An ERK5-PFKFB3 axis regulates glycolysis and represents a therapeutic vulnerability in pediatric diffuse midline glioma.

Metabolic reprogramming in pediatric diffuse midline glioma is driven by gene expression changes induced by the hallmark histone mutation H3K27M, which results in aberrantly permissive activation of oncogenic signaling pathways. Previous studies of diffuse midline glioma with altered H3K27 (DMG-H3K27a) have shown that the RAS pathway, specifically through its downstream kinase, extracellular-signal-related kinase 5 (ERK5), is critical for tumor growth. Further downstream effectors of ERK5 and their role in DMG-H3K27a metabolic reprogramming have not been explored. We establish that ERK5 is a critical regulator of cell proliferation and glycolysis in DMG-H3K27a. We demonstrate that ERK5 mediates glycolysis through activation of transcription factor MEF2A, which subsequently modulates expression of glycolytic enzyme PFKFB3. We show that in vitro and mouse models of DMG-H3K27a are sensitive to the loss of PFKFB3. Multi-targeted drug therapy against the ERK5-PFKFB3 axis, such as with small-molecule inhibitors, may represent a promising therapeutic approach in patients with pediatric diffuse midline glioma.

Targeting mitochondrial energetics reverses panobinostat- and marizomib-induced resistance in pediatric and adult high-grade gliomas.

In previous studies, we demonstrated that panobinostat, a histone deacetylase inhibitor, and bortezomib, a proteasomal inhibitor, displayed synergistic therapeutic activity against pediatric and adult high-grade gliomas. Despite the remarkable initial response to this combination, resistance emerged. Here, in this study, we aimed to investigate the molecular mechanisms underlying the anticancer effects of panobinostat and marizomib, a brain-penetrant proteasomal inhibitor, and the potential for exploitable vulnerabilities associated with acquired resistance. RNA sequencing followed by gene set enrichment analysis (GSEA) was employed to compare the molecular signatures enriched in resistant compared with drug-naïve cells. The levels of adenosine 5'-triphosphate (ATP), nicotinamide adenine dinucleotide (NAD)+ content, hexokinase activity, and tricarboxylic acid (TCA) cycle metabolites required for oxidative phosphorylation to meet their bioenergetic needs were analyzed. Here, we report that panobinostat and marizomib significantly depleted ATP and NAD+ content, increased mitochondrial permeability and reactive oxygen species generation, and promoted apoptosis in pediatric and adult glioma cell lines at initial treatment. However, resistant cells exhibited increased levels of TCA cycle metabolites, which required for oxidative phosphorylation to meet their bioenergetic needs. Therefore, we targeted glycolysis and the electron transport chain (ETC) with small molecule inhibitors, which displayed substantial efficacy, suggesting that resistant cell survival is dependent on glycolytic and ETC complexes. To verify these observations in vivo, lonidamine, an inhibitor of glycolysis and mitochondrial function, was chosen. We produced two diffuse intrinsic pontine glioma (DIPG) models, and lonidamine treatment significantly increased median survival in both models, with particularly dramatic effects in panobinostat- and marizomib-resistant cells. These data provide new insights into mechanisms of treatment resistance in gliomas.

Predictors of first pass effect and effect on outcomes in mechanical thrombectomy for basilar artery occlusion.

INTRODUCTION: For patients undergoing endovascular thrombectomy (EVT), those who are recanalized with a successful single pass (first pass effect, FPE) have better functional outcomes than those who do not. There is a scarcity of data regarding predictors of FPE in basilar artery occlusion (BAO). We aim to determine what characteristics may predict FPE for posterior circulation thrombectomies in a cohort of patients undergoing EVT for BAO. METHODS: We reviewed prospectively-collected data for patients presenting to a comprehensive stroke center with BAO between December 2015 and April 2019. Patients were included in this study if they underwent manual aspiration thrombectomy for BAO. Patients were excluded if they had occlusions of the posterior cerebral or vertebral arteries or if they had tandem lesions. Patients were stratified by whether FPE or modified FPE (mFPE) was achieved, and multivariate logistic regression analyses were performed to identify predictors of FPE and the effect of FPE on clinical outcome. RESULTS: 100 patients with BAO underwent thrombectomy. Mean age was 64.7 ± 16.7, 42% were female, and median NIHSS was 20 (IQR 11-27). 33% met criteria for FPE and 60% for mFPE. Univariate analysis identified female gender, lack of IV-tPA use, pcASPECTS, atrial fibrillation, and hyperlipidemia as possible predictors of FPE. On multivariate analysis, age, pcASPECTs, atrial fibrillation, hyperlipidemia and IV-tPA use were not independent predictors of FPE or mFPE. Female gender was an independent predictor of mFPE (p = 0.02), but not FPE (p = 0.18). FPE was a predictor of mRS 0-2 at 90 days (p = 0.04). Predictors of mortality were age (p < 0.01), baseline NIHSS (p < 0.01) and mFPE (p = 0.01). CONCLUSION: In this cohort analysis of 100 patients with basilar artery occlusion undergoing manual aspiration thrombectomy, female gender was associated with mFPE but not FPE. Previously-reported anterior circulation FPE predictors including age, ASPECTS and atrial fibrillation were not predictors of FPE in this cohort of patients with BAO.

