Journal: Frontiers in Oncology

Article Title: Intratumor Heterogeneity of MIF Expression Correlates With Extramedullary Involvement of Multiple Myeloma

doi: 10.3389/fonc.2021.694331

Figure Lengend Snippet: Identification of MIF High and MIF Low MM cells in patient-derived xenografts. (A) Patient #16-derived BM CD138 + cells were intravenously injected into B-NDG mice to generate a PDX model (n = 4). They were euthanized to collect fresh tissue cells for flow cytometry within four to five weeks after inoculation. Mice received MIF −/− ARD cells were used as negative controls. Representative plots show the expression profiles of CD138 and MIF in isolated cells. Green represents CD138 − cells, namely non-MM cells; light blue represents MIF − MM cells, dark blue represents MIF Low MM cells, red represents MIF High MM cells. (B) Percentage of infiltrating CD138 + MM cells varies considerably in collected samples (n = 4). P = 0.0017 (two-way ANOVA). (C) Compare mean fluorescence intensity (MFI) of MIF between MIF High and MIF Low populations in diverse samples. The mean value ± standard deviation is 362.5 ± 81.8 and 19.5 ± 5.5 respectively. **** p < 0.0001 (two-way ANOVA). (D) Ratio of MIF High /MIF Low MM cells is notably higher in BM compared to extramedullary samples. **** p < 0.0001 (student’s t -test). (E) Representative IHC staining images of paired FFPE samples show sheets of MIF Low CD138 + cells surrounded by MIF High ones, and more evident loss of MIF expression in EMM (spleen) than in IMM (the blue and red boxes indicate representative areas of MIF Low and MIF High MM cells, respectively).

Article Snippet: To establish xenograft models, patient-derived primary MM cells or luciferase-expressing ARD cell line were implanted into six-week-old female immunocompromised B-NDG mice (NOD- Prkdc scid IL2rg tm1 /Bcgen, Biocytogen Jiangsu CO., Ltd.) via tail vein injection.

Techniques: Derivative Assay, Injection, Flow Cytometry, Expressing, Isolation, Fluorescence, Standard Deviation, Immunohistochemistry

Journal: Frontiers in Oncology

Article Title: Intratumor Heterogeneity of MIF Expression Correlates With Extramedullary Involvement of Multiple Myeloma

doi: 10.3389/fonc.2021.694331

Figure Lengend Snippet: Identification of MIF High and MIF Low MM cells in ARD cell line-derived xenografts. (A) B-NDG mice were intravenously injected with ARD cell line to establish another xenograft model (n = 5). Control mice received vehicle (PBS) or MIF −/− ARD cells. Three to four weeks after inoculation, samples were processed in the same way as described in the PDX model. Representative flow cytometry plots show MIF and CD138 expression in IMM and EMM (green, CD138 - cells, namely non-MM cells; light blue, MIF - MM cells; dark blue, MIF Low MM cells; red, MIF High MM cells). (B) CD138 + cells (ARD cells) infiltrate all tested samples with varying degrees. P = 0.0001 (two-way ANOVA). (C) MFI of MIF differs greatly between MIF High and MIF Low MM cells in diverse samples, with 373.4 ± 59.3 (MIF High ) versus 24.0 ± 8.1 (MIF Low ). **** p < 0.0001 (two-way ANOVA). (D) Significantly higher ratio of MIF High /MIF Low MM cells in BM versus extramedullary samples. **** p < 0.0001 (student’s t -test). (E) MIF High and MIF Low ARD cells in vivo are also identified by IHC. Representative images show their coexistence and more evident loss of MIF expression in spleen than in paired BM (the blue and red boxes indicate representative areas of MIF Low and MIF High MM cells, respectively).

Article Snippet: To establish xenograft models, patient-derived primary MM cells or luciferase-expressing ARD cell line were implanted into six-week-old female immunocompromised B-NDG mice (NOD- Prkdc scid IL2rg tm1 /Bcgen, Biocytogen Jiangsu CO., Ltd.) via tail vein injection.

Techniques: Derivative Assay, Injection, Flow Cytometry, Expressing, In Vivo

Journal: The Journal of Biological Chemistry

Article Title: Endorepellin, the Angiostatic Module of Perlecan, Interacts with Both the ?2?1 Integrin and Vascular Endothelial Growth Factor Receptor 2 (VEGFR2)

doi: 10.1074/jbc.M111.243626

Figure Lengend Snippet: Effects of endorepellin on PAE cells and their transgenic counterparts expressing either VEGFR1 or VEGFR2. A, fluorescent images of PAE cells following staining with rhodamine phalloidin to visualize the actin cytoskeleton (red) and DAPI to visualize the nuclei (blue). Note that only PAE-VEGFR2 cells respond to endorepellin (100 nm, 30 min as seen through actin cytoskeleton disassembly (white arrows)) and this effect could be blocked by 1 μm Na3VO4. The experiments were repeated three times with similar results. Bar, 10 μm. B, representative immunoblots with antibodies against the α2 integrin subunit of total lysates from various PAE cells following treatment with endorepellin (100 nm) at various time points. Note that the α2 integrin subunit is down-regulated only in the PAE-VEGFR2 cells at 30 min with further decreases at 40 min. C, endorepellin induces down-regulation of VEGFR2 in a dose-dependent manner. PAE-VEGFR2 cells were treated with endorepellin (0–200 nm) for 10 min. D, in-cell binding assays using IR800-labeled endorepellin (50 nm) in the presence or absence of VEGFA (500 nm) on PAE, PAE-VEGFR1 (PAE-R1), or PAE-VEGFR2 (PAE-R2). The bound endorepellin fluorescence (800 nm) was normalized on far-red dye DRAQ5 (700 nm), which binds DNA. E, in-cell binding using a 10-min preincubation with a neutralizing mouse monoclonal antibody directed toward the ectodomain of VEGFR2 (10 μg/ml) followed by a 1-h incubation with IR800-endorepellin. In C–E, values represent the mean ± S.E. of three experiments run in triplicate. *, p < 0.05; **, p < 0.01.

Article Snippet: The cells were then washed again and incubated with the far-red fluorescent DNA dye DRAQ5 TM (1:10,000) from Biostatus Limited (Leicestershire, UK) in 0.1% BSA/PBS.

Techniques: Transgenic Assay, Expressing, Staining, Western Blot, Binding Assay, Labeling, Fluorescence, Incubation