SUMMARY Lack of sensitive single-cell analysis tools has limited the characterization of metabolic activity in cancer stem cells. By hyperspectral stimulated Raman scattering imaging of single living cells and mass spectrometry analysis of extracted lipids, we report here significantly increased levels of unsaturated lipids in ovarian cancer stem cells (CSCs) as compared to non-CSCs. Higher lipid unsaturation levels were also detected in CSC-enriched spheroids compared to monolayer cultures of ovarian cancer cell lines or primary cells. Inhibition of lipid desaturases effectively eliminated CSCs, suppressed sphere formation in vitro, and blocked tumor initiation capacity in vivo. Mechanistically, we demonstrate that NF-κB directly regulates the expression levels of lipid desaturases and that inhibition of desaturases blocks NF-κB signaling. Collectively, our findings reveal that increased lipid unsaturation is a metabolic marker for ovarian CSCs and a target for CSC-specific therapy.

PURPOSE To evaluate the addition of the humanized monoclonal antiprogrammed death ligand-1 (PD-L1) antibody, atezolizumab, to platinum-based chemotherapy and bevacizumab in newly diagnosed stage III or IV ovarian cancer (OC). METHODS This multicenter placebo-controlled double-blind randomized phase III trial (ClinicalTrials.gov identifier: NCT03038100 ) enrolled patients with newly diagnosed untreated International Federation of Gynecology and Obstetrics (FIGO) stage III or IV OC who either had undergone primary cytoreductive surgery with macroscopic residual disease or were planned to receive neoadjuvant chemotherapy and interval surgery. Patients were stratified by FIGO stage, Eastern Cooperative Oncology Group performance status, tumor immune cell PD-L1 staining, and treatment strategy and randomly assigned 1:1 to receive 3-weekly cycles of atezolizumab 1,200 mg or placebo (day 1, cycles 1-22), with paclitaxel plus carboplatin (day 1, cycles 1-6) plus bevacizumab 15 mg/kg (day 1, cycles 2-22), omitting perioperative bevacizumab in neoadjuvant patients. The co-primary end points were investigator-assessed progression-free survival and overall survival in the intention-to-treat and PD-L1–positive populations. RESULTS Between March 8, 2017, and March 26, 2019, 1,301 patients were enrolled. The median progression-free survival was 19.5 versus 18.4 months with atezolizumab versus placebo, respectively (hazard ratio, 0.92; 95% CI, 0.79 to 1.07; stratified log-rank P = .28), in the intention-to-treat population and 20.8 versus 18.5 months, respectively (hazard ratio, 0.80; 95% CI, 0.65 to 0.99; P = .038), in the PD-L1–positive population. The interim (immature) overall survival results showed no significant benefit from atezolizumab. The most common grade 3 or 4 adverse events were neutropenia (21% with atezolizumab v 21% with placebo), hypertension (18% v 20%, respectively), and anemia (12% v 12%). CONCLUSION Current evidence does not support the use of immune checkpoint inhibitors in newly diagnosed OC. Insight from this trial should inform further evaluation of immunotherapy in OC.

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OBJECTIVES: Lack of sensitive single-cell analysis tools has limited the characterization of the metabolic activity of cancer stem cells (CSCs). The objectives of this study were to identify and target metabolic pathways specifically deregulated in ovarian CSCs. METHODS: We developed a new technique based on hyperspectral stimulated Raman scattering imaging of single living cells coupled with electrospray ionization mass spectrometry (ESI-MS) analysis of extracted lipids to identify unique metabolic spectra of ovarian CSCs. SiRNA knockdown and pharmacological inhibitors were used to block fatty acid desaturases and to measure effects on the ovarian CSCs charcateristics. RESULTS: We report significantly increased levels of unsaturated lipids in flow-sorted ovarian CSCs (ALDH+CD133+) compared to non-CSCs (ALDH-CD133-). Higher lipid unsaturation levels were also detected in CSC-enriched spheroids compared to monolayer cultures of ovarian cancer cell lines or primary cells derived from human ovarian tumors. ESI-MS identified that the elevated unsaturation in lipid droplets involved an enhanced conversion from saturated fatty acids (18:0) to unsaturated fatty acids (18:1). Expression of fatty acid desaturases was increased in ovarian CSCs compared to non-CSCs. Pharmacological inhibition or siRNA knockdown of lipid desaturases Δ9 (SCD1) or Δ6 effectively eliminated CSCs, suppressed sphere formation in vitro, and blocked tumor initiation capacity in vivo, as measured by a tumor initiation dilution assay. Mechanistically, the lipid desaturase inhibitors suppressed ovarian cancer cell stemness by blocking the NF-κB survival pathway. Chromatin immunoprecipitation and overexpression of RelA demonstrated that NF-κB transcriptionally regulates SCD1 expression. Collectively, our data suggest that a positive feedback loop involving lipid desaturases, NF-κB, and the stem cell marker ALDH1 can be effectively blocked by SCD1 inhibitors, suppressing ovarian CSCs. CONCLUSIONS: These results demonstrate that increased lipid unsaturation is a novel metabolic marker for ovarian CSCs and a target for CSC-specific therapy. Citation Format: Junjie Li, Salvatore Condello, Jessica Thomes-Pepin, Xiaoxiao Ma, Yu Xia, Thomas D. Hurley, Ji-Xin Cheng, Daniela Matei. LIPID DESATURATION IS A METABOLIC MARKER AND THERAPEUTIC TARGET OF OVARIAN CANCER STEM CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP21.