N-Cadherin Regulates 3D Tumor Spheroid Migration and Enhances Sensitivity to Tyrosine Kinase Inhibition in Neurofibromatosis Type 2

Introduction: : Vestibular Schwannoma (VS) is the 4th most common brain tumor that causes significant morbidities including deafness and brainstem compression. Current treatment options are limited and inadequate. Surgery carries significant risks and long-term morbidities, while tyrosine kinase inhibitors (TKIs) such as brigatinib and dasatinib are efficacious in only 10% of patients and their cumulative toxicity limits widespread use. These limitations underscore the urgent need for more effective and safer therapies. N-cadherin, a key cell–cell and cell-matrix adhesion molecule, regulates cancer cell cohesive migration and therapeutic responsiveness, yet its role in VS remains poorly defined. We hypothesize that N-cadherin preserves intercellular cohesion and coordinates intracellular signaling, and that its loss disrupts collective behavior and alters cellular response to tyrosine kinase inhibition. This study explores how N-cadherin regulates VS cell behavior and therapeutic efficacy of TKIs in a 3D tumor spheroid model and patient-derived primary VS cultures.

Materials and

Methods: : Primary VS cultures were analyzed for N-cadherin expression using immunofluorescence. N-cadherin knockdown was achieved in human NF2 schwannoma cells using lentiviral-shRNA. A 3D schwannoma spheroid model was established to quantify schwannoma migration in ECM and in astrocyte co-culture using time-lapse microscopy. Primary VS was analyzed for leader–follower dynamics and N-cadherin vesicle content. Recombinant IL-6 was used to induce stemness marker expression and assessed using qRT-PCR. Dasatinib, and brigatinib, synergy scores were assessed with concurrent N-cadherin inhibition (BZA, GC-4). The mechanistic effects N-cadherin inhibition on AKT, FAK and SRC signaling was quantified by western blot.

Results, Conclusions, and Discussions:: N-cadherin was variably expressed across VS, with tumors located in the internal auditory canal showing increased proportion of leader cells in the migrating front evidenced by increased intracellular vesicular staining and higher intracellular/punctate. This heterogeneous expression pattern may reflect the diverse molecular spectrum of NF2 tumors. (Fig. 1 A-B). In a 3D tumor spheroid model N-cadherin inhibition significantly enhanced migration of tumor spheroid on Matrigel but inhibited on co-cultured astrocytes (Fig. 1C-E). This suggests that N-cadherin may differentially regulate schwannoma cell migration in a context-dependent manner influenced by their tumor microenvironment. In primary VS culture, N-cadherin exhibited intracellular vesicle/puncta staining in leader cells compared to follower cells (Fig. 1 F-G), suggesting N-cadherin stabilizes leader/follower positioning in collective migration. Schwannoma cells treated with IL-6 exhibited a robust induction (> 4-fold) of stemness markers including OCT3/4, SOX2, KLF4, and cMYC. In contrast, N-cadherin knockdown by shRNA abrogated this induction (Fig. 1H-I). Drug synergy screening revealed that the combination of dasatinib and brigatinib exhibited strong synergistic effects. Notably, schwannoma cells expressing shNCad showed significant increased sensitivity to these drugs compared to shCtrl cells (dasatinib IC50: 0.57 μM vs. 1.58 μM; brigatinib IC50: 0.19 μM vs. 0.60 μM, Fig. 2A-B). TKI treatment downregulated AKT, SRC and FAK expression, which is further enhanced by N-cadherin shRNA knockdown, suggesting that N-cad inhibition enhanced cellular sensitivity to TKIs by suppressing phosphorylation and activation of AKT, SRC and FAK (Fig. 2C). Cell viability assays in 3 distinct primary VS demonstrated that simultaneous pharmacologic N-cadherin inhibition significantly enhanced anti-tumor efficacy of dasatinib and brigatinib (Fig. 2D-E). In conclusion, N-cadherin governs both structural and signaling dynamics in NF2 schwannoma cells by mediating their collective migration and maintaining stemness potential. Targeting N-cadherin sensitize schwannoma cells to tyrosine kinase inhibitors while disrupting adhesion-dependent survival, offering a potentially promising strategy to overcome therapeutic resistance in NF2.

Acknowledgements and/or References (Optional):: This work was supported by the National Institute on Deafness and Other Communication Disorders (NIDCD) grant 1K08DC020761-01 (Y.R.) and the American Neurotology Society Research Grant (Y.R.)