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The first consensus guidelines for scoring the histopathological growth patterns (HGPs) of liver metastases were established in 2017. Since then, numerous studies have applied these guidelines, have further substantiated the potential clinical value of the HGPs in patients with liver metastases from various tumour types and are starting to shed light on the biology of the distinct HGPs. In the present guidelines, we give an overview of these studies, discuss novel strategies for predicting the HGPs of liver metastases, such as deep-learning algorithms for whole-slide histopathology images and medical imaging, and highlight liver metastasis animal models that exhibit features of the different HGPs. Based on a pooled analysis of large cohorts of patients with liver-metastatic colorectal cancer, we propose a new cut-off to categorise patients according to the HGPs. An up-to-date standard method for HGP assessment within liver metastases is also presented with the aim of incorporating HGPs into the decision-making processes surrounding the treatment of patients with liver-metastatic cancer. Finally, we propose hypotheses on the cellular and molecular mechanisms that drive the biology of the different HGPs, opening some exciting preclinical and clinical research perspectives., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Cancer is first a localized tissue disorder, whose soluble and exosomal molecules and invasive cells induce a host response providing the stromal components of the primary tumor microenvironment (TME). Once the TME is developed, cancer-derived molecules and cells can more efficiently spread out and a whole-body response takes place, whose pathophysiological changes may result in a paraneoplastic syndrome. Remote organ-specific prometastatic reactions may also occur at this time, facilitating metastatic activities of circulating tumor cells (CTCs) through premetastatic niche development at targeted organs. However, additional signaling factors from the inter-organ communication network involved in the pathophysiology and comorbidities of cancer patients may also regulate prometastatic reaction-stimulating effects of cancer and non-cancer tissue factors. This article provides a conceptual overview of our ongoing clinical research on the liver prometastatic reaction (LPR) of patients with colorectal cancer (CRC), their portal vein- and hepatic artery-driven LPR-Stimulating Factors (LPR-SF), and their resulting LPR-derived Metastasis-Stimulating Factors (LPR-MSF) acting on liver-invading CRC cells. In addition, we also provide new insights on the molecular subtyping of LPR-responsive cancer phenotypes in patients with CRC and melanoma; and on how to investigate and interpret the prometastatic infrastructure in the real pathophysiological context of patients with cancer undergoing surgical procedures and receiving pharmacological treatments with multiple side effects, including those affecting the LPR, its stimulating factors and responsive cancer phenotypes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Liver metastases present with distinct histopathological growth patterns (HGPs), including the desmoplastic, pushing and replacement HGPs and two rarer HGPs. The HGPs are defined owing to the distinct interface between the cancer cells and the adjacent normal liver parenchyma that is present in each pattern and can be scored from standard haematoxylin-and-eosin-stained (H&E) tissue sections. The current study provides consensus guidelines for scoring these HGPs., Methods: Guidelines for defining the HGPs were established by a large international team. To assess the validity of these guidelines, 12 independent observers scored a set of 159 liver metastases and interobserver variability was measured. In an independent cohort of 374 patients with colorectal liver metastases (CRCLM), the impact of HGPs on overall survival after hepatectomy was determined., Results: Good-to-excellent correlations (intraclass correlation coefficient >0.5) with the gold standard were obtained for the assessment of the replacement HGP and desmoplastic HGP. Overall survival was significantly superior in the desmoplastic HGP subgroup compared with the replacement or pushing HGP subgroup (P=0.006)., Conclusions: The current guidelines allow for reproducible determination of liver metastasis HGPs. As HGPs impact overall survival after surgery for CRCLM, they may serve as a novel biomarker for individualised therapies.

