#METABOLOMICS WORKBENCH indranil_paul_20210622_074124 DATATRACK_ID:2711 STUDY_ID:ST001861 ANALYSIS_ID:AN003017 VERSION 1 CREATED_ON 01-20-2022 #PROJECT PR:PROJECT_TITLE A multi-tiered map of EMT defines major transition points and identifies PR:PROJECT_TITLE vulnerabilities PR:PROJECT_SUMMARY Epithelial to mesenchymal transition (EMT) is a complex cellular program PR:PROJECT_SUMMARY proceeding through a hybrid E/M state linked to cancer-associated stemness, PR:PROJECT_SUMMARY migration and chemoresistance. Deeper molecular understanding of this dynamic PR:PROJECT_SUMMARY physiological landscape is needed to define events which regulate the transition PR:PROJECT_SUMMARY and entry into and exit from the E/M state. Here, we quantified >60,000 PR:PROJECT_SUMMARY molecules across ten time points and twelve omic layers in human mammary PR:PROJECT_SUMMARY epithelial cells undergoing TGFβ-induced EMT. Deep proteomic profiles of whole PR:PROJECT_SUMMARY cells, nuclei, extracellular vesicles, secretome, membrane and phosphoproteome PR:PROJECT_SUMMARY defined state-specific signatures and major transition points. Parallel PR:PROJECT_SUMMARY metabolomics showed metabolic reprogramming preceded changes in other layers, PR:PROJECT_SUMMARY while single-cell RNA sequencing identified transcription factors controlling PR:PROJECT_SUMMARY entry into E/M. Covariance analysis exposed unexpected discordance between the PR:PROJECT_SUMMARY molecular layers. Integrative causal modeling revealed co-dependencies governing PR:PROJECT_SUMMARY entry into E/M that were verified experimentally using combinatorial inhibition. PR:PROJECT_SUMMARY Overall, this dataset provides an unprecedented resource on TGFβ signaling, EMT PR:PROJECT_SUMMARY and cancer. PR:INSTITUTE Boston University PR:LAST_NAME Paul PR:FIRST_NAME Indranil PR:ADDRESS 71 East Concord Street, Room # K320 PR:EMAIL indranil@bu.edu PR:PHONE 6177929631 PR:DOI http://dx.doi.org/10.21228/M8Z98Q #STUDY ST:STUDY_TITLE Parallelized multidimensional analytic framework, PAMAF, applied to mammalian ST:STUDY_TITLE cells uncovers novel regulatory principles in EMT ST:STUDY_SUMMARY Painting a holistic picture of disease etiology will require longitudinal ST:STUDY_SUMMARY systems-scale reconstruction of the multitiered architecture of eukaryotic ST:STUDY_SUMMARY signaling. As opposed to ‘one omic at a time’, which provides an incomplete ST:STUDY_SUMMARY view on disease mechanisms, here we developed an experimental and analytics ST:STUDY_SUMMARY framework, PAMAF, to simultaneously acquire and analyze twelve omic modalities ST:STUDY_SUMMARY from the same set of samples, i.e., protein abundance from whole-cells, nucleus, ST:STUDY_SUMMARY exosomes, secretome and membrane; peptidome; N-glycosylation, phosphorylation; ST:STUDY_SUMMARY metabolites; mRNA, miRNA; and, in parallel, single-cell transcriptomes. We ST:STUDY_SUMMARY applied PAMAF in a well-studied in vitro model of TGFβ-induced EMT to generate ST:STUDY_SUMMARY the EMT-ExMap dataset, cataloguing >61,000 expression profiles (>10,000 ST:STUDY_SUMMARY differential) over 12 days. PAMAF revealed that EMT is more complex than ST:STUDY_SUMMARY currently understood and identified numerous stage-specific mechanisms and ST:STUDY_SUMMARY vulnerabilities not captured in literature. Broad application of PAMAF will ST:STUDY_SUMMARY provide unprecedented insights into multifaceted biological processes relevant ST:STUDY_SUMMARY to human health and disease. ST:INSTITUTE Boston University ST:LAST_NAME Paul ST:FIRST_NAME Indranil ST:ADDRESS 71 East Concord St ST:EMAIL indranil@bu.edu ST:PHONE 6177929632 ST:SUBMIT_DATE 2021-06-22 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Female #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta1_1 Replicate:1 | Treatment:Control | Treatment:0_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta1_1 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta10_1 Replicate:1 | Treatment:TGFbeta | Treatment:12_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta10_1 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta3_1 Replicate:1 | Treatment:TGFbeta | Treatment:1_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta3_1 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta4_1 Replicate:1 | Treatment:TGFbeta | Treatment:2_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta4_1 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta5_1 Replicate:1 | Treatment:TGFbeta | Treatment:3_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta5_1 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta6_1 Replicate:1 | Treatment:TGFbeta | Treatment:4_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta6_1 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta2_1 Replicate:1 | Treatment:TGFbeta | Treatment:4_hours RAW_FILE_NAME=20190525_Indranil_pos_spme_meta2_1 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta7_1 Replicate:1 | Treatment:TGFbeta | Treatment:5_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta7_1 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta8_1 Replicate:1 | Treatment:TGFbeta | Treatment:6_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta8_1 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta9_1 Replicate:1 | Treatment:TGFbeta | Treatment:8_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta9_1 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta1_2 Replicate:2 | Treatment:Control | Treatment:0_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta1_2 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta10_2 Replicate:2 | Treatment:TGFbeta | Treatment:12_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta10_2 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta3_2 Replicate:2 | Treatment:TGFbeta | Treatment:1_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta3_2 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta4_2 Replicate:2 | Treatment:TGFbeta | Treatment:2_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta4_2 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta5_2 Replicate:2 | Treatment:TGFbeta | Treatment:3_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta5_2 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta6_2 Replicate:2 | Treatment:TGFbeta | Treatment:4_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta6_2 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta2_2 Replicate:2 | Treatment:TGFbeta | Treatment:4_hours RAW_FILE_NAME=20190525_Indranil_pos_spme_meta2_2 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta7_2 Replicate:2 | Treatment:TGFbeta | Treatment:5_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta7_2 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta8_2 Replicate:2 | Treatment:TGFbeta | Treatment:6_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta8_2 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta9_2 Replicate:2 | Treatment:TGFbeta | Treatment:8_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta9_2 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta1_3 Replicate:3 | Treatment:Control | Treatment:0_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta1_3 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta10_3 Replicate:3 | Treatment:TGFbeta | Treatment:12_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta10_3 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta3_3 Replicate:3 | Treatment:TGFbeta | Treatment:1_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta3_3 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta4_3 Replicate:3 | Treatment:TGFbeta | Treatment:2_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta4_3 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta5_3 Replicate:3 | Treatment:TGFbeta | Treatment:3_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta5_3 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta6_3 Replicate:3 | Treatment:TGFbeta | Treatment:4_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta6_3 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta2_3 Replicate:3 | Treatment:TGFbeta | Treatment:4_hours RAW_FILE_NAME=20190525_Indranil_pos_spme_meta2_3 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta7_3 Replicate:3 | Treatment:TGFbeta | Treatment:5_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta7_3 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta8_3 Replicate:3 | Treatment:TGFbeta | Treatment:6_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta8_3 SUBJECT_SAMPLE_FACTORS - 20190525_Indranil_pos_spme_meta9_3 Replicate:3 | Treatment:TGFbeta | Treatment:8_day RAW_FILE_NAME=20190525_Indranil_pos_spme_meta9_3 #COLLECTION CO:COLLECTION_SUMMARY Human breast epithelial MCF10A cells were kindly provided by Prof. Senthil CO:COLLECTION_SUMMARY Muthuswamy (Beth Israel Deaconess Medical Center, Harvard Medical School). Cells CO:COLLECTION_SUMMARY were cultured in DMEM/F-12 supplemented with 5% Horse serum, EGF 20 ng/mL CO:COLLECTION_SUMMARY (Sigma), Insulin 10 μg/mL (Sigma), Hydrocortisone 0.5 mg/mL (Sigma), Cholera CO:COLLECTION_SUMMARY toxin 100 ng/mL (Sigma), 100 units/mL Penicillin and 100 μg/mL Streptomycin CO:COLLECTION_SUMMARY (HyClone) and grown at 37C in a humidified incubator with 5% CO2. To induce CO:COLLECTION_SUMMARY EMT, cells were stimulated with 10 