Journal: The Journal of Clinical Investigation

Article Title: ACAT1 regulates tertiary lymphoid structures and correlates with immunotherapy response in non–small cell lung cancer

doi: 10.1172/JCI181517

Figure Lengend Snippet: ( A ) Tertiary lymphoid structures (TLS) in 305 human lung cancer specimens were observed using H&E. ( B and C ) IHC analyses of ACAT1 levels of 305 human lung cancer specimens were performed. ( D ) Expression of ACAT1 in the TLS_high group and TLS_low group in A. ( E – G ) TLS maturity in 305 human lung cancer specimens were observed using immunofluorescence staining. Purple, CD8; red, CD19; yellow, CD21; blue, CD23; and gray, DAPI for nucleus. ( E ) Immature early TLS (eTLS). ( F ) Primary follicle-like TLS (pTLS). ( G ) Secondary follicle-like TLS (sTLS). ( H ) Correlation analysis between ACAT1 levels and TLS class in 305 human lung cancer specimens. ( I – L ) Kaplan-Meier plots of the overall survival durations of the patients ( n = 194). ( I ) The patients were divided into 2 groups with TLS+ ( n = 91) and TLS– ( n = 103). ( J ) The patients were divided into 4 groups with eTLS ( n = 38)/pTLS ( n = 24)/sTLS ( n = 29)/TLS– ( n = 103). ( K ). The patients were divided into 2 groups with ACAT1 low expression ( n = 91) and ACAT1 high expression ( n = 103) ( L ). The patients were divided into 8 groups with ACAT1 lo TLS– ( n = 37); ACAT1 lo eTLS ( n = 20); ACAT1 lo pTLS ( n = 15); ACAT1 lo sTLS ( n = 19); ACAT1 hi TLS– ( n = 66); ACAT1 hi eTLS ( n = 18); ACAT1 hi pTLS ( n = 9); ACAT1 hi sTLS ( n = 10). ( M ) IHC analyses of ACAT1 expression of 29 human lung cancer specimens that received immunotherapy were performed. Representative images are shown. Data are shown as the mean ± SD. ** P < 0.05; ** P < 0.01; **** P < 0.0001 with Mann-Whitney test ( C , D , and M ) and log-rank test ( I – L ).

Article Snippet: For IHC, sections were incubated with an anti-ACAT1 antibody (1:100, no. 44276, Cell Signaling Technology) in phosphate-buffered saline with 3% BSA, followed by horseradish peroxidase–conjugated secondary antibodies according to the manual of the SABC-HRP Kit (P0615, Beyotime), and analyzed by Olympus Slideview VS200.

Techniques: Expressing, Immunofluorescence, Staining, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: ACAT1 regulates tertiary lymphoid structures and correlates with immunotherapy response in non–small cell lung cancer

doi: 10.1172/JCI181517

Figure Lengend Snippet: ( A – C ) Tumor development was measured in orthotopic lung tumor mice models. Schematic diagram ( A ). Acat1 negative control (NC) and knockdown (KD) lung carcinoma cells were inoculated on the lungs of C57/BL6. LLC model, NC and KD n = 10 and 12, respectively, experimental replicates = 3. ( B ). KP model, n = 4 per group, experimental replicates = 2. ( C ). ( D and E ) Tertiary lymphoid structures (TLS) in mice lung tissues of KP model were observed using H&E and immunofluorescence. Purple, CD8; red, CD19; yellow, CD21; blue, CD23; and gray, DAPI for nucleus. Representative staining images are shown ( D ). TLS score of lung tissues in NC and KD groups were determined by the number and maturity of TLS as well as the ratio of the total area of all TLS to the total tumor area, n = 14 per group ( E ). ( F ) Tumor development for NC and KD groups with or without anti-PD1 treatment was measured in LLC model, NC and KD n = 4 and 6, respectively, experimental replicates = 2. ( G and H ) Tumor development for NC sg and Acat1 sg groups with or without anti-PD1 treatment was measured in KP-cas9 model, n = 6 per group. ( I and J ) TLS in G were observed using H&E and immunofluorescence. Purple: CD8; red: CD19; yellow: CD21; blue: CD23; and grey: DAPI. Representative staining images are shown, n = 4 per group. ( I ). TLS score was determined by the number and maturity of TLS as well as the ratio of the total area of all TLS to the total tumor area ( J ). Data are shown as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 with 2-tailed unpaired Student’s t tests. Please also see .

