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: bioRxiv

Article Title: Induction of long-term allogeneic cell acceptance and formation of immune privileged tissue in immunocompetent hosts

doi: 10.1101/716571

Figure Lengend Snippet: Schema of piggyBa c ( pB) and Sleeping Beauty (SB) -transposable plasmid used to express immunomodulatory factors in mESCs. cDNA encoding Ccl21, Pdl1, Fasl, Serpinb9, H2-M3, Cd47, Cd200, and Mfge8 were each cloned into separate pB and SB transposon vectors and integrated into the genome of safe-cell C57BL/6 mESCs by transposition in sequential transfection and selection (as described in ). From the polyclonal transgenic pools, 47 clonal lines were isolated and expanded Expression levels of immunomodulatory transgenes in the clonal lines as measured by RT-qPCR. Each colored wedge shows the value of a specific transgene. All values relative to the expression in isolated splenocytes from C57BL/6 mice (control, thick black circle in each radial graph) activated ex vivo with antibodies against CD3εand CD28. Concentric circles; log10 scale. Non-immune modified safe-cell mESCs (parental line) shown in upper left box. Clones outlined in red and blue boxes were later screened in vivo for acceptance or rejection in allogeneic hosts . Red boxes; clones accepted in allogeneic hosts (clone 1 and clone 43 renamed Klg-1 and Klg-2, respectively). Blue boxes; clones rejected in allogeneic hosts. c . Expression of transgene-encoded factors in Clone 1 (Klg-1) mESCs.

Article Snippet: For both mESCs on coverslips and teratoma tissue sections, primary antibodies were used against mouse PD-L1 (Novus NBP1-43262), CD47 (BD Biosciences 740055), CD200 (BD Biosciences 565544), H2-M3 (BD Biosciences 551769), FASL (Novus NBP1-97519), SERPINB9 (Hycultbiotech HP8035), CCL21 (R&D Systems AF457), and MFGE8 (Biolegend 518603).

Techniques: Plasmid Preparation, Clone Assay, Transfection, Selection, Transgenic Assay, Isolation, Expressing, Quantitative RT-PCR, Ex Vivo, Modification, In Vivo

Journal: bioRxiv

Article Title: Induction of long-term allogeneic cell acceptance and formation of immune privileged tissue in immunocompetent hosts

doi: 10.1101/716571

Figure Lengend Snippet: a . Map of representative piggyBac (pB) and Sleeping Beauty (SB) plasmid used to express eight immunomodulatory transgenes ( Ccl21, Pdl1, Fasl, Serpinb9, H2-M3, Cd47, Cd200 , and Mfge8) and luciferase in safe-cell C57BL/6 mESCs. Transgene expression driven by a CAG promoter and linked by an IRES to an eGFP fluorophore, or Puromycin r or Neomycin r genes. Expression cassette flanked by long-terminal repeat piggyBac or Sleeping Beauty sequences. b . Two sequential methods were used to generate safe-cell mESCs with expression of all eight immunomodulatory transgenes: Method 1) safe-cell C57BL/6 mESCs transfected with eGFP-linked Pdl1 , eGFP high clone was isolated, followed by simultaneous transfection of Ccl21, Fasl, Serpinb9, H2-M3, Cd47, Cd200, Mfge8 and luciferase (each containing Puromycin r ) and drug selection with Puromycin. Method 2) safe-cell C57BL/6 mESCs simultaneously transfected with Pdl1, Fasl, Cd47, and Cd200 , each with IRES-linked Puromycin r , followed by selection with Puromycin and flow cytometry sorting for the highest 5% expression of each factor using factor-specific antibodies. Bulk sorted cells then transfected with Ccl21, Mfge8, SerpbinB9 , and H2-M3 containing Neomycin r , followed by selection with Neomycin. Both methods resulted in a polyclonal pool subcloned by limited dilution plating, Clones were analyzed by RT-qPCR for transgene expression levels ( in main text).

Article Snippet: For both mESCs on coverslips and teratoma tissue sections, primary antibodies were used against mouse PD-L1 (Novus NBP1-43262), CD47 (BD Biosciences 740055), CD200 (BD Biosciences 565544), H2-M3 (BD Biosciences 551769), FASL (Novus NBP1-97519), SERPINB9 (Hycultbiotech HP8035), CCL21 (R&D Systems AF457), and MFGE8 (Biolegend 518603).