Outcome Prediction in Patients with Severe Traumatic Brain Injury Using Deep Learning from Head CT Scans.

Background After severe traumatic brain injury (sTBI), physicians use long-term prognostication to guide acute clinical care yet struggle to predict outcomes in comatose patients. Purpose To develop and evaluate a prognostic model combining deep learning of head CT scans and clinical information to predict long-term outcomes after sTBI. Materials and Methods This was a retrospective analysis of two prospectively collected databases. The model-building set included 537 patients (mean age, 40 years ± 17 [SD]; 422 men) from one institution from November 2002 to December 2018. Transfer learning and curriculum learning were applied to a convolutional neural network using admission head CT to predict mortality and unfavorable outcomes (Glasgow Outcomes Scale scores 1-3) at 6 months. This was combined with clinical input for a holistic fusion model. The models were evaluated using an independent internal test set and an external cohort of 220 patients with sTBI (mean age, 39 years ± 17; 166 men) from 18 institutions in the Transforming Research and Clinical Knowledge in Traumatic Brain Injury (TRACK-TBI) study from February 2014 to April 2018. The models were compared with the International Mission on Prognosis and Analysis of Clinical Trials in TBI (IMPACT) model and the predictions of three neurosurgeons. Area under the receiver operating characteristic curve (AUC) was used as the main model performance metric. Results The fusion model had higher AUCs than did the IMPACT model in the prediction of mortality (AUC, 0.92 [95% CI: 0.86, 0.97] vs 0.80 [95% CI: 0.71, 0.88]; P < .001) and unfavorable outcomes (AUC, 0.88 [95% CI: 0.82, 0.94] vs 0.82 [95% CI: 0.75, 0.90]; P = .04) on the internal data set. For external TRACK-TBI testing, there was no evidence of a significant difference in the performance of any models compared with the IMPACT model (AUC, 0.83; 95% CI: 0.77, 0.90) in the prediction of mortality. The Imaging model (AUC, 0.73; 95% CI: 0.66-0.81; P = .02) and the fusion model (AUC, 0.68; 95% CI: 0.60, 0.76; P = .02) underperformed as compared with the IMPACT model (AUC, 0.83; 95% CI: 0.77, 0.89) in the prediction of unfavorable outcomes. The fusion model outperformed the predictions of the neurosurgeons. Conclusion A deep learning model of head CT and clinical information can be used to predict 6-month outcomes after severe traumatic brain injury. © RSNA, 2022 Online supplemental material is available for this article. See also the editorial by Haller in this issue.

Disrupted Surfaces of Porous Membranes Reduce Nuclear YAP Localization and Enhance Adipogenesis through Morphological Changes.

The disrupted surface of porous membranes, commonly used in tissue-chip and cellular coculture systems, is known to weaken cell-substrate interactions. Here, we investigated whether disrupted surfaces of membranes with micron and submicron scale pores affect yes-associated protein (YAP) localization and differentiation of adipose-derived stem cells. We found that these substrates reduce YAP nuclear localization through decreased cell spreading, consistent with reduced cell-substrate interactions, and in turn enhance adipogenesis while decreasing osteogenesis.

Loss of MAT2A compromises methionine metabolism and represents a vulnerability in H3K27M mutant glioma by modulating the epigenome.

