Journal: Oncology Letters

Article Title: Investigation of bone invasion and underlying mechanisms of oral cancer using a cell line-derived xenograft model

doi: 10.3892/ol.2022.13502

Figure Lengend Snippet: H&E staining to assess the effects of G-SCs, P-SCs and HDFs on bone invasion in OSCC in vivo . (A) H&E staining was used to assess bone mass. Black line, bone resorption. (B) Quantification of length of bone resorption. Data are presented as mean ± SD (n=4). Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. (C) H&E staining was used to assess the type of OSCC bone invasion. Black arrow, erosive area; white arrow, infiltrative area. H&E, hematoxylin and eosin; G-SCs, gingival tissue-derived stromal cells; P-SCs, periodontal ligament tissue-derived stromal cells; HDFs, human dermal fibroblasts; OSCC, oral squamous cell carcinoma; ns, not significant.

Article Snippet: The human OSCC HSC-3 cell line (cat. no. JCRB0623), HDFs (cat. no. CC-2511) and the murine macrophage RAW264.7 cell line (cat. no. RCB0535) were purchased from the Japanese Collection of Research Bioresources Cell Bank, Lonza Group, Ltd. and RIKEN BioResource Center respectively.

Techniques: Staining, In Vivo, Derivative Assay

Journal: Oncology Letters

Article Title: Investigation of bone invasion and underlying mechanisms of oral cancer using a cell line-derived xenograft model

doi: 10.3892/ol.2022.13502

Figure Lengend Snippet: Effect of G-SCs, P-SCs and HDFs on invasion and EMT of HSC-3 cells in OSCC bone invasion region. Immunohistochemical staining was used to (A) assess and (B) quantify effect of G-SCs, P-SCs and HDFs on protein expression levels of MMP9. Immunohistochemical staining was used to (C) assess and (D) quantify effect of G-SCs, P-SCs and HDFs on protein expression levels of MT1-MMP. Immunohistochemical staining was used to (E) assess and (F) quantify effect of G-SCs, P-SCs and HDFs on protein expression levels of Snail. Black arrow, osteoclast. Data are presented as median and interquartile range (n=4). Statistical analysis was performed using Kruskal-Wallis followed by Dunn's test. *P<0.05 and ****P<0.0001. G-SCs, gingival tissue-derived stromal cells; P-SCs, periodontal ligament tissue-derived stromal cells; HDFs, human dermal fibroblasts; OSCC, oral squamous cell carcinoma; ns, not significant; EMT, epithelial-mesenchymal transition; MMP, matrix metalloproteinase; MT1-MMP, membrane type 1 MMP.

Article Snippet: The human OSCC HSC-3 cell line (cat. no. JCRB0623), HDFs (cat. no. CC-2511) and the murine macrophage RAW264.7 cell line (cat. no. RCB0535) were purchased from the Japanese Collection of Research Bioresources Cell Bank, Lonza Group, Ltd. and RIKEN BioResource Center respectively.

Techniques: Immunohistochemical staining, Staining, Expressing, Derivative Assay, Membrane

Journal: Oncology Letters

Article Title: Investigation of bone invasion and underlying mechanisms of oral cancer using a cell line-derived xenograft model

doi: 10.3892/ol.2022.13502

Figure Lengend Snippet: Effects of G-SCs, P-SCs and HDFs on the protein expression levels of RANKL and PTHrP in HSC-3 cells in the erosive area of OSCC bone invasion region. Immunohistochemical staining was used to (A) assess and (B) quantify effect of G-SCs, P-SCs and HDFs on protein expression levels of RANKL. Immunohistochemical staining was used to (C) assess and (D) quantify effect of G-SCs, P-SCs and HDFs on protein expression levels PTHrP in HSC-3 cells in the erosive area of OSCC bone invasion region. Data are presented as median and interquartile range (n=4). Statistical analysis was performed by Kruskal-Wallis followed by Dunn's test. *P<0.05, ***P<0.001 and ****P<0.0001. IHC, immunohistochemistry; G-SCs, gingival tissue-derived stromal cells; P-SC, periodontal ligament tissue-derived stromal cell; HDFs, human dermal fibroblasts; OSCC, oral squamous cell carcinoma; ns, not significant.

Article Snippet: The human OSCC HSC-3 cell line (cat. no. JCRB0623), HDFs (cat. no. CC-2511) and the murine macrophage RAW264.7 cell line (cat. no. RCB0535) were purchased from the Japanese Collection of Research Bioresources Cell Bank, Lonza Group, Ltd. and RIKEN BioResource Center respectively.