Id1 promotes carcinogenesis and metastasis, and predicts prognosis of non-small cell lung cancer (NSCLC)-adenocarcionoma patients. We hypothesized that Id1 may play a critical role in lung cancer colonization of the liver by affecting both tumor cells and the microenvironment. Depleted levels of Id1 in LLC (Lewis lung carcinoma cells, LLC shId1) significantly reduced cell proliferation and migration in vitro. Genetic loss of Id1 in the host tissue (Id1 -/- mice) impaired liver colonization and increased survival of Id1 -/- animals. Histologically, the presence of Id1 in tumor cells of liver metastasis was responsible for liver colonization. Microarray analysis comparing liver tumor nodules from Id1 +/+ mice and Id1 -/- mice injected with LLC control cells revealed that Id1 loss reduces the levels of EMT-related proteins, such as vimentin. In tissue microarrays containing 532 NSCLC patients' samples, we found that Id1 significantly correlated with vimentin and other EMT-related proteins. Id1 loss decreased the levels of vimentin, integrinβ1, TGFβ1 and snail, both in vitro and in vivo. Therefore, Id1 enables both LLC and the host microenvironment for an effective liver colonization, and may represent a novel therapeutic target to avoid NSCLC liver metastasis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Random homozygous gene perturbation (RHGP), in combination with liver sinusoidal endothelial cell (LSEC) adhesion screening of clonal colon cancer cells with perturbed genes, was used to identify genes contributing to the hepatic microvascular adhesion of colon cancer cells. Plasmid vector encoding transactivator and gene search vector were transfected into HT-29 human colorectal cancer cells to create a HT-29 RHGP cell library; the adhesion of these library cells to primary cultured mouse LSEC significantly decreased in the presence of RSL1 ligand (inducer), indicating that most of the genes contributing to HT-29 adhesion to LSEC were altered. Next, HT-29 RHGP cell library fractions with upregulated or silenced LSEC adhesion-related genes were isolated. Around 160 clones having altered expression in LSEC adhesion-related genes were obtained, and nine relevant protein-coding genes were identified. Some were proadhesive genes detected because of their overexpression in adherent HT-29 cells (DGCR8 and EFEMP1 genes) and their silenced status in nonadherent HT-29 cells (DGKE, DPY19L1, KIAA0753, PVR and USP11 genes). Others were antiadhesive genes detected because of their overexpression in nonadherent HT-29 cells (ITPKC gene) and their silenced status in adherent HT-29 cells (PPP6R2 gene). Silencing of PVR, DGCR8 and EFEMP1 genes decreased adhesion to LSEC and hepatic microvascular retention of HT-29 cells. The results conclude that RHGP was a valuable strategy for the discovery of mechanisms regulating microvascular adhesion of circulating colon cancer cells before hepatic metastasis formation. Identified genes may contribute to understand the metastatic process of colon cancer and to discovering molecular targets for hepatic metastasis therapeutics., (© 2013 UICC.)

