Journal: Oncotarget

Article Title: The inhibition of malignant melanoma cell invasion of bone by the TLR7 agonist R848 is dependent upon pro-inflammatory cytokines produced by bone marrow macrophages

doi: 10.18632/oncotarget.25711

Figure Lengend Snippet: MTS assays of cultures of B16F10 cells, the mammary tumor cell line MMT060562 (MMT), and the lung carcinoma cell line Ex-3LL (3LL) treated with the indicated doses of IL-6, IL-12, and IFN-γ for one day. The data are representative of more than three independent experiments. * p <0.01.

Article Snippet: The mouse spontaneous mammary tumor (MMT) cell line MMT060562 and the Ex-3LL (3LL) mouse Lewis lung carcinoma cell line was purchased from the JCRB Cell Bank (Osaka, JP).

Techniques:

Journal: Cancer Immunology Research

Article Title: Rescue of Notch-1 Signaling in Antigen-Specific CD8+ T Cells Overcomes Tumor-Induced T-cell Suppression and Enhances Immunotherapy in Cancer

doi: 10.1158/2326-6066.cir-14-0021

Figure Lengend Snippet: Figure 1. Induction of Notch-1 and Notch-2 regulates CD8þ T-cell functions and is inhibited in tumor-infiltrating T cells. A, CD3þ T cells were activated with plate-bound anti-CD3/CD28 (0.5 mg each) in the presence of increasing concentrations of GSI, Z-Ile-Leu-CHO. Proliferation was determined after 72 hours by [3H]-thymidine uptake. Activated T cells cultured with DMSO and nonstimulated T cells (NS) were used as controls. Results represent mean SD from three similar independent experiments. , P < 0.001. B, CFSE-labeled CD4þ or CD8þ T cells were activated as shown in A with 30 mmol/L GSI, and proliferation was determined 72 hours later by flow cytometry. Histograms are a representative result from three experiments. C, Notch isoform mRNAs were measured in T cells activated for 48 hours. Results represent mean SD from two experiments. , P < 0.001. D, CD3þ, CD4þ, or CD8þ T cells were activated with anti-CD3/CD28, and whole-cell extracts were harvested after 48 and 72 hours. Western blot analyses are representative results of four repeats. E andF, CFSE- labeled CD3þ T cells from floxed and conditional-null Notch-1 and Notch-2 mice were activated as shown in A and monitored for cell proliferation by CFSE. Supernatants were harvested and IFNg levels were measured by ELISA. Results represent mean SD from three independent experiments. , P < 0.001. G, T lymphocytes were isolated from tumors and spleen of mice bearing s.c. 3LL tumors for 17 days or spleens from mice without tumors. T cells were activated with anti-CD3/CD28 for 24 hours and tested for Notch-1 and Notch-2 mRNA by real-time PCR. Results represent mean SD from four different animals, tested in triplicates. , P < 0.001. TBM, tumor-bearing mice.

Article Snippet: Lewis lung carcinoma (3LL) and EL-4 thymoma cells were obtained from the American Type Culture Collection and maintained in RPMI-1640 (Lonza-BioWhittaker) supplemented with 10% fetal calf serum (Hyclone), 25 mmol/L HEPES (Invitrogen, Life Technologies), 4 mmol/L L-glutamine (Invitrogen, Life Technologies), and 100 U/mL of penicillin, streptomycin (Invitrogen, Life Technologies).

Techniques: Cell Culture, Labeling, Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, Isolation, Real-time Polymerase Chain Reaction

Journal: Cancer Immunology Research

Article Title: Rescue of Notch-1 Signaling in Antigen-Specific CD8+ T Cells Overcomes Tumor-Induced T-cell Suppression and Enhances Immunotherapy in Cancer

doi: 10.1158/2326-6066.cir-14-0021

Figure Lengend Snippet: Figure 5. Transgenic N1IC in activated antigen-specific CD8þ T cells block tumor growth. A, 106 3LL or 3LL-OVA cells were s.c. injected in N1IC or N1ICf/f mice. Tumor volumes were measured using calipers, as described in Materials and Methods. Results represent mean SD from two independent experiments (N1ICf/f n ¼ 7; N1ICf/f n ¼ 7). ns, nonstatistical significance, P < 0.001; , P < 0.001. B, CD45.1þ mice were injected s.c. with 3LL-OVA for 7 days, after which they were adoptively transferredwith naïve CD8þ T cells from N1IC or N1ICf/f mice (CD45.2þ), and immunizedwith siinfekl. Results represent mean SD from three independent experiments. N1ICf/f n ¼ 22; N1ICf/f n ¼ 22. , P < 0.001; , P < 0.01. C, lymph nodes collected 10 days after immunization from B were challenged with siinfekl and the production of IFNg was measured using ELISpot. Results represent mean SD from three independent experiments. , P < 0.01.

