NASH limits anti-tumour surveillance in immunotherapy-treated HCC methods
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Methods
Standard mouse diet feeding (ad libitum water and food access) and treatment regimens were as described previously. Male mice were housed at the German Cancer Research Center (DKFZ) (constant temperature of 20-24 °C and 45-65% humidity with a 12-h light-dark cycle). Mice were maintained under specific pathogen-free conditions and experiments were performed in accordance with German law and the governmental bodies, and with approval from the Regierungspräsidium Karlsruhe (G11/16, G129/16, G7/17). Tissues from inducible knock-in mice expressing the human unconventional prefoldin RPB5 interactor were received from N. Djouder,. The plasmids for hydrodynamic tail-vein delivery have been described previously -. For interventional studies, male mice fed a CD-HFD were treated with bi-weekly for 8 weeks by intravenous injection of 25 µg CD8...
Magnetic resonance Imaging
MRI was done in the small animal imaging core facility in DKFZ using a Bruker BioSpec 9.4 Tesla (Ettlingen). Mice were anaesthetized with 3.5% sevoflurane, and imaged with T2-weighted imaging using a T2_TurboRARE sequence: TE = 22 ms, TR = 2,200 ms, field of view (FOV) 35 × 35 mm, slice thickness 1 mm, averages = 6, scan time 3 min 18 s, echo spacing 11 ms, rare factor 8, slices 20, image size 192 × 192 pixels, resolution 0.182 × 0.182 mm.
Flow cytometry and FACS
After perfusion and mechanical dissection, livers were incubated for up to 35 min at 37 °C with collagen IV (60 U final concentration (f.c.)) and DNase I (25 µg/ml f.c.), filtered at 100 µm, and washed with RPMI1640 (11875093, Thermo Fisher). Next, samples underwent a two-step Percoll gradient (25%/50% Percoll/HBSS) and centrifugation for 15 min at 1,800 g and 4 °C. Enriched leukocytes were then collected, washed, and counted. For re-stimulation, cells were incubated for 2 h at 37 °C under 5% CO 2 with 1:500 Biolegend´s Cell Activation Cocktail (with brefeldin A) (423304) and 1:1,000 Monensin Solution (420701). Live/dead discrimination was done using DAPI or ZombieDyeNIR according to the manufacturer's instructions with subsequent staining of titrated antibodies (Supplementary Tables - ). Samples for flow cytometric-activated...
Preparation for mass spectrometry, data acquisition, and data analysis
After FACS purification, cells were resuspended in 50% (vol/vol) 2,2,2-trifluoroethanol in PBS pH 7.4 buffer and lysed by repeated sonication and freeze-thaw cycles. Proteins were denatured at 60 °C for 2 h, reduced using dithiothreitol at a final concentration of 5 mM (30 min at 60 °C), cooled to room temperature, alkylated using iodoacetamide at 25 mM (30 min at room temperature in the dark), and diluted 1:5 using 100 mM ammonium bicarbonate, pH 8.0. Proteins were digested overnight by trypsin (1:100 ratio, 37 °C), desalted using C18-based stage-tips, dried under vacuum, resuspended in 20 µl HPLC-grade water with 0.1% formic acid, and measured using A380.
Preparation for mass spectrometry, data acquisition, and data analysis
We used 0.5 µg of peptides for proteomic analysis on a C18 column using a nano liquid chromatography system (EASY-nLC 1200, Thermo Fisher Scientific). Peptides were eluted using a gradient of 5-30% buffer B (80% acetonitrile and 0.1% formic acid) at a flow rate of 300 nl/min at a column temperature of 55 °C. Data were acquired by data-dependent Top15 acquisition using a high-resolution orbitrap tandem mass spectrometer (QExactive HFX, Thermo Scientific). All MS1 scans were acquired at 60,000 resolution with AGC target of 3 × 10 6, and MS2 scans were acquired at 15,000 resolution with AGC target of 1 × 10 5 and maximum injection time of 28 ms. Analyses were performed using MaxQuant (1.6.7.0), mouse UniProt Isoform fasta (Version: 2019-02-21, number of sequences 25,233) as a source for protein sequences. One per cent FDR was used for...
Flow cytometry of human biopsies
Analysis of patient material (Supplementary Table ) was performed on liver tissue (needle biopsies or resected tissue, BIOFACS Study KEK 2019-00114), which were obtained from the patient collection nAC-2019-3627 (CRB03) from the biological resource centre of CHU Grenoble-Alpes (nBRIF BB-0033-00069). Tissue samples were minced using scalpels, incubated (with 1 mg/ml collagenase IV (Sigma Aldrich), 0.25 µg/ml DNase (Sigma Aldrich), 10% FCS (Thermo Fisher Scientific), RPMI 1640 (Seraglob)) for 30 min at 37 °C, stopping enzymatic reactions with 2 mM EDTA (StemCell Technologies, Inc.) in PBS. After filtering through a 100-µm cell strainer, cells were resuspended in FACS buffer (PBS, EDTA 2 mM, FCS 0.5%) with Human TruStain FcX (Fc Receptor Blocking Solution) (Biolegend), incubated for 15 min at 4 °C and stained with antibodies (Supplementary Table ).
