Lucia Fallavollita

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.

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.

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.

Loss of Tumorigenicity and Metastatic Potential in Carcinoma Cells Expressing the Extracellular Domain of the Type 1 Insulin-Like Growth Factor Receptor

The receptor for the type 1 insulin-like growth factor (IGF-IR) was identified as a major regulator of the malignant phenotype and a target for cancer therapy. In the present study, a novel IGF-IR mutant consisting of the entire extracellular domain of the receptor (IGFIR933) was genetically engineered and expressed in highly metastatic H-59 murine lung carcinoma cells. We show here that the cells expressed a truncated heterotetramer (βm-α-α-βm) that was secreted into the medium and could neutralize the effects of exogenous IGF-I, thus diminishing IGF-I-induced signaling and blocking IGF-I-mediated cellular functions such as cell proliferation, invasion, and survival. In vivo, tumor incidence and growth rate were markedly reduced in mice inoculated s.c. with H-59/IGFIR933 cells. Moreover, after the intrasplenic/portal inoculation of these cells, there was a 90% reduction in the incidence of hepatic metastases and a significant increase in the long-term, disease-free survival of the mice compared with controls. Our results identify the IGFIR933 as a potent antitumorigenic and antimetastatic agent with potential applications for cancer gene therapy.

The Secretory Leukocyte Protease Inhibitor Is a Type 1 Insulin-Like Growth Factor Receptor–Regulated Protein that Protects against Liver Metastasis by Attenuating the Host Proinflammatory Response

The secretory leukocyte protease inhibitor (SLPI) can attenuate the host proinflammatory response by blocking nuclear factor kappaB (NF-kappaB)-mediated tumor necrosis factor alpha (TNF-alpha) production in macrophages. We have previously shown that highly metastatic human and mouse carcinoma cells, on their entry into the hepatic microcirculation, trigger a rapid host proinflammatory response by inducing TNF-alpha production in resident Kupffer cells. Using GeneChip microarray analysis, we found that in mouse Lewis lung carcinoma subclones, SLPI expression was inversely correlated with tumor cell ability to induce a proinflammatory response and metastasize to the liver and with type 1 insulin-like growth factor receptor expression levels. To establish a causal relationship between SLPI expression and the metastatic phenotype, we generated, by transfection, multiple clones of the highly metastatic subline (H-59) that overexpress SLPI. We show here that the ability of these cells to elicit a host proinflammatory response in the liver was markedly decreased, as evidenced by reduced TNF-alpha production and vascular E-selectin expression, relative to controls. Moreover, these cells formed significantly fewer hepatic metastases (up to 80% reduction) as compared with mock-transfected controls. Our findings show that SLPI can decrease the liver-metastasizing potential of carcinoma cells and that this protective effect correlates with a decrease in the production of hepatic TNF-alpha and E-selectin. They suggest that factors that attenuate the host proinflammatory response may have a therapeutic potential in the prevention of liver metastasis.

Regulation of urokinase plasminogen activator/plasmin- mediated invasion of melanoma cells by the integrin vitronectin receptor αVβ3

The integrin vitronectin receptor αvβ3 is a mediator of cellular migration and invasion and has been identified as a marker of progression in malignant melanoma. Using a human melanoma model, we have previously shown that this receptor was coordinately expressed with the receptor for the urokinase plasminogen activator (uPAR). In our present study, the link between these receptors was further investigated by assessing the effect of αvβ3 ligation on uPAR transcription and function. Using the reverse transcription‐polymerase chain reaction, we found that receptor ligation by immobilized monoclonal antibodies (MAbs) induced a rapid increase (up to 4.5 fold) in uPAR mRNA levels, which was maximal 4 hr after cell attachment. An increase was also noted in plasminogen activator inhibitor type‐1 (PAI‐1) mRNA levels (2.7‐fold), but none was noted in uPA levels. In addition, ligation of αvβ3 resulted in a significant increase in cell surface‐associated plasmin levels, which coincided with a 2‐ to 3‐fold increase in cell invasion as measured in the Matrigel invasion assay. This increase in invasion could in turn be abolished by antibodies directed to uPA and uPAR and by the plasmin inhibitors ϵ‐aminocaproic acid and aprotinin. Furthermore, ligation of the integrin αvβ3 triggered a rapid increase of up to 12‐fold in total cellular PKC activity, and this coincided with the redistribution of PKCβ, but not PKCα, from the cytosol to the membrane. Treatment of the cells with the PKCβ‐specific inhibitor LY379196 blocked uPAR and PAI‐1 mRNA induction and reduced the increase in cell invasion due to αvβ3 ligation, confirming the involvement of this isoform in the response. The results provide evidence that the vitronectin receptor can enhance invasion by regulating the uPAR/uPA/plasmin system of proteolysis and implicate PKCβ as an intermediate in the activation pathway.

