Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: SPI1 was highly expressed in macrophages within the metastatic lesions of gastric cancer. ( A ) UMAP representation colored according to different cell types. ( B ) Density plot of SPI1 expression distribution in scRNA sequencing data. ( C ) UMAP indicated SPI1 staining of macrophages in primary and metastasis gastric cancer (GC). ( D ) Differential analysis of SPI1 expression in the primary and metastasis tumor of GC in scRNA sequencing data. ( E ) Infiltration of SPI1 + CD68 + TAMs in normal tissue, primary tumor, and metastasis sites of GC. ( F ) Differential analysis of SPI1 + CD68 + TAMs infiltration between primary and metastasis tumor according to double immunohistochemical staining. ( G ) SPI1 + CD68 + TAMs were screened by flow cytometry in primary and metastasis tumor of patients with GC. ( H ) Quantitative analysis of SPI1 + CD68 + TAMs infiltration based on flow cytometry. scRNA, single-cell RNA; SPI1, Spi-1 proto-oncogene; TAMs, tumor-associated macrophages; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: Expressing, Sequencing, Staining, Immunohistochemical staining, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: SPI1 + CD68 + TAMs was an independent prognostic factor in patients with metastatic gastric cancer. ( A ) Overall survival (OS) and disease-free survival (DFS) of patients with gastric cancer (GC) in different SPI1 + CD68 + TAMs groups in the training cohort. ( B ) OS and DFS of patients with GC in different SPI1 + CD68 + TAMs groups in the external validation cohort. ( C ) OS and DFS of patients with GC in different SPI1 − CD68 + TAMs groups in the training cohort. ( D ) OS and DFS of patients with GC in different SPI1 − CD68 + TAMs groups in the external validation cohort. ( E ) Univariate cox regression analysis of patients with GC in the training cohort. ( F ) SPI1 + CD68 + TAMs was an independent prognostic factor of patients with GC in the training cohort. ( G ) Univariate cox regression analysis of patients with GC in the external validation cohort. ( H ) SPI1 + CD68 + TAMs was an independent prognostic factor of patients with GC in the external validation cohort. SPI1, Spi-1 proto-oncogene; TAMs, tumor-associated macrophages.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: Biomarker Discovery

