#METABOLOMICS WORKBENCH kwessendorf_20251208_105849 DATATRACK_ID:6857 STUDY_ID:ST004457 ANALYSIS_ID:AN007463 PROJECT_ID:PR002810
VERSION             	1
CREATED_ON             	December 17, 2025, 8:18 am
#PROJECT
PR:PROJECT_TITLE                 	Modeling Lipid Homeostasis Using Stable Isotope Tracing and Flux Analysis
PR:PROJECT_SUMMARY               	Lipids represent the most diverse pool of metabolites found in cells,
PR:PROJECT_SUMMARY               	facilitating compartmentation, signaling, and other functions. Dysregulation of
PR:PROJECT_SUMMARY               	lipid metabolism is linked to disease states ranging from cancer to
PR:PROJECT_SUMMARY               	neurodegeneration. However, limited tools are available for quantifying
PR:PROJECT_SUMMARY               	metabolic fluxes across the lipidome. To directly measure reaction fluxes
PR:PROJECT_SUMMARY               	encompassing membrane lipid homeostasis, we apply stable isotope tracing, liquid
PR:PROJECT_SUMMARY               	chromatography-high-resolution mass spectrometry, and network-based mass
PR:PROJECT_SUMMARY               	isotopomer modeling to non-small cell lung cancer (NSCLC) models. Lipid
PR:PROJECT_SUMMARY               	metabolic flux analysis (MFA) enables the concurrent quantitation of fatty acid
PR:PROJECT_SUMMARY               	synthesis, elongation, headgroup assembly, and salvage reactions within
PR:PROJECT_SUMMARY               	virtually any biological system. Lipid-MFA highlights distinct changes in fatty
PR:PROJECT_SUMMARY               	acid synthase and very long-chain fatty acid (VLCFA) elongation fluxes in
PR:PROJECT_SUMMARY               	typical culture conditions. Using this approach, we resolve differences in
PR:PROJECT_SUMMARY               	sphingolipid recycling in p53-deficient versus liver kinase B1 (LKB1)-deficient
PR:PROJECT_SUMMARY               	NSCLC tumors using precision-cut lung slice culture. Finally, Lipid-MFA
PR:PROJECT_SUMMARY               	demonstrates the unique trafficking of long-chain versus very long-chain
PR:PROJECT_SUMMARY               	ceramide fluxes as well as the isozyme specificity of a classical ceramide
PR:PROJECT_SUMMARY               	synthase inhibitor. These results illustrate the ability of Lipid-MFA to
PR:PROJECT_SUMMARY               	quantify lipid homeostasis and elucidate molecular mechanisms in membrane lipid
PR:PROJECT_SUMMARY               	metabolism.
PR:INSTITUTE                     	Salk Institute for Biological Studies
PR:LAST_NAME                     	Wessendorf-Rodriguez
PR:FIRST_NAME                    	Karl
PR:ADDRESS                       	10010N Torrey Pines Rd, La Jolla, California, 92037, USA
PR:EMAIL                         	kwessendorf@salk.edu
PR:PHONE                         	7874490440
#STUDY
ST:STUDY_TITLE                   	Compare lipid metabolism in lung tumors from mice harboring Kras G12D/+ and
ST:STUDY_TITLE                   	either STK1-/- (KL) or Tp53 -/- mutations (KP).
ST:STUDY_SUMMARY                 	Lung from mice with KL or KP tumors were inflated using a solution of 0.3% low
ST:STUDY_SUMMARY                 	melt agarose. The lobes were then sliced using a vibratome instrument to a
ST:STUDY_SUMMARY                 	thickness of 300µM and allowed to recover overnight in RPM1640 media containing
ST:STUDY_SUMMARY                 	2% FBS. After recovery, slices were washed with 0.9% w/V saline twice and
ST:STUDY_SUMMARY                 	tracing media was added. The tracing media was DMEM containing 1either 10mM 13C
ST:STUDY_SUMMARY                 	glucose or 0.4mM 13C serine and 2% FBS. At the end of the 24 hours of tracing,
ST:STUDY_SUMMARY                 	wells were washed three times with 0.9% w/V saline, then placed on a metal block
ST:STUDY_SUMMARY                 	chilled in a slurry of liquid nitrogen and ethanol to flash freeze the slices. A
ST:STUDY_SUMMARY                 	3mM dermal punch was used to isolate portions of the slice with tumors and
ST:STUDY_SUMMARY                 	transferred to eppendorfs and kept frozen until extracted. At the time of
ST:STUDY_SUMMARY                 	extracting, 3 ceramic beads were added to the punches along with 500µL of
ST:STUDY_SUMMARY                 	methanol and internal standards. Punches were homogenized using a Retsch
ST:STUDY_SUMMARY                 	instrument for 10 minutes at a setting of 30Hz. 50µL of the homogenate was
ST:STUDY_SUMMARY                 	taken for protien measurement, while the rest was spun down at 21000gs for
ST:STUDY_SUMMARY                 	15minutes. The supernatant was transferred to a new tube and dried under
ST:STUDY_SUMMARY                 	nitrogen. The dried metabolites were kept in a -80C freezer until they were
ST:STUDY_SUMMARY                 	resuspended in 60uL of methanol and analyzed on a C18 column. MS data was
ST:STUDY_SUMMARY                 	analyzed using EL-Maven v0.12.1 with a mass error of 5ppm and maximum retention
ST:STUDY_SUMMARY                 	time shift between parent ions and isotopomer ions of 5 seconds. An in-house
ST:STUDY_SUMMARY                 	matlab script was used for natural abundance correction.
