HLA-A*0201-restricted CTL epitope of a novel osteosarcoma antigen, papillomavirus binding factor

<p>Abstract</p> <p>Background</p> <p>To develop peptide-based immunotherapy for osteosarcoma, we previously identified papillomavirus binding factor (PBF) as a CTL-defined osteosarcoma antigen in the context of HLA-B55. However, clinical application of PBF-based immunotherapy requires identification of naturally presented CTL epitopes in osteosarcoma cells in the context of more common HLA molecules such as HLA-A2.</p> <p>Methods</p> <p>Ten peptides with the HLA-A*0201 binding motif were synthesized from the amino acid sequence of PBF according to the BIMAS score and screened with an HLA class I stabilization assay. The frequency of CTLs recognizing the selected PBF-derived peptide was determined in peripheral blood of five HLA-A*0201⁺patients with osteosarcoma using limiting dilution (LD)/mixed lymphocyte peptide culture (MLPC) followed by tetramer-based frequency analysis. Attempts were made to establish PBF-specific CTL clones from the tetramer-positive CTL pool by a combination of limiting dilution and single-cell sorting. The cytotoxicity of CTLs was assessed by ⁵¹Cr release assay.</p> <p>Results</p> <p>Peptide PBF A2.2 showed the highest affinity to HLA-A*0201. CD8+ T cells reacting with the PBF A2.2 peptide were detected in three of five patients at frequencies from 2 × 10^-7to 5 × 10^-6. A tetramer-positive PBF A2.2-specific CTL line, 5A9, specifically lysed allogeneic osteosarcoma cell lines that expressed both PBF and either HLA-A*0201 or HLA-A*0206, autologous tumor cells, and T2 pulsed with PBF A2.2. Five of 12 tetramer-positive CTL clones also lysed allogeneic osteosarcoma cell lines expressing both PBF and either HLA-A*0201 or HLA-A*0206 and T2 pulsed with PBF A2.2.</p> <p>Conclusion</p> <p>These findings indicate that PBF A2.2 serves as a CTL epitope on osteosarcoma cells in the context of HLA-A*0201, and potentially, HLA-A*0206. This extends the availability of PBF-derived therapeutic peptide vaccines for patients with osteosarcoma.</p

Analysis of cell cycle-related proteins in gastric intramucosal differentiated-type cancers based on mucin phenotypes: a novel hypothesis of early gastric carcinogenesis based on mucin phenotype

<p>Abstract</p> <p>Background</p> <p>Abnormalities of cell cycle regulators are common features in human cancers, and several of these factors are associated with the early development of gastric cancers. However, recent studies have shown that gastric cancer tumorigenesis was characterized by mucin expression. Thus, expression patterns of cell cycle-related proteins were investigated in the early phase of differentiated-type gastric cancers to ascertain any mechanistic relationships with mucin phenotypes.</p> <p>Methods</p> <p>Immunostaining for Cyclins D1, A, E, and p21, p27, p53 and β-catenin was used to examine impairments of the cell cycle in 190 gastric intramucosal differentiated-type cancers. Mucin phenotypes were determined by the expressions of MUC5AC, MUC6, MUC2 and CD10. A Ki-67 positive rate (PR) was also examined.</p> <p>Results</p> <p>Overexpressions of p53, cyclin D1 and cyclin A were significantly more frequent in a gastric phenotype than an intestinal phenotype. Cyclin A was overexpressed in a mixed phenotype compared with an intestinal phenotype, while p27 overexpression was more frequent in an intestinal phenotype than in a mixed phenotype. Reduction of p21 was a common feature of the gastric intramucosal differentiated-type cancers examined.</p> <p>Conclusions</p> <p>Our results suggest that the levels of some cell cycle regulators appear to be associated with mucin phenotypes of early gastric differentiated-type cancers.</p

Analysis of Molecular Alterations in Left- and Right-Sided Colorectal Carcinomas Reveals Distinct Pathways of Carcinogenesis : Proposal for New Molecular Profile of Colorectal Carcinomas

To clarify distinct genetic profiles of colorectal cancers based on tumor location (left- and right-sided), we evaluated the status of loss of heterozygosity (LOH), CpG islands methylation phenotype (CIMP), microsatellite instability (MSI), and mutations of p53, Ki-ras, and APC genes in 119 colorectal cancers. Statuses of LOH (at 5q, 8p, 17p, 18q, and 22q), MSI, and CIMP (MINT1, MINT2, MINT31, MLH-1, MGMT, p14, p16, and RASSF1A) were determined using microsatellite polymerase chain reaction and methylation-specific polymerase chain reaction coupled with a crypt isolation method, respectively. In addition, mutations of p53, Ki-ras, and APC genes were also examined. LOH, MSI, and CIMP status allowed us to classify samples into two groups: low or negative and high or positive. Whereas the frequency of p53 mutations in the LOH-high status was significantly higher in left-sided cancers than in right-sided cancers, CIMP-high in the LOH-high status and MSI-positive status were more frequently found in right-sided cancers compared with left-sided cancers. Finally, location-specific methylated loci were seen in colorectal cancers: type I (dominant in right-sided cancer) and type II (common in both segments of cancer). Our data confirm that distinct molecular pathways to colorectal cancer dominate in the left and right sides of the bowel

High Expression of CD109 Antigen Regulates the Phenotype of Cancer Stem-Like Cells/Cancer-Initiating Cells in the Novel Epithelioid Sarcoma Cell Line ESX and Is Related to Poor Prognosis of Soft Tissue Sarcoma

