Research ArticleExperimental Studies
Open Access

Development of a Glymphatic Pathway-based Rat Model for Cancer Metastasis from Brain to Lung

ABHIJITH SREEJITH, HAIJUN XIAO, IYLAN HOWSON, MAURIZIO BOCCHETTA and MUNA ARYAL
In Vivo March 2026, 40 (2) 784-794; DOI: https://doi.org/10.21873/invivo.14236

Abstract

Background/Aim: Brain-to-lung metastasis is clinically rare but represents an underexplored route of cancer dissemination. Understanding this process may reveal novel mechanisms involving cerebrospinal fluid (CSF) and glymphatic clearance pathways. Existing preclinical models fail to replicate this directional spread or allow controlled investigation of central nervous system (CNS)-driven metastasis. This study aimed to develop a reproducible rat model to examine whether tumor cells introduced into the CNS can disseminate to peripheral organs, specifically the lungs.

Materials and Methods: We established a brain-to-lung metastatic model in immunocompromised nude rats using intrathecal injection of A549-LUC human lung adenocarcinoma cells. This approach enabled precise tumor placement within the subarachnoid space for controlled modeling of metastatic progression. In vitro bioluminescence assays confirmed robust luciferase activity, peaking at 4 minutes post D-luciferin addition. In vivo imaging using the In Vivo Imaging System (IVIS) was employed to track tumor localization and dissemination over time.

Results: IVIS imaging revealed early tumor localization in the CNS, followed by progressive and asymmetric spread to the lungs, with higher radiance in the right lung. Intracranial tumor detection was limited by poor signal penetration through the skull. Post-mortem hematoxylin and eosin staining confirmed tumor lesions in brain and lung tissues. Physiological monitoring showed initial weight gain followed by decline, and survival analysis indicated a median survival of 36 days, with complete mortality by day 40.

Conclusion: This intrathecal model overcomes limitations of systemic injection techniques by enabling stepwise investigation of brain-to-lung metastasis. While immune interactions are restricted, this reproducible platform supports therapeutic testing – including ultrasound-enhanced glymphatic drug delivery – and provides a foundation for studying rare but clinically significant metastatic routes.

Keywords:

Introduction

Metastasis is the primary cause of cancer-related mortality (1) with brain-to-lung metastatic progression being particularly challenging to model pre-clinically (2). While brain-to-lung metastasis is uncommon compared to lung-to-brain spread, rare metastatic routes are increasingly recognized in advanced disease and in patients with prolonged survival due to improved therapies. Understanding this pathway is essential for uncovering mechanisms of cancer dissemination and identifying potential therapeutic targets. Existing preclinical models, such as intravenous injection, often produce diffuse tumor dissemination (3, 4), complicating the investigation of organ-specific metastasis and therapeutic responses (3, 5). Alternative methods such as intracardiac or stereotactic tumor cell injections partially address this issue but bypass critical steps of the metastatic cascade, limiting translational relevance (4-7). Notably, intracardiac injection of cancer cells serves more as a model of tumor establishment rather than authentic metastatic spread, since it circumvents the initial steps of tumor cell detachment from a primary site and intravasation into the circulation (7). Genetically engineered and spontaneous models offer physiological context but are slow, unpredictable, and poorly reproducible (4, 8), whereas patient-derived xenografts retain tumor heterogeneity but require immunocompromised hosts and are technically demanding, restricting their widespread use (9-11).

Recent evidence has highlighted the glymphatic system, a brain-wide perivascular network facilitating cerebrospinal fluid (CSF) flow and solute clearance, as a potential route for metastatic dissemination (11-16). Tumor cells may exploit glymphatic pathways to migrate from the central nervous system to peripheral organs, such as the lungs, this representing an underexplored mechanism of metastasis. Moreover, the glymphatic system presents an opportunity for targeted therapeutic delivery (13, 16). Specifically, recent studies demonstrate that ultrasound-mediated glymphatic modulation can significantly enhance CSF dynamics within the central nervous system (17). By applying focused ultrasound, this technique increases convective CSF flow and accelerates solute transport along perivascular spaces. This improvement facilitates more uniform distribution of therapeutic agents throughout the brain parenchyma, overcoming barriers to drug penetration in deep regions. Importantly, enhanced CSF movement may also promote clearance toward peripheral compartments via glymphatic pathways, potentially extending drug delivery to distant metastatic sites such as the lungs. Thus, ultrasound-assisted glymphatic activation represents a promising strategy for improving treatment of both primary brain tumors and secondary metastases by leveraging natural clearance routes. However, its role in metastatic dissemination remains largely unexplored because of the lack of suitable preclinical models.

