Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A-B, CAOV3, TYKNU, TOV21G, and TOV112D cells were treated with ( A ) 10µM erastin or ( B ) 250nM RSL3 in the presence or absence of 2-8% ascites from three OVCA patients. Liproxtasin-1 (Lipro-1) (2µM) was used as a control ( n =3/cell line, 24 hours). C-D , cells were treated with 5µM erastin for 20 hours or 2µM RSL3 for 2 hours in the presence or absence of 2% ascites. Lipid peroxidation was measured via flow cytometry analysis of BODIPY TM 581/591 C11 staining ( n provided in panel). E-F , cells were either pre-treated with 8% OVCA ascites (Day 0, 16 hours) or left untreated and subsequently treated with 10µM erastin and 8% OVCA ascites. Cell death was visualized ( E ) and measured ( F ) via the CellTox TM Green assay (Days 2-4) in which green fluorescence indicates cell death ( n =3, scale=80µm). G , cells were treated with 10µM erastin in the presence or absence of 2-8% ascites from liver cirrhosis (LC) or OVCA patients for 24 hours ( n provided in panel, 24 hours). H , micro-organospheres (MOS) were treated with 50µM IKE in the presence or absence of 10-40% OVCA ascites for 72 hours ( n =5). I-J , CAOV3 cells transduced with lentivirus expressing luciferase were resuspended in PBS or human OVCA ascites and IP injected into 6-week-old SCID beige mice. Tumor growth ( I ) was visualized and ( J ) measured as total photon flux (p/s) 10 days after tumor injection via IVIS bioluminescent imaging ( n =6). Cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Statistical significance was assessed using correlated-samples one-way or two-way ( F ) ANOVA, and multiple comparisons were adjusted using Holm-Šídák’s method ( D-H ). Statistical significance for J was assessed using the two-tailed Student’s t -test method.

Article Snippet: Imidazole Ketone Erastin (IKE) was purchased from MedChemExpress (HY-114481) and RSL3 was purchased from Cayman Chemical (19288).

Techniques: Control, Flow Cytometry, Staining, CellTox Assay, Fluorescence, Transduction, Expressing, Luciferase, Injection, Imaging, Glo Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A-B , CAOV3, TYKNU, TOV21G, and TOV112D cells were treated with ( A ) 5µM IKE or ( B ) 2.5µM JKE-1674 in the presence or absence of 2-8% ascites from three OVCA patients. Liproxtasin-1 (Lipro-1) (2µM) was used as a control ( n =3/cell line, 24 hours). C , validation of ascites rescue against si GPX4 (100nM) with three different ascites samples in CAOV3 cells ( n =3, 72 hours). D , si GPX4 knockdown confirmation via qRT-PCR ( n =3). E , CAOV3 cells were treated with 10µM erastin in the presence of either 2% ascites or an additional 2% FBS ( n =3, 24 hours). F-H , CAOV3 cells were treated with ( F ) 200nM staurosporine (STS) for 24 hours, ( G ) 400nM actinomycin D (AMD) for 48 hours, or ( H ) 100nM rapamycin for 48 hours in the presence or absence of 2% ascites ( n =3). I-J , qRT-PCR analysis of ( I ) CHAC1 and ( J ) SLC7A11 expression in CAOV3 cells treated with 5µM erastin in the presence or absence of 10% ascites ( n =3, 16 hours). K-L , western blot analysis of lipid peroxidation levels in tumors harvested 10 days after injection of PBS-resuspended and ascites-resuspended CAOV3 cells in SCID beige mice. Signal quantification is depicted in L ( n =6). Cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Statistical significance for C , I , and J was assessed using one-way ANOVA, and multiple comparisons were adjusted using Holm-Šídák’s method. Statistical significance for L was assessed using the two-tailed Student’s t -test method.

Article Snippet: Imidazole Ketone Erastin (IKE) was purchased from MedChemExpress (HY-114481) and RSL3 was purchased from Cayman Chemical (19288).