ST:INSTITUTE                     	Salk Institute for Biological Studies
ST:LAST_NAME                     	Wessendorf-Rodriguez
ST:FIRST_NAME                    	Karl
ST:ADDRESS                       	10010 N Torrey Pines Rd
ST:EMAIL                         	kwessendorf@salk.edu
ST:PHONE                         	7874490440
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	KL_Gluc_1	KL_Gluc_1	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Gluc_1.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Gluc_2	KL_Gluc_2	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Gluc_2.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Gluc_3	KL_Gluc_3	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Gluc_3.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Gluc_4	KL_Gluc_4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Gluc_4.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Gluc_5	KL_Gluc_5	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Gluc_5.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Gluc_6	KL_Gluc_6	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Gluc_6.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Gluc_7	KL_Gluc_7	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Gluc_7.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Gluc_8	KL_Gluc_8	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Gluc_8.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Gluc_9	KL_Gluc_9	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Gluc_9.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Gluc_10	KL_Gluc_10	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Gluc_10.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Gluc_11	KL_Gluc_11	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Gluc_11.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Gluc_12	KL_Gluc_12	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Gluc_12.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Ser_1	KL_Ser_1	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Ser_1.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Ser_2	KL_Ser_2	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Ser_2.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Ser_3	KL_Ser_3	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Ser_3.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Ser_4	KL_Ser_4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Ser_4.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Ser_5	KL_Ser_5	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Ser_5.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Ser_6	KL_Ser_6	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Ser_6.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Ser_7	KL_Ser_7	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Ser_7.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Ser_8	KL_Ser_8	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Ser_8.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Ser_9	KL_Ser_9	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Ser_9.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Ser_10	KL_Ser_10	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Ser_10.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Ser_11	KL_Ser_11	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Ser_11.mzXML
SUBJECT_SAMPLE_FACTORS           	KL_Ser_12	KL_Ser_12	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KL_Ser_12.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Gluc_1	KP_Gluc_1	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Gluc_1.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Gluc_2	KP_Gluc_2	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Gluc_2.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Gluc_3	KP_Gluc_3	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Gluc_3.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Gluc_4	KP_Gluc_4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Gluc_4.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Gluc_5	KP_Gluc_5	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Gluc_5.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Gluc_6	KP_Gluc_6	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Gluc_6.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Gluc_7	KP_Gluc_7	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Gluc_7.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Gluc_8	KP_Gluc_8	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Gluc_8.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Gluc_9	KP_Gluc_9	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Gluc_9.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Gluc_10	KP_Gluc_10	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Gluc_10.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Gluc_11	KP_Gluc_11	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Gluc_11.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Gluc_12	KP_Gluc_12	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Gluc_12.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Ser_1	KP_Ser_1	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Ser_1.