The research team, led by Leandro Castañeyra-Ruiz and colleagues at CHOC Children’s Research Institute, integrated three lines of evidence: quantitative magnetic resonance imaging in a cohort of forty-two preterm and term infants, multiplex profiling of inflammatory proteins in cerebrospinal fluid, and laboratory experiments using primary human choroid plexus cells. Their central hypothesis was that inflammatory cerebrospinal fluid, rich in growth factors such as vascular endothelial growth factor and epidermal growth factor, could reactivate proliferative programs that are normally active only during fetal development, causing the choroid plexus to enlarge in infants who go on to develop hydrocephalus.

The imaging findings were striking in their consistency. On T2-weighted MRI scans, choroid plexus thickness increased in a stepwise fashion across clinical categories. In the axial plane, mean thickness measured 3.44 millimeters in control infants, 4.53 millimeters in preterm infants with intraventricular hemorrhage managed without surgery, and 5.39 millimeters in infants who ultimately required cerebrospinal fluid diversion. Sagittal measurements showed an even more pronounced gradient, rising from 3.51 millimeters in controls to 6.19 millimeters in the diversion group. Statistical analysis revealed that clinical category alone explained roughly 38 to 41 percent of the variance in thickness, and the differences between the most severely affected infants and controls were highly significant in both imaging planes.

Critically, the researchers went beyond simple group comparisons. Because infants requiring diversion were also the most premature at birth, with a mean gestational age of just over 27 weeks compared with nearly 38 weeks in controls, the team used multivariable regression to adjust for gestational age at birth and postmenstrual age at imaging. The association between clinical progression and choroid plexus thickness survived these adjustments, indicating that the enlargement was not simply a byproduct of earlier birth or a different maturational stage at the time of scanning. The team also controlled for ventricular size using the frontal and occipital horn ratio, a standard measure of ventricular dilation, and for hemorrhage severity grade. Even after full adjustment, clinical category remained independently associated with thickness, while ventricular size itself showed no independent relationship with axial thickness. In other words, the choroid plexus appeared to be enlarging as part of the disease process itself, not merely being stretched by expanding ventricles.

To probe the cellular basis of this remodeling, the researchers turned to laboratory experiments with primary human choroid plexus epithelial cells and endothelial cells, the two principal cell types of the tissue. They exposed the cells for seventy-two hours to different cerebrospinal fluid conditions: an artificial fluid control, a low-inflammatory fluid obtained from patients with spinal muscular atrophy, and a growth factor-enriched inflammatory fluid derived from patients with severe traumatic brain injury. Multiplex cytokine analysis confirmed that the inflammatory fluid carried significantly elevated concentrations of vascular endothelial growth factor, and that hemorrhage-derived and injury-derived inflammatory fluids shared this growth factor signature.

The proliferative response was substantial. In epithelial cells, the proportion of nuclei staining positive for proliferating cell nuclear antigen, a marker of active DNA synthesis, rose from 23.7 percent under artificial fluid conditions to 42.1 percent under inflammatory exposure. In endothelial cells the shift was even larger, climbing from 44.26 percent to 77.22 percent. When the researchers supplemented the culture media with recombinant epidermal growth factor, epithelial proliferation surged to 60 percent in artificial fluid and 71.7 percent in low-inflammatory fluid, while recombinant vascular endothelial growth factor likewise boosted endothelial proliferation. These results demonstrated that growth factors alone are sufficient to drive robust proliferation in these immature tissue cells.

The team then tested whether the response depended on specific growth factor receptors. Blocking the epidermal growth factor receptor with erlotinib numerically reduced epithelial proliferation back toward baseline, though the reduction did not reach statistical significance, so the authors were careful to note that epidermal growth factor receptor dependence was not formally established. In contrast, pharmacological inhibition of the vascular endothelial growth factor receptor significantly reduced endothelial proliferation, cutting it by nearly 40 percentage points relative to inflammatory fluid alone and restoring it toward control levels. Taken together, the pharmacological evidence was stronger for vascular endothelial growth factor pathway involvement than for the epidermal growth factor arm, but both experiments supported the general principle that inflammatory fluid acts through growth factor signaling.

