Influence of host defenses on the hepatic colonization of B16F10 melanoma cells

To investigate the significance of host immunity in metastasis we have simultaneously evaluated metastatic development and the tumoricidal action of host defenses in an experimental system for liver metastasis which involves the intrasplenic injection of B16F10 melanoma cells in syngeneic mice. In addition, three experimental groups were treated with immunosuppressive doses of cyclosporin A (CsA) during the following periods of the malignant process: 1st–5th days, 1st–12th days and 7th–12th days. Analysis of cytolytic effects of macrophages, NK cells and T-lymphocytes on tumor cells reveals a decay in antitumor immunity from the 7th day to the 12th day and a marked resistance of B16F10 melanoma cells derived from hepatic metastases to T-lymphocytes and NK cells. The 1st–5th day CsA treatment of tumor-bearing mice produced a reduction in both T-lymphocyte and macrophage reactions against tumor cells and a significant increase in the 7th day micrometastasis incidence in the liver. Once micrometastases have been established the CsA-treatment suppression on the 5th day allows the tumor growth rate in these mice to become the same as in controls. However, the 7th–12th day CsA treatment produces a clear inhibitory effect on focal metastatic development which may correspond to thein vitro antiproliferative effect of CsA, detected on cultured B16F10 melanoma cells.Recipient of financial support and Head of Department.     

Mouse Colon Carcinoma Cells Established for High Incidence of Experimental Hepatic Metastasis Exhibit Accelerated and Anchorage-Independent Growth

Highly metastatic variants of mouse colon 38 colon carcinoma cells were established by repeated selection in vivo for liver metastasis and designated as SL4 cells. The SL4 cells formed colonies in the liver of 100% of syngenic mice when injected intrasplenically, while the incidence of liver metastasis was 27% of mice injected with parental cells. The weight of livers, which is an indicator of experimental hepatic metastasis formation, was significantly higher after intrasplenic injection and subsequent splenoctomy with SL4 cells than colon 38 cells. The incidence of hepatic metastasis after intracecal injection of SL4 cells was significantly higher than that of colon 38 cells. The SL4 cells were tested in vitro for their properties. Differences were not detected in the motility and invasive behavior between colon 38 cells and SL4 cells. SL4 cells showed a higher proliferation rate than colon 38 cells under adherent conditions. SL4 cells maintained a capacity to proliferate under non-adherent conditions whereas parental cells did not. SL4 cells should be a useful tool to study the mechanism of hepatic metastasis of colon carcinoma cells and to develop methods to prevent hepatic metastasis.     

Macrophage Populations in Different Stages of Induced Hepatic Metastases in Rats: an Immunohistochemical Analysis

Macrophages play a role in the host defence against cancer. Little is known about changes in macrophage populations during early metastatic growth. To evaluate the distribution, number and phenotype of macrophages in the development of hepatic metastases in a rat model (Wag/Rij rats and syngeneic CC531 colon carcinoma cell line), an immunohistochemical study was performed with the monoclonal antibodies ED1 (monocytes, and all macrophages), ED2 (resident tissue macrophages, like Kupffer cells) and ED3 (a subpopulation of macrophages which may play a role in the recruitment of lymphocytes). OX19 and Hisl4 were used to identify lymphocytes. In this study a new monoclonal antibody CC52 is described, which recognizes the CC531 tumour cell line. Liver metastases were induced by injection of CC53I colon carcinoma cells into a mesenteric vein. Rats were killed at various intervals. Results show three major macrophage populations during hepatic tumour growth: (1) on day 3, infiltrates are observed around the micrometastases, which contain mainly newly recruited macrophages (ED1⁺ and ED2⁻); (2) after 7 days, ED3-positive (ED3 ⁺) macrophages together with T lymphocytes are found in the infiltrates; (3) an increase in the number of ED2-positive (ED2⁺) Kupffer cells is observed in the liver parenchyma after 14 days. In conclusion, the present results suggest that various populations of macrophages, newly recruited (ED1⁺) as well as resident Kupffer cells (ED2⁺), are involved in the immune response against tumour cell deposits in the liver.     

