Organ-site dependence for the production of urokinase-type plasminogen activator and metastasis by human renal cell carcinoma cells.

We examined the role of urokinase-type plasminogen activator (u-PA) in the metastasis of the human renal cell carcinoma (HRCC) implanted in athymic nude mice. Cells from a HRCC KG-2 line were implanted in orthotopic (kidney) and ectopic (subcutaneous) organs. The KG-2 cells implanted in the kidney produced local tumors and lung metastases, whereas those implanted subcutaneously produced only local tumors. The production of u-PA was determined by immunohistochemistry and an enzyme-linked immunosorbent assay (ELISA). High levels of u-PA were produced by the metastatic kidney tumors and lung metastases, whereas the subcutaneous tumors produced low levels. KG-2 cells co-cultured with mouse kidney or lung fibroblasts produced higher levels of u-PA than KG-2 cells co-cultured with mouse skin fibroblasts. Furthermore, KG-2 cells cultured with the conditioned medium from mouse kidney or lung fibroblasts produced higher levels of u-PA than KG-2 cells cultured with the conditioned medium from mouse skin fibroblasts. The results indicate that the expression of u-PA by KG-2 cells is one of the important factors that determine their metastatic potential and that the production of u-PA is influenced by the organ microenvironment, including soluble factors produced by surrounding fibroblasts.     

In vivo selection of human renal cell carcinoma cells with high metastatic potential in nude mice

Studies were made to determine whether the orthotopic implantation of human renal cell carcinoma cells (HRCC) into nude mice will produce distant metastases, thus allowing for the selection of variant cells with high metastatic potential. The parental SN12C line was established in culture from a surgical specimen of HRCC. The renal subcapsule (RSC) of adult nude mice was injected with SN12C cells; the mice were killed when they became moribund. Cell lines were established from either single or multiple lung HRCC metastases. The intravenous injection of many (but not all) of the metastasis-derived lines produced significantly more experimental metastases than did the parental cells. The injection of cells into the RSC demonstrated that, in general, cells derived from spontaneous metastases were more metastatic than cells of the parental line. Hence adult nude mice can be used to select HRCC cells with high metastatic potential. These HRCC variant lines offer a good model for studying the cell properties of metastatic HRCC.Abbreviations EMEM Eagle's minimum essential medium - HBSS Hank's balanced salt solution without Ca²⁺ and Mg²⁺ - HRCC human renal cell carcinoma - RSC renal subcapsule     

Expression of vascular endothelial growth factor by human renal cancer cells enhances angiogenesis of primary tumors and production of ascites but not metastasis to the lungs in nude mice

We determined the role that vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), plays in the progression of human renal cell cancer in nude mice. Low metastatic and low VEGF/VPF-expressing human renal cancer cells SN12C were transfected with the VEGF165 cDNA or plasmid alone as control. VEGF165-transfected SN12C cells produced large amounts of biologically active VEGF in culture that did not affect cell doubling time or confluence. Subsequent to implantation into the renal subcapsule of nude mice, the VEGF165-transfected SN12C cells produced fast-growing (PCNA labeling), large tumors that expressed high levels of VEGF/VPF and were well vascularized (CD3-positive vessels). The tumors produced hyperpermeability of peritoneal blood vessels (Evans blue dye-leak assay), bloody ascites, and short survival time. Parental or control transfected SN12C cells produced less vascularized, slower growing tumors with no ascites. Regardless of in vivo expression level of VEGF, the incidence of spontaneous lung metastasis was low, suggesting that in itself, the expression of VEGF/VPF by renal cancer cells is not sufficient to produce metastasis. This revised version was published online in July 2006 with corrections to the Cover Date.     