Techniques: Control, Knockdown, Quantitative RT-PCR, Expressing, Western Blot, Injection, Glo Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A-C , ( A ) triglyceride, ( B ) cholesterol, and ( C ) protein levels were measured in regular, dialyzed, and delipidated ascites via Triglyceride-Glo TM , Amplex TM Red Cholesterol, and Pierce TM BCA assays respectively. Protein levels in dialyzed and delipidated ascites were adjusted using regular ascites as control ( n =3). D-E , CAOV3, TYKNU, TOV21G, and TOV112D cells were treated with 2% regular, dialyzed, heat-inactivated, or delipidated ascites collected from three metastatic OVCA patients in the presence or absence of ( D ) 10µM erastin or ( E ) 250nM RSL3 ( n =3/cell-line, 24 hours). F-G , DEG Volcano plots of the RNA-seq pairwise comparison analyses of ( F ) CAOV3 and ( G ) TOV21G cells that were treated with 10% ascites for 16 hours ( n =3, adj. P =0.01). H , Venn diagram comparison of lipid species detected in ascites versus lipid species found to be increased in cells treated with the same ascites ( n =5). I , heat map for sum total of unsaturated fatty acid (UFA) present in increased triglycerides in CAOV3 cells treated with 10% ascites and 5µM erastin for 16 hours. The UFA composition of each triglyceride was recorded and the same UFAs were added together. The sum of each UFA is presented as a number on the heat map ( n =5). All Cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Statistical significance was assessed using correlated-samples one-way ANOVA, and multiple comparisons were adjusted using Holm-Šídák’s method. All statistical tests were two-tailed where applicable.

Article Snippet: Imidazole Ketone Erastin (IKE) was purchased from MedChemExpress (HY-114481) and RSL3 was purchased from Cayman Chemical (19288).

Techniques: Control, RNA Sequencing Assay, Comparison, Glo Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A , cell viability was assessed in CAOV3 cells treated with 2% regular, dialyzed, heat-inactivated, or delipidated OVCA ascites and 10µM erastin ( n =3, 24 hours). B , Volcano plot of the differentially expressed genes (DEGs) of RNA-seq analysis for the transcriptome changes of CAOV3 and TOV21G cells treated with 10% ascites for 16 hours. DEGs from each cell line were combined for a pairwise comparison of total DEGs under ascites-treatment ( n =3/cell-line, adj. P =0.01). C , Gene Set Enrichment Analysis (GSEA) for the combined DEGs for the indicated gene-sets from CAOV3 and TOV21G. D , structural lipidomic pairwise comparison analysis for CAOV3 cells treated with 5µM erastin with or without 10% ascites for 16 hours ( n =5, adj. P =0.01). E , pie-chart of the lipid composition and relative abundance of all significantly increased intracellular lipids in the ascites-treated cells. F-G , heat map of all ( F ) significant lipid semiquantitative concentration increases and ( G ) top 50 specific lipid species increases (nmol lipid/mg protein) in erastin and ascites treated cells. H-I , CAOV3 cells were treated with 10% ascites and lipid droplet levels were measured via flow cytometry analysis of BODIPY TM 493/503 staining ( n =3, 16 hours). Cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Statistical significance was assessed using the two-tailed Student’s t -test method ( D , I ), and P values were adjusted using the Benjamini-Hochberg correction method ( D ).

Article Snippet: Imidazole Ketone Erastin (IKE) was purchased from MedChemExpress (HY-114481) and RSL3 was purchased from Cayman Chemical (19288).