Ultrastructure of early phase hepatic metastasis of human colon carcinoma cells with special reference to desmosomal junctions with hepatocytes

To demonstrate ultrastructural events in the early phase of hepatic metastasis of human colon carcinoma, we intrasplenically injected a highly metastasizable, human colon carcinoma cell line LM-H3 (1 x 10(6) cells) into nude mice, and electron microscopically investigated the hepatic metastasis. At 24 h, tumor cells adhered to the endothelial wall of terminal portal venules and periportal sinusoids. At 48-72 h, after extravasation, they deeply invaded the hepatic cell plate and the interstitial tissue of the portal tract, in which they underwent proliferation and made the metastatic foci. Tumor cells were linked with each other or with surrounding hepatocytes by desmosomes. Desmosomes were maintained during the mitosis. When invading tumor cells were exposed to the bile canaliculi, they generated microvilli on the surface. Microvilli were also formed at the luminal surface of intracytoplasmic inclusions. In the interstitial tissue of the portal tract, tumor cells were closely associated with fibroblasts. However, no junctional specializations were seen between them. The present study demonstrated that human colon carcinoma cell line LM-H3 formed desmosomes with hepatocytes soon after invasion of the hepatic cell plate, suggesting the regulatory role of an interaction with hepatocytes in the growth of metastatic foci within the liver parenchyma.     

Changes in the macrophage content of lung metastases at different stages in tumor growth.

The macrophage content of experimental B16 melanoma metastases at different stages of their growth has been quantified with the use of morphometry in conjunction with a recently developed histochemical method for selectively staining intratumoral macrophages. Data are presented from analyses of 954 sections of 155 individual lung metastases, showing that the macrophage content of individual B16 melanoma lung metastases not only varies significantly but also falls dramatically once metastases contain more than 700 tumor cells. In addition to providing new information on host response reactions of micrometastases, these experiments also indicate that conclusions on intratumoral macrophages derived from studies of large primary tumors and metastases in advanced stages of growth may have little or no relevance to events in micrometastases.     

Aberrant metastatic behavior and particular features of early gastric cancer

In this paper, we have focused on the metastatic behavior of EGC and its particularities. The main factors that are currently considered as predictors of the metastatic behavior and that are used in the therapeutic decision (endoscopic resection vs surgical removal) are the tumor size (upper or bellow 2 cm), depth of infiltration, angiolymphatic invasion, the presence or absence of ulceration, and histologic type (undifferentiated vs differentiated carcinomas). However, most of the metastatic cases were published as case reports or case series. This is the reason why a proper estimation of metastatic risk in EGC is not well known. To date, 79 cases presenting bone metastases, three reports of brain metastases, and one EGC that was associated with skin metastasis were published. However, occult metastasis, lymph node micrometastasis, and skip metastasis can also occur and should be identified. Making a synthesis of the literature data that is correlated with our experience, we finally proposed the inclusion of the six Japanese subgrouping system, tumor size, angiolymphatic invasion, and micrometastasis as components of the pTNM staging system, which should be particularly adapted for EGC.     

Correlation between expression of MMP-7 in gastric submucosal invasive carcinoma and lymph node micrometastasis

To clarify the mechanism of tumor metastasis, lymph nodes micrometastasis and expression of MMP-7, which is related to lymph node metastases, were investigated in 45 submucosal, invasive gastric carcinomas. Metastases to lymph nodes were detected in 12 of 45 cases(26.7%) by HE stain. In 13 of 45 cases (28.9%), only micrometastases were detected by immunostaining with anti-cytokeratin antibody. The incidence of micrometastases was higher in poorly-differentiated carcinoma than well-differentiated carcinoma. Expression of MMP-7 was higher in metastasis positive cases than in negative cases. In poorly-differentiated cases, expression of MMP-7 was associated with micrometastasis. In conclusion, expression of MMP-7 may play an important role not only in tumor metastasis but in micrometastasis to lymph node especially in poorly-differentiated cases.     

Can immunohistochemistry enhance the detection of micrometastases in pelvic lymph nodes from patients with high-grade urothelial carcinoma of the bladder?