<div><p>Epithelioid sarcoma (ES) is a relatively rare, highly malignant soft tissue sarcoma. The mainstay of treatment is resection or amputation. Currently other therapeutic options available for this disease are limited. Therefore, a novel therapeutic option needs to be developed. In the present study, we established a new human ES cell line (ESX) and analyzed the characteristics of its cancer stem-like cells/cancer-initiating cells (CSCs/CICs) based on ALDH1 activity. We demonstrated that a subpopulation of ESX cells with high ALDH1 activity (ALDH^high cells) correlated with enhanced clonogenic ability, sphere-formation ability, and invasiveness <i>in vitro</i> and showed higher tumorigenicity <i>in vivo</i>. Next, using gene expression profiling, we identified CD109, a GPI-anchored protein upregulated in the ALDH^high cells. CD109 mRNA was highly expressed in various sarcoma cell lines, but weakly expressed in normal adult tissues. CD109-positive cells in ESX predominantly formed spheres in culture, whereas siCD109 reduced ALDH1 expression and inhibited the cell proliferation <i>in vitro</i>. Subsequently, we evaluated the expression of CD109 protein in 80 clinical specimens of soft tissue sarcoma. We found a strong correlation between CD109 protein expression and the prognosis (<i>P</i> = 0.009). In conclusion, CD109 might be a CSC/CIC marker in epithelioid sarcoma. Moreover, CD109 is a promising prognostic biomarker and a molecular target of cancer therapy for sarcomas including ES.</p> </div

CD109 positively regulates ALDH activity and negatively regulates the TGFβ/Smad signaling pathway.

<div><p>A. The cell proliferation curve of ESX treated with SiCD109 or control siRNA. Bars represent mean±SEM (n=4). ^※<i>p</i><0.05, determined by the Mann-Whitney test.</p> <p>B, C. mRNA expression of ALDH1A1 (D) and TGFβ1R1 (E). The expression of ALDH1A1 and TGFβ1R1 was evaluated by real-time PCR two days after transfection.</p> <p>D. FACS analysis of ALDH activity.</p> <p>E. The proportion of ALDH^high cells. Bars represent mean±SEM (n=4). ^※<i>p</i><0.05, determined by the Mann-Whitney test.</p></div

Cancer-initiating ability of ALDH^high cells <i>in vitro</i> and <i>in vivo</i>.

<div><p>A. The features of spherical colonies derived from resultant ALDH^high cells and ALDH^low cells of ESX.</p> <p>B. The number of spherical colonies from ALDH^high and ALDH^low cells of ESX. Bars represent mean±SEM (n=6). ^※※※<i>p</i><0.001, determined by the Mann-Whitney test. </p> <p>C. The mRNA expression of stem/progenitor cell-related genes. RNA was isolated from freshly sorted spheroid cells (1x10⁵) on day 7. Bars represent mean±SEM. ^※<i>p</i><0.05, determined by the Mann-Whitney test.</p> <p>D. The features of xenotransplanted cells <i>in </i><i>vivo</i>. Macroscopic features of 1×10⁴ ALDH^high and ALDH^low cells of ESX in an NOD/SCID mouse at 10 weeks after xenotransplantation.</p> <p>E. Tumor growth curve of ALDH^high and ALDH^low　cells of ESX. Bars represent the mean±SD (ALDH^high n= 4, ALDH^low n=2).</p> <p>F. Tumorigenesis of ALDH^high and ALDH^low cells of ES cell lines in NOD/SCID mice.</p> <p>G. H&E of the xenotransplanted tumors derived from ALDH^high and ALDH^lowcells of ESX (1×10⁴) (above; scale bar 50μm, below; 20μm).</p></div

CD109 is a representative marker for the features of cancer-initiating ability.

<div><p>A. FACS analysis of CD109 positive cells in ESX was shown. </p> <p>B. The mRNA expression of CD109 and ALDH1A1. Bars represent mean±SEM. ^※※※<i>p</i><0.001, determined by the Mann-Whitney test.</p> <p>C. The features of spherical colonies (indicated by arrows) derived from resultant CD109-positive cells and CD109-negative cells of ESX (original magnification ×40).</p> <p>D. The number of spherical colonies from CD109-positive cells and CD109-negative cells of ESX. Bars represent mean±SEM (n=3). ^※※<i>p</i><0.01, determined by the Mann-Whitney test. </p> <p>E. The mRNA expression of stem/progenitor cell-related genes. Bars represent mean±SEM. ^※<i>p</i><0.05, determined by the Mann-Whitney test.</p></div

Identification of an ALDH^high population in ES cell lines.

<div><p>A. All 3 ES cell lines, ESX, VA-ES-BJ and FU-EPS-1, demonstrated ALDH activity. FACS analysis of ALDH1 expression in cells and the DEAB control.</p> <p>B. The proportions of ALDH^high cells in ESX, VA-ES-BJ and FU-EPS-1. Bars represent mean±SEM (n=4) of multiple experiments. ^※※※<i>p</i><0.001, determined by the Mann-Whitney test. </p> <p>C. Differentiation of ALDH^high and ALDH^low cells in vitro. Sorted ALDH^high and ALDH^low cells were analyzed on days 0 (immediately after sorting), 9 and 12. Representative fluorescence-activated cell sorting analysis is shown.</p></div