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: SPI1 was associated with M2 polarization of macrophages. ( A ) Screening M1/M2 macrophages used flow cytometry with CD45, CD68, CD80, CD206 and SPI1 in patients with gastric cancer (GC). ( B ) Quantitative analysis of SPI1 + CD68 + TAMs infiltration in M1 and M2 type based on flow cytometry of patients with GC. ( C ) The markers of induced M1 and M2-type macrophages were detected by qRT-PCR. ( D ) Detecting the macrophage-related markers with qRT-PCR after SPI1 knockdown. ( E ) qRT-PCR was used to detect the macrophage-associated markers after SPI1 overexpression. ( F ) Western blot was used to detect the macrophage-related markers after SPI1 knockdown. ( G ) Macrophage-related markers were detected with western blot after SPI1 overexpression. ( H ) Flow cytometry was used to detect the macrophage-related markers after SPI1 knockdown. ( I ) Quantitative analysis of flow cytometry in SPI1 knockdown macrophages. ( J ) Flow cytometry was used to detect the macrophage-related markers after SPI1 overexpression. ( K ) Quantitative analysis of flow cytometry in SPI1 overexpression macrophages. IL-10, interleukin-10; mRNA, messenger RNA; qRT-PCR, quantitative Reverse Transcription Polymerase Chain Reaction; SPI1, Spi-1 proto-oncogene; TAMs, tumor-associated macrophages.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: Flow Cytometry, Quantitative RT-PCR, Knockdown, Over Expression, Western Blot, Reverse Transcription, Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: SPI1 + CD68 + TAMs promoted tumor angiogenesis through VEGF pathway. ( A ) GSVA analysis between SPI1-high and SPI1-low TAMs groups in scRNA sequencing data. ( B ) Incoming and outgoing interaction strength in different types of cells. ( C ) Cell–cell communication between different cell types in the VEGF signaling pathway. ( D ) Response to bevacizumab in patients with different TAMs infiltrates in the external validation cohort. ( E ) Immunofluorescence revealed the infiltration of SPI1 + CD68 + TAMs around the tumor blood vessels (CD31 marked). ( F ) Difference in the number of SPI1 + CD68 + TAMs and SPI1 − CD68 + TAMs around tumor vessels. ( G ) Difference in distance between blood vessels with SPI1 + CD68 + TAMs and SPI1 − CD68 + TAMs. DAPI, 4',6-Diamidino-2-Phenylindole; GSVA, gene set variation analysis; NK, Natural Killer cells; scRNA, single-cell RNA; SPI1, Spi-1 proto-oncogene; TAMs, tumor-associated macrophages; VEGFA, vascular endothelial growth factor A.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: Sequencing, Biomarker Discovery, Immunofluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: The role of SPI1 in GC cell growth and metastasis in vivo. ( A ) The representative images of the xenograft tumor. ( B ) Quantitative analysis of tumor growth curve. ( C ) Quantitative analysis of tumor weight in different SPI1 expression group. ( D ) Bioluminescence images of tumor-bearing mice individually treated with shNC, shSPI1, Vector, SPI1 transfected macrophages at day 5, 10, 15, 20, 25, 30, 40, and 50. ( E ) Representative photographs of peritoneum and mesentery metastasis lesions in different SPI1 expression groups. ( F ) The fluorescence intensity of tumors in various groups of mice. ( G ) Quantitative analysis of peritoneum nodules. ( H ) Survival curves of mice in different groups. GC, gastric cancer; SPI1, Spi-1 proto-oncogene.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: In Vivo, Expressing, Plasmid Preparation, Transfection, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: SPI1 closely interacted with endothelial cells and regulated VEGFA transcription. ( A ) Wound healing assay was used to detect the migration ability of HUVEC cells cultured with conditioned medium of SPI1 knockdown macrophages. ( B ) Compared with the control group, the overexpression of SPI1 enhanced the migration ability of HUVEC cells. ( C ) Tube formation assay was used to detect the angiogenic ability of HUVEC cells after cultured with conditioned medium of SPI1 knockdown macrophages. ( D ) On overexpressing SPI1, the angiogenesis ability of HUVEC cells was augmented. ( E ) The mRNA level of VEGFA changed after knockdown or overexpression of SPI1. ( F ) The protein level of VEGFA changed after knockdown or overexpression of SPI1. ( G ) Motif sequence logo plot of SPI1 according to JASPAR database. ( H ) Putative SPI1 and VEGFA promoter binding sites. ( I ) ChIP analysis of the direct interaction between SPI1 and the promoter of VEGFA. ( J ) The binding site of SPI1 to the VEGFA promoter was assessed using ChIP-qPCR. ChIP, chromatin immunoprecipitation; HUVEC, Human Umbilical Vein Endothelial Cells; mRNA, messenger RNA; qPCR, quantitative PCR; SPI1, Spi-1 proto-oncogene; VEGFA, vascular endothelial growth factor A.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: Wound Healing Assay, Migration, Cell Culture, Knockdown, Control, Over Expression, Tube Formation Assay, Sequencing, Binding Assay, ChIP-qPCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: In vivo response to anti-PD-1 immunotherapy and antiangiogenesis treatment. ( A ) Response to immunotherapy in patients with different CPS scores and SPI1 + CD68 + TAMs infiltration. ( B ) Difference analysis of immunotherapy response in patients with different SPI1 + CD68 + TAMs infiltration. ( C ) Correlation analysis of SPI1 and PD-L1. ( D–G ) ROC curve of CPS score, CD68 + cells, SPI1 + CD68 + TAMs, and CPS score plus SPI1 + CD68 + TAMs. ( H ) Multiple immunofluorescences staining of PD-1 + CD8 + T cells and SPI1 + CD68 + TAMs. ( I ) Spatial distribution of SPI1 + CD68 + TAMs, SPI1 − CD68 + TAMs and PD-1 + CD8 + T cells analyzed by HALO. ( J ) Average distance from PD-1 + CD8 + T cells to SPI1 + CD68 + TAMs and SPI1 − CD68 + TAMs (p<0.05). ( K ) The discrepancy in the quantity of SPI1 + CD68 + TAMs and SPI1 − CD68 + TAMs surrounding PD-1 + CD8 + T cells (p<0.05). ( L ) Bioluminescence images of NOD/SCID mice with different treatment in week 1, 3, and 6. ( M ) The fluorescence intensity of intraperitoneal tumors in various groups of mice. CPS, Combined Positive Score; DAPI, 4',6-Diamidino-2-Phenylindole; PD-1, programmed cell death protein-1; PD-L1, programmed death-ligand 1; ROC, receiver operating characteristic; SPI1, Spi-1 proto-oncogene; TAMs, tumor-associated macrophages.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: In Vivo, Staining, Fluorescence

