Takayuki Mizoi

Selective infiltration of CCR5(+)CXCR3(+) T lymphocytes in human colorectal carcinoma.

T cell infiltration in colorectal cancer is associated with a favorable prognosis, suggesting an occurrence of a certain degree of anti‐tumor immunity. T helper type 1 (Th1) and Th2 cells are now known to selectively express CC‐chemokine receptor 5 (CCR5)/CXC‐chemokine receptor 3 (CXCR3) and CCR4, respectively. To clarify the mechanism of T cell infiltration, we examined in situ expression of these chemokine receptors and their respective chemokine ligands in 40 cases of human colorectal cancer. Immunohistochemistry showed a predominant accumulation of T cells expressing CCR5 and CXCR3 mainly along the invasive margin, whereas those expressing CCR4 were rare. Flow cytometric analysis showed that more than half of CD8+ T cells and a fraction of CD4+ cells isolated from fresh tumor tissues co‐expressed CCR5 and CXCR3, and CD8+ T cells and CD4+ cells predominantly produced interferon‐γ (IFN‐γ) over interleukin‐4 (IL‐4) after in vitro stimulation. RANTES/CCL5, a ligand of CCR5, was localized within infiltrating CD8+ T cells in a granular pattern, whereas IP‐10/CXCL10, a ligand of CXCR3, was localized in cancer cells and macrophages along the invasive margin. These data were consistent with an active recruitment of T cells expressing CCR5 or CXCR3 into the invasive margin of colorectal cancer. With the previous clinicopathological studies showing a favorable prognostic impact of T cell infiltration in colorectal cancer, our study supports the occurrence of a certain level of Th1‐shifted cellular immune responses in human colorectal cancer.

Introduction of antisense CD44s cDNA down‐regulates expression of overall CD44 isoforms and inhibits tumor growth and metastasis in highly metastatic colon carcinoma cells

We created antisense CD44 transfectants using LS174T, a colon adenocarcinoma cell line and assessed the effects of overall CD44 down‐regulation on colorectal tumor growth and metastasis. The expression of antisense CD44s (the standard form of CD44) cDNA markedly inhibited the overall expression of CD44 variants. In vitro studies showed a significantly reduced ability of the stable antisense transfectants (LS174TAS1 and LS174TAS2) to bind hyaluronate and osteopontin, ligands for CD44. These cells developed tumors more slowly than controls (parental LS174T and mock transfectants) when the cells were subcutaneously injected into SCID mice. However, in vitro proliferation assays demonstrated no significant difference between the antisense transfectants and the controls on a hyaluronate‐coated surface, suggesting the participation of ligands other than hyaluronate in tumor growth in vivo. Intrasplenic injection of parental LS174T cells produced colonies in the liver in 10 of 11 mice, whereas mice injected with the antisense transfectants were completely free of metastasis. In peritoneal dissemination, the weight of nodules and amount of ascites were significantly reduced in LS174TAS1 and AS2 compared with the controls. In vitro adhesion assays between the transfectants or controls and human peritoneal mesothelial cells revealed that the binding of LS174T cells to mesothelial cells was partly mediated by CD44‐hyaluronate interaction. These data suggest that CD44‐hyaluronate interaction plays a crucial role in peritoneal dissemination in colorectal carcinoma. The results of our study demonstrate the possible application of antisense CD44s to the treatment of colorectal carcinoma. Int. J. Cancer 91:67–75, 2001.

Differing expression of MMPs-1 and-9 and urokinase receptor between diffuse-and intestinal-type gastric carcinoma

Gastric cancer is classified into intestinal and diffuse types, which exhibit different biological behavior. Urokinase‐type plasminogen activator (uPA), its receptor (uPAR) and matrix metalloproteinases (MMPs)‐1 and ‐9 are considered to play important roles in cancer invasion and metastasis. We have already suggested a functional duality of these matrix‐degrading enzymes/factors; they may also be involved in the matrix turnover (remodeling) or host immune/inflammatory reactions as far as they are expressed by host cells. We performed a retrospective study on the immuno‐histochemical expression of these enzymes/factors in surgical specimens from patients with gastric cancer, including 26 with the diffuse and 78 with the intestinal type. We also evaluated macrophages since they are major sources of uPAR. The positivity rate for uPA in cancer cells was significantly lower in diffuse‐type than in intestinal‐type. Stromal expression was seen mainly along the invasive margin (tumor–host interface). The degree of stromal expression of uPAR and MMP‐9 and the macrophage number were markedly decreased in diffuse‐type compared with intestinal‐type. Stromal expression of uPAR and macrophage number in intestinal‐type were higher in patients without liver metastasis than in patients with liver metastasis, while uPA expression in cancer cells was more pronounced in patients with liver metastasis. Studies using frozen sections revealed that the expression of MMP‐1, restricted to the stromal area, was more decreased in diffuse‐type (18 patients) than in intestinal‐type (21 patients). Our results show that the in situ expression of matrix‐degrading enzymes/factors in gastric cancer is significantly more diminished in diffuse‐type than in intestinal‐type, suggesting a multifunctional aspect of the matrix‐degradation process in cancer tissue. Int. J. Cancer (Pred. Oncol.) 84:74–79, 1999.