Independent published datasets lent further biological plausibility to the model. Reanalysis of a publicly available RNA sequencing dataset from a neonatal rat hemorrhage model revealed a 12.7-fold elevation of Mki67, a marker of actively cycling cells, in choroid plexus tissue from hemorrhaged animals compared with controls. Separate work has shown that hemoglobin, the primary injury mediator in intraventricular hemorrhage, directly induces choroid plexus epithelial proliferation at concentrations 37 to 65 percent above control levels. Clinical proteomic studies have also documented a temporal arc: inflammatory cytokines such as interleukin-6 and tumor necrosis factor alpha are elevated during the acute phase of post-hemorrhagic hydrocephalus and decline with treatment, while transthyretin, a constitutive secretory protein of the choroid plexus, is sharply suppressed during the acute phase and recovers as treatment progresses. This pattern is consistent with a two-stage process in which acute inflammatory injury triggers proliferative activation and altered epithelial identity, followed by persistence of a structurally remodeled tissue state.

The developmental timing of these events may be the key to why prematurity matters so much. During late gestation, roughly 24 to 28 weeks of human development, the epithelial and endothelial compartments of the choroid plexus remain structurally immature and retain proliferative competence. The tissue expresses receptors for insulin-like growth factor 1, epidermal growth factor, and vascular endothelial growth factor, and it responds to these signals. In a full-term infant, these growth programs have largely quieted. But in a baby born at the edge of viability, an inflammatory surge from hemorrhage or infection may find a tissue still poised to grow, effectively prolonging developmental programs that should have been time-limited and producing structural expansion that persists beyond the acute inflammatory phase.

The authors are careful about what their findings do and do not show. Cerebrospinal fluid secretion rates and transporter activity were not directly measured, so choroid plexus enlargement on MRI should not be read as evidence of fluid overproduction, and hydrocephalus remains a multifactorial disease involving impaired fluid resorption, scarring, and pathway obstruction. The in vitro experiments used injury-derived inflammatory fluid as a model of growth factor-enriched exposure rather than hemorrhage-specific neonatal fluid, and the cross-sectional imaging design cannot establish whether enlargement persists over time. Still, if the findings are validated prospectively, choroid plexus thickness could become an adjunct imaging biomarker for identifying which infants with hemorrhage are most likely to progress to shunt dependency, and modulation of growth factor pathways within defined developmental windows could emerge as a testable therapeutic strategy for a condition that currently offers clinicians few options beyond surgery.

Subject of Research: Inflammatory cerebrospinal fluid exposure and choroid plexus remodeling in preterm acquired hydrocephalus

Article Title: Choroid plexus remodeling associated with inflammatory CSF exposure in preterm acquired hydrocephalus

Article References: Jison, G., Ramos, N., Cotrim-Gomes, F., Lee, S., Hanak, B. W., Muhonen, M., & Castañeyra-Ruiz, L. (2026). Choroid plexus remodeling associated with inflammatory CSF exposure in preterm acquired hydrocephalus. iScience, 29(10), Article 117759. https://doi.org/10.1016/j.isci.2026.117759

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117759

Keywords: hydrocephalus, choroid plexus, preterm infants, intraventricular hemorrhage, cerebrospinal fluid, inflammation, VEGF, EGF, MRI, proliferation, neonatology, iScience

The research, led by Franziska Köhler and Sonja Kallendrusch of Leipzig University and the HMU Health and Medical University, together with colleagues in Erfurt and Potsdam, compared two complementary patient-derived culture systems side by side. The first, patient-derived neurospheres (PDNS), are compact, free-floating spheroids grown from dissociated tumor cells under serum-free conditions. They offer a controlled, tumor-cell-intrinsic system in which variables can be manipulated with relative ease. The second, patient-derived tissue slice cultures (PDTC), preserve the tumor’s native architecture: thin, 350-micrometer slices of fresh surgical tissue are cultured at an air–liquid interface, retaining the extracellular matrix, vascular structures, immune cells, and stromal compartments that neurospheres inevitably discard. The team systematically exposed both models to two AAV serotypes, AAV2 and AAV6, each carrying a green fluorescent protein (GFP) reporter gene, and then tracked transduction with live confocal microscopy, quantitative PCR, flow cytometry, and immunofluorescence.

