Journal: Cancer Immunology, Immunotherapy : CII

Article Title: A new small cell lung cancer (SCLC)-specific marker discovered through antigenic subtraction of neuroblastoma cells

doi: 10.1007/s00262-003-0376-9

Figure Lengend Snippet: IHC staining of normal lung tissue and SCLC tissue with VAC51.2 MoAb. b Summary of IHC relative staining of sections derived from normal lung, SCLC tumor, and lung metastases (each section represents a distinct normal individual or patient). c The upper panel depicts a summary of IHC relative staining of sections derived from normal pancreas in comparison with pancreatic cancer tissue. The lower panel represents IHC staining of sections from normal pancreas and pancreatic cancer tissue probed with VAC51.2. d The left panel depicts a summary of IHC relative staining of sections derived from normal colon in comparison with colon cancer tissue. The right panel represents IHC staining of sections from normal colon and colon cancer tissue probed with VAC51.2. e The upper panel depicts a summary of IHC relative staining of sections derived from normal breast in comparison with breast cancer tissue. The lower panel represents IHC staining of sections from normal breast and breast cancer tissue probed with VAC51.2. The level of staining was graded based on relative intensity from 0 to 4, where 0 represents no staining, 1 represents low staining, and 4 represents strong staining

Article Snippet: Internalization of MoAb Human SCLC (NCI-H209) and human neuroendocrine tumor [Be (2)-M17] cells were Fc receptor blocked (BD PharMingen, San Diego, Calif.) before incubation for 30 min with MoAb on ice at a concentration of 10 μg/ml.

Techniques: Immunohistochemistry, Staining, Derivative Assay, Comparison

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: A new small cell lung cancer (SCLC)-specific marker discovered through antigenic subtraction of neuroblastoma cells

doi: 10.1007/s00262-003-0376-9

Figure Lengend Snippet: FACS analysis of intracellular staining with VAC51.2 of an SCLC cell line (upper panel) in comparison with a neuroblastoma cell line (lower panel). Shaded curves represent binding of isotype control to differential cell lines. Bold-lined curves represent binding of VA51.2 MoAb to the differential cell lines

Article Snippet: Internalization of MoAb Human SCLC (NCI-H209) and human neuroendocrine tumor [Be (2)-M17] cells were Fc receptor blocked (BD PharMingen, San Diego, Calif.) before incubation for 30 min with MoAb on ice at a concentration of 10 μg/ml.

Techniques: Staining, Comparison, Binding Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: A new small cell lung cancer (SCLC)-specific marker discovered through antigenic subtraction of neuroblastoma cells

doi: 10.1007/s00262-003-0376-9

Figure Lengend Snippet: Immunoaffinity purification of SCLC antigen from sera of SCLC patients with grade III–IV disease. a The purification scheme using a sequence of three immunoaffinity columns to purify the SCLC antigen from SCLC patients in comparison with control (normal) human serum. b Results of a western blot analysis with the final product of the immunoaffinity purification procedure of normal sera versus SCLC sera following staining with VAC109.12 MoAb. Two thick fuzzy bands (with median Mr of approximately 215 and 122 kDa, respectively) were detected in the SCLC sera. No band was detected in the normal sera sample. Membrane preparation from SCLC cell line was used as a positive control, which exhibited a distinct thin band at an Mr of about 215 kDa. c A serial dilution of immunoaffinity-purified fractions extracted from sera of normal individuals versus SCLC patients tested in an ELISA. A strong binding of SCLC MoAb (VAC109.12) to the specific antigen eluted from SCLC patients was shown to exceed six logs as compared to the normal serum {calculated as [(O.D. SCLC−O.D. Normal)/O.D. normal]×100}

Article Snippet: Internalization of MoAb Human SCLC (NCI-H209) and human neuroendocrine tumor [Be (2)-M17] cells were Fc receptor blocked (BD PharMingen, San Diego, Calif.) before incubation for 30 min with MoAb on ice at a concentration of 10 μg/ml.