Article Snippet: Lewis lung carcinoma (3LL) and EL-4 thymoma cells were obtained from the American Type Culture Collection and maintained in RPMI-1640 (Lonza-BioWhittaker) supplemented with 10% fetal calf serum (Hyclone), 25 mmol/L HEPES (Invitrogen, Life Technologies), 4 mmol/L L-glutamine (Invitrogen, Life Technologies), and 100 U/mL of penicillin, streptomycin (Invitrogen, Life Technologies).

Techniques: Transgenic Assay, Blocking Assay, Injection, Enzyme-linked Immunospot

Journal: Cancer Immunology Research

Article Title: Rescue of Notch-1 Signaling in Antigen-Specific CD8+ T Cells Overcomes Tumor-Induced T-cell Suppression and Enhances Immunotherapy in Cancer

doi: 10.1158/2326-6066.cir-14-0021

Figure Lengend Snippet: Figure 6. Expression of N1IC in antigen-specific T cells enhances the efficacy of T cell–based immunotherapy. A, 5 106 CD8þ T cells N1IC or N1ICf/f preactivated in vitro for 48 hours were adoptively transferred into mice bearing 3LL-OVA tumors for 7 days. Tumor volume was monitored, as described in Materials and Methods. Results represent mean SD from two similar experiments. N1ICf/f n ¼ 8; N1ICf/f n ¼ 8. , P < 0.001. B and C, single-cell suspensions from tumors represented in A were collected and monitored for the percentage of CD45.2þ CD8þ T cells (B) and CD44 and CD62L in CD45.2þ cells by flow cytometry. C, results represent mean SD from three independent experiments. N1ICf/f n ¼ 6; N1ICf/f n ¼ 6. , P < 0.001. D and E, spleens (D) and lymph nodes (E) were harvested 10 days after T-cell transfer and challenged with siinfekl for 24 hours, after which they were tested for CD107a (D) and production of IFNg (E) by flow cytometry and ELISpot, respectively. Results represent mean SD from two similar independent experiments. , P < 0.001.

Article Snippet: Lewis lung carcinoma (3LL) and EL-4 thymoma cells were obtained from the American Type Culture Collection and maintained in RPMI-1640 (Lonza-BioWhittaker) supplemented with 10% fetal calf serum (Hyclone), 25 mmol/L HEPES (Invitrogen, Life Technologies), 4 mmol/L L-glutamine (Invitrogen, Life Technologies), and 100 U/mL of penicillin, streptomycin (Invitrogen, Life Technologies).

Techniques: Expressing, In Vitro, Cytometry, Enzyme-linked Immunospot

Journal: Cancer Immunology Research

Article Title: Rescue of Notch-1 Signaling in Antigen-Specific CD8+ T Cells Overcomes Tumor-Induced T-cell Suppression and Enhances Immunotherapy in Cancer

doi: 10.1158/2326-6066.cir-14-0021

Figure Lengend Snippet: Figure 7. N1IC in T cells overcomes the tolerogenic effect induced by MDSC. A, MDSC were isolated from tumors and spleens of mice bearing s.c. 3LL cells for 17 days using anti-Gr1 kits. Then, total RNA was isolated and tested for Notch ligands by quantitative PCR. Results represent mean SD from four independent animals and tested in triplicate. , P < 0.001. B, activated CD3þ T cells were cocultured at different ratios with tumor-infiltrating MDSC for 48 hours. Then, T cells were negatively isolated using anti-CD11b beads and whole-protein extracts were harvested and used for detection of Notch-1 and Notch-2 isoforms by Western blotting. A representative experiment of three repeats is shown. C, activated T cells cocultured with MDSC at a 1:1/2 ratio were treated with L-NMMA (500 mmol/L), D-NMMA (500 mmol/L), and NN (200 mmol/L) for 48 hours. Then, extracts were isolated and used as shown in B. Representative results are from three similar experiments. D, CD8þ T cells from CD45.2þ N1IC or N1ICf/f mice were adoptively transferred into CD45.1þ

Article Snippet: Lewis lung carcinoma (3LL) and EL-4 thymoma cells were obtained from the American Type Culture Collection and maintained in RPMI-1640 (Lonza-BioWhittaker) supplemented with 10% fetal calf serum (Hyclone), 25 mmol/L HEPES (Invitrogen, Life Technologies), 4 mmol/L L-glutamine (Invitrogen, Life Technologies), and 100 U/mL of penicillin, streptomycin (Invitrogen, Life Technologies).