人绒毛膜癌裸鼠原位移植模型的建立

目的建立人绒毛膜癌裸鼠原位移植瘤模型。方法培养人绒毛膜癌细胞系JAR,制备JAR单细胞悬液,给5只8周龄BALB/c裸鼠经皮下注射建立皮下移植瘤模型。待裸鼠皮下成瘤后,无菌条件下取瘤组织并切成1 mm^3组织块,通过手术方式植入10只10周龄BALB/c裸鼠子宫腔内,裸鼠濒死状时4%水合氯醛(10 g/kg)腹腔注射麻醉处死,观察子宫成瘤及腹腔转移情况。解剖取子宫原位移植瘤、腹腔内转移瘤、腹腔淋巴结及其他脏器组织标本,通过组织病理学检查进行鉴定。结果10只BALB/c裸鼠中共有7只裸鼠子宫内可见移植瘤肿块形成,其中2只可同时观察到子宫移植瘤和腹膜转移瘤。在病理学形态和结构上,皮下移植瘤模型、原位移植模型和腹膜转移瘤的瘤细胞与人绒毛膜癌细胞系JAR一致。结论成功建立人绒毛膜癌JAR细胞的BALB/c裸鼠原位移植瘤模型。     

Multi-organ metastatic capability of Chinese hamster ovary cells revealed by green fluorescent protein (GFP) expression

Stable high-level green fluorescent protein (GFP)-expressing Chinese hamster ovary cells (CHO) were used to visualize the degree of metastatic behavior of this cell line in nude and SCID mice. A stable GFP high-expression CHO clone, selected in 1.5 M methotrexate, was injected subcutaneously in nude and severe combined immunodeficient (SCID) mice and implanted orthotopically in the ovary of nude mice. CHO proved to be highly metastatic from both the subcutaneous and orthotopic sites as brightly visualized by GFP fluorescence. High-level GFP-expression allowed the visualization of metastatic tumor in fresh live host tissue in great detail. Metastases were visualized by GFP expression in the lung, pleural membrane, spleen, kidney, ovary, adrenal gland, and peritoneum after orthotopic implantation in nude mice. Metastases were visualized by GFP expression mainly in the lung, pleural membrane after subcutaneous implantation in nude mice. Metastases were visualized in the lung and pleural membrane, liver, kidney, and ovary after subcutaneous implantation in SCID mice. The construction of highly fluorescent stable GFP transfectants of CHO has revealed the multi-organ metastatic capability of CHO cells. CHO has such a high degree of malignancy that it is metastatic from both the orthotopic and subcutaneous transplant sites. This highly malignant GFP-expressing cell-line with multi-organ metastatic affinity should serve as a powerful tool to study tumor-host interaction.     

Blockade of epidermal growth factor receptor signaling on tumor cells and tumor-associated endothelial cells for therapy of human carcinomas

The purpose of this study was to determine whether the expression of epidermal growth factor receptor (EGF-R) and activated EGF-R by tumor-associated endothelial cells is influenced by interaction with specific growth factors in the microenvironment. Different human carcinoma cell lines expressing EGF-R with low or high levels of EGF/transforming growth factor (TGF)-alpha were implanted into orthotopic organs of nude mice. In the EGF/TGF-alpha-positive bladder cancer (253J-BV), pancreatic cancer (L3.6pl), and renal cancer (RBM1-IT) but not in the EGF/TGF-alpha-negative renal cancer SN12-PM6, tumor-associated endothelial cells expressed EGF-R and activated EGF-R. Mice were implanted with human 253J-BV bladder tumors (EGF+) or human SN12-PM6 renal tumors (EGF-). Treatment with oral PKI 166 (a specific inhibitor of EGF-R phosphorylation) alone, intraperitoneal paclitaxel alone (253J-BV), gemcitabine alone (SN12-PM6), or combination of PKI 166 and chemotherapy produced a 60%, 32%, or 81% reduction in the volume of 253J-BV bladder tumors, respectively, and 26%, 23%, or 51% reduction in the volume of SN12-PM6 kidney tumors, respectively. Immunohistochemical analyses demonstrated down-regulation of activated EGF-R in EGF/TGF-alpha-positive and EGF/TGF-alpha-negative lesions from mice treated with PKI 166, although apoptosis of tumor-associated endothelial cells was found only in EGF/TGF-alpha-positive tumors. Collectively, these data suggest that expression of activated EGF-R by tumor-associated endothelial cells provides an important target for therapy.     

