We utilized Fas21, a resveratrol analog, to modulate the function of hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs) during the angiogenic phase of murine liver metastasis by B16 melanoma and 51b colorectal carcinoma. Preangiogenic micrometastases were treated with Fas21 (1 mg/kg/day) or vehicle during the development of intra-angiogenic tracts. Mice treated with Fas21 showed reduced liver tumor foci in both liver metastasis models. Micrometastases were classified immunohistochemically, as well as according to their position coordinates and connection to local microvasculature. The volume of liver occupied by sinusoidal-type foci, containing infiltrating angiogenic capillaries, decreased by ~50% in Fas21-treated mice compared to vehicle-treated ones in both tumor metastasis models. The volume of portal foci, containing peripheral neoangiogenesis within a discontinuous layer of myofibroblasts, was similar in all experimental groups in both tumor metastasis models, but displayed enhanced necrotic central areas devoid of angiogenesis following Fas21 treatment. As a result, sinusoidal tumors from mice treated with Fas21 showed a 50% reduction in desmin(+)/asma(+) HSCs and CD31(+) vessel density, and a 45% reduction in intrametastatic VEGF mRNA compared with sinusoidal tumors from vehicle-treated mice. Necrotic portal metastases increased 2-4-fold in treated mice. In vitro, Fas21 reduced VEGF secretion by HSCs and 51b cells dose-dependently. Additionally, HSCs migration in response to tumor soluble factors was dose-dependently diminished by Fas21, as was LSEC migration in response to HSCs and tumor soluble factors. Resveratrol analog Fas21 inhibits the proangiogenic response of HSCs and LSECs during the development of murine liver metastasis.

• Angiogenesis
• Cancer
• Hepatic stellate cell
• Liver metastasis
• Liver sinusoidal endothelial cell
• Tumor microenvironment

• capsule
• carcinoma-associated fibroblasts
• desmoplasia
• hepatic stellate cells
• histologic growth patterns
• liver
• microenvironment

• Biomarkers
• Biopsy
• Cancer-Associated Fibroblasts/metabolism
• Cancer-Associated Fibroblasts/pathology
• Colorectal Neoplasms/pathology
• Endothelial Cells/metabolism
• Female
• Hepatic Stellate Cells/metabolism
• Humans
• Immunohistochemistry
• Liver/pathology
• Liver Neoplasms/diagnosis
• Liver Neoplasms/secondary
• Lymphocytes, Tumor-Infiltrating/metabolism
• Lymphocytes, Tumor-Infiltrating/pathology
• Male
• Tumor Microenvironment

Cancer cell migration, tumor angiogenesis, and activated hepatic stellate cells (a-HSCs) promote the development of colorectal liver metastases (CLM). Wiskott–Aldrich syndrome protein family verprolin-homologous protein 2 (WAVE2) has been associated with CLM, although the underlying molecular mechanisms remain unclear.

In the current study, we evaluated the relationship between WAVE2 and CLM in 103 CLM patients who underwent liver resection. Immunohistochemistry (IHC) staining was performed to determine the association between WAVE2 protein expression and hepatic micro-metastasis in human CLM tissues. WAVE2 knockout was performed in hepatic stellate cells (HSC) to explore the function and signaling pathways of WAVE2 in colorectal cancer progression.

Significantly higher levels of WAVE2 were detected in portal-associated relative to sinusoid-associated micro-metastasis. A strong correlation was identified between WAVE2 levels and microvessel density (MVD) in hepatic metastasis. Similarly, expression of WAVE2 was closely associated with activation of HSCs. Mechanistically, WAVE2 regulated the progression of human CLM acts by regulating the growth factor β (TGF-β) and Hippo pathways via effector yes-associated protein (YAP1).

Overall, our results demonstrated that WAVE2 participates in CLM tumor microenvironment, and can be a potential latent therapeutic target for CLM.

• WAVE2
• colorectal cancer
• hepatic stellate cells
• liver metastasis

• Biomarkers
• Colorectal cancer
• Exosomal proteins
• Gene expression profiling
• Inter-organ prometastatic communication
• Liver metastasis
• Liver prometastatic reaction
• Melanoma
• Premetastatic niche
• Tumor microenvironment