Article Snippet: For IHC, sections were incubated with an anti-ACAT1 antibody (1:100, no. 44276, Cell Signaling Technology) in phosphate-buffered saline with 3% BSA, followed by horseradish peroxidase–conjugated secondary antibodies according to the manual of the SABC-HRP Kit (P0615, Beyotime), and analyzed by Olympus Slideview VS200.

Techniques: Negative Control, Knockdown, Immunofluorescence, Staining

Journal: The Journal of Clinical Investigation

Article Title: ACAT1 regulates tertiary lymphoid structures and correlates with immunotherapy response in non–small cell lung cancer

doi: 10.1172/JCI181517

Figure Lengend Snippet: ( A – D ) CD45 + immune cells were sorted by flow cytometry from tumor isolated from orthotopic lung tumor mice models. Single-cell sequencing was performed ( A ), canonical markers of different cell types were used to identify cell clusters ( B ), and cell clusters are displayed ( C ). The proportion of different immune cell subsets in the 2 groups (pool of 3 mice per group) ( D ). ( E ) The proportion of different immune cell subsets in mouse tumor were verified by flow cytometry, n = 7 per group, experimental replicates = 2. ( F – I ) Gene-expression levels related to B cell activation ( F ), proliferation ( G ), antigen processing and presentation ( H ), and B-cell receptor signaling pathway of Acat1 negative control (NC) and knockdown (KD) groups. ( J – M ) Anti-CD20 antibodies were used to eliminate B cells in the KP model, n = 4 per group, experimental replicates = 2. ( J ), followed by determination of the B cell depletion effect ( K ), tumor volume ( L ), tumor weight ( M ). ( N and O ) Tertiary lymphoid structures (TLS) in mouse lung tissues of J were observed using H&E and immunofluorescence. Purple, CD8; red, CD19; yellow, CD21; blue, CD23; and grey, DAPI for nucleus. Representative staining images are shown ( O ). TLS score was determined by the number and maturity of TLS as well as the ratio of the total area of all TLS to the total tumor area, n = 5 per group. ( N ). Data are shown as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 with 2-tailed unpaired Student’s t tests. Please also see .

Article Snippet: For IHC, sections were incubated with an anti-ACAT1 antibody (1:100, no. 44276, Cell Signaling Technology) in phosphate-buffered saline with 3% BSA, followed by horseradish peroxidase–conjugated secondary antibodies according to the manual of the SABC-HRP Kit (P0615, Beyotime), and analyzed by Olympus Slideview VS200.

Techniques: Flow Cytometry, Isolation, Sequencing, Gene Expression, Activation Assay, Negative Control, Knockdown, Immunofluorescence, Staining

Journal: The Journal of Clinical Investigation

Article Title: ACAT1 regulates tertiary lymphoid structures and correlates with immunotherapy response in non–small cell lung cancer

doi: 10.1172/JCI181517

Figure Lengend Snippet: ( A ) Protein interaction and enrichment analysis of ACAT1. The protein-protein interaction network of ACAT1 was constructed by the STRING/Proteins Database. ( B ) IP and IB analyses were performed with the indicated antibodies. ( C ) Workflow of the proteomic and succinylation modification proteomic detection. ( D ) Pathway schematic showing proteins with upregulated succinylation sites (Wilcoxon test, FDR P < 0.05) mapped onto key metabolic pathways. ( E – G ) Mitochondria were extracted from carcinoma (T) and adjacent tissue (N) from lung cancer patients, and mitochondria lysates of the mitochondria were prepared. The total mitochondrial-protein succinylation levels were analyzed by IB with the indicated antibodies. A comparison of mitochondrial-protein succinylation modification levels ( E ) and ACAT1 expression level ( F ) between carcinoma (T) and adjacent tissue (N) in , n = 10 per group. Paired t tests were performed. A correlation analysis between the ACAT1 levels and of mitochondrial-protein succinylation levels in ( G ). ( H ) ACAT1-mediated HADHA succinylation was analyzed by mixing purified ACAT1, HADHA, and succinyl-CoA. Heat-inactived ACAT1 was used as a negative control. The succinylation level of HADHA was detected by IB. * P < 0.05; *** P < 0.001. Please also see .