Journal: International journal of molecular sciences

Article Title: Monocyte/Macrophage-Specific Loss of ARNTL Suppresses Chronic Kidney Disease-Associated Cardiac Impairment.

doi: 10.3390/ijms252313009

Figure Lengend Snippet: Figure 2. The effect of monocyte/macrophage-specific transcription factor PU.1 on the induction of GPR68 expression. (A) The 5/6Nx-derived serum did not increase the transcriptional activity upstream of Gpr68 in NIH3T3. NIH3T3 was transfected with Gpr68 (-1734)-Luc or pGL4.18 and incubated with 10% serum from Sham and 5/6Nx mice for 24 h. (B) Transcription factors binding upstream of Gpr68 analyzed by previous transcriptome analyses. The blue waveform shows the sequenced tags in ChIP sequence analysis for each transcription factor. The numbers on the horizontal axis indicate the distance from the transcription start site (kbp). (C) The PU.1 protein in RAW264.7 incubated with 10% serum from Sham and 5/6Nx mice for 24 h. (D) High-PU.1-expressing NIH3T3 was created by introducing a PU.1 expression plasmid. PU.1 expression levels were measured using Western blotting. (E,F) The mRNA levels of Gpr68 (E) and Arntl (F) in NIH3T3-transfected pcDNA3.1 or PU.1-expressing vectors were measured after incubation with 10% serum from Sham and 5/6Nx mice for 24 h. (G) A schematic of mouse Gpr68. The numbers indicate the distance from the transcription start site (+1). Black rectangles, E-box. The arrow symbols indicate the location on the gene where the primer sets localize for the analysis of ChIP. (H) The binding of endogenous ARNTL to the Gpr68 upstream region in NIH3T3-transfected pcDNA3.1 or PU.1-expressing vectors. Values are expressed as the mean ± S.D. (n = 3–5). **, p < 0.01 indicates significant differences between the two groups (two-way ANOVA with Tukey–Kramer post hoc tests or Student’s t-test).

Article Snippet: Mouse PU.1-expressing plasmid (MR203632, OriGene) was transfected into NIH3T3 cells using LipofectamineTM LTX and PLUSTM reagents (Thermo Fisher Scientific).

Techniques: Expressing, Derivative Assay, Activity Assay, Transfection, Incubation, Binding Assay, Sequencing, Plasmid Preparation, Western Blot

Journal: eLife

Article Title: Convergent organization of aberrant MYB complex controls oncogenic gene expression in acute myeloid leukemia

doi: 10.7554/eLife.65905

Figure Lengend Snippet:

Article Snippet: Antibody , anti-SPI1 (Rabbit monoclonal) , Cell Signaling Technology , Cat# 2266, RRID: AB_10692379 , WB (1:1000).

Techniques: Recombinant, CRISPR, Knock-Out, Plasmid Preparation, Sequencing, Cell Viability Assay, Software, Genome Wide, Flow Cytometry