Antigen expression of metastasizing and non-metastasizing human melanoma cells xenografted into nude mice

In order to study differences in antigen expression related to the different stages of the process of metastasis of human melanoma cell lines, we determined the expression pattern of a series of well-characterized genes in a set of human melanoma cell lines with different metastatic behavior in nude mice. This set included non-metastatic (IF6, 530), sporadically metastatic (M14, Mel 57), and frequently metastatic (BLM, MV3) cell lines after subcutaneous inoculation. To study the phenotype of these cell lines both the cultured cells and representative samples of local tumors at the inoculation site and their metastases in the lungs were immunostained with a panel of monoclonal antibodies directed against melanocytic differentiation or progression antigens. Although most cell lines (IF6, 530, M14 and Mel 57) showed HLA-DR expressionin vitro, these antigens were lacking in all xenografted lesions studied with exception of the 530 cell line. 530 Xenografts, however, showed a dramatic down-regulation of HLA-DR compared with the cell linein vitro. The same phenomenon was seen with respect to ICAM-1 expression. The expression of all other antigens studied in xenografts, both in subcutaneous tumors and in lung lesions, was in general comparable to that in the melanoma cell linesin vitro, with exception of the 530 cell line. In all melanoma cell lines except 530 the degree of intra- and interlesional heterogeneity regarding the expression of all antigens studied was limited. Remarkably, comparison of the immunophenotype of the frequently metastasizing (BLM, MV3) and the sporadically (M14, Mel 57) or non-metastasizing (IF6, 530) cell lines showed that the two frequently metastasizing cell lines had marked expression of the progression antigens VLA-2 and epidermal growth factor receptor, and lack of expression of the differentiation antigen NKI-beteb. These findings warrant further studies on the role of these antigens in the process of metastasis of human melanoma cells in nude mice.     

Regulation of carcinoembryonic antigen expression in human colon carcinoma cells by the organ microenvironment.

The expression of carcinoembryonic antigen (CEA) is thought to be involved in homotypic adhesion and has been associated with the progression of human colon carcinomas (HCC) to the metastatic state. Three cell lines established from surgical specimens of Dukes' stage D (KM20) or Dukes' stage B (KM12C, KM12SM) with high and low preoperative CEA serum levels, respectively, were studied subsequent to growth in culture, in the subcutis (ectopic) or cecal wall (orthotopic) of nude mice. In all cell lines, CEA expression was higher in cecal tumors than in subcutaneous lesions. The degree of differentiation and CEA expression by HCC growing in the cecal wall of nude mice correlated with the pathological diagnosis and preoperative CEA level of the original patients. To better understand the regulation of CEA expression, the HCC cells were grown in culture as sparse and confluent monolayers or as multicell spheroids. The CEA expression level increased in all three cell lines growing as confluent monolayers and was highest in multicell spheroids. Treatment of sparse monolayer cultures of KM12SM cells with mitomycin-C inhibited cell division and was associated with higher production of CEA protein. Moreover, conditioned media from confluent monolayer cultures or from spheroids up-regulated CEA production in sparse monolayer cells. These data show that CEA expression in HCC cells may be regulated by cell density and by factors from the organ environment.     

Establishment of human osteosarcoma cell lines with high metastatic potential to lungs and their utilities for therapeutic studies on metastatic osteosarcoma