Article Snippet: For IHC, sections were incubated with an anti-ACAT1 antibody (1:100, no. 44276, Cell Signaling Technology) in phosphate-buffered saline with 3% BSA, followed by horseradish peroxidase–conjugated secondary antibodies according to the manual of the SABC-HRP Kit (P0615, Beyotime), and analyzed by Olympus Slideview VS200.

Techniques: Construct, Modification, Comparison, Expressing, Purification, Negative Control

Journal: The Journal of Clinical Investigation

Article Title: ACAT1 regulates tertiary lymphoid structures and correlates with immunotherapy response in non–small cell lung cancer

doi: 10.1172/JCI181517

Figure Lengend Snippet: ( A – C ) Targeted metabolic assays of ACAT1 overexpression (OE) and negative control (NC) in HEK293T cells, n = 5 per group. Heatmap representing metabolite changes in OE compared with NC cells ( A ). Content differences of key metabolites in TCA ( B ). Content differences of key metabolites in glycolysis ( C ). ( D – G ) The oxygen consumption rate measurement of specific cells with indicated treatment: Acat1 OE, NC, and knockdown (KD) Lewis lung carcinoma (LLC) cells, n = 6 per group ( D ); NC LLC cells, NC LLC cells pretreated with Succinyl-CoA (SCoA), OE LLC cells, OE LLC cells pretreated with ACAT1 inhibitor AH, n = 3 per group ( E ); OE LLC cells, OE LLC cells pretreated with Succinyl-CoA or Acetyl-CoA (ACoA), n = 6 per group ( F ); OE LLC cells, OE LLC cells with overexpression of Hadha or K305R mutant HADHA (mut Hadha ), OE LLC cells with Hadha knockdown (si Hadha ), n = 5 per group ( G ). Data are shown as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 with 2-tailed unpaired Student’s t tests.

Article Snippet: For IHC, sections were incubated with an anti-ACAT1 antibody (1:100, no. 44276, Cell Signaling Technology) in phosphate-buffered saline with 3% BSA, followed by horseradish peroxidase–conjugated secondary antibodies according to the manual of the SABC-HRP Kit (P0615, Beyotime), and analyzed by Olympus Slideview VS200.

Techniques: Over Expression, Negative Control, Knockdown, Mutagenesis

Journal: The Journal of Clinical Investigation

Article Title: ACAT1 regulates tertiary lymphoid structures and correlates with immunotherapy response in non–small cell lung cancer

doi: 10.1172/JCI181517

Figure Lengend Snippet: ( A ) B cell culture in vitro with indicated tumor culture medium (TCM) from Acat1 knockdown (KD)/negative control (NC)/overexpression (OE) LLC cells. BBCM, basic B cell culture medium. ( B ) Viability of B cells cultured in TCM at 36 hours determined by a CCK8 assay, n = 4 per group. ( C and D ) Representative images of B cell clumps cultured under TCM culture conditions, obtained by fluorescence microscopy, n = 9 per group ( C ). The B cell colony index was determined by the number and size of B cell colonies observed under microscope ( D ). ( E and F ) CD69 ( E ) and GL7 ( F ) levels of B cells cultured in TCM were determined by flow cytometry. ( G ) Viability of B cells cultured in TCM with or without NAC appending at 36 hours determined by a CCK8 assay, n = 4 per group. ( H – J ) NAC administered in drinking water was used to eliminate ROS in the KP models, followed by determination of tumor volume ( H and I ) and ROS clear effect (Tumor cells were labeled with DCFH-DA and detected by flow cytometry) ( J ). ( K and L ) Tertiary lymphoid structures (TLS) in mouse lung tissues of KP model were observed using H&E and immunofluorescence. Purple. CD8; red, CD19; yellow, CD21; blue, CD23; and gray, DAPI for nucleus. Representative staining images are shown ( L ). TLS score was determined by the number and maturity of TLS and the ratio of the total area of all TLS to the total tumor area, n = 5 per group ( K ). Data are representative of at least 2 independent experiments. Data are shown as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 with 2-tailed unpaired Student’s t tests. Please also see .

