#METABOLOMICS WORKBENCH Stopka28_20220627_104706_mwtab.txt DATATRACK_ID:3321 STUDY_ID:ST002203 ANALYSIS_ID:AN003606 PROJECT_ID:PR001406 VERSION 1 CREATED_ON June 27, 2022, 11:39 am #PROJECT PR:PROJECT_TITLE Single Cell Spatial Analysis Reveals the Topology of Immunomodulatory Purinergic PR:PROJECT_TITLE Signaling in Glioblastoma PR:PROJECT_TYPE Single Cell Spatial Analysis Reveals the Topology of Immunomodulatory Purinergic PR:PROJECT_TYPE Signaling in Glioblastoma PR:PROJECT_SUMMARY Abstract from manuscript "Glioblastoma develops an immunosuppressive PR:PROJECT_SUMMARY microenvironment that fosters tumorigenesis and resistance to current PR:PROJECT_SUMMARY therapeutic strategies. Here we use multiplexed tissue imaging and single-cell PR:PROJECT_SUMMARY RNA-sequencing to characterize the composition, spatial organization, and PR:PROJECT_SUMMARY clinical significance of extracellular purinergic signaling in glioblastoma. We PR:PROJECT_SUMMARY show that glioblastoma exhibit strong expression of CD39 and CD73 ectoenzymes, PR:PROJECT_SUMMARY correlating with increased adenosine levels. Microglia are the predominant PR:PROJECT_SUMMARY source of CD39, while CD73 is principally expressed by tumor cells, particularly PR:PROJECT_SUMMARY in tumors with amplification of EGFR and astrocyte-like differentiation. PR:PROJECT_SUMMARY Spatially-resolved single-cell analyses demonstrate strong spatial correlation PR:PROJECT_SUMMARY between tumor CD73 and microglial CD39, and that their spatial proximity is PR:PROJECT_SUMMARY associated with poor clinical outcomes. Together, this data reveals that tumor PR:PROJECT_SUMMARY CD73 expression correlates with tumor genotype, lineage differentiation, and PR:PROJECT_SUMMARY functional states, and that core purine regulatory enzymes expressed by PR:PROJECT_SUMMARY neoplastic and tumor-associated myeloid cells interact to promote a distinctive PR:PROJECT_SUMMARY adenosine-rich signaling niche and immunosuppressive microenvironment PR:PROJECT_SUMMARY potentially amenable to therapeutic targeting. " 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, Boston, MA PR:EMAIL wahassan@bwh.harvard.edu PR:PHONE 6175259746 #STUDY ST:STUDY_TITLE Single Cell Spatial Analysis Reveals the Topology of Immunomodulatory Purinergic ST:STUDY_TITLE Signaling in Glioblastoma ST:STUDY_SUMMARY Abstract from manuscript "Glioblastoma develops an immunosuppressive ST:STUDY_SUMMARY microenvironment that fosters tumorigenesis and resistance to current ST:STUDY_SUMMARY therapeutic strategies. Here we use multiplexed tissue imaging and single-cell ST:STUDY_SUMMARY RNA-sequencing to characterize the composition, spatial organization, and ST:STUDY_SUMMARY clinical significance of extracellular purinergic signaling in glioblastoma. We ST:STUDY_SUMMARY show that glioblastoma exhibit strong expression of CD39 and CD73 ectoenzymes, ST:STUDY_SUMMARY correlating with increased adenosine levels. Microglia are the predominant ST:STUDY_SUMMARY source of CD39, while CD73 is principally expressed by tumor cells, particularly ST:STUDY_SUMMARY in tumors with amplification of EGFR and astrocyte-like differentiation. ST:STUDY_SUMMARY Spatially-resolved single-cell analyses demonstrate strong spatial correlation ST:STUDY_SUMMARY between tumor CD73 and microglial CD39, and that their spatial proximity is ST:STUDY_SUMMARY associated with poor clinical