Here, we describe a brain-to-lung metastatic rat model using intrathecal injection of luciferase-labeled human lung adenocarcinoma (A549-LUC) cells. This model allows precise tumor seeding in the subarachnoid space, enabling controlled dissemination along glymphatic pathways to the lungs. Investigating this mechanism may yield critical insights into metastatic dissemination and uncover novel therapeutic targets for rare but clinically significant routes. Furthermore, the proposed model offers a robust and clinically relevant platform for studying metastatic progression, assessing treatment strategies, and advancing glymphatic-targeted drug delivery approaches.

Materials and Methods

Cell culture. A549 human lung adenocarcinoma cells [A549-LUC, luciferase-labeled (18)] were cultured in Dulbecco’s modified Eagle’s medium (Gibco-ThermoFisher Scientific, Waltham, MA, USA) supplemented with GlutaMAX-I and 10% fetal bovine serum (Gibco) at 37°C in a humidified atmosphere containing 5% CO2. Cells were maintained in logarithmic growth phase by passaging every 2-3 days at a 1:2 split ratio. Early-passage cultures were expanded and cryopreserved in liquid nitrogen in complete medium containing 10% dimethyl sulfoxide. Frozen vials were rapidly thawed in a 37°C water bath, transferred to T75 flasks with prewarmed medium, and cultured with a medium change at 24 h to remove residual dimethyl sulfoxide. Confluent cultures (~80%) were washed with calcium- and magnesium-free phosphate-buffered saline (PBS), detached with 0.25% trypsin-EDTA, neutralized with complete medium, centrifuged twice at 180 × g for 5 min, resuspended in fresh medium, and counted using a hemocytometer. Cells were seeded at appropriate densities and returned to the incubator until use in subsequent in vivo experiments.

In vitro bioluminescence imaging. Bioluminescence activity of A549-LUC cells was assessed using an IVIS Spectrum system (PerkinElmer, Waltham, MA, USA). Cells were seeded in black-walled, clear-bottom 24-well plates at 1×104 to 1×105 cells per well and cultured overnight. On the day of imaging, D-luciferin potassium salt (GoldBio, St Louis, MO, USA) was prepared in sterile PBS (15 mg/ml) and added to each well at a final concentration of 150 μg/ml. Plates were placed into the IVIS chamber, and bioluminescence signals were acquired periodically (0, 5-, 10-, 15-, 20-, and 25-min post-addition) under auto-exposure settings (binning: medium, f/stop: 1). Photon flux was quantified in photons per second (p/s) using Living Image software (PerkinElmer), and peak luminescence values were used for normalization.

Animal model. Adult male immunocompromised SRG rats (Sprague Dawley, Rag2−/−, Il2rg−/−; 250-300 g) were used in this study. A total of eight animals were used. All of the procedures were approved by the Institutional Animal Care and Use Committee at Loyola University Chicago (APLAC Number: LUC215925) and North Carolina A&T State University (IACUC-LA23-0054). All rats were acclimated for 3 days prior to experiments. Physiological parameters were monitored using PhysioSuite (Kent Scientific Corporation, Torrington, CT, USA) during cell inoculation and in vivo imaging. After a 3-day acclimation period, tumor cells were injected on day 0 to establish the model as described below. In vivo imaging was initiated immediately after injection and performed weekly to monitor tumor size until study completion. The study lasted 40 days, during which body weight and survival were monitored as defined endpoints.

Intrathecal cisterna magna injection. Rats were anesthetized with 4% isoflurane for ~5 min using an electronic vaporizer (SomnoFlo; Kent Scientific) and maintained at 2% isoflurane during the procedure. Animals were weighed and secured in a custom 3D-printed stereotaxic frame (19) (Figure 1A). A heating pad was used to maintain body temperature, and eye lubricant (OptixCare, Durham, NC, USA) was applied. Hair over the dorsal scalp and upper neck was removed using an electric trimmer followed by Nair lotion (Walmart, Chicago, IL, USA). A549-LUC cells (1×106 in 20 μl PBS) mixed with 60 μl artificial CSF (Tocris, Minneapolis, MN, USA) were injected into the cisterna magna (Figure 1B) via a 27-gauge butterfly needle connected to polyurethane tubing and a 1 ml syringe, allowing direct access to the CSF for dissemination to the brain and peripheral organs.

