| Overall design |
Orthotopic MMTV-PyMT mammary tumors were generated as follows. Late stage, primary MMTV-PyMT tumor-derived cells were obtained from 14- to 15-week old female MMTV-PyMT mice, following tumor digestion with collagenase IV (0.2 mg/ml, Worthington), dispase (2 mg/ml, Lifetechnologies), and DNaseI (0.1 mg/ml, Lifetechnologies) treatment in IMDM medium for 30 min at 37°C. The cell suspensions were filtered using a cell strainer (70 μm) and washed in PBS containing 2 mM EDTA and 2% FBS. Tumor-derived cells were then cultured in 15-cm petri dishes for 48 h in IMDM supplemented with 10% FBS, 2 mM L-glutamine (Amimed) and a combination of 50 units/ml penicillin and 50 μg/ml streptomycin (Gibco). Non-adherent/dead cells and debris were then washed away, and 2x106 tumor-derived cells (in PBS) were orthotopically injected in the fourth mammary fat pad of 8-10–week-old female FVB/n mice. When tumors reached a size of 300 mm3, mice were injected i.p with the following monoclonal antibodies:
1) mouse IgG1 (clone MOPC-21, 20 mg/kg) + rat IgG2a (clone 2A3, 4 mg/kg), designated as “IgG”; or
2) anti-VEGFA/ANGPT2 (A2V, 20 mg/kg) + rat IgG2a (clone 2A3, 4 mg/kg), designated as “A2V”; or
3) anti-CD40 (clone FGK45, 4 mg/kg) + mouse IgG1 (MOPC21, 20 mg/kg), designated as “anti-CD40”; or
4) anti-VEGFA/ANGPT2 (A2V, 20 mg/kg) + anti-CD40 (clone FGK45, 4 mg/kg), designated as “A2V + anti-CD40”.
Five days later, mice were euthanized and tumors harvested and prepared for fluorescence-activated cell sorting (FACS). Cells were stained with CD45, CD8, CD4, CD31, CD11b, F480 and 7-AAD. After live-dead and single-cell discrimination, cell populations were discriminated according to the following markers:
Endothelial cells (ECs): CD45–CD31+;
Tumor associated macrophages (TAMs): CD45+CD11b+F480+.
CD8+ T cells: CD45+CD11b–CD8+;
CD4+ T cells: CD45+CD11b–CD4+.
Cell sorting was performed using FACS Aria II apparatus (BD). Each cell population (n=4) was isolated from an independent tumor. Typically, 2x104 to 2x105 cells were obtained for each cell population.
Citation(s):
Schmittnaegel M, et al. Dual angiopoietin-2 and VEGFA inhibition elicits antitumor immunity that is enhanced by PD-1 checkpoint blockade. Sci Transl Med 2017 Apr 12;9(385). PMID: 28404865
Kashyap AS, et al., Optimized antiangiogenic reprogramming of the tumor microenvironment potentiates CD40 immunotherapy. in press
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