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.

Autologous bone marrow stromal cells genetically engineered to secrete an igf-I receptor decoy prevent the growth of liver metastases.

Liver metastases respond poorly to current therapy and remain a frequent cause of cancer-related mortality. We reported previously that tumor cells expressing a soluble form of the insulin-like growth factor-I receptor (sIGFIR) lost the ability to metastasize to the liver. Here, we sought to develop a novel therapeutic approach for prevention of hepatic metastasis based on sustained in vivo delivery of the soluble receptor by genetically engineered autologous bone marrow stromal cells. We found that when implanted into mice, these cells secreted high plasma levels of sIGFIR and inhibited experimental hepatic metastases of colon and lung carcinoma cells. In hepatic micrometastases, a reduction in intralesional angiogenesis and increased tumor cell apoptosis were observed. The results show that the soluble receptor acted as a decoy to abort insulin-like growth factor-I receptor (IGF-IR) functions during the early stages of metastasis and identify sustained sIGFIR delivery by cell-based vehicles as a potential approach for prevention of hepatic metastasis.

The host inflammatory response promotes liver metastasis by increasing tumor cell arrest and extravasation.

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-alpha 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 three-dimensional reconstruction. E-selectin expression was evident mainly on sinusoidal vessels by 6 and 10 hours, respectively, following H-59 and CX-1 inoculation, and this corresponded to a stabilization of the number of tumor cells within the sinuses. Tumor cells arrested in E-selectin(+) vessels and appeared to flatten and traverse the vessel lining, away from sites of intense E-selectin staining. This process was evident by 8 (H-59) and 12 (CX-1) hours after inoculation, coincided with increased endothelial vascular cell adhesion molecule-1 expression, and involved tumor cell attachment in areas of intense vascular cell adhesion molecule-1 and platelet endothelial cell adhesion molecule-1 expression. Nonmetastatic (human) MIP-101 and (murine) M-27 cells induced a weaker response and could not be seen to extravasate. The results show that metastatic tumor cells can alter the hepatic microvasculature and use newly expressed endothelial cell receptors to arrest and extravasate.

Increased tumor growth in mice with diet-induced obesity: impact of ovarian hormones.

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.

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.

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.

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 (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 (beta(m)-alpha-alpha-beta(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.

PADMA-28, a traditional Tibetan herbal preparation, blocks cellular responses to bFGF and IGF-I.

The growth factors basic fibroblast growth factor (bFGF) and insulin-like growth factor 1 (IGF-I) have been implicated in the pathophysiology of atherosclerosis and restenosis. The Tibetan herbal preparation PADMA-28 (a mixture of 22 plants which is used as an anti-atherosclerosis agent) was tested for its ability to inhibit the mitogenic activity of bFGF and IGF-I, growth factors involved in restenosis, atherosclerosis and tumour progression. DNA synthesis and proliferation of vascular smooth muscle cells, in response to serum bFGF, thrombin, or combinations thereof, were abrogated in the presence of microgram amounts of both the aqueous and organic, partially purified, extracts of PADMA-28. These fractions also inhibited IGF-I-mediated proliferation, migration and invasion of tumour cells responsive to IGF-I. The inhibition by PADMA 28 was reversible upon removal of the PADMA extracts, indicating that the effects were not related to cell toxicity. These and other properties (i.e., anti-oxidant activity) of PADMA-28 may be responsible for its beneficial effect as an anti-atherosclerotic agent, suggesting that this herbal preparation may have potential applications in the prevention of intimal hyperplasia and arterial stenosis secondary to coronary angioplasty and bypass surgery, as well as in the prevention and treatment of other vascular diseases and tumour growth and metastasis.

Vascular endothelial growth factor C expression and lymph node metastasis are regulated by the type I insulin-like growth factor receptor.

Vascular endothelial growth factor (VEGF)-C is a lymphangiogenic factor implicated in lymphatic metastasis. In this study, we investigated the role of the type I insulin-like growth factor receptor (IGF-IR) in the regulation of VEGF-C expression. We used Lewis lung carcinoma subline M-27 cells transfected with human IGF-IR cDNA. These cells, but not the wild-type cells, expressed VEGF-C mRNA, produced a M(r) 58,000 VEGF-C precursor protein, and secreted a M(r) 29,000 processed form in response to IGF-I. In vivo, they acquired a lymph node metastasizing potential. VEGF-C induction was abolished in cells expressing an IGF-IR with tyrosine-phenylalanine substitutions in the kinase domain, but not in the COOH-terminal domain. The induction was phosphatidylinositol 3'-kinase dependent and, to a lesser extent, mitogen-activated protein kinase signaling dependent, as determined by the use of the respective inhibitors LY294002 (84.6% reduction) and PD98059 (38% reduction). The results identify the IGF-IR as a positive regulator of VEGF-C expression and implicate it in the control of lymphatic metastasis.

Inhibition of hepatic endothelial E-selectin expression by C-raf antisense oligonucleotides blocks colorectal carcinoma liver metastasis.

Cytokine-dependent induction of E-selectin expression is mediated through cooperative signaling involving the Ras/Raf/mitogen-activated protein kinase pathway. We previously reported that metastatic tumor cells entering the hepaticcirculation rapidly induce a cytokine cascade leading to E-selectin induction (A-M. Khatib, et al., Cancer Res., 59:1356-1361, 1999).Here, we investigated the effect of a blockade of E-selectin induction on colorectal carcinoma metastasis using rodent (host)-specific C-raf antisense oligonucleotides and human colorectal carcinoma CX-1 cells. Pretreatment of hepatic endothelial cells in vitro with the antisense oligonucleotides abrogated E-selectin-dependent CX-1 adhesion. In vivo, pretreatment of nude mice with these oligonucleotides abrogated E-selectin induction in response to intrasplenic/portal inoculation of CX-1 cells, and this reduced the number of liver metastases by 86% relative to controls. The results suggest that the inhibition of tumor-induced, hepatic microvessel E-selectin expression may provide a useful strategy for the prevention of hepatic 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-alpha, and produced detectable levels of IGF-I, EGF and PDGF-alpha, 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.