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 GFPlabeled 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. ' 2009 UICC Key words: cancer; metastasis; lung; rodent; adhesion molecules; e-selectin; neutrophil; sialyl Lewis X Lung cancer is the leading cause of cancer-related deaths worldwide. 1 A high proportion of patients harbor metastasis to regional lymph nodes upon presentation, and over half of all patients who undergo curative intent surgical resection of the lung will develop metastatic recurrence. 2 The liver is a common site of metastasis for lung cancer, with autopsy studies showing hepatic metastases in over 50% of lung cancer patients. 3 The hematogenous dissemination of cancer cells from a primary tumor to distant organs involves a cascade of sequential steps (reviewed in Refs. 4,5). The adhesion of circulating tumor cells to the vascular endothelium of distant organs is a key step in the metastatic cascade that may determine the organ tropism and extent of metastasis. Interactions between endothelial selectins and tumor cell selectin ligands have been implicated in the metastatic process. [5][6][7][8] Selectins are a family of transmembrane glycoproteins possessing an extracellular lectin domain that binds to carbohydrate moieties present on their ligands. 9 The three known selectins that function as cell adhesion molecules are expressed on the surface of endothelial cells (E-selectin and P-selectin), platelets (P-selectin) and leukocytes (L-selectin). E-selectin has been shown to contribute to the metastatic spread of multiple cancers, including lung and colon carcinoma, and m...

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 basal level of adhesion to the sinusoidal endothelial cells, which could be significantly and specifically augmented by pre-activation of the endothelial cells with rTNFa. 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.r 1997 Wiley-Liss, Inc.The ability of disseminating cancer cells to establish metastases in secondary organs is regulated by a combination of factors, including access to the organ microvasculature (hemodynamic factors) and specific host-tumor cell interactions (Radinsky and Fidler, 1992). The attachment of circulating tumor cells to the vascular endothelium of the target organ is thought to be one key step in the metastatic cascade. The evidence suggests that this attachment precedes and is required for tumor cell extravasation and subsequent invasion into the target organ parenchyma (Brodt, 1996). Organ-specific receptors have been identified on the luminal surface of the microvascular endothelium which are specifically recognized by tumor cell ligands, thereby facilitating tumor cell arrest and transmigration into the extravascular space (Pauli et al., 1992). Studies of leukocyte transmigration have suggested that the specificity of the interaction between a circulating leukocyte and the microvascular endothelium may be determined by the outcome of a series of sequential receptor-ligand interactions rather than the complementarity of a single receptor-ligand system (Springer, 1994). It appears likely that the process of tumor extravasation is also mediated through a series of adhesive interactions (reviewed in B...

The liver is a major site of metastases for some of the most common human malignancies, carcinomas of the gastrointestinal tract in particular. Liver metastases are frequently inoperable and are associated with poor prognosis. 1 The metastatic cascade involves a sequence of steps that can lead to tumor cell arrest in the vascular bed of an invaded organ such as the liver and subsequently to tumor extravasation into the extravascular space.2 These events are regulated by, and in turn, can induce host proinflammatory responses that involve tumor-and host-derived chemokines and cytokines. A key mediator of the inflammatory response is tumor necrosis factor (TNF)-␣. This cytokine can play a dual role in tumor progression and metastasis. On one hand it can inhibit tumor growth through its cytocidal and proapoptotic activities but on the other, it can promote tumor progression through different mechanisms such as the induction of vascular endothelial adhesion receptors and the promotion of growth, invasion, and metastasis. The ultimate effect of TNF-␣ may depend on its concentrations, on tumor cell susceptibility, and on the stage of the disease. 3,4

Obesity increases the risk of many cancers in both males and females. This study describes a link between obesity, obesity-associated metabolic alterations, and the risk of developing cancer in male and female mice. The goal of this study was to evaluate the relationship between gender and obesity and to determine the role of estrogen status in obese females and its effect on tumor growth. We examined the susceptibility of C57BL/6 mice to diet-induced obesity, insulin resistance/glucose intolerance, and tumors. Mice were injected sc with one of two tumorigenic cell lines, Lewis lung carcinoma, or mouse colon 38-adenocarcinoma. Results show that tumor growth rate was increased in obese mice vs. control mice irrespective of the tumor cell type. To investigate the effect of estrogen status on tumor development in obese females, we compared metabolic parameters and tumor growth in ovariectomized (ovx) and intact obese female mice. Obese ovx female mice developed insulin resistance and glucose intolerance similar to that observed in obese males. Our results demonstrate that body adiposity increased in ovx females irrespective of the diet administered and that tumor growth correlated positively with body adiposity. Overall, these data point to more rapid tumor growth in obese mice and suggest that endogenous sex steroids, together with diet, affect adiposity, insulin sensitivity, and tumor growth in female mice.

