Pnina Brodt

Liver endothelial E‐selectin mediates carcinoma cell adhesion and promotes liver metastasis

E‐selectin is a cytokine‐inducible endothelial cell adhesion receptor which is involved in the process of leukocyte rolling, the first in a cascade of interactions leading to leukocyte transmigration. Several studies have implicated this receptor in carcinoma cell adhesion to the endothelium, an interaction thought to be required for tumor extravasation during metastasis. To study the role of this receptor in the process of metastasis, we utilized a murine carcinoma line H‐59 which is highly metastatic to the liver in vivo. When adhesion of H‐59 cells to primary cultures of murine hepatic endothelial cells was measured, it was found that the tumor cells had a low which basall of adhesion to the sinusoidal endothelial cells, which could be significantly and specifically augmented by pre‐activation of the endothelial cells with rTNFα. This incremental increase in adhesion to the activated endothelium could be completely and specifically abolished by a neutralizing monoclonal antibody to murine E‐selectin (MAb 9A9). Similar results were obtained with 2 highly metastatic human colorectal carcinoma lines, HM 7 and CX‐1, but not with a second murine subline, M‐27, which is poorly metastatic to the liver. To assess the role of E‐selectin in metastasis to the liver in vivo, the effect of MAb 9A9 on experimental liver metastasis was evaluated using the syngeneic H‐59 model. We show here that this antibody caused a marked, specific and Fc‐independent inhibition of experimental liver metastasis, reducing the median number of metastases by 97% relative to the control groups. Our results provide evidence that endothelial E‐selectin is a mediator of carcinoma metastasis to the liver. Int. J. Cancer 71:612‐619, 1997.

Type 1 insulin-like growth factor regulates MT1-MMP synthesis and tumor invasion via PI 3-kinase/Akt signaling

The membrane type 1 matrix metalloproteinase (MT1-MMP) has been identified as a major activator of MMP-2 – a process involving the formation of a trimolecular complex with TIMP-2. We previously identified the IGF-I receptor as a positive regulator of MMP-2 synthesis. Here, we investigated the role of IGF-IR in the regulation of MT1-MMP. Highly invasive Lewis lung carcinoma subline H-59 cells express MT1-MMP and utilize it to activate their major extracellular matrix degrading proteinase-MMP-2. These cells were transiently transfected with a plasmid vector expressing a luciferase reporter gene downstream of the mouse MT1-MMP promoter. IGF-I treatment increased luciferase activity in the transfected cells by up to 10-fold and augmented endogenous MT1-MMP mRNA and protein synthesis by up to 2–3-fold, relative to controls. MT1-MMP induction and invasion were blocked by the PI 3-kinase inhibitors LY294002 and wortmannin and by rapamycin, but not by the MEK inhibitor PD98059. Overexpression of a dominant negative Akt mutant or of the tumor suppressor phosphatase and tensin homologue, PTEN, in these cells also caused a significant reduction in MT1-MMP expression and invasion. The results demonstrate that IGF-IR controls tumor cell invasion by coordinately regulating MMP-2 expression and its MT1-MMP-mediated activation and identify PI 3-kinase/Akt/mTOR signaling as critical to this regulation.

Systemic inflammation increases cancer cell adhesion to hepatic sinusoids by neutrophil mediated mechanisms

Interactions between endothelial selectins and selectin ligands expressed on tumor cells have been implicated in the binding of circulating metastatic cancer cells to the vascular endothelium during extravasation. Moreover, there is mounting evidence that inflammatory environments can accelerate the progression of metastasis by neutrophil mediated mechanisms. In this study, a physiologically relevant in vivo model of early metastasis coupled with intravital microscopy was used to visualize the trafficking of tumor cells within the liver vasculature in real time. Using GFP‐labeled Lewis lung carcinoma subline H‐59 cells, we show here that disrupting the interactions between endothelial selectins and tumor cell selectin ligands diminished tumor cell recruitment to the liver. Furthermore, systemic inflammation induced by intravenous injection of lipopolysaccharide significantly enhanced the metastatic potential of these lung carcinoma cells by increasing their propensity to adhere to the liver sinusoidal endothelium. Confocal microscopy revealed frequent colocalization of cancer cells with neutrophils and neutrophil depletion in vivo significantly attenuated the lipopolysaccharide‐induced increase in H‐59 cell adhesion. Although direct selectin–selectin ligand interactions contributed significantly to tumor cell adhesion to sinusoidal endothelial cells, we show here that in addition, interactions between adherent neutrophils within the inflamed sinusoids and circulating tumor cells may further increase tumor cell arrest in the liver.