Isolation of cells for scRNA-seq data analysis (human)
Analyses used liver samples from patients undergoing bariatric surgery at the Department of Surgery at Heidelberg University Hospital (S-629/2013). Samples were preserved by FFPE for pathological evaluation and single cells were generated by mincing, using the Miltenyi tumour dissociation kit (130-095-929) per the manufacturer's instructions, filtering through a 70-µm cell strainer and washing. ACK lysis using the respective buffer (Thermo Fischer Scientific A1049201) was performed, and samples were stored in FBS with 20% DMSO until further processing (scRNA-seq analysis and mass cytometry).
Isolation of cells for scRNA-seq data analysis (human)
Cells were thawed in a 37 °C water bath, washed with PBS + 0.05 mM EDTA (10 min, 300 g at 4 °C), Fc receptor-block (10 min at 4 °C), stained with CD45-PE (3 µl, Hl30, 12-0459-42) and Live/Dead discrimination (1:1,000, Thermofischer, L34973 ), washed and sorted on a FACSAria FUSION in collaboration with the DKFZ FACS. Library generation was performed according to the manufacturer's protocol (Chromium Next EM Single Cell 3′GEM, 10000128), and sequencing was performed on an Illumina NovaSeq 6000. De-multiplexing and barcode processing were performed using the Cell Ranger Software Suite (Version 4.0.0) and reads were aligned to human GRCh38. A gene-barcode matrix containing cell barcodes and gene expression counts was generated by counting the single-cell 3′ UMIs, which were imported into R (v4.0.2), where quality control and...
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Standard mouse diet feeding (ad libitum water and food access) and treatment regimens were as described previously. Male mice were housed at the German Cancer Research Center (DKFZ) (constant temperature of 20-24 °C and 45-65% humidity with a 12-h light-dark cycle). Mice were maintained under specific pathogen-free conditions and experiments were performed in accordance with German law and the governmental bodies, and with approval from the Regierungspräsidium Karlsruhe (G11/16, G129/16, G7/17). Tissues from inducible knock-in mice expressing the human unconventional prefoldin RPB5 interactor were received from N. Djouder,. The plasmids for hydrodynamic tail-vein delivery have been described previously -. For interventional studies, male mice fed a CD-HFD were treated with bi-weekly for 8 weeks by intravenous injection of 25 µg CD8-depleting antibody (Bioxcell, 2.43), 50 µg NK1.1-depleting antibody (Bioxcell, PK136), 300 µg anti-PDL1 (Bioxcell, 10F.9G2), 200 µg anti-TNF (Bioxcell, XT3.11), 100 µg anti-CD4 (Bioxcell, GK1.5), or 150 µg anti-PD1 (Bioxcell, RMP1-14). PD1 -/- mice were kindly p...
MRI was done in the small animal imaging core facility in DKFZ using a Bruker BioSpec 9.4 Tesla (Ettlingen). Mice were anaesthetized with 3.5% sevoflurane, and imaged with T2-weighted imaging using a T2_TurboRARE sequence: TE = 22 ms, TR = 2,200 ms, field of view (FOV) 35 × 35 mm, slice thickness 1 mm, averages = 6, scan time 3 min 18 s, echo spacing 11 ms, rare factor 8, slices 20, image size 192 × 192 pixels, resolution 0.182 × 0.182 mm.
After perfusion and mechanical dissection, livers were incubated for up to 35 min at 37 °C with collagen IV (60 U final concentration (f.c.)) and DNase I (25 µg/ml f.c.), filtered at 100 µm, and washed with RPMI1640 (11875093, Thermo Fisher). Next, samples underwent a two-step Percoll gradient (25%/50% Percoll/HBSS) and centrifugation for 15 min at 1,800 g and 4 °C. Enriched leukocytes were then collected, washed, and counted. For re-stimulation, cells were incubated for 2 h at 37 °C under 5% CO 2 with 1:500 Biolegend´s Cell Activation Cocktail (with brefeldin A) (423304) and 1:1,000 Monensin Solution (420701). Live/dead discrimination was done using DAPI or ZombieDyeNIR according to the manufacturer's instructions with subsequent staining of titrated antibodies (Supplementary Tables - ). Samples for flow cytometric-activated cell sorting (FACS) were sorted and samples for flow cytometry were fixed using eBioscience IC fixation (00-8222-49) or FOXP3 Fix/Perm kit (00-5523-00) according to the manufacturer's instructions. Intracellular staining was performed in eBioscience Perm buffer (00-8333-56). Cells were analys...
