Journal: JCI Insight

Article Title: Increased fatty acid delivery by tumor endothelium promotes metastatic outgrowth

doi: 10.1172/jci.insight.187531

Figure Lengend Snippet: ( A ) Schematic of transendothelial transport assay. ( B ) Representative images of BODIPY-C16 (green) in LLC tumor cells after 1 hour. VEGFR1-Fc was used in control samples to bind tumor cell–derived VEGF-B. Endothelial cells act as a physical barrier for BODIPY-C16 access, demonstrated in a representative image from an endothelial cell–free control (“No ECs”). A control performed without tumor cells (“No TCs”) is shown to confirm removal of endothelial cells prior to fluorescence imaging of basolateral tumor cells. Scale bar: 100 μm. ( C ) BODIPY-C16 fluorescence in LLC tumor cells, normalized to WT plus VEGFR1-Fc control ( n = 3 per group). ( D ) Representative confocal images of WT or Rptor -KO endothelial cells treated with BODIPY-C16 for 5 minutes. Nuclear (Hoechst, blue) and actin (phalloidin, red) staining was used to detect perinuclear and cell boundaries, respectively. Total (all z -planes) and basolateral (bottom 10% of z -planes) BODIPY-C16 (green) staining are also shown, with the basolateral BODIPY presented at higher exposure (HE). Scale bar: 20 μm. BODIPY-C16 intensities at the basolateral surface were normalized to total signal in each cell. WT ( n = 16) and Rptor -KO ( n = 10) cells were analyzed from 2 independent experiments. ( E and F ) WT ( n = 4) or Rptor ECKO ( n = 5) mice were inoculated with LLC tumor cells as described in A. One hour prior to tumor harvest, animals were injected with BODIPY-C16 (50 μg). Median fluorescence intensity (MFI) was determined by flow cytometry in ( E ) CD45 – CD31 + endothelial cells and ( F ) CD45 – EpCAM + FSC hi tumor cell–enriched populations and normalized to littermate WT controls. * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001 by 2-way ANOVA with Tukey’s post hoc test ( B ) or unpaired, 2-tailed Student’s t test ( D – F ).

Article Snippet: For VEGFR1 neutralization in vivo, animals were treated with 2.5 mg/kg of normal rabbit IgG (R&D Systems, AB-108-C) or αVEGFR1 (R&D Systems, AF471) every 3–4 days by i.p. injection.

Techniques: Transport Assay, Control, Derivative Assay, Fluorescence, Imaging, Staining, Injection, Flow Cytometry

Journal: Journal of Immunotherapy

Article Title: TIMP-2 Targets Tumor-associated Myeloid Suppressor Cells With Effects in Cancer Immune Dysfunction and Angiogenesis

doi: 10.1097/cji.0b013e3182619c8e

Figure Lengend Snippet: FIGURE 1. Enhanced angiogenesis and accelerated tumor growth in timp2/ mice. A, Growth rate of Lewis lung carcinoma in timp2/ mice versus tumors in wild-type (WT) mice (mean ± SEM, n = 10 in each group, *P = 0.007, 14 day, ** P = 0.003, 16 day, ***P = 0.002, 18 day, ****P = 0.005, 20 day postinjection). B, Vascular endothelial growth factor-A (VEGF-A) serum levels in naive, tumor-free WT versus timp2/ mice (n = 5 in each group, mean ± SD *P = not significant), tumor-bearing WT versus timp2/ mice (n = 10 in each group, mean ± SD ****P = 0.0004), naive WT mice versus tumor-bearing WT (n = 10 in each group, mean ± SD, **P = 0.004), naive timp2/ mice versus tumor bearing timp2/ (n = 10, mean ± SD, ***P = 0.0001). C, Infrared molecular imaging of avb3 in Lewis lung carcinoma-2 tumor-bearing WT and timp2/ mice. D, Accumulated avb3 at the tumor site, infrared signal in pixels/tumor area of duplicate experiment (n = 4 in each group, mean ± SD). E, Colocalization of infrared avb3 with FITC-CD31 in blood vessels in tumor sections from timp2/ mice versus tumor in WT mice. Scale bar = 50 mm.

Article Snippet: Frozen tumor sections were cut 10–12mm in a cryostat (Leica Microsystems, Germany), fixed in 4% paraformaldehyde for 30 minutes and stained at room temperature for 2 hours with the following antibodies: Alexa488-CD31, PE-CD11b, APC-F4/80 (Biolegend), APC-VEGF-R1 (R&D Systems, MN) and counter-stained with mounting media containing DAPI (InVitrogen).

