#METABOLOMICS WORKBENCH Stopka28_20240201_065322 DATATRACK_ID:4622 STUDY_ID:ST003065 ANALYSIS_ID:AN005021 PROJECT_ID:PR001911 VERSION 1 CREATED_ON February 2, 2024, 12:49 pm #PROJECT PR:PROJECT_TITLE Investigative needle core biopsies for multi-omics in Glioblastoma PR:PROJECT_TYPE Single Cell Spatial Analysis Reveals the Topology of Immunomodulatory Purinergic PR:PROJECT_TYPE Signaling in Glioblastoma PR:PROJECT_SUMMARY Glioblastoma (GBM) is a primary brain cancer with an abysmal prognosis and few PR:PROJECT_SUMMARY effective therapies. The ability to investigate the tumor microenvironment PR:PROJECT_SUMMARY before and during treatment would greatly enhance both our understanding of PR:PROJECT_SUMMARY disease response and progression, as well as the delivery and impact of PR:PROJECT_SUMMARY therapeutics. Stereotactic biopsies are a routine surgical procedure performed PR:PROJECT_SUMMARY primarily for diagnostic histopathologic purposes. The role of investigative PR:PROJECT_SUMMARY biopsies – tissue sampling for the purpose of understanding tumor PR:PROJECT_SUMMARY microenvironmental responses to treatment using integrated multi-modal molecular PR:PROJECT_SUMMARY analyses (‘Multi-omics”) has yet to be defined. Here we adapt stereotactic PR:PROJECT_SUMMARY needle core biopsy tissue for highly resolved multi-omics analysis methods PR:PROJECT_SUMMARY including single cell RNA sequencing, spatial-transcriptomics, metabolomics, PR:PROJECT_SUMMARY proteomics, phosphoproteomics, T-cell clonotype analysis, and MHC Class I PR:PROJECT_SUMMARY immunopeptidomics. Biopsy tissue was obtained from a single patient with PR:PROJECT_SUMMARY recurrent GBM during one procedure. In a second patient, we analyzed PR:PROJECT_SUMMARY multi-regional core biopsies to decipher spatial and genomic variance. Finally PR:PROJECT_SUMMARY in a separate cohort of patients we investigated the utility of stereotactic PR:PROJECT_SUMMARY biopsies as a method for generating patient derived xenograft models. Dataset PR:PROJECT_SUMMARY integration across modalities showed good correspondence between spatial PR:PROJECT_SUMMARY modalities and highlighted immune cell associated metabolic pathways and poor PR:PROJECT_SUMMARY correlation between RNA expression and the tumor MHC Class I immunopeptidome. In PR:PROJECT_SUMMARY conclusion, stereotactic needle biopsy cores are of sufficient quality for the PR:PROJECT_SUMMARY purposes of investigative biopsy and can generate multi-omics data, providing PR:PROJECT_SUMMARY data rich insight into a patient’s disease process and tumor immune PR:PROJECT_SUMMARY microenvironment and could be of potential value in evaluating treatment PR:PROJECT_SUMMARY responses. PR:INSTITUTE Brigham and Women's Hospital PR:DEPARTMENT Department of Neurosurgery PR:LABORATORY Nathalie Y.R. Agar PR:LAST_NAME Stopka PR:FIRST_NAME Sylwia PR:ADDRESS 60 Fenwood Rd, PR:EMAIL sstopka@bwh.harvard.edu PR:PHONE 617-525-9746 #STUDY ST:STUDY_TITLE Investigative needle core biopsies for multi-omics in Glioblastoma ST:STUDY_SUMMARY Glioblastoma (GBM) is a primary brain cancer with an abysmal prognosis and few ST:STUDY_SUMMARY effective therapies. The ability to investigate the tumor microenvironment ST:STUDY_SUMMARY before and during treatment would greatly enhance both our understanding of