Techniques: Immunoaffinity Purification, Purification, Sequencing, Comparison, Western Blot, Staining, Membrane, Positive Control, Serial Dilution, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Journal of Radiation Research

Article Title: hTERT promoter methylation promotes small cell lung cancer progression and radiotherapy resistance

doi: 10.1093/jrr/rraa052

Figure Lengend Snippet: The promoter of hTERT is methylated and correlates with radiotherapy in SCLC. ( A ) mRNA levels of hTERT in normal and SCLC tissues. ( B ) mRNA levels of hTERT in normal tissues and SCLC patients with or without radiotherapy. ( C ) Methylation status of the hTERT promoter in normal and SCLC tissues. ( D ) Methylation status of the hTERT promoter in normal tissues and SCLC patients with or without radiotherapy. ( E ) The correlation of hTERT and DNMT3B was analyzed in the TCGA lung cancer dataset. ( F ) mRNA expression of DNMT3B in H20 cells transfected with DNMT3B shRNA was determined by qPCR. ( G ) Methylation status of the hTERT promoter in H20 cells transfected with DNMT3B shRNA. Data are shown as mean ± S.D. * P < 0.05; * * P < 0.01; * * * P < 0.001; ns, not significant (Student’s t-test).

Article Snippet: H20 and H446 human SCLC cell lines were purchased from the China Center for Type Culture Collection.

Techniques: Methylation, Expressing, Transfection, shRNA

Journal: Journal of Radiation Research

Article Title: hTERT promoter methylation promotes small cell lung cancer progression and radiotherapy resistance

doi: 10.1093/jrr/rraa052

Figure Lengend Snippet: The methylation of hTERT promotes SCLC proliferation. ( A ) mRNA expression of hTERT in H20 cells transfected with hTERT expression plasmid was determined by qPCR. ( B ) Protein expression of hTERT in H20 cells transfected with hTERT expression plasmid was determined by western blot. Cell viability of H20 cells ( C ) or H446 cells ( D ) transfected with hTERT expression plasmid was determined by cell count assay. Cell viability of H20 cells ( E ) or H446 cells ( F ) transfected with hTERT expression plasmid was determined by MTT assay. ( G ) Methylation status of the hTERT promoter in the normal pulmonary epithelium BEAS-2B cells or SCLC cells H20 and H446. ( H ) Methylation status of the hTERT promoter in H20 and H446 cells treated with 5-Aza. ( I ) mRNA expression of hTERT in H20 and H446 cells treated with 5-Aza. Cell viability of H20 cells ( J ) or H446 cells ( K ) treated with 5-Aza and transfected with or without hTERT expression plasmid was determined by cell count assay. ( L ) mRNA expression of hTERT in H20 cells transfected with hTERT shRNA was determined by qPCR. ( M ) Cell viability of H20 and H446 cells transfected with hTERT shRNA was determined by cell count assay. Data are shown as mean ± S.D. * P < 0.05; * * P < 0.01; * * * P < 0.001; ns, not significant (ANOVA test in E and G, others Student’s t-test).

Article Snippet: H20 and H446 human SCLC cell lines were purchased from the China Center for Type Culture Collection.

Techniques: Methylation, Expressing, Transfection, Plasmid Preparation, Western Blot, Cell Counting, MTT Assay, shRNA

Journal: Journal of Radiation Research

Article Title: hTERT promoter methylation promotes small cell lung cancer progression and radiotherapy resistance

doi: 10.1093/jrr/rraa052

Figure Lengend Snippet: The methylation of hTERT promotes SCLC migration and invasion. ( A ) Transwell assay of migration and invasion of H20 cells transfected with hTERT expression plasmid or vector. ( B ) Statistical results of the Transwell assay in (A). ( C ) qPCR analysis of the expression of epithelioid markers OCLN and JUP or the mesenchymal markers ZEB1, ZEB2, TWIST1 and FN1 in H20 cells transfected with hTERT expression plasmid or vector. ( D ) Transwell assay of migration and invasion of H20 cells treated with 5-Aza. ( E ) Statistical results of the Transwell assay in (D). ( F ) qPCR analysis of the expression of epithelioid markers OCLN and JUP or the mesenchymal markers ZEB1, ZEB2, TWIST1 and FN1 in H20 cells treated with 5-Aza. Data are shown as mean ± S.D. * P < 0.05; * * P < 0.01; * * * P < 0.001; ns, not significant (Student’s t-test).