Colon cancer is one of the most frequently occurring types of cancers in the world. Primary tumours are treated very efficiently, but the metastatic cases are known to have severe outcomes. Therefore, the aim of the present study was to obtain a greater understanding of the transformation of primary colon cancer cells into metastatic phenotypes. Small changes in protein expression provoke the metastatic phenotype transformation. More sensitive methods to detect small variations are required. A murine colon cancer cell line with metastatic characteristics in a very early phase was created in order to investigate the first steps of transformation using a murine liver metastasis model. The protein expression patterns of metastatic and non-metastatic cells were compared using the stable isotope labelling by amino acids in cell culture method in combination with mass spectrometry. Quantitative proteomics data indicated that nicotinamide adenine dinucleotide hydride (NADH) dehydrogenase complex I was overexpressed in metastatic cells with respect to non-metastatic cells. Since the NADH dehydrogenase complex catalyses the oxidation of NADH to NAD⁺, the functionality of the complex was studied by measuring the amount of NADH. The results revealed that metastatic cells accumulate more NADH and reactive oxygen species. In addition, the mitochondrial membrane potential of metastatic cells was lower than that of non-metastatic cells, indicating that the activity of NADH dehydrogenase and the mitochondrial oxidative chain were decreased in metastatic cells. During the incipient transformation of primary cancer cells, NADH dehydrogenase complex I was overexpressed but then became inactive due to the Warburg effect, which inhibits mitochondrial activity. In the first step of transformation, the high energy demand required in an adverse environment is fulfilled by overexpressing components of the respiratory chain, a fact that should be considered for future anti-metastatic therapies.

• Chemotherapy
• Cyclooxygenase-2
• Drug delivery system
• Metastatic niche
• Oxaliplatin

The purpose of this study was to characterise growth patterns, proteolysis, and angiogenesis in colorectal liver metastases from chemonaive patients with multiple liver metastases. Twenty-four patients were included in the study, resected for a median of 2.6 metastases. The growth pattern distribution was 25.8% desmoplastic, 33.9% pushing, and 21% replacement. In 20 patients, identical growth patterns were detected in all metastases, but in 8 of these patients, a second growth pattern was also present in one or two of the metastases. In the remaining 4 patients, no general growth pattern was observed, although none of the liver metastases included more than two growth patterns. Overall, a mixed growth pattern was demonstrated in 19.3% of the liver metastases. Compared to metastases with pushing, those with desmoplastic growth pattern had a significantly up-regulated expression of urokinase-type plasminogen activator receptor (P = 0.0008). Angiogenesis was most pronounced in metastases with a pushing growth pattern in comparison to those with desmoplastic (P = 0.0007) and replacement growth pattern (P = 0.021). Although a minor fraction of the patients harboured metastases with different growth patterns, we observed a tendency toward growth pattern uniformity in the liver metastases arising in the same patient. The result suggests that the growth pattern of liver metastases is not a random phenomenon.

The recruitment of vascular stromal and endothelial cells is an early event occurring during cancer cell growth at premetastatic niches, but how the microenvironment created by the initial three-dimensional (3D) growth of cancer cells affects their angiogenesis-stimulating potential is unclear.

The proangiogenic profile of CT26 murine colorectal carcinoma cells was studied in seven-day cultured 3D-spheroids of <300 μm in diameter, produced by the hanging-drop method to mimic the microenvironment of avascular micrometastases prior to hypoxia occurrence.

Spheroid-derived CT26 cells increased vascular endothelial growth factor (VEGF) secretion by 70%, which in turn increased the in vitro migration of primary cultured hepatic sinusoidal endothelium (HSE) cells by 2-fold. More importantly, spheroid-derived CT26 cells increased lymphocyte function associated antigen (LFA)-1-expressing cell fraction by 3-fold; and soluble intercellular adhesion molecule (ICAM)-1, given to spheroid-cultured CT26 cells, further increased VEGF secretion by 90%, via cyclooxygenase (COX)-2-dependent mechanism. Consistent with these findings, CT26 cancer cells significantly increased LFA-1 expression in non-hypoxic avascular micrometastases at their earliest inception within hepatic lobules in vivo; and angiogenesis also markedly increased in both subcutaneous tumors and hepatic metastases produced by spheroid-derived CT26 cells.

3D-growth per se enriched the proangiogenic phenotype of cancer cells growing as multicellular spheroids or as subclinical hepatic micrometastases. The contribution of integrin LFA-1 to VEGF secretion via COX-2 was a micro environmental-related mechanism leading to the pro-angiogenic activation of soluble ICAM-1-activated colorectal carcinoma cells. This mechanism may represent a new target for specific therapeutic strategies designed to block colorectal cancer cell growth at a subclinical micrometastatic stage within the liver.