Relevant animal models for metastasis of osteosarcoma is needed to understand the biology and to develop the treatment modality of metastasis of human osteosarcoma. Therefore, we screened six human osteosarcoma cell lines for metastatic ability in nude mice. The HuO9 cell line was identified as being metastatic to the lung after intravenous injection. We established two sublines, HuO9-M112 and HuO9-M132, with high metastatic potential to the lung from the parental HuO9 cells by in vivo selection. There were no differences between these two sublines and the parental cells in the growth rate in vitro and the tumorigenicity after subcutaneous injection in nude mice, however, mice injected with the metastatic sublines became moribund earlier than mice injected with the parental HuO9 cells did. Thus, adriamycin (ADR) and recombinant interleukin-12 (IL-12) were administered to mice injected with the HuO9-M112 subline to suppress experimental lung metastases. Production of lung colonies was significantly suppressed and the prognoses of mice were significantly improved by both ADR and IL-12 treatments. These results indicate that both ADR and IL-12 are effective agents against pulmonary metastatic osteosarcoma, and that these sublines are useful for studies on the biological behavior and treatment of pulmonary metastatic osteosarcoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

Growth and metastatic behavior of human tumor cells implanted into nude and beige nude mice

The growth and metastatic behavior of several human tumor lines grown in adult nude mice, nude mice pretreated with antiserum against asialo GM1 glycoprotein, and beige nude mice were studied. The cell lines were all injected s.c. and i.v. A human colon carcinoma line was also injected into the spleen, and two human renal carcinoma lines were injected into renal subcapsule. All the tumor lines grew as fast or faster in adult nude mice compared with beige nude mice. There were no discernible differences in the production of experimental lung metastasis among the three groups of mice, but human colorectal carcinoma cells and human renal carcinoma cells produced more metastases in nude mice than in beige nude mice after intrasplenic or renal subcapsule injection, respectively. In vitro cytotoxicity assays confirmed that adult nude mice had high levels of natural killer (NK) cell activity whereas nude mice pretreated with anti-asialo GM1 serum and beige nude mice did not. The in vitro NK cell activity of nude mice was demonstrable against mouse lymphoma cells but not against human leukemia cells which were sensitive to lysis by human NK cells. These results suggest that the implantation of human tumor cells into beige nude mice, which are deficient in NK cell activity does not provide an advantageous model for the study of growth and metastasis of human neoplasms.Abbreviations NK natural killer - HCC human colon carcinoma - HRCC human renal cell carcinoma - anti-asGM1 anti-asialo GM1 - i.s. intrasplenic - RSC renal subcapsule - HBSS Ca²⁺- and Mg²⁺-free Hanks' balanced salt solution     

Metastatic and non-metastatic colorectal cancer (CRC) cells induce host metalloproteinase production in vivo

Numerous studies have demonstrated the persistent localization of matrix metalloproteinase (MMP) expression to the interface between invading human colorectal cancer (CRC) cells and surrounding stroma supporting a role for MMPs in CRC invasion and metastasis. The present study sought to determine whether CRC cells of varying metastatic potential would have differential effects on host MMP release. Subcutaneous CRC tumors were generated in BALB/c nude mice using three CRC cell lines: SW480, SW620, and the highly metastatic SW620S5 clone. Representative samples from the subcutaneous CRC were then orthotopically implanted on the cecum of recipient nude mice. Subcutaneous and cecal tumors were analyzed for MMP expression via zymography, western blot, and RT-PCR. In vitro, none of the three cell lines expressed MMP-2 nor MMP-9. In contradistinction, the subcutaneous tumors expressed limited amounts of MMP-2 and MMP-9 while the cecal tumors expressed significant amounts of MMP-2 and MMP-9 as well as other smaller members of the MMP family. MMP-9 mRNA and protein was confirmed as host in origin by RT-PCR with mouse specific primers and a mouse MMP-9 molecular weight of 105 kDa as determined by zymography and western blot analysis. In situ hybridization also localized the mRNA for MMP-9 to the host stromal cells. In conclusion, CRC cells appear incapable of producing MMP-2 and MMP-9 in vitro but are capable of up-regulating host MMP production in vivo. Enhanced host MMP-9 production in metastatic CRC cell-derived subcutaneous and cecal tumors suggests that metastatic colon cells may acquire the expression of important MMP regulating factor(s) in vivo.     

