GO-CRISPR screens implicate axon guidance pathways as supporting HGSOC spheroid cell viability.

A) iOvCa147, TOV1946, or OVCAR8 cells were cultured under adherent conditions (Adh) or in suspension to induce spheroid formation (Sph). Lysates were prepared and analyzed by western blotting for the proliferation marker Ki67 and the quiescence marker p130, and phosphorylated and total levels of ERK and p38. Tubulin was blotted as a loading control. B) Flow chart of GO-CRISPR screening used for each of iOvCa147, TOV1946, or OVCAR8 control cells and derivatives of each 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 (0 hours) 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. Red arrows indicate the relevant comparisons of sgRNA sequence abundance that were made to analyze screen outcomes. Genes with relatively greater effect on viability in suspension were selected by comparing their scores between adherent and suspension conditions and considering genes with an enrichment ratio (ER) of <1. C) Scatter plots representing the spheroid score on y-axis and adherent score on the 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 (paired t-test). 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. Tiers of Reactome categories are indicated by shading.

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 (Wald test), 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. 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 (Wald test), FDR 10%, highlighted in red) and 96 genes were upregulated (log2 fold change > 1, padj < 0.05 (Wald test), FDR 10%, highlighted in grey). C) Top 15 most significantly enriched pathways 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 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 compared with adherent. All experiments were performed in at least triplicate biological replicates. Means were compared with the same gene in adherent culture using a one way anova (*P<0.05, ****P<0.0001).

Netrins are expressed in dormant patient derived spheroids.

Spheroids were isolated from ascites of HGSOC patients and processed for immunohistochemical staining. A-B) Serial sections from solid HGSOC tumors were stained with antibodies to p130, Ki67, or a no primary antibody control (NP). Scale bar = 100μm for upper panels and 2mm for lower panels. C-E) Serial spheroid sections from the indicated patients were stained with Ki67 and p130. Sections with dense positive nuclear staining are indicated with white arrows. Scale bar = 100μm. F-H). Immunohistochemical staining was performed for the indicated proteins on serial sections of ascites derived spheroids. Omission of primary antibody was used a control for background staining for each patient sample (NP). 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 an enrichment ratio <1 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 one way anova and significance levels are illustrated by bubble size. Inviable cell-gene combinations are depicted as empty spaces. C) Cultures of the indicated cell lines were cultured in suspension for five days and stimulated with 0.5 μg/mL Netrin-1. Netrin-1 signaling was analyzed by SDS-PAGE and western blotting for phospho-ERK, ERK, and tubulin. D) Quiescent adherent OVCAR8 cells were stimulated with 0.5 μg/mL Netrin-1 or 0.5 μg/mL EGF, and compared with OVCAR8 cells in suspension stimulated as in C. Extracts were prepared and blotted for phospho-ERK, ERK, and tubulin. E) 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 serve as expression and loading controls. F) Netrin-1 signaling in OVCAR8, UNC5 4KO and DDN 3KO derivatives was tested by transferring cells to suspension and stimulating with Netrin-1 as before. Western blotting for phospho-ERK, ERK were also as before. G) OVCAR8 cells were seeded in suspension culture and treated with the MEK inhibitors PD184352, Trametinib or DMSO vehicle for 72 hours. Mean viability was determined by re-attachment and compared by one way anova (****P<0.0001). H) OVCAR8 cells were cultured in suspension and treated with Trametinib or DMSO vehicle as in G. Viability was determined by trypan blue dye exclusion and compared by one way anova (**P<0.01). I) Extracts were prepared from Trametinib and control treated spheroid cells and blotted for phospho-ERK, ERK, and tubulin. J) Model summarizing the roles of Netrin ligands, receptors, and downstream targets MEK and ERK in dormant survival signaling.

Loss of Netrin signaling reduces spheroids and prolongs survival.

A) Control OVCAR8L (negative control CRISPR targeted cells) and UNC5 4KO cells were injected into the peritoneal space of female NOD/SCID mice. After two weeks, animals were euthanized and engrafted cells were collected with abdominal washes. B) RT-qPCR was used to compare gene expression of the indicated cell cycle markers in RNA extracted from proliferating cells in culture before engraftment and compared with RNA obtained from spheroids in xenografted mice. Technical replicates of RNA derived from three different mice is shown. C) Spheroids obtained from abdominal washes from each mouse were plated in adherent conditions to compare their abundance between control and UNC5 4KO genotypes. One example of each genotype of cell is shown. Crystal violet staining and dye extraction were used to quantitate biomass and averages were compared by one way anova (n=6, *P<0.05). D) DNA was extracted from cells collected in abdominal washes and human Alu repeats were detected by qPCR to quantitate and compare human cancer cells. Means were compared by one way anova (n=6, **P<0.01). E) Control OVCAR8 and UNC5 4KO cells were xenografted as above and mice were monitored until humane endpoint. F) Kaplan-Meier analysis of survival for mice engrafted with the indicated genotypes of cells. Survival was compared by logrank test.

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). 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 Actin 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. One way 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 nodules.

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 plot radius illustrates 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 one way 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 one way anova (** P<0.01, *** P<0.001). 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

CRISPR screen analysis details and internal controls.

