#METABOLOMICS WORKBENCH juliehaines_20230221_084725 DATATRACK_ID:3753 STUDY_ID:ST002487 ANALYSIS_ID:AN004060 PROJECT_ID:PR001607
VERSION             	1
CREATED_ON             	February 21, 2023, 9:03 am
#PROJECT
PR:PROJECT_TITLE                 	Simultaneous targeting of PD-1 and IL2Rβγ with radiation therapy to inhibit
PR:PROJECT_TITLE                 	pancreatic cancer growth and metastasis
PR:PROJECT_SUMMARY               	In pancreatic ductal adenocarcinoma (PDAC) patients, we show that response to
PR:PROJECT_SUMMARY               	radiation therapy (RT) is characterized by increased IL2Rβ and IL2Rγ and
PR:PROJECT_SUMMARY               	decreased ILR2α expression. The bispecific aPD1-IL2v is a PD-1-targeted IL-2
PR:PROJECT_SUMMARY               	variant (IL2v) immunocytokine with engineered IL-2 cis-targeted to PD-1 and
PR:PROJECT_SUMMARY               	abolished IL2Rα binding, which enhances tumor-antigen specific T cell
PR:PROJECT_SUMMARY               	activation while reducing regulatory T cell (Treg) suppression. Using aPD1-IL2v
PR:PROJECT_SUMMARY               	in orthotopic PDAC KPC-driven tumor models, we show marked improvement in local
PR:PROJECT_SUMMARY               	and metastatic survival along with profound increase in tumor-infiltrating
PR:PROJECT_SUMMARY               	polyfunctional CD8+ T cell subsets with a transcriptionally and metabolically
PR:PROJECT_SUMMARY               	active phenotype, and preferential activation of antigen-specific CD8+ T cells.
PR:PROJECT_SUMMARY               	In combination with single dose RT, aPD1-IL2v treatment results in a robust,
PR:PROJECT_SUMMARY               	durable expansion of polyfunctional CD8+ T cells, T cell stemness,
PR:PROJECT_SUMMARY               	tumor-specific memory immune response, natural killer (NK) cell activation, and
PR:PROJECT_SUMMARY               	decreased Tregs. These data show that aPD1-IL2v leads to profound local and
PR:PROJECT_SUMMARY               	distant response in PDAC.
PR:INSTITUTE                     	University of Colorado Denver
PR:LABORATORY                    	Lab of Angelo D'Alessandro in collaboration with lab of Sana Karam
PR:LAST_NAME                     	Haines
PR:FIRST_NAME                    	Julie
PR:ADDRESS                       	12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
PR:EMAIL                         	julie.haines@cuanschutz.edu
PR:PHONE                         	3037243339
#STUDY
ST:STUDY_TITLE                   	Simultaneous targeting of PD-1 and IL2Rβγ with radiation therapy to inhibit
ST:STUDY_TITLE                   	pancreatic cancer growth and metastasis - mouse spleen and lymph node T-cell
ST:STUDY_TITLE                   	metabolomics
ST:STUDY_SUMMARY                 	Mice were orthotopically implanted with PK5L1940 cells then radiation therapy (8
ST:STUDY_SUMMARY                 	Gy) administered 7 days post-implantation. PD1-IL2v and aCD25 dosed once per
ST:STUDY_SUMMARY                 	week beginning day 7 post-implantation. Spleen and associated lymph nodes were
ST:STUDY_SUMMARY                 	collected at time of sacrifice and sorted for CD8+ T-cells. The obtained T-cells
ST:STUDY_SUMMARY                 	were profiled by mass spectrometry-based metabolomics.
