Summary of Study ST003341
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002077. The data can be accessed directly via it's Project DOI: 10.21228/M84R60 This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST003341 |
| Study Title | Untargeted Lipidomic Profiling of Canine Cancer Cell Lines |
| Study Summary | Cell sensitivity to ferroptosis is closely associated with cellular lipid composition and unsaturation. To further characterize the 31 canine cell lines tested for ferroptosis sensitivity, we collected untargeted lipidomic data for each line in duplicate. Omics characterizations of cancer cell lines and integration with viability data in efforts such as the Cancer Cell Line Encyclopedia (CCLE) and the Cancer Dependency Map (www.depmap.org) have yielded powerful resources to explore causal and predictive biomarkers of response to genetic and small molecule perturbations. Our data facilitate a similar approach for the canine lines included in this study. Lipidomics data covered over 750 individual species falling into 18 main lipid classes. Total concentrations varied widely between individual cell lines, as did relative lipid composition. Likewise, the fraction of cellular polyunsaturated fatty acids (PUFAs), previously reported as a contributor to ferroptosis sensitivity, varied from 20% to almost 70%. Thus, the panel of tested canine cell lines represented a wide spectrum of cellular lipid composition. |
| Institute | Kojin Therapeutics, Inc. |
| Last Name | Wawer |
| First Name | Mathias |
| Address | 451 D St., Suite 502, Boston, MA 02210 |
| mwawer@kojintx.com | |
| Phone | 617-952-0636 |
| Submit Date | 2024-07-12 |
| Total Subjects | 31 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-09-12 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002077 |
| Project DOI: | doi: 10.21228/M84R60 |
| Project Title: | Validation of ferroptosis in canine cancer cells to enable comparative oncology and translational medicine |
| Project Summary: | Ferroptosis is a cell death mechanism that has attracted significant attention as a potential basis for the development of new cancer therapies. Validation of ferroptosis biology in species commonly used in translation and pre-clinical development is a necessary foundation for enabling the advancement of such ferroptosis modulating drugs. Here, we demonstrate that canine cancer cells exhibit sensitivity to a wide range of ferroptosis-inducing perturbations in a manner indistinguishable from human cancer cells, and recapitulate characteristic patterns of ferroptotic response across tumor types seen in the human setting. We further performed lipidomic profiling of all canine cancer cell lines to enable the discovery of lipid biomarkers for ferroptosis sensitivity. The foundation provided herein establishes the dog as a relevant efficacy and toxicology model for ferroptosis and creates new opportunities to leverage the canine comparative oncology paradigm to accelerate the development of ferroptosis-inducing drugs for human cancer patients. |
| Institute: | Kojin Therapeutics, Inc. |
| Last Name: | Wawer |
| First Name: | Mathias |
| Address: | 451 D St., Suite 502, Boston, MA 02210 |
| Email: | mwawer@kojintx.com |
| Phone: | 617-952-0636 |
| Publications: | https://www.biorxiv.org/content/10.1101/2024.04.28.591561v1 |
Subject:
| Subject ID: | SU003462 |
| Subject Type: | Cultured cells |
| Subject Species: | Canis lupus familiaris |
| Taxonomy ID: | 9615 |
Factors:
Subject type: Cultured cells; Subject species: Canis lupus familiaris (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Cell_Line |
|---|---|---|---|
| SA364571 | 46 | Canine cancer cells | 1771 |
| SA364572 | 45 | Canine cancer cells | 1771 |
| SA364573 | 60 | Canine cancer cells | 17CM98 |
| SA364574 | 59 | Canine cancer cells | 17CM98 |
| SA364575 | 31 | Canine cancer cells | Abram |
| SA364576 | 32 | Canine cancer cells | Abram |
| SA364577 | 23 | Canine cancer cells | Angus |
| SA364578 | 24 | Canine cancer cells | Angus |
| SA364579 | 29 | Canine cancer cells | Bliley |
| SA364580 | 30 | Canine cancer cells | Bliley |
| SA364581 | 11 | Canine cancer cells | C2 |
| SA364582 | 12 | Canine cancer cells | C2 |
| SA364595 | 48 | Canine cancer cells | Cindy-HSA |
| SA364596 | 47 | Canine cancer cells | Cindy-HSA |
| SA364583 | 17 | Canine cancer cells | CLL1390 |
| SA364584 | 18 | Canine cancer cells | CLL1390 |
| SA364585 | 28 | Canine cancer cells | CML10C2 |
| SA364586 | 27 | Canine cancer cells | CML10C2 |
| SA364587 | 43 | Canine cancer cells | CML6M |
| SA364588 | 44 | Canine cancer cells | CML6M |
| SA364589 | 21 | Canine cancer cells | CMT12 |
| SA364590 | 22 | Canine cancer cells | CMT12 |
| SA364591 | 61 | Canine cancer cells | CMT27 |