Colorectal cancer (CRC) frequently metastasizes to the liver, a phenomenon that involves the participation of transforming-growth-factor-β(1) (TGFβ(1)). Blockade of the protumorigenic effects elicited by TGFβ(1) in advanced CRC could attenuate liver metastasis. We aimed in the present study to assess the antimetastatic effect of TGFβ(1)-blocking peptides P17 and P144, and to study mechanisms responsible for this activity in a mouse model. Colon adenocarcinoma cells expressing luciferase were pretreated with TGFβ(1) (Mc38-luc(TGFβ1) cells), injected into the spleen of mice and monitored for tumor development. TGFβ(1) increased primary tumor growth and liver metastasis, whereas systemic treatment of mice with either P17 or P144 significantly reduced tumor burden (p<0.01). In metastatic nodules, mitotic/apoptotic ratio, mesenchymal traits and angiogenesis (evaluated by CD-31, as well as circulating endothelial and progenitor cells) induced by TGFβ(1) were consistently reduced following injection of peptides. In vitro experiments revealed a direct effect of TGFβ(1) in Mc38 cells, which resulted in activation of Smad2, Smad3 and Smad1/5/8, and increased invasion and transendothelial migration, whereas blockade of TGFβ(1)-signaling reverted these features. Because TGFβ(1)-mediated epithelial-mesenchymal transition (EMT) has been suggested to induce a cancer stem cell (CSC) phenotype, we analyzed the ability of this cytokine to induce tumorsphere formation and the expression of CSC markers. In TGFβ(1)-treated cells, tumorspheres were enriched in CD44 and SOX2, which were diminished in the presence of P17. Our data provide a preclinical rationale to evaluate P17 and P144 as potential therapeutic options for the treatment of metastatic CRC., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Previous studies identified the human nonmetastatic gene 23 (NME1, hereafter Nm23-H1) as the first metastasis suppressor gene. An inverse relationship between Nm23-H1 and expression of lysophosphatidic acid receptor 1 gene (LPAR1, also known as EDG2 or hereafter LPA1) has also been reported. However, the effects of LPA1 inhibition on primary tumor size, metastasis, and metastatic dormancy have not been investigated., Methods: The LPA1 inhibitor Debio-0719 or LPA1 short hairpinned RNA (shRNA) was used. Primary tumor size and metastasis were investigated using the 4T1 spontaneous metastasis mouse model and the MDA-MB-231T experimental metastasis mouse model (n = 13 mice per group). Proliferation and p38 intracellular signaling in tumors and cell lines were determined by immunohistochemistry and western blot to investigate the effects of LPA1 inhibition on metastatic dormancy. An analysis of variance-based two-tailed t test was used to determine a statistically significant difference between treatment groups., Results: In the 4T1 spontaneous metastasis mouse model, Debio-0719 inhibited the metastasis of 4T1 cells to the liver (mean = 25.2 liver metastases per histologic section for vehicle-treated mice vs 6.8 for Debio-0719-treated mice, 73.0% reduction, P < .001) and lungs (mean = 6.37 lesions per histologic section for vehicle-treated mice vs 0.73 for Debio-0719-treated mice, 88.5% reduction, P < .001), with no effect on primary tumor size. Similar results were observed using the MDA-MB-231T experimental pulmonary metastasis mouse model. LPA1 shRNA also inhibited metastasis but did not affect primary tumor size. In 4T1 metastases, but not primary tumors, expression of the proliferative markers Ki67 and pErk was reduced by Debio-0719, and phosphorylation of the p38 stress kinase was increased, indicative of metastatic dormancy., Conclusion: The data identify Debio-0719 as a drug candidate with metastasis suppressor activity, inducing dormancy at secondary tumor sites.

Background: Implantation and growth of metastatic cancer cells at distant organs is promoted by inflammation-dependent mechanisms. A hepatic melanoma metastasis model where a majority of metastases are generated via interleukin-18-dependent mechanisms was used to test whether anti-inflammatory properties of resveratrol can interfere with mechanisms of metastasis., Methods: Two experimental treatment schedules were used: 1) Mice received one daily oral dose of 1 mg/kg resveratrol after cancer cell injection and the metastasis number and volume were determined on day 12. 2) Mice received one daily oral dose of 1 mg/kg resveratrol along the 5 days prior to the injection of cancer cells and both interleukin-18 (IL-18) concentration in the hepatic blood and microvascular retention of luciferase-transfected B16M cells were determined on the 18th hour. In vitro, primary cultured hepatic sinusoidal endothelial cells were treated with B16M-conditioned medium to mimic their in vivo activation by tumor-derived factors and the effect of resveratrol on IL-18 secretion, on vascular cell adhesion molecule-1 (VCAM-1) expression and on tumor cell adhesion were studied. The effect of resveratrol on melanoma cell activation by IL-18 was also studied., Results: Resveratrol remarkably inhibited hepatic retention and metastatic growth of melanoma cells by 50% and 75%, respectively. The mechanism involved IL-18 blockade at three levels: First, resveratrol prevented IL-18 augmentation in the blood of melanoma cell-infiltrated livers. Second, resveratrol inhibited IL-18-dependent expression of VCAM-1 by tumor-activated hepatic sinusoidal endothelium, preventing melanoma cell adhesion to the microvasculature. Third, resveratrol inhibited adhesion- and proliferation-stimulating effects of IL-18 on metastatic melanoma cells through hydrogen peroxide-dependent nuclear factor-kappaB translocation blockade on these cells., Conclusions: These results demonstrate multiple sites for therapeutic intervention using resveratrol within the prometastatic microenvironment generated by tumor-induced hepatic IL-18, and suggest a remarkable effect of resveratrol in the prevention of inflammation-dependent melanoma metastasis in the liver.

