Fig 2: High SPHK1 expression is associated with reduced survival of patients with HGSCA. Quantification of SPHK1 mRNA in benign ovaries (n = 7) and ovarian cancer (n = 77) patient samples by OpenArray Real-Time PCR. Expression levels were normalized to RPLP0. Statistical significance was determined by Mann Whitney U test.*p < 0.05. B. Kaplan-Meier plot analysis of progression-free and overall survival of patients stratified by SPHK1 transcript levels (Affymetrix ID: 219257_s_at) in a combined cohort of 13 gene expression datasets. Low and high SPHK1 expression were defined by the auto-calculated best cutoff. Significance values were determined by log-rank test. HR indicates the hazard ratio, and “Low” and“High” in parentheses indicate the number of cases per group.

Fig 3: Overexpression of SPHK1 in ovarian fibroblasts enhances TGF-ß1-induced myofibroblast marker expression and their ability to promote tumor cell migration and invasionA. Western blot and B. qRT-PCR analysis of CAF markers in TRS3-GFP and TRS3-SPHK1 cells 48 hours after stimulation with TGF-ß1. The mRNA level of each gene is expressed relative to its level in vehicle treated TRS3-GFP cells. *p < 0.05 related to vehicle treated control; #p < 0.05 related to TGF-ß1-stimulated expression values as indicated. C. Immunofluorescence staining of aSMA in TRS3-GFP and TRS3-SPHK1 cells with and without TGF-ß1 stimulation. Scale bar, 100 µm. D. Transwell migration of TRS3-GFP and TRS3-SPHK1 cells. Data are shown as fold change normalized to TRS3-GFP cells. E. Representative images of collagen gel contraction by TRS3-GFP and TRS3-SPHK1 cells at 24 hours. F. Quantification of collagen gel contraction. Data are presented as mean percent contraction compared to the total area of the well. G. SKOV3 or OVCAR3 cells were cocultured with TRS3-GFP or TRS3-SPHK1 cells for 48 hours. Migration and invasion of isolated ovarian cancer cells was assessed by transwell and matrigel invasion assay, respectively. Data are expressed as fold change normalized to ovarian cancer cells cocultured with TRS3-GFP cells. All data are presented as the mean ± SEM of at least three independent experiments. *p < 0.05. NS, not significant.

Fig 4: Ovarian cancer cells stimulate SPHK1 expression in ovarian fibroblasts via TGF-ß1 signalingA, B. qRT-PCR analysis of SPHK1 mRNA expression in TRS3 ovarian fibroblasts cells A. cultured alone or cocultured with fluorescently labeled ovarian cancer cells for 48 hours, followed by FACS or B. stimulated with non-conditioned media (control) or conditioned media from ovarian cancer cells for 48 hours. C. Transcript levels of CAF markers in TRS3 cells incubated in ovarian cancer cell conditioned media for 48 hours. The mRNA level of each gene is indicated relative to its level in TRS3 cells incubated in non-conditioned media. D. Transcript levels of CAF markers in TRS3 cells incubated in ovarian cancer cell conditioned media for 6 hours with either anti-TGF-ß antibody (a TGF-ß) or IgG control. E. Induction of CAF markers by ovarian cancer conditioned media in TRS3 cells with or without pretreatment with the TGF-ß type I receptor inhibitor A83-01. The mRNA level of each gene is expressed relative to its level in vehicle treated TRS3 cells (control). F. TRS3 cells were treated with TGF-ß1 for the indicated times and mRNA (upper panel) and protein (lower panel) were harvested and evaluated for SPHK1 expression. G. Transcript levels of CAF-associated genes in TRS3 cells stimulated with TGF-ß1 for 48 hours with or without pretreatment with A83-01. The mRNA level of each gene is expressed relative to its level in vehicle treated TRS3 cells. Data are presented as the mean ± SEM of at least three independent experiments. *p < 0.05. CM, conditioned media.

Fig 5: Pharmacological inhibition of SPHK1 or S1PR2/3 signaling inhibits TGF-ß1-induced myofibroblast differentiationA. Western blot and B. qRT-PCR analysis of CAF markers in TRS3 cells pretreated with the SPHK1 inhibitor SKI-5C (5 µM) for 1 hour, and then stimulated with TGF-ß1 for 48 hours. The mRNA level of each gene is expressed relative to its level in vehicle treated cells. *p < 0.05 related to vehicle treated control; #p < 0.05 related to TGF-ß1-stimulated expression values as indicated. C. Representative images of TGF-ß-induced collagen gel contraction at 24 hours by TRS3 cells with and without pretreatment with SKI-5C. Data are presented as mean percent contraction compared to the total area of the well. D. TRS3 cells were incubated with S1PR1 antagonist W146 (10 µM), S1PR2 antagonist JTE-013 (10 µM), or S1PR3 antagonist CAY10444 (10 µM) for 1 hour before TGF-ß1 stimulation for 48 hours. qRT-PCR analysis of CAF markers where the mRNA level of each gene is expressed relative to its level in vehicle treated cells. *p < 0.05 related to vehicle treated control; #p < 0.05 related to TGF-ß1-stimulated expression.