Journal: PLoS ONE

Article Title: Overexpression of CDCA2 in Human Squamous Cell Carcinoma: Correlation with Prevention of G1 Phase Arrest and Apoptosis

doi: 10.1371/journal.pone.0056381

Figure Lengend Snippet: ( A ) Quantification of CDCA2 mRNA expression in OSCC-derived cell lines and the HeLa cell line by qRT-PCR analysis. Significant up-regulation of CDCA2 mRNA is seen in all cell lines compared with that in HNOKs (* P <0.05, Mann-Whitney's U test). Data are expressed as the means ± SEM of triplicate results. ( B ) Western blot analysis of CDCA2 in the OSCC cell lines, the HeLa cell line, and the HNOKs. CDCA2 protein expression is up-regulated in all cell lines compared with HNOKs. Densitometric CDCA2 protein data are normalized to α-tubulin protein levels. The values are expressed as a percentage of the HNOKs. p.c., positive control (HepG2 (Human hepatocellular liver carcinoma cell line) Nuclear Lysate).

Article Snippet: The human OSCC cell lines (HSC-2, HSC-3, HSC-4, Ca9-22, Ho-1-u-1, and Sa3) were purchased from the RIKEN BioResource Center through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology, Tsukuba, Japan.

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, MANN-WHITNEY, Western Blot, Positive Control

Journal: Frontiers in Immunology

Article Title: Generation of Human Immunosuppressive Myeloid Cell Populations in Human Interleukin-6 Transgenic NOG Mice

doi: 10.3389/fimmu.2018.00152

Figure Lengend Snippet: Development of human tumor-associated macrophages (TAMs) in tumor-engrafted HSC-NOG-hIL-6 Tg mice. (A) Schema of generation of tumor-bearing humanized mice. HSC-NOG-hIL-6 Tg or HSC-NOG non-Tg mice were inoculated with HSC4 (1.5 × 10 6 ) at 12–14 weeks after HSC transplantation. Human leukocytes in tumor, spleen and peripheral blood (PB) were analyzed at 30–51 days after HSC4 engraftment. (B) Immunohistochemistry of human macrophages in tumor and spleen. Tumor-engrafted HSC-NOG-hIL-6 Tg or HSC-NOG non-Tg mice were analyzed at 36–51 days after tumor transplantation when the tumor size reached 2,000 mm 3 . Serial sections from tumor or spleen were stained with peroxidase-conjugated anti-CD68 or anti-CD163 antibodies. Each panel shows a representative image from three independent sections. (C) Enumeration of CD68 + or CD163 + macrophages. The number of signals in a whole tissue section was counted and divided by the area of the section using a BZ-9000 microscope (Keyence, Tokyo, Japan). Three independent sections were used, and the average number per unit area (cm 2 ) is shown. The ratio of CD163 + to CD68 + cells was calculated using the average numbers. An asterisk indicates statistical significance based on Student’s t -test (* p < 0.05). (D) Expression of IL-4Rα in human macrophages. (Upper panels) FACS plots of IL-4Rα in hCD45 + CD14 + CD68 + macrophages in tumor, spleen, and PB of HSC4-engrafted HSC-NOG-hIL-6 Tg. (Bottom panel) Histogram of hIL-4Rα expression in hCD68 + macrophages in various tissues in HSC4-engrafted HSC-NOG-hIL-6 Tg and HSC-NOG non-Tg mice. Representative data from three experiments are shown.

Article Snippet: A human tumor cell line, HSC4, derived from human head and neck squamous cell carcinoma, was provided by Y. Kawakami (Keio University School of Medicine, Tokyo, Japan).

Techniques: Transplantation Assay, Immunohistochemistry, Staining, Microscopy, Expressing

Journal: Frontiers in Immunology

Article Title: Generation of Human Immunosuppressive Myeloid Cell Populations in Human Interleukin-6 Transgenic NOG Mice