The liver is a major metastasis-susceptible site and majority of patients with hepatic metastasis die from the disease in the absence of efficient treatments. The intrahepatic circulation and microvascular arrest of cancer cells trigger a local inflammatory reaction leading to cancer cell apoptosis and cytotoxicity via oxidative stress mediators (mainly nitric oxide and hydrogen peroxide) and hepatic natural killer cells. However, certain cancer cells that resist or even deactivate these anti-tumoral defense mechanisms still can adhere to endothelial cells of the hepatic microvasculature through proinflammatory cytokine-mediated mechanisms. During their temporary residence, some of these cancer cells ignore growth-inhibitory factors while respond to proliferation-stimulating factors released from tumor-activated hepatocytes and sinusoidal cells. This leads to avascular micrometastasis generation in periportal areas of hepatic lobules. Hepatocytes and myofibroblasts derived from portal tracts and activated hepatic stellate cells are next recruited into some of these avascular micrometastases. These create a private microenvironment that supports their development through the specific release of both proangiogenic factors and cancer cell invasion- and proliferation-stimulating factors. Moreover, both soluble factors from tumor-activated hepatocytes and myofibroblasts also contribute to the regulation of metastatic cancer cell genes. Therefore, the liver offers a prometastatic microenvironment to circulating cancer cells that supports metastasis development. The ability to resist anti-tumor hepatic defense and to take advantage of hepatic cell-derived factors are key phenotypic properties of liver-metastasizing cancer cells. Knowledge on hepatic metastasis regulation by microenvironment opens multiple opportunities for metastasis inhibition at both subclinical and advanced stages. In addition, together with metastasis-related gene profiles revealing the existence of liver metastasis potential in primary tumors, new biomarkers on the prometastatic microenvironment of the liver may be helpful for the individual assessment of hepatic metastasis risk in cancer patients.

• Animals
• Contrast Media/pharmacology
• Diagnostic Imaging/methods
• Female
• Image Processing, Computer-Assisted
• Liver Neoplasms/pathology
• Mice
• Models, Statistical
• Neoplasm Metastasis
• Neoplasm Transplantation
• Splenic Neoplasms/pathology
• Tomography, X-Ray Computed/methods

• Cell Line, Tumor
• Cells, Cultured
• Chemotaxis
• Colorectal Neoplasms/immunology
• Colorectal Neoplasms/metabolism
• Colorectal Neoplasms/pathology
• Endothelial Cells/physiology
• Fibroblasts/metabolism
• Fibroblasts/pathology
• Humans
• Inflammation/pathology
• Interleukin-8/biosynthesis
• Liver/immunology
• Liver/metabolism
• Liver/pathology
• Liver Neoplasms/immunology
• Liver Neoplasms/metabolism
• Liver Neoplasms/secondary
• NF-kappa B/antagonists & inhibitors
• Stromal Cells/metabolism
• Stromal Cells/pathology
• Tumor Necrosis Factor-alpha/physiology
• Up-Regulation

• Animals
• Apoptosis/physiology
• Cell Division/physiology
• Cell Line, Tumor
• Cell Movement/physiology
• Endostatins/genetics
• Endothelial Cells/cytology
• Enteroendocrine Cells/pathology
• Enteroendocrine Cells/physiology
• Female
• Humans
• Intestinal Neoplasms/physiopathology
• Intestinal Neoplasms/secondary
• Liver Neoplasms, Experimental/physiopathology
• Liver Neoplasms, Experimental/secondary
• Mice
• Mice, Nude
• Neoplasm Transplantation
• Neovascularization, Pathologic/pathology
• Neovascularization, Pathologic/physiopathology
• Spleen
• Transfection
• Transplantation, Heterologous
• Umbilical Veins/cytology

AIM: To investigate the value and prospect of RT-PCR in detecting micrometastasis in regional lymph nodes of gastric cancer.

METHODS: Histopathology was used and K19 mRNA expression was detected by RT-PCR in tumor tissues and lymph nodes from gastric cancer patients undergoing radical resection of gastric carcinoma.

RESULTS: K19 mRNA was expressed in all tumor specimens of 30 cases; of the 126 lymph nodes, 26 were histopathologically positive (20.6%), and 42 positive (33.3%) by RT-PCR. Amplification fragments of 460 and 540 bp were shown in all the tumor tissues and metastatic lymph nodes after K19 and β-actin RT-PCR, while only a 540 bp fragment appeared in the lymph nodes of non-tumor patients.

CONCLUSION: K19 mRNA RT-PCR is sensitive and specific in testing micrometastasis in regional lymph nodes of gastric cancer, and it is superior to routine histopathology.