Immunohistochemistry for keratin has enhanced our ability to detect micrometastases in certain cancer patients with negative lymph nodes by routine histologic examination of H&E-stained sections. However, there is no information about micrometastasis of bladder cancer. We performed immunohistochemistry for keratins on 159 pelvic lymph nodes, which were negative for metastatic tumors on routine H&E-stained sections, from 19 patients with high-grade muscle invasive urothelial bladder cancer. In 1 man, 1 lymph node contained a keratin-positive micrometastasis that was not present on the original H&E-stained slide. However, the metastasis was seen readily on a new H&E-stained section prepared from the paraffin block adjacent to the keratin-stained section. Immunohistochemical analysis for keratins revealed no additional case of micrometastasis of urothelial carcinoma of the bladder. The perinodal fibroadipose tissue of a lymph node from a woman contained a few keratin-positive benign glands of endosalpingiosis. A thorough examination of the H&E-stained sections is the best method for detecting lymph node metastases of urothelial carcinoma from the bladder. There is a potential risk for misdiagnosis of metastases by using immunohistochemistry or polymerase chain reactions for keratins because of the occasional presence of benign epithelial cells in pelvic lymph nodes and associated connective tissue.     

Neutrophil-mediated experimental metastasis is enhanced by VEGFR inhibition in a zebrafish xenograft model

Inhibition of VEGF signalling effectively suppresses localized tumour growth but accelerates tumour invasiveness and micrometastasis by unknown mechanisms. To study the dynamic and reciprocal interactions between tumour cells and their microenvironment during these processes, we established a xenograft model by injecting tumour cells into the blood circulation of transparent zebrafish embryos. This reproducibly results in rapid simultaneous formation of a localized tumour and experimental micrometastasis, allowing time-resolved imaging of both processes at single-cell resolution within 1 week. The tumour vasculature was initiated de novo by remodelling of primitive endothelial cells into a functional network. Roles of myeloid cells in critical tumourigenesis steps such as vascularization and invasion were revealed by genetic and pharmaceutical approaches. We discovered that the physiological migration of neutrophils controlled tumour invasion by conditioning the collagen matrix and forming the metastatic niche, as detected by two-photon confocal microscopy and second harmonic generation. Administration of VEGFR inhibitors blocked tumour vascularization and a localized tumour growth but enhanced migration of neutrophils, which in turn promoted tumour invasion and formation of micrometastasis. This demonstrates the in vivo cooperation between VEGF signalling and myeloid cells in metastasis and provides a new mechanism underlying the recent findings that VEGFR targeting can promote tumour invasiveness.     

Characterization of the invasive and metastatic phenotype in human renal cell carcinoma

The purpose of these studies was to identify some characteristics of metastatic cells and deficiencies of non-metastatic cells in the heterogeneous SN12 human renal cell carcinoma. The SN12 parental line and several isolated variants with different metastatic potential were studied bothin vivo andin vitro. We compared the ability of metastatic and non-metastatic cells to adhere to components of the extracellular matrix or to endothelial cells, to migrate and invade, to form multicell aggregates, to survive in the circulation, and to produce experimental and spontaneous lung metastases. In general, highly metastatic SN12 cells capable of producing spontaneous lung metastases demonstrated invasion through reconstituted basement membranecoated filters; the cells also released diffusible collagenolytic activity into the culture medium that could enhance invasion by otherwise non-invasive and non-metastatic SN12 cells. In addition to enhanced invasion, metastatic cells produced more homotypic aggregation then non-metastatic cells and survived to produce experimental metastasis. Collectively, these data confirm that metastatic cells must complete all steps of the process; in this process, failure to produce metastasis is probably due to one or more deficiencies.     

Nuclear profiles of cancer cells reveal the metastatic potential of gastric cancer