After FACS purification, cells were resuspended in 50% (vol/vol) 2,2,2-trifluoroethanol in PBS pH 7.4 buffer and lysed by repeated sonication and freeze-thaw cycles. Proteins were denatured at 60 °C for 2 h, reduced using dithiothreitol at a final concentration of 5 mM (30 min at 60 °C), cooled to room temperature, alkylated using iodoacetamide at 25 mM (30 min at room temperature in the dark), and diluted 1:5 using 100 mM ammonium bicarbonate, pH 8.0. Proteins were digested overnight by trypsin (1:100 ratio, 37 °C), desalted using C18-based stage-tips, dried under vacuum, resuspended in 20 µl HPLC-grade water with 0.1% formic acid, and measured using A380.
We used 0.5 µg of peptides for proteomic analysis on a C18 column using a nano liquid chromatography system (EASY-nLC 1200, Thermo Fisher Scientific). Peptides were eluted using a gradient of 5-30% buffer B (80% acetonitrile and 0.1% formic acid) at a flow rate of 300 nl/min at a column temperature of 55 °C. Data were acquired by data-dependent Top15 acquisition using a high-resolution orbitrap tandem mass spectrometer (QExactive HFX, Thermo Scientific). All MS1 scans were acquired at 60,000 resolution with AGC target of 3 × 10 6, and MS2 scans were acquired at 15,000 resolution with AGC target of 1 × 10 5 and maximum injection time of 28 ms. Analyses were performed using MaxQuant (1.6.7.0), mouse UniProt Isoform fasta (Version: 2019-02-21, number of sequences 25,233) as a source for protein sequences. One per cent FDR was used for controlling at the peptide and protein levels, with a minimum of two peptides needed for consideration of analysis. GSEA was performed using ClusterProfiler (3.18) and gene sets obtained from WikiPathway ( https://www.wikipathways.org/ ) and MSigDB ( https://broadinstitute.org/msigdb ) -.
Analysis of patient material (Supplementary Table ) was performed on liver tissue (needle biopsies or resected tissue, BIOFACS Study KEK 2019-00114), which were obtained from the patient collection nAC-2019-3627 (CRB03) from the biological resource centre of CHU Grenoble-Alpes (nBRIF BB-0033-00069). Tissue samples were minced using scalpels, incubated (with 1 mg/ml collagenase IV (Sigma Aldrich), 0.25 µg/ml DNase (Sigma Aldrich), 10% FCS (Thermo Fisher Scientific), RPMI 1640 (Seraglob)) for 30 min at 37 °C, stopping enzymatic reactions with 2 mM EDTA (StemCell Technologies, Inc.) in PBS. After filtering through a 100-µm cell strainer, cells were resuspended in FACS buffer (PBS, EDTA 2 mM, FCS 0.5%) with Human TruStain FcX (Fc Receptor Blocking Solution) (Biolegend), incubated for 15 min at 4 °C and stained with antibodies (Supplementary Table ).
Analyses used liver samples from patients undergoing bariatric surgery at the Department of Surgery at Heidelberg University Hospital (S-629/2013). Samples were preserved by FFPE for pathological evaluation and single cells were generated by mincing, using the Miltenyi tumour dissociation kit (130-095-929) per the manufacturer's instructions, filtering through a 70-µm cell strainer and washing. ACK lysis using the respective buffer (Thermo Fischer Scientific A1049201) was performed, and samples were stored in FBS with 20% DMSO until further processing (scRNA-seq analysis and mass cytometry).
Cells were thawed in a 37 °C water bath, washed with PBS + 0.05 mM EDTA (10 min, 300 g at 4 °C), Fc receptor-block (10 min at 4 °C), stained with CD45-PE (3 µl, Hl30, 12-0459-42) and Live/Dead discrimination (1:1,000, Thermofischer, L34973 ), washed and sorted on a FACSAria FUSION in collaboration with the DKFZ FACS. Library generation was performed according to the manufacturer's protocol (Chromium Next EM Single Cell 3′GEM, 10000128), and sequencing was performed on an Illumina NovaSeq 6000. De-multiplexing and barcode processing were performed using the Cell Ranger Software Suite (Version 4.0.0) and reads were aligned to human GRCh38. A gene-barcode matrix containing cell barcodes and gene expression counts was generated by counting the single-cell 3′ UMIs, which were imported into R (v4.0.2), where quality control and normalization were executed using Seurat v3. Cells with more than 10% mitochondrial genes, fewer than 200 genes per cell, or more than 6,000 genes per cell were excluded. Matrices from 10 samples were integrated with Seurat v3 to remove batch effects across samples. PCA analysis of filtered gene...
Machine-readable layer
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