Journal: Oncoimmunology
Article Title: Selective BRAF inhibition decreases tumor-resident lymphocyte frequencies in a mouse model of human melanoma
doi: 10.4161/onci.20226
Figure Lengend Snippet: Figure 2. PLX4720 treatment leads to a decreased frequency of immune cells in BRAF V600E /PTEN −/− melanomas and this cannot be restored by CTLA-4 blockade. (A) Tumor-bearing Tyr::CreER T2 PTEN F−/− BRAF F-V600E/+ mice were mock or PLX4720 treated for 2, 7, 14 or 21 d. Tumors were removed directly following euthanasia and single cell suspensions were analyzed by use of flow cytometry. Dead cells were removed from all analyses (except for intracellular stainings) by discarding propidium iodide positive cells. Leukocytes were defined as being CD45 + cells. CD4 + , CD8 + and regulatory T cells were respectively defined as the CD4 + CD8 - , CD4 - CD8 + or the CD4 + CD25 + FoxP3 + population. B cells were characterized by their expression of B220 and CD19 while NK-cells were distinguished by the expression of NK1.1 in the absence of CD4 or CD8 expression. Myeoloid derived suppressor cells (MDSCs) and macrophages were respectively defined as the CD11b + GR1 + or CD11b + F4/80 + cell population. The shown values represent the frequency of the assessed cell population as a percentage of all living cells in the single cell suspension of the tumor for individually analyzed mice. As the T reg population was distinguished by use of an intracellular stain the values in this plot are shown as a percentage of all cells in the tumor suspension. (B) A tumor from a Tyr::CreER T2 PTEN F−/− BRAF F-V600E/+ mouse was placed on PLX4720 treatment and tumor appearance was followed photographically over time. Depicted is the tumor phenotype at start, day 5, day 14 and day 35 of PLX4720 treatment (C) Tyr::CreER T2 PTEN F−/− BRAF F-V600E/+ mice received a mock-treatment, PLX4720-treatment, anti-CTLA-4 mAb treatment (twice weekly for 6 weeks) or the combination of PLX4720 and CTLA-4 blockade treatment. The frequency of immune cells as a percentage of living cells in the tumor was assessed by flow cytometry as described for panel A.
Article Snippet: Dead cells were excluded from the analysis by addition of propidium iodide (eBioscience, 00–6990–50) during the sample acquisition.
Techniques: Flow Cytometry, Expressing, Derivative Assay, Suspension, Staining
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