Figure 1.
Figure 1.

Intrathecal injection of A549-LUC cells in SRG rats. (A) Custom-made stereotactic frame used to secure rats during intrathecal injection. (B) Schematic representation of the cisterna magna injection procedure, showing delivery of 1×106 A549-LUC cells mixed with artificial cerebrospinal fluid into the cerebrospinal fluid for brain and peripheral organ dissemination.

In vivo bioluminescence imaging. Tumor progression was monitored weekly using IVIS imaging. D-Luciferin (15 mg/ml) was administered intraperitoneally at 150 mg/kg, and animals were returned to their cages for 25 min for systemic distribution. Rats were then anesthetized with 2% isoflurane and placed supine in the IVIS chamber on a heated platform. Images were acquired under auto-exposure (medium binning, f/stop=1) until a stable maximal signal was recorded. Regions of interest (ROIs) were drawn over the cranium and thoracic cavities, and photon flux was quantified in photons per second (p/s) using Living Image software, with background subtraction and normalization to body weight and baseline (day 0). Imaging was performed by personnel blinded to experimental groups.

Body weight and neurological assessment. Rats were monitored weekly for body weight and neurological function. Body weight was recorded, and the percentage change from baseline was calculated. Neurological assessments included evaluation of gait, posture, coordination, spontaneous activity, and limb reflexes. Animals showing ≥10% body weight loss, severe neurological deficits (e.g., inability to ambulate, seizures, extreme lethargy), or other signs of distress were humanely euthanized according to Institutional Animal Care and Use Committee-approved protocols using CO2 inhalation followed by a secondary method to confirm death.

Tumor volume and survival analysis. Tumor volume was inferred from bioluminescence intensity obtained via IVIS imaging, and longitudinal measurements were used to assess growth kinetics in the brain and lungs. Animal survival was recorded daily, and Kaplan–Meier survival curves were generated using GraphPad Prism (Boston, MA, USA) to estimate median survival and compare distributions between experimental groups.

Ex vivo brain and lung tissue sectioning. Brains and lungs were fixed in formalin for ≥24 h, sectioned into 10 slices (~2 mm thickness), and stored at −4°C until cryosectioning. For cryosectioning, tissues were embedded in OCT compound on the cryostat mounting plate (Epredia CryoStar NX50 Kalamazoo, MI, USA) and rapidly frozen. Tissue blocks were trimmed to expose the surface and sectioned at 20 μm thickness. Sections were transferred promptly onto microscope slides (two sections per slide), labeled, and stored at −20°C until further analysis.

Hematoxylin and eosin (H&E) staining. The slides were removed from the freezer and placed in a glass slide container. The following reagents were used for staining and mounting: 100% filtered hematoxylin (VWR, Radnor, PA), 1% hydrochloric acid, and PBS (Thermo Fisher, Waltham, MA, USA); 100% ethanol (Azer Scientific, Morgantown, PA, USA); Scott’s tap water substitute and eosin Y (Sigma Aldrich, Burlington, MA, USA); 70% ethanol, 95% ethanol, and 100% xylene (Fisher Scientific, Pittsburgh, PA, USA). All solutions were prepared in glass containers that contained 100 ml to fully cover the slides.

For staining, slides were immersed using tweezers in 100% filtered hematoxylin for 4 min, then transferred to the rinse container and rinsed with deionized water over a sink for 3 min or until the water ran clear. Slides were differentiated by immersion into 1% hydrochloric acid in 70% ethanol for 30 s. Slides were rinsed again and placed in Scott’s tap water substitute for 45 s to neutralize acidity and enhance the nuclear contrast until the color was apparent, followed by another rinse. For counterstaining, slides were immersed in eosin Y solution for 3 min, and briefly dipped in 70% ethanol for 5 s to remove excess eosin. The eosin container was protected from light with aluminum foil when not in use. Dehydration was performed by transferring slides sequentially through a graded alcohol series: 95% ethanol, and 100% ethanol for two changes, 30 s each. Following dehydration, the slides were cleared in xylene for 3 min.