Article Snippet: For IHC, sections were incubated with an anti-ACAT1 antibody (1:100, no. 44276, Cell Signaling Technology) in phosphate-buffered saline with 3% BSA, followed by horseradish peroxidase–conjugated secondary antibodies according to the manual of the SABC-HRP Kit (P0615, Beyotime), and analyzed by Olympus Slideview VS200.

Techniques: Cell Culture, In Vitro, Knockdown, Negative Control, Over Expression, CCK-8 Assay, Fluorescence, Microscopy, Flow Cytometry, Labeling, Immunofluorescence, Staining

Journal: Cells

Article Title: Modulating Cholesterol Metabolism via ACAT1 Knockdown Enhances Anti-B-Cell Lymphoma Activities of CD19-Specific Chimeric Antigen Receptor T Cells by Improving the Cell Activation and Proliferation

doi: 10.3390/cells13060555

Figure Lengend Snippet: ACAT1 shRNA can effectively downregulate the expression of ACAT1 in Jurkat cell lines. ( a ) Schematic diagram showing the ACAT1-shRNA gene expression under U6 promoter in lentiviral vector pLL3.7. ( b , c ) Relative ACAT1 expression analysis in Jurkat cell line. Jurkat cells were transduced with lentiviruses expressing ACAT1-shRNAs and the relative gene and protein expression was confirmed by qPCR ( b ) and Western blot analysis ( c ). ( d ) The constant interfering ability of shACAT1-A was analyzed by qPCR with 2-day intervals until 8 days after transduction in Jurkat cells. GAPDH was used as an internal normalization control. Results are representative of three independent experiments. For all panels, the bars represent the mean ± SD. ns, not significant, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: After being transferred to the nitrocellulose membrane, ACAT1 protein was detected using an antibody (Cat # 44276, Cell signaling technology, MA, USA or Cat # ab154396, Abcam, Cambridge, UK) and visualized. β-actin (Cat# 4967, Cell signaling technology) or GAPDH (Cat# 14C10, Cell signaling technology) was used as a reference.

Techniques: shRNA, Expressing, Gene Expression, Plasmid Preparation, Transduction, Western Blot, Control

Journal: Cells

Article Title: Modulating Cholesterol Metabolism via ACAT1 Knockdown Enhances Anti-B-Cell Lymphoma Activities of CD19-Specific Chimeric Antigen Receptor T Cells by Improving the Cell Activation and Proliferation

doi: 10.3390/cells13060555

Figure Lengend Snippet: Silencing of the ACAT1 gene enhances the anti-tumor effect of CD19-CAR-transduced T cells. ( a ) Schematic diagram showing CD19-CAR (19CAR), CD19-CAR with ACAT1-shRNA (shACAT1-19CAR), and CD19-CAR with negative control shRNA (NC-19CAR). ( b ) The significant downregulation of the ACAT1 gene in CD19-CAR T cells was analyzed by Western blot ( left panel ). The relative expression level of ACAT1 was statistically analyzed ( right panel ). β-actin was used as an internal normalization control. ( c , d ) Cytotoxic activity of control, CD19-CAR, NC-CD19-CAR, or shACAT1-CD19-CAR-transduced T cells. All the T cells (effector cells) were mixed with Raji cells or Daudi cells at the E:T ratio of 1:1 for 16 h and CD19-positive cells were evaluated by flow cytometry. The representative pseudocolor plots are shown on the left side and the cytotoxicity rate was statistically analyzed, as presented on the right side. Results are representative of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After being transferred to the nitrocellulose membrane, ACAT1 protein was detected using an antibody (Cat # 44276, Cell signaling technology, MA, USA or Cat # ab154396, Abcam, Cambridge, UK) and visualized. β-actin (Cat# 4967, Cell signaling technology) or GAPDH (Cat# 14C10, Cell signaling technology) was used as a reference.