Development of a high metastatic orthotopic model of human renal cell carcinoma in nude mice: benefits of fragment implantation compared to cell-suspension injection

In this study we compared the metastatic rate of human renal cell carcinoma SN12C in two orthotopic nude mouse models: surgical orthotopic implantation (SOI) of histologically intact tumor tissue and cellular orthotopic injection (COI) of cell suspensions in the kidney. The primary tumors resulting from SOI were larger and much more locally invasive than primary tumors resulting from COI. SOI generated higher metastatic expression than COI. The differences in metastatic rates in the involved organs (lung, liver, and mediastinal lymph nodes) were 2–3 fold higher in SOI compared to COI (P<0.05). Median survival time in the SOI model was 40 days, which was significantly shorter than that of COI (68 days) (P<0.001). Histological observation of the primary tumors from the SOI model demonstrated a much richer vascular network than the COI model. Lymph node and lung metastases were larger and more cellular in the SOI model compared to COI. We conclude that the tissue architecture of the implanted tumor tissue in the SOI model plays an important role in the initiation of primary tumor growth, invasion, and distant metastasis. This study directly demonstrates that the implantation of histologically intact tumor tissue orthotopically allows accurate expression of the clinical features of human renal cancer in nude mice. This model should be of value in cancer research and antimetastatic drug discovery for renal cancer, a currently very poorly responding malignancy.     

In situ mRNA hybridization technique for analysis of metastasis-related genes in human colon carcinoma cells.

The purpose of this study was to determine whether the expression level of several genes that regulate different steps of the metastatic process correlates with the metastatic potential of human colon carcinoma cells. The mRNA expression level for epidermal growth factor receptor (growth), basic fibroblast growth factor and interleukin-8 (angiogenesis), type IV collagenase (invasion), E-cadherin and carcinoembryonic antigen (adhesion), and the multidrug resistance gene mdr-1 (drug resistance) in the human KM12 colon carcinoma cell lines and clones with different metastatic potential was measured by Northern blot analysis and by in situ hybridization technique. Highly metastatic KM12SM and KM1214 cells growing in culture uniformly expressed high levels of epidermal growth factor receptor, basic fibroblast growth factor, and carcinoembryonic antigen mRNA, whereas cultures of low metastatic KM12C, clone 1, clone 3, and clone 6 cells displayed heterogeneous patterns of expression. KM12C (low metastatic) and KM12SM (highly metastatic) cells were implanted into the subcutis (ectopic) or the wall of the cecum (orthotopic) of nude mice. The mRNA expression level for epidermal growth factor receptor, basic fibroblast growth factor, interleukin-8, type IV collagenase, carcinoembryonic antigen, and mdr-1 was increased in the cecal wall tumors as compared with subcutaneous tumors or in vitro cultures. These data demonstrate a direct correlation between constitutive and inducible expression of several metastasis-related genes and the metastatic potential of human colon carcinoma cells.     

Transformed NIH 3T3 cells expressing human melanoma N-ras oncogene metastasize to lymph node in nude mice

The effect of the N-ras oncogene on the propensity of transformed cells to disseminate from the tumor and to metastasize, using NIH 3T3 cells transformed either with human melanoma DNA containing the N-ras oncogene or with the cloned N-ras from human neuroblastoma, was investigated. The results show that NIH 3T3 expressing these genes readily formed tumors after subcutaneous injection in nude mice. Spontaneous lymph node metastasis was observed after a first cycle of transfection in one animal inoculated with cells containing human melanoma N-ras oncogene, and in 95 per cent of the animals after the second and third rounds of transfection, indicating that the metastatic capacity was transferred. In all cases human N-ras oncogene was found in both the metastases and the associated tumors. No control NIH 3T3 cells formed tumors or metastases in nude mice, and NIH 3T3 cells transfected with cloned N-ras activated oncogene formed tumors in 100 per cent of injected mice, but no spontaneous metastases. Thus human activated N-ras gene may not be sufficient to confer metastatic behavior in nude mice and the metastatic ability of human melanoma DNA transfected cells may be due to, among other possibilities, expression of other gene sequences from melanoma DNA co-transfected with the N-ras oncogene, or to specific activated murine sequences switched on during the initial process of transfection.     