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Ser_2	KP_Ser_2	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Ser_2.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Ser_3	KP_Ser_3	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Ser_3.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Ser_4	KP_Ser_4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Ser_4.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Ser_5	KP_Ser_5	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Ser_5.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Ser_6	KP_Ser_6	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Ser_6.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Ser_7	KP_Ser_7	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Ser_7.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Ser_8	KP_Ser_8	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Ser_8.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Ser_9	KP_Ser_9	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Ser_9.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Ser_10	KP_Ser_10	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Ser_10.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Ser_11	KP_Ser_11	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Ser_11.mzXML
SUBJECT_SAMPLE_FACTORS           	KP_Ser_12	KP_Ser_12	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	RAW_FILE_NAME(Raw_File_Name)=KP_Ser_12.mzXML
#COLLECTION
CO:COLLECTION_SUMMARY            	Lung from mice with KL or KP tumors were inflated using a solution of 0.3% low
CO:COLLECTION_SUMMARY            	melt agarose. The lobes were then sliced using a vibratome instrument to a
CO:COLLECTION_SUMMARY            	thickness of 300µM and allowed to recover overnight in RPM1640 media containing
CO:COLLECTION_SUMMARY            	2% FBS. After recovery, slices were washed with 0.9% w/V saline twice and
CO:COLLECTION_SUMMARY            	tracing media was added. The tracing media was DMEM containing 1either 10mM 13C
CO:COLLECTION_SUMMARY            	glucose or 0.4mM 13C serine and 2% FBS. At the end of the 24 hours of tracing,
CO:COLLECTION_SUMMARY            	wells were washed three times with 0.9% w/V saline, then placed on a metal block
CO:COLLECTION_SUMMARY            	chilled in a slurry of liquid nitrogen and ethanol to flash freeze the slices. A
CO:COLLECTION_SUMMARY            	3mM dermal punch was used to isolate portions of the slice with tumors and
CO:COLLECTION_SUMMARY            	transferred to eppendorfs and kept frozen until extracted. At the time of
CO:COLLECTION_SUMMARY            	extracting, 3 ceramic beads were added to the punches along with 500µL of
CO:COLLECTION_SUMMARY            	methanol and internal standards. Punches were homogenized using a Retsch
CO:COLLECTION_SUMMARY            	instrument for 10 minutes at a setting of 30Hz. 50µL of the homogenate was
CO:COLLECTION_SUMMARY            	taken for protein measurement, while the rest was spun down at 21000gs for
CO:COLLECTION_SUMMARY            	15minutes. The supernatant was transferred to a new tube and dried under
CO:COLLECTION_SUMMARY            	nitrogen. The dried metabolites were kept in a -80C freezer until they were
CO:COLLECTION_SUMMARY            	resuspended in 60uL of methanol and analyzed on a C18 column10 nanomoles of
CO:COLLECTION_SUMMARY            	norvaline was added to each sample along with a mixture of Equisplash (Avanti
CO:COLLECTION_SUMMARY            	Polar Lipids, Cat# 330731), and SPB 18:0;O2 [D7] (Avanti Polar Lipids, Cat#
CO:COLLECTION_SUMMARY            	860658), SPB 18:1;O2 [D7] (Avanti Polar Lipids, Cat# 860657), glucosylceramide
CO:COLLECTION_SUMMARY            	18:1;O2[D7]-15:0 (Avanti Polar Lipids, Cat# 330729), lactosylceramide
CO:COLLECTION_SUMMARY            	18:1;O2[D7] (Avanti Polar Lipids, Cat# 330727), and GM3-d3 18:1;O2/18:0 [D3]
CO:COLLECTION_SUMMARY            	(Cayman Chemicals, item No. 39226). The Eppendorfs with 100% methanol, used for
CO:COLLECTION_SUMMARY            	polar lipid analysis on C18, were centrifuged at 21,000 g for 15 min at 4°C.
CO:COLLECTION_SUMMARY            	300 μL of methanol were collected and evaporated under nitrogen and stored at
CO:COLLECTION_SUMMARY            	-80°C until resuspended for analysis.
CO:SAMPLE_TYPE                   	Lung
#TREATMENT
TR:TREATMENT_SUMMARY             	After overnight recovery in RPMI 1640, slices were washed twice with PBS and
TR:TREATMENT_SUMMARY             	then cultured in DMEM containing 10mM glucose and 2% FBS for 24 hours. Media
TR:TREATMENT_SUMMARY             	used contained either 13C glucose or 13C serine to enable stable isotope
TR:TREATMENT_SUMMARY             	tracing.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Lipids were extracted from cell pellets using 100% methanol for polar lipids on
SP:SAMPLEPREP_SUMMARY            	a C18 column. Lipid containing phases were dried under nitrogen and stored at
SP:SAMPLEPREP_SUMMARY            	-80C until they were resuspended for the appropriate analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	All metabolites abundances were calculated from C18 analysis.