For mounting, slides were removed from xylene, and three drops of PBS were applied by 0.5-10 μl micropipette at the center of the tissue sections, before applying the coverslip. Excess mounting medium was removed as needed with a xylene-dipped tissue. Slides were left to dry flat in a fume hood for ≤10 minimum, followed by drying overnight at room temperature to allow the PBS to harden. Once dried, they were stored in containers until imaging.

Brightfield microscopy imaging. H&E-stained tissue sections were imaged to confirm the presence or absence of tumor cells or masses in the lungs and brain regions that exhibited bioluminescent signals during in vivo IVIS imaging. This histological assessment represents a critical step in validating the integrity of the tumor model. For imaging, we used a brightfield microscope (Axio Imager Z2; Zeiss, White Plains, NY, USA) equipped with a digital camera (Axiocam 712; Zeiss) and controlled through Zen 3.3 software (Blue Edition, Zen Pro; Zeiss). Prior to imaging, the microscope was powered on, and the software was initialized. Slides were placed on the microscope stage, and the stage position was auto calibrated through the software. The transmitted light mode was selected as the illumination source. Tissue sections were first examined under low magnification objectives (5× to 20×) to evaluate overall tissue architecture, followed by higher magnification (50×) imaging for detailed visualization of individual tumor cells. Images were acquired under optimized exposure and focus settings using the “Snap” function within the acquisition module. Representative high-magnification fields and comprehensive low-magnification scans were captured to document tissue morphology.

For whole-section coverage, tiled imaging was performed at 10× magnification using the “Tile” function in the acquisition module. The Brightfield (transmitted light) channel was selected, and tile boundaries were defined by marking the outer edges of the tissue section. A minimum of four marker positions were used to generate a grid of tiles encompassing the full sample area. The focus strategy was set to Software Autofocus. Once configured, imaging was initiated using the “Start Experiment” button, and sequential tile images were automatically acquired.

Following the acquisition, tiled images were processed using the “Stitching” option within the processing module to generate a seamless composite image. All images were saved automatically in CZI format, with additional JPEG versions generated for convenient visualization and downstream analysis.

Results

Temporal bioluminescence of A549-LUC cells. The bioluminescence activity of A549-LUC cells was monitored over 25 min following the addition of D-luciferin. Photon flux was highest at 4 min post-substrate addition (136.6±43.3 p/s) and decreased progressively over time, measuring 128.2±41.5 p/s at 11 min, 121.1±37.7 p/s at 17 min, and 104.9±33.2 p/s at 25 min (Figure 2). This trend demonstrates a rapid initial reaction between luciferase and D-luciferin, producing strong luminescence that gradually diminishes, likely due to substrate consumption and enzymatic stabilization (20). These results confirm that A549-LUC cells exhibit robust in vitro luciferase activity, providing a reliable signal for subsequent temporal monitoring in in vivo experiments.

Figure 2.
Figure 2.

In vitro bioluminescence activity of A549-LUC cells. Bioluminescence of A549-LUC cells was monitored over 25 min following the addition of D-luciferin. Photon flux increased rapidly after substrate addition, reaching a peak early, then gradually declining over time. The data demonstrate robust luciferase activity in vitro, providing a reliable signal for subsequent temporal monitoring in in vivo experiments.

Longitudinal tumor growth in brain and lungs. Tumor growth in rats was monitored following intrathecal injection of A549-LUC cells over 40 days using IVIS bioluminescence imaging (Figure 3A). Photon flux, reported as radiance (106 p/s/cm2/sr), progressively increased in the subarachnoid space, indicating tumor proliferation in the brain (Figure 3B). Average radiance values were 1.14×106 at day 7, 4.07×106 at day 15, 4.03×106 at day 18, 3.67×106 at day 26, 6.19×106 at day 33, and 1.33×107 at day 40. Tumor progression in the lungs was also observed (Figure 3B), with the right lung increasing from 1.997×104 at day 7 to 1.71×107 at day 40, and the left lung from 1.926×104 at day 7 to 7.98×106 at day 40. These findings indicate initial tumor establishment in the central nervous system, followed by progressive dissemination to the lungs. The consistently higher radiance in the right lung compared to the left suggests potential asymmetrical metastatic spread (21, 22). Overall, these results demonstrate that the intrathecal A549-LUC model reliably recapitulates brain-to-lung metastasis, providing a robust platform for longitudinal evaluation of therapeutic interventions.

Figure 3.
Figure 3.