ST:INSTITUTE                     	University of Colorado Denver
ST:LAST_NAME                     	Haines
ST:FIRST_NAME                    	Julie
ST:ADDRESS                       	12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
ST:EMAIL                         	julie.haines@cuanschutz.edu
ST:PHONE                         	3037243339
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Mus musculus
#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           	-	MSL-1	factor:Untrx	RAW_FILE_NAME=AG-23-MSL-1-r4
SUBJECT_SAMPLE_FACTORS           	-	MSL-2	factor:Untrx	RAW_FILE_NAME=AG-23-MSL-2-r5
SUBJECT_SAMPLE_FACTORS           	-	MSL-3	factor:Untrx	RAW_FILE_NAME=AG-23-MSL-3-r6
SUBJECT_SAMPLE_FACTORS           	-	MSL-4	factor:RT	RAW_FILE_NAME=AG-23-MSL-4-r13
SUBJECT_SAMPLE_FACTORS           	-	MSL-5	factor:RT	RAW_FILE_NAME=AG-23-MSL-5-r9
SUBJECT_SAMPLE_FACTORS           	-	MSL-6	factor:RT	RAW_FILE_NAME=AG-23-MSL-6-r7
SUBJECT_SAMPLE_FACTORS           	-	MSL-7	factor:IL2v	RAW_FILE_NAME=AG-23-MSL-7-r12
SUBJECT_SAMPLE_FACTORS           	-	MSL-8	factor:IL2v	RAW_FILE_NAME=AG-23-MSL-8-r15
SUBJECT_SAMPLE_FACTORS           	-	MSL-9	factor:IL2v	RAW_FILE_NAME=AG-23-MSL-9-r14
SUBJECT_SAMPLE_FACTORS           	-	MSL-10	factor:RT+IL2v	RAW_FILE_NAME=AG-23-MSL-10-r1
SUBJECT_SAMPLE_FACTORS           	-	MSL-11	factor:RT+IL2v	RAW_FILE_NAME=AG-23-MSL-11-r8
SUBJECT_SAMPLE_FACTORS           	-	MSL-12	factor:RT+IL2v	RAW_FILE_NAME=AG-23-MSL-12-r11
SUBJECT_SAMPLE_FACTORS           	-	MSL-13	factor:aCD25	RAW_FILE_NAME=AG-23-MSL-13-r10
SUBJECT_SAMPLE_FACTORS           	-	MSL-14	factor:aCD25	RAW_FILE_NAME=AG-23-MSL-14-r3
SUBJECT_SAMPLE_FACTORS           	-	MSL-15	factor:aCD25	RAW_FILE_NAME=AG-23-MSL-15-r2
#COLLECTION
CO:COLLECTION_SUMMARY            	Spleens of C57BL/6 mice were homogenized to a single cell suspension by mashing
CO:COLLECTION_SUMMARY            	the spleen through a 70 uM cell strainer, and the erythrocytes were lysed with
CO:COLLECTION_SUMMARY            	ACK (ammonium-chloride-potassium) lysis buffer for 3 min at RT. CD8 T cells were
CO:COLLECTION_SUMMARY            	sorted with a CD8-negative selection Stemcell isolation kit following
CO:COLLECTION_SUMMARY            	manufacturer instructions. Cells were washed with PBS at 1200RPM, 4degC for 10
CO:COLLECTION_SUMMARY            	minutes and pellets were flash frozen.
CO:SAMPLE_TYPE                   	Spleen; T-cells
#TREATMENT
TR:TREATMENT_SUMMARY             	Mice were orthotopically implanted with PK5L1940 cells. RT administered 7 days
TR:TREATMENT_SUMMARY             	post-implantation. PD1-IL2v and aCD25 dosed once per week beginning day 7
TR:TREATMENT_SUMMARY             	post-implantation.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Metabolites from frozen T-cell pellets were extracted with cold 5:3:2
SP:SAMPLEPREP_SUMMARY            	MeOH:acetonitrile:water at a concentration of 4 million cells/mL. Samples were
SP:SAMPLEPREP_SUMMARY            	vortexed 30 min at 4 degrees C then supernatants clarified by centrifugation (10
SP:SAMPLEPREP_SUMMARY            	min, 10,000 g, 4 degrees C) and transferred to autosampler vials.
SP:PROCESSING_STORAGE_CONDITIONS 	4℃
SP:EXTRACT_STORAGE               	-80℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Negative C18
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Phenomenex Kinetex C18 2.1 x 150 mm, 1.7 um
CH:SOLVENT_A                     	95% water 5% acetonitrile 1 mM ammonium acetate
CH:SOLVENT_B                     	95% acetonitrile 5% water 1 mM ammonium acetate
CH:FLOW_GRADIENT                 	0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min
CH:FLOW_GRADIENT                 	100-0% B, 3-5 min hold at 0% B
CH:FLOW_RATE                     	450 uL/min
CH:COLUMN_TEMPERATURE            	45
CH:SAMPLE_INJECTION              	10 uL
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Orbitrap Exploris 480
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	We use a Thermo Orbitrap Exploris 120 (not available in the drop down box).
