DOI: 10.5061/dryad.4tmpg4fj7
title: N-cadherin dynamically regulates pediatric glioma cell migration in complex environments

description: ## 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 &gt;0.5, FDR &lt;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 represents no count. ###
 Description of the data and file structure The RNA sequencing data
 comprises two sheets in one Excel file. Table S2 is Control and N-cad
 shRNA samples, and Table S3 is Leader and follower cells. The first sheet
 presents the differential gene expression analysis results for paired
 samples from four biological replicates. Adjusted p-values are set to NA
 due to zero count or extreme count outliers. In the second sheet,
 normalized gene counts for each sample are provided. The first row in each
 sheet contains the sample names, with corresponding paired sample numbers
 indicated. The first column represents gene symbols.
description: 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 leaders and followers change position, leading to
 altered N-cadherin levels and organization. Together, the results suggest
 that pediatric glioma cells adapt to different microenvironments by
 regulating N-cadherin dynamics and cell-cell contacts.
description: 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 the Agilent Tapestation to assess cDNA product. To
 construct RNA sequencing libraries, we used Illumina’s Nextera XT kit to
 fragment the cDNA and add barcoded sequencing adapters. Differential gene
 expression analysis was performed with the DEseq2 for paired sample R
 package (Love et al., 2014). Genes with a Benjamini-Hochberg
 adjusted<em> p</em>-value &lt; 0.05 were defined as
 differentially expressed.