Nuclear profiles have been reported as useful prognostic predictors in various cancers. Data from computerized morphometry are objective and can be quickly derived using conventional microscopic analysis, but image analysis of nuclear features has only rarely been applied to investigations of gastric cancer. The aim of this study was to evaluate the correlation between one of these morphological nuclear features and the clinicopathological parameters in patients with gastric cancer. The morphometric nuclear feature (nuclear area) was analysed in 400 patients with gastric cancer. In each case, 300 cancer nuclei on routine haematoxylin and eosin-stained slides were analysed through the use of a computer-assisted image analysis system which traced the nuclear profiles (magnificationx400) on a computer monitor. The morphometric data were compared with the patients' clinicopathological status and survival rate. The mean nuclear area (NA) of cancer cells from 400 cases of gastric cancer was 47.2 microm(2). The NAs of cancer cells from tumours with microvessel invasion (lymphatic or venous invasion), lymph node metastasis or hepatic metastasis at the time of operation were significantly larger than those of cancer cells from tumours without such invasion or metastases. Cytokeratin (CK) immunostaining was performed on 2577 lymph nodes from 91 patients with advanced gastric cancer (pT3, pN0, pM0, stage II) to detect micrometastases. CK-positive lymph nodes were detected in 350 of 2577 lymph nodes (13. 6%) and in 62 of 91 patients (68.1%). The mean NA of cancer cells from 62 tumours with micrometastases (44 microm(2)) was larger than that of cancer cells from 29 tumours without micrometastases (38.8 microm(2), p=0.043), and a significant positive correlation was detected between the NAs of cancer cells from 91 tumours and the number of micrometastatic lymph nodes of 91 patients (rho=0.278, p=0. 008). Cancer cells with large NA correlated strongly with haematogenous and lymph node recurrence or relapse after gastrectomy and the NA of cancer cells was identified as an independent prognostic factor in gastric cancer. Nuclear morphometry is an objective, reproducible, and technically uncomplicated procedure. The NA of cancer cells correlates closely with the metastatic potential of gastric cancer. Nuclear morphometry may therefore be useful for the selection of patients who are at risk of haematogenous or lymph node metastatic recurrence after surgery.     

Multiparameter flow cytometry as a tool for the detection of micrometastatic tumour cells in the sentinel lymph node procedure of patients with breast cancer

AIM: To investigate whether multiparameter flow cytometry (MP-FCM) can be used for the detection of micrometastasis in sentinel lymph nodes (SLNs) in breast cancer. METHODS: Formalin fixed, paraffin wax embedded sentinel lymph nodes (n = 238) from 98 patients were analysed. For each lymph node, sections for haematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for cytokeratin (MNF116) were cut at three levels with a distance of 500 microm. The intervening material was used for MP-FCM. Cells were immunostained with MNF116, followed by an incubation with fluorescein isothiocyanate (FITC) labelled goat antimouse immunoglobulin. DNA was stained using propidium iodide. From each lymph node 100,000 cells were analysed on the flow cytometer. RESULTS: Thirty eight of the 98 patients with breast carcinoma showed evidence of metastatic disease in the SLN by one ore more of the three methods. In 37 of 38 cases where metastatic cells were seen in the routine H&E and/or IHC, more than 1% cytokeratin positive cells were detected by MP-FCM. In 24 patients, metastatic foci were more than 2 mm (macrometastasis) and in 14 these foci were smaller than 2 mm (micrometastasis). In three of these 14 cases, MP-FCM revealed positive SLNs, although this was not seen at first glance in the H&E or IHC sections. After revision of the slides, one of these three remained negative. However, MP-FCM analysis of the cytokeratin positive cells showed an aneuploid DNA peak, which was almost identical to that of the primary breast tumour. Duplicate measurements, done in 41 cases, showed a 99% reproducibility. In five of 14 patients with micrometastasis, one or two metastatic foci were found in the non-SLN. However, in 15 of 24 macrometastases multiple non-SLNs were found to have metastatic tumour. All micrometastases except for the remaining negative one mentioned above showed only diploid tumour cells, despite the fact that their primary tumours contained both diploid and aneuploid tumour cells. In primary tumours with more than 60% aneuploid cells, predominantly aneuploid macrometastasis were found, whereas diploid primary tumours only showed diploid micrometastases or macrometastases in their SLN. Aneuploid SLN macrometastases were associated with non-SLN metastases in five of seven patients, whereas diploid cases showed additional non-SLN metastases in only seven of 16 patients. CONCLUSION: In all cases, MP-FCM was sufficient to detect micrometastatic tumour cells in a large volume of lymph node tissue from SLNs. In some cases it was superior to H&E and IHC staining. Approximately 30% of SLN micrometastases are accompanied by additional non-SLN metastases. The size of the aneuploid fraction (> 60%) in the primary tumour may influence the risk of having both SLN and non-SLN metastases.     