| SA364592 | 62 | Canine cancer cells | CMT27 |
| SA364593 | 38 | Canine cancer cells | CTAC |
| SA364594 | 37 | Canine cancer cells | CTAC |
| SA364597 | 50 | Canine cancer cells | D17 |
| SA364598 | 49 | Canine cancer cells | D17 |
| SA364601 | 8 | Canine cancer cells | Den-HSA |
| SA364602 | 7 | Canine cancer cells | Den-HSA |
| SA364599 | 56 | Canine cancer cells | DH82 |
| SA364600 | 55 | Canine cancer cells | DH82 |
| SA364603 | 33 | Canine cancer cells | Gracie |
| SA364604 | 34 | Canine cancer cells | Gracie |
| SA364605 | 15 | Canine cancer cells | HMPOS |
| SA364606 | 16 | Canine cancer cells | HMPOS |
| SA364607 | 3 | Canine cancer cells | Jones |
| SA364608 | 4 | Canine cancer cells | Jones |
| SA364609 | 39 | Canine cancer cells | Kinsey |
| SA364610 | 40 | Canine cancer cells | Kinsey |
| SA364611 | 19 | Canine cancer cells | McKinley |
| SA364612 | 20 | Canine cancer cells | McKinley |
| SA364613 | 25 | Canine cancer cells | Moresco |
| SA364614 | 26 | Canine cancer cells | Moresco |
| SA364615 | 6 | Canine cancer cells | Nike |
| SA364616 | 5 | Canine cancer cells | Nike |
| SA364617 | 36 | Canine cancer cells | OS24 |
| SA364618 | 35 | Canine cancer cells | OS24 |
| SA364619 | 9 | Canine cancer cells | OSA8 |
| SA364620 | 10 | Canine cancer cells | OSA8 |
| SA364621 | 42 | Canine cancer cells | PARKS |
| SA364622 | 41 | Canine cancer cells | PARKS |
| SA364623 | 57 | Canine cancer cells | SB |
| SA364624 | 58 | Canine cancer cells | SB |
| SA364625 | 2 | Canine cancer cells | STSA |
| SA364626 | 1 | Canine cancer cells | STSA |
| SA364627 | 14 | Canine cancer cells | Tyler1 |
| SA364628 | 13 | Canine cancer cells | Tyler1 |
| SA364629 | 53 | Canine cancer cells | Tyler2 |
| SA364630 | 54 | Canine cancer cells | Tyler2 |
| SA364631 | 51 | Canine cancer cells | Yamane |
| SA364632 | 52 | Canine cancer cells | Yamane |
| Showing results 1 to 62 of 62 |
Collection:
| Collection ID: | CO003455 |
| Collection Summary: | All cell lines were obtained through the Flint Animal Cancer Center (FACC) and were part of the published FACC tumor cell line panel. [1] Cell lines were maintained in RPMI 1640 culture medium containing 10% fetal bovine serum (FBS), penicillin (100 units/mL), streptomycin (100 μg/mL) and incubated at 37 °C in a humidified atmosphere of 5% CO2:95% air. Cell medium was removed by vacuum aspiration and cells were washed with 4 mL cold PBS (no Mg2+/Ca2+). After vacuum aspiration of PBS, 0.25% Trypsin-EDTA (Gibco™25200056) was added and incubated for 5 minutes at 37°C. Cells were resuspended in media (RPMI 1640) to stop the digestion and transferred to 15 ml conical tubes. Cell pellets were collected by centrifuging at 3500 rpm for 10 min (4°C). Cells were then washed twice by resuspending them in cold PBS and repeating centrifugation. Before the final centrifugation, cells were counted and aliquoted to 1,000,000 cells per tube. Cell pellets were stored at -80°C before lipid extraction and LC-MS analysis. [1] Fowles, J. S., Dailey, D. D., Gustafson, D. L., Thamm, D. H. & Duval, D. L. The Flint Animal Cancer Center (FACC) Canine Tumour Cell Line Panel: a resource for veterinary drug discovery, comparative oncology and translational medicine. Vet. Comp. Oncol. 15, 481–492 (2017). |
| Sample Type: | Cultured cells |
| Tissue Cell Quantity Taken: | 1,000,000 |
Treatment:
| Treatment ID: | TR003471 |
| Treatment Summary: | No treatment |
Sample Preparation:
| Sampleprep ID: | SP003469 |
| Sampleprep Summary: | After thawing, cells were mixed with PBS to make a suspension of 1 million cells in 200 μL, utilizing vortexing and sonication. A methyl-tert-butyl ether (MTBE)-based liquid-liquid extraction protocol was used. The cell suspensions were transferred to 8 mL screw-cap glass tubes containing 1.2 mL methanol, 10 μL internal standard mix (see Sampleprep Protocol Comments) and 4 mL MTBE. Samples were vortexed, sonicated for 15 min, and incubated on a tabletop shaker at 500 rpm at room temperature for 1 h. After incubation, phase separation was induced by adding 0.75 mL water to each sample before vortexing and centrifuging at 2000 x g for 20 min. The upper organic phase of each sample was carefully removed using a Pasteur pipette and transferred into a clean glass tube. The remaining aqueous phase was re-extracted with 2 mL of the upper phase of a mixture MTBE/methanol/water 10:3:2.5 (v/v/v). After vortexing and centrifuging, the organic phase was collected and combined with the initial organic phase. The extracted lipids were dried overnight in a SpeedVac vacuum concentrator. The dried lipid extracts were reconstituted in 200 μL n-butanol/methanol 1:1 (v/v) and transferred into autosampler vials for analysis by LC-MS/MS. |
