Figures and data
GO-CRISPR screens implicate axon guidance pathways as supporting EOC spheroid cell viability.
A) iOvCa147, TOV1946, or OVCAR8 cells were cultured under adherent conditions (Adh) or in suspension to induce spheroids (Sph). Lysates were prepared and analyzed by western blotting for phosphorylated and total levels of ERK and p38. B) Flow chart of GO-CRISPR screening used for each of iOvCa147, TOV1946, or OVCAR8 control cells or that express Cas9. Cas9-positive cells (top row) and Cas9-negative cells (bottom row) were transduced with the GeCKO v2 pooled sgRNA library. After antibiotic selection, cells were expanded under adherent culture conditions (T0) before being transferred to suspension culture conditions to induce spheroid formation and select for cell survival. After 48 hours spheroids were transferred to standard plasticware to isolate viable cells (Tf). Red arrows indicate the relevant comparisons of sgRNA sequence abundance that were made to analyze screen outcomes. C) Scatter plots representing spheroid score (Tf) on y-axis and adherent (T0) score on x-axis calculated by TRACS for each gene in each cell line (iOvCa147, TOV1946, OVCAR8). Colored data points represent genes with ER < 1 and padj < 0.05. D) Venn diagram illustrating overlap of genes identified as supporting cell viability in suspension culture from iOvCa147, TOV1946, and OVCAR8 cells. E) Graph depicting enriched pathways from ConsensusPathDB using the 1382 commonly identified genes from D. Categories are ranked by q-value.
Axon guidance pathway components are upregulated in iOvCa147 spheroid cells in a DYRK1A dependent manner.
A) RNA was isolated from iOvCa147 cells following culture under adherent conditions or in suspension conditions to induce spheroid formation for 6 hours. Triplicate independent cultures were processed for RNA-seq. A volcano plot shows differentially expressed genes in spheroid cells compared to adherent. 1,937 genes were found to be downregulated in iOvCa147 spheroid cells (log2 fold change < 1, padj < 0.05, FDR 10%, highlighted in grey) and 1,834 genes were upregulated (log2 fold change > 1, padj < 0.05, FDR 10%, highlighted in blue). B) Top 15 most significantly enriched pathways (padj < 0.05) whose genes were upregulated in suspension culture compared to adherent in RNA-seq analysis. C) A volcano plot showing differentially expressed genes in DYRK1A−/− spheroid cells compared to iOvCa147 spheroid cells. 744 genes were found to be downregulated in DYRK1A−/− spheroid cells (log2 fold change < 1, padj < 0.05, FDR 10%, highlighted in red) and 96 genes were upregulated (log2 fold change > 1, padj < 0.05, FDR 10%, highlighted in grey). D) Top 15 most significantly enriched pathways (padj < 0.05) that were represented by downregulated genes in DYRK1A-/- suspension culture compared to control cells in suspension. E) Venn diagram depicting overlapping enriched pathways identified in GO-CRISPR screens in green; enriched pathways identified in upregulated genes in parental iOvCa147 spheroid cells in blue; and enriched pathways identified in downregulated genes in DYRK1A−/− spheroid cells in red. 78 pathways were commonly enriched in all three datasets (shown in yellow). F) Top 10 most significantly enriched pathways (padj < 0.05) among the 78 identified in C.
Expression of Netrin ligands and their dependence receptors is increased in suspension culture.
A-C) RT-qPCR was performed to quantitate mRNA expression levels of Netrin ligands and receptors in three different HGSOC cell lines. Relative expression of the indicated transcripts is shown for suspension culture conditions. All experiments for performed in at least triplicate replicates. D-F). Immunohistochemical staining was performed for the indicated proteins using spheroids isolated from HGSOC patient ascites that was fixed and paraffin embedded. Omission of primary antibody was used a control for background staining for each patient sample. Scale bar = 100μm
Netrin ligands and their receptors are required for spheroid cell survival.
