anti-cd74 mouse monoclonal antibody  (Santa Cruz Biotechnology)

Journal: Neuro-Oncology

Article Title: MIF-CD74 signaling drives immune modulation in medulloblastoma

doi: 10.1093/neuonc/noag020

Figure Lengend Snippet: Cell-to-cell communications within the TIME for human Group3 and Group4 MB. (A) The UMAP plot of cell type clusters show the identification of 15 distinct cell clusters from the single-cell RNA-seq data of Group3 MB samples. OPC: Oligodendrocyte Precursor Cell. APC: Astrocyte Precursor Cell. M2_1: M2 macrophages cluster 1. M2_2: M2 macrophages cluster 2. NSC: Neural stem cell. Complement-M, complement macrophage. inflam dendritic cell: inflammatory dendritic cell. (B) The violin plot of the marker gene expression for each cell cluster. (C) CellCrossTalker predicted ligand-receptor-mediated tumor-immune cell communications using scRNA-seq data from Group3 MB samples. The vertical axis represents ligands and their corresponding receptors, while the horizontal axis indicates the sender cell type associated with the ligand and the receiver cell type associated with the receptor. (D) CellCrossTalker predicted ligand-receptor-mediated communications between M2 macrophages and tumor cells, M2 macrophages and T cells, M2 macrophages and B cells, as well as M2 macrophages and myeloid cells, using scRNA-seq data from Group3 MB samples. The vertical axis represents ligands and their corresponding receptors, while the horizontal axis shows the sender cell type associated with the ligand and the receiver cell type associated with the receptor. (E) The UMAP plot of cell type clusters show the identification of 13 distinct cell clusters. Single-cell RNA-seq data from Group4 MB samples were obtained from Hendrikse et al. (F) The heatmap illustrates the expression of marker genes used to annotate each cell cluster. (G) CellCrossTalker predicted ligand-receptor-mediated cell-cell communications using scRNA-seq data from Group4 MB samples. The vertical axis represents ligands and their corresponding receptors, while the horizontal axis shows the sender cell type associated with the ligand and the receiver cell type associated with the receptor. (H) The heatmap shows the interaction strength of MIF-CD74 between various pairs of cell types, as predicted by CellCrossTalker using scRNA-seq data from Group4 MB samples. (I) CellCrossTalker predicted ligand-receptor-mediated communication co-receptors between tumor cells and immune compartment, using scRNA-seq data from Group3 and Group4 MB samples. The vertical axis represents ligands and their corresponding receptors and co-receptors, while the horizontal axis shows the sender cell type associated with the ligand and the receiver cell type associated with the receptor.

Article Snippet: In brief, tissue sections were incubated in Tris-EDTA buffer (cell conditioning 1; CC1) at 95 ̊C for 1-h to retrieve antigenicity, followed by incubation with CD74 antibody (Origene CF507339 ) at 1:500 for 1-h.

Techniques: Single Cell, RNA Sequencing, Marker, Gene Expression, Expressing

Journal: Neuro-Oncology

Article Title: MIF-CD74 signaling drives immune modulation in medulloblastoma

doi: 10.1093/neuonc/noag020

Figure Lengend Snippet: Restricted expression of CD74 and MIF in human normal tissues and MB subgroups. (A) Graph depicting the expression of CD74 (ENSG00000019582.14) in normal human tissue RNA-sequencing data obtained from the GTEx consortium. The dataset comprises 7859 samples across 31 distinct normal tissues, with sample sizes ranging from 5 to 1152 samples per tissue. Expression levels are presented as relative expression levels in transcripts per million (TPM). (B) CD74 protein expression in normal human cerebellum. Scale bar, 100 um. (C) Graph depicting the expression of MIF (ENSG00000240972.1) in normal human tissue RNA-sequencing data obtained from the GTEx consortium. The dataset comprises 7859 samples across 31 distinct normal tissues, with sample sizes ranging from 5 to 1152 samples per tissue. Expression levels are presented as relative expression levels in transcripts per million (TPM). (D) MIF protein expression in normal human cerebellum. Scale bar, 100 um. (E-J) Protein levels across human MB subgroups and subtypes (based on DNA methylome classification) from Ayrault cohort for the three selected proteins: CD74, CD68 and MIF. Boxplots show median (line), upper and lower quartiles (boxes), and lines extending to highest and lowest observations (whiskers). (K) CD74 immunohistochemistry staining analysis of paired human pediatric diagnostic (left) and relapse (right) MB samples. Black arrows depict CD74 positivity. Scale bar represents 100 µM. (L) Immunohistochemistry membrane staining depicting CD74 expression across a subgrouped human diagnostic MB tissue microarray. Scale bar represents 200 µM.