| Sampleprep Protocol Filename: | standards.xlsx |
| Sampleprep Protocol Comments: | Internal Standard Cayman Item # Concentration in Mix (µg/mL) Polarity Used to Normalize TG(16:0-d9/16:0/16:0) 30181 10 Positive TG, pos-ion features not specified below and unidentified features DG(16:0-d9/16:0/0:0) 28781 5 Positive DG SM(d18:1/16:0-d9) 30141 10 Positive SM Cer(d18:1-d7/16:0) 22787 5 Positive Cer CE(16:0-d9) 30134 100 Positive CE CAR(17:0-d3) 35459 5 Positive CAR PE(16:0-d9/16:0) 30597 5 Negative PE, neg-ion features not specified below and unidentified features FA(16:0-d9) 30557 40 Negative FA PI(16:0-d9/16:0) 31102 10 Negative PI PS(16:0-d9/16:0) 30706 10 Negative PS PC(16:0-d9/16:0) 30581 10 Negative PC LPC(16:0-d9/0:0) 30580 10 Negative LPC |
Combined analysis:
| Analysis ID | AN005475 | AN005476 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Dionex Ultimate 3000 | Thermo Dionex Ultimate 3000 |
| Column | Thermo Accucore C30 (150 x 2.1mm,2.6um) | Thermo Accucore C30 (150 x 2.1mm,2.6um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Plus Orbitrap | Thermo Q Exactive Plus Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | area ratio (analyte peak area / internal class standard peak area) | area ratio (analyte peak area / internal class standard peak area) |
Chromatography:
| Chromatography ID: | CH004159 |
| Chromatography Summary: | Solvent A: Acetonitrile/water/formic acid 60:40:0.1 (v/v/v), 10 mM ammonium formate; Solvent B: Acetonitrile/isopropanol/formic acid 10:90:0.1 (v/v/v), 10 mM ammonium formate |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Thermo Accucore C30 (150 x 2.1mm,2.6um) |
| Column Temperature: | 40 |
| Flow Gradient: | (minute, % Pump B): (0,30),(5,43),(5.1,50),(14,70),(14.1,70),(21,99),(28,99),(28.1,30),(33,30) |
| Flow Rate: | 300 uL/min |
| Solvent A: | 60% Acetonitrile/40% water; 0.1% formic acid; 10 mM ammonium formate |
| Solvent B: | 10% Acetonitrile/90% isopropanol; 0.1% formic acid; 10 mM ammonium formate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS005201 |
| Analysis ID: | AN005475 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Lipostar software (Version 2.1.2.; Molecular Discovery) was used for feature detection, noise and artifact reduction, peak alignment, normalization, and lipid identification. Data were processed as follows: 1. Perform peak picking, smoothing, and retention time alignment to generate a list of all features. 2. Apply retention time window filter, retaining data from 1 to 28 min. 3. Remove peaks that did not contain MS/MS spectra and removed noise and high-signal background. 4. Perform automated lipid identification by querying the Lipid Maps database. The identification algorithm uses the detected accurate-mass m/z with a 5 ppm mass tolerance filter, as well as the corresponding MS/MS spectrum to identify lipids, and it ranks the identifications via a 1-to-4-star scale of increasing confidence. 5. The integrated areas of all identified lipids were normalized to the integrated area of their corresponding class-specific internal standard within each sample when possible. 6. Identifications and integration of the five most abundant molecular species in each class (sorted by their average peak area ratio) were manually verified using their MS/MS spectra and chromatograms. |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | Internal_Standard.pdf |
| MS ID: | MS005202 |
| Analysis ID: | AN005476 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Lipostar software (Version 2.1.2.; Molecular Discovery) was used for feature detection, noise and artifact reduction, peak alignment, normalization, and lipid identification. Data were processed as follows: 1. Perform peak picking, smoothing, and retention time alignment to generate a list of all features. 2. Apply retention time window filter, retaining data from 1 to 28 min. 3. Remove peaks that did not contain MS/MS spectra and removed noise and high-signal background. 4. Perform automated lipid identification by querying the Lipid Maps database. The identification algorithm uses the detected accurate-mass m/z with a 5 ppm mass tolerance filter, as well as the corresponding MS/MS spectrum to identify lipids, and it ranks the identifications via a 1-to-4-star scale of increasing confidence. 5. The integrated areas of all identified lipids were normalized to the integrated area of their corresponding class-specific internal standard within each sample when possible. 6. Identifications and integration of the five most abundant molecular species in each class (sorted by their average peak area ratio) were manually verified using their MS/MS spectra and chromatograms. |
| Ion Mode: | NEGATIVE |
| Analysis Protocol File: | Internal_Standard.pdf |