Diffuse midline gliomas (DMGs) bearing driver mutations of histone 3 lysine 27 (H3K27M) are incurable brain tumors with unique epigenomes. Here, we generated a syngeneic H3K27M mouse model to study the amino acid metabolic dependencies of these tumors. H3K27M mutant cells were highly dependent on methionine. Interrogating the methionine cycle dependency through a short-interfering RNA screen identified the enzyme methionine adenosyltransferase 2A (MAT2A) as a critical vulnerability in these tumors. This vulnerability was not mediated through the canonical mechanism of MTAP deletion; instead, DMG cells have lower levels of MAT2A protein, which is mediated by negative feedback induced by the metabolite decarboxylated S-adenosyl methionine. Depletion of residual MAT2A induces global depletion of H3K36me3, a chromatin mark of transcriptional elongation perturbing oncogenic and developmental transcriptional programs. Moreover, methionine-restricted diets extended survival in multiple models of DMG in vivo. Collectively, our results suggest that MAT2A presents an exploitable therapeutic vulnerability in H3K27M gliomas.

First Female Neurosurgeon in the United States: Dorothy Klenke Nash, MD.

Dr Dorothy Klenke Nash (1898-1976) became the first female neurosurgeon in the United States in 1928 and maintained her status as the country's only female neurosurgeon until 1960. She graduated with her medical degree from the Columbia College of Physicians and Surgeons in 1927 and then trained at the Neurologic Institute of New York under Dr Byron Stookey. During her training, she contributed to the advancement of neurosurgical practice through academic research. In 1931, she married Charles B. Nash, and together they had 2 children, George (1932) and Dorothy Patricia (1937). Dr Nash became a senior surgeon at St. Margaret's Hospital in Pittsburgh in 1942. Shortly thereafter, she joined the inaugural University of Pittsburgh Department of Neurosurgery led by Dr Stuart N. Rowe and became an instructor of neurosurgery at the University of Pittsburgh School of Medicine. In acknowledgment of her advocacy for public access to services for mental health and cerebral palsy, Dr Nash was recognized as a Distinguished Daughter of Pennsylvania (1953) and honored by Mercy Hospital (1957), Bryn Mawr College (1960), and Columbia University (1968). She retired from neurosurgical practice in 1965, at which time she devoted herself to her grandchildren and her Catholic faith. She died on March 5, 1976 at the age of 77. With unwavering tenacity, Dr Nash paved the way for all women in neurosurgery.

Socioeconomic Implications of Professional Relationships within Modern Care Delivery Systems.

No physician can successfully deliver high-value patient care in the modern-day health care system in isolation. Delivery of effective patient care requires integrated and collaborative systems that depend on dynamic professional relationships among members of the health care team. An overview of the socioeconomic implications of professional relationships within modern care delivery systems and potential employment models is presented.

Does stroke etiology influence outcome in the posterior circulation? An analysis of 107 consecutive acute basilar occlusion thrombectomies.

OBJECTIVE: Acute basilar artery occlusion (BAO) harbors a more guarded prognosis after thrombectomy compared with anterior circulation large-vessel occlusion. Whether this is a function of a greater proportion of atherosclerotic/intrinsic lesions is not well studied. The authors aimed to elucidate the prevalence and predictors of intracranial intrinsic atherosclerotic disease in patients with acute BAO and to compare angiographic and clinical outcomes between patients with BAO secondary to embolic versus intrinsic disease. METHODS: A prospectively maintained stroke database was reviewed for all patients presenting between January 2013 and December 2019 to a tertiary care academic comprehensive stroke center with acute, nontandem BAO. Patient data were extracted, subdivided by stroke mechanism and treatment modality (embolic [thrombectomy only] and intrinsic [thrombectomy + stenting]), and angiographic and clinical results were compared. RESULTS: Of 107 patients, 83 (78%) had embolic occlusions (thrombectomy only) and 24 (22%) had intrinsic disease (thrombectomy + stenting). There was no significant difference in patient age, presenting National Institutes of Health Stroke Scale score, time to presentation, selected medical comorbidities (hypertension, hyperlipidemia, diabetes, and atrial fibrillation), prior stroke, and posterior circulation Alberta Stroke Program Early CT Score. Patients with intrinsic disease were more likely to be active smokers (50% vs 26%, p = 0.04) and more likely to be male (88% vs 48%, p = 0.001). Successful recanalization, defined as a modified Thrombolysis in Cerebral Infarction (mTICI) grade of 2b or 3, was achieved in 90% of patients and did not differ significantly between the embolic versus intrinsic groups (89% vs 92%, p > 0.99). A 90-day good outcome (modified Rankin Scale [mRS] score 0-2) was found in 37% of patients overall and did not differ significantly between the two groups (36% vs 41%, p = 0.41). Mortality was 40% overall and did not significantly differ between groups (41% vs 36%, p = 0.45). CONCLUSIONS: In the current study, demographic and clinical results for acute BAO showed that compared with intrinsic disease, thromboembolic disease is a more common mechanism of acute BAO, with 78% of patients undergoing thrombectomy alone. However, there was no significant difference in revascularization and outcome results between patients with embolic disease and those with intrinsic disease.