doi: 10.3389/fimmu.2018.00152

Figure Lengend Snippet: Immunosuppressive function of human tumor-associated macrophages (TAMs) in tumor-engrafted HSC-NOG-hIL-6 Tg mice. (A) Expression of human arginase-1 (Arg-1) in TAMs. Tumor-infiltrating cells and mononuclear cells (MNCs) from spleen and peripheral blood (PB), prepared from HSC4-engrafted HSC-NOG-hIL-6 Tg mice, were analyzed by intracellular staining with anti-hARG1 or isotype control. The expression of Arg-1 in CD14 + CD68 + cells in tumor, spleen, and PB is shown. A representative fluorescence-activated cell sorting (FACS) plot from three independent experiments is shown. (B) Reverse transcription polymerase chain reaction (RT-PCR) for vascular endothelial growth factor (VEGF) and interleukin (IL)-10. hCD45 + CD11b + myeloid cells were purified from tumor and spleen in tumor-engrafted HSC-NOG-hIL-6 Tg or HSC-NOG non-Tg mice. After isolation of total RNA and synthesis of cDNA, VEGF and IL-10 were detected by PCR. The intensity of each band was measured using ImageJ software; normalized values are shown. Representative data from two independent experiments are shown. (C) Suppression of T cell activation by TAMs in vitro . hCD45 + CD11b + myeloid cells were sorted from tumor or spleen in tumor-engrafted HSC-NOG-hIL-6 Tg or HSC-NOG non-Tg mice. The cells were cultured with carboxyfluorescein succinimidyl ester (CFSE)-labeled CD3 + T cells from another non-tumor engrafted HSC-NOG non-Tg mouse in a 96-well plate followed by stimulation with or without immobilized anti-CD3 and anti-CD28 antibodies. T cell proliferation was analyzed by FACS on day 7 after staining with anti-CD3 and CD8. The number in each quadrant shows the mean fluorescence intensity (MFI) value. Representative data from three independent experiments are shown. To determine the degree of suppression, the MFI value of CD8 + T cells or CD4 + T cells, which at least proliferated one time, in the first quadrant (left top quadrant) or in the third quadrant (left bottom quadrant), respectively, was normalized by those of T cells in a control group, which were stimulated with anti-CD3/CD28 antibodies alone without CD11b + cells. The mean ± SDs from three independent experiments are shown. Student’s t -test was performed to analyze the statistical significance (** p < 0.01). (D) Tumor growth in HSC-NOG-hIL-6 Tg or HSC-NOG non-Tg mice. HSC4 were transplanted into HSC-NOG-hIL-6 Tg mice (black, n = 3) or HSC-NOG non-Tg mice (gray, n = 3) at 14 weeks after HSC transplantation. Tumor growth was monitored over 36 days after tumor engraftment. The human CD34 + HSC cells came from different donor from Figure S3 in Supplementary Material. An asterisk indicates statistical significance based on Student’s t -test (* p < 0.05).

Article Snippet: A human tumor cell line, HSC4, derived from human head and neck squamous cell carcinoma, was provided by Y. Kawakami (Keio University School of Medicine, Tokyo, Japan).

Techniques: Expressing, Staining, Control, Fluorescence, FACS, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Purification, Isolation, Software, Activation Assay, In Vitro, Cell Culture, Labeling, Transplantation Assay

Journal: Frontiers in Oncology

Article Title: Inhibition of ATM or ATR in combination with hypo-fractionated radiotherapy leads to a different immunophenotype on transcript and protein level in HNSCC

doi: 10.3389/fonc.2024.1460150

Figure Lengend Snippet: Treatment scheme of HNSCC cell lines. HPV-negative (HSC4, Cal-33) and HPV-positive (UM-SCC-47, UD-SCC-2) HNSCC cells were seeded on day 1 and treated with 1µM AZD0156 or 0,1 µM VE-822 24 hours later. Irradiation with 5 Gy was performed 3 hours after treatment with smKI and repeated 24 hours later. Cells were harvested and analyzed 24 hours after the last treatment.

Article Snippet: Two HPV-negative HSC4 (RRID: CVCL_1289), Cal-33 (RRID: CVCL_1108) and two HPV-positive UM-SCC-47 (RRID: CVCL_7759), UD-SCC-2 (RRID: CVCL_E325) HNSCC cell lines were generously provided by Dr. Thorsten Rieckmann at the University Medical Center Hamburg-Eppendorf.

Techniques: Irradiation

Journal: Frontiers in Oncology

Article Title: Inhibition of ATM or ATR in combination with hypo-fractionated radiotherapy leads to a different immunophenotype on transcript and protein level in HNSCC

doi: 10.3389/fonc.2024.1460150

Figure Lengend Snippet: Cell death analysis of HNSCC treated with ATMi or ATRi w/o 2x5Gy RT. RT combined with ATRi shows the highest toxicity in HNSCC cells, regardless of the HPV status. 48 hours post-RT, HPV-negative HSC4 (A) and Cal-33 (B) cells as well as HPV-positive UM-SCC-47 (C) and UD-SCC-2 (D) cells were identified as viable (AnnexinV-, PI-), apoptotic (AnnexinV+, PI-) or necrotic (AnnexinV+, PI+). Percentages of viable, apoptotic, and necrotic cells are shown as stacked bars representing the mean ± SD. A one-tailed Mann-Whitney U test was performed to compare the different treatment approaches within one cell line: *p ≤ 0.05, n=4.