ng/mL TGF-β1 (Invivogen) and treatments were CO:COLLECTION_SUMMARY staggered such that all cells (plates) were harvested at the same time. To CO:COLLECTION_SUMMARY minimize cross-contamination (EV & Sec) and promiscuous background signaling CO:COLLECTION_SUMMARY (particularly for Phos), cells were cultured in serum-free conditions for 16 CO:COLLECTION_SUMMARY hours prior to harvesting. At the time of harvest, conditioned media were first CO:COLLECTION_SUMMARY transferred to fresh 50 mL tubes and kept on ice. Cells were washed once with CO:COLLECTION_SUMMARY ice-cold PBS and scraped off the plates in ice-cold PBS. Each sample was then CO:COLLECTION_SUMMARY distributed into multiple aliquots for multi-omics extractions, centrifuged at CO:COLLECTION_SUMMARY 800×g for 5 minutes at 4°C and stored as dry pellets at –80°C. Live cells CO:COLLECTION_SUMMARY were imaged in their culture vessels before harvesting using ZOE fluorescent CO:COLLECTION_SUMMARY cell imager (Bio-Rad). CO:SAMPLE_TYPE Breast cancer cells #TREATMENT TR:TREATMENT_SUMMARY Human breast epithelial MCF10A cells were kindly provided by Prof. Senthil TR:TREATMENT_SUMMARY Muthuswamy (Beth Israel Deaconess Medical Center, Harvard Medical School). Cells TR:TREATMENT_SUMMARY were cultured in DMEM/F-12 supplemented with 5% Horse serum, EGF 20 ng/mL TR:TREATMENT_SUMMARY (Sigma), Insulin 10 μg/mL (Sigma), Hydrocortisone 0.5 mg/mL (Sigma), Cholera TR:TREATMENT_SUMMARY toxin 100 ng/mL (Sigma), 100 units/mL Penicillin and 100 μg/mL Streptomycin TR:TREATMENT_SUMMARY (HyClone) and grown at 37°C in a humidified incubator with 5% CO2. To induce TR:TREATMENT_SUMMARY EMT, cells were stimulated with 10 ng/mL TGF-β1 (Invivogen) and treatments were TR:TREATMENT_SUMMARY staggered such that all cells (plates) were harvested at the same time. To TR:TREATMENT_SUMMARY minimize cross-contamination (EV & Sec) and promiscuous background signaling TR:TREATMENT_SUMMARY (particularly for Phos), cells were cultured in serum-free conditions for 16 TR:TREATMENT_SUMMARY hours prior to harvesting. At the time of harvest, conditioned media were first TR:TREATMENT_SUMMARY transferred to fresh 50 mL tubes and kept on ice. Cells were washed once with TR:TREATMENT_SUMMARY ice-cold PBS and scraped off the plates in ice-cold PBS. Each sample was then TR:TREATMENT_SUMMARY distributed into multiple aliquots for multi-omics extractions, centrifuged at TR:TREATMENT_SUMMARY 800×g for 5 minutes at 4°C and stored as dry pellets at –80°C. Live cells TR:TREATMENT_SUMMARY were imaged in their culture vessels before harvesting using ZOE fluorescent TR:TREATMENT_SUMMARY cell imager (Bio-Rad). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Each cell pellet was thawed on ice and resuspended in 500 μL ice-cold water by SP:SAMPLEPREP_SUMMARY vortexing for 3 seconds and 500 μL of chilled (–80°C) 90% methanol + 10% SP:SAMPLEPREP_SUMMARY chloroform solution was immediately added and vortexed for another 10 seconds SP:SAMPLEPREP_SUMMARY and then kept on ice. Samples were incubated for 30 minutes at 4°C while SP:SAMPLEPREP_SUMMARY rotating and then centrifuged at 800×g for 10 mins at 4°C. The supernatants SP:SAMPLEPREP_SUMMARY were transferred to fresh tubes and centrifuged at 16000×g for 45 minutes at SP:SAMPLEPREP_SUMMARY 4°C. The cleared supernatant containing metabolites were cleaned using a SPME SP:SAMPLEPREP_SUMMARY (solid phase microextraction) protocol adopted from Mousavi et. al. (Mousavi et SP:SAMPLEPREP_SUMMARY al., 2019), vacufuged to dryness and stored at –80°C. The cell pellets were SP:SAMPLEPREP_SUMMARY used for protein extraction using GuHCl lysis method as described below. #CHROMATOGRAPHY CH:INSTRUMENT_NAME Thermo Scientific EASY-nLC 1200 System CH:COLUMN_NAME Thermo Easy Spray CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS For metabolite identifications we used the R package MAIT (Fernández-Albert et MS:MS_COMMENTS al., 2014), which integrates peak detection, peak annotation and statistical MS:MS_COMMENTS analysis. Briefly, XCMS (Tautenhahn et al., 2012) is used to detect and align MS:MS_COMMENTS peaks followed by annotation with CAMERA (Kuhl et al., 2012). A special function MS:MS_COMMENTS ‘Biotransformations’ is applied to refine annotations and measured ions are MS:MS_COMMENTS then putatively identified by matching mass-to-charge ratios to a reference list MS:MS_COMMENTS of calculated masses of metabolites listed in the Human Metabolome Database MS:MS_COMMENTS (HMDB, http://www.hmdb.ca, 2019). MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST001861_AN003017_Results.txt UNITS:Neutral Mass Has RT:Yes RT units:Minutes #END