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

We investigated effects of endostatin (ES) in the prometastatic microenvironment of inflammation occurring during the microvascular phase of cancer cell infiltration in the liver. We used a model of intrasplenic injection of B16 melanoma (B16M) cells leading to hepatic metastasis through vascular cell adhesion molecule-(VCAM-1)-mediated capillary arrest of cancer cells via interleukin-18 (IL-18)-dependent mechanism. We show that administration of 50 mg/kg recombinant human (rh) ES 30 min before B16M, plus repetition of same dose for 3 additional days decreased metastasis number by 60%. A single dose of rhES before B16M injection reduced hepatic microvascular retention of luciferase-transfected B16M by 40% and inhibited hepatic production of tumor necrosis factor alpha (TNF-alpha) and IL-18 and VCAM-1 expression by hepatic sinusoidal endothelia (HSE). Consistent with these data, rhES inhibited VCAM-1-dependent B16M cell adhesion to primary cultured HSE receiving B16M conditioned medium, and it abolished the HSE cell production of TNF-alpha and IL-18 induced by tumor-derived vascular endothelial cell growth factor (VEGF). rhES abrogated recombinant murine VEGF-induced tyrosine phosphorylation of KDR/flk-1 receptor in HSE cells, preventing the proinflammatory action of tumor-derived VEGF on HSE. rhES also abolished hepatic production of TNF-alpha, microvascular retention of luciferase-transfected B16M, and adhesion of B16M cells to isolated HSE cells, all of them induced in mice given 5 micro g/kg recombinant murine VEGF for 18 h. This capillary inflammation-deactivating capability constitutes a nonantiangiogenic antitumoral action of endostatin that decreases cancer cell arrest within liver microvasculature and prevents metastases promoted by proinflammatory cytokines induced by VEGF.

Myofibroblasts infiltrate malignant liver tumors, although their pathogenic implications are unclear. Immunohistochemical detection of alpha-smooth muscle actin, glial fibrillary acidic protein (GFAP), and CD31 and CD34 expression was used to analyze the contribution of myofibroblasts to angiogenesis in hepatic metastasis produced by intrasplenically-injected B16 melanoma (B16M). Because activated hepatic stellate cells (HSCs) are oxygen-sensing myofibroblasts producing vascular endothelial growth factor (VEGF), the effect of B16M and human A375 melanoma supernatants on VEGF production by immortalized rat HSC line T6 and primary cultured human HSCs also was studied under an hypoxic atmosphere mimicking a tumor microenvironment. Myofibroblast infiltration preceded endothelium recruitment in avascular micrometastasis and generated specific stroma for sinusoidal-type and portal-type angiogeneses. Thereafter, myofibroblasts and endothelial cells colocalized within both angiogenic patterns and their numerical densities correlated with metastasis development. Myofibroblasts often were GFAP-positive, suggesting an HSC origin. Melanoma supernatants stimulated VEGF messenger RNA and protein synthesis by HSCs. These effects were potentiated by hypoxia. VEGF up-regulation was accompanied by increased expression of cyclooxygenase type 2 (COX-2) and PGE2 synthesis. HSC production of VEGF decreased under COX-2 inhibition, whereas it was increased by exogenous PGE2. The high VEGF expression in HSCs induced by melanoma factors and hypoxia resulted in mitogenic, antiapoptotic, and motogenic stimulation of both murine hepatic sinusoidal endothelium and human umbilical vein endothelium. In conclusion, temporal and positional relationships evolve between myofibroblast and endothelium recruitment during metastasis development. Mechanistically, hypoxic induction of VEGF in tumor-activated HSCs may create a proangiogenic microenvironment, facilitating endothelial cell recruitment and survival during hepatic metastasis transition from an avascular to a vascular stage.