MS:MS_COMMENTS                   	Resolution 120,000, scan range 65-975 m/z, maximum injection time 100 ms,
MS:MS_COMMENTS                   	microscans 1, automatic gain control (AGC) detection duration 20 msec, source
MS:MS_COMMENTS                   	voltage 2.0 kV, capillary temperature 320 C, vaporizer temp 200 C, and sheath
MS:MS_COMMENTS                   	gas 50, auxiliary gas 10, and sweep gas 1 (all nitrogen). Data converted to
MS:MS_COMMENTS                   	mzXML using RawConverter. Metabolites were annotated and integrated using Maven
MS:MS_COMMENTS                   	in conjunction with the KEGG database.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	peak area
MS_METABOLITE_DATA_START
Samples	MSL-1	MSL-2	MSL-3	MSL-4	MSL-5	MSL-6	MSL-7	MSL-8	MSL-9	MSL-10	MSL-11	MSL-12	MSL-13	MSL-14	MSL-15
Factors	factor:Untrx	factor:Untrx	factor:Untrx	factor:RT	factor:RT	factor:RT	factor:IL2v	factor:IL2v	factor:IL2v	factor:RT+IL2v	factor:RT+IL2v	factor:RT+IL2v	factor:aCD25	factor:aCD25	factor:aCD25
L-cystine	43963	1650	4752	3545	6025	3670	12223	4893	3792	4950	10809	15111	7941	2203	0
ADP	268482	30510	93663	14227	79423	12500	230306	92940	105986	123161	50742	83275	145680	64482	263940
GDP	35639	6734	36295	360	26530	12109	22063	24500	16242	30445	9147	20965	40322	25215	37779
CDP	107886	49467	66513	88669	90670	146967	5490	27948	17564	23735	18211	12614	10113	26497	48640
Cytidine	158019	9365	6463	8801	19624	5067	114368	59088	79671	61754	28903	79336	25621	50802	8065
UDP	994415	200388	330875	368497	808117	411942	627753	724402	743880	549227	535269	591840	632855	537286	368416
Xanthine	854929	2233	3804	3970	83823	18958	294676	40276	152937	51494	29625	235279	3276	42807	15782
5-Hydroxyisourate	3550	3689	6030	6810	7391	11411	5249	15853	8350	6091	14096	6199	7325	7867	6219
Urate	460284	148057	14623	171208	81371	313290	185330	89638	138233	203689	34998	384746	16744	80784	0
Adenylosuccinic acid	78239	0	312	0	7909	0	47606	2734	11053	10171	2608	17572	0	2478	1274
UDP-glucose	171044	0	20854	0	70996	7839	46399	33558	47870	41442	19143	20154	20514	24934	34668
Phosphate	22142932	16221733	37146324	3111541	43834328	25736296	14884719	33527174	27821616	36012456	39138028	35079608	40429132	34556316	46475004
Diphosphate	3667087	2550076	7267859	66317	8337971	5144761	2694636	6821682	4359119	7878210	7766020	5942359	6332894	7616070	11751784
D-Glucose	423444	3386060	2333913	7882769	1783717	2982387	142759	378147	180006	538100	279463	107138	192408	180171	422578
D-Glucose 6-phosphate	1382984	18496	93627	2643	303839	39850	253166	306171	228304	251532	141755	197013	192631	247020	239756
D-Fructose 1-6-bisphosphate	1732566	2373	81177	400	165035	15680	289306	20398	102296	86691	31209	263823	15678	47718	32423
D-Glyceraldehyde 3-phosphate/Glycerone phosphate	521648	1042	65176	461	293329	28174	169459	31419	143359	200704	99253	278310	9236	94816	9090
1-3-Bisphosphoglycerate	1170615	3928	252538	2201	664318	12833	154847	15398	270714	88973	47941	198234	20541	61160	206121
2/3-Phospho-D-glycerate	123872	1120	81885	2185	290003	12164	29772	102983	102710	222862	104448	62357	23951	135145	58093