Techniques: Plasmid Preparation, Luciferase, Expressing, Transfection, Isolation, Selection, Flow Cytometry, Clone Assay, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Osteoprotegerin-dependent M cell self-regulation balances gut infection and immunity

doi: 10.1038/s41467-019-13883-y

Figure Lengend Snippet: a Enrichment analysis based on KEGG functional hierarchy for gene expression in M cells relative to their expression in enterocytes. Node size indicates the false-discovery rate of the parametric enrichment analysis. Red and blue nodes indicate respective significantly upregulated and downregulated pathways in M cells. b Gene expression profiles of enterocytes and M cells are shown. The heat map colors represent logFC for expression levels of genes compared with the mean expression value of each gene in enterocytes. c Increased expression of Tnfrsf11b ( Opg ) in the FAE of Peyer’s patches, relative to expression in villus epithelium (VE). Results are normalized to Gapdh expression and are presented relative to the expression in the mean of VE. Values are presented as the mean ± standard error. *** p < 0.005 (Student’s t -test, n = 4 animals). d Stereomicroscopic image of a whole-mount specimen of FAE monolayer and associated crypts, which was separated from the follicle by manipulation after treatment with EDTA as described in the Methods. The FAE area was surrounded by 12 crypts, as indicated by arrows. Scale bar: 200 µm. e OPG (red) is expressed in Tnfaip2 (blue)-positive and GP2 (green)-positive M cells in the FAE. Scale bars: 50 µm. f OPG (green) is expressed in Spi-B (red)-positive M cells of a FAE-associated crypt. The lower panel is an enlarged view of the area within the square in the upper panel. Cell nuclei were stained with DAPI (blue, left panel). F-actin was stained with phalloidin (gray, right panel). DIC differential interference contrast image. Scale bars: upper panel, 20 µm; lower panel, 10 µm. g Opg is an early expressing gene in the ileal epithelium after RANKL administration. Results were normalized to Gapdh expression and are presented relative to the expression in the epithelium without GST-RANKL treatment (time 0). Values are presented as the mean ± standard error. All data are representative of two ( c ) or three independent experiments ( a , b , d , e – g ). The source data underlying panels c and g are provided as a Source Data file.

Article Snippet: The primary antibodies used for immunofluorescence staining are as follows: anti-OPG antibody (catalog number AF549; R&D systems, 1:400); anti-GP2 antibody (D278-3; MBL, 1:400); anti-Tnfaip2 antibody (Kimura et al. : 200); anti-SpiB antibody (AF7204; R&D Systems, 1:200); anti-RelB antibody (sc-226; Santa Cruz Biotechnology, 1:100); and anti-RANKL antibody (14-5952; eBioscience, 1:100).

Techniques: Functional Assay, Expressing, Staining

Journal: Nature Communications

Article Title: Osteoprotegerin-dependent M cell self-regulation balances gut infection and immunity

doi: 10.1038/s41467-019-13883-y

Figure Lengend Snippet: a The numbers of M cells labeled with Tnfaip2 (green) and GP2 (red) are prominently increased in the follicle-associated epithelium (FAE) of Payer’s patches from Opg −/− mice. Each indicated channel is shown separately in gray scale. Scale bars: 200 µm. b The numbers of Tnfaip2-positive or GP2-positive M cells in the FAE of Peyer’s patches from WT and Opg −/− mice were quantified. Values are presented as the mean ± standard deviation. *** p < 0.005 (Student’s t -test, n = 4 of WT and 9 of Opg −/− mice). c The expression levels of M cell-associated genes in the FAE of Peyer’s patches were normalized to Gapdh expression and are presented relative to the expression in the villus epithelium (VE). Values are presented as the mean ± standard deviation. ** p < 0.01, *** p < 0.005 (Student’s t -test, n = 4 animals). d Whole-mount staining images of GP2 (red), Spi-B (green), and DAPI (blue) in cecal FAE. Scale bars: 100 µm. e The numbers of Tnfaip2-positive or GP2-positive M cells in the FAE of cecal patches from WT and Opg −/− mice were quantified. Values are presented as the mean ± standard deviation. * p < 0.05 (Student’s t-test, n = 4 of WT and 9 of Opg −/− mice). f The uptake of latex beads into Peyer’s patches and cecal patches was measured. Data from three independent experiments are presented as the mean ± standard deviation. *** p < 0.005. n.s. not significant (Student’s t -test, n = 7 animals). The source data underlying panels b , c , e and f are provided as a Source Data file.

Article Snippet: The primary antibodies used for immunofluorescence staining are as follows: anti-OPG antibody (catalog number AF549; R&D systems, 1:400); anti-GP2 antibody (D278-3; MBL, 1:400); anti-Tnfaip2 antibody (Kimura et al. : 200); anti-SpiB antibody (AF7204; R&D Systems, 1:200); anti-RelB antibody (sc-226; Santa Cruz Biotechnology, 1:100); and anti-RANKL antibody (14-5952; eBioscience, 1:100).

Techniques: Labeling, Standard Deviation, Expressing, Staining