Journal: Oncotarget

Article Title: Combining BRAF inhibitor and anti PD-L1 antibody dramatically improves tumor regression and anti tumor immunity in an immunocompetent murine model of anaplastic thyroid cancer

doi: 10.18632/oncotarget.7839

Figure Lengend Snippet: The graph is representative of 3 different experiments using technical triplicate and is plotted as fold change normalized to the expression of A375. PD-L1 mRNA expression of BRAF V600E (8505c, BCPAP, SW1736) thyroid cell lines showed higher base line expression compared to BRAF WT cell lines (TPC-1, HTh-7 and the normal thyroid cells HTORi) (Mann-Whitney U, P≤0.05), thyroid cells as a group showed higher base line expression compared to 4 melanoma cell lines (A375, A2085, MEL-Juso and UACC-903) (Mann-Whitney U, P≤0.05). Western blot (B) analysis showed protein levels corresponded to mRNA levels in thyroid cells.

Article Snippet: PD-L1 gene expression was measured using TaqMan Gene Expression Assays (Invitogen, Primer i.d Hs-01125301_m1, Mm00452054_m1) by real-time reverse transcriptase polymerase chain reaction (RT-PCR) with technical triplicates and repeated at least twice.

Techniques: Expressing, MANN-WHITNEY, Western Blot

Journal: Oncotarget

Article Title: Combining BRAF inhibitor and anti PD-L1 antibody dramatically improves tumor regression and anti tumor immunity in an immunocompetent murine model of anaplastic thyroid cancer

doi: 10.18632/oncotarget.7839

Figure Lengend Snippet: Patients samples carrying BRAF V600E (n=16) showed higher induction of PD-L1 compared to BRAF WT tumors (n=12) (medians: 0.51 vs. −0.70, P = 0.015).

Article Snippet: PD-L1 gene expression was measured using TaqMan Gene Expression Assays (Invitogen, Primer i.d Hs-01125301_m1, Mm00452054_m1) by real-time reverse transcriptase polymerase chain reaction (RT-PCR) with technical triplicates and repeated at least twice.

Techniques:

Journal: Oncotarget

Article Title: Combining BRAF inhibitor and anti PD-L1 antibody dramatically improves tumor regression and anti tumor immunity in an immunocompetent murine model of anaplastic thyroid cancer

doi: 10.18632/oncotarget.7839

Figure Lengend Snippet: IFNγ. alone B. PLX4720 or PD03250901 alone or in combination with IFNγ C. Quantitative PCR fold change of PD-L1 mRNA was normalized to control (A, B) or to IFNγ treatment state (C). D. Western blot analysis for PD-L1, total ERK, phospho ERK and β-actin were done for all treatment combination mentioned above (* indicate p<0.01, ** indicate p<0.001, ns- not significant).

Article Snippet: PD-L1 gene expression was measured using TaqMan Gene Expression Assays (Invitogen, Primer i.d Hs-01125301_m1, Mm00452054_m1) by real-time reverse transcriptase polymerase chain reaction (RT-PCR) with technical triplicates and repeated at least twice.

Techniques: Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: Oncotarget

Article Title: Combining BRAF inhibitor and anti PD-L1 antibody dramatically improves tumor regression and anti tumor immunity in an immunocompetent murine model of anaplastic thyroid cancer

doi: 10.18632/oncotarget.7839

Figure Lengend Snippet: Schedule for the experiment using Orthotropic SCID mouse model for 2 human thyroid cancer cell lines (8505c; ATC derived, and BCPAP; PTC derived cells) is outlined in A. In vivo results of PLX4720 treatment for both cell lines showing obvious tumor volume reduction B. PD-L1 mRNA expression of tumors pooled for each group showing significant reduction of PD-L1 (2 and 37 fold for 8505c and BCPAP respectively) when treated with PXL4720. C. IHC analysis for human PD-L1 showed a marked reduction of staining with PLX4720 treatment for both cell lines D.

Article Snippet: PD-L1 gene expression was measured using TaqMan Gene Expression Assays (Invitogen, Primer i.d Hs-01125301_m1, Mm00452054_m1) by real-time reverse transcriptase polymerase chain reaction (RT-PCR) with technical triplicates and repeated at least twice.