Techniques: RNA Sequencing Assay, Comparison, Concentration Assay, Flow Cytometry, Staining, Glo Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A , Cell viability was assessed in CAOV3 cells treated with 10µM erastin, 2% ascites, and 1mM bezafibrate (PPARα agonist), 0.8mM C-75 Trans (FASN inhibitor), 100µM Sulfo-N-succinimidyl oleate (SSO) (CD36 inhibitor), 50µM MF-438 (SCD1 inhibitor), and 80µM BMS-309403 (FABP4 inhibitor) ( n provided in panel, 48 hours). B-D , ( B ) TYKNU, ( C ) TOV21G, and ( D ) TOV112D cells were treated with 10µM erastin, 2% ascites, and 200µM ( B , D ) or 400µM ( C ) bezafibrate ( n =3, 48 hours). E-F , CAOV3 cells were treated with ( E ) 5µM IKE or ( F ) 2.5µM JKE-1674, 2% ascites, and 200µM bezafibrate ( n =3, 48 hours). G-H , CAOV3 cells were treated with ( G ) 10µM erastin, 2% ascites, 200µM bezafibrate, 60µM fenofibrate, 500µM ciprofibrate, or ( H ) 250nM GW7647 ( n provided in panel, 48 hours). I , CAOV3 cells were transduced with a PPRE-luciferase reporter constructed and treated with 2% ascites in the presence or absence of 800µM bezafibrate. Luminescence was measured after 1mM D-luciferin addition ( n =3, 16 hours). J , The log-transformed changes in CAOV3 PPARA target DEGs after 10% ascites exposure ( n =3, 16 hours, adj. P =0.01). The target genes were determined via the PPARGene database ( http://www.ppargene.org/index.php ), which provides all verified PPARα target genes. K , ascites from 7-week-old NSG mice were collected after IVIS imaging. All ascites was pooled together and 8% was added to CAOV3 cells with or without 2.5µM JKE-1674 treatment to assess ferroptosis protection ( n =3). Cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Statistical significance was assessed using correlated-samples one-way (ANOVA), and multiple comparisons were adjusted using Holm-Šídák’s method. All statistical tests were two-tailed where applicable.

Article Snippet: Imidazole Ketone Erastin (IKE) was purchased from MedChemExpress (HY-114481) and RSL3 was purchased from Cayman Chemical (19288).

Techniques: Transduction, Luciferase, Construct, Transformation Assay, Imaging, Glo Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A-B , cell viability of CAOV3 cells was assessed via treatment with ( A ) 10µM erastin or ( B ) 250 nM RSL3, together with 2% ascites, in the presence or absence of 200µM bezafibrate ( n =3, 48 hours). C-D , Volcano plots of the different lipid species from lipidomic comparison analyses for CAOV3 cells treated 10% ascites ( C ) with or ( D ) without 200µM bezafibrate for 16 hours ( n =5, adj. P =0.01). E , Venn diagram comparison of lipid changes between ascites-only versus ascites-and-bezafibrate treated cells. The heat map depicts the class composition of the 243 lipids that remain unchanged with the ascites-and-bezafibrate treatment. F-G , cells were treated with 10% ascites in the presence or absence of 200µM bezafibrate and lipid droplet levels were measured via flow cytometry analysis of BODIPY TM 493/503 staining ( n =3, 16 hours). H , micro-organospheres (MOS) developed from an OVCA patient were treated with 20% ascites and 50µM IKE, and 1mM bezafibrate or 1mM ciprofibrate to assess resensitivity to ferroptosis ( n =5, 72 hours). I-J , CAOV3 transduced with lentivirus expressing luciferase were treated with either 800µM bezafibrate, 10µM JKE-1674, or both, 5 hours before being injected IP into 6-week-old NSG mice. Tumor growth was then ( I ) visualized and ( J ) measured as total photon flux (p/s) 7 days after injection via IVIS bioluminescent imaging ( n =7). Cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Statistical significance for C-D was assessed using the two-tailed Student’s t -test method ( D , I ) and P values were adjusted using the Benjamini-Hochberg correction method ( C-D ). All other statistical significance was assessed using correlated-samples one-way ANOVA, and multiple comparisons were adjusted using Holm-Šídák’s method. All statistical tests were two-tailed where applicable.

Article Snippet: Imidazole Ketone Erastin (IKE) was purchased from MedChemExpress (HY-114481) and RSL3 was purchased from Cayman Chemical (19288).

