enrichment of common EV-associated markers, downregulation of markers associated with cancer progression and resistance and modulation of inflammation-associated markers. When DCs were pulsed with GEVs, we noted that naïve GEVs suppressed IL-12p70 production by DCs in a GEV dose-dependent manner. In contrast, GEVs from cells exposed to FUS hyperthermia promoted a significant upregulation in IL-12p70 production by DCs, consistent with a pro-inflammatory stimulus. Conclusion: FUS hyperthermia triggers release of proteomically distinct GEVs that are capable of facilitating an important component of innate immune activation, lending both to a potential mechanism by which FUS interfaces with the tumor-immune landscape and to a role for GEV-associated biomarkers in monitoring response to FUS.

Keywords: Focused ultrasound; cytokine; exosome; extracellular vesicles; glioma.

Figure 1

Focused ultrasound hyperthermia application in PetakaG3 cell culture chambers. A. Schematic representation of focused ultrasound system. All in vitro experiments were performed in a degassed water tank warmed to 37^⁰C. For hyperthermia treatments, a 1.1 MHz single-element focused transducer was oriented directly across from the Petaka chamber. Position of the chamber with respect to the fixed transducer was controlled by a 3D linear motion controller. B. Representative layout of sonications applied to Petaka chamber. A 14x18 grid of sonications was applied to GL261-luc2 cells seeded within each chamber, with 5mm spacing between each insonation. C. Simulated spatial temperature profile for selected FUS hyperthermia parameters. The radius of hyperthermia (>40 °C) at the focus, as determined by simulation was approximately 2.5 mm. D. Simulated temporal evolution of focal temperature. Peak temperature at the focus was expected to reach 50°C at the surface (i.e. on the wall) of the Petaka for each sonication based on in silico predictions.

Figure 2

Size characterization of GEVs. A. Average size of GEVs isolated via differential ultracentrifugation according to mode diameter measured by NanoSight NTA. n=15-16 per group. B. Representative 62kx cryo-EM image of a 70.96 nm FUS hyperthermia-exposed GEV isolated by differential ultracentrifugation. C. Representative 29kx cryo-EM image of several GEVs treated with FUS hyperthermia. Particle diameters in this image range from 56.27 to 79.98 nm. Statistical significance assessed by unpaired two-tailed t-test. “n.s.” = not significant.

Figure 3

FUS hyperthermia incites greater release of GEVs in vitro. A. Overall concentration of EVs by Nanosight NTA profiling revealing a significant increase in EVs isolated 15 minutes following FUS hyperthermia exposure. B. Comparison of EV size distribution across experimental groups, indicating enrichment for particles ~110 nm in size on average. **p<0.01 vs. Control. n=8-9 per group. Statistical significance assessed by unpaired two-tailed t-test.

Figure 4

FUS hyperthermia alters proteomic profile of GEVs. Volcano plot depicting significantly regulated murine proteomic markers expressed by GEVs isolated via differential ultracentrifugation. Fold changes were tabulated as treated over control. Vertical black dotted lines denote fold change of ±2. Statistical significance was determined by Fisher's Exact Test with Benjamini-Hochberg correction. Horizontal black dotted line denotes p=0.05, which was considered the threshold for statistical significance. n=3 per group. Select proteomic markers are labeled with alternate protein IDs defined in the text and/or tables.

Figure 5

Immortalized murine dendritic cells decrease IL-12p70 production in a GEV dose-dependent manner, and FUS hyperthermia-exposed GEVs promote a restoration of these levels. A. Overview of experimental design. Briefly, GL261-luc2 cells were seeded in Petaka chambers. Supernatants from control or FUS hyperthermia-exposed cells were collected for GEV isolation by differential ultracentrifugation. Immortalized murine DC2.4 dendritic cells were exposed to GEVs for 24 hours, following which supernatants were collected for quantification of IL12-p70 production. B. Fold change in IL-12p70 production by DC2.4 cells following exposure to GEV doses ranging from 0 to 20 µg. *p<0.05, **p<0.01 vs. Control (0 µg GEV). ^†p<0.05 vs. 0.5 µg GEV. 0-10 µg: n=4 per group, 20 µg: n=2. C. Linear regression analysis of GEV dose escalation data demonstrating a significantly nonzero slope (p<0.05, R² = 0.6945) of -0.0254 ± 0.008423 and y-intercept of 0.7391 ± 0.07888. D. Fold change in IL-12p70 production by unstimulated DC2.4 cells, FUS^- GEV (1 µg dose), and FUS⁺ GEV (1 µg dose) groups. *p<0.05, **p<0.01 vs. group indicated. FUS^- GEV: n=2, GEV^-, FUS⁺ GEV: n=4 per group. Statistical significance (for B,D) assessed by one-way ANOVA followed by Tukey multiple comparison correction.