Journal: Cancer immunology research

Article Title: Stromal Fibroblasts Mediate Anti–PD-1 Resistance via MMP-9 and Dictate TGFβ Inhibitor Sequencing in Melanoma

doi: 10.1158/2326-6066.CIR-18-0086

Figure Lengend Snippet: A. Mice treated with either anti–PD-1(250 μg i.p.) or IgG isotype control (250 μg i.p.) every 3 days±TEW-7197 (25 mg/kg po daily) when tumors reach 60–80 mm3. Tumor volumes monitored every 3 days. 6 mice/group. Representative of 2 independent experiments. B. Mice treated with either anti–PD-L1(200 μg i.p.) or IgG isotype control (200 μg i.p.) every 3 days ±TEW-7197(25 mg/kg p.o. daily). Tumor volumes monitored every 3 days. 6 mice/group. Representative of 2 independent experiments. C. Left: autochthonous and syngeneic transplant BrafV600EPten–/– melanoma tissues were analyzed with CD8 immunofluorescence (10x) and PD-L1 IHC (20x). Representative of 3 tumors. Right top: flow cytometry of infiltrating CD45+CD3+CD8+ T cells in both autochthonous and syngeneic BrafV600EPten–/– melanoma tissues. Gated on viable CD45+ cells. Right bottom: CD3+CD8+ T cells (% of CD45+ cells). D. Syngeneic BrafV600EPten–/– melanoma model treated with either anti–PD-1 (250 μg i.p.), anti–CTLA-4(100 μg i.p.), or IgG isotype control (250 μg i.p.) every 3 days±TEW-7197 (25 mg/kg p.o. daily). Tumor volumes were monitored every 3 days. 7 mice/group. Representative of 2 independent experiments. E. Flow cytometry for TILs at the conclusion of the experiment in D. F. Mice from the autochthonous BrafV600EPten–/– melanoma model were treated with TEW-7197 (25 mg/kg po daily x 2 weeks). Primary melanoma tissues were resected, scored (Top), and analyzed for PD-L1 expression by IHC (red, Bottom). Representative of 5 tumors (40x). IHC scores were calculated for 10 random fields in 5 tumors/group. G. Autochthonous BrafV600EPten–/– melanoma model was treated with TEW-7197 (25 mg/kg p.o. daily x 2 weeks). Primary melanoma tissues were resected and flow cytometry performed to quantitate PD-L1 surface expression on CD45–EpCAM–CD90.2– cells. 3 tumors/group. Representative of 2 independent experiments. See Supplementary Fig. S2. All data is mean±SEM. Significance calculated using an unpaired t-test or a one-way ANOVA. *p<0.05, **p<0.005.

Article Snippet: After five passages, cells were trypsinized and enriched by CD90.2 microbeads (Stemcell Technologies, cat. number 18951) according to the manufacture’s protocol.

Techniques: Immunofluorescence, Flow Cytometry, Expressing

Journal: Cancer immunology research

Article Title: Stromal Fibroblasts Mediate Anti–PD-1 Resistance via MMP-9 and Dictate TGFβ Inhibitor Sequencing in Melanoma

doi: 10.1158/2326-6066.CIR-18-0086

Figure Lengend Snippet: Autochthonous BrafV600EPten–/– melanoma model treated with TEW-7197 (25 mg/kg po daily x 2 weeks), and tumors resected for A. trichrome staining (blue: connective tissue), B. Vimentin IHC, and C. α-SMA IHC. All histology are representative of 3 tumors/group and ≥6 sections/tumor. D. Autochthonous BrafV600EPten–/– melanoma model was treated with TEW-7197 (25 mg/kg p.o. daily x 2 weeks) versus a vehicle control, resected, and single-cell suspensions were generated. Flow cytometry used to quantitate CD45–EpCAM–CD90.2+ melanoma-associated fibroblasts (MAFs). 3 tumors/group. Representative of 2 independent experiments. E. Left: in vitro transwell migration assay. The BrafV600EPten–/– melanoma cell line treated with TEW-7197 and BrafV600EPten–/–/MAF chemotaxis measured. 5 wells/condition. Representative of 2 independent experiments. Right: in vitro proliferation assay. BrafV600EPten–/–/MAFs treated with increasing concentrations of TEW-7197 and cellular proliferation was monitored by MTT assay. 3 wells/condition. Representative of 3 independent experiments. F. Schematic of the in vivo MAF proliferation assay. 4-HT: 4-hydroxytamoxifen, EdU: 5-ethynyl-2’-deoxyuridine DNA incorporation dye. G. Quantitation of EdU-FAM+CD45–EpCAM–CD90.2+ proliferating MAFs in vehicle control and TEW-7197-treated autochthonous BrafV600EPten–/– melanomas. 4 mice/group. Representative of 2 independent experiments. See Supplementary Fig. S3 and S4. All data is mean±SEM. Significance calculated using an unpaired t-test or a one-way ANOVA. *p<0.05, **p<0.005. ns: non-significant.

Article Snippet: After five passages, cells were trypsinized and enriched by CD90.2 microbeads (Stemcell Technologies, cat. number 18951) according to the manufacture’s protocol.

Techniques: Staining, Generated, Flow Cytometry, In Vitro, Transwell Migration Assay, Chemotaxis Assay, Proliferation Assay, MTT Assay, In Vivo, Quantitation Assay

Journal: Cancer immunology research

Article Title: Stromal Fibroblasts Mediate Anti–PD-1 Resistance via MMP-9 and Dictate TGFβ Inhibitor Sequencing in Melanoma

doi: 10.1158/2326-6066.CIR-18-0086

Figure Lengend Snippet: A.Top: delayed dosing scheme of anti–PD-1 and TEW-7197. Bottom: tumor measurements before and after TEW-7197 initiation following previous anti–PD-1 therapy. 6 mice/group. B. Final tumor volume measurements from A. C. Flow cytometry for TILs performed at the conclusion of the experiment in A. D. IFNγ ELISPOT analysis of TRP2-specific T cells performed in A. Splenocytes harvested from 5 mice/group. Representative of 2 independent experiments. E. Quantitation of EdU-FAM+CD45–EpCAM–CD90.2+ proliferating MAFs in autochthonous BrafV600EPten–/– melanomas following the indicated treatments as performed in A. 3 tumors/group. Representative of 2 independent experiments. F. Tumor tissues resected for trichrome staining and G. α-SMA IHC. All histology representative of 3 tumors/group and at least 6 sections/tumor. H. Mice treated as indicated by the experiment described in A. Primary melanoma tissues resected at the same time points as in A-D. Flow cytometry utilized to quantitate PD-L1 surface expression on CD45–EpCAM–CD90.2– cells. 3 tumors/group. See Supplementary Fig. S7. All data is mean±SEM. Significance calculated using a one-way ANOVA. *p<0.05.

Article Snippet: After five passages, cells were trypsinized and enriched by CD90.2 microbeads (Stemcell Technologies, cat. number 18951) according to the manufacture’s protocol.

Techniques: Flow Cytometry, Enzyme-linked Immunospot, Quantitation Assay, Staining, Expressing