Techniques: Derivative Assay, In Vivo, Expressing, Staining

Journal: Oncotarget

Article Title: Combining BRAF inhibitor and anti PD-L1 antibody dramatically improves tumor regression and anti tumor immunity in an immunocompetent murine model of anaplastic thyroid cancer

doi: 10.18632/oncotarget.7839

Figure Lengend Snippet: PD-L1 mRNA expression was measured against GAPDH expression and compared to non treated cells. While IFNγ robustly increased PD-L1 mRNA expression in all cell lines, none of the four cell lines exhibited a significant change in PD-L1 expression (<2 fold change) when treated with PLX4720 (compared to control) or with the combination of IFNγ + PLX4720 (compared to IFNγ alone) A. Cell surface analysis of PD-L1 using FACS showed a saturation of PD-L1 + cells while PLX4720 treatment did not differ from DMSO alone. The same result was seen for all four murine cell lines. A representative of one cell line (3743) is shown B.

Article Snippet: PD-L1 gene expression was measured using TaqMan Gene Expression Assays (Invitogen, Primer i.d Hs-01125301_m1, Mm00452054_m1) by real-time reverse transcriptase polymerase chain reaction (RT-PCR) with technical triplicates and repeated at least twice.

Techniques: Expressing, Control

Journal: Oncotarget

Article Title: Combining BRAF inhibitor and anti PD-L1 antibody dramatically improves tumor regression and anti tumor immunity in an immunocompetent murine model of anaplastic thyroid cancer

doi: 10.18632/oncotarget.7839

Figure Lengend Snippet: A. TBP: T POCreER; B raf tm1Mmcm/WT; Tr p 53 tm1Brn/tm1Brn (TBP). IP: intraperitoneal. Tumor volume represents in cubic millimeters as mean±SD. The combination of PLX4720 and Anti PD-L1 monoclonal antibody had a dramatic effect on tumor volume reduction compared to control and to either treatment alone (147±61g, P<0.05) B, C. The presence of CD8 + -CTL, the cytotoxicity marker; Granzyme B, Treg marker; FoxP3, T cell exhaustion marker; TIM3 and the expression of mouse PD-L1 and mouse PD-1 were analyzed using IHC. CD8 + -CTL infiltration and cytoxicity were evident with anti PD-L1 treatment alone and were intensively stained when combined with PLX4720. The exhaustion markers TIM3 and PD-1 were evident with anti PD-L1 treatment but were highly induced when combining with PLX4720 D.

Article Snippet: PD-L1 gene expression was measured using TaqMan Gene Expression Assays (Invitogen, Primer i.d Hs-01125301_m1, Mm00452054_m1) by real-time reverse transcriptase polymerase chain reaction (RT-PCR) with technical triplicates and repeated at least twice.

Techniques: Control, Marker, Expressing, Staining

Journal: Oncotarget

Article Title: Combining BRAF inhibitor and anti PD-L1 antibody dramatically improves tumor regression and anti tumor immunity in an immunocompetent murine model of anaplastic thyroid cancer

doi: 10.18632/oncotarget.7839

Figure Lengend Snippet: A. In the naïve environment the lack of migration of T-cells into the tumor site and the mild reactivity that drives T-cells to produce cytokine (among them IFNγ) has substantial impact on the expression of PD-L1 which tempers the already weak anti tumor reaction. B. BRAFi treatment results in increased T cell infiltration into the tumor microenvironment. Although BRAFi potentially down-regulates the expression of PD-L1 on BRAF V600E tumor cells by down regulating MAP kinase activity in these cells, it paradoxically up-regulates MAP kinase in the BRAF WT T-cells which results in IFNγ production and intensive up-regulation of PD-L1 on tumor cells. Immune response with BRAFi alone therefore does not come to its full potential. C. Adding anti PD-L1 antibody is an additional step necessary to allow the immune response facilitated by BRAFi to reach its full potential.

Article Snippet: PD-L1 gene expression was measured using TaqMan Gene Expression Assays (Invitogen, Primer i.d Hs-01125301_m1, Mm00452054_m1) by real-time reverse transcriptase polymerase chain reaction (RT-PCR) with technical triplicates and repeated at least twice.

Techniques: Migration, Expressing, Activity Assay