Inhibition of colon carcinoma cell lung colony formation by a soluble form of E-selectin.

During metastasis, tumor cells adhere to vascular endothelia. E-selectin is an adhesive protein expressed by cytokine-activated endothelium that can support adhesion of colon cancer cells through the recognition of specific carbohydrate ligands. Using a series of colon carcinoma cell lines that displayed E-selectin adhesiveness and an increased metastatic capacity in cytokine-treated mice, we examined possible inhibition of cytokine-dependent experimental lung metastasis by a soluble form of E-selectin, the recombinant fusion protein E-selectin-immunoglobulin. We found that E-selectin-immunoglobulin bound to the surfaces of HT-29 colon carcinoma cells and blocked the formation of cytokine-inducible experimental lung metastases; control L-selectin-immunoglobulin also bound to HT-29 cells but had no effect on tumor cell lung colonization. E-selectin-immunoglobulin was found to interfere with E-selectin-dependent adhesion of HT-29 cells to activated vascular endothelium and to block the retention of these cells in the lung, a process that implies tumor cell adhesive interactions with the host vasculature. Our results demonstrate that E-selectin-immunoglobulin inhibits adhesion and formation of lung metastases by colon carcinoma cells and suggest that impairment of tumor cell-endothelium adhesion might represent a therapeutic approach to the metastatic diffusion of tumors.     

Differential expression of protease-activated receptors-1 and -2 in stromal fibroblasts of normal, benign, and malignant human tissues

The serine proteases thrombin and trypsin are among many factors that malignant cells secrete into the extracellular space to mediate metastatic processes such as cellular invasion, extracellular matrix degradation, angiogenesis, and tissue remodeling. The degree of protease secretion from malignant cells has been correlated to their metastatic potential. Protease activated receptors (PAR)-1 and -2, which are activated by thrombin and trypsin respectively, have not been extensively characterized in human tumors in situ. We investigated the presence of PAR-1 and PAR-2 in human normal, benign and malignant tissues using immunohistochemistry and in situ hybridization. Our results demonstrate PAR-1 and PAR-2 expression in the tumor cells, mast cells, macrophages, endothelial cells, and vascular smooth muscle cells of the metastatic tumor microenvironment. Most notably, an up-regulation of PAR-1 and PAR-2 observed in proliferating, smooth muscle actin (SMA)-positive stromal fibroblasts surrounding the carcinoma cells was not observed in normal or benign conditions. Furthermore, in vitro studies using proliferating, SMA-positive, human dermal fibroblasts, and scrape-wounded human dermal fibroblasts demonstrated the presence of PAR-1 and PAR-2 not detected in quiescent, SMA-negative cultures. PAR-1 and PAR-2 in the cells forming the tumor microenvironment suggest that these receptors mediate the signaling of secreted thrombin and trypsin in the processes of cellular metastasis.     

Experimental and clinicopathologic studies on the function of the HGF receptor in human colon cancer metastasis

The organ-specific metastasis characterizes several human cancers, including colon carcinoma, a disease that frequently involves metastases in the liver. The data on the molecular mechanisms of liver metastasis would therefore be highly useful for prognostic purposes. Although the upregulation/amplification of the hepatocyte growth factor (HGF) receptor, c-met, has been frequently observed in colon cancer metastasis, the actual functional significance of the feature in the liver metastatization is not yet known. We have used three human colon carcinoma cell lines (HT29, HT25 and WiDr), characterized by different liver metastatic potentials in SCID mice, to analyze the expression of c-met and the biological effects of HGF. We found that HGF induces scattering in in vitro liver-metastatic cell lines (HT25 and WiDr) only at doses which are non-mitogenic (1–20 ng/ml). Analysis of the c-met expression revealed that the metastatic cell lines express authentic c-met gene and protein material, unlike the non-metastatic HT29 cell line, which expresses only the c-terminal cytoplasmic domain of the c-met β-chain. Interestingly, c-met was found to be localized in the substrate-attached peripheral membrane and partially colocalized with phosphotyrosine-proteins in the metastatic cells only when kept on fibronectin. On the other hand, we have analyzed 86 primary human colon cancers in Dukes' B (invasive but non-metastatic) and C (invasive and lymph node metastatic) stages. Western blotting of the proteins isolated from the tumor tissues and immunohistochemical control study on the paraffin samples of a third of these cases (25/86) all indicated a significant upregulation of the c-met protein in the Dukes' C tumor glands compared to the Dukes' B stages (P<0.001 and P<0.05, respectively). Since the two stages differ in the involvement of the regional lymph nodes but not in the invasion depth, the clinicopathological data and our experimental findings further support the notion that the c-met expression in human colon cancer can be considered as a marker of the metastatic potential due to its involvement in the generation of the motility signal. This revised version was published online in July 2006 with corrections to the Cover Date.     