Techniques: Comparison, Flow Cytometry, Staining, Transduction, Expressing, Luciferase, Injection, Imaging, Glo Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A , Volcano plot of the DEGs of bezafibrate treatment (200µM, 16 hours) from RNA-seq analysis of CAOV3 cells ( n =3, adj. P =0.01). B , Venn diagram comparison of a list of oppositely expressed genes from ascites-treated vs. bezafibrate-treated cells. C-D , western blot analysis of HMGCS2 protein expression upon 10% ascites treatment from three patients with or without 200µM bezafibrate for 16 hours ( n =3). E-F , ferroptosis sensitivity was compared between CAOV3 wildtype (WT) and HMGCS2 KO cells with ( E ) erastin or ( F ) RSL3 treatment ( n =3, 24 hours). G , HMGCS2 expression was restored in HMGCS2 KO cells with hmgcs2 OE plasmid and ferroptosis sensitivity was compared with 10µM erastin or 125nM RSL3 treatment for 24 hours ( n =3). H-I , lipid peroxidation was measured via flow cytometry analysis of BODIPY TM 581/591 C11 staining in HMGCS2 KO cells that were treated with 5µM erastin for 20 hours ( n =3). J-K , HMGCS2 OE cells were treated with 2% ascites for 16 hours and lipid droplet levels were measured via flow cytometry analysis of BODIPY TM 493/503 staining ( n =3). L , cell viability was assessed via treatment with 10µM erastin in the presence or absence of 20µM baicalin with 2% ascites ( n =3, 24 hours). M-N , cells were treated with 10% ascites in the presence or absence of 25µM baicalin (24 hours) and lipid droplet levels were measured as described ( N ) ( n =3). O , cell viability was assessed in cells treated with 10µM erastin in the presence or absence of 100µM malonyl CoA lithium for 24 hours ( n =3). P-Q , cells were treated with 100µM malonyl CoA lithium (16 hours) and lipid droplet levels were measured as described ( Q ) ( n =3). Cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Statistical significance for D-O was assessed using correlated-samples one-way ANOVA, and multiple comparisons were adjusted using Holm-Šídák’s method. Student’s t -test was performed for Q . All statistical tests were two-tailed where applicable.

Article Snippet: Imidazole Ketone Erastin (IKE) was purchased from MedChemExpress (HY-114481) and RSL3 was purchased from Cayman Chemical (19288).

Techniques: RNA Sequencing Assay, Comparison, Western Blot, Expressing, Plasmid Preparation, Flow Cytometry, Staining, Glo Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A-B , Cell viability was measured in CAOV3 cells transfected with ( A ) 50 nM si HMGCS2 or ( B ) 50 nM si TFRC and treated with 10µM erastin for 24 hours ( n =3). C-D , ( C ) HMGCS2 and ( D ) TFRC silencing was validated via qRT-PCR ( n =3). E-F , ( E ) HMGCS2 and ( F ) TFRC downregulation upon 10% ascites exposure for 16 hours was validated via qRT-PCR ( n =3). G-H , knockout of HMGCS2 was validated in HMGCS2 KO cells via ( G ) qRT-PCR and ( H ) western blot analysis ( n =3). I , HMGCS2 overexpression was validated via western blot analysis ( n =3). J-K , cells overexpressing HMGCS2 were treated with 5µM erastin for 20 hours and ( J ) lipid peroxidation was visualized and ( K ) measured with flow cytometry analysis of BODIPY TM 581/591 C11 staining ( n =3). L , patient overall survival data was extracted from the TCGA database and analyzed via the GEPIA 2 survival analysis tool. All cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Excluding L , statistical significance was assessed using correlated-samples one-way ANOVA, and multiple comparisons were adjusted using Holm-Šídák’s method. All statistical tests were two-tailed where applicable.

Article Snippet: Imidazole Ketone Erastin (IKE) was purchased from MedChemExpress (HY-114481) and RSL3 was purchased from Cayman Chemical (19288).

Techniques: Transfection, Quantitative RT-PCR, Knock-Out, Western Blot, Over Expression, Flow Cytometry, Staining, Glo Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Ascitic fluid protects against ferroptosis and enables the peritoneal spread of ovarian cancer