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

Journal: Journal of Translational Medicine

Article Title: Combined IL-21 and Low-Dose IL-2 therapy induces anti-tumor immunity and long-term curative effects in a murine melanoma tumor model

doi: 10.1186/1479-5876-4-24

Figure Lengend Snippet: Tumor-specific protective immunity in long-term survivor mice. IL-21 + IL-2 treated mice that survived disease free for 150 days were rechallenged with B16F10 (5 × 10 5 ) or 3LL(5 × 10 4 ) tumor cells. All 3LL challenged mice (4/4) exhibited mean tumor growth to ≥ 200 mm 2 by day 32; all B16F10 challenged mice (5/5) were protected. Naïve mice challenged with the same B16F10 (5/5) or 3LL (5/5) tumor cells had rapid mean tumor growth to ≥ 200 mm 2 by day 25.

Article Snippet: The B16 F10 melanoma and 3LL (Lewis Lung) cell lines were obtained from ATCC.

Techniques:

Journal: PLoS ONE

Article Title: An In Situ Autologous Tumor Vaccination with Combined Radiation Therapy and TLR9 Agonist Therapy

doi: 10.1371/journal.pone.0038111

Figure Lengend Snippet: The immunomodulatory effect of TLR agonist and RT was tested in both short term (A) and long term animal studies (B). Experiments were carried out on both C57BL/6 wildtype and cogenic B cell deficient mice. Mice were inoculated with 1×10 5 3LL cells on day 0 and were divided into 5 groups (n = 15/group for wildtype and n = 10/group for B cell deficient mice) that received either no treatment, 20 Gy RT of the primary tumor on day 14 post-tumor inoculation, only control oligo, only TLR9 agonist, or the combination of tumor RT, followed immediately by TLR9 agonist injection on day 14. For short term studies, 25 wildtype or B cell deficient mice were sacrificed on day 15 and splenocytes were harvested for flow cytometry. For long term studies, 75 wildtype and 50 B cell deficient mice were used. TLR9 agonist was injected subcutaneously, twice a week for three weeks (from day 14 to 35). The tumor volume was measured at 1–3-day intervals with a vernier caliper. Of the 75 wildtype mice, 40 mice were sacrificed on day 35 for immunological studies and the remaining 35 mice were used for survival studies. For B cell deficient mice, all of them were sacrificed on day 35 and were used for immunological studies.

Article Snippet: Lewis lung carcinoma (3LL) cells (American Type Culture Collection, Manassas, VA) was propagated in high glucose DMEM supplemented with 10% FBS, sodium pyruvate, non-essential amino acids and 100 U/ml of penicillin and streptomycin (Invitrogen, Carlsbad, CA).

Techniques: Control, Injection, Flow Cytometry

Journal: PLoS ONE

Article Title: An In Situ Autologous Tumor Vaccination with Combined Radiation Therapy and TLR9 Agonist Therapy

doi: 10.1371/journal.pone.0038111

Figure Lengend Snippet: TLR9 agonist induces the proliferation of TLR9-expression splenocytes in mice bearing an ectopic 3LL footpad tumor.

Article Snippet: Lewis lung carcinoma (3LL) cells (American Type Culture Collection, Manassas, VA) was propagated in high glucose DMEM supplemented with 10% FBS, sodium pyruvate, non-essential amino acids and 100 U/ml of penicillin and streptomycin (Invitrogen, Carlsbad, CA).

Techniques: Control

Journal: PLoS ONE

Article Title: An In Situ Autologous Tumor Vaccination with Combined Radiation Therapy and TLR9 Agonist Therapy

doi: 10.1371/journal.pone.0038111

Figure Lengend Snippet: C57BL/6 mice bearing 3LL tumor were divided into 5 groups receiving either PBS, control oligo, TLR9 agonist, RT (20 Gy) or RT+TLR9 agonist. Splenocytes and sera were harvested 24 hours after treatment. The activation of CD8+ T cells, B cells, pDCs and NKDCs was analyzed by flow cytometry (A) and the release of IFN-γ and IL-10 was analyzed by ELISA (B).

Article Snippet: Lewis lung carcinoma (3LL) cells (American Type Culture Collection, Manassas, VA) was propagated in high glucose DMEM supplemented with 10% FBS, sodium pyruvate, non-essential amino acids and 100 U/ml of penicillin and streptomycin (Invitrogen, Carlsbad, CA).