Keratin immunohistochemistry does not contribute to correct lymph node staging in patients with invasive lobular carcinoma

Studies suggest that immunohistochemistry improves rate of detecting sentinel lymph node metastases and is needed for adequate staging in invasive lobular carcinoma. Our study evaluates the use of cytokeratin immunohistochemistry in detecting sentinel lymph node metastases and its effect on staging patients with invasive lobular carcinoma. Material from 76 patients with invasive lobular carcinoma was reviewed. Cytokeratin immunostaining was performed on negative nodes, and deposits were classified as macrometastasis (>2.0 mm), micrometastasis (>0.2-2 mm), or isolated tumor cells (相似文献   

Primary tumour expression of the cysteine cathepsin inhibitor Stefin A inhibits distant metastasis in breast cancer

Using the clinically relevant 4T1-derived syngeneic murine model of spontaneous mammary metastasis to bone, we have identified the cysteine cathepsin inhibitor Stefin A as a gene differentially expressed in primary and metastatic mammary tumours. In primary tumours, Stefin A expression correlated inversely with metastatic potential in 4T1-derived lines and was not detected in tumour cells in culture, indicating induction only within the tumour microenvironment. Enforced expression of Stefin A in the highly metastatic 4T1.2 cell line significantly reduced spontaneous bone metastasis following orthotopic injection into the mammary gland. Consistent with the mouse data, Stefin A expression correlated with disease-free survival (absence of distant metastasis) in a cohort of 142 primary tumours from breast cancer patients. This was most significant for patients with invasive ductal carcinoma expressing Stefin A, who were less likely to develop distant metastases (log rank test, p = 0.0075). In a multivariate disease-free survival analysis (Cox proportional hazards model), Stefin A expression remained a significant independent prognostic factor in patients with invasive ductal carcinoma (p = 0.0014), along with grade and progesterone receptor (PR) status. In human lung and bone metastases, we detected irregular Stefin A staining patterns, with expression often localizing to micrometastases (<0.2 mm) in direct contact with the stroma. We propose that Stefin A, as a cysteine cathepsin inhibitor, may be a marker of increased cathepsin activity in metastases. Using immunohistology, the cathepsin inhibitor was detected co-expressed with cathepsin B in lung and bone metastases in both the murine model and human tissues. We conclude that Stefin A expression reduces distant metastasis in breast cancer and propose that this may be due to the inhibition of cysteine cathepsins, such as cathepsin B.     

A Comparative Study of Metastatic Renal Cell Carcinoma with Correlation to Subtype and Primary Tumor

