Journal: Oncoimmunology

Article Title: A novel MDSC-induced PD-1 − PD-L1 + B-cell subset in breast tumor microenvironment possesses immuno-suppressive properties

doi: 10.1080/2162402X.2017.1413520

Figure Lengend Snippet: The concurrent increases of MDSCs and B cells in breast cancer mice and surface molecules of splenic B cells and MDSCs in tumor or normal mice. Wide-type BALB/c mice were injected subcutaneously with 1 × 106 4T1 murine breast cancer cells, and were sacrificed at day 21 after tumor inoculation. (A) The brief descriptions of tumor-bearing status were shown in the left and middle panel. The overall splenic cells were analyzed from tumor-bearing and tumor-free mice (right panel). (B) Paraffin-embedded spleen tissues were collected from tumor-bearing mice to detect CD19+B cells (left panel) and CD11b+ MDSCs (right panel) by IHC staining (original magnification × 200 and × 400). (C) The percentage of B cells was detected using anti-CD19 Ab from tumor and normal splenic cells. (D) Splenic MDSCs were stained for CD11b and Gr-1, and detected by FC (right panel). The percentages are indicated in the representative dot plots (left two panels). (E) Isolated splenic MDSCs were assessed for their ability to suppress CD3+ T-cell proliferation at a 1:1, 3:1 or 5:1 of Gr-1+CD11b+cell: T-cell ratio. (F) FC analysis of immune checkpoint molecules including PD-1, PD-L1, CTLA-4, Tim-3, CD200R and CEACAM showing their expression frequencies in B cells. (G) Expression of the co-stimulatory molecules and activation markers including CD80, CD86, MHC II, CCR6 and CD62L on the surface of splenic CD19+ B cells from 4T1 tumor-bearing and control mice. (H) Immune checkpoint molecules on MDSCs were measured by FC from normal and tumor-bearing mice. The results are shown as the mean±SEM from five independent experiments with 8 mice per group per experiment. The t test or One-way ANOVA analysis were used for statistical analysis.* = P < 0.05, ** = P < 0.01, *** = P < 0.001, ns = not significant. FC, flow cytometry; MDSC, Myeloid-derived suppressor cells; IHC, immunohistochemistry.

Article Snippet: Cell lines 4T1 mammary cells are of BALB/C origin and were acquired from the American Type Culture Collection (ATCC).

Techniques: Injection, Immunohistochemistry, Staining, Isolation, Expressing, Activation Assay, Flow Cytometry, Derivative Assay

Journal: Oncoimmunology

Article Title: A novel MDSC-induced PD-1 − PD-L1 + B-cell subset in breast tumor microenvironment possesses immuno-suppressive properties

doi: 10.1080/2162402X.2017.1413520

Figure Lengend Snippet: A typical PD-1−PD-L1+CD19+ B-cell subtype exert immuno-regulatory properties. (A) The left two panels show a representative dot plots of PD-1−PD-L1+ B cells gated on CD19+ cells from normal or tumor-bearing spleen. The right panel depicted values from 8 independent experiments. (B) Representative FC analysis showing the percentage of PD-1−PD-L1+CD19+ B cells at 48 h in the presence or absence of MDSCs in the culture system (B1). B2 shows summarized data from 5 independent experiments. (C) The percentage of PD-1−PD-L1+CD19+ B cells in BrdU-labeled B cells at 48 h with or without MDSCs from 8 independent experiments. (D) The percentage of CD5+ and CD5+CD1dhi B cells in tumor-bearing or tumor-free mice (D1) and (D2) in the MDSC-co-cultured system was evaluated by FC. (E) The intra-cellular IL-10 level of B cells was analyzed by FC in 4T1 or normal mice (E1) and (E2) in the MDSC-co-cultured group. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, ns = not significant as determined with the t test. MDSC, myeloid-derived suppressor cells; FC, flow cytometry; IL, interleukin.

Article Snippet: Cell lines 4T1 mammary cells are of BALB/C origin and were acquired from the American Type Culture Collection (ATCC).