具有不同转移潜能的前列腺癌细胞亚系的分离鉴定

目的 克隆具有不同转移潜能的癌细胞亚系,并应用体内外实验对各亚系的侵袭转移能力进行检测。方法 应用倍比稀释法对前列腺癌细胞系ＰＣ－３Ｍ进行单细胞克隆,并应用体内外实验包括生长曲线,人工基底膜侵袭实验,软琼脂集落形成,裸鼠异种接种及自发性转移实验检测各亚系的生物学特性及侵袭转移能力。结果 从前列腺癌细胞系ＰＣ－３Ｍ成功分离建立了４个具有不同转移潜能的亚系,经体内外多项实验检测证明：１Ｅ８为高转移亚系     

Recombined CC chemokine ligand 2 into B16 cells induces production of Th2-dominant [correction of dominanted] cytokines and inhibits melanoma metastasis

This study is aimed to verify whether CCL2 can induce Th2 polarization in vivo and subsequently inhibit tumor metastasis. B16 cells (a murine melanoma cell line) highly expressing CCL2 (CCL2-B16 cells) were obtained by transfection with recombinant plasmid CCL2-pcDNA3. Primary thymocytes were co-cultured with CCL2-B16 cells and STAT-6-mediated Th2 polarization was noticed after co-culture. Caudal vein injection of CCL2-B16 cells effectively inhibited pulmonary metastasis in C57BL/6 mice, but not in nude mice, indicating that T cells play a role in CCL2-induced inhibition of tumor metastasis. We found that high level of CCL2 up-regulated the expression of Th2-related cytokine (IL-4) in tumor microenvironment and increased CD4+, CD8+, and CD45RB+ cells in the peripheral blood and tumor tissues. We also demonstrated that inoculation of mice with CCL2-B16 cells prolonged mice survival time when they were reinjected with wildtype B16 cells, implying that CCL2 can activate immuno-memory in mice. It is concluded that high expression of CCL2 can induce Th2 polarization in tumor microenvironment and can effectively inhibit tumor metastasis, which casts new lights on the role of chemokines in reconstruction of immune surveillance in patients suffering from tumors.     

Metastatic conversion of chemically transformed human cells

A linear model for human cell metastasis has been developed in vitro from chemically transformed normal human cells. The chemically transformed cells are nontumorigenic in nude mice, but can be converted to a tumorigenic phenotype by transfection with a nondirectional cDNA library or antisense cDNA to the ML-1 gene. The primary transfected cell line (TR1T) forms localized, progressively growing tumors in nude mice that do not invade into the surrounding tissue. This tumorigenic TR1T cell line could be advanced into a metastatic stage following an additional transfection (TR2M cell line) with the cDNA expression library or antisense cDNA to the ML-1 gene. Metastatic cells, selected from tumors that were attached to internal organs, exhibited an increase in invasiveness as measured in vitro using an invasion chamber. The metastatic cells also exhibited an increased expression of matrix metalloproteinase-1 (MMP-1), although MMP-1 was not part of the cDNA that was transfected into either the TR1T cells or the doubly transfected metastatic TR2M cells. These data suggest that the increase in MMP-1 expression was a secondary downstream event responding to an upstream genetic change that initiated the conversion of cells from a tumorigenic to a metastatic stage. In summary, human cell lines representing premalignant, malignant, and metastatic phenotypes have been established in culture that can be used to identify gene changes that occur as normal human cells progress to a metastatic stage during tumor development. One gene, ML-1, that is found in the expression library appears to be involved in malignant progression, because ML-1 antisense cDNA will convert chemically transformed cells to both tumorigenic and metastatic stages, and cells from both local and metastatic tumors have a reduced or complete loss of expression of the ML-1 gene.     