Techniques: Imaging

Journal: Journal of Immunotherapy

Article Title: TIMP-2 Targets Tumor-associated Myeloid Suppressor Cells With Effects in Cancer Immune Dysfunction and Angiogenesis

doi: 10.1097/cji.0b013e3182619c8e

Figure Lengend Snippet: FIGURE 5. Increased vascular endothelial growth factor (VEGF)-R1 + myeloid-derived suppressor cells (MDSCs) and Annexin A1 in tumor-bearing timp2/mice. A, A representative fluorescence activated cell sorting analysis of VEGF-R1 expression by CD11b + Gr-1 + (MDSC, gate R4) splenocytes from tumor-bearing wild-type (WT) and timp2/ mice. B, Increased percentage of VEGF-R1 + MDSCs in spleens from tumor-bearing timp2/ mice versus tumor-bearing WT mice (n = 6 spleens in each group, means ± SD). C, Immune staining of VEGF-R1 + Gr-1 + inflammatory cells in timp2/ tumor sections versus WT tumors. Scale bar = 20 mm. D, Number of Gr-1 + VEGF-R1 + cells per hpf (40) in timp2/ tumors versus WT tumors (4 tumors per group, n = 16 hpf per tumor, mean ± SD). E, Expression of Annexin A in spleens from tumor-free WT versus timp2/ mice over Tubulin-a protein loading control, integrated density values are shown later. F, Expression of Annexin A1 (37 kDa) and bioactive peptides in spleens (S) and tumors (T) from tumor- bearing WT mice versus timp2/ mice over loading control Tubulin-a, integrated density values are shown.

Article Snippet: Frozen tumor sections were cut 10–12mm in a cryostat (Leica Microsystems, Germany), fixed in 4% paraformaldehyde for 30 minutes and stained at room temperature for 2 hours with the following antibodies: Alexa488-CD31, PE-CD11b, APC-F4/80 (Biolegend), APC-VEGF-R1 (R&D Systems, MN) and counter-stained with mounting media containing DAPI (InVitrogen).

Techniques: Derivative Assay, Fluorescence, FACS, Expressing, Staining, Control

Journal: Journal of Immunotherapy

Article Title: TIMP-2 Targets Tumor-associated Myeloid Suppressor Cells With Effects in Cancer Immune Dysfunction and Angiogenesis

doi: 10.1097/cji.0b013e3182619c8e

Figure Lengend Snippet: FIGURE 6. TIMP-2 upregulation decreases tumor myeloid-derived suppressor cells (MDSCs) and angiogenesis. A, Fluorescence mo- lecular tomography (FMT) showing decreased angiogenesis indicators Angiosense (green) and Integrinsense (yellow) in A549TIMP-2 tumor versus Control. B, Angiogenesis indicators by FMT in A549TIMP-2 xenograft versus Control (n = 5 in each group, mean ± SD **P = 0.002). C, Gross and hematoxylin and eosin staining showed low angiogenesis and tumor necrosis (*) in A549TIMP-2 tumor versus A549Control angiogenic blood vessels (arrows), scale bar = 50 mm. D, A549TIMP-2 xenograft showing low Angiosense (red) and blood vessels FITC-CD31 + versus Control xenograft, nuclei counter-stained with DAPI, scale bar = 50 mm. E, TIMP-2 in myeloid CD11b + cells, tumor cells (arrows) and stroma, scale bar = 20 mm. F, MDSCs in A549TIMP-2 xenograft versus Control xenograft, scale bar = 50 mm. G, Number of recruited MDSCs per hpf (40) in tumor sections (n = 12 in each group, mean ± SD, **P < 0.0001). H, Vascular endothelial growth factor (VEGF)-R1 + Gr-1 + cells recruited by tumors, scale bar = 20 mm. I, Number of VEGF-R1 + Gr-1 + cells per hpf (60) in A549TIMP-2 versus A549Control (n = 24 in each group, mean ± SD, **P < 0.004.

Article Snippet: Frozen tumor sections were cut 10–12mm in a cryostat (Leica Microsystems, Germany), fixed in 4% paraformaldehyde for 30 minutes and stained at room temperature for 2 hours with the following antibodies: Alexa488-CD31, PE-CD11b, APC-F4/80 (Biolegend), APC-VEGF-R1 (R&D Systems, MN) and counter-stained with mounting media containing DAPI (InVitrogen).

Techniques: Derivative Assay, Fluorescence, Tomography, Control, Staining