Techniques: Expressing, Immunohistochemical staining, Staining, Immunohistochemistry, Derivative Assay

Journal: Oncology Letters

Article Title: Investigation of bone invasion and underlying mechanisms of oral cancer using a cell line-derived xenograft model

doi: 10.3892/ol.2022.13502

Figure Lengend Snippet: Identification of potential genes underlying the differential effects of G-SCs and P-SCs on OSCC bone invasion following crosstalk with HSC-3 cells in vivo . (A) Biological processes associated with upregulated differentially expressed genes in P-SCs was assess using Gene Ontology enrichment analysis (only presenting the potential biological process underlying the differential effects of G-SCs and P-SCs on OSCC bone invasion following crosstalk with HSC-3 in vivo ). (B) Protein-protein interaction network was used to identify the hub genes in biological process of ‘cell differentiation’. G-SC, gingival tissue-derived stromal cells; P-SCs, periodontal ligament tissue-derived stromal cells; OSCC, oral squamous cell carcinoma; CDK1, cylin-dependent kinase 1; INS, insulin; AURKA, aurora kinase A; CCNB1, cyclin B1; TOP2A, DNA topoisomerase IIα; NUSAP1, nucleolar and spindle-associated protein 1; CENPF, centromere protein F; ASPM, assembly factor for spindle microtubules; ANLN, anillin actin binding protein; RACGAP1, Rac GTPase activating protein 1.

Article Snippet: The human OSCC HSC-3 cell line (cat. no. JCRB0623), HDFs (cat. no. CC-2511) and the murine macrophage RAW264.7 cell line (cat. no. RCB0535) were purchased from the Japanese Collection of Research Bioresources Cell Bank, Lonza Group, Ltd. and RIKEN BioResource Center respectively.

Techniques: In Vivo, Cell Differentiation, Derivative Assay, Binding Assay

Journal: Cancer Medicine

Article Title: Development of a complete human anti-human transferrin receptor C antibody as a novel marker of oral dysplasia and oral cancer

doi: 10.1002/cam4.267

Figure Lengend Snippet: DNA copy number analysis of oral dysplasia and oral squamous cell carcinoma (OSCC) showed an amplified genomic region at chromosome 3q. Recurrent genetic changes are depicted according to the copy number analyzer for the GeneChip (CNAG) output of the single-nucleotide polymorphism array analysis of eight oral dysplasia and eight OSCC samples. A summary of all of the identified genetic alteration patterns in the (A) oral dysplasia and (B) OSCC samples is shown. Regions with copy number gains are indicated by red lines and losses are indicated by green lines. Red arrows show the common amplified regions of chromosome 3q in the dysplasia and OSCC samples. (C) A precise genomic map of the amplified regions of 3q. Several genes in the TFRC region were identified in the OSCC expression profiles as being expressed at levels that were more than twofold higher than those in the control oral tissues. (D) Scatter plot of the DNA copy number versus the TFRC mRNA expression rate. Correlations were quantified with Spearman's rank correlation coefficient. TFRC, transferrin receptor C.

Article Snippet: Eight human OSCC cell lines (Ca922, HO-1-u-1, HSC2, HSC3, HSC4, SAS, HSQ89, and Sa-3) were purchased from the RIKEN BioResource Center (Ibaraki, Japan).

Techniques: Amplification, Expressing

Journal: Cancer Medicine

Article Title: Development of a complete human anti-human transferrin receptor C antibody as a novel marker of oral dysplasia and oral cancer

doi: 10.1002/cam4.267

Figure Lengend Snippet: TFRC expression in OSCC. (A) The TFRC mRNA level was examined in five primary oral dysplasia tissues, eight OSCC primary tissues, and eight OSCC cell lines (Ca9-22, Ho1u1, HSC2, HSC3, HSC4, HSQ89, SAS, and Sa-3) by quantitative real-time PCR. mRNA samples from the oral tissues of normal healthy volunteers were used as controls. * P < 0.05, ** P < 0.01. (B) TFRC expression was evaluated in primary OSCC tissues compared with normal oral tissues (right panel) and eight OSCC cell lines compared with control keratinocyte HaCaT cells (left panel) by immunoblotting. (C) TFRC expression was evaluated on OSCC cells and control HaCaT cells by flow cytometry using the anti-TFRC antibody. The cells were stained with the phycoerythrin-labeled anti-TFRC antibody, followed by flow cytometry analysis. The figure shows representative flow cytometry histogram profiles of the control keratinocyte cell line (HaCaT) and seven OSCC cell lines (Ca9-22, HSC2, HSC3, HSC4, SAS HSQ89, SAS, and Sa-3). Open histograms represent cells stained with IgG isotype controls, and filled histograms indicate cells stained with the anti-TFRC antibody. * P < 0.01. OSCC, oral squamous cell carcinoma; TFRC, transferrin receptor C.

