Intraepithelial CD8+ T-cell-count becomes a prognostic factor after a longer follow-up period in human colorectal carcinoma: possible association with suppression of micrometastasis.

T-cell infiltration into human cancer tissues can be a manifestation of host immune responses to cancer cells. The present study was undertaken to explore the clinicopathological significance of intraepithelial CD8(+) T cells using 371 consecutively sampled human colorectal carcinomas. By univariate analysis, we noted that the survival curves by intraepithelial CD8(+) T cells became separated only after 1 to 2 years postoperation. Multivariate analyses revealed that the beneficial effect of this factor becomes significant only after a longer (more than 2 year), but not after a shorter (less than 2 year) follow-up period. Furthermore, the number of intraepithelial CD8(+) T cells was significantly higher in patients alive for more than 5 years than in patients who either died of cancer after a curative operation or patients who underwent a noncurative operation. Patients' cancer-specific death long after a curative operation is thought to be caused by the growth of micrometastases in other organs or near the primary sites. The effects of intraepithelial CD8(+) T cells, therefore, may be mediated by suppression of micrometastasis, rather than suppression of growth in the primary tumour. In conclusion, our data support a hypothesis on the presence of systemic immunosurveillance against micrometastasis of cancer cells.

Allelic loss of the NF1 gene in anal malignant melanoma in a patient with neurofibromatosis type 1.

A 64-year-old man with neurofibromatosis type 1 (NF1) developed a primary malignant melanoma of the anus. Genetic analysis of the resected tumor confirmed loss of heterozygosity (LOH) of the NF1 gene. Anorectal malignant melanoma in NF1 is extremely rare, and genetic studies of the NF1 gene in such patients have not been reported. The allelic loss detected in the present patient supports the previously raised idea that NF1 can function as a tumor suppressor gene in the development of malignant melanoma in patients with NF1.

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.

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.

Role of nitric oxide and superoxide anion in elimination of low metastatic human colorectal carcinomas by unstimulated hepatic sinusoidal endothelial cells.

Human colorectal carcinoma (CRC) cell survival for the first 24 h after implantation in the hepatic sinusoid determines its potential to colonize the liver. Nearly 10-fold more highly metastatic CX-1 cells survive within the livers of nude mice 24 h after intrasplenic injection than weakly metastatic clone A cells. Because CRCs contact sinusoidal endothelial cells (SECs) during implantation, we sought to determine whether SECs were more toxic to clone A than to CX-1 cells. When 2 x 10(4) vital dye-labeled CRC cells were added to murine SEC monolayers, more than 30% of clone A cells lost calcein AM fluorescence compared to fewer than 5% of CX-1 cells after 24 h of coculture with SECs. Kupffer cells did not mediate this effect, because neither enriched Kupffer cells nor SECs treated with a Kupffer cell inhibitor altered the SEC-mediated toxic effect to clone A cells. Pretreatment with a nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine, superoxide dismutase, or dexamethasone, blocked SEC-mediated toxicity to clone A cells, whereas calcium chelation and catalase did not. In addition, clone A cells were more sensitive to a superoxide donor, 3-morpholinosydnonimine N-ethylcarbamide, than were CX-1 cells, and neither cell line was sensitive to sodium nitroprusside, a nitric oxide donor. Thus, unstimulated murine SECs produce reactive oxygen species that are selectively toxic to weakly metastatic clone A cells. This may be a mechanism by which host liver cells eliminate weakly metastatic neoplastic cells.

CD44H participates in the intrahepatic growth of murine colon 26 adenocarcinoma cells.