Techniques: Control, Activation Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: An In Situ Autologous Tumor Vaccination with Combined Radiation Therapy and TLR9 Agonist Therapy

doi: 10.1371/journal.pone.0038111

Figure Lengend Snippet: Sera from tumor bearing mice treated with either PBS, control oligo, TLR9 agonist, RT (20 Gy) or combined RT and TLR9 agonist were analyzed for the presence of tumor specific antibodies by indirect ELISA coated with total 3LL tumor lysate (A). The total IgG titer was evaluated by an Easy-Titer Mouse IgG Assay Kit (B) and the infiltration of IgG antibody was visualized by confocal microscope after stained with FITC conjugated anti-mouse IgG (C).

Article Snippet: Lewis lung carcinoma (3LL) cells (American Type Culture Collection, Manassas, VA) was propagated in high glucose DMEM supplemented with 10% FBS, sodium pyruvate, non-essential amino acids and 100 U/ml of penicillin and streptomycin (Invitrogen, Carlsbad, CA).

Techniques: Control, Indirect ELISA, Microscopy, Staining

Journal: PLoS ONE

Article Title: An In Situ Autologous Tumor Vaccination with Combined Radiation Therapy and TLR9 Agonist Therapy

doi: 10.1371/journal.pone.0038111

Figure Lengend Snippet: Wild type (C57BL/6) or congenic B cell deficient mice (Igh-6 tm1Cgn ) bearing 3LL tumors were divided into 5 groups receiving either PBS, control oligo, TLR9 agonist, RT (20 Gy) or RT+TLR9 agonist. Tumor growth curves were generated by measuring three orthogonal tumor diameters at 1–3-day intervals with a vernier caliper.

Article Snippet: Lewis lung carcinoma (3LL) cells (American Type Culture Collection, Manassas, VA) was propagated in high glucose DMEM supplemented with 10% FBS, sodium pyruvate, non-essential amino acids and 100 U/ml of penicillin and streptomycin (Invitrogen, Carlsbad, CA).

Techniques: Control, Generated

Journal: eLife

Article Title: CD95/Fas ligand mRNA is toxic to cells

doi: 10.7554/eLife.38621

Figure Lengend Snippet:

Article Snippet: Cell line (Mus musculus) , 3LL , ATCC , ATCC #CRL-1642 , Mouse Lewis lung carcinoma.

Techniques: Derivative Assay, Produced, CRISPR, Knock-Out, Isolation, Western Blot, Recombinant, Fluorescence, Flow Cytometry, Selection, Expressing, Plasmid Preparation, Control, Infection, Mutagenesis, Transfection, Construct, Synthesized, Blocking Assay, Sequencing, PCR Cloning, Gene Expression, Magnetic Beads

Journal: Cancer Gene Therapy

Article Title: Tumor vessel-injuring ability improves antitumor effect of cytotoxic T lymphocytes in adoptive immunotherapy

doi: 10.1038/cgt.2012.85

Figure Lengend Snippet: Antitumor efficacy of CTL expressing anti-flk1 cTCR. CTL were purified from C57BL/6 or BALB/c mice and transduced with anti-flk1 cTCR or anti-flk1 scFv-expressing retroviral vectors. ( a ) C57BL6 mice bearing B16BL6 or 3LL and BALB/c mice bearing Meth-A, or CT26 tumors were i.v. injected with 5 × 10 6 anti-flk1 cTCR or anti-flk1 scFv-expressing CTL (cTCR–CTL or scFv–CTL). As a control, PBS was i.v. injected into tumor-bearing mice. The tumor volume was calculated after measuring the major and minor axes of the tumor at indicated points. Each point represents the mean±s.d. from six mice. ( b ) C57BL/6 mice were i.v. injected with 3 × 10 5 B16BL6 cells (day 0). Eight days later, 5 × 10 6 or 1 × 10 6 CTL were i.v. injected. At 14 days after metastasis, the lungs were collected from these mice, and the number of metastatic nodules was counted. Data are presented as means±s.d. from six mice. Statistical analysis was performed using one-way analysis of variance: * P <0.01 versus the scFv–CTL (1 × 10 6 )-injected group; † P <0.01 versus the scFv–CTL (5 × 10 6 )-injected group; ‡ P <0.01 versus the cTCR–CTL (1 × 10 6 )-injected group.

Article Snippet: Lewis lung carcinoma 3LL cells (H-2 b ) were purchased from RIKEN BioResource Center (Ibaraki, Japan) and cultured in DMEM supplemented with 10% FBS.

Techniques: Expressing, Purification, Transduction, Retroviral, Injection, Control