Light and electron microscopic immunolocalization of endothelial leucocyte adhesion molecule-1 in inflammatory bowel disease

Endothelial leucocyte adhesion molecule-1 (ELAM-1) is a rapidly inducible adhesion molecule for neutrophils in vascular endothelial cells. We investigated its immunohistochemical localization in 17 cases of inflammatory bowel disease. ELAM-1 was preferentially expressed in venules in actively inflamed mucosa and granulation tissue. Most capillaries were negative for ELAM-1. In areas with mild inflammation its expression diminished markedly and in normal mucosa of the colon and small intestine its expression was sparse. Electron microscopically, venules in active inflammation had swollen endothelial cells with well-developed rough endoplasmic reticulum. Immunoelectron microscopy revealed ELAM-1 localization along the luminal plasma membrane and in rough endoplasmic reticulum and round granules, findings suggestive of active production in endothelial cells. Furthermore, exocytosis of immuno-reactive substance into the lumen was confirmed. Our study suggests that venules in actively inflamed area play an important role in eliciting and/or maintaining acute inflammatory processes by active permeation of neutrophils from the blood stream into the tissue, and that ELAM-1 may be a secretory protein as well as a transmembrane receptor protein.

Outcomes after Pylorus-preserving Gastrectomy for Early Gastric Cancer: A Prospective Multicenter Trial

The aim of the present study was to compare in a prospective, multicenter trial the results early and late after pylorus-preserving gastrectomy (PPG) versus conventional distal gastrectomy (CDG) with Billroth I anastomosis for early gastric cancer. Eighty-one patients with early gastric cancer were randomized and then underwent either PPG or CDG. Duration of operation, intraoperative blood loss, days until removal of the nasogastric tube, days until start of oral intake, and decrease in body weight were studied as parameters for outcomes early after the surgery. Late results were studied in patients followed for longer than 3 years. Change in body weight, status of oral intake, symptoms suggesting early dumping syndrome, and overall satisfaction were addressed in the questionnaire. The presence of gallstones was examined with ultrasonography. There were no differences in early results between PPG and CDG. The incidence of early dumping syndrome was lower in PPG (8%) than in CDG (33%). Other late results including the incidence of gallstones were not different between the 2 groups. These results indicate that PPG is as safe as CDG and has an advantage in terms of early dumping syndrome.

Cell Adhesion Molecule Expression by Vascular Endothelial Cells as an Immune/Inflammatory Reaction in Human Colon Carcinoma

The cell adhesion of inflammatory cells to vascular endothelial cells is an important process in the recruitment of inflammatory cells to the site. In cancer tissue, infiltration of inflammatory cells has been suggested to be a mechanism of host resistance. To clarify this infiltration mechanism, we investigated cell adhesion molecule expression (E‐selectin, P‐selectin, and ICAM‐1) in vascular endothelial cells by immunohistochemistry in colon carcinoma. Venules distributed along the invasive margin expressed E‐ and P‐selectins and ICAM‐1. These phenotypical features are identical to those of endothelial cells observed in active inflammatory lesions, and the vessels can, therefore, be designated as immunologically activated vessels. Nevertheless, the majority of blood vessels within the tumor lacked immunoreactivity for all these adhesion molecules and, therefore, could be designated as immunologically inactive vessels. Granulocytes, lymphocytes and macrophages, bearing the counter‐receptors of these adhesion molecules, were more densely distributed along the invasive margin. In contrast, few inflammatory cells were present within the tumor. In conclusion, the present study has demonstrated the phenotypical heterogeneity of tumor vessels; those for inflammatory cell infiltration to the tumor and those for the nutrient supply to the tumor.

Orthotopic implantation mouse model and cDNA microarray analysis indicates several genes potentially involved in lymph node metastasis of colorectal cancer

In colorectal cancer (CRC) patients, metastasis to the regional lymph node (LN) is an important first step in the dissemination of cancers. To identify the genes possibly involved in LN metastasis of CRC, we analyzed LN metastases in an orthotopic implantation mouse model with 22 CRC cell lines using Matrigel, an extracellular matrix protein derived from mice sarcoma, and combined the data with gene expression profiles of cDNA microarray of those cell lines. With this implantation analysis, the incidence of LN metastasis was 60% in 228 orthotopically implanted mice and varied from 100% to 0% among the cell lines. KM12c and Clone A showed LN metastasis in all orthotopically implanted mice, but DLD‐1, HCT‐8, and SW948 did not show LN metastases at all. In contrast, the incidence of liver and lung metastasis in 22 CRC cell lines was 13% and 1%, respectively. Combining those data with cDNA microarray in vitro, we isolated 636 genes that were differentially expressed depending on the incidence of LN metastasis. Among those genes, the expression level of ring finger protein 125 (RNF125), previously known as an E3 ubiquitin ligase in T cell activation, was significantly different between primary tumors in Stage III CRC patients with LN metastasis and Stage II patients without LN metastasis. In conclusion, the orthotopic implantation mice model with Matrigel was useful, and we isolated candidate genes such as RNF125 that possibly play an important role in LN metastasis of CRC cells. (Cancer Sci 2008; 99: 711–719)