Metastatic patterns of lung cancer visualized live and in process by green fluorescence protein expression

We demonstrate here the visualization of human lung cancer metastasis live and in process in nude mice by green fluorescent protein (GFP) expression. The human lung adenocarcinoma cell line Anip 973 stably transfected with the humanized GFP-S65T cDNA was selected for very bright green fluorescence. GFP-transfected lung cancer cells were initially inoculated subcutaneously in nude mice. Five weeks after transplantation, the resulting tumor had reached over 1 cm in diameter and had very bright GFP fluorescence. Fragments of subcutaneous tumor were implanted onto the visceral pleura of the left lung of nude mice by surgical orthotopic implantation (SOI) of histologically-intact tissue via transverse thoracotomy. The ipsilateral resulting tumor was highly fluorescent due to GFP expression. GFP expression allowed the visualization of the advancing margin of the ipsilateral tumor into the fresh normal lung tissue. Lymphogenous and direct-seeding metastases in the pulmonary hilum, cervical lymph nodes, the mediastinum and contralateral pleural cavity and contralateral lung in the SOI-treated mice were brightly visualized by GFP expression in fresh tissue. GFP-transfected and untransfected tumor had similar metastatic characteristics suggesting that GFP expression had no effect on metastasis itself. The results with the GFP-transfected tumor cells, combined with the use of SOI, demonstrate a fundamental advance in the visualization and study of lung cancer metastasis in process.     

Effect of host microenvironment on the microcirculation of human colon adenocarcinoma.

It is generally accepted that the host microenvironment influences tumor biology. There are discrepancies in growth rate, metastatic potential, and efficacy of systemic treatment between ectopic and orthotopic tumors. Liver is the most common and critical site of distant metastasis of colorectal carcinoma. Tumorigenicity and efficacy of chemotherapeutic agents in colorectal tumors are different in liver and subcutaneous sites. Thus, we hypothesize that the liver (orthotopic) versus subcutaneous (ectopic) microenvironment would have different effects on the angiogenesis and maintenance of the microcirculation of colorectal tumor. To this end, we developed a new method to monitor and to quantify microcirculatory parameters in the tumor grown in the liver. Using this approach, we compared the microcirculation of LS174T, a human colon adenocarcinoma, metastasized to the liver with that of the host liver vessels and that of the same tumor grown in the subcutaneous space. In the liver metastasis model, 5 x 10(6) LS174T cells were injected into the spleen of nude mice. Four to eight weeks later, the liver with metastatic tumors was exteriorized and placed on a special stage and observed under an intravital fluorescence microscope. The dorsal skinfold chamber model was used to study the subcutaneous tumors. Red blood cell velocity, vessel diameter, density, permeability, and leukocyte-endothelial interactions were measured using fluorescence microscopy and image analysis. Vascular endothelial growth factor/ vascular permeability factor (VEGF/VPF) mRNA expression was determined by the Northern blot analysis. LS174T tumor foci in the liver had tortuous vascular architecture, heterogeneous blood flow, significantly lower vascular density, and significantly higher vascular permeability than normal liver tissue. Tumors grown in the liver had significantly lower vessel density, especially in the center coincident with central necrosis, than the subcutaneous tumors. The frequency distribution of vessel diameters of liver tumor was slightly shifted to smaller size compared with that of subcutaneous tumor. Leukocyte rolling in liver tumor was twofold lower than that in subcutaneous tumor. These physiological findings were consistent with the measurement of VEGF/VPF in that the VEGF/VPF mRNA level was lower in the liver tumor than that in the subcutaneous tumor. However, macromolecular vascular permeability in the liver tumor was significantly higher than in the subcutaneous tumor. Liver sinusoidal endothelial cells, the origin of liver tumor vessel endothelium, are known to be fenestrated and not to have a basement membrane, suggesting that the difference in endothelial cell origin may explain the difference in tumor vascular permeability in two sites. These findings demonstrate that liver microenvironment has different effects on some aspects of the tumor angiogenesis and microcirculation compared with the subcutaneous tissues. The new model/method described in this paper has significant implications in two research areas: 1) the liver microenvironment and its effect on tumor pathophysiology in conjunction with cytokine/ growth factor regulation and 2) the delivery of drugs, cells, and genes to liver tumors.