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Choi et al. Cancer Drug Resist. 2026;9:12 Page 9 of 20

both CSF influx and interstitial efflux by activating the mechanosensitive channel transient receptor potential
vanilloid-4 (TRPV4) in astrocytes . Using a combination of in vivo transcranial fluorescence imaging, ex
[64]
vivo optical clearing, and molecular assays, they visualized a marked increase in tracer entry into periarterial
spaces and deeper cortical layers [Figure 3C] while simultaneously observing accelerated clearance toward
the deep cervical lymph nodes (dcLNs) [Figure 3D]. At the cellular level, low-intensity US induced
TRPV4-dependent Ca influx, which activated calmodulin (CaM) and promoted AQP4 translocation to the
2+
astrocyte surface, thereby increasing water permeability along the glymphatic pathway. Pharmacological
experiments using TRPV4 agonists and antagonists confirmed this mechanism: activating TRPV4
reproduced the US-induced augmentation, whereas blocking TRPV4 abolished both influx and clearance
effects [Figure 3E]. The study further demonstrated improved amyloid-β clearance, showing reduced
residues at the injection site and increased accumulation in dcLNs, again in a TRPV4-dependent manner.
High-resolution confocal imaging revealed transient astrocyte swelling (20-40 min) due to increased
AQP4-mediated water influx, followed by complete recovery by 65 min, indicating a reversible and
non-injurious physiological response. Overall, the results demonstrate that low-intensity US augments
glymphatic transport through a TRPV4-CaM-AQP4 water-transport mechanism, supporting both CSF
influx and interstitial clearance.

In contrast to studies relying primarily on ex vivo imaging or MRI-based snapshots, Gong et al. directly
visualized the moment US was applied, using a custom two-photon imaging platform built around a
ring-shaped transducer that allowed US delivery and optical imaging simultaneously . This configuration
[39]
enabled real-time observation of how microbubble-assisted sonication deforms vessel walls and alters
glymphatic transport in the living mouse brain. Using a cranial window preparation and a dual-modality
setup, the authors simultaneously tracked CSF tracer movement and vessel wall behavior during and after US
exposure. This approach revealed that US induced rapid, cyclic vessel deformation, characterized by
alternating invagination and dilation synchronized with sonication. These mechanical changes were
substantially larger than in sham conditions, with a 7.25-fold greater diameter change and a 3.09-fold faster
deformation rate. Concurrently, the perivascular tracer signal exhibited a marked decline, indicating
accelerated local clearance during US exposure. Quantitatively, tracer intensity decreased 1.86-fold more
than in controls, and the maximum clearance rate increased 4.57-fold, demonstrating that US enhances not
only CSF influx pathways but also downstream efflux dynamics. A strong correlation between the rate of
vessel deformation and the rate of tracer clearance (R = 0.82) further established a direct biophysical
2
coupling between US-driven vascular motion and glymphatic transport efficiency. By classifying vessels as
small (10-40 μm), medium (40-70 μm), or large (70-100 μm), the study also showed that while small vessels
exhibited the greatest absolute deformation, larger vessels generated much stronger clearance responses.
These findings indicate that US can modulate glymphatic flow across multiple vascular scales and that efflux
along larger draining vessels may be particularly sensitive to US-induced mechanical forces.

Curley et al. investigated how microbubble-assisted focused US can enhance ISF transport within brain
tumors, thereby improving the delivery of locally and systemically administered therapeutics . Although
[65]
this work does not target glymphatic transport from the CSF, it is noteworthy because interstitial flow
enhancement represents a complementary mechanism by which US can facilitate solute movement from
perivascular pathways into tumor parenchyma. This study focuses on both the BTB and BBB, which together
restrict the penetration of gene-therapy vectors and other macromolecular agents. The BTB limits
convection from blood to tumor tissue due to elevated ISF pressure, whereas the BBB becomes relevant when
infiltrating tumor cells extend beyond the tumor core. While microbubble-assisted focused US is already
known to transiently and reversibly open both barriers, Curley et al. further evaluated its impact in glioma
disease models by examining nanoparticle delivery and functional gene expression following US with
microbubbles [Figure 4A] . Specifically, fluorescence imaging of whole brains and excised tumors
[65]

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