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Page 16 of 20 Choi et al. Cancer Drug Resist. 2026;9:12

transport toward mLVs. Second, injection volume had a dose-dependent inhibitory effect on contraction
frequency; volumes of 4, 10, and 20 μL showed progressively reduced activity, with 20 μL producing the
lowest frequency. Excessive volume may elevate intraluminal pressure or shear stress, triggering negative
feedback mechanisms that suppress lymphatic contractions and potentially diverting tracer flow toward
low-resistance pathways. Thus, minimizing injection volume to the effective threshold is essential for
experimental consistency. Third, removal of skin and the resulting loss of external mechanical support
significantly impaired lymphatic contraction. As contraction frequency decreased following skin removal,
the authors suggested that external confinement contributes to maintaining physiological lymphatic
pumping. Moreover, anesthesia has been shown to adversely affect glymphatic function and reduce transport
toward the mLV , underscoring the need for protocols that incorporate adequate wake periods or
[81]
standardized anesthetic depth. Collectively, these observations highlight the importance of methodological
consistency, transparent reporting, and careful control of experimental variables in studies evaluating
glymphatic and lymphatic transport.

Despite these advances, several limitations remain. Most studies have been conducted in rodents with
normal brain physiology, and the extent to which tumor-associated abnormalities - elevated interstitial
pressure, vessel immaturity, heterogeneous extracellular matrix, or altered AQP4 localization - modulate US
responsiveness is not yet known. Moreover, current findings on molecular-size dependence suggest that
while US can enhance delivery of small agents via glymphatic pathways, the transport of large biological
therapeutics may require additional strategies or optimized modulation paradigms.

Looking forward, the convergence of US neuromodulation, lymphatic biology, and intrathecal drug delivery
opens several compelling research directions. Future work is needed to define optimal US parameters for
tumor-bearing brains, evaluate therapeutic distribution under pathological glymphatic impairment, and
determine how localized vs. global glymphatic modulation influences tumor microenvironments. Clinical
translation will further require systematic evaluation of safety, repeatability, and long-term effects,
particularly as US-based BBB opening technologies are already being tested in patients.

CONCLUSION
The growing body of research on the glymphatic system has fundamentally reshaped our understanding of
how the brain regulates fluid transport, metabolic waste removal, and therapeutic distribution. Although
glymphatic dysfunction has been extensively examined in neurodegenerative diseases, its role in brain
cancers - and its relevance to drug delivery - has remained comparatively underexplored. This review
synthesizes emerging evidence showing that US, across a range of frequencies and exposure conditions, can
actively augment glymphatic influx, enhance CSF–ISF exchange, and accelerate interstitial solute clearance.
These effects collectively highlight US as a promising tool for overcoming key barriers to therapeutic
penetration in brain tumors.

Across the studies reviewed here, US was shown to modulate glymphatic transport through complementary
biophysical and molecular mechanisms: amplification of perivascular pulsatility, vessel wall micron-scale
deformation, TRPV4–CaM–AQP4–mediated fluid regulation, and microbubble-enhanced perivascular
streaming. Importantly, these effects were demonstrated using diverse imaging modalities - including MRI,
optical clearing–based 3D microscopy, and real-time two-photon imaging - providing converging evidence
that US can influence multiple stages of glymphatic flow, from periarterial influx to parenchymal transport
and perivenous efflux.

Overall, the studies reviewed here suggest that US has the potential to reshape therapeutic strategies for brain
tumors by unlocking an underutilized fluid pathway - the glymphatic system - for targeted drug delivery. By

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