#METABOLOMICS WORKBENCH Rossella97_20250429_024247 DATATRACK_ID:5871 STUDY_ID:ST003892 ANALYSIS_ID:AN006394 PROJECT_ID:PR002437 VERSION 1 CREATED_ON May 1, 2025, 2:37 am #PROJECT PR:PROJECT_TITLE Dual Ribosome Profiling reveals metabolic limitations of cancer and stromal PR:PROJECT_TITLE cells in the tumor microenvironment PR:PROJECT_SUMMARY Cancer cells, immune cells, and stromal cells within the tumor microenvironment PR:PROJECT_SUMMARY (TME) collaboratively influence disease progression and therapeutic responses. PR:PROJECT_SUMMARY The nutrient-limited conditions of the TME, particularly the scarcity of PR:PROJECT_SUMMARY glucose, amino acids, and lipids, challenge cancer cell survival. However, the PR:PROJECT_SUMMARY metabolic constraints faced by immune and stromal cells in comparison to cancer PR:PROJECT_SUMMARY cells, and how these limitations affect therapeutic outcomes, remain poorly PR:PROJECT_SUMMARY understood. Here, we introduce Dual Ribosome Profiling (DualRP), a method that PR:PROJECT_SUMMARY allows for simultaneous analysis of translation and identification of ribosome PR:PROJECT_SUMMARY stalling, revealing amino acid shortages in different cell types within tumors. PR:PROJECT_SUMMARY Using DualRP, we uncover that interactions between cancer cells and fibroblasts PR:PROJECT_SUMMARY trigger an inflammatory response, mitigating amino acid limitations during PR:PROJECT_SUMMARY glucose starvation. In immunocompetent mouse models, we observe that immune PR:PROJECT_SUMMARY checkpoint blockade therapy induces serine and glycine restrictions specifically PR:PROJECT_SUMMARY in T cells, but not in cancer cells. We further demonstrate that these amino PR:PROJECT_SUMMARY acids are essential for optimal T cell function both in vitro and in vivo, PR:PROJECT_SUMMARY highlighting their critical role in effective immunotherapy. Our findings show PR:PROJECT_SUMMARY that therapeutic interventions create distinct metabolic demands across PR:PROJECT_SUMMARY different tumor cell types, with nutrient availability significantly influencing PR:PROJECT_SUMMARY the success of immunotherapy. DualRP’s ability to explore cell type-specific PR:PROJECT_SUMMARY metabolic vulnerabilities offers a promising tool for advancing our PR:PROJECT_SUMMARY understanding of tumor biology and improving therapeutic strategies. PR:INSTITUTE DKFZ PR:LAST_NAME Del Pizzo PR:FIRST_NAME Rossella PR:ADDRESS Im Neuenheimer Feld 280 69120 Heidelberg PR:EMAIL rossella.delpizzo@dkfz-heidelberg.de PR:PHONE +49 6221 42 2856 #STUDY ST:STUDY_TITLE 13C-glucose tracing into serine and glycine in OT-1 CD8+ T cells and E0771-OVA ST:STUDY_TITLE co-cultures analyzed by GC-MS ST:STUDY_SUMMARY We investigated the incorporation of U-[13C]-glucose-derived carbon into serine ST:STUDY_SUMMARY and glycine in OT-1 CD8+ T cells and E0771-OVA breast cancer cells. Mono- and ST:STUDY_SUMMARY co-cultures were treated with U-[13C]-glucose, and metabolite labeling patterns ST:STUDY_SUMMARY were quantified using gas chromatography-mass spectrometry (GC-MS) to assess ST:STUDY_SUMMARY metabolic differences between culture conditions. ST:INSTITUTE DKFZ ST:LAST_NAME Del Pizzo ST:FIRST_NAME Rossella ST:ADDRESS Im Neuenheimer Feld 280 69120 Heidelberg ST:EMAIL rossella.delpizzo@dkfz-heidelberg.de ST:PHONE +49 6221 42 2856 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENDER Female #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 - SC-283 Sample source:Spleen | Treatment:control RAW_FILE_NAME(Raw file name)=SC-283_Naive_T_cells_6h_RPMI_1.D SUBJECT_SAMPLE_FACTORS - SC-284 Sample source:Spleen | Treatment:control RAW_FILE_NAME(Raw file name)=SC-284_Naive_T_cells_6h_RPMI_2.D SUBJECT_SAMPLE_FACTORS - SC-285 Sample source:Spleen | Treatment:control RAW_FILE_NAME(Raw file name)=SC-285_Naive_T_cells_6h_RPMI_3.D SUBJECT_SAMPLE_FACTORS - SC-286 Sample source:Spleen | Treatment:co-culture RAW_FILE_NAME(Raw file name)=SC-286_T_cells_co-culture_6h_RPMI_1.D SUBJECT_SAMPLE_FACTORS - SC-287 Sample source:Spleen | Treatment:co-culture RAW_FILE_NAME(Raw file name)=SC-287_T_cells_co-culture_6h_RPMI_2.D SUBJECT_SAMPLE_FACTORS - SC-288 Sample source:Spleen | Treatment:co-culture RAW_FILE_NAME(Raw file name)=SC-288_T_cells_co-culture_6h_RPMI_3.D SUBJECT_SAMPLE_FACTORS - SC-289 Sample source:SUM159PT cells | Treatment:co-culture RAW_FILE_NAME(Raw file name)=SC-289_E0771-OVA_co-culture_6h_RPMI_1.D SUBJECT_SAMPLE_FACTORS - SC-290 Sample source:SUM159PT cells | Treatment:co-culture RAW_FILE_NAME(Raw file name)=SC-290_E0771-OVA_co-culture_6h_RPMI_2.D SUBJECT_SAMPLE_FACTORS - SC-291 Sample source:SUM159PT cells | Treatment:co-culture RAW_FILE_NAME(Raw file name)=SC-291_E0771-OVA_co-culture_6h_RPMI_3.D SUBJECT_SAMPLE_FACTORS - SC-292 Sample source:Spleen | Treatment:control RAW_FILE_NAME(Raw file name)=SC-292_T_cells_alone_6h_RPMI_1.D SUBJECT_SAMPLE_FACTORS - SC-293 Sample source:Spleen | Treatment:control RAW_FILE_NAME(Raw file name)=SC-293_T_cells_alone_6h_RPMI_2.D SUBJECT_SAMPLE_FACTORS - SC-294 Sample source:Spleen | Treatment:control RAW_FILE_NAME(Raw file name)=SC-294_T_cells_alone_6h_RPMI_3.D SUBJECT_SAMPLE_FACTORS - SC-295 Sample source:SUM159PT cells | Treatment:control RAW_FILE_NAME(Raw file name)=SC-295_E0771-OVA_alone_6h_RPMI_1.D SUBJECT_SAMPLE_FACTORS - SC-296 Sample source:SUM159PT cells | Treatment:control RAW_FILE_NAME(Raw file name)=SC-296_E0771-OVA_alone_6h_RPMI_2.D SUBJECT_SAMPLE_FACTORS - SC-297 Sample source:SUM159PT cells | Treatment:control RAW_FILE_NAME(Raw file name)=SC-297_E0771-OVA_alone_6h_RPMI_3.D SUBJECT_SAMPLE_FACTORS - SC-298 Sample source:Spleen | Treatment:co-culture serine deprivation RAW_FILE_NAME(Raw file name)=SC-298_T_cells_co-culture_6h_RPMI_no_serine_1.D SUBJECT_SAMPLE_FACTORS - SC-299 Sample source:Spleen | Treatment:co-culture serine deprivation RAW_FILE_NAME(Raw file name)=SC-299_T_cells_co-culture_6h_RPMI_no_serine_2.D SUBJECT_SAMPLE_FACTORS - SC-300 Sample source:Spleen | Treatment:co-culture serine deprivation RAW_FILE_NAME(Raw file name)=SC-300_T_cells_co-culture_6h_RPMI_no_serine_3.D SUBJECT_SAMPLE_FACTORS - SC-301 Sample source:SUM159PT cells | Treatment:co-culture serine deprivation RAW_FILE_NAME(Raw file name)=SC-301_E0771-OVA_co-culture_6h_RPMI_no_serine_1.D SUBJECT_SAMPLE_FACTORS - SC-302 Sample source:SUM159PT cells | Treatment:co-culture serine deprivation RAW_FILE_NAME(Raw file name)=SC-302_E0771-OVA_co-culture_6h_RPMI_no_serine_2.D SUBJECT_SAMPLE_FACTORS - SC-303 Sample source:SUM159PT cells | Treatment:co-culture serine deprivation RAW_FILE_NAME(Raw file name)=SC-303_E0771-OVA_co-culture_6h_RPMI_no_serine_3.D SUBJECT_SAMPLE_FACTORS - SC-304 Sample source:Spleen | Treatment:serine deprivation RAW_FILE_NAME(Raw file name)=SC-304_T_cells_alone_6h_RPMI_no_serine_1.D SUBJECT_SAMPLE_FACTORS - SC-305 Sample source:Spleen | Treatment:serine/glycine deprivation RAW_FILE_NAME(Raw file name)=SC-305_T_cells_alone_6h_RPMI_no_serine_2.D SUBJECT_SAMPLE_FACTORS - SC-306 Sample source:Spleen | Treatment:serine/glycine deprivation RAW_FILE_NAME(Raw file name)=SC-306_T_cells_alone_6h_RPMI_no_serine_3.D SUBJECT_SAMPLE_FACTORS - SC-307 Sample source:SUM159PT cells | Treatment:serine/glycine deprivation