ST:STUDY_SUMMARY disease response and progression, as well as the delivery and impact of ST:STUDY_SUMMARY therapeutics. Stereotactic biopsies are a routine surgical procedure performed ST:STUDY_SUMMARY primarily for diagnostic histopathologic purposes. The role of investigative ST:STUDY_SUMMARY biopsies – tissue sampling for the purpose of understanding tumor ST:STUDY_SUMMARY microenvironmental responses to treatment using integrated multi-modal molecular ST:STUDY_SUMMARY analyses (‘Multi-omics”) has yet to be defined. Here we adapt stereotactic ST:STUDY_SUMMARY needle core biopsy tissue for highly resolved multi-omics analysis methods ST:STUDY_SUMMARY including single cell RNA sequencing, spatial-transcriptomics, metabolomics, ST:STUDY_SUMMARY proteomics, phosphoproteomics, T-cell clonotype analysis, and MHC Class I ST:STUDY_SUMMARY immunopeptidomics. Biopsy tissue was obtained from a single patient with ST:STUDY_SUMMARY recurrent GBM during one procedure. In a second patient, we analyzed ST:STUDY_SUMMARY multi-regional core biopsies to decipher spatial and genomic variance. Finally ST:STUDY_SUMMARY in a separate cohort of patients we investigated the utility of stereotactic ST:STUDY_SUMMARY biopsies as a method for generating patient derived xenograft models. Dataset ST:STUDY_SUMMARY integration across modalities showed good correspondence between spatial ST:STUDY_SUMMARY modalities and highlighted immune cell associated metabolic pathways and poor ST:STUDY_SUMMARY correlation between RNA expression and the tumor MHC Class I immunopeptidome. In ST:STUDY_SUMMARY conclusion, stereotactic needle biopsy cores are of sufficient quality for the ST:STUDY_SUMMARY purposes of investigative biopsy and can generate multi-omics data, providing ST:STUDY_SUMMARY data rich insight into a patient’s disease process and tumor immune ST:STUDY_SUMMARY microenvironment and could be of potential value in evaluating treatment ST:STUDY_SUMMARY responses. ST:INSTITUTE Brigham and Women's Hospital ST:DEPARTMENT Department of Neurosurgery ST:LABORATORY Nathalie Y.R. Agar ST:LAST_NAME Stopka ST:FIRST_NAME Sylwia ST:ADDRESS 60 Fenwood Rd ST:EMAIL sstopka@bwh.harvard.edu ST:PHONE 617-525-9746 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #FACTORS #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 - btc_pilot_tissue-total ion count sample_id:btc_pilot_tissue-total ion count RAW_FILE_NAME=btc_pilot_tissue-total ion count #COLLECTION CO:COLLECTION_SUMMARY Biopsy Core Four underwent a preservation process, being rapidly frozen on dry CO:COLLECTION_SUMMARY ice. For MALDI MSI the core was then cryo-sectioned into 10 µm-thick slices. CO:SAMPLE_TYPE Brain #TREATMENT TR:TREATMENT_SUMMARY N/A #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Biopsy Core Four underwent a preservation process, being rapidly frozen on dry SP:SAMPLEPREP_SUMMARY ice. For MALDI MSI the core was then cryo-sectioned into 10 µm-thick slices. SP:SAMPLEPREP_SUMMARY These slices were subsequently thaw-mounted onto an indium tin oxide (ITO) SP:SAMPLEPREP_SUMMARY slide. Serial sections were prepared for microscopy staining. For the mass SP:SAMPLEPREP_SUMMARY spectrometry analysis, we utilized a 1,5-diaminonaphthalene hydrochloride MALDI SP:SAMPLEPREP_SUMMARY matrix, with 15N glutamate spiked in as an internal standard. The