Lactate	1187587	643679	516314	1434072	699642	808649	626420	200982	230343	515729	180679	565893	216768	266606	195455
Mannitol	104343	79164	140639	186710	165222	56986	31674	25624	23295	30585	52353	13994	64459	41092	94737
Citrate	5169674	140011	704377	158148	2492591	456076	1441253	441889	2236255	1187263	1487048	1974994	394735	2528073	1012813
Succinate	43891	87244	102929	188573	115919	49054	30868	34480	28222	54458	49256	28789	42687	79875	39469
Fumarate	216076	9425	11784	9401	38558	13803	116732	18750	39907	47923	29651	46433	14178	31946	16545
Malate	1519210	52980	285720	82742	791836	176143	820943	341185	571117	616442	555192	1371193	243279	563212	416602
2-Hydroxyglutarate/Citramalate*** (%CV=31.25)	55351	7973	13491	3580	51255	10951	18012	7546	28285	19374	26368	18626	9671	16142	11668
alpha-D-Ribose 1-phosphate	447637	3992	50025	1246	437049	79087	188967	140696	319761	312659	173006	272401	44127	330837	95685
5-Phospho-alpha-D-ribose 1-diphosphate	93406	1561	10482	0	44796	2801	55655	46207	132169	97751	84420	15374	4298	67861	34253
Dehydroascorbate	263050	12095	26703	10733	108426	39754	68821	31611	103567	52077	63534	63367	15317	106263	25836
UDP-N-acetyl-D-glucosamine	452523	3026	34350	0	165200	14082	272366	109719	184396	175684	117416	159171	32455	99584	68432
Glycolate	109868	137976	235394	138131	208824	142567	134789	207014	121138	100005	155292	197305	173847	125993	161497
N-Acetyl-L-citrulline	42022	34469	28490	71228	20316	25999	58906	28378	55933	37477	22991	32792	34980	32969	7466
L-Cysteate	13409	12226	5532	9531	6456	8655	10071	6226	10712	7839	9364	14256	6741	5910	18764
Glycerol 3-phosphate	46830	2456	15972	1428	51215	23981	20126	43401	57300	106154	34001	38191	2655	57549	19904
Ethanolamine phosphate	2559881	35574	252671	13404	941046	64999	1582107	698503	1046622	1189261	710176	1308192	314610	601378	370270
Octanoic acid (caprylate)	77843	131529	95118	114379	78266	85022	127971	133138	87083	78605	81493	115745	73490	106209	67534
Nonanoic acid (pelargonate)	922756	1075030	654945	1285880	1098707	930976	977252	1363611	1251925	1052921	1349510	937572	762380	976898	795306
Decanoic acid (caprate)	323268	483444	467808	668549	619130	475015	610371	683007	714245	291091	433444	676287	631825	488500	523059
Dodecanoic acid (lauric acid)	301332	436007	293821	364273	365145	391439	387215	467962	401798	288683	284638	333689	313480	348170	238048
Tetradecanoic acid (myristic acid)	704732	1320614	1112186	1320130	1665798	1628543	975353	1368791	1057957	997143	775041	1616647	1098642	704700	926216
Hexadecanoic acid (palmitic acid)	15198033	29100870	20738458	27146342	37355700	24802094	13409557	33964160	13580519	15771707	14503209	29664570	11176117	15528353	14597779
Hexadecenoic acid (Palmitoleic acid)	537994	510838	362585	376660	1063939	585439	454392	502132	448454	372645	313579	679807	308250	364913	523875
Octadecenoic acid (Oleic acid)	6926603	2361502	2047029	2379165	10650884	6421995	3116907	5633439	3122430	4365384	2404150	4803618	1324841	2002717	2614855
Linoleic acid ((9Z,12Z)-Octadecadienoic acid)	5268920	616418	1362268	1391346	9321381	7764527	2722649	3583602	4300749	3037445	2616824	5210467	770324	1495486	2181361
