Infectious complications resulting from resection of colorectal cancer (CRC) elevates the risk of cancer recurrence and metastasis, but the reason for this risk relationship is unknown. Defining the mechanisms responsible may offer opportunities to improve outcomes in a majority of patients whose tumors are resected as part of their therapy. The complex formed between Toll receptor TLR4 and myeloid differentiation factor MD2 defines a major cell surface receptor for lipopolysaccharide (LPS), a gram-negative bacterial antigen that has been implicated in infectious complications after CRC resection. As the TLR4/MD2 complex is expressed on CRC cells, we hypothesized that LPS may promote liver metastasis in CRC by stimulating TLR4 signaling. In support of this hypothesis, we report here that LPS enhances liver metastasis of human CRC cells that express TLR4/MD2 after intrasplenic graft of immunocompromised nude mice. Compared with TLR4 nonexpressing, nonmetastatic CRC cells, we observed increased in vitro adherence to different extracellular matrices and human umbilical vein endothelial cells (HUVEC). Furthermore, we observed an increased likelihood of in vivo capture within hepatic sinusoids after LPS treatment. No differences were apparent in phosphorylation of p38 and MAPK isoforms, but in metastatic CRC cells expressing surface TLR4 treatment with LPS increased Ser473 phosphorylation of AKT kinase. We showed that enhanced adherence elicited by LPS in these cells could be blocked at three different levels, using Eritoran (TLR4 small molecule antagonist), PI-103 (PI3K inhibitor), or anti-b1 integrin blocking antibodies. Taken together, the results indicate that stimulation of the TLR4/MD2 complex by LPS activates PI3K/AKT signaling and promotes downstream b1 integrin function, thereby increasing the adhesiveness and metastatic capacity of CRC cells. Our findings suggest that inhibiting LPS-induced TLR4 signaling could improve therapeutic outcomes by preventing cancer metastasis during the perioperative period of CRC resection. Cancer Res; 71(5); 1989-98. Ó2011 AACR.

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Objective: Infectious complications in colorectal cancer patients undergoing curative resection are associated with cancer recurrence, but the exact mechanism is not well understood. Toll-like receptor 4 (TLR4) is the sole receptor for lipopolysaccharide (LPS), a gram-negative bacterial antigen involved in such infectious complication. TLR4 expression has been found on several cancer types. We hypothesize that LPS promotes cancer cell metastatic potential via TLR4 signaling in colorectal cancer cells. Methods: We compared the effects of LPS stimulation on two well-characterized colorectal cancer cell lines: Caco-2 (non-metastatic) and HT-29 (metastatic). Flow cytometry was performed to detect cell surface TLR4 and various integrin expression. Cancer cells were incubated with or without 0.1 µg/mL LPS for 4 hours. In vitro static adhesion assay was used to study the ability of cell adhesion to extracellular matrix proteins (fibronectin, collagen I and collagen IV). Intravital microscopy, a technique used to study microcirculation in vivo, was carried out to observe dynamic attachment of intra-splenic injected cancer cells to C57/BL6 mouse liver sinusoids. Results: Caco-2 cells lack TLR4 receptors and are unresponsive to LPS treatment. In contrast, HT-29 cells were shown to express TLR4 receptors and HT-29 responds to LPS by a 2-fold increase of β1, but not α5, integrin expression in LPS-treated HT-29 cells (Mean Fluorescence Intensity:1041 vs. 458(untreated)). LPS-treated HT-29 cells showed enhanced attachment to fibronectin (1.6-fold; p < 0.01), collagen I (3-fold; p < 0.01) and collagen IV (2-fold; P < 0.01) relative to controls in vitro. Similarly, adherence of HT-29 cells to hepatic sinusoids in vivo was increased by LPS treatment (2 fold; p < 0.05). Conclusion: These data suggest that LPS-TLR4 signaling in colorectal cancer cells increases the metastatic potential. LPS-TLR4 mediated signaling cascade may be a valuable therapeutic target for the prevention of cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5279.

Background: Emerging data suggest that post-operative infections may increase esophageal cancer recurrence, possibly accounting for the poor prognosis associated with this malignancy. We have previously shown that lipopolysaccharide (LPS), a gram-negative bacterial antigen, injected into mice increases in vivo cancer cell adhesion to the hepatic sinusoids. However, the influence of LPS directly on the cancer cell is unknown. We thus sought to elucidate the direct impact of LPS and its sole trans-membrane receptor, Toll-Like Receptor 4 (TLR4) on human esophageal cancer cell metastatic potential. Methods: C57BL/6 mice were prepared for hepatic intravital microscopy and received intra-arterial inoculations with highly metastatic human esophageal squamous cells (HKESC-2; 1.5×106 cells/100μl) pre-incubated in LPS (0.1 μg/ml x 48hrs) or control media. HKESC-2-sinusoidal endothelial cell interactions were quantified with direct in vivo visualization of the hepatic sinusoids. In vitro static adhesion assay was use to measure cancer cell adhesion to fibronectin, collagen I and IV. SB203580, a p38 antagonist, was use to block LPS signaling. Two important adhesion molecules we have previously shown to be involved in the adhesion of these cancer cells to hepatic sinusoids, selectins and sialyl Lewis X (sLeX), were blocked using murine i.v. injection of fucoidin and incubation of cancer cells with blocking antibodies respectively. Results are presented as mean +/− SEM and MWU-test determined significance (* p<0.05). Results: HKESC-2 cells express TLR4 by flow cytometry and RT-PCR respectively. Immunoblots of LPS incubated HKESC-2 cells revealed increased phosphorylation of p38 MAP Kinase, an intracellular TLR4 downstream signaling molecule. Thus treated cells had a 2-fold increase in vitro adhesion to fibronectin but not to Collagen 1 or 4, an effect that was reversed by inhibition of p38 phosphorylation. Murine hepatic intravital microscopy demonstrated that LPS treated esophageal cancer cells had a 2.3 fold increase in adhesion to sinusoidal endothelium as compared to control (cells/field of view: 33.2+/−3.6 vs. 14.2 +/−0.5)*. This LPS-facilitated in vivo adhesion was attenuated by blockade of selectins and sLeX (7.7 +/− 0.4*; 14.37 +/− 0.8*). Conclusion: Using a physiologically relevant in vivo model of the early steps of cancer metastasis we have demonstrated that LPS-TLR4 mediated inflammation increased the metastatic potential of human esophageal cancer cells, at least partially through increased selectin-sialyl Lewis X binding. These findings identify LPS-TLR4 binding as a potential therapeutic target for the treatment and prevention of metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3434.