Close Association between Fas Ligand (FasL; CD95L)-positive Tumor-associated Macrophages and Apoptotic Cancer Cells along Invasive Margin of Colorectal Carcinoma: A Proposal on Tumor-Host Interactions

Anti‐tumor immune responses are considered to be one of the key host reactions in human colorectal cancer, with T cells as important effector cells. For the induction of tumor‐specific immunity, processing of cancer cells and pruning of T cells by antigen‐presenting cells are important. The present study was designed to clarify the relationship between Fas ligand (FasL; CD95L) expression and apoptotic cancer cells. Immunohistochemistry using frozen sections taken from 58 patients with colorectal cancer revealed that stromal cells composed mainly of tumor‐associated macrophages expressed FasL, leaving cancer cells negative for FasL. These macrophages were abundantly distributed along the invasive margin. In situ hybridization revealed that these macrophages as well as cancer cells expressed FasL mRNA, whereas macrophages in the normal colon mucosa rarely expressed FasL. Apoptotic cancer cells recognized by monoclonal antibody M30 CytoDEATH were localized not only in cancer cell nests, but also in the stroma along the invasive margin showing a dissociated pattern, which was particularly evident in the areas of FasL+ macrophages. Furthermore, these macrophages, phenotypically similar to dendritic cells, occasionally contained M30+ apoptotic cancer cells in the cytoplasm. Clinicopathologic analyses in 123 cases revealed 1) a positive correlation between the degree of dissociated M30+ apoptotic cancer cells and the number of macrophages along the invasive margin and 2) an inverse association between the degree of dissociated M30+ apoptotic cancer cells and the occurrence of hematogenous metastasis after surgical resection of the primary tumor. In conclusion, the present study shows the impor‐ tance of FasL+ activated macrophages as one of the host defense mechanisms against cancer cell spread in human colorectal cancer.

Intercellular adhesion molecule-1 expression by macrophages in human gastrointestinal carcinoma: Possible roles as host immune/inflammatory reaction

Tumor‐associated macrophages (TAM) are one of the factors which modulate the carcinoma progression. The present study described immunohistochemical expression of intercellular adhesion molecule‐1 (ICAM‐1) in stromal cells in human gastrointestinal carcinoma identifying the cell types by immunoelectron microscopy. In colon and gastric carcinomas, ICAM‐1‐positive cells were mostly stromal cells, and major cell types were identified as macrophages and fibroblasts by immunoelectron microscopy. Macrophages were characterized by their ovoid shape, cytoplasmic projections, abundant vacuoles, phagocytosis, and paucity of rough endoplasmic reticulum. Fibroblasts contained stacks of rough endoplasmic reticulum. Macrophages were major cells among ICAM‐1‐positive cells along the invasive margin, while fibroblasts were predominant in the stroma within carcinoma in colon and intestinal‐type gastric carcinomas. Lymphocytes positive for lymphocyte function associated antigen (LFA‐1), a counter‐receptor of ICAM‐1, were densely distributed along the invasive margin, and sparsely in the stroma within carcinoma. In diffuse‐type gastric carcinoma, most macrophages were dendriticshaped and negative for ICAM‐1. Our study suggests that the invasive margin is an area similar to active inflammation, where the antigen presenting cells (macrophages) and lymphocytes may interact via the ICAM‐1/LFA‐1 adhesion.

Cancer-associated splicing variants of the CDCA1 and MSMB genes expressed in cancer cell lines and surgically resected gastric cancer tissues

BACKGROUND Alternative splicing is a molecular mechanism by which different combinations of exons can be alternatively spliced to produce different mRNA isoforms. Recently, several databases have been published to predict the alternative splicing of mRNA; cancer-specific alternative splicing has also been predicted with these databases. Those variants may be potentially useful targets for cancer therapy, however, the accuracy and veracity of these databases have yet to be confirmed. METHODS In this study, we analyzed 17 genes by reverse transcriptase-polymerase chain reaction (RT-PCR) that were predicted to have cancer-specific alternative splicing by using the splicing database, the Alternative Splicing Annotation Project (ASAP) by Lee et al, between 38 cancer cell lines from various organs and 9 corresponding normal tissues. By designing 2 types of primer sets for RT-PCR including (1) primers flanking the alternatively spliced exons and (2) primers spanning the exon/exon junctions, cancer-associated splicing variants were investigated. RESULTS The alternatively splicing events were detected in 15 of 17 genes (88%); 35 of 43 variants (81%) were detected successfully with RT-PCR. Among these variants, M-RIP, HYAL2, CDCA1, and MSMB genes showed differential expressions between cancer cell lines and corresponding normal tissues. Furthermore, RT-PCR with surgically resected gastric cancer tissues (diffuse type, 6; intestinal type, 4) confirmed that 2 variants of CDCA1 were upregulated in cancer tissues, whereas both variants of MSMB were expressed predominantly in normal tissues. CONCLUSION Alternative splicing variants, especially in CDCA1, were detected in this study and may be potentially useful as diagnostic markers and/or novel targets for anticancer therapy.