Techniques: shRNA, Negative Control, Western Blot, Expressing, Control, Activity Assay, Flow Cytometry

Journal: Cells

Article Title: Modulating Cholesterol Metabolism via ACAT1 Knockdown Enhances Anti-B-Cell Lymphoma Activities of CD19-Specific Chimeric Antigen Receptor T Cells by Improving the Cell Activation and Proliferation

doi: 10.3390/cells13060555

Figure Lengend Snippet: Downregulation of the ACAT1 gene improves the cell activation and degranulation of CD19-CAR-transduced T cells. ( a – c ) CD19-CAR-transduced T cells were co-cultured with Raji cells at the E:T ratio of 1:1 and the expression of ( a ) CD69, ( b ) IFN-γ, ( c ) GzmB, and ( d ) CD107a was evaluated by flow cytometry. Transduced T cells were stained with anti-human CD69-APC, anti-human IFN-γ-APC, anti-human GzmB-PE/Cy7, and anti-human CD107a-PE/Cy7 antibodies in separate experiments and analyzed by flow cytometry. The mean fluorescent intensity (MFI) of these proteins was statistically analyzed and is shown in the column chart. One-way ANOVA was used to measure the statistical significance indicated as * p < 0.05, ** p < 0.01.

Article Snippet: After being transferred to the nitrocellulose membrane, ACAT1 protein was detected using an antibody (Cat # 44276, Cell signaling technology, MA, USA or Cat # ab154396, Abcam, Cambridge, UK) and visualized. β-actin (Cat# 4967, Cell signaling technology) or GAPDH (Cat# 14C10, Cell signaling technology) was used as a reference.

Techniques: Activation Assay, Cell Culture, Expressing, Flow Cytometry, Staining

Journal: Cells

Article Title: Modulating Cholesterol Metabolism via ACAT1 Knockdown Enhances Anti-B-Cell Lymphoma Activities of CD19-Specific Chimeric Antigen Receptor T Cells by Improving the Cell Activation and Proliferation

doi: 10.3390/cells13060555

Figure Lengend Snippet: Downregulation of the ACAT1 gene improves the cell proliferation of CD19-CAR-transduced T cells. ( a , b ) CD19-CAR-transduced T cells or non-transduced T cells were stained with 2 μM/mL CFSE for 10 min and cultured for 5 days. CFSE dilution was analyzed by flow cytometry on day 0 ( a ) and day 5 ( b ), respectively. ( c , d ) CD19-CAR-transduced T cells or non-transduced T cells were stained with 2 μM/mL CFSE staining for 10 min and co-cultured with Daudi cells for 5 days. CFSE dilution was analyzed by flow cytometry on day 0 ( c ) and day 5 ( d ), respectively. The MFI of CFSE was statistically analyzed. Data are representative of three different experiments. *** p < 0.001, ns, not significant.

Article Snippet: After being transferred to the nitrocellulose membrane, ACAT1 protein was detected using an antibody (Cat # 44276, Cell signaling technology, MA, USA or Cat # ab154396, Abcam, Cambridge, UK) and visualized. β-actin (Cat# 4967, Cell signaling technology) or GAPDH (Cat# 14C10, Cell signaling technology) was used as a reference.

Techniques: Staining, Cell Culture, Flow Cytometry

Journal: Cells

Article Title: Modulating Cholesterol Metabolism via ACAT1 Knockdown Enhances Anti-B-Cell Lymphoma Activities of CD19-Specific Chimeric Antigen Receptor T Cells by Improving the Cell Activation and Proliferation

doi: 10.3390/cells13060555

Figure Lengend Snippet: Silencing of ACAT1 enhances in vivo anti-B-cell lymphoma of CD19-CAR-transduced T cells. ( a ) Schematic diagram of in vivo experiment using NSG mice model. NSG mice (5 mice/group) were infused subcutaneously with FFLuc-labeled Raji cells and mice were i.v. injected with PBS, CD19-CAR T (19CAR-T), NC-CD19-CAR T (NC-19CAR-T), or shACAT1-CD19-CAR T (shACAT1-19CAR-T) cells ( b ) on day 7, Kaplan–Meier survival analysis of Raji-challenged mice (n = 3) after treatment with CD19-CAR-transduced T cells. ( c ) IVIS imaging (n = 2) was performed to monitor tumor burden at days 6, 13, 20, 27, and 34. The bioluminescence of tumor cells was measured and the light intensity (p/s) was analyzed. * p < 0.05, ** p < 0.01.

Article Snippet: After being transferred to the nitrocellulose membrane, ACAT1 protein was detected using an antibody (Cat # 44276, Cell signaling technology, MA, USA or Cat # ab154396, Abcam, Cambridge, UK) and visualized. β-actin (Cat# 4967, Cell signaling technology) or GAPDH (Cat# 14C10, Cell signaling technology) was used as a reference.

Techniques: In Vivo, Labeling, Injection, Imaging