Theophylline-treated B16-F10 melanoma cells show a lower experimental metastatic potential in vivo. To identify the possible mechanism(s) involved and on the basis of previous reports, we tested the induction of apoptosis in B16-F10 cells. Fluorescence activated cell sorter (FACS) analysis and p53 overexpression in theophylline-treated B16-F10 melanoma cells appeared to suggest enhanced cell death by apoptosis. The in vivo effects of orally administered theophylline in mice were investigated using different treatment schedules in mice that had undergone hepatic or pulmonary colonization with tumour cells. Mice received theophylline in their drinking water according to different protocols: (i) from 3 days before tumour cell inoculation until animal sacrifice ('early treatment'); (ii) from 3 days before until 3 days after tumour cell inoculation ('short treatment'); or (iii) from 3 days after tumour cell inoculation until animal sacrifice ('late treatment'). In the 'early treatment' group, the number of melanoma foci was reduced by 92.3% in the liver and 81.4% in the lung compared with control animals (P < 0.001). In the 'short treatment' group, there was an 80.2% and 72.2% reduction in liver and lung metastases, respectively (P < 0.001). In the 'late treatment' group, the inhibition of metastasis was 59.7% for liver and 45.3% for lung (P < 0.005). Survival studies showed that 50% of the 'early' theophylline-treated animals died 33.2 +/- 2.0 days after intrasplenic injection (control group: 23.1 1.8 days; P < 0.001) and 33.9 +/- 2.5 days after tail vein injection (control group: 24.1 +/- 1.4 days; P < 0.001). Taken together, these observations provide useful information for the potential clinical application of theophylline as a chemotherapeutic agent against malignant melanoma.

Proinflammatory cytokines, including IL-1beta and tumor necrosis factor-alpha (TNF-alpha), promote cancer cell adhesion and liver metastases by up-regulating the expression of vascular cell adhesion molecule-1 (VCAM-1) on hepatic sinusoidal endothelium (HSE). In this study, hepatic metastasis after intrasplenically injected mouse B16 melanoma (B16M) cells was reduced 84-95% in mice with null mutations for either IL-1beta or the IL-1beta-converting enzyme (ICE, caspase-1) compared with wild-type mice. On day 12, 47% of wild-type mice were dead compared with 19% of either IL-1beta or ICE-deficient mice. In vitro, conditioned medium from B16M cells (B16M-CM) induced the release of TNF-alpha and IL-1beta from cultures of primary murine HSE. The effect of B16M-CM on HSE resulted in increased numbers of B16M cells adhering to HSE, which was completely abrogated by a specific inhibitor of ICE, anti-IL-18 or IL-18-binding protein. Exogenous IL-18 added to HSE also increased the number of adhering melanoma cells; however, this was not affected by IL-1 receptor blockade or TNF neutralization but rather by anti-VCAM-1. These results demonstrate a role for IL-1beta and IL-18 in the development of hepatic metastases of B16M in vivo. In vitro, soluble products from B16M cells stimulate HSE to sequentially release TNF-alpha, IL-1beta, and IL-18. The IL-18 cytokine increases expression of VCAM-1 and the adherence of melanoma cells.

B16 melanoma (B16M) was used to study the relationship between glutathione (GSH) metabolism and the metastatic activity of malignant cells. GSH content increased in B16M cells during the initial period of exponential growth in vitro, to reach a maximum of 37 +/- 3 nmol/10(6) cells 12 h after plating, and then gradually decreased to control values (10 +/- 2 nmol/10(6) cells) when cultures approached confluency. On the contrary, glutathione disulphide (GSSG) levels (0.5 +/- 0.2 nmol/10(6) cells) and the rate of glutathione efflux (GSH + GSSG) (2.5 +/- 0.4 nmol/10(6) cells per h) remained constant as B16M grew. Changes in enzyme activities involved in GSH synthesis or the glutathione redox cycle did not explain shifts in the glutathione status (GSH/GSSG). However, two facts contributed to explain why GSH levels changed within B16M cells: a) high intracellular levels of GSH induced a feed-back inhibition of its own synthesis in B16M cells from cultures with low cellular density (LD cells); b) transport of cyst(e)ine, whose availability is the major rate-limiting step for GSH synthesis, was limited by cell-cell contact in cultures with high cellular density (HD cells). Intrasplenic injection of B16M cells with high GSH content (exponentially-growing cultures) showed higher metastatic activity in the liver than cells with low GSH content (cells at confluency). However, when low GSH-content cells (HD cells) were incubated in the presence of GSH ester, which rapidly enters the cell and delivers free GSH, their metastatic activity significantly increased. Our results demonstrate that changes in GSH content regulate the metastatic behaviour of B16M cells.

