N-cadherin dynamically regulates pediatric glioma cell migration in complex environments

## N-cadherin dynamically regulates pediatric glioma cell migration in complex environments

Table S2. Control vs. N-cadherin shRNAs. We compared RNA transcriptomes from bulk cell populations, control or N-cadherin-depleted pediatric high-grade glioma cells. N-cad depletion decreased RNA expression but unaffected the expression of other cadherins and integrins except for CDH3. Differential gene expression analysis was performed with the DEseq2 for paired sample R package.

Table S3. Leader vs. follower cells. We compared RNA transcriptomes from migrating glioma leader and follower cells that we isolated by photoconversion and flow cytometry. 44 gene transcripts increased and 36 decreased in leader relative to followers out of 19,729 genes that were quantified (log2 fold-change >0.5, FDR <0.05). YAP-response genes and wound-healing genes were higher in leader than follower cells. Differential gene expression analysis was performed with the DEseq2 for paired sample R package. NA r..., Pediatric high-grade gliomas are highly invasive and essentially incurable. Glioma cells migrate between neurons and glia, along axon tracts, and through extracellular matrix surrounding blood vessels and underlying the pia. Mechanisms that allow adaptation to such complex environments are poorly understood. N-cadherin is highly expressed in pediatric gliomas and is associated with shorter survival. We found that inter-cellular homotypic N-cadherin interactions differentially regulate glioma migration according to the microenvironment, stimulating migration on cultured neurons or astrocytes but inhibiting invasion into reconstituted or astrocyte-deposited extracellular matrix. N-cadherin localizes to filamentous connections between migrating leader cells but to epithelial-like junctions between followers. Leader cells have more surface and recycling N-cadherin, increased YAP1/TAZ signaling, and increased proliferation relative to followers. YAP1/TAZ signaling is dynamically regulated as..., To compare RNA transcriptomes from bulk cell populations, control or N-cad shRNA cells were dissociated using Accutase at room temperature for 10 min and resuspended in HBSS. 500 cells were collected in the center of a 1.5 ml centrifuge tube containing 4.75 μl SMART-seq reaction buffer (Takara) using a BD FACSymphony S6 (BD Bioscience), avoiding cell loss on the tube walls. To compare RNA transcriptomes from leader and follower cells, approximately 90 spheroids of PBT-05 cells expressing histone H2B-Dendra2 cells were allowed to migrate for 24 hrs on laminin and photoconverted as above. After photoconversion, cells were dissociated with Accutase at room temperature, resuspended in HBSS and transferred to ice, and approximately 200 leader and follower cells were sorted into SMART-seq reaction buffer as above. Each experiment was performed on four different occasions.
RNA was prepared and cDNA was synthesized with the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara) and ran th...,