outcomes. Together, this data reveals that tumor ST:STUDY_SUMMARY CD73 expression correlates with tumor genotype, lineage differentiation, and ST:STUDY_SUMMARY functional states, and that core purine regulatory enzymes expressed by ST:STUDY_SUMMARY neoplastic and tumor-associated myeloid cells interact to promote a distinctive ST:STUDY_SUMMARY adenosine-rich signaling niche and immunosuppressive microenvironment ST:STUDY_SUMMARY potentially amenable to therapeutic targeting. " 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 1619 Commonwealth Avenue, apt 32 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 - mouse_CD73 sample_id:Dataset1 RAW_FILE_NAME=MALDI-MSI_adenosine_CD73-Total Ion Count SUBJECT_SAMPLE_FACTORS - human_tumor_CD73 sample_id:Dataset2 RAW_FILE_NAME=MALDI-MSI_adenosine_CS-Total Ion Count_Tumor_4 #COLLECTION CO:COLLECTION_SUMMARY As stated in the manuscript "Frozen resection tissue from 9 glioblastomas, CO:COLLECTION_SUMMARY including 4 cases with high CD73 and 5 cases with low CD73 by IHC, were selected CO:COLLECTION_SUMMARY for MALDI MSI." CO:SAMPLE_TYPE Brain #TREATMENT TR:TREATMENT_SUMMARY N/A #SAMPLEPREP SP:SAMPLEPREP_SUMMARY As stated in the manuscript "Frozen resection tissue from 9 glioblastomas, SP:SAMPLEPREP_SUMMARY including 4 cases with high CD73 and 5 cases with low CD73 by IHC, were selected SP:SAMPLEPREP_SUMMARY for MALDI MSI. Tissue was sectioned to 10 µm, thaw mounted onto SP:SAMPLEPREP_SUMMARY indium-tin-oxide (ITO) slides, and serial sections were obtained for H&E SP:SAMPLEPREP_SUMMARY staining. A high-resolution image of the whole H&E tissues was obtained through SP:SAMPLEPREP_SUMMARY image stitching (Zeiss Observer Z.1, Oberkochen, Germany) using a SP:SAMPLEPREP_SUMMARY plan-apochromat lens (20×) using an AxioCam MR3 camera. Matrix preparation of SP:SAMPLEPREP_SUMMARY 1,5-diaminonaphthalene hydrochloride was prepared to a concentration of 4.3 SP:SAMPLEPREP_SUMMARY mg/mL in 4.5/5/0.5 HPLC grade water/ethanol/1 M HCl (v/v/v). The matrix was SP:SAMPLEPREP_SUMMARY sprayed using a TM-sprayer (HTX imaging, Carrboro, NC) with parameters of a flow SP:SAMPLEPREP_SUMMARY rate (0.09 mL/min), spray nozzle velocity (1200 mm/min), spray nozzle SP:SAMPLEPREP_SUMMARY temperature (75°C), nitrogen gas pressure (10 psi), track spacing (2 mm) and a SP:SAMPLEPREP_SUMMARY four pass spray." #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE None (Direct infusion) CH:INSTRUMENT_NAME Bruker CH:COLUMN_NAME 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:MS_RESULTS_FILE ST002203_AN003606_Results.txt UNITS:Da Has m/z:Yes Has RT:No RT units:No RT data #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Da MS_METABOLITE_DATA_START Samples human_tumor_CD73 mouse_CD73 Factors sample_id:Dataset2 sample_id:Dataset1 adenosine triphosphate 505.9897 505.9897 adenosine diphosphate 426.0232 426.0232 adenosine monophosphate 346.0567 346.0567 adenosine 302.0669 302.0669 inosine 303.0507 303.0507 hypoxanthine 171.0077 171.0077 xanthine 151.0261 151.0261 urate 167.0213 167.0213 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name human_tumor_CD73 mouse_CD73 adenosine triphosphate 505.9897 505.9897 adenosine diphosphate 426.0232 426.0232 adenosine monophosphate 346.0567 346.0567 adenosine 302.0669 302.0669 inosine 303.0507 303.0507 hypoxanthine 171.0077 171.0077 xanthine 151.0261 151.0261 urate 167.0213 167.0213 METABOLITES_END #END