The role of mannose receptors from hepatic sinusoidal endothelium (HSE) in liver colonization by B16 melanoma (B16M) cells was studied. The expression of high mannose-type oligosaccharides on the surface of B16M cells was enhanced by in vitro treatment with 1-deoximannojirimycin (1-DMM). There was a significant (P < 0.01) enhancement of hepatic metastasis when B16M cells were 1-DMM-treated before being intrasplenically injected into C57BL/6J mice. Intraperitoneal administration of 5 mg/kg recombinant human interleukin-1 receptor antagonist (rHuIL-1Ra) inhibited the 1-DMM-induced enhancement of metastasis. Expression of high mannose-type oligosaccharides on the surface of 1-DMM-treated B16M cells and their in vitro adhesion to the HSE was significantly correlated (R = 0.82). The addition of either 100 microg/ml mannan or paraformaldehyde (PFA)-fixed 1-DMM-treated B16M cells to cultured HSE for a period of 12 h significantly (P < 0.01) increased the release of IL-1beta from the HSE compared to that liberated by the HSE incubated with either basal medium or PFA-fixed untreated B16M cells. The same HSE treatments also significantly (P < 0.01) increased the degree of adhesion of other B16M cells to HSE, being abrogated by anti-mouse vascular cell adhesion molecule-1 (VCAM-1) antibodies. The conditioned media from HSE cultures, activated by PFA-fixed, 1-DMM-treated B16M cells significantly (P < 0.01) increased B16M cell proliferation when compared to conditioned media from HSE cultures incubated with PFA-fixed, untreated B16M cells. Thus, 1-DMM treatment of B16M cells enhanced the development of hepatic metastasis by IL-1-dependent mechanisms. The mechanism is consistent with in vitro mannose receptor-mediated melanoma cell attachment to the HSE, which subsequently upregulates IL-1beta release, VCAM-1-dependent adherence, and melanoma growth factor(s) release by HSE.

In this work we report the presence of intrametastatic smooth-muscle iso-alpha-actin (SMA)-expressing cells which appeared from the early stages of the hepatic metastasis process of intrasplenically injected B16 melanoma (B16M) cells. They formed a network of stromal cells among B16M cells, a very low percentage of them expressing desmin. In contrast, those parts of liver tissue unaffected by metastasis had perisinusoidal desmin-expressing quiescent hepatic stellate cells (qHSC) which did not express SMA. Exposure of freshly isolated rat quiescent hepatic stellate cells (qHSC) to B16M cell-conditioned medium (B16M-CM) leads to a progressive increase (P < .01) in the number of SMA-expressing cells, which was accompanied by a parallel reduction in the number of desmin-expressing cells. In addition, B16M-CM also contained chemotactic factor(s) which significantly (P < .01) increased (50%) in vitro qHSC migration and stimulated both [3H]thymidine and [3H]glucosamine uptake in qHSC. Moreover, B16M-CM also significantly (P < .01) enhanced qHSC secretion of matrix metalloproteinase-2 (MMP-2), and unknown chemotactic factor(s) enhancing in vitro migration of B16M cells. The results suggest that B16 melanoma releases qHSC-activating factors, which induce the appearance of metastasis-infiltrating myofibroblasts by a paracrine mechanism. Such cells showed cytoskeletal alterations which are associated with enhanced proliferation, glycosaminoglycan synthesis, MMP-2 secretion, and tumor-chemotactic factor production. Thus, tumor-activated qHSC may play an important role in melanoma cell motility and invasion during hepatic metastasis progression.