Article Snippet: Eight human OSCC cell lines (Ca922, HO-1-u-1, HSC2, HSC3, HSC4, SAS, HSQ89, and Sa-3) were purchased from the RIKEN BioResource Center (Ibaraki, Japan).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Flow Cytometry, Staining, Labeling

Journal: Cancer Medicine

Article Title: Development of a complete human anti-human transferrin receptor C antibody as a novel marker of oral dysplasia and oral cancer

doi: 10.1002/cam4.267

Figure Lengend Snippet: Effects of TFRC knockdown and overexpression on OSCC cell growth. (A) A TFRC expression vector was introduced into HSC4 cells, and cell growth was analyzed by MTT assay. The data are shown as the mean ± SD of triplicate samples. * P < 0.05 versus parental HSC4 cells. The TFRC expression level in parental, mock-, and TFRC-transfected HSC4 cells was determined by FACS (B) analysis and (C) immunoblotting. (D) Retroviral vectors containing TFRC shRNA or mock shRNA (control) were transfected into SAS cells. Forty-eight hours after transfection, the ZsGreen-positive cells were sorted and cell growth was analyzed by MTT assay. The data are shown as the mean ± SD of triplicate samples. * P < 0.05 versus parental SAS cells. OSCC, oral squamous cell carcinoma; TFRC, transferrin receptor C; MTT, methyl thiazolyl tetrazolium.

Article Snippet: Eight human OSCC cell lines (Ca922, HO-1-u-1, HSC2, HSC3, HSC4, SAS, HSQ89, and Sa-3) were purchased from the RIKEN BioResource Center (Ibaraki, Japan).

Techniques: Over Expression, Expressing, Plasmid Preparation, MTT Assay, Transfection, Western Blot, shRNA

Journal: Cancer Medicine

Article Title: Development of a complete human anti-human transferrin receptor C antibody as a novel marker of oral dysplasia and oral cancer

doi: 10.1002/cam4.267

Figure Lengend Snippet: Effects of anti-TFRC antibody treatment on OSCC cells. (A) Cell growth curves of various OSCC cell lines (HSC2, HSC3, HSC4, and SAS) and control HaCaT cells after treatment with the indicated concentration of anti-TFRC antibody. The various cell lines were analyzed by MTT assay. * P < 0.05, ** P < 0.01. (B) The percentages of viable cells at 48 or 72 h after treatment with the indicated concentrations of the anti-TFRC antibody were compared between the OSCC and control HaCaT cells. The experiments were performed in triplicate and repeated independently at least three times. * P < 0.05, ** P < 0.01 versus HaCaT cells. (C) Following anti-TFRC antibody treatment for 72 or 96 h, SAS/OSCC and HSC2/OSCC cells were labeled with Annexin-V and propidium iodide, and the percentage of apoptotic cells was determined by flow cytometry. The experiments were performed in triplicate and repeated independently at least three times. (D) The number of viable SAS and HSC2 cells was determined at the indicated time points after treatment with 1.0 mg/mL of the anti-TFRC antibody or PBS. * P < 0.05, ** P < 0.01. (E) Identification of cleaved caspase-3 (arrowheads) in OSCC cells after anti-TFRC antibody treatment. SAS/OSCC cells were treated with the anti-TFRC antibody (1.0 mg/mL) for 48 h, and immunoblotting analysis was performed with an anti-caspase-3 antibody. The asterisk indicates a nonspecific band. (F) The cell cycle phase distribution was determined for SAS and HSC2/OSCC cells at 48 h after anti-TFRC antibody treatment. The cells were labeled with propidium iodide and analyzed on a FACScan flow cytometer. OSCC, oral squamous cell carcinoma; TFRC, transferrin receptor C; MTT, methyl thiazolyl tetrazolium; PBS, phosphate-buffered saline.

Article Snippet: Eight human OSCC cell lines (Ca922, HO-1-u-1, HSC2, HSC3, HSC4, SAS, HSQ89, and Sa-3) were purchased from the RIKEN BioResource Center (Ibaraki, Japan).