Inhibition of carcinoma cell growth and metastasis by a vesicular stomatitis virus G-pseudotyped retrovector expressing type I insulin-like growth factor receptor antisense.

A replication-defective, vesicular stomatitis virus G-pseudotyped, Moloney murine leukemia virus retroviral vector (vLTR-IGF-IR(AS)) was generated in which a type I insulin-like growth factor receptor (IGF-IR) antisense fragment is expressed in a bicistronic mRNA with an enhanced green fluorescent protein (EGFP) reporter under the control of a potent long terminal repeat (LTR). The suitability of these retroparticles for gene therapy was tested with highly metastatic, carcinoma H-59 cells, which depend on IGF-IR expression for tumorigenicity and metastasis. Transduction with these, but not with control retroviral particles expressing EGFP only, resulted in a 70% reduction in IGF-IR levels and the loss of IGF-IR-regulated functions. Moreover, the ability of vLTR-IGF-IR(AS) retroparticle-transduced tumor cells to form experimental hepatic metastases was significantly reduced relative to controls. The results identify retrovector-mediated delivery of IGF-IR antisense as a potential strategy for cancer gene therapy.

Loss of responsiveness to IGF-I in cells with reduced cathepsin L expression levels

The lysosomal cysteine proteinase cathepsin L is involved in proteolytic processing of internalized proteins. In transformed cells, where it is frequently overexpressed, its intracellular localization and functions can be altered. Previously, we reported that treatment of highly metastatic, murine carcinoma H-59 cells with small molecule cysteine proteinase inhibitors altered the responsiveness of the type I insulin-like growth factor (IGF-I) receptor and consequently reduced cell invasion and metastasis. To assess more specifically the role of cathepsin L in IGF-I-induced signaling and tumorigenicity, we generated H-59 subclones with reduced cathepsin L expression levels. These clonal lines showed an altered responsiveness to IGF-I in vitro, as evidenced by (i) loss of IGF-I-induced receptor phosphorylation and Shc recruitment, (ii) reduced IGF-I (but not IGF-II)-induced cellular proliferation and migration, (iii) decreased anchorage-independent growth and (iv) reduced plasma membrane levels of IGF-IR. These changes resulted in increased apoptosis in vivo and an impaired ability of the cells to form liver metastases. The results demonstrate that cathepsin L expression levels regulate cell responsiveness to IGF-I and thereby identify a novel function for cathepsin L in the control of the tumorigenic/metastatic phenotype.

TUMOR CELL ADHESION TO FROZEN LYMPH NODE SECTIONS : A CORRELATE OF LYMPHATIC METASTASIS IN BREAST CARCINOMA MODELS OF HUMAN AND RAT ORIGIN

SummaryThe role of tumor cell adhesion in lymphatic metastasis of breast cancer was investigatedin vitro using a rat mammary carcinoma model of four cell lines with different metastatic phenotypes, two human breast cancer cell lines, and cryostast sections of normal rat or human lymph nodes, respectively. A positive correlation was found between the adhesion levels obtained with three metastatic rat mammary cell lines (TMT-081 > MT-100M & TMT-50) and a non-metastatic line MT-W9B, the latter being 3–4 fold less adhesive to the lymph node sections than the metastatic tumors. This selective adhesion was specific, as it was not found with cryostat sections of rat liver and brain. Enzyme assays indicated that cell surface glycoproteins bearing terminal β-galactoside residues were involved in the adhesion of the rat tumors.Adhesion of the human breast carcinoma cells Hs578T to sections of human lymph nodes was significantly higher than that of the normal breast epithelial cell line Hs578Bst, and comparable to adhesion of a second breast carcinoma line, MCF-7. Moreover, Hs578T cells isolated from regional lymph nodes of tumor-bearing nude mice were significantly more adhesive to human lymph node sections than the parental line.Adhesion of both human and rat tumors could be partially blocked by the addition of the synthetic peptide GRGDSPK and by antibodies directed to the β1 chain of integrin, suggesting that an integrin receptor may played a role in the adhesion. The results suggest that tumor cell adhesion to cryostat sections of lymph nodes is a correlate of the malignant phenotype in mammary tumors of diverse origins, and could be used to delineate the adhesion factors mediating lymphatic metastasis.