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. 2022 Oct 10;21(2):170–186. doi: 10.1158/1541-7786.MCR-22-0486

Figure 4.

Figure 4. TWEAK in combination with carboplatin enriches for CSCs and eliminates non-CSCs. A, Stem cell marker flow cytometry of HGSOC cells treated with vehicle, TWEAK (100 ng/mL), carboplatin (125 μmol/L), or TWEAK and carboplatin for 72 hours. Representative gating for CAOV4 (left), percent positive cells representing mean plus SD (right). One-way ANOVA, Tukey post hoc test (n = 3). B, qRT-PCR for select stemness genes in HGSOC cells with vehicle, TWEAK (100 ng/mL), carboplatin (125 μmol/L), or TWEAK and carboplatin for 72 hours represented relative to the vehicle. One-way ANOVA, Tukey post hoc test (n = 3). C, Spheroid efficiency of HGSOC cells treated with vehicle, TWEAK (100 ng/mL), carboplatin (125 μmol/L), or TWEAK and carboplatin for 4 days represented relative to vehicle. One-way ANOVA, Tukey post hoc test (n = 4). D, Flow cytometry analysis of CD117 expression on Fn14+ cells treated with vehicle, TWEAK (100 ng/mL), carboplatin (125 μmol/L), or TWEAK and carboplatin for 72 hours. Representative gating (left) and percent positive cells representing mean plus SD (right). One-way ANOVA, Tukey post hoc test (n = 3). E, Viability of sorted OV90 CD117+ or CD117− cells grown for 8 days normal media (top). Two-way repeated-measures ANOVA, Sidak post hoc test (n = 4). Spheroid efficiency of OV90 CD117+ or CD117− cells treated with carboplatin (90 μmol/L) for 4 days represented relative to vehicle (bottom). Unpaired t test (n = 4). F, qRT-PCR of select genes for sorted CD117+ or CD117− cells treated with vehicle or TWEAK (100 ng/mL) for 72 hours. One-way ANOVA, Tukey post hoc test (n = 4). G, Viability of CD117+ sorted CAOV4 cells treated with either carboplatin (125 μmol/L), carboplatin and TWEAK (100 ng/mL), TWEAK then carboplatin, and carboplatin then TWEAK for 120 hours. One-way ANOVA, Tukey post hoc test (n = 3). H, Flow cytometry analysis of CD117 and Fn14 expression CAOV4 cells mock sorted or CD117− sorted cells treated with vehicle or TWEAK (100 ng/mL) and carboplatin (30 μmol/L) for two cycles of 7 days, where initial treatment is for 24 hours with subsequent 2-fold dilution with media. Representative gating (top) and percent Fn14+CD117+ cells representing mean plus SD. Two-way ANOVA, Sidak post hoc test (n = 4 mock, 5 CD117− sorted). Data represent mean and SEM. *, P < 0.05.

TWEAK in combination with carboplatin enriches for CSCs and eliminates non-CSCs. A, Stem cell marker flow cytometry of HGSOC cells treated with vehicle, TWEAK (100 ng/mL), carboplatin (125 μmol/L), or TWEAK and carboplatin for 72 hours. Representative gating for CAOV4 (left), percent positive cells representing mean plus SD (right). One-way ANOVA, Tukey post hoc test (n = 3). B, qRT-PCR for select stemness genes in HGSOC cells with vehicle, TWEAK (100 ng/mL), carboplatin (125 μmol/L), or TWEAK and carboplatin for 72 hours represented relative to the vehicle. One-way ANOVA, Tukey post hoc test (n = 3). C, Spheroid efficiency of HGSOC cells treated with vehicle, TWEAK (100 ng/mL), carboplatin (125 μmol/L), or TWEAK and carboplatin for 4 days represented relative to vehicle. One-way ANOVA, Tukey post hoc test (n = 4). D, Flow cytometry analysis of CD117 expression on Fn14+ cells treated with vehicle, TWEAK (100 ng/mL), carboplatin (125 μmol/L), or TWEAK and carboplatin for 72 hours. Representative gating (left) and percent positive cells representing mean plus SD (right). One-way ANOVA, Tukey post hoc test (n = 3). E, Viability of sorted OV90 CD117+ or CD117− cells grown for 8 days normal media (top). Two-way repeated-measures ANOVA, Sidak post hoc test (n = 4). Spheroid efficiency of OV90 CD117+ or CD117− cells treated with carboplatin (90 μmol/L) for 4 days represented relative to vehicle (bottom). Unpaired t test (n = 4). F, qRT-PCR of select genes for sorted CD117+ or CD117− cells treated with vehicle or TWEAK (100 ng/mL) for 72 hours. One-way ANOVA, Tukey post hoc test (n = 4). G, Viability of CD117+ sorted CAOV4 cells treated with either carboplatin (125 μmol/L), carboplatin and TWEAK (100 ng/mL), TWEAK then carboplatin, and carboplatin then TWEAK for 120 hours. One-way ANOVA, Tukey post hoc test (n = 3). H, Flow cytometry analysis of CD117 and Fn14 expression CAOV4 cells mock sorted or CD117− sorted cells treated with vehicle or TWEAK (100 ng/mL) and carboplatin (30 μmol/L) for two cycles of 7 days, where initial treatment is for 24 hours with subsequent 2-fold dilution with media. Representative gating (top) and percent Fn14+CD117+ cells representing mean plus SD. Two-way ANOVA, Sidak post hoc test (n = 4 mock, 5 CD117− sorted). Data represent mean and SEM. *, P < 0.05.