a-Linolenic acid (Octadecatrienoic acid)	244431	33667	50737	59122	315451	352006	111096	111680	96167	101475	50465	96665	24450	61578	87814
Arachidonic acid (Eicosatetraenoic acid)	11127387	714745	1936727	1266878	5430224	3336222	4888341	4674585	3110253	6195247	2332813	8718033	1699648	1742870	3483754
Docosahexaenoic acid	3440235	216377	969481	408371	2634631	1832777	2249247	1888406	2134761	1510435	1263660	2439894	542649	961734	1436957
Eicosapentaenoic acid	181174	23087	36536	37366	106035	199346	82512	67285	35909	39964	26269	56373	11611	19960	45186
(8Z-11Z-14Z)-Icosatrienoic acid	902185	41722	136933	59659	712681	296866	500543	369977	313612	312544	257551	649019	116159	141875	290132
(7Z-10Z-13Z-16Z-19Z)-Docosa-7-10-13-16-19-pentaenoic acid	642325	55219	129934	99537	456159	433277	354249	338696	325429	316011	221894	471053	93531	169707	303623
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	KEGG ID	parent	medRt
L-cystine	C00491	239.0150	0.6830
ADP	C00008	426.020447	0.57
GDP	C00035	442.015167	0.538
CDP	C00112	402.004822	2.145
Cytidine	C00475	242.077789	0.656
UDP	C00015	402.997437	0.777
Xanthine	C00385	151.025558	0.696
5-Hydroxyisourate	C11821	183.013046	0.772
Urate	C00366	167.020386	0.645
Adenylosuccinic acid	C03794	462.064911	0.516
UDP-glucose	C00029	565.045288	0.541
Phosphate	C00009	96.969231	0.596
Diphosphate	C00013	176.935074	0.587
D-Glucose	C00031	179.055389	0.666
D-Glucose 6-phosphate	C02965	259.021118	0.57
D-Fructose 1-6-bisphosphate	C00354	338.987335	0.53
D-Glyceraldehyde 3-phosphate/Glycerone phosphate	C00118	168.989685	0.576
1-3-Bisphosphoglycerate	C00236	264.950928	0.528
2/3-Phospho-D-glycerate	C00631	184.984894	0.535
Lactate	C01432	89.024048	0.644
Mannitol	C00392	181.070953	0.684
Citrate	C00158	191.018982	0.573
Succinate	C00042	117.018814	0.575
Fumarate	C00122	115.003181	0.546
Malate	C00149	133.013672	0.567
2-Hydroxyglutarate/Citramalate*** (%CV=31.25)	C02630	147.029297	0.593
alpha-D-Ribose 1-phosphate	C00620	229.010925	0.578
5-Phospho-alpha-D-ribose 1-diphosphate	C00119	388.942932	0.516
Dehydroascorbate	C05422	173.008423	0.556
UDP-N-acetyl-D-glucosamine	C00043	606.071838	0.54
Glycolate	C00160	75.008469	0.724
N-Acetyl-L-citrulline	C15532	216.095154	0.704
L-Cysteate	C00506	167.996643	0.818
Glycerol 3-phosphate	C00093	171.005554	0.579
Ethanolamine phosphate	C00346	140.011261	0.617
Octanoic acid (caprylate)	C06423	143.107178	2.087
Nonanoic acid (pelargonate)	C01601	157.122787	2.184
Decanoic acid (caprate)	C01571	171.138336	2.273
Dodecanoic acid (lauric acid)	C02679	199.169525	2.481
Tetradecanoic acid (myristic acid)	C06424	227.200729	2.775
Hexadecanoic acid (palmitic acid)	C00249	255.231918	3.207
Hexadecenoic acid (Palmitoleic acid)	C08362	253.216293	2.84
Octadecenoic acid (Oleic acid)	C00712	281.247528	3.249
Linoleic acid ((9Z,12Z)-Octadecadienoic acid)	C01595	279.231903	2.885
a-Linolenic acid (Octadecatrienoic acid)	C06427	277.216217	2.645
Arachidonic acid (Eicosatetraenoic acid)	C00219	303.231842	2.769
Docosahexaenoic acid	C06429	327.231781	2.66
Eicosapentaenoic acid	C06428	301.216156	2.574
(8Z-11Z-14Z)-Icosatrienoic acid	C03242	305.247437	2.975
(7Z-10Z-13Z-16Z-19Z)-Docosa-7-10-13-16-19-pentaenoic acid	C16513	329.247803	2.802
METABOLITES_END
#END