Evaluation of free-hand screw placement in cervical, thoracic, and lumbar spine by neurosurgical residents.

BACKGROUND: Knowledge of free-hand screw technique remains critical to adequately train neurosurgical residents. The purpose of this study was to evaluate the accuracy of screw placement via the free-hand technique in lumbar, thoracic, and cervical spine by neurosurgical residents completing an enfolded spine fellowship. METHODS: Medical records of all patients who underwent free-hand screw placement at all spinal levels over a 6-month period by senior neurosurgical residents enrolled in an in-folded spine fellowship were retrospectively reviewed. Postoperative CT images were assessed for presence and direction of cortical breach. RESULTS: Twenty-six patients underwent 162 free-hand screw placements. The most commonly placed screws were cervical lateral mass screws (n = 69), thoracic (n = 41), and lumbar pedicle screws (n = 41). The most common indication for surgery was deformity (n = 22), followed by infection (n = 2) and trauma (n = 2). Fifty-five breaches were identified in 44 (27 %) screws placed in 21 patients (81 %). Anterior breach was identified in 22 cases (40.0 %), lateral in 12 (23.6 %), superior in 7 (12.7 %), and inferior in 7 (12.7 %), and medial in 6 (10.9 %). The most common level of breach was observed in cervical lateral mass screws (n = 19, 43 %) and least common in C2 pars screws (n = 1, 2%). With an average length of follow up of 12.1 ± 7.7 months of follow-up, no clinical sequalae of screw breach was observed. CONCLUSIONS: Despite the high prevalence of screw breach using the free-hand technique by neurosurgical residents, the absence of clinical sequelae implies safety and emphasizes the importance of early exposure to this technique during neurosurgical residency training.

Biographies of international women leaders in neurosurgery.

We received so many biographies of women neurosurgery leaders for this issue that only a selection could be condensed here. In all of them, the essence of a leader shines through. Many are included as "first" of their country or color or other achievement. All of them are included as outstanding-in clinical, academic, and organized neurosurgery. Two defining features are tenacity and service. When faced with shocking discrimination, or numbing indifference, they ignored it or fought valiantly. When choosing their life's work, they chose service, often of the most neglected-those with pain, trauma, and disability. These women inspire and point the way to a time when the term "women leaders" as an exception is unnecessary.-Katharine J. Drummond, MD, on behalf of this month's topic editors.

Telescoping flow diverters for a pediatric fusiform distal anterior cerebral artery aneurysm: technical case report.

BACKGROUND AND IMPORTANCE: Pediatric intracranial aneurysms are rare, with fusiform aneurysms of the distal anterior circulation even more so. A limited number of prior reports detail the management of this pathology. CLINICAL PRESENTATION: We report a case of a 15-year-old boy presenting with new-onset headache found to have a 3.1 cm × 1.4 cm × 1.4 cm fusiform partially thrombosed aneurysm of the A2 segment of the left anterior cerebral artery. Subsequent treatment with flow diversion resulted in the placement of five telescoping PEDs (2.5 mm × 20 mm, 2.75 mm × 20 mm, 2.75 mm × 20 mm, 3 mm × 25 mm, and 3 mm × 20 mm) from the left pericallosal artery to the left A1 segment. Catheter-based angiography at 6 months post-placement indicated normal vessel caliber, no residual aneurysm, and no in-stent stenosis. The patient's headache resolved after treatment. CONCLUSION: Telescoping PEDs are a feasible low-risk treatment option for long-segment fusiform distal ACA aneurysms in children that can have optimal clinical and radiographic outcomes.

Rare Transsellar Collateral Artery and Secondary Ophthalmic Artery Aneurysm in a Young Adult with Internal Carotid Artery Aplasia.