The purpose of this study was to determine if CD44, a metastasis-associated cell adhesion molecule, is involved in the hepatic colonization by murine colon 26 adenocarcinoma cells. Indirect membrane immunofluorescence and FACS analysis showed strong expressions of CD44 and integrin beta 1 on colon 26 cells. Injection of 1 x 10(5) colon 26 cells into the superior mesenteric vein of syngeneic BALB/c mice produced macroscopic hepatic nodules in 92% (22/24) of the mice 14 days after inoculation. When colon 26 cells were pretreated with an anti-CD44 monoclonal antibody (mAb), IM7, only 30% (3/10) of the mice produced minute nodules in the liver on day 14 (P < 0.001), though IM7 did not inhibit growth of the cells in vitro. Pretreatment of colon 26 cells with an anti-integrin beta 1 mAb did not significantly block the hepatic metastasis. Histologically, microcolonies of tumor cells were detected in all of the livers on day 14 including the IM7-pretreatment mice that were free of gross nodules. However, percentages of tumor-occupied areas in the liver were consistently lower in IM7-pretreatment mice than in control mice (0.82% vs. 5.0% on day 14; P < 0.005). Reverse transcription-polymerase chain reaction (RT-PCR) amplification of mRNA revealed that colon 26 cells and splenocytes only expressed the hematopoietic isoform of CD44 (CD44H), which had no insertion of variant exons, while normal colonocytes expressed possible variant isoforms. These data suggest that malignant transformation of murine colonic epithelium altered the expression pattern of CD44 isoforms and that CD44H participates in the intrahepatic growth of colon 26 cells.

Implantation of human colorectal carcinoma cells in the liver studied by in vivo fluorescence videomicroscopy.

In vivo fluorescence videomicroscopy (IVFM) was used to analyse the behavior of weakly and highly metastatic human colorectal carcinoma (CRC) cells during implantation in the liver. A highly metastatic human CRC cell line, CX-1, and a weakly metastatic line, Clone A, were double-labeled with rhodamine B isothiocyanate-dextran (Rd-Dx) to locate cells and with calcein AM to assess cell metabolic activity in an experimental metastasis model. Double-labeled CRC cells (2.0 x 10(6)) were injected into the spleens of groups of nude mice and the livers observed by IVFM over the next 72 h. CRC cells were implanted within 30 s after injection into either portal venules or the proximal third of hepatic sinusoids. Approximately 0.5% of CRC cells traversed the liver through portal-central venous shunts and implanted in the lung. The number of CX-1 cells in the liver was similar to that of Clone A cells during the first 12 h. However, more CX-1 cells than Clone A cells remained in the liver at 4 h and were in groups of 8-12 cells whereas only a few, single Clone A cells were detected in the liver at 72 h. Not all Clone A cells are committed to die within 4 h of implantation because cells harvested 4 h after hepatic implantation proliferated normally in vitro when removed from the hepatic microenvironment. Since the stress of mechanical deformation during implantation may cause differences in cell survival, CX-1 and Clone A cells were passed through filters with 8 microM pores in vitro at 10-15 cm of water pressure to recreate the trauma of hepatic implantation. Approximately 50% of both CX-1 and Clone A cells were lysed. Furthermore, both CRC lines remained metabolically active when co-cultivated with liver cells for at least 24 h in vitro. Thus, the difference in metastatic potential between the two CRC lines may reside in their response to the combination of mechanical implantation and subsequent growth in the liver parenchyma.

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 dendritic-shaped 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.

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.

Immunocytochemical localization of basic fibroblast growth factor in carcinomas and inflammatory lesions of the human digestive tract.

BACKGROUND: Basic fibroblast growth factor (bFGF) is one of the important angiogenic peptides. Clarification of its localization in human cancer and inflammatory diseases is important for the study of angiogenesis. EXPERIMENTAL DESIGN: Sliced human gastrointestinal carcinomas and inflammatory lesions were frozen after fixation. Immunocytochemical localization of bFGF was studied at the light and electron microscopic levels. RESULTS: In gastric carcinoma of the diffuse type, bFGF was mainly observed in some of the endothelial cells of the microvasculature and in fibroblasts. Gastric carcinoma of the intestinal type and colonic carcinoma showed the immunoreactivity in fibroblasts, vascular endothelial cells, and in the extracellular matrix (ECM). Carcinoma cells were occasionally positive. In inflammatory lesions, bFGF was most prominent in the ECM. Areas of deposition of bFGF in the ECM partly corresponded to areas of increased immunoreactivity for heparan sulphate proteoglycan. Immunoelectron microscopically, bFGF was localized in the cytosol of fibroblasts and cancer cells but no localization was observed in the lumen of rough endoplasmic reticulum or perinuclear space in any of the cells observed. bFGF was also localized along the luminal surface of vascular endothelial cells, in nuclei of fibroblasts, vascular endothelial cells, smooth muscle cells, and some cancer cells, and in the ECM. CONCLUSIONS: Extensive localization of bFGF in the ECM was related to the areas of increased vascular permeability and active angiogenesis. In cancer tissues, the distribution pattern of bFGF supports a paracrine mechanism among mesenchymal cells rather than a direct paracrine mechanism on vascular cells from carcinoma cells. Nuclear localization of bFGF may support its activity as a transcriptional factor.

