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Ioannides et al. J Cancer Metastasis Treat 2020;6:15 I http://dx.doi.org/10.20517/2394-4722.2020.22 Page 3 of 7

Administration of EV
All animal experimentation described in this study was per the guidelines provided by the NIH and
approved by all Institutional Animal Care and Use Committees (IACUC #AUP-18-032). Male wild type
mice (C57Bl6/J, Jackson) were maintained in standard housing conditions, respectively (20 °C ± 1 °C;
70% ± 10% humidity; 12 h:12 h light and dark cycle) and had free access to standard rodent chow and water.
Four-month-old wild-type C57Bl/6J male mice were divided into the following three groups receiving
EV: Intracranial (IC), retro-orbital (RO), and intranasal (IN). For each route of administration, a total of
6
6.70 × 10 EV were delivered in a total volume of 8 µL (4 sites), 50 µL and 20 µL for IC, RO and IN respectively.

IC delivery: Mice were anesthetized (4%) and maintained on 2% (v/v) isoflurane/oxygen for the stereotaxic
implantation of EV. Surgical injections were performed using a 32G microsyringe at an injection rate of
0.25 µL/min. Each hippocampus received 2 distinct injections of EV per hemisphere in an injection volume
of 2 µL (EV in sterile hibernation buffer) per site for a total of 4 injections (8 µL) per animal. Stereotactic
coordinates from the bregma were anterior-posterior (AP): -1.94, mediolateral (ML): -1.25, dorsal-ventral
(DV): -1.50 for the first site and AP: -2.60, ML: -2.0, and DV: -1.5 for the second site.

RO delivery: Mice were anesthetized with 4% isoflurane and gentle pressure was applied using the fingers
against the skin that is ventral and dorsal to the eye. A dermal syringe with a 29G needle, bevel down, was
inserted gently to the retro-orbital vein by the corner of the eye. The 50 µL EV injection was applied in a
slow, smooth fashion. The needle was carefully removed and the mouse was monitored until fully recovered
(within 3-5 min) from anesthesia.

[18]
IN delivery: Mice were lightly anesthetized (2% isoflurane) and prepared for IN administration of EV .
Manual restraint was used to hold the mouse in a supine position with the head elevated. The end of a 20 µL
micropipette was placed at the external nares, and then the 20 µL solution was poured in slowly by the
nasal tip. Mice were held for about 10 s before returning to the holding cage to recover.

Intracranial imaging
Two days post-EV treatment, mice were deeply anesthetized using isoflurane and euthanized via
intracardiac perfusion using 4% paraformaldehyde (Sigma) in 100 mmol/L phosphate-buffered saline (PBS;
pH 7.4, Gibco). Brains were cryoprotected using a sucrose gradient (10%-30%) and sectioned coronally into
30 µm thick sections using a cryostat (Microm, Thermo Scientific, US). For each endpoint 4 representative
coronal brain sections from each experimental group were selected at approximately 15 section intervals
to encompass the rostrocaudal axis from the middle of hippocampus including regions of the prefrontal
cortex (PFC), the subventricular zone (SVZ) and the dentate gyrus (DG), which were then stored in PBS.
Tissues (n = 54) were DAPI nuclear counterstained mounted onto slides and sealed in slow fade/antifade
mounting medium (Life Technologies). Confocal analyses were carried out using multiple Z-stacks taken
at 5-mm intervals using a confocal laser scanning microscope (Nikon Eclipse TE2000-U, EZ-C2 interface).
Individual Z-sections were then analyzed using Nikon Elements software (version 3.0). Images were
deconvoluted using AutoQuant X3 and surface analysis was performed with Imaris (v8.5, BitPlane, Inc.,
Switzerland).

Statistical analysis
Statistical analyses were carried out using GraphPad Prism (v6) software. One-way analyses of variance
(ANOVA) were used to assess significance between the control, irradiated, and irradiated group
receiving EV. When overall group effects were found to be statistically significant, a Bonferroni’s multiple
comparisons test was used to compare the 9 Gy with individual experimental groups. Data in the text are
presented as means ± SEM, and all analyses considered a value of P ≤ 0.05 to be statistically significant.