doi: 10.1101/2024.11.23.624998

Figure Lengend Snippet: A-B , TFRC membrane expression was measured via flow cytometry analysis in cells treated with 2% ascites for 18 hours and 2µM RSL3 and 800µM bezafibrate for 2 hours ( n =3). C , labile iron levels were measured in cells treated with 2% ascites for 18 hours, and 2µM RSL3 and 800µM bezafibrate for 2 hours ( n =3). Labile iron was measured via calculating the change in Calcein-AM mean fluorescence intensity after treatment with 0.05µM Calcein-AM (15 minutes) and 100µM deferoxamine mesylate (DFO) (1 hour). D , heat map of the free fatty acid (FFA) profile in OVCA ascites, measured via FFA lipidomic profiling ( n =8). E , cell viability was assessed in CAOV3 cells that were treated with 10µM erastin in the presence of either 250µM oleic acid, 150µM linoleic acid or both (250µM, 150µM) ( n =3, 24 hours). F , cell viability was assessed in cells treated with 10µM erastin with or without 200µM bezafibrate and 50µM oleic acid for 24 hours ( n =3). G-I , TFRC membrane expression ( G-H ) and labile iron levels ( I ) were measured with using 50µM oleic acid in place of ascites ( n =3). J , mechanism of action model for ascites protection against ferroptosis and its mitigation by bezafibrate. Cell viability tests were measured with the CellTiter-Glo® assay. All data represent mean ± s.d. Statistical significance was assessed using correlated-samples one-way ANOVA, and multiple comparisons were adjusted using Holm-Šídák’s method. All statistical tests were two-tailed where applicable.

Article Snippet: Imidazole Ketone Erastin (IKE) was purchased from MedChemExpress (HY-114481) and RSL3 was purchased from Cayman Chemical (19288).

Techniques: Membrane, Expressing, Flow Cytometry, Fluorescence, Glo Assay, Two Tailed Test

Journal: JCI insight

Article Title: Metabolism of PLTP, CETP, and LCAT on multiple HDL sizes using the Orbitrap Fusion Lumos.

doi: 10.1172/jci.insight.143526

Figure Lengend Snippet: Figure 2. An interinstrument platform comparison of PLTP and CETP tracer enrichment data. (A) PRM scans of the same CETP peptide from the Q Exactive and the Lumos. The relative peak intensities of the fragment ions are conserved between the 2 instruments. The y7 2HM3 (tracer) peak environment is zoomed in. R = 120 K for the Q Exactive and 240 K for the Lumos. (B) Loess regression plots showing that the standard error (gray) of the fitted curves is lower on the Lumos. PLTP data are from alpha1 and CETP from alpha2. Legend: the PRM ions’ m/z values. (C) Box plots depicting the distribution of the enrichment data in B. Each data point is the average enrichment (n = 3 PRM ions’ measurements) per time point. The box plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range. The gray lines indicate the relative shift in enrichment value per given time point. Variance was calculated using individual PRM ion enrichment data in B, not the averages.

Article Snippet: Each protein was quantified using the following peptide standards: APOA1 (THLAPYSDEL[R-labeled]), APOE (LGPLVEQG[R-labeled]), LCAT (SSGLVSNAPGVQI[R-labeled]), CETP (ASYPDITGE[K-labeled]), and PLTP (AVEPQLQEEE[R-labeled]) (New England Peptides, NEP, Supplemental Table 2).

Techniques: Comparison

Journal: JCI insight

Article Title: Metabolism of PLTP, CETP, and LCAT on multiple HDL sizes using the Orbitrap Fusion Lumos.

doi: 10.1172/jci.insight.143526

Figure Lengend Snippet: Figure 3. Compartmental models and kinetics parameters for PLTP and CETP in the larger alpha HDL size fractions. (A) Compartmental model for PLTP and CETP (the average of n = 6 participants). PLTP flux into alpha1 is approximately 45% from the source (0.010 mg/kg/d) and 55% from the smaller alpha2 (0.012 mg/kg/d). Approximately 75% of PLTP on alpha2 is transferred to alpha0 and alpha1 (0.10 and 0.59 pool/d, respectively) while the remaining 25% is removed from the model system (0.20 pool/d). CETP appears on alpha1 and alpha2 via direct secretion. (B) Enrichment curve fits generated from the models in A, participant 1. (C) FCR and production rate (PR) for PLTP and CETP. Bar graphs represent the mean value for n = 6 participants, error bars represent SD, and open circles represent values per participant.

Article Snippet: Each protein was quantified using the following peptide standards: APOA1 (THLAPYSDEL[R-labeled]), APOE (LGPLVEQG[R-labeled]), LCAT (SSGLVSNAPGVQI[R-labeled]), CETP (ASYPDITGE[K-labeled]), and PLTP (AVEPQLQEEE[R-labeled]) (New England Peptides, NEP, Supplemental Table 2).

Techniques: Generated