Article Snippet: H20 and H446 human SCLC cell lines were purchased from the China Center for Type Culture Collection.

Techniques: Methylation, Migration, Transwell Assay, Transfection, Expressing, Plasmid Preparation

Journal: Journal of Radiation Research

Article Title: hTERT promoter methylation promotes small cell lung cancer progression and radiotherapy resistance

doi: 10.1093/jrr/rraa052

Figure Lengend Snippet: The methylation of hTERT promotes SCLC radiotherapy resistance. Methylation status of the hTERT promoter in H20 ( A ) and H446 ( B ) cells treated with a single dose of 5 Gy irradiation. mRNA level of hTERT in H20 ( C ) and H446 ( D ) cells treated with a single dose of 5 Gy irradiation. Cell viability of H20 cells ( E ) or H446 cells ( F ) transfected with hTERT expression plasmid and treated with a gradient dose of irradiation. Cell viability of H20 cells ( G ) or H446 cells ( H ) treated with 5-Aza and treated with a gradient dose of irradiation. Data are shown as mean ± S.D. * * * P < 0.001; ns, not significant (ANOVA test in (E) and (H), others Student’s t-test).

Article Snippet: H20 and H446 human SCLC cell lines were purchased from the China Center for Type Culture Collection.

Techniques: Methylation, Irradiation, Transfection, Expressing, Plasmid Preparation

Journal: Journal of Radiation Research

Article Title: hTERT promoter methylation promotes small cell lung cancer progression and radiotherapy resistance

doi: 10.1093/jrr/rraa052

Figure Lengend Snippet: EZH2 acted as the downstream effector of hTERT. ( A ) Correlation of hTERT and EZH2 analysed in the TCGA lung cancer dataset. ( B ) mRNA levels of EZH2 in H20 or H446 cells transfected with hTERT expression plasmid were determined by qPCR. ( C ) mRNA levels of EZH2 in H20 or H446 cells treated with 5-Aza and transfected with or without hTERT expression plasmid were determined by qPCR. ( D ) mRNA levels of EZH2 in H20 cells transfected with EZH2 expression plasmid were determined by qPCR. ( E ) Cell viability of H20 cells transfected with EZH2 expression plasmid was determined by cell count assay. ( F ) Cell viability of H20 cells transfected with EZH2 expression plasmid was determined by MTT assay. ( G ) Transwell assay of migration and invasion of H20 cells transfected with EZH2 expression plasmid or vector. ( H ) Cell viability of H20 cells transfected with EZH2 expression plasmid and treated with a gradient dose of irradiation. Data are shown as mean ± S.D. * * P < 0.01; * * * P < 0.001; ns, not significant (ANOVA test in F and H, others Student’s t-test).

Article Snippet: H20 and H446 human SCLC cell lines were purchased from the China Center for Type Culture Collection.

Techniques: Transfection, Expressing, Plasmid Preparation, Cell Counting, MTT Assay, Transwell Assay, Migration, Irradiation

Journal: Cell reports

Article Title: WEE1 inhibition enhances the antitumor immune response to PD-L1 blockade by the concomitant activation of STING and STAT1 pathways in SCLC

doi: 10.1016/j.celrep.2022.110814

Figure Lengend Snippet: (A) Cell viability IC 50 values were determined in response to treatment with AZD1775 (0–10 μM) for 5 days in 16 human and three murine SCLC cell lines. The data shown represent the means ± SD of three individual experiments. (B) Cells were treated with 1 μM AZD1775 for 24 or 48 h. Then, the proportion of apoptotic cells was detected using annexin V-propidium iodide-based flow cytometry. Bars represent mean ± SD of triplicate. Statistical significance was determined using Student’s t test (**p < 0.01, ***p < 0.001). (C) Cells were treated with 1 μM AZD1775 for 16 h. Cell-cycle states were detected with EdU-DAPI-based flow cytometry. (D) Cells were treated with 1 μM AZD1775 for 8, 24, or 48 h. Western blots show protein expression of phospho-WEE1, phospho- and total CDK1, γH2AX, cleaved PARP, and actin (loading control) at each time indicated. (E) Tumor-growth curves of subcutaneous tumors in nude mice with conditional loss of Trp53 , p130 , and Rb1 (RPP; top) and Trp53 , Rb1 , and MYC T58A (RPM; bottom) treated with vehicle or 60 mg/kg of AZD1775 (n = 5 per group). Bars represent mean ± SE. Statistical significance was determined using Student’s t test (**p < 0.01). (F) Western blots showing phosho-WEE1, γH2AX, and actin (loading control) of RPP or RPM tumors from (E). See also .