In vivo tumor progression monitored by IVIS bioluminescence imaging. (A) Representative IVIS images show tumor growth in the brain (subarachnoid space) and lungs of SRG rats following intrathecal injection of A549-LUC cells. (B) Tumor progression in the brain was detected early, followed by dissemination to the lungs over time. Bioluminescence intensity reflects tumor burden, demonstrating that the intrathecal A549-LUC model reliably recapitulates brain-to-lung metastasis for longitudinal therapeutic studies. Data are expressed as absolute bioluminescence values (photons/s) and presented as the mean±standard deviation (error bars) for each time point. In the graphs, the data depict temporal trends in tumor progression across the study period. D: Day.

Longitudinal body weight and survival analysis. Body weight of SRG rats injected intrathecally with A549-LUC cells was monitored over 40 days. Rats showed progressive weight gain from an average of 174.8 g on day 0 to a peak of 275 g on day 26, followed by a decline to 225 g by day 40 (Figure 4A).

Figure 4.
Figure 4.

Body weight changes and survival of tumor-bearing SRG rats. (A) Body weight of rats was monitored over time following intrathecal injection of A549-LUC cells. Animals showed progressive weight gain during the early phase, followed by a decline as disease progressed. Data are expressed as absolute body weight values (g) and presented as the mean±standard deviation (SD) for each time point. In the graph, data depict temporal trends in body weight changes. (B) Kaplan–Meier survival curve showing the survival of the cohort over the study period. The median survival time of the cohort (n=6) was 36 days. These data demonstrate that the intrathecal A549-LUC model produces a reproducible disease course suitable for longitudinal therapeutic studies.

Two animals were censored from the survival analysis for reasons unrelated to tumor burden, resulting in six being included in the Kaplan–Meier analysis. Survival remained at 100% until day 33, then decreased sharply, with 33% survival by day 36 and 0% by day 40 (all animals had reached the study endpoint, resulting in 0% survival) (Figure 4B). The median survival time was 36 days, reflecting the progression of tumor burden in this intrathecal A549-LUC model. These results demonstrate a predictable disease course, supporting the model’s utility for longitudinal therapeutic studies.

Metastatic tumor localization along glymphatic pathways. Histological analysis of rat brains following intrathecal injection of tumor cells revealed multiple metastatic foci distributed throughout the brain. Initial identification on H&E sections marked these lesions as yellow-circled ROI in Figure 5A. Subsequent nonlinear registration with a standardized rat brain atlas allowed precise localization of ROIs to specific neuroanatomical structures and facilitated classification into glymphatic-associated versus non-specific regions. A substantial proportion of metastatic colonies were localized along glymphatic-relevant structures, including periventricular zones, the aqueduct, and subarachnoid-adjacent regions. These sites correspond to recognized pathways of glymphatic transport, suggesting that tumor dissemination exploited existing CSF-glymphatic routes. Notably, clusters of metastatic cells were observed adjacent to the lateral ventricles, periaqueductal gray, and fissural boundaries, highlighting the role of perivascular and periventricular conduits in tumor spread.

Figure 5.
Figure 5.

Overlay of hematoxylin and eosin-stained section of rat brain with atlas boundaries. Yellow circles indicate locations where tumor-like cells were observed at lower magnification (5×). Representative regions indicated by colored boxes are shown at higher magnification (20×), including the hippocampal fissure, medial cerebellar nucleus, ventral hippocampal commissure, and crus 1 of the ansiform lobule.

Figure 5B, examination at 20× revealed tumor-like cells within specific regions, including the hippocampal fissure, medial cerebellar nucleus, ventral hippocampal commissure, and crus 1 of the ansiform lobule. In these areas, tumor cells exhibited typical metastatic morphology, including increased nuclear-to-cytoplasmic ratios, irregular nuclear contours, and clustering along perivascular spaces. These observations further support the preferential localization of metastatic cells along glymphatic-associated pathways.

In contrast, a subset of metastatic lesions occurred within non-glymphatic regions of the parenchyma, including the cortical plate and cerebellar hemispheres. These locations lacked direct association with ventricular or perivascular compartments and likely represent secondary infiltration or hematogenous dissemination rather than primary glymphatic transport. Quantitative assessment revealed that the majority of metastatic foci were glymphatic-associated, whereas a smaller fraction was confined to non-specific parenchymal regions, emphasizing the preferential spread of tumor cells along glymphatic pathways.