matrix was SP:SAMPLEPREP_SUMMARY prepared at a concentration of 4.3 mg/mL in a mixture of 4.5 parts HPLC grade SP:SAMPLEPREP_SUMMARY water, 5 parts ethanol, and 0.5 parts 1 M HCl (v/v/v). A TM-sprayer from HTX SP:SAMPLEPREP_SUMMARY Imaging was used to spray the matrix, with the following parameters: a flow rate SP:SAMPLEPREP_SUMMARY of 0.09 mL/min, spray nozzle velocity of 1200 mm/min, spray nozzle SP:SAMPLEPREP_SUMMARY temperature of 75 °C, nitrogen gas pressure of 10 psi, track spacing of SP:SAMPLEPREP_SUMMARY 2 mm, and a four-pass spray. The mass spectrometry analysis was carried out SP:SAMPLEPREP_SUMMARY using a 15 Tesla SolariX XR FT-ICR MS (Bruker Daltonics, Billerica, MA). The SP:SAMPLEPREP_SUMMARY instrument was set to negative ion mode, and the mass range scanned was from m/z SP:SAMPLEPREP_SUMMARY 46.07 to 3000, with a sampling step size of 30 µm. Each sampling point SP:SAMPLEPREP_SUMMARY consisted of 200 laser shots at a laser power of 21% (arbitrary scale), with a SP:SAMPLEPREP_SUMMARY laser repetition rate of 1,000 Hz. We employed the Continuous Accumulation of SP:SAMPLEPREP_SUMMARY Selected Ions (CASI) mode, setting Q1 to m/z 150 with an isolation window of SP:SAMPLEPREP_SUMMARY 200. The mass range was calibrated using a tune mix solution from Agilent SP:SAMPLEPREP_SUMMARY Technologies with the electrospray source. Additionally, the internal standard SP:SAMPLEPREP_SUMMARY 15N glutamate was used for on-line calibration during the acquisition. For data SP:SAMPLEPREP_SUMMARY analysis, SCiLS Lab software (version 2023c Pro, Bruker Daltonics, Billerica, SP:SAMPLEPREP_SUMMARY MA) was used to view and process ion images and mass spectra. The dataset was SP:SAMPLEPREP_SUMMARY normalized to the total ion current (TIC), and peaks were annotated using SP:SAMPLEPREP_SUMMARY Metaboscape (2021b,Bruker Daltonics, Billerica, MA)). Metabolites were SP:SAMPLEPREP_SUMMARY putatively annotated based on an accurate mass with a Δppm < 0.5 and MSMS SP:SAMPLEPREP_SUMMARY measurements. MetaboanalystR (85) was used to perform pathway enrichment SP:SAMPLEPREP_SUMMARY analysis at every pixel. The resulting enrichment ratios were displayed SP:SAMPLEPREP_SUMMARY spatially with an in-house R script. Metabolite differential expression was SP:SAMPLEPREP_SUMMARY tested with a t-test and FDR correction. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE None (Direct infusion) CH:INSTRUMENT_NAME none CH:COLUMN_NAME none CH:SOLVENT_A none CH:SOLVENT_B none CH:FLOW_GRADIENT none CH:FLOW_RATE none CH:COLUMN_TEMPERATURE none #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker Solarix FT-ICR-MS MS:INSTRUMENT_TYPE FT-ICR MS:MS_TYPE MALDI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Bruker software #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Total Ion Count MS_METABOLITE_DATA_START Samples btc_pilot_tissue-total ion count Factors sample_id:btc_pilot_tissue-total ion count Acetyaspartic acid 424822.5 Acetylphosphate 217920.094 Acetylputrescine 23420.4492 Adenine 301557.25 Adenosine diphosphate 87756.2344 Adenosine monophosphate 350348.188 Alanine 55727.6836 Alanylasparagine 86445.9297 alky acylglycerophosphoethanolamine 266219.594 Amino-carboline 54734.8477 amino-carboxyethyl hydroxybenzothiazole 180788.109 Aminoadipic acid 228410.125 apo-methylcrotonoyCoA:carbon-dioxide