Inflammation can play a regulatory role in cancer progression and metastasis. Previously, we have shown that metastatic tumor cells entering the liver trigger a proinflammatory response involving Kupffer cell-mediated release of tumor necrosis factor-␣ and the up-regulation of vascular endothelial cell adhesion receptors, such as E-selectin. Here, we analyzed spatio-temporal aspects of the ensuing tumor-endothelial cell interaction using human colorectal carcinoma CX-1 and murine carcinoma H-59 cells and a combination of immunohistochemistry, confocal microscopy, and threedimensional reconstruction. E-selectin expression was evident mainly on sinusoidal vessels by 6 and 10 hours, The host microenvironment plays an important role in the regulation of tumor progression at the primary site. It can also facilitate tumor dissemination by promoting neovascularization and providing growth-enhancing factors to the metastatic cells at the secondary sites of growth.

The receptor for the type 1 insulin-like growth factor (IGF-I) regulates multiple cellular functions impacting on the metastatic phenotype of tumor cells, including cellular proliferation, anchorage-independent growth, survival, migration, synthesis of the 72-kDa type IV collagenase and invasion. We have used site-directed mutagenesis to generate domain-specific mutants of the receptor ␤ subunit to analyze the role of specific tyrosines in the regulation of the invasive/metastatic phenotype. Poorly invasive M-27 carcinoma cells expressing low receptor numbers were transfected with a plasmid vector expressing IGF-I receptor cDNA in which single or multiple tyrosine codons in the kinase domain, namely Tyr-1131, Tyr-1135, and Tyr-1136 or the C-terminal tyrosines 1250 and 1251 were substituted with phenylalanine. Changes in the invasive and metastatic properties were analyzed relative to M-27 cells expressing the wild type receptor. We found that cells expressing the Y1131F,Y1135F,Y1136F or Y1135F receptor mutants lost all IGF-IR-dependent functions and their phenotypes were indistinguishable from, or suppressed relative to, the parent line. The Y1250F,Y1251F substitution abolished anchorage-independent growth, cell spreading, and the anti-apoptotic effect of IGF-I whereas all other IGF-IR-dependent phenotypes were either unperturbed (i.e. mitogenicity) or only partially reduced (migration and invasion). The results identify three types of receptor-dependent functions in this model: those dependent only on an intact kinase domain (DNA synthesis), those dependent equally on kinase domain and Tyr-1250/1251 signaling (e.g. apoptosis, soft agar cloning) and those dependent on kinase domain and enhanced through Tyr-1250/1251 signaling (migration, invasion). They suggest that signals derived from both regions of the receptor cooperate to enhance tumor metastasis.Cancer metastasis is a complex multistep process involving sequential interactions between the disseminating tumor cells and a continuously changing host microenvironment. These interactions include cell-cell and cell-extracellular matrix communication mediated through specific adhesion receptors, such as integrins, which regulate tumor cell attachment, spreading, and migration (1, 2), the dissolution of tissue barriers through the degradative activity of enzymes such as metallo-and serine-proteinases (3, 4) and growth modulation by paracrine, host-derived peptide growth factors (5, 6).The human receptor for the type 1 insulin-like growth factor (IGF-IR) 1 is a heterotetramer consisting of two 130-to 135-kDa ␣ and two 90-to 95-kDa ␤ subunits with several ␣-␣ and ␣-␤ disulfide bridges (7). It is synthesized as a single polypeptide chain, which is then glycosylated and proteolytically cleaved into the ␣ and ␤ subunits (8). The ligand-binding domain is located on the extracellular ␣ subunit. Approximately one-third of the ␤ subunit is extracellular and is connected to the intracellular portion by a single transmembrane domain. The intracellular region of the ␤ subuni...

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 (IGFIR 933 ) was genetically engineered and expressed in highly metastatic H-59 murine lung carcinoma cells. We show here that the cells expressed a truncated heterotetramer (␤ m -␣-␣-␤ m ) 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/IGFIR 933 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 IGFIR 933 as a potent antitumorigenic and antimetastatic agent with potential applications for cancer gene therapy.

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.