The technical rigor of the workflow is central to the study’s credibility. Thirteen surgical specimens from 25 collected samples ultimately entered the analysis, twelve of them IDH-wildtype glioblastomas under the WHO 2021 classification. Tissue not required for diagnosis was obtained with informed consent and ethical approval. For neurospheres, tumor tissue was mechanically and enzymatically dissociated with Accutase, filtered, and seeded at 100,000 cells per well; after 10 to 15 days, spheroids of 100 to 150 micrometers in diameter were ready for experiments. Slice cultures were punched into 2 to 3 millimeter discs and maintained on membrane inserts. After testing several media formulations, the researchers selected NeuroCult-XF, a serum-free proliferation medium, because it preserved tissue architecture and histopathological features better than serum-containing alternatives or other defined formulations. Blinded pathological review confirmed that fibrillary architecture and even characteristic gemistocytic features—enlarged cell bodies with eccentric nuclei—were retained in both models.

The cellular composition analysis revealed stark differences between the two systems, exactly as the team had hypothesized. Slice cultures retained expression of immune markers such as CD68, the vascular marker CD31, and the hypoxia marker HIF1α in organotypic patterns closely mirroring the original tumors. Neurospheres, by contrast, showed virtually no CD31, scarce and disorganized CD68-positive cells, and only weak HIF1α restricted to larger spheroids. More subtly, sphere-forming conditions selectively enriched stem-like populations: the mean SOX2-positive cell fraction rose from 0.114 in native tissue to 0.328 in PDNS, and Nestin positivity climbed from 0.132 to 0.393, while the mesenchymal marker CD44 fell. This finding matters because it confirms that neurosphere culture is not a neutral act of preservation but an active selection process that reshapes the cellular landscape before any vector is ever introduced.

When the vectors were applied, the serotype differences were unambiguous. In neurospheres, AAV6 drove significantly greater GFP expression than AAV2 across a dose range of 10^6 to 10^9 particles, with significant signal appearing as early as day 2 at higher doses, while AAV2 reached significance only at the highest dose after 5 days. Quantitative PCR corroborated the fluorescence data: five days after exposure to 10^8 particles, PLAT-normalized vector genome abundance averaged 146.7 copies per cell for AAV2 but 1,074.0 for AAV6 in EGF-containing medium. Notably, epidermal growth factor supplementation itself proved to be a critical experimental variable. Because AAV6 is known to use EGFR as a co-receptor for cellular entry, the team deliberately compared EGF-containing and EGF-free conditions. In neurospheres, EGF presence modified both the magnitude and temporal stability of AAV6-mediated GFP expression, and marker-defined analysis showed that EGF deprivation broadened GFP positivity across GFAP-, CD44-, Nestin-, and SOX2-positive populations, whereas under EGF-containing conditions only the Nestin-positive fraction showed significant GFP signal.

Exploratory flow cytometry from two independent experiments added an intriguing layer. GFP positivity was markedly enriched among SOX2-positive cells (40.8 percent versus 7.2 percent in SOX2-negative cells) and among cells triple-positive for CD133, Nestin, and SOX2 (41.0 percent versus 7.5 percent). The authors are careful to stress that these figures, based on limited sample numbers, are descriptive and cannot distinguish preferential vector uptake from differences in population abundance, survival, or promoter activity. Still, the pattern suggests that the most stem-like, aggressive cellular states within a glioblastoma may be among those most efficiently engaged by AAV6—a hypothesis with obvious therapeutic implications if it can be confirmed with larger datasets and mechanistic studies.

