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Choi et al. Cancer Drug Resist. 2026;9:12 Page 15 of 20

Among various modulation modalities, US is uniquely capable of delivering focused energy to highly
localized regions within the brain. This feature is advantageous for minimizing off-target toxicity in
surrounding tissues - an especially critical consideration in oncology. However, most glymphatic modulation
studies to date have been designed to influence broad cortical or subcortical regions simultaneously, and thus
the potential benefit of localized glymphatic modulation remains underexplored. This raises an important
question: which brain pathologies, beyond tumors, exhibit spatially localized glymphatic impairment, and to
what extent can localized modulation improve therapeutic outcomes? Addressing this question may broaden
the clinical relevance of US-based glymphatic modulation to conditions such as traumatic brain injury,
stroke, or focal epileptic lesions, although these applications have not yet been systematically evaluated to the
best of our knowledge.

At the same time, several key challenges remain for the effective translation of US-based glymphatic
modulation. These include the lack of standardized US parameters across studies, incomplete understanding
of dose-response relationships between acoustic exposure and glymphatic transport, and limited ability to
noninvasively and quantitatively monitor glymphatic modulation in real-time. In transcranial applications,
skull-induced acoustic attenuation, phase aberration, and focal distortion further complicate accurate energy
delivery, potentially limiting penetration depth and spatial precision of US-based glymphatic modulation. In
addition, disease-specific variability in vascular integrity, interstitial pressure, and perivascular
architecture–together with inter-individual differences in skull thickness and geometry–may further
complicate parameter optimization, particularly in heterogeneous tumor environments. Addressing these
technical and biological challenges will be critical for advancing US-based glymphatic modulation toward
reliable and reproducible clinical applications.

Observation of the glymphatic flow alterations has primarily relied on MRI and optical imaging, both of
which enable dynamic visualization of contrast transport at different spatial scales. Contrast-enhanced MRI
provides whole-brain, macroscale characterization of glymphatic pathways in both small animals and
humans, whereas optical imaging allows microscale resolution in superficial regions of rodent brains through
optical windows. An emerging complementary approach is photoacoustic imaging (PAI), which detects
optically absorptive chromophores with ultrasonic resolution [67,68] . Because PAI can be deployed across a
wide range of imaging depths - from photoacoustic microscopy to photoacoustic tomography - it effectively
bridges the resolution-depth gap between pure optical imaging and tomographic modalities such as MRI or
PET [69-71] . Although its application to glymphatic imaging is still recent , PAI’s scalability and compatibility
[72]
with US-based modulation systems suggest that it could become a valuable tool for monitoring
US-augmented glymphatic flow [73-75] . Moreover, functional vessel-imaging methods such as ultrafast US
Doppler could help identify pulsatile vascular segments and clarify their mechanistic relationship with
glymphatic flow [76-78] .

Accurate assessment of glymphatic function, however, requires careful attention to experimental factors that
directly influence lymphatic and glymphatic dynamics . Bouta et al. systematically evaluated how key
[79]
physical variables - animal posture, contrast agent injection volume, and mechanical tissue support - alter
lymphatic contraction physiology . Rather than treating these variables as procedural details, they should be
[80]
recognized as critical control parameters that shape the reproducibility and interpretability of lymphatic
imaging studies. First, mouse posture relative to gravity significantly affected lymphatic contraction
frequency: upright positioning preserved normal contraction rates, whereas the supine position markedly
reduced them, despite unchanged ejection fraction (EF). This finding implies that gravitational forces impose
resistance that alters lymphatic pumping rhythms, a consideration especially relevant when imaging contrast

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