• K-19 mRNA
• RT-PCR
• Micrometastasis
• Gastric cancer

• Actins/genetics
• Biomarkers, Tumor
• Carcinoma/pathology
• DNA Primers/chemistry
• Humans
• Keratin-19/genetics
• Lymphatic Metastasis
• Neoplasm Metastasis
• Prognosis
• RNA, Messenger/metabolism
• Reverse Transcriptase Polymerase Chain Reaction
• Stomach Neoplasms/genetics
• Stomach Neoplasms/pathology

• endostatin
• gene
• adeno-associated virus
• hepatocellular carcinoma

• Adenoviridae/genetics
• Carcinoma, Hepatocellular
• Cell Line, Tumor
• Endostatins/genetics
• Gene Transfer Techniques
• Humans
• Liver Neoplasms

Endostatin, a carboxy-terminal fragment of collagen XVIII, potently inhibits angiogenesis and tumour growth, presumably through induction of apoptosis in endothelial cells and/or inhibition of their migration. Here we have tested how the timing of recombinant human endostatin (rh-E) administration affects its antitumour activity in a liver metastasis model of mouse C26 colorectal carcinoma cells. The effects of rh-E treatment on hepatic tumour load and on early tumour cell seeding were evaluated. Recombinant human endostatin was most effective in reducing intrahepatic tumour growth when administered prior to tumour cell inoculation. Analysis of early tumour cell seeding by using [¹²⁵I]iododeoxyuridine-labelled C26 cells or by in vivo microscopy showed that rh-E reduced tumour cell seeding in the liver sinusoids. Recombinant human endostatin did not inhibit tumour growth when administered later than 4 days after tumour injection. Pretreatment of human umbilical vein endothelial cells with rh-E in vitro reduced C26 tumour cell adhesion under flow conditions two-fold as assessed by video microscopy and multiphoton laser scanning microscopy. Our results show that rh-E, in addition to antiangiogenic effects, reduces tumour cell adhesion in the liver sinusoids during the very early phases of metastasis formation. These data point towards a previously unknown mode of action of endostatin, that is, its ability to interfere with tumour cell seeding. Such insights may be helpful in the design of trials to improve (surgical) treatment of colorectal carcinoma and liver metastases.

• Animals
• Antineoplastic Agents/pharmacology
• Cell Adhesion
• Colorectal Neoplasms/drug therapy
• Colorectal Neoplasms/pathology
• Disease Models, Animal
• Endostatins/pharmacology
• Endothelial Cells
• Humans
• Liver Neoplasms/prevention & control
• Liver Neoplasms/secondary
• Mice
• Microscopy, Confocal
• Microscopy, Video
• Neoplasm Seeding
• Recombinant Proteins/pharmacology
• Time Factors
• Umbilical Veins

• Adenoviridae/genetics
• Angiogenesis Inhibitors
• Animals
• Antineoplastic Agents
• Carcinoma, Squamous Cell/pathology
• Carcinoma, Squamous Cell/therapy
• Down-Regulation
• Endostatins/genetics
• Genetic Therapy
• Genetic Vectors/genetics
• Humans
• Lymph Nodes/pathology
• Lymphangiogenesis
• Lymphatic Metastasis
• Lymphatic Vessels/metabolism
• Mice
• Neoplasm Transplantation
• Tongue Neoplasms/pathology
• Tongue Neoplasms/therapy
• Tumor Cells, Cultured
• Vascular Endothelial Growth Factor C/genetics
• Vascular Endothelial Growth Factor C/metabolism

• Animals
• Apolipoproteins/biosynthesis
• Apolipoproteins/genetics
• Apoprotein(a)
• Apoptosis/genetics
• Cell Line, Tumor
• Cell Movement/genetics
• Colorectal Neoplasms/blood supply
• Colorectal Neoplasms/metabolism
• Colorectal Neoplasms/pathology
• Colorectal Neoplasms/therapy
• DNA, Complementary/administration & dosage
• DNA, Complementary/genetics
• Disease Models, Animal
• Endothelium, Vascular/pathology
• Genetic Therapy/methods
• Kringles/genetics
• Lipoprotein(a)/biosynthesis
• Lipoprotein(a)/genetics
• Liver Neoplasms, Experimental/genetics
• Liver Neoplasms, Experimental/metabolism
• Liver Neoplasms, Experimental/prevention & control
• Liver Neoplasms, Experimental/secondary
• Male
• Mice
• Mice, Inbred BALB C
• Neovascularization, Pathologic/genetics
• Neovascularization, Pathologic/metabolism
• Neovascularization, Pathologic/therapy

