Fig 1.
Sensitivity of canine cell lines to VDX-111.
(A) Survival of canine cell lines after 72 hours of VDX-111 exposure. MCT = mast cell tumor, STS = soft tissue sarcoma. Cell lines of the FACC Canine Cell Line panel cover a variety of cancer types and display a variety of sensitivities at 1 μM and 100 nM VDX-111. Error bars represent SD. (B) Heat map displaying the percent of cells surviving compared to ethanol control after 72 hours in VDX-111 exposure, as measured via resazurin fluorescence.
Fig 2.
(A) Bliley cells expressing NucLight Red were imaged over time under VDX-111 and YOYO-1 exposure. An object count of red fluorescence indicates robust Bliley cell growth under low concentrations and some growth even under the highest VDX-111 concentrations (left). A green fluorescence object count, compared to total red cells indicates a low but present rate of apoptosis at 23 hours of drug exposure (right). (B) CTAC cells expressing NucLight Red were imaged over time under VDX-111 and YOYO-1 exposure. An object count of red fluorescence shows CTAC sensitivity to high VDX-111 concentrations. All groups are statistically different by two-way ANOVA (****p<0.0001) except 1 uM compared to 500 nM (left). A green fluorescence object count, compared to total red cells indicates high rates of apoptosis at 23 hours of drug exposure (right). (C) DH82 cells expressing NucLight Red were imaged over time under VDX-111 and YOYO-1 exposure. An object count of red fluorescence shows DH82 sensitivity to high VDX-111 concentrations. All groups are statistically different by two-way ANOVA (****p<0.0001)(left). A green fluorescence object count, compared to total red cells indicates moderate rates of apoptosis at 23 hours of drug exposure (right). Error bars represent SEM. ***<0.001 **<0.01.
Fig 3.
(A) Cells were allowed to migrate across a membrane with 8.0 μm pores for 18 hours with or without VDX-111. Cells were then stained, imaged, and counted. (B) Number of migrated cells on the membrane per imaged 10x field of view were normalized to the control condition. Ten fields were quantified for each condition. Wells without membrane were also quantified via resazurin fluorescence to account for any cell death and reduced proliferation in the 18-hour period, and the percent difference in live cells was removed from the control counts. Error bars represent SD. ****<0.0001 ***<0.001 **<0.01.
Fig 4.
Canine cell line gene expression correlation with VDX-111 sensitivity identifies components of the PI3K-AKT pathway.
(A) An expression matrix was created correlating gene expression measured by RNAseq with the percent survival of the canine cells under VDX-111 exposure. Displayed on the chart are the genes with correlation (+/-) 0.6 with 1 uM treatment, showing the correlation at 1 μM, 100 nM, the p-values of each and gene name. A positive correlation indicates greater survival with higher expression, while a negative correlation indicates lower survival with higher expression. (B) Graphs showing the FACC canine cell line panel expression of MAX and SETD9 correlating to survival at 1 μM.
Fig 5.
Pathway analysis of gene expression correlating with VDX-111 survival.
(A) Pathway analysis using Metascape (metascape.org) with an input of 1261 significantly correlated genes (p<0.05) including either a positive or negative correlation with 1 μM treatment. Multiple signaling pathways were identified among the significantly correlated genes. (B) Heatmap of expression levels in each cell line of genes in the Metascape analysis of the PI3K-AKT pathway. Cell lines with >50% survival at 1 μM treatment are indicated as resistant, <50% survival at 1 μM treatment are indicated as sensitive.
Fig 6.
Phosphorylation of growth promoting pathways is reduced with VDX-111 treatment.
Cells were treated with ethanol control, 100 nM, or 1 μM VDX-111 for 4 hours before harvesting lysates for western blot. Images were quantified with ImageJ and corrected to cofilin levels. Ratios were then normalized to the control condition. Three separate experiments were quantified to obtain averages shown in graphs. Error bars represent SD.