Immunoelectron microscopic localization of transforming growth factor beta 1 and latent transforming growth factor beta 1 binding protein in human gastrointestinal carcinomas: qualitative difference between cancer cells and stromal cells.

Transforming growth factor beta 1 (TGF-beta 1) is secreted as an inactive complex associated with latent TGF-beta 1 binding protein (LTBP). Tissue localization of these proteins has not been fully understood in human pathological conditions. We examined the immunohistochemical localization of TGF-beta 1 precursor (proTGF-beta 1) and LTBP in carcinomas and granulation tissue in the human gastrointestinal tract at the light and electron microscopic levels. In normal tissue, endothelial cells and granulocytes sporadically showed immunoreactivity for proTGF-beta 1, while epithelial cells were all negative. In cancer tissue, both cancer cells and stromal cells (fibroblasts, macrophages, and endothelial cells) were positive for proTGF-beta 1, more frequently in diffuse-type gastric carcinomas than in differentiated-type adenocarcinomas. Immunoelectron microscopy revealed that proTGF-beta 1 was localized in rough endoplasmic reticulum and perinuclear cisternae in fibroblasts, macrophages, and endothelial cells in cancer stroma and in fibrous granulation tissue. In contrast, the intracellular localization of proTGF-beta 1 in carcinoma cells was predominantly observed in the cytosol (cytoplasmic matrix). This finding suggests disarranged or blocked intracellular transportation of proTGF-beta 1 in cancer cells. The immunoreactivity for LTBP was not observed in the normal epithelial cells. It was localized in cancer stroma, not in cancer cells. Ultrastructurally, LTBP was located in the extracellular matrix around fibroblasts and smooth muscle cells. The intracellular immunoreactivity for LTBP was observed in rough endoplasmic reticulum of fibroblasts and smooth muscle cells, the same as in granulation tissue. These results suggest that gastrointestinal carcinoma cells produce no or a small amount of LTBP in vivo. Our investigation suggests that extensive fibrosis in both cancer stroma and granulation tissues may be promoted by TGF-beta 1 mainly secreted from stromal cells.

Immunohistochemical identification of transforming growth factor-beta and its binding protein in human gastrointestinal carcinoma.

We attempted to clarify the tissue localization of TGF-beta and latent TGF-beta binding protein (LTBP) in human gastrointestinal carcinomas by immunohistochemistry. The immunoreactivity for TGF-beta was observed in both carcinoma cells and stromal cells, particularly in diffuse-type gastric carcinoma. The immunoreactivity for LTBP was observed only in stromal cells. These results suggest that both carcinoma cells and stromal cells may produce TGF-beta, and that only stromal cells may produce LTBP in gastrointestinal carcinoma.

[Relationship between venous invasion and hematogenous metastasis in the colorectal cancer patients].

We investigated the correlation between venous invasion and hematogenous metastasis. In thirteen patients with colorectal carcinoma, depth of invasion (ss.s or a1.a2) was the same. Four cases had hepatic metastasis at operation (group A), 3 cases were identified with hematogenous recurrence after operation (group B), and 6 cases were disease free over 10 years after operation (group C). Specimens were all step-sectioned and serial sections were stained both with HE and EM. Results were as follows. 1. Venous invasion was found in all cases and average rates of venous invasion (ARVI) of groups A and B were higher than that of group C (A; 7.8%, B; 6.3%, C; 2.1%, p less than 0.05). 2. In ss.s layer, ARVI of group A and B were higher than that of group C (A; 6.1%, B; 6.2%, C; 1.5%, p less than 0.05). 3. Rates of venous invasion to larger veins (diameter greater than 200 microns) were higher in group A than in others (A; 53.5%, B; 25.6%, C; 7.9%, p less than 0.05), and those to the middle-sized veins (diameter; 50-200 microns) were higher in group B than in others. These facts suggest that cancer invasion to the veins located in ss.s (a1.a2) layer or ranged in diameter over 200 microns relates to hematogenous metastasis.

Light and electron microscopic immunolocalization of endothelial leucocyte adhesion molecule-1 in inflammatory bowel disease. Morphological evidence of active synthesis and secretion into vascular lumen.

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 immunoreactive 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.