Article Snippet: Two HPV-negative HSC4 (RRID: CVCL_1289), Cal-33 (RRID: CVCL_1108) and two HPV-positive UM-SCC-47 (RRID: CVCL_7759), UD-SCC-2 (RRID: CVCL_E325) HNSCC cell lines were generously provided by Dr. Thorsten Rieckmann at the University Medical Center Hamburg-Eppendorf.

Techniques: One-tailed Test, MANN-WHITNEY

Journal: Frontiers in Oncology

Article Title: Inhibition of ATM or ATR in combination with hypo-fractionated radiotherapy leads to a different immunophenotype on transcript and protein level in HNSCC

doi: 10.3389/fonc.2024.1460150

Figure Lengend Snippet: Flow cytometric analysis of immune checkpoint surface marker on HNSCC after ATMi or ATRi w/o 2x5Gy RT. Combining RT with ATRi leads to the upregulation of immunostimulatory ICOS-L and CD137-L on HNSCC cells, irrespective of the HPV status. 48 hours after the last treatment, the HNSCC cells were harvested and the expression of immunostimulatory ICMs ICOS-L and CD137-L was examined by flow cytometry. The gating strategy is presented in (A) ICM expression is presented as ΔMFI (delta of mean fluorescence intensity of stained samples – background fluorescence) (B, C) . The combined treatment of RT and ATRi increased ICOS-L expression in all four cell lines compared to ATRi or RT alone, as well as in comparison to RT combined with ATMi (B) . Dual treatment with RT and ATMi tended to even decrease ICOS-L surface expression compared to RT or RT with ATRi (B) . RT and ATRi led to enhanced expression of immunostimulatory CD137-L in HPV-negative HSC4 cells compared to ATRi or RT alone, or the combined treatment of RT and ATMi (C) . In HPV-positive UM-SCC-47 and UD-SCC-2 cells, the combination of RT and ATRi also demonstrated higher surface expression of CD137-L compared to RT alone or treatment with RT and ATMi, respectively (C) . A one-tailed Mann-Whitney U test was performed to compare the different treatment approaches within one cell line: *p ≤ 0.05, n=4.

Article Snippet: Two HPV-negative HSC4 (RRID: CVCL_1289), Cal-33 (RRID: CVCL_1108) and two HPV-positive UM-SCC-47 (RRID: CVCL_7759), UD-SCC-2 (RRID: CVCL_E325) HNSCC cell lines were generously provided by Dr. Thorsten Rieckmann at the University Medical Center Hamburg-Eppendorf.

Techniques: Marker, Expressing, Flow Cytometry, Fluorescence, Staining, Comparison, One-tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: A predominant enhancer co-amplified with the SOX2 oncogene is necessary and sufficient for its expression in squamous cancer

doi: 10.1038/s41467-021-27055-4

Figure Lengend Snippet: a BRD4 ChIP-seq profile at the SOX2 locus in KYSE140 cells with and without e1 repression. b Averaged BRD4 ChIP-seq profile, across BRD4 sites that harbor high-confidence SOX2 binding (*SOX2 ChIP-seq peaks containing SOX motifs) or the other BRD4 sites, in KYSE140 cells with and without e1 repression. c BRD4 ChIP-seq profile at the e1–e8 locus in LK2, NCI-H520, and HSC4 cells with and without e1 repression. d Top: PhastCons scores (0:1 range) representing the conservation level of DNA sequences in the e1 enhancer. Middle: distribution of JASPAR DNA motifs identified in the e1 enhancer. Bottom: CRISPR cutting sites that overlap with the identified DNA motifs. e RT-qPCR measuring expression changes of SOX2 in KYSE140 and LK2 cells after CRISPR-mediated disruption of each of the identified DNA motifs. The expression level was normalized to the sgAAVS1 control. n = 2 biologically independent experiments. *: combinatorial CRISPR cutting of SOX (2nd), AP1, RUNX, and STAT (2nd) motifs in KYSE140 cells, or SOX (2nd), AP1, SNAIL, and TCF motifs in LK2 cells. f ChIP-qPCR showing the relative enrichment of BRD4 at e1–e5 in KYSE140 and LK2 cells after combinatorial CRISPR cutting of the selected motifs. ChIP enrichment was normalized to DNA concentration of each sample (measured by Qubit) and then to sonicated genomic input. n = 2 biologically independent experiments. g Presence of the candidate functional motifs in the e1–e5 enhancers. Source data are provided as a Source Data file.

Article Snippet: Squamous cancer cell lines KYSE140, KYSE70, LK2, NCI-H520, HSC4, TE1, TE10, SKMES1, and RERFLCAI were obtained from the Broad Institute Cancer Cell Line Encyclopedia (CCLE) project , .

Techniques: ChIP-sequencing, Binding Assay, CRISPR, Quantitative RT-PCR, Expressing, Disruption, Control, ChIP-qPCR, Concentration Assay, Sonication, Functional Assay