Techniques: Labeling, Cell Culture, Derivative Assay, Flow Cytometry

Journal: Oncoimmunology

Article Title: A novel MDSC-induced PD-1 − PD-L1 + B-cell subset in breast tumor microenvironment possesses immuno-suppressive properties

doi: 10.1080/2162402X.2017.1413520

Figure Lengend Snippet: The distribution of different B cell subsets in BM, peripheral blood, spleen and TDLNs. (A) PD-1−PD-L1+CD19+ B cells was expanded in the BM, peripheral blood, spleen, LNs and primary tumor tissue in tumor-bearing or normal mice . Representative dot plots of FC in LNs were shown. (B) The percentage of Gr-1+CD11b+MDSCs and representative dot plots in tumor-bearing or tumor-free mice from 5 independent experiments with triplicate samples was shown. (C) Normal splenic B cells were cultured with 4T1 cells or 4T1-supernatant, and the percentage of PD-1−PD-L1+CD19+ B cells was detected by FC. (D) The percentage of CD5+CD19+ B cells (E) CD5+CD1dhi CD19+B cells and (F) intra-cellular staining was with PE-anti-IL-10 antibody to detect IL-10+ B cells determined by FC. Data show mean±SEM of 7 independent experiments. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, ns = not significant as determined by t test. BM, bone marrow; LN, lymph node; TDLN, tumor-draining lymph node; MDSC, Myeloid-derived suppressor cells.

Article Snippet: Cell lines 4T1 mammary cells are of BALB/C origin and were acquired from the American Type Culture Collection (ATCC).

Techniques: Cell Culture, Staining, Derivative Assay

Journal: European journal of nuclear medicine and molecular imaging

Article Title: Targeting angiogenesis for radioimmunotherapy with a 177 Lu-labeled antibody

doi: 10.1007/s00259-017-3793-2

Figure Lengend Snippet: Therapeutic study groups. Mice bearing 4T1 mammary tumors were administered a one-time injection of the given agent.

Article Snippet: Murine breast cancer models 4T1 murine breast cancer cells were purchased from the American Type Culture Collection and maintained in RPMI 1640 medium (Invitrogen) with 10% fetal bovine serum supplementation.

Techniques: Injection

Journal: European journal of nuclear medicine and molecular imaging

Article Title: Targeting angiogenesis for radioimmunotherapy with a 177 Lu-labeled antibody

doi: 10.1007/s00259-017-3793-2

Figure Lengend Snippet: Characterization of labeling conditions and binding properties. (A) DOTA and DTPA chelators were compared for radiolabeling of TRC105 with 177Lu at 0.15 μg of protein per 1 μCi of isotope. DTPA exhibited higher labeling yields at all time points. (B) Varying concentrations of conjugated antibody were used for radiolabeling with DTPA, with similar results observed at all concentrations. A final ratio of 0.15 μg of protein per 1 μCi of isotope was chosen for in vivo studies. (C) Flow cytometry showed no binding of the as-developed tracers to 4T1 tumor cells. (D) TRC105 and DTPA-TRC105 were found to bind at similar levels to HUVECs.

Article Snippet: Murine breast cancer models 4T1 murine breast cancer cells were purchased from the American Type Culture Collection and maintained in RPMI 1640 medium (Invitrogen) with 10% fetal bovine serum supplementation.

Techniques: Labeling, Binding Assay, Radioactivity, In Vivo, Flow Cytometry

Journal: European journal of nuclear medicine and molecular imaging

Article Title: Targeting angiogenesis for radioimmunotherapy with a 177 Lu-labeled antibody

doi: 10.1007/s00259-017-3793-2

Figure Lengend Snippet: (A) Biodistribution studies were performed at 1 and 7 days post-injection of 177Lu-DTPA-TRC105 or 177Lu-DTPA-IgG in 4T1 tumor-bearing mice. Gradual clearance was seen in most organs, with tumor uptake of the targeted tracer remaining high over this time period at approximately 11–14 %ID/g (n = 3). Notable differences in uptake for the nonspecific IgG were observed in the spleen and bone at 7 d p.i., as compared to the targeted tracer. (B) As mice were sacrificed, biodistribution studies were performed in other control groups. These data were collected at an average of 13 days post-injection for the 177Lu only group, and 14 days post-injection for the low dose group (n = 4–6 per group).

Article Snippet: Murine breast cancer models 4T1 murine breast cancer cells were purchased from the American Type Culture Collection and maintained in RPMI 1640 medium (Invitrogen) with 10% fetal bovine serum supplementation.

Techniques: Injection