A) Flowchart of data analysis used to identify genes that are most relevant to ovarian cancer cell survival in suspension. B) Mathematical formulas to define gene scores, enrichment scores (n=guides per gene), and enrichment ratios used by TRACS. C) Reader-operator curves were generated to assess TRACS categorization of 1000 non-targeting control sgRNAs in the GeCKOv2 library for each of the three cell lines screened. D) Spheroid cell viability levels from low throughput siRNA gene knock downs for 35 genes in iOvCa147 and OVCAR8 cells was plotted against the enrichment ratio for the same genes in the same two cell lines from this screen. These independent approaches to measure viability were compared using linear regression.

Generation of iOvCa147 cells deficient for DYRK1A and loss of viability in suspension.

A) Strategy to generate DYRK1A-/- cells using a pair of sgRNAs that flank exon 2. PCR For and PCR Rev primers flank exon 2 and are used to detect deletion events. B) Agarose gel showing PCR products for iOVCA147 cells and putative DYRK1A-/- cells. The full length amplicon containing exon 2 was detected in parental iOvCa147 cells (1,348 bp). A smaller amplicon (1,026 bp) was detected in DYRK1A-/- cells, indicting successful excision of the 322 bp region encompassing exon 2. C) Western blot comparing DYRK1A expression in iOVCA147 cells and DYRK1A-/- cells. D) IP kinase assay to evaluate DYRK1A activity in DYRK1A-/- cells. Anti-DYRK1A antibodies or IgG was used to immunoprecipitate from iOVCA147 and DYRK1A-/- cells. Precipitates were incubated with ATP and Tau protein. Samples were resolved by SDS-PAGE and probed with pSer404-Tau antibody. E) Parental iOvCa147 or DYRK1A-/- cells were incubated in suspension conditions for 24 hours, 72 hours, or 4 days to induce spheroid formation, and then re-plated in adherent conditions for 24 hours to allow reattachment. Reattached spheroid cells were stained with crystal violet and absorbance was quantified. Alternatively, cells in suspension were isolated and utilized for Cell TitreGLO cell viability measurements. DYRK1A-/-spheroid cells had impaired survival compared to parental iOvCa147 spheroid cells at each time point and reattached and crystal violet stained cell viability matched Cell TitreGLO measurements of viability (one way anova, ****P<0.0001).

Specificity of Netrin-1 and Netrin-3 IHC staining of OVCAR8 spheroids.

A) OVCAR8 cells were allowed to form spheroids for six hours before fixation in formalin and embedding in paraffin for sectioning. Serial sections were stained with Netrin-1 Abs or a no primary antibody control. B) OVCAR8 cells overexpressing Netrin-1 were used to prepare spheroids as in A and stained in parallel to samples in A. C) OVCAR8 deleted for Netrin-1 expression were used to prepare spheroids as in A and serial sections were stained for Netrin-1 or with a no primary antibody control and counter stained. D) OVCAR8 cells were allowed to form spheroids for six hours before fixation in formalin and embedding in paraffin for sectioning. Serial sections were stained with Netrin-3 Abs or a no primary antibody control. E) OVCAR8 cells overexpressing Netrin-3 were used to prepare spheroids as in D and stained in parallel to samples in D. F) OVCAR8 deleted for Netrin-3 expression were used to prepare spheroids as in D and serial sections were stained for Netrin-3 or with a no primary antibody control. All scale bars = 100μm

Evaluation of target gene transcript levels in multigene knock out cells.

A) RT-qPCR was performed on RNA isolated from control sgRNA expressing OVCAR8 cells and UNC5 4KO to assess expression of UNC5 family members. Graphs depict relative expression levels for each indicted gene in control and knock out cell lines. Mean expression levels of targeted cells were compared to background measurements using one way anova (****P<0.0001). B) RT-qPCR was used to measure mRNA levels of NTN1 and NTN3 transcripts in their respective CRISPR targeted, and OVCAR8 controls. Mean values were compared by one way anova (****P<0.0001). C) Levels of mRNA corresponding to DCC, DSCAM, and NEO1 were determined by RT-qPCR in DDN 3KO cells and control OVCAR8. Mean values were compared by one way anova (****P<0.0001). D) The indicated genotypes of cells were subjected to suspension culture and protein extracts were prepared at the indicated times. Proteins were resolved by SDS-PAGE, blotted, and probed with the indicated antibodies.

Evaluation of dormancy arrest in cell culture by RT-qPCR.

RNA was extracted from asynchronously proliferating cells and suspension cultures 72 hours post transfer from adherent. RT-qPCR was used to compare gene expression of the indicated cell cycle markers in iOvCa147, OVCAR8, TOV1946.

Frequency of Netrin ligand and receptor deletions, mutations or expression changes in high grade serous ovarian cancer.

A) Oncoprint of genetic alterations in 10 netrin signaling related genes in 398 patients. Alteration types are defined on the right. Only the first 154 patients are shown. B) Oncoprint illustrating gene expression outliers among 10 netrin signaling related genes in 201 patients (first 122 patients shown). Genomic data used is from Ovarian Serous Cystadenocarcinoma (TCGA, PanCancer Atlas) (201 samples with RNA seqV2, z-score cut off of 1.5). C) 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). Progression free survival was used to construct Kaplan-Meier plots and survival was compared using a logrank test.