Characterization of the Host Proinflammatory Response to Tumor Cells during the Initial Stages of Liver Metastasis

The influx of metastatic tumor cells into the liver triggers a rapid proinflammatory cytokine cascade. To further analyze this host response, we used intrasplenic/portal inoculation of green fluorescent protein-marked human and murine carcinoma cells and a combination of immunohistochemistry and confocal microscopy. The metastatic murine lung carcinoma H-59 or human colorectal carcinoma CX-1 cells triggered tumor necrosis factor (TNF)-alpha production by Kupffer cells located in sinusoidal vessels around the invading tumor cells. H-59 cells rapidly elicited a fourfold increase in the number of TNF-alpha(+) Kupffer cells relative to basal levels within 2 hours and this response declined gradually after 6 hours. Increased cytokine production in these mice was confirmed by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay performed on isolated Kupffer cells. CX-1 cells elicited a more gradual response that peaked at 10 to 16 hours, remained high up to 48 hours, and involved CX-1-Kupffer cell attachment. Furthermore, the rapidly induced production of TNF-alpha was followed by increased expression of the vascular adhesion receptors E-selectin P-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 on sinusoidal endothelial cells. This proinflammatory response was tumor-specific and was not observed with nonmetastatic murine M-27 or human MIP-101 carcinoma cells. These results identify Kupffer cell-mediated TNF-alpha production as an early, tumor-selective host inflammatory response to liver-invading tumor cells that may influence the course of metastasis.

Inhibition of the type I insulin-like growth factor receptor expression and signaling: Novel strategies for antimetastatic therapy

The receptor for the type 1 insulin-like growth factor (IGF-1R) plays a critical role in the acquisition of the malignant phenotype. Using a highly metastatic murine lung carcinoma model, it was demonstrated that this receptor regulates several cellular functions that can impact on the metastatic potential of the cells, including cellular proliferation, anchorage-independent growth, cell migration, and invasion. The tumor model was used to develop several strategies for altering receptor expression and function as means of abrogating the metastatic potential of the cells. They include stable expression in the tumor cells of IGF-1R antisense RNA and dominant negative receptor mutants in which tyrosines in the kinase domain were substituted with phenylalanine. In addition, a novel strategy was used based on altering post ligand-binding receptor turnover. This led to inhibition of receptor re-expression and signaling and resulted in increased tumor cell apoptosis. When combined with the development of viral vectors designed to deliver genetic information with high efficiency, these strategies could form the basis for development of highly specific, antimetastatic therapy in tumors with known IGF-IR involvement.

The Multifaceted Role of the Microenvironment in Liver Metastasis: Biology and Clinical Implications

The liver is host to many metastatic cancers, particularly colorectal cancer, for which the last 2 decades have seen major advances in diagnosis and treatment. The liver is a vital organ, and the extent of its involvement with metastatic disease is a major determinant of survival. Metastatic cells arriving in the liver via the bloodstream encounter the microenvironment of the hepatic sinusoid. The interactions of the tumor cells with hepatic sinusoidal and extrasinusoidal cells (endothelial, Kupffer, stellate, and inflammatory cells) determine their fate. The sinusoidal cells can have a dual role, sometimes fatal to the tumor cells but also facilitatory to their survival and growth. Adhesion molecules participate in these interactions and may affect their outcome. Bone marrow-derived cells and chemokines also play a part in the early battle for survival of the metastases. Once the tumor cells have arrested and survived the initial onslaught, tumors can grow within the liver in 3 distinct patterns, reflecting differing host responses, mechanisms of vascularization, and proteolytic activity. This review aims to present current knowledge of the interactions between the host liver cells and the invading metastases that has implications for the clinical course of the disease and the response to treatment.

Peripheral lymph node stromal cells can promote growth and tumorigenicity of breast carcinoma cells through the release of IGF-I and EGF

The regional lymph nodes draining primary breast carcinomas are generally the first site to be invaded by disseminating tumor cells. The extent of lymph node involvement remains the most reliable indicator for staging and prognosis of breast cancer. We have investigated host–tumor interactions between breast carcinoma cells and the lymph node stroma, which may control the outcome of lymph node infiltration. In a previous study, we identified integrin‐mediated cell adhesion as a correlate of the metastatic potential of human and rat carcinoma cells. Our present objective was to determine whether lymphatic stromal cells can affect cancer cell growth through the elaboration of growth‐modulating factors. Two lymphatic stromal cell lines, ST‐A4 and ST‐B12, were established from normal rat lymph node stromal cell cultures. SFM conditioned by these cells increased the proliferation of human (Hs578T and MCF‐7) and rat (TMT‐081) breast carcinoma cells by up to 7‐fold and augmented their ability to form colonies in semisolid agar by up to 41‐fold. This effect was specific as normal, diploid human breast epithelial cells (Hs578Bst), a nontumorigenic, immortalized human breast epithelial cell line (MCF‐10A) and a nonmetastatic rat mammary carcinoma cell line (MT‐W9B) had either no or reduced responses. RT‐PCR analysis revealed that both lymph node stromal cell lines expressed mRNA transcripts for multiple growth factors, including IGF‐I, EGF, HGF and PDGF‐α, and produced detectable levels of IGF‐I, EGF and PDGF‐α, as assessed by Western blotting. Antibody‐mediated depletion assays identified IGF‐I and EGF as the major mitogenic factors in the CM. The identification of these cells raises the possibility that the lymph node microenvironment may contribute actively to the process of cancer cell dissemination.