Liver sinusoids, in contrast with the capillaries of other tissues, contain large numbers of sequestered lymphocytes. These blood-borne cells preferentially home in the liver. The mechanism regulating the recruitment of these cells and molecular regulation of the recognition of endothelial cells is as yet unclear. The present study sought to evaluate the cell adhesion molecules on human liver-associated lymphocytes and their ligands on sinusoidal lining cells in 29 patients undergoing partial hepatectomy for liver tumors. Liver-associated lymphocytes and peripheral blood lymphocytes were analyzed by flow cytometry using monoclonal antibodies. Frozen sections of liver tissue were stained according to alkaline phosphatase anti-alkaline phosphatase method. Cytometric analysis showed that virtually all liver-associated lymphocytes expressed on their surface the cell adhesion molecules LFA-1 and VLA-4. This liver-associated lymphocyte population also presented a significantly higher percentage of Mac 1, ICAM-1, and LFA-3 and an increased surface expression of LFA-1, LFA-2, and NCAM in comparison with peripheral blood lymphocytes. It was likewise shown that sinusoidal cells express ICAM-1, ICAM-2, ICAM-3, VCAM-1 and LFA-3 ligands. Liver-associated lymphocytes thus strongly express a number of different adhesion molecules. The corresponding ligands were also detected on sinusoidal lining cells. LFA-1 and VLA-4 would seem to be important pathways of temporary lymphocyte-endothelial adhesion in liver sinusoids.

We evaluated the expression of the cell-adhesion molecules (CAM) that might be involved in liver-associated lymphocyte (LAL) contacts with other sinusoidal cells and/or be responsible for natural-killer(NK)- and lymphokine-activated killer(LAK) activity in patients with liver metastasis. The LAL population was isolated by sinusoidal high-pressure lavage from partial hepatectomies obtained from patients operated for metastases (n = 13) and benign liver tumors (n = 9). Surface expression of the beta-2-integrin chains (CD11a, CD11b, CD11c and CD18), and the beta-I-integrin chains (CD49b, CD49d, CD49f and CD29), as well as that of members of the immunoglobulin superfamily (CD2, CD54, CD56 and CD58), were analyzed by one- or two-color flow cytometry. Quantitative and qualitative differences were observed in both groups of patients in the expression of CAM between LAL and peripheral blood lymphocytes (PBL). LAL were characterized by an increase in the percentage of CD11b-, CD49b-, CD49d-, CD54-, CD56- and CD58-positive cells in comparison with PBL. Fluorescence values for CD2, CD11a, CD18 and CD56 were higher in LAL than in PBL. Moreover, the population expressing these antigens of differentiation presented a bimodal distribution (dim and bright): in LAL, as opposed to PBL, the percentage of cells with a bright phenotype was greater than of those with a dim one. The increase in CAM expression on LAL could be due to the influence of the liver sinusoidal micro-environment. Results were more unexpected for the comparison between benign and malignant tumors. No difference was found in CAM expression on LAL between these 2 categories. Consequently, it cannot be this factor that explains the decrease in LAK activity of LAL in patients with metastasis.

Hepatic metastasis in melanoma is affected by processes of tumor cell adhesion to sinusoidal cells, avoidance of cytotoxic cells, and local growth-promoting activity. A role for interleukin-1 (IL-1) in these events was evaluated in vitro and in vivo by specifically blocking IL-1 receptors using the naturally occurring IL-1 receptor antagonist (IL-1Ra). At 10- and 100-fold molar excess, IL-1Ra reduced the IL-1-mediated adhesion of B16 melanoma cells to cultured hepatic sinusoidal endothelial cells by 60 and 100%, respectively. Conditioned media derived from murine hepatic sinusoidal endothelial cells contain growth-promoting activity for B16 cells, and IL-1 increased its production 2.5-fold (P < 0.01). The addition of IL-1Ra to sinusoidal cells reduced the spontaneous release of the growth-promoting activity by 32% (P < 0.05). In addition, blocking IL-1 receptors on melanoma cells reduced their responsiveness to endothelial-conditioned medium (P < 0.05). A single dose of IL-1Ra (0.5 mg/kg) given to mice i.p. 2 h prior to intrasplenic injection of melanoma cells reduced the number of hepatic metastases by 50% and the metastatic volume by 70% compared to vehicle-injected controls (P < 0.01). When given 2, 4, and 6 days after the injection of tumor cells, IL-1Ra reduced the volume of metastases by 58% (P < 0.01). Fifty % of mice pretreated with 0.5 mg/kg IL-1Ra and given 3 additional doses on days 2, 4, and 6 died after 13.5 +/- 0.4 days compared to 11.3 +/- 0.2 days for controls (P < 0.01). In mice pretreated and given 10 daily doses at 5 mg/kg, there was an 80% reduction in hepatic metastases. Using this regimen, survival was 18.1 +/- 2.4 days in the IL-1Ra group and 11.2 +/- 1.5 in controls (P < 0.001). These studies demonstrate a significant role for IL-1 in implantation and growth of metastatic melanoma in the liver.