A) Illustration of netrin ligands, receptors, and other intracellular signaling molecules that are included in the Axon Guidance pathway category. The frequency of their identification in CRISPR screens is illustrated by shading and indicates how many cell lines had a negative enrichment ratio for a given component. B) The indicated ovarian cancer cell lines were infected with lentiviruses expressing sgRNAs directed against the indicated Netrin signaling genes. Cells were transferred to suspension culture conditions to induce spheroid formation for 72 hours and then returned to adherent conditions for 24 hours to facilitate reattachment. Re-attached cells were stained with Crystal Violet and retained dye was extracted and quantitated to measure relative survival. Each cell-gene combination was assayed in at least three biological replicates, averaged, and viability is displayed as a bubble plot. Mean survival for a given cell-gene combination was compared with GFP control gRNA transduced cells using anova and significance levels are illustrated by bubble size. Inviable cell-gene combinations are depicted as empty white spaces. C) Cultures of the indicated cell lines were serum starved and transferred to suspension culture conditions and stimulated. Netrin-1 signaling was analyzed by SDS-PAGE and western blotting for phospho-ERK, ERK, and tubulin. D) Suspension cultures of OVCAR8, or knock out derivatives, were harvested and analyzed for relative phosphorylation levels of ERK by western blotting. Total ERK and Tubulin blotting serves as expression and loading controls. E) Netrin-1 signaling in OVCAR8 cells deleted for all UNC5 receptors (UNC5 4KO) was tested by serum starving cells, transferring them to suspension, and stimulating with Netrin-1 as before. Western blotting for phospho-ERK, ERK, and tubulin were as before. F) OVCAR8 cells were seeded in suspension culture and treated with the MEK inhibitor PD184352 (PD) or DMSO vehicle for up to 72 hours. Mean viability was compared by anova (***P<0.005). G) Model summarizing the roles of Netrin ligands, receptors, and downstream targets MEK and ERK in dormant survival signaling.
Netrin ligand overexpression is associated with poor clinical outcome in HGSOC.
A) TCGA RNA-seq data for HGSOC patients (TCGA PanCancer Atlas study) was used to identify high Netrin-1 or -3 and low Netrin-1 or -3 expressing patients (high expressing are above z-score 1.2 and low are below 1.2). Overall survival was used to construct Kaplan-Meier plots and survival was compared using a logrank test. B) OVCAR8 cells were stably transduced with lentiviral constructs to overexpress epitope tagged Netrin-1 or -3. Western blotting for Netrins, Myc-tags, and tubulin were used to determine relative expression levels of both Netrins in these cell populations and vector controls. C) Control and Netrin overexpressing cells were transferred to suspension culture conditions to form spheroids, and replated to assay for viability. Mean viability and standard deviation is shown for each. Anova was used to compare survival (* P<0.05, *** P<0.001). D) Reattached spheroids were fixed and stained with Crystal Violet to examine size and abundance in control and Netrin-1 or -3 overexpression.
Netrin overexpression causes increased dissemination of tumor nodels.
A) Netrin overexpressing and control OVCAR8 cells were injected into the intraperitoneal space of female NOD/SCID mice. Mice were euthanized following 35 days and analyzed for disease burden by necropsy and histopathology. B) The spread of cancer to the diaphragm, liver, omentum, and mesometrium was determined from necropsies and colors used in the anatomical schematic correspond with petal plots for each genotype of cells. Petal plots illustrate frequency of mice bearing disease spread to a particular location. The radius of color fill is proportional to the total number of animals with tumor nodules found in that location. C) Photographs of necropsy findings in the mesometrium. Locations of ovaries (*), oviduct (yellow arrow), and uterine horn (green arrow) are indicated in each case. Tumor nodules are indicated by white arrows. D) The number of mesometrium associated tumor nodules was determined for each mouse. Mean values are indicated and differences between genotype were determined by anova (* P<0.05, ** P<0.01). E) Photographs of necropsy findings in the liver. Location of sternum is indicated (#) in each image. Tumor nodules are indicated by white arrows. F) The number of liver associated tumor nodules was determined for each mouse. Mean values are indicated and differences between genotype were determined by anova (* P<0.05, ** P<0.01). G and H) Histology of mesometrial tumor nodules are shown. Serial sections were stained with H&E, or with the indicated antibodies for immunohistochemistry. Ovaries are indicated (*), as are the oviduct (yellow arrow), the uterus (green arrow), and the tumor nodule (white arrow). Scale bar = 2mm