Insulin-like growth factor-I regulates the liver microenvironment in obese mice and promotes liver metastasis

Among the mechanisms implicated in the tumor-promoting effects of obesity, signaling by insulin-like growth factor-I (IGF-I) and insulin has received considerable attention. However, the emerging realization that obesity is associated with chronic inflammation has prompted other consideration of how the IGF-I axis may participate in cancer progression. In the present study, we used two mouse models of chronic (LID) and inducible (iLID) igf-1 gene deficiency in the liver to investigate the role of IGF-I in regulating the host microenvironment and colorectal carcinoma growth and metastasis in obese mice. Obese mice had a heightened inflammatory response in the liver, which was abolished in mice with chronic IGF-I deficiency (LID). In control animals changes to the hepatic microenvironment associated with obesity sustained the presence of tumor cells in the liver and increased the incidence of hepatic metastases after intrasplenic/portal inoculation of colon carcinoma cells. These changes did not occur in LID mice with chronic IGF-1 deficiency. In contrast, these changes occurred in iLID mice with acute IGF-1 deficiency, in the same manner as the control animals, revealing a fundamental difference in the nature of the requirement for IGF-1 on tumor growth and metastasis. In the setting of obesity, our findings imply that IGF-1 is critical to activate and sustain an inflammatory response in the liver that is needed for hepatic metastasis, not only through direct, paracrine effect on tumor cell growth, but also through indirect effects involving the tumor microenvironment.

Suppression of basement membrane type IV collagen degradation and cell invasion in human melanoma cells expressing an antisense RNA for MMP-1

During progression from benign nevus to vertical growth phase melanoma, melanocytes acquire the ability to invade into the dermis. This process requires rupture of the basal lamina and dissolution of dermal type I collagen. Metastases-derived human melanoma MIM cells have an invasive ability in vitro which is dependent on metalloproteinases. In the present study we analysed the role of type I collagenase (MMP-1) in melanoma invasion using MIM cells in which the constitutive expression of MMP-1 was suppressed by stable transfection with a plasmid vector expressing a 777 bp antisense fragment of MMP-1 genomic DNA. Two clones were isolated in which MMP-1 mRNA expression was blocked by 90-96% with a corresponding loss in protein synthesis. In their morphological appearance and growth rate in vitro these cells were indistinguishable from wild type cells or control cells transfected with the same vector expressing the MMP-1 fragment in the sense orientation. Their mRNA and protein levels for type IV collagenase (MMP-2) were unchanged as assessed by Northern and Western blot analyses and by gelatin zymography. However, when the invasive ability of the cells was measured, we found that in addition to type I collagen, invasion through type IV collagen and a reconstituted, type IV collagen-containing basement membrane (Matrigel) were also significantly inhibited as compared to normal or sense-transfected cells. The results indicate that despite the presence of functional MMP-2, degradation of type IV collagen matrices by the melanoma cells was dependent on expression of MMP-1.

Integrin α3β1 can promote adhesion and spreading of metastatic breast carcinoma cells on the lymph node stroma

We have reported that metastatic human melanoma cells utilize the αvβ3 integrin to adhere to lymph node vitronectin (VN). In the present study, the adhesion of human and rat breast carcinoma cells to lymph node tissue was analyzed. We have previously shown a correlation between the metastatic potential of breast carcinoma cells and an RGD‐mediated adhesion to cryostat sections of peripheral lymph nodes; this adhesion could be blocked by an antibody to the integrin β1 subunit. Here, we show that the metastatic breast carcinoma cell were significantly more adherent to fibronectin (FN) expressed by lymph node‐derived stromal cells than non‐metastatic cells. Metastatic cells also spread more rapidly than non‐metastatic cells on FN‐coated substrates. Using a combination of immunofluorescence microscopy, immunoprecipitation and blocking assays with integrin‐specific antibodies, we found (i) that expression of the α3β1 integrin on metastatic mammary carcinoma cells was specifically increased in comparison to non‐metastatic cells and (ii) that the α3β1 receptor was involved in the increased adhesion of metastatic cells to lymph node FN and in cell spreading on FN‐coated substrates. Our data also suggest that the α5β1 integrin, which is also expressed on the metastatic cells, did not contribute to this increase in adhesion. Our data implicate the α3β1 integrin in adhesion to lymph node stromal cell FN and suggest that metastatic cells of different tissue origins (e.g., melanoma and breast carcinoma) may utilize distinct integrin‐ligand combinations to colonize the same target organ.