The most sobering results came from the tissue slice cultures. Overall GFP readouts were comparable to, or in some cases higher than, those in neurospheres, but interpatient variability was dramatically greater. Some patient specimens transduced robustly; others showed little or no detectable GFP despite identical conditions. Mean fluorescence intensities of 4,989 for AAV2, 4,232 for AAV6, and 6,448 for AAV6 without EGF in slice cultures contrasted with far lower values in matched neurospheres, but the spread between patients was wide enough that no single-patient result could be generalized. The authors argue that this variability should not be dismissed as experimental noise. Instead, it may represent a biologically faithful reflection of the heterogeneity that any real-world gene therapy for glioblastoma would confront. A vector that performs uniformly in a homogeneous spheroid panel may still fail in a substantial fraction of patients, and only multi-patient tissue-based validation can expose that vulnerability before clinical translation.

Spatial analysis added another dimension. In neurospheres, GFP-positive cells clustered predominantly at the spheroid periphery, consistent with diffusion-limited vector transport through densely packed three-dimensional tissue, although the authors caution that GFP fluorescence reflects the downstream consequence of delivery and expression rather than physical particle penetration itself. Slice cultures showed a more homogeneous distribution of fluorescence across the tissue, an effect not associated with preferential vascular localization and therefore likely driven by preserved microarchitecture rather than vascular entry routes. Equally notable was the observation of occasional GFP-positive cells in two samples of adjacent non-neoplastic brain tissue, a preliminary but cautionary signal that off-target transduction of tumor-adjacent cells can occur under experimental conditions, consistent with the broad central nervous system tropism of naturally occurring AAV serotypes.

The study’s self-imposed limitations are candidly acknowledged. Vector dose escalation could not be performed in slice cultures because of limited tissue availability, preventing fully matched comparisons between models. Flow cytometric findings rested on only two experiments. Vector genome quantification cannot distinguish entry, trafficking, persistence, or processing, and GFP expression additionally depends on transcription and translation. Slice cultures themselves, while architecturally faithful, remain ex vivo systems that cannot reproduce systemic immunity, vascular dynamics, or long-term tumor evolution. The significant increase in SOX2 expression observed in neurospheres after AAV6 treatment without EGF likewise supports an association rather than a demonstrated causal effect of vector exposure on tumor-cell state.

What the study ultimately delivers is a framework rather than a verdict. The authors explicitly do not claim that both models are mandatory for every preclinical AAV program, nor that either predicts in vivo performance. Instead, they advocate a question-driven approach: neurospheres, with their lower variability and experimental tractability, are well suited to initial serotype screening and dissection of tumor-cell-intrinsic effects; candidate vectors that pass that filter can then be challenged against panels of slice cultures from multiple patients, where preserved architecture, stromal and vascular compartments, and honest interpatient heterogeneity reveal how performance holds up in tissue that more closely resembles what a surgeon would leave behind in a resection cavity. In a disease where nearly every promising preclinical result has eventually collided with clinical reality, the insistence that model selection itself is a determinant of apparent vector efficacy may prove to be one of the more consequential methodological messages glioblastoma gene therapy has received in years.

Subject of Research: Preclinical evaluation of AAV-mediated gene delivery in patient-derived glioblastoma cultures

Article Title: Patient-derived tissue cultures complement neurospheres for preclinical evaluation of AAV-mediated gene delivery in glioblastoma

Article References: Köhler, F., Hess, K., Koloske, C., Gaunitz, F., Rosahl, S. K., Gerlach, R., & Kallendrusch, S. (2026). Patient-derived tissue cultures complement neurospheres for preclinical evaluation of AAV-mediated gene delivery in glioblastoma. Journal of Neuro-Oncology, 179(3), Article 93. https://doi.org/10.1007/s11060-026-05807-w

Image Credits: AI Generated

DOI: 10.1007/s11060-026-05807-w

Keywords: glioblastoma, AAV vectors, gene therapy, patient-derived models, neurosphere cultures, tissue slice cultures, tumor microenvironment, viral vectors, EGF, transduction, preclinical testing, gene delivery