Although angiogenesis is a prerequisite for the growth of most human solid tumours, alternative mechanisms of vascularisation can be adopted. We have previously described a non-angiogenic growth pattern in liver metastases of colorectal adenocarcinomas (CRC) in which tumour cells replace hepatocytes at the tumour–liver interface, preserving the liver architecture and co-opting the sinusoidal blood vessels. The aim of this study was to determine whether this replacement pattern occurs during liver metastasis of breast adenocarcinomas (BC) and whether the lack of an angiogenic switch in such metastases is due to the absence of hypoxia and subsequent vascular fibrinogen leakage. The growth pattern of 45 BC liver metastases and 28 CRC liver metastases (73 consecutive patients) was assessed on haematoxylin- and eosin-stained tissue sections. The majority of the BC liver metastases had a replacement growth pattern (96%), in contrast to only 32% of the CRC metastases (P<0.0001). The median carbonic anhydrase 9 (CA9) expression (M75 antibody), as a marker of hypoxia, (intensity × % of stained tumour cells) was 0 in the BC metastases and 53 in the CRC metastases (P<0.0001). There was CA9 expression at the tumour–liver interface in only 16% of the BC liver metastases vs 54% of the CRC metastases (P=0.002). There was fibrin (T2G1 antibody) at the tumour-liver interface in only 21% of the BC metastases vs 56% of the CRC metastases (P=0.04). The median macrophage count (Chalkley morphometry; KP-1 anti-CD68 antibody) at the interface was 4.3 and 7.5, respectively (P<0.0001). Carbonic anhydrase 9 score and macrophage count were positively correlated (r=0.42; P=0.002) in all metastases. Glandular differentiation was less in the BC liver metastases: 80% had less than 10% gland formation vs only 7% of the CRC metastases (P<0.0001). The liver is a densely vascularised organ and can host metastases that exploit this environment by replacing the hepatocytes and co-opting the vasculature. Our findings confirm that a non-angiogenic pattern of liver metastasis indeed occurs in BC, that this pattern of replacement growth is even more prevalent than in CRC, and that the process induces neither hypoxia nor vascular leakage.

• Adenocarcinoma/metabolism
• Adenocarcinoma/pathology
• Antigens, CD/analysis
• Antigens, Differentiation, Myelomonocytic/analysis
• Breast Neoplasms/metabolism
• Breast Neoplasms/pathology
• Carbonic Anhydrases/analysis
• Cell Hypoxia
• Colorectal Neoplasms/metabolism
• Colorectal Neoplasms/pathology
• Fibrin/analysis
• Glycoproteins/analysis
• Humans
• Immunohistochemistry
• Liver Neoplasms/pathology
• Liver Neoplasms/secondary
• Macrophages/pathology
• Neovascularization, Pathologic
• Vesicular Transport Proteins

• Angiogenesis Inhibitors/pharmacology
• Animals
• Disease Models, Animal
• Endostatins/pharmacology
• Humans
• Liver Neoplasms/blood supply
• Liver Neoplasms/prevention & control
• Liver Neoplasms/secondary
• Male
• Melanoma, Experimental/blood supply
• Mice
• Mice, Inbred C57BL
• Microcirculation/drug effects
• Recombinant Proteins/pharmacology
• Tumor Cells, Cultured
• Vascular Cell Adhesion Molecule-1/analysis

• Animals
• Apoptosis
• Cell Division
• Cell Hypoxia
• Cell Line
• Cell Movement
• Cyclooxygenase 2
• Endothelial Growth Factors/analysis
• Endothelial Growth Factors/pharmacology
• Endothelium, Vascular/pathology
• Glial Fibrillary Acidic Protein/analysis
• Intercellular Signaling Peptides and Proteins/analysis
• Intercellular Signaling Peptides and Proteins/pharmacology
• Isoenzymes/analysis
• Liver/pathology
• Liver Neoplasms/pathology
• Liver Neoplasms/secondary
• Lymphokines/analysis
• Lymphokines/pharmacology
• Male
• Melanoma, Experimental/pathology
• Mice
• Mice, Inbred C57BL
• Neoplasm Transplantation
• Neovascularization, Pathologic
• Prostaglandin-Endoperoxide Synthases/analysis
• Rats
• Rats, Sprague-Dawley
• Reverse Transcriptase Polymerase Chain Reaction
• Vascular Endothelial Growth Factor A
• Vascular Endothelial Growth Factors

• DK56621 NIDDK NIH HHS