Internal carotid artery (ICA) aplasia is a rare congenital abnormality in which the third aortic arch fails to develop. Most collateral circulation will originate from the circle of Willis, with other sites being rare. Changes in flow through the collateral vasculature result in aneurysm formation at a young age. A 38-year-old woman had an incidental diagnosis of left ICA aplasia and right ophthalmic artery aneurysm. Arteriography demonstrated that the left supraclinoid ICA originated from the right cavernous ICA and appeared as an anomalous hypertrophied transsellar artery on angiography. Her flow-related aneurysm was treated uneventfully. The present case demonstrates an extremely rare Lie type D collateralization pattern and secondary flow-rated aneurysm in the context of ICA aplasia. Patients identified with this anatomic variant should be monitored for subsequent aneurysm formation. Identification of this variant before endovascular and transsphenoidal procedures is crucial to prevent major intracranial vessel injury.

Successful reperfusion, rather than number of passes, predicts clinical outcome after mechanical thrombectomy.

INTRODUCTION: For patients undergoing mechanical thrombectomy, numerous (>3) thrombectomy passes may be harmful. However, non-recanalization leads to poor outcomes. For patients requiring multiple thrombectomy passes to achieve reperfusion, it remains unclear if the risk/benefit ratio favors recanalization. OBJECTIVE: To test the hypothesis that the benefits afforded by successful reperfusion outweigh the risk conveyed by the numerous passes required. METHODS: We retrospectively reviewed prospectively collected data for patients presenting to a comprehensive stroke center with anterior circulation large vessel occlusion (ACLVO) and undergoing thrombectomy requiring more than one pass over 24 months. We stratified patients into three groups: group 1 (successful reperfusion in 2-3 passes), group 2 (successful reperfusion in ≥4 passes), and group 3 (unsuccessful reperfusion). RESULTS: 250 patients with ACLVO constituted the study cohort. Despite similar demographics, group 2 patients had better clinical outcomes than those in group 3 at 24 hours (National Institutes of Health Stroke Scale (NIHSS) score 13.5 vs 19.1, p<0.001) and at 90 days (modified Rankin Scale score 0-2 rates of 31.1% vs 0.0%, p=0.006) On multivariate logistic regression analysis, age (p=0.034), Alberta Stroke Program Early CT Score (p<0.01), NIHSS score (p=0.02), and parenchymal hematoma type 2 (p=0.015) were significant predictors of functional independence among those who achieved successful reperfusion, but the number of passes required did not predict outcome for these patients (p=0.74). CONCLUSION: Patients who achieve successful reperfusion after many passes have better clinical outcomes than those who do not, despite the number of passes and procedural time required. The number of passes required to achieve successful reperfusion beyond the first pass is not a predictor of functional independence.

A History of the Lobes of the Brain.

The division of the cerebrum into 5 lobes is widely accepted in the scientific community. Despite this, a history of the lobes of the brain has not been discussed in the literature. Therefore, this article recounts this history with emphasis on the contributions of Thomas Willis (1664), Felix Vicq d'Azyr (1796), Johann-Christian Reil (1796), François Chaussier (1807), and Louis Pierre Gratiolet and François Leuret (1857) into one of the most widely accepted concepts in neuroanatomy.

Stentriever salvage after failed manual aspiration thrombectomy.

INTRODUCTION: Manual aspiration thrombectomy (MAT) and stent retriever mediated aspiration thrombectomy (SMAT) are well described reperfusion strategies for large vessel occlusions. This study aims to identify predictors of successful crossover to SMAT after failed first pass MAT. METHODS: Prospectively collected data for patients with acute large vessel occlusions undergoing thrombectomy over a 23 month period at a comprehensive stroke center were reviewed. The primary outcome was successful removal of the index clot with resultant Thrombolysis in Cerebral Infarction 2b or greater reperfusion at any point after a failed initial MAT attempt, and multivariate logistic regression analyses were performed to determine predictors of successful crossover to SMAT. RESULTS: Of 433 large vessel thrombectomies, 319 underwent first pass MAT, and 113 patients required a repeated pass for the index thrombus. Second pass MAT was performed in 77% of cases and was successful in 54%; second pass SMAT was performed in 23% of cases and was successful in 73% (P=0.11). Third pass MAT was employed in 45% of cases and was successful in 43% while SMAT was performed in 55% of cases and was successful in 77% (P=0.03). Overall, 12% of patients undergoing MAT on the first pass crossed over to successful SMAT. Predictors of successful crossover were internal carotid artery (ICA) location and higher presenting National Institutes of Health Stroke Scale (NIHSS) score. CONCLUSION: After failed first pass MAT, subsequent passes with SMAT had higher rates of successful index clot removal; patients with a higher initial NIHSS score and ICA clot location should be considered for early crossover or even initial SMAT for their clots.