Quantitative microdensitometry and computerised interactive image analysis were used to compare the expression of endogenous lectins by cells of mouse colon 26 carcinomas, growing either as primary tumours or metastases, in five different anatomic sites (caecum, liver, lung, spleen, s.c.). Endogenous lectins were visualised in tissue sections using the ABC peroxidase technique with a panel of 17 biotinylated neoglycoproteins representing a variety of carbohydrates found in glycoproteins, glycolipids and proteoglycans. Clear-cut site-associated differences in endogenous lectin expression were detected in cancer cells growing in all five sites. The patterns of these changes were complex and shifts in expression of different lectins were independently variable in both direction and amount. In addition to site-associated variations, differences in lectin expression were also detected in the liver and lungs, between cells in spontaneous metastases and cells in colonies generated by direct injection of cancer cells into the bloodstream. The results demonstrate quantitative, as distinct from qualitative, differences developing in cancer cell populations after delivery of cells to different target organs. The differences between liver and lung metastases are in accord with analogous site-associated differences in metastatic patterns produced by colon carcinoma cells in mice and in humans.

We have examined the anatomical-functional sites within mouse liver where phenylhydrazine (PHZ)-induced hematopoietic foci, and M5076 reticulum cell sarcoma, B16F10 melanoma and Lewis lung-carcinoma cells specifically develop as colonies after intrasplenic injection. Cancer foci occurred predominantly in the 2.4 to 4.0 segment of the sinusoidal pathway, corresponding to hepatic acinar zone I. No significant differences were detected between different types of tumor, including their different tendencies to spontaneously metastasize liver, or as a result of the different procedures used for obtaining foci or metastases. In addition, PHZ-treatment of mice previously injected with tumor cells, resulted in double colonization of the liver tissue by both hematopoietic and cancer cells, predominantly in zone I. This spatial coincidence indicates that non-cancer-specific mechanisms operate in zone I, either promoting implantation and/or growth of cell colonies or, alternatively, inhibiting these processes in the region surrounding the central vein (Rappaport zone 3). Our observations failed to reveal mutual displacement of cancer or hematopoietic foci by potential competition for development sites in zone I. Enumeration and diameter measurements of cancer foci in PHZ-treated animals showed that the presence of hepatic hematopoietic foci coincided with a significant increase in the hepatic metastasis volume. However, the fact that no significant differences in pulmonary metastases occurred in both the PHZ-treated and control mice given tail-vein injection of cancer cells, and that PHZ reduces cancer cell proliferation in vitro, reveal evidence of local interactions with hematopoietic foci which promote growth of cancer foci in liver.

The functional zonation of liver tissue provides a framework for studying the implantation and growth of metastatic colonies within given zones of the hepatic acini. A very exact method for calibrating the position of metastatic foci in the hepatic acini was made possible by using the succinate-dehydrogenase reaction, which reveals the functional differences of the acinar zones. In mouse livers, metastasis was induced by intrasplenic injection of both high and low metastatic-capacity B16 melanoma and Lewis lung carcinoma cells. In rats, liver metastasis resulting from rhabdomyosarcoma was studied by injecting these cells into the s.c. tissue. All cases of metastasis occurred in hepatic acinar zone 1, and no significant differences were detected resulting from the type of tumor, its metastatic potential, or the procedure used for obtaining the metastasis. In subsequent experiments, metastasis was induced after first altering the zonal distribution of the hepatic extracellular matrix; distribution of the sinusoidal macrophages; and the sinusoidal diameter. However, even under these conditions, metastasis continues to occur exclusively in hepatic acinar zone 1. Thus, metastatic predilection for hepatic acinar zone 1 cannot be explained solely in terms of hemodynamic causes or the influence of extracellular matrix. Although it may still turn out that these elements play some secondary role in the phenomenon, our research points to the sinusoidal endothelial cells as a factor directly responsible for metastatic predilection for zone 1.