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

Journal: International Journal of Molecular Sciences

Article Title: Effects of Survival Motor Neuron Protein on Germ Cell Development in Mouse and Human

doi: 10.3390/ijms22020661

Figure Lengend Snippet: Deficiency of SMN in SSC maintenance in mice. ( A ) Graphic flow chart of SSC isolation. Testis cells for SSCs in vitro culture are obtained from male neonatal pups fractionated by magnetic-activated cell sorting (MACS) with magnetic microbeads conjugated to anti-THY1 (CD 90.2) antibody. ( B , C ) Detection of SSC marker PLZF in fractionated SSCs by immunofluorescent staining. 81.75% of PLZF positive cells (red color) is detected in THY1 + cells. DAPI is used for DNA stain (blue color). In contrast, fewer (18.09%) THY1 − cells presented PLZF ( n = 3). Scale bar: 200 μm. t -test analysis is performed and significant differences is shown (*** p < 0.001). ( D ) PLZF expression in SSCs from SMA-like mice after in vitro culture. Bright field (BF) shows the morphology of SSC culture in mouse Smn1 knockout (KO) and heterozygous littermate control (HT) group. The KO SSCs express less PLZF protein (green color) compared with the HT control. DAPI used for nuclei stain is showed as blue color. Scale bar: 100 μm. ( E ) The percentage of expressing PLZF is quantified in SSCs from KO, HT and wild-type (WT) SMA littermate mice. * Indicates a statistically significant difference, p < 0.05. ns indicates no significance.

Article Snippet: After Collagenase IV and Trypsin digestion, testicle cells were incubated with anti-CD90.2 (THY1) antibody and then labeled with anti-biotin microbeads (130-090-485, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Isolation, In Vitro, FACS, Marker, Staining, Expressing, Knock-Out, Control

Journal: Cancer research

Article Title: Armored Chimeric Antigen Receptor-T Cell Therapy Targets Antigen-Heterogeneous Glioma

doi: 10.1158/0008-5472.CAN-26-1515

Figure Lengend Snippet: (A–B) C57BL/6J mice were implanted intracranially with a mixture of 5e4 muIL-13Rα2+ CT-2A cells and 5e4 muIL-13Rα2− CT-2A cells. Six days later, mice were injected intratumorally with 5e5 CAR+ murine T cells per mouse. N = 7 per group. (A) Timeline of anti–TNF-α antibody injection. (B) Tumor growth as monitored by bioluminescence imaging. (C) CD90.2+ C57BL/6J mice were injected with tumor and CD90.1+ T cells as described in Fig. 2A and sacrificed two or nine days post ACT. N = 3 per group. CD90.2+ cells isolated from each brain were co-cultured with IL-13Rα2− CT-2A cells at a 1:1 effector-to-target ratio for 44 hours. Activation was assessed by cytokine release using FluoroSpot plate. The assay contained biological triplicates (i.e., 3 mice) per treatment group, with each biological replicate seeded in technical duplicates (i.e., 2 wells per mouse; 6 wells per treatment group). Each dot in the plot represents 1 mouse, with a value equal to the mean number of spots between technical duplicate wells. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant. (D) C57BL/6J mice were implanted intracranially with a mixture of 5e3 muIL-13Rα2+ SB28-GKO cells and 5e3 muIL-13Rα2− SB28-GKO cells. Six days later, mice were injected intratumorally with 5e5 CAR+ murine T cells per mouse. Tumor growth as monitored by bioluminescence imaging. N = 10 per group.

Article Snippet: Subsequently, red blood cells were lysed using Miltenyi 1x Red Blood Cell Lysis solution, and T cells were enriched with a REAlease ® CD90.2 MicroBead Kit (Miltenyi, Cat# 130-126-706).

Techniques: Injection, Imaging, Isolation, Cell Culture, Activation Assay

Journal: Cell Death & Disease

Article Title: The histone methyltransferase ESET is required for the survival of spermatogonial stem/progenitor cells in mice

doi: 10.1038/cddis.2014.171

Figure Lengend Snippet: MACS and RNAi efficiency and SSC culture in vitro . ( a ) Immunofluorescence of Thy1 (red) and DAPI (blue) in presorted and two-step sorted cells. Bar=80 μ m. ( b ) Proportion of Thy1-positive cells in presorted and two-step sorted fraction ( n =4). More than 350 cells were counted for each sample. Asterisks indicate statistically significant differences ( **P<0.01 ). ( c ) qRT-PCR analysis of Eset expression in the ESET-KD, NC and Mock SSCs group. Values were normalized to that of Gapdh, **P<0.01 compared with NC or Mock controls. ( d ) ESET and global H3K9me3 level were slightly decreased in the ESET-KD SSCs, examined by western blot. ( e ) Relative number of Lin28-positive cells in ESET-KD, NC and Mock SSC groups after 2 weeks culture. Five thousands of SSCs for each treatment were plated in multiple wells of a 96-well plate and cultured for 2 weeks ( n =3). The number of Lin28-positive cells in ESET-KD group was lower than that in NC and Mock group ( **P<0.01 ). ( f – k ) Culture of SSCs after transduced with lentiviral. ESET-KD SSCs ( f and i ), NC group ( g and j ) and Mock group ( h and k ) were analyzed after 2 weeks culture. Immunofluorescence for Lin28 (red) and DAPI (blue) were performed to count the number of SSCs ( i – k ). Bar=80 μ m

Article Snippet: Magnetic microbeads conjugated to anti-Thy1 antibody (30-H12; Miltenyi Biotec, Bergisch Gladbach, Germany) were used for MACS to enrich Thy1-positive cells.

Techniques: In Vitro, Immunofluorescence, Quantitative RT-PCR, Expressing, Western Blot, Cell Culture, Transduction