Large-bore aspiration catheter selection does not influence reperfusion or outcome after manual aspiration thrombectomy.

INTRODUCTION: Various large-bore catheters can be employed for manual aspiration thrombectomy (MAT); clinical differences are rarely explored. METHODS: Prospectively collected demographic, angiographic, and clinical data for patients with acute internal carotid artery, middle cerebral artery M1, or basilar occlusions undergoing MAT over 23 months at a comprehensive stroke center were reviewed. We excluded patients in stentriever-based randomized trials/registries. The four most commonly utilized aspiration catheters were analyzed, and multivariate logistic regression analyses were performed to determine the effect of primary aspiration catheter choice on first-pass success, final reperfusion, and modified Rankin Scale (mRS) score at 90 days. RESULTS: Of 464 large vessel thrombectomies, 180 were performed via MAT on the first pass with one of four catheters. First-pass success was achieved in 42% of cases overall; this rate did not differ significantly between catheters: 50% for Sofia, 45% for CAT6, 40% for 0.072 inch Navien, and 36% for ACE68, p=0.67. Final Thrombolysis in Cerebral Infarction 2b or 3 reperfusion was achieved in 94% of cases overall: 97% of cases with CAT6, 95% with Sofia, 92% with Navien, and 92% with ACE68, p=0.70. Mean number of passes for index thrombus (2.0 overall), median procedure time (32 min overall), 90-day good outcome (mRS 0-2, mean 36%), and 90-day mortality (mean 27%) did not differ significantly between patients treated with different initial catheters. CONCLUSION: Among large-bore aspiration catheters, catheter selection is not an independent predictor of first-pass success, final reperfusion, or clinical outcome.

Porous Substrates Promote Endothelial Migration at the Expense of Fibronectin Fibrillogenesis.

Porous substrates have gained increased usage in cell studies and tissue mimetic applications because they can partition distinct cell types while still allowing important biochemical crosstalk. In the presented work, we investigated how porous substrates with micron and submicron features influence early cell migration and the associated ECM establishment, which can critically affect the rate of cell coverage on the substrate and the ensuing tissue organization. We showed through time-lapse microscopy that cell speed and migratory distance on membranes with 0.5 µm pores were nearly two-fold of those observed on nonporous membranes, while values on membranes with 3.0 µm pores fell in between. Although the cell directionality ratio and the persistence time was unaffected by the presence of pores, the cells did exhibit directionality preferences based on the hexagonal pore patterning. Fibronectin fibrillogenesis exhibited a distinct inverse relationship to cell speed, as the fibrils formed on the nonporous control were significantly longer than those on both types of porous substrates. We further confirmed on a per cell basis that there is a negative correlation between fibronectin fibril length and cell speed. The observed trade-off between early cell coverage and ECM establishment thus warrants consideration in the selection or the engineering of the ideal porous substrate for tissue mimetic applications and may help guide future cell studies.

Membrane Pore Spacing Can Modulate Endothelial Cell-Substrate and Cell-Cell Interactions.

Mechanical cues and substrate interaction affect the manner in which cells adhere, spread, migrate and form tissues. With increased interest in tissue-on-a-chip and co-culture systems utilizing porous membranes, it is important to understand the role of disrupted surfaces on cellular behavior. Using a transparent glass membrane with defined pore geometries, we investigated endothelial fibronectin fibrillogenesis and formation of focal adhesions as well as development of intercellular junctions. Cells formed fewer focal adhesions and had shorter fibronectin fibrils on porous membranes compared to non-porous controls, which was similar to cell behavior on continuous soft substrates with Young's moduli seven orders of magnitude lower than glass. Additionally, porous membranes promoted enhanced cell-cell interactions as evidenced by earlier formation of tight junctions. These findings suggest that porous membranes with discontinuous surfaces promote reduced cell-matrix interactions similarly to soft substrates and may enhance tissue and barrier formation.