Article Snippet: In brief, tissue sections were incubated in Tris-EDTA buffer (cell conditioning 1; CC1) at 95 ̊C for 1-h to retrieve antigenicity, followed by incubation with CD74 antibody (Origene CF507339 ) at 1:500 for 1-h.

Techniques: Expressing, RNA Sequencing, Immunohistochemistry, Staining, Diagnostic Assay, Membrane, Microarray

Journal: Neuro-Oncology

Article Title: MIF-CD74 signaling drives immune modulation in medulloblastoma

doi: 10.1093/neuonc/noag020

Figure Lengend Snippet: Inhibition of CD74 via the lateral ventricle demonstrates immune-modulation and decreased tumor burden in primary and relapsed MB. (A) IHC of treatment-naïve primary (top row) and radiation-treated (bottom row) GTML allografts displaying H&E, CD74, and MIF expression within the tumor. Results representative of 3 independent replicates. Much of the tumor outside the cerebellum was removed for downstream RNA sequencing analyses, Scale bar, 100 µm. (B) Experimental schematic for evaluating the CD74-MIF blocking peptide C36L1. GTML primary or relapse cells expressing firefly luciferase were allografted into the cerebellum of FVBNRJ mice. C36L1 was administered either intraperitoneally or locoregionally through the lateral ventricle at time of engraftment and 1-week after, C36L1 vehicle was used as a control. Bioluminescence imaging was conducted to monitor tumor engraftment, progression, and/or regression up to 14 days post-therapy. At the endpoint, the tumor and CNS were harvested and assessed for immune infiltration and tumor burden. (C) Growth rate of mice bearing primary GTML allografts ( n = 3-4 per group) treated with vehicle control (black), C36L1 peptide through the lateral ventricle (red), or C36L1 peptide intraperitoneally (blue) was determined by calculating the slope of tumor growth between day 7 and 14 (endpoint). Line represents mean growth rate with individual points representing individual mice. Points below the dashed line indicate tumor regression. Statistical significance was calculated by two-way ANOVA with Tukey’s post-test. (D) UMAP projection of flow cytometry analysis of tumor/cerebellum of mice treated vehicle control or the C36L1 peptide via the lateral ventricle. The left panel display cells colored by experimental group (Control - black, C36L1 delivered via lateral ventricle - red, C36L1 delivered intraperitoneally - blue), highlighting the distribution and intensity target expression across different cell populations within the tumor and cerebellum of treated and control mice. The visualization provides insights into the immune cell infiltration and its association with the treatment groups. (E) UMAP projection of flow cytometry results, colored by the expression of CD74. (F) UMAP projection of flow cytometry results, colored by the expression of MHCII. (G) UMAP projection of flow cytometry results, colored by the expression of CD11b. (H) IHC of control (top row) and peptide-treated (bottom row) GTML primary tumor allografts displaying H&E, CD3, F4/80 and CD74 within the tumor. Results representative of 3 independent replicates, Scale bar, 100 um. (I) Growth rate of mice bearing recurrent GTML allografts ( n = 5 per group) treated with vehicle control (black) or C36L1 peptide through the lateral ventricle (blue) was calculated by calculating the slope of tumor growth between day 7 and 14 (endpoint). Line represents mean growth rate with individual points representing individual mice. Points below the dashed line indicate tumor regression. Statistical significance was calculated by two-way ANOVA with Tukey’s post-test. (J) Bar chart to illustrate the proportion of tumor-associated immune cells identified as microglia. A higher proportion of microglia is observed in the TME of CD36L1 peptide treatment versus scrambled control. (K) Bar chart to illustrate the level of CD74 expression in the TME of mice treated with CD36L1 versus scrambled control. (L) Bar charts displaying the proportion of CD38+ cells in the microglia population. Statistical analysis was performed using a two-way ANOVA with Tukey’s post-test to compare the groups. Error bars represent the SD. Significant differences between groups are indicated by * P < .05, ** P < .01, and *** P < .001.

Article Snippet: In brief, tissue sections were incubated in Tris-EDTA buffer (cell conditioning 1; CC1) at 95 ̊C for 1-h to retrieve antigenicity, followed by incubation with CD74 antibody (Origene CF507339 ) at 1:500 for 1-h.

Techniques: Inhibition, Expressing, RNA Sequencing, Blocking Assay, Luciferase, Control, Imaging, Flow Cytometry