ligase 35466.9766 Arachidonic acid 747459.563 Arginine 36122.3672 Ascorbic acid 617553.063 Asparaginylglutamic acid 49101.3555 AsparaginyMethionine 143647.719 AsparaginyProline 99109.6875 AsparaginyValine 106507.711 Aspartic acid 1165955.38 aspartyalanine 38705.125 Asymmetric dimethylarginine 30156.1016 Azelaic acid 33608.5586 Butyrolactone 19186.9668 Caffeine 21908.2285 Carboline 115116.109 Carboxy-aminopropyhistidine 218028.875 Carnosine 70159.3203 Chlorotryptophan 120525.781 Citramalic acid 253888.719 Citrulline 47153.4336 Citryglutamic acid 350752.281 CPA(18:0/0:0) 394687.656 Creatine 147777.734 Cyanomethyhydroxy-indoone 53882.5859 Cystathionine 774093.563 Cysteine 104271.844 CysteinyGlutamine 72571.9844 Diacetylspermidine 20104.1836 Dimethy dithiolane 313623.563 Docosahexaenoic acid 247204.859 Dodecylbenzenesulfonic acid 169353.859 Dopamine 26368.4238 Erythronic acid 324575.406 Erythrose phosphate 50793.3984 Ethosuximide 22429.6094 Ethylamino dihydroxybenzamide 18162.4512 Etomidate 29052.5488 Fosfomycin 524999.438 Fructose phosphate 546009.375 Fucose phosphate 72976.9063 Furoic acid 232873.453 Garcinia acid 29595.502 Gentisic acid 222614.125 Glucuronic acid 170768.422 Glutamic acid 4892345 Glutaminylproline 37821.5352 Glutamylalanine 49245.3867 Glutamylhydroxyproline 17032.543 Glutarylglycine 421187.906 Glutathione 1824109.38 Glycerylphosphorylethanolamine 492146.219 Glycylproline 165610.281 Guanadrel 41373.2227 Guanine 100453.789 Hexenedioic acid 64986.5352 Histidine 304277.156 Histidine trimethylbetaine 28675.4785 HistidinyIsoleucine 18043.6582 HistidinySerine 21893.8828 Homogentisic acid 29847.3379 HPPH 22881.8848 Hydantoin-propionic acid 38369.1172 Hydroxy-oxoglutaric acid 234788.078 Hydroxybenzoic acid 26790.25 Hydroxymethyl pteridinedione 9388.17383 Hydroxymethymethyluracil 29567.3711 Hydroxyminaline 24197.9492 Hydroxyphenylacetic acid 408450.563 Hydroxyproline 85656.5234 Hypogeic acid 67379.7031 I 85816.7109 Indoleacetonitrile 4140482.5 Inosine 672129 Isobutyrylglycine 49350.6758 Isopropy dimethoxy-methylpyrazine 19959.5059 Ketobutyric acid 15661.5371 Ketoisovaleric acid 4728.43262 Kynurenic acid 48840.4414 Lactic acid 26178.1738 Levetiracetam 48294.3438 Lidocaine 87810.125 Linoleic acid 170627.641 Lycoperdic acid 279614.375 Malic acid 1692631.88 Methoxytyramine 21000.5547 Methy-thiophenecarboxaldehyde 17674.7363 Methy(furfurylthio)pyrazine 19373.9629 Methylhistidine 57948.7422 Methylindole 143231.813 Methylquinoline 86869.6484 Methylquinoxaline 30227.8457 Methythienymethylene-furanone 116725.633 Metronidazole 58306.4883 Monoethyl malonic acid 393930.906 Muconic acid 29167.3047 N-acetamidopropyl pyrrolidin-one 42327.3477 N-acety aminoadipate 62371.0273 N-Acetycitrulline 71884.6016 N-Nitrosoproline 101076.742 Nepafenac 4572.64941 Nicotinuric acid 20494.9707 Norophthalmic acid 258962.188 Oleic acid 1069957.38 oxoethoxy sulfonic acid 27320.9492 Oxoglutaric acid 72139.3203 Palmitic acid 609174.625 Pentanesulfenothioic acid 203498.719 Pentaporphyrin I 320733.563 Phosphate 1420816.75 Phosphodimethylethanolamine 1029447.13 Phosphoglyceric acid 321402.813 Phosphoserine 78365.1875 Phthalic acid 106712.211 Propenyl propenylthio propenyl sulfate 16727.0645 Purine 17275.0762 Pyridoxamine 59651.6367 Pyroglutamylglycine 64695.1641 Pyrophosphate 238193.609 