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
CH:SOLVENT_A                     	98:2 v/v water: methanol with 5 mM ammonium acetate
CH:SOLVENT_B                     	50:50 v/v methanol: isopropanol with 5 mM ammonium acetate
CH:FLOW_GRADIENT                 	0 min, 30% B; 1 min, 30% B; 2 min, 70% B; 11 min, 95%B; 17 min, 30%B; 21.5 min,
CH:FLOW_GRADIENT                 	30%B; 27 min, 30% B
CH:FLOW_RATE                     	0.2 mL/min
CH:COLUMN_TEMPERATURE            	35
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Lipids were analyzed in positive mode using spray voltage 3.5 kV. Sweep gas
MS:MS_COMMENTS                   	flow was 1 arbitrary units, auxiliary gas flow 10 arbitrary units and sheath gas
MS:MS_COMMENTS                   	flow 50 arbitrary units, with a capillary temperature of 325 °C. Full mass
MS:MS_COMMENTS                   	spectrometry (scan range 220–2,500 m/z) was used at 140,000 resolution with
MS:MS_COMMENTS                   	10E6 automatic gain control and a maximum injection time of 100 ms. Data were
MS:MS_COMMENTS                   	analyzed using EI-Maven. Mass error was set to 5 ppm for metabolite
MS:MS_COMMENTS                   	identification. Isotopologues were filtered to elute within 5 seconds of the
MS:MS_COMMENTS                   	unlabeled parent ion to ensure all labeled species came from the same
MS:MS_COMMENTS                   	metabolite. Mass isotopologue distributions were analyzed with an in-house
MS:MS_COMMENTS                   	MATLAB script which integrated the metabolite fragment ions and corrected for
MS:MS_COMMENTS                   	natural isotope abundances.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Relative Abundance
MS_METABOLITE_DATA_START
Samples	KL_Gluc_1	KL_Gluc_2	KL_Gluc_3	KL_Gluc_4	KL_Gluc_5	KL_Gluc_6	KL_Gluc_7	KL_Gluc_8	KL_Gluc_9	KL_Gluc_10	KL_Gluc_11	KL_Gluc_12	KL_Ser_1	KL_Ser_2	KL_Ser_3	KL_Ser_4	KL_Ser_5	KL_Ser_6	KL_Ser_7	KL_Ser_8	KL_Ser_9	KL_Ser_10	KL_Ser_11	KL_Ser_12	KP_Gluc_1	KP_Gluc_2	KP_Gluc_3	KP_Gluc_4	KP_Gluc_5	KP_Gluc_6	KP_Gluc_7	KP_Gluc_8	KP_Gluc_9	KP_Gluc_10	KP_Gluc_11	KP_Gluc_12	KP_Ser_1	KP_Ser_2	KP_Ser_3	KP_Ser_4	KP_Ser_5	KP_Ser_6	KP_Ser_7	KP_Ser_8	KP_Ser_9	KP_Ser_10	KP_Ser_11	KP_Ser_12
Factors	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Stk11-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C6]glucose | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4	Sample source:Precision Cut Lung Slices (Mus musculus) | Genotype:Kras G12D/+ and Tp53-/- | Treatment:2% FBS and [U-13C3]serine | Injection order:B4
SPB 18:0;O2	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	0.004805415	0.013163608	0.00503661	0.002578243	0.010057833	0.005704277	0.007641641	0.0089827	0.006145181	0.012134208	0.009507139	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	0.000898203	0.001864916	0.001606755	0.001104122	0.00092921	0*	0.00262002	0.002149735	0.0023236	0.001865172	0.001742266	0.002075694
SPB 18:1;O2	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	0.004440099	0.006390236	0.003434006	0.003391566	0.006695492	0.005614739	0.00484374	0.007777379	0.005039846	0.007149676	0.005989356	0.007564248	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	0.004165024	0.005519616	0.008396966	0.004188517	0.002770795	0.00495471	0.008128712	0.005009761	0.006569908	0.00507843	0.005495067	0.005373683
Cer 18:0;O2/24:0	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	0.053126988	0.071077365	0.056625151	0.048468497	0.121453941	0.109912911	0.0847571	0.158924456	0.086663859	0.100941714	0.102194925	0.074107	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	0.017568763	0.01984134	0.040834065	0.014814919	0.022038371	0.028858044	0.035871927	0.044226411	0.045124946	0.042930199	0.040485557	0.050552738
Cer 18:1;O2/24:0	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	0.084536811	0.100557687	0.07760791	0.071791122	0.146627218	0.119741961	0.11426528	0.177067862	0.072756072	0.10050144	0.091625267	0.100207392	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	0.06380459	0.070727769	0.183508948	0.060652227	0.063634915	0.070880564	0.098834664	0.095694071	0.10210429	0.096125449	0.094747404	0.102912536
SM 42:1;O2	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	0.321974925	0.359661062	0.244391533	0.268759864	0.505608007	0.413448038	0.377063878	0.622686178	0.207642907	0.35255172	0.243064268	0.311305904	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	0.163391062	0.183982919	0.438082734	0.153561162	0.166815652	0.219085152	0.268185089	0.254594747	0.314060494	0.257272921	0.259896902	0.294223063
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Retention time	m/z ratio
SPB 18:0;O2	7.47	302.3050
SPB 18:1;O2	7.22	300.2893
Cer 18:0;O2/24:0	15.52	652.6604
Cer 18:1;O2/24:0	15.12	650.6445
SM 42:1;O2	14.09	815.7004
METABOLITES_END
#END