Metastatic tumor distribution in rat lung. Histological examination of rat lung tissue sections stained with H&E revealed multiple ROIs (yellow circles) consistent with A549 metastatic lung cancer (Figure 6). Each slide contained approximately 20 distinct ROIs, visually identified based on abnormal cellular morphology and clustering patterns. These metastatic regions displayed characteristic histopathological features, including increased cellular density relative to surrounding normal lung parenchyma, disruption of alveolar architecture with tumor cells forming compact nests or sheets, hyperchromatic and pleomorphic nuclei indicative of neoplastic transformation, and perivascular and peribranchial localization, suggesting preferential colonization sites.

Figure 6.
Figure 6.

Metastatic lung cancer lesions in rat lung tissue. (A) Hematoxylin and eosin-stained section with yellow-circled regions of interest. (B) Magnified views (20×) of representative regions of interest, showing dense cellular clusters, disrupted alveolar architecture, and hyperchromatic nuclei.

The ROIs were distributed throughout the lung tissue in a non-random pattern, potentially reflecting the influence of vascular or lymphatic dissemination pathways. Collectively, these findings confirm the presence of metastatic lesions in the rat brain-lung metastasis model.

Discussion

We successfully demonstrated a reproducible rat model of brain-to-lung metastasis using intrathecal injection of A549-LUC cells. The model overcomes limitations of conventional intravenous metastasis models by enabling organ-specific tumor seeding in the central nervous system and controlled dissemination to the lungs, reflecting clinically observed metastatic patterns.

In vitro bioluminescence assays confirmed robust luciferase activity in A549-LUC cells, providing reliable longitudinal monitoring. IVIS imaging demonstrated progressive tumor growth in the subarachnoid space and metastasis to the lungs, with higher radiance in the right lung suggesting potential asymmetrical spread influenced by vascular or lymphatic factors. Body weight and survival analyses further validated the model, with median survival of 36 days, supporting reproducible disease progression suitable for longitudinal studies.

Histology confirmed tumor localization along glymphatic pathways, including periventricular regions, aqueduct, and subarachnoid-adjacent structures, highlighting CSF-mediated dissemination. Lung metastases were widely distributed, with necrotic areas indicating high tumor proliferation. The model provides a unique platform for testing emerging therapeutic strategies, including ultrasound-mediated glymphatic drug delivery, which could enhance penetration into the central nervous system and peripheral metastatic sites. Noninvasive longitudinal monitoring improves experimental throughput and reduces animal stress.

Conclusion

This study offers a reproducible brain-to-lung metastasis model in rats that mirrors key clinical features of metastatic disease. It enables mechanistic studies of metastatic dissemination, evaluation of therapeutic interventions, and testing of glymphatic-targeted drug delivery strategies. Future applications of this model might provide critical insights into metastatic biology and support translational therapeutic development.

Acknowledgements

This work was supported by the National Academy of Medicine and NC A&T START-UP and Provost Seed Funds. We would like to thank Dr. Boyce E. Collins, Research Operations Director at the College of Engineering, NC A&T, for their assistance in providing access to the imaging facilities for H&E section analysis.

Footnotes

  • Authors’ Contributions

    Abhijith Sreejith and Haijun Xiao performed experiments and contributed to manuscript editing. Iylan Howson assisted with experimental procedures. Maurizio Bocchetta provided cancer cells, offered guidance on cell handling, and contributed to manuscript editing. Muna Aryal conceptualized the study, performed experiments, analyzed and interpreted data, and wrote the manuscript.

  • Conflicts of Interest

    All Authors have no conflicts of interest to disclose.

  • Artificial Intelligence (AI) Disclosure

    During the preparation of this manuscript, a large language model (Microsoft Copilot, accessed through the University) was used solely for language editing and stylistic improvements in select paragraphs. No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the Authors. Additionally, no figures or visual data were generated or modified using generative AI or machine-learning-based image enhancement tools.

  • Received November 5, 2025.
  • Revision received December 17, 2025.
  • Accepted February 2, 2026.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Development of a Glymphatic Pathway-based Rat Model for Cancer Metastasis from Brain to Lung
ABHIJITH SREEJITH, HAIJUN XIAO, IYLAN HOWSON, MAURIZIO BOCCHETTA, MUNA ARYAL
In Vivo Mar 2026, 40 (2) 784-794; DOI: 10.21873/invivo.14236
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