Clear cell (CRCC), papillary (PRCC) and chromophobe (CHRC) renal cell carcinoma (RCC) are the three most frequent subtypes of RCC. The rate and distribution of their metastatic lesions have not been well documented. We compared metastatic RCC according to subtype and primary tumor characteristics to better understand their behavior and to aid in the diagnosis of metastatic RCC. Pathology reports and clinical charts related to 283 CRCC, 48 PRCC and 13 CHRCC, including their respective sarcomatoid variants, were reviewed. A hundred and thirty-seven CRCC, 5 PRCC and 1 CHRCC with metastases were identified. CRCC and non-CRCC (PRCC and CHRCC) had different patterns of metastasis and primary tumor growth. CRCC metastases were predominantly distributed in lungs, bone, brain, lymph nodes, and adrenal glands. The associated primary CRCC measured 1.5 to 15 cm, were of all grades and stages, and were often associated with invasion of small or large veins. Three PRCC had regional lymph node metastases, 1 PRCC had both regional and mediastinal lymph node metastases. Bone metastasis was present in 1 case each of PRCC and CHRCC. One PRCC with metastasis solely to regional nodes measured 4 cm. The other 4 cases of PRCC with regional lymph node and/or distant metastases as well as the CHRCC with distant metastases were greater than 8 cm in diameter. In metastasizing and non-metastasizing non-CRCC, invasion of small veins was rare and invasion of renal veins was not seen. We cannot comment with any certainty on the metastatic behavior of CHRCC. In our experience, PRCC tend to loco-regional invasion with lymph node spread. They have a low potential for vascular invasion and distant metastases that likely occur only at late stages of the disease. CRCC has a propensity for vascular invasion and may be associated with distant metastasis at an early stage. Therefore, metastatic RCC at a distant location are most likely to be of CRCC origin than PRCC origin.     

Lymphatic spread is related to VEGF-C expression and D2-40-positive myofibroblasts in intrahepatic cholangiocarcinoma.

Lymph node metastasis via lymphatic vessels is related with an adverse outcome in many tumors. It is unclear whether lymphatic spread needs the development of the new lymphatic vessels or the expression of lymphangiogenetic factor in intrahepatic cholangiocarcinoma. The aim of this study was to assess the role of lymphangiogenesis, vascular endothelial growth factor-C (VEGF-C) expression, and D2-40-positive myofibroblastic cells for lymphatic spread and patient outcome in 88 cases of intrahepatic cholangiocarcinoma. We also assessed VEGF-C expression in 15 cases of metastatic lymph nodes. There was a significant correlation between lower lymphatic vessel density in the tumor center and positive lymphatic invasion (P=0.0100). Poorly differentiated cholangiocarcinoma showed higher lymphatic vessel density in the tumor periphery and in the peritumoral area (P=0.0315 and P=0.0360, respectively). Lymphatic invasion was observed higher in the peritumoral area (63%, 24/38) and in the tumor periphery (79%, 30/38) than in the tumor center (27%, 9/38). There was no significant correlation between the proliferative lymphatic vessels and pathologic features; however, lymphatic invasion was significantly associated with VEGF-C expression (P=0.0006), and the VEGF-C expression was seen in 12 of 15 cases (80%) of metastatic lymph node. Nodal metastasis was correlated with D2-40-positive myofibroblasts (P=0.0161). VEGF-C expression was an independent prognostic factor by multivariate survival analysis (P=0.0131). Our findings suggest that VEGF-C has an important role in lymphatic invasion via the preexisting lymphatic vessels in the tumor margin, and that lymphangiogenesis does not play a direct role in lymphatic metastasis. D2-40-positive myofibroblasts may contribute to lymphatic metastasis.     

Cellular localization of urokinase-type plasminogen activator,its inhibitors,and their mRNAs in breast cancer tissues

The plasminogen activation (PA) system may participate in cancer invasion and metastasis. A series of breast cancer tissue specimens was analysed using in situ hybridization and immunohistochemistry. Urokinase-type plasminogen activator (u-PA) mRNA was detected in cancer cells and fibroblasts adjacent to them and its expression was found to be more intense in invasive than in intraductal regions. In invasive but not in intraductal regions, especially those with abundant stroma, plasminogen activator inhibitor-1 (PAI-1) mRNA was observed in cancer cells, fibroblasts, macrophages, and endothelial cells, and PAI-2 mRNA was present in cancer cells, and fibroblasts, macrophages, and lymphocytes around them. These PAI-1- and PAI-2-positive cancer cells were localized at the periphery of the invasive front. Immunohistochemistry yielded basically similar results. A retrospective study of surgically resected breast cancers from 73 patients revealed significant clinical differences associated with u-PA and PAI-2 expression in cancer cells, associated with a poor and a good prognosis, respectively. These findings indicate that breast cancer cells and fibroblasts express u-PA initially and then its inhibitors, and that this process is related to invasion. Expression of u-PA and PAI-2 in cancer cells themselves may serve to up-regulate and limit PA-mediated invasion and metastasis, respectively. © 1997 John Wiley & Sons, Ltd.