Techniques: Concentration Assay, MTT Assay, Labeling, Flow Cytometry, Western Blot

Journal: Cancer Medicine

Article Title: Development of a complete human anti-human transferrin receptor C antibody as a novel marker of oral dysplasia and oral cancer

doi: 10.1002/cam4.267

Figure Lengend Snippet: The induction of CDC and ADCC activities by anti-TFRC antibody treatment. (A) Anti-TFRC antibody-mediated CDC activity in various OSCC cells. The panel shows the percentage of viable cells. After the cells were incubated with human serum complement and anti-TFRC antibody at concentrations ranging from 0.01 to 10 mg/mL, the extent of cell lysis was measured by LDH release and is shown as the percentage of the value obtained from the untreated cells. The experiments were repeated independently at least three times. (B) Anti-TFRC antibody-mediated ADCC activity in various OSCC cells. ADCC assays were performed with the same series of cells used in (A). After incubating the cells with peripheral blood mononuclear cells from a normal donor and 0.01 to 10 mg/mL of the anti-TFRC antibody, the ADCC activity was measured by LDH release. Two OSCC cell lines (HSC2 and SAS) exhibited dose-dependent effects, whereas two other OSCC lines (HSC3 and HSC4) and control HaCaT cells showed no effects. (C) Anti-TFRC antibody-mediated ADCC activity in HSC4/TFRC cells compared with mock or parental HSC4 cells. (D) Anti-TFRC antibody-mediated ADCC activity in SAS/shTFRC cells compared with sh mock or parental SAS cells. The results are shown as the mean ± the for each sample, and all were independently repeated in triplicate. * P < 0.05, ** P < 0.01. CDC, complement-dependent cytotoxicity; ADCC, antibody-dependent cellular cytotoxicity; OSCC, oral squamous cell carcinoma; TFRC, transferrin receptor C; LDH, lactate dehydrogenase.

Article Snippet: Eight human OSCC cell lines (Ca922, HO-1-u-1, HSC2, HSC3, HSC4, SAS, HSQ89, and Sa-3) were purchased from the RIKEN BioResource Center (Ibaraki, Japan).

Techniques: Activity Assay, Incubation, Lysis

Journal: Cancer Medicine

Article Title: Development of a complete human anti-human transferrin receptor C antibody as a novel marker of oral dysplasia and oral cancer

doi: 10.1002/cam4.267

Figure Lengend Snippet: Anti-TFRC antibody treatment blocks the incorporation of transferrin into OSCC cells. Transferrin uptake in the high-TFRC-expressing SAS and HSC2 cell lines at (A) 24 h and (B) 72 h after anti-TFRC antibody treatment. Transferrin was rapidly internalized after 5 min and continuously internalized up to 60 min in the control cells, but the anti-TFRC antibody-treated cells showed inhibited transferrin incorporation. These experiments were performed with a FACScan flow cytometer, and the quantity of internalized transferrin is represented by the MFI. The left panels show the results for the SAS cells and the right panels show the results for the HSC2 cells. The experiments were performed in triplicate and repeated independently at least three times. * P < 0.05, ** P < 0.01. OSCC, oral squamous cell carcinoma; TFRC, transferrin receptor C; MFI, mean fluorescence intensity.

Article Snippet: Eight human OSCC cell lines (Ca922, HO-1-u-1, HSC2, HSC3, HSC4, SAS, HSQ89, and Sa-3) were purchased from the RIKEN BioResource Center (Ibaraki, Japan).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: Cancer Medicine

Article Title: Development of a complete human anti-human transferrin receptor C antibody as a novel marker of oral dysplasia and oral cancer

doi: 10.1002/cam4.267

Figure Lengend Snippet: In vivo antitumor activity of the anti-TFRC antibody against OSCC in subcutaneously xenografted mice. (A) The anti-TFRC antibody reduced tumor growth in Rag-2/Jak3 double-deficient ( Rag-2 −/− Jak3 −/− ) immunodeficient mice that were xenografted with SAS cells. The anti-TFRC antibody was intravenously administered at 15 or 7.5 mg/kg twice per week for 3 weeks (arrowheads). Tumor growth was assessed by measuring the volume of each tumor at twice per week. Each group contained five mice. * P < 0.05, ** P < 0.01. (B) Body weights were measured at each time point. The body weights of the treated mice gradually increased during the treatment. (C) The xenografted mice were sacrificed 29 days after treatment, and the weights of the isolated tumors from each group were directly measured. Two photos show typical cases of mice treated with PBS (left) or 15 mg/kg of the anti-TFRC antibody (right). The right panel shows the comparison of the average tumor weights ± standard errors among the three groups. * P < 0.05, ** P < 0.01. OSCC, oral squamous cell carcinoma; TFRC, transferrin receptor C; PBS, phosphate-buffered saline.

Article Snippet: Eight human OSCC cell lines (Ca922, HO-1-u-1, HSC2, HSC3, HSC4, SAS, HSQ89, and Sa-3) were purchased from the RIKEN BioResource Center (Ibaraki, Japan).

Techniques: In Vivo, Activity Assay, Isolation