Ribothymidine 23060.9922 Saccharopine 14183.1455 Sinalexin 100159.547 Stearic acid 578371.938 Succinic anhydride 24399.3184 Succinyglutamic acid semialdehyde 28319.1797 Sulfate 89183.8438 Sulfinoalanine 64012.3984 Tartaric acid 6933.96484 Taurine 2651377.5 Tetrahydro-carboline-carboxylic acid 39562.9531 Threonine 190020.922 Thymine 102251.234 trihydroxy-hydroxymethyl oxolan-yl methoxy sulfonic acid 50618.7305 Triphosphate 43337.918 Tryptophan 23256.2617 Undecylbenzenesulfonic acid 175846.188 Urate radical 71950.875 Uridine 27083.293 Urolithin D 29870.3477 Xanthine 372827.219 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Acetyaspartic acid Acetylphosphate Acetylputrescine Adenine Adenosine diphosphate Adenosine monophosphate Alanine Alanylasparagine alky acylglycerophosphoethanolamine Amino-carboline amino-carboxyethyl hydroxybenzothiazole Aminoadipic acid apo-methylcrotonoyCoA:carbon-dioxide ligase Arachidonic acid Arginine Ascorbic acid Asparaginylglutamic acid AsparaginyMethionine AsparaginyProline AsparaginyValine Aspartic acid aspartyalanine Asymmetric dimethylarginine Azelaic acid Butyrolactone Caffeine Carboline Carboxy-aminopropyhistidine Carnosine Chlorotryptophan Citramalic acid Citrulline Citryglutamic acid CPA(18:0/0:0) Creatine Cyanomethyhydroxy-indoone Cystathionine Cysteine CysteinyGlutamine Diacetylspermidine Dimethy dithiolane Docosahexaenoic acid Dodecylbenzenesulfonic acid Dopamine Erythronic acid Erythrose phosphate Ethosuximide Ethylamino dihydroxybenzamide Etomidate Fosfomycin Fructose phosphate Fucose phosphate Furoic acid Garcinia acid Gentisic acid Glucuronic acid Glutamic acid Glutaminylproline Glutamylalanine Glutamylhydroxyproline Glutarylglycine Glutathione Glycerylphosphorylethanolamine Glycylproline Guanadrel Guanine Hexenedioic acid Histidine Histidine trimethylbetaine HistidinyIsoleucine HistidinySerine Homogentisic acid HPPH Hydantoin-propionic acid Hydroxy-oxoglutaric acid Hydroxybenzoic acid Hydroxymethyl pteridinedione Hydroxymethymethyluracil Hydroxyminaline Hydroxyphenylacetic acid Hydroxyproline Hypogeic acid I Indoleacetonitrile Inosine Isobutyrylglycine Isopropy dimethoxy-methylpyrazine Ketobutyric acid Ketoisovaleric acid Kynurenic acid Lactic acid Levetiracetam Lidocaine Linoleic acid Lycoperdic acid Malic acid Methoxytyramine Methy-thiophenecarboxaldehyde Methy(furfurylthio)pyrazine Methylhistidine Methylindole Methylquinoline Methylquinoxaline Methythienymethylene-furanone Metronidazole Monoethyl malonic acid Muconic acid N-acetamidopropyl pyrrolidin-one N-acety aminoadipate N-Acetycitrulline N-Nitrosoproline Nepafenac Nicotinuric acid Norophthalmic acid Oleic acid oxoethoxy sulfonic acid Oxoglutaric acid Palmitic acid Pentanesulfenothioic acid Pentaporphyrin I Phosphate Phosphodimethylethanolamine Phosphoglyceric acid Phosphoserine Phthalic acid Propenyl propenylthio propenyl sulfate Purine Pyridoxamine Pyroglutamylglycine Pyrophosphate Ribothymidine Saccharopine Sinalexin Stearic acid Succinic anhydride Succinyglutamic acid semialdehyde Sulfate Sulfinoalanine Tartaric acid Taurine Tetrahydro-carboline-carboxylic acid Threonine Thymine trihydroxy-hydroxymethyl oxolan-yl methoxy sulfonic acid Triphosphate Tryptophan Undecylbenzenesulfonic acid Urate radical Uridine Urolithin D Xanthine METABOLITES_END #END