RAW_FILE_NAME(Raw file name)=SC-307_E0771-OVA_alone_6h_RPMI_no_serine_1.D SUBJECT_SAMPLE_FACTORS - SC-308 Sample source:SUM159PT cells | Treatment:serine/glycine deprivation RAW_FILE_NAME(Raw file name)=SC-308_E0771-OVA_alone_6h_RPMI_no_serine_2.D SUBJECT_SAMPLE_FACTORS - SC-309 Sample source:SUM159PT cells | Treatment:serine/glycine deprivation RAW_FILE_NAME(Raw file name)=SC-309_E0771-OVA_alone_6h_RPMI_no_serine_3.D #COLLECTION CO:COLLECTION_SUMMARY After six hours of culture in media with or without serine and in the presence CO:COLLECTION_SUMMARY of U-[13C]-glucose, CD8+ T cells and E0771-OVA tumor cells were collected for CO:COLLECTION_SUMMARY metabolic analysis. Cells were rapidly quenched using a 60% methanol and 10 mM CO:COLLECTION_SUMMARY ammonium acetate solution precooled to −40 °C, following a protocol adapted CO:COLLECTION_SUMMARY from Sellick et al. After two sequential washes with quenching solution, the CO:COLLECTION_SUMMARY cell pellets were stored at −80 °C until further processing. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY CD8+ T cells were isolated from the spleens of eight-week-old OT-1 female mice TR:TREATMENT_SUMMARY and activated for 72 hours on plates coated with anti-CD3 and anti-CD28 TR:TREATMENT_SUMMARY antibodies. Activated T cells were maintained in RPMI 1640 supplemented with 10% TR:TREATMENT_SUMMARY FBS, 1% penicillin-streptomycin, sodium pyruvate, HEPES, β-mercaptoethanol, and TR:TREATMENT_SUMMARY murine IL-2. For co-culture assays, pre-activated OT-1 CD8+ T cells were TR:TREATMENT_SUMMARY combined with E0771-OVA tumor cells at a 1:5 ratio (T cells:tumor cells) after TR:TREATMENT_SUMMARY tumor cell adherence. T cell-only and tumor cell-only conditions were also TR:TREATMENT_SUMMARY prepared. Metabolic assays were conducted in complete RPMI or RPMI lacking TR:TREATMENT_SUMMARY serine, in which natural glucose was replaced by U-[13C6]-glucose. After six TR:TREATMENT_SUMMARY hours of culture under these conditions, cells were collected for metabolic TR:TREATMENT_SUMMARY analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted from cell pellets by adding 800 µl 60% methanol SP:SAMPLEPREP_SUMMARY containing 6.67 µg/ml glutaric acid in LC/MS grade water. After addition of 500 SP:SAMPLEPREP_SUMMARY µl chloroform, samples were vortexed for 10 minutes at 4°C and then spun at SP:SAMPLEPREP_SUMMARY 17,000 x g for 10 minutes at 4°C. The upper phase was transferred into a fresh SP:SAMPLEPREP_SUMMARY tube and dried down in a Vacufuge plus speed-vac at 4°C. The lower organic SP:SAMPLEPREP_SUMMARY phase was also dried and used for further analysis. Protein pellets were SP:SAMPLEPREP_SUMMARY resuspended in 0.2 M NaOH, heated at 90°C for 15 minutes, and quantified using SP:SAMPLEPREP_SUMMARY the Pierce BCA Protein Assay Kit. Polar metabolites were derivatized with 20 mg SP:SAMPLEPREP_SUMMARY ml−1 methoxyamine in pyridine for 90 min at 37°C and subsequently with SP:SAMPLEPREP_SUMMARY N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide, with 1% SP:SAMPLEPREP_SUMMARY tert-butyldimethylchlorosilane for 60 min at 60°C. Fatty acids were esterified SP:SAMPLEPREP_SUMMARY with sulfuric acid/methanol for 72 h at 60°C and then extracted with hexane. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890A CH:COLUMN_NAME Agilent DB5-MS (30m x 0.25mm, 0.25um) CH:SOLVENT_A Not applicable (GC-MS analysis) CH:SOLVENT_B Not applicable (GC-MS analysis) CH:FLOW_GRADIENT Not applicable (GC-MS analysis) CH:FLOW_RATE 1 ml/min CH:COLUMN_TEMPERATURE 100°C for 3 min, ramp at 2.5°C/min to 300°C for polar metabolites; 80°C for CH:COLUMN_TEMPERATURE 1 min, ramp at 5°C/min to 300°C for fatty acids #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 5975C MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS Mass distributions and metabolite concentrations were measured with a 7890A GC MS:MS_COMMENTS system (Agilent Technologies) combined with a 5975C Inert MS system (Agilent MS:MS_COMMENTS Technologies). 7890A GC system generated D.files. D. files were imported in MSD MS:MS_COMMENTS ChemStation Data Analysis Application software and exported to generate data to MS:MS_COMMENTS AIA format (.CDF file). Mass distributions were extracted from the raw ion MS:MS_COMMENTS chromatograms using a custom Matlab M-file, which applies consistent integration MS:MS_COMMENTS bounds and baseline correction to each ion (Young et al., 2008). In addition, we MS:MS_COMMENTS corrected for naturally occurring isotopes using the method of Fernandez et al. MS:MS_COMMENTS (Fernandez et al., 1996). Negative values and mass overlaps were considered as MS:MS_COMMENTS zero. We corrected the mass spectra for potential metabolite contamination in a MS:MS_COMMENTS blank extraction. All labeling fractions were transformed to a natural abundance MS:MS_COMMENTS corrected mass distribution vector (MDV) (Buescher et al., 2015). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Mass isotopomer abundances (%) MS_METABOLITE_DATA_START Samples SC-283 SC-284 SC-285 SC-286 SC-287 SC-288 SC-289 SC-290 SC-291 SC-292 SC-293 SC-294 SC-295 SC-296 SC-297 SC-298 SC-299 SC-300 SC-301 SC-302 SC-303 SC-304 SC-305 SC-306 SC-307 SC-308 SC-309 Factors Sample source:Spleen | Treatment:control Sample source:Spleen | Treatment:control Sample source:Spleen | Treatment:control Sample source:Spleen | Treatment:co-culture Sample source:Spleen | Treatment:co-culture Sample source:Spleen | Treatment:co-culture Sample source:SUM159PT cells | Treatment:co-culture Sample source:SUM159PT cells | Treatment:co-culture Sample source:SUM159PT cells | Treatment:co-culture Sample source:Spleen | Treatment:control Sample source:Spleen | Treatment:control Sample source:Spleen | Treatment:control Sample source:SUM159PT cells | Treatment:control Sample source:SUM159PT cells | Treatment:control Sample source:SUM159PT cells | Treatment:control Sample source:Spleen | Treatment:co-culture serine deprivation Sample source:Spleen | Treatment:co-culture serine deprivation Sample source:Spleen | Treatment:co-culture serine deprivation Sample source:SUM159PT cells | Treatment:co-culture serine deprivation Sample source:SUM159PT cells | Treatment:co-culture serine deprivation Sample source:SUM159PT cells | Treatment:co-culture serine deprivation Sample source:Spleen | Treatment:serine deprivation Sample source:Spleen | Treatment:serine/glycine deprivation Sample source:Spleen | Treatment:serine/glycine deprivation Sample source:SUM159PT cells | Treatment:serine/glycine deprivation Sample source:SUM159PT cells | Treatment:serine/glycine deprivation Sample source:SUM159PT cells | Treatment:serine/glycine deprivation Glycine 1.26 4.45 2.21 27.65 28.96 30.29 27.67 27.05 28.56 34.19 33.92 33.86 37.46 35.62 36.13 55.03 54.71 56.03 54.51 56.62 52.42 50.77 50.97 51.10 61.96 56.01 52.04 Serine 0.12 1.34 0.25 26.80 27.77 28.51 30.82 29.99 29.38 28.21 29.30 29.77 37.79 37.09 36.68 59.82 58.33 63.76 62.57 63.53 59.16 46.18 48.29 48.16 69.48 68.79 69.62 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Isotopomer Retention index Quantified m/z PubChem ID KEGG ID Glycine M+2 16,66 246 750 C00037 Serine M+3 28,09 390 5951 C00065 METABOLITES_END #END