Article Snippet: SCLC human-derived cell lines were obtained from the American Type Culture Collection (ATCC) and European Collection of Authenticated Cell Cultures (ECACC) ( ).

Techniques: Flow Cytometry, Western Blot, Expressing, Control

Journal: Cell reports

Article Title: WEE1 inhibition enhances the antitumor immune response to PD-L1 blockade by the concomitant activation of STING and STAT1 pathways in SCLC

doi: 10.1016/j.celrep.2022.110814

Figure Lengend Snippet: (A) Quantification of cells containing micronuclei (MN) after 1 μM AZD1775 treatment for 24 h. Bars represent mean ± SD of eight areas. Statistical significance was determined using Student’s t test (***p < 0.001). (B) Western blots showing the protein expression of STING pathway, phospho (p)- and total (t)-STING, p- and t-TBK1, p- and t-IRF3, cGAS, and actin (loading control) in SCLC cells treated with 1 μM AZD1775 for 8, 24, and 48 h. (C) Quantitative mRNA expression of IFN-α , IFN-β after treatment with 1 μM AZD1775 for 48 h in SCLC cells (H526, H82, H446, RPP, and RPM). Bars represent mean ± SD of triplicate. Statistical significance was determined using Student’s t test (**p < 0.01, ***p < 0.001). (D and E) Quantitative mRNA expression of (D) CXCL10 and (E) CCL5 after treatment with 1 μM AZD1775 for 48 h in SCLC cells (H526, H82, H446, RPP, and RPM). Bars represent mean ± SD of triplicate. Statistical significance was determined using Student’s t test (**p < 0.01, ***p < 0.001). See also – .

Article Snippet: SCLC human-derived cell lines were obtained from the American Type Culture Collection (ATCC) and European Collection of Authenticated Cell Cultures (ECACC) ( ).

Techniques: Western Blot, Expressing, Control

Journal: Cell reports

Article Title: WEE1 inhibition enhances the antitumor immune response to PD-L1 blockade by the concomitant activation of STING and STAT1 pathways in SCLC

doi: 10.1016/j.celrep.2022.110814

Figure Lengend Snippet: (A and B) HALLMARK pathway enrichment analyses of differentially expressed genes (DEGs) from RPP tumors on the mice treated with vehicle, AZD1775, or AZD1775 plus anti-PD-L1 antibody for 21 days. (A) AZD1775 versus vehicle and (B) AZD1775 plus anti-PD-L1 antibody versus vehicle (n = 5 per group). (C and D) HALLMARK pathway enrichment analyses of DEGs from RPM tumors on the mice treated with vehicle, AZD1775, or AZD1775 plus anti-PD-L1 antibody for 18 days. (C) AZD1775 versus vehicle and (D) AZD1775 plus anti-PD-L1 antibody versus vehicle (n = 4 per group). (E) Quantitative mRNA expression of IFN-γ after treatment with 1 μM of AZD1775 for 48 h in SCLC cells (H526, H82, H446, RPP, and RPM cells). The data shown represent the means ± SD of triplicate. p values were calculated by Student’s t test (**p < 0.01, ***p < 0.001). (F) Western blots show expression of p-WEE1, p- and total STAT1, and actin (loading control) in SCLC cells (H526, H82, H446). Cells were treated with 1 μM AZD1775 for 24 h. (G) Quantitative mRNA expression of IRF1 after treatment with 1 μM AZD1775 for 48 h in SCLC cells (H82, H446). The data shown represent the means ± SD of triplicate. p values were calculated by Student’s t test (*p < 0.05). See also .

Article Snippet: SCLC human-derived cell lines were obtained from the American Type Culture Collection (ATCC) and European Collection of Authenticated Cell Cultures (ECACC) ( ).

Techniques: Expressing, Western Blot, Control