The Role of Soluble Fibrin in Lymphocyte and LAK Cell Adherence to and Migration across Vascular Endothelial Cells: Implications for Immunotherapy and Cancer

Although conventional therapies for metastatic cancers have made significant progress in recent years, they are relatively nonspecific and have many deleterious side-effects. Recently, novel therapies, including adoptive cellular immune therapies have had sporadic, but spectacular success in cancers such as malignant melanoma and renal cell carcinoma: tumors in which an immune response has been demonstrated. However, other physiological mechanisms, such as blood coagulation inhibit the immune response against cancers. Our previous work has shown that one of these coagulation proteins, soluble fibrin (sFn), inhibits unstimulated and activated lymphocyte adherence to tumor cells by blocking leukocyte integrin (CD11a/CD18) binding to tumor cell CD54, suggesting that sFn is an immunosuppressive agent in cancer. Since these receptors are also involved in lymphocyte/endothelial cell adherence and diapedesis (a necessary step in the immune response to cancer), it was hypothesized that sFn inhibits these functions, and that blockade of this inhibition using specific peptides would restore these immune responses. Fluorescently labeled lymphocytes and Interleukin-2 activated lymphocytes (LAK cells) were incubated with sFn (or its components; fibrinogen, Gly-Pro-Arg-Pro, or thrombin) in the presence or absence of specific blocking peptides. Lymphocyte and LAK cell adherence to endothelial cell monolayers was measured by perfusion at physiological shear rates in a microscope-mounted closed perfusion chamber, followed by image analysis using Image Pro Plus software. Diapedesis was measured by detection of fluorescence in 24-well microplates following immune cell incubation (18 h) with endothelial cell monolayers grown in transwells. SFn inhibited lymphocyte (54.1 + 11.3 %) and LAK cell (43.9 + 4.4 %) adherence to sFn pretreated endothelial cells, and intermediate values were obtained from sFn pre-treatment of only one cell type. Adherence was restored by peptide mediated blockade of sFn/CD54 binding, but not by CD11b blocking peptides. Diapedesis was also inhibited by sFn (lymphocyte 29.6 + 7.7 %; LAK 12.2 + 4.9 %) and restoration was observed using blocking peptides. These results confirm the stated hypotheses, and if physiologically relevant, suggest that sFn is an etiological agent in tumor growth and metastasis, and that blockade using fibrin specific peptides may enhance the effectiveness of adoptive immunotherapies

Nest expansion assay: a cancer systems biology approach to in vitro invasion measurements

<p>Abstract</p> <p>Background</p> <p>Traditional <it>in vitro </it>cell invasion assays focus on measuring one cell parameter at a time and are often less than ideal in terms of reproducibility and quantification. Further, many techniques are not suitable for quantifying the advancing margin of collectively migrating cells, arguably the most important area of activity during tumor invasion. We have developed and applied a highly quantitative, standardized, reproducible Nest Expansion Assay (NEA) to measure cancer cell invasion <it>in vitro</it>, which builds upon established wound-healing techniques. This assay involves creating uniform circular "nests" of cells within a monolayer of cells using a stabilized, silicone-tipped drill press, and quantifying the margin expansion into an overlaid extracellular matrix (ECM)-like component using computer-assisted applications.</p> <p>Findings</p> <p>The NEA was applied to two human-derived breast cell lines, MCF10A and MCF10A-CA1d, which exhibit opposite degrees of tumorigenicity and invasion <it>in vivo</it>. Assays were performed to incorporate various microenvironmental conditions, in order to test their influence on cell behavior and measures. Two types of computer-driven image analysis were performed using Java's freely available <it>ImageJ </it>software and its <it>FracLac </it>plugin to capture nest expansion and fractal dimension, respectively – which are both taken as indicators of invasiveness. Both analyses confirmed that the NEA is highly reproducible, and that the ECM component is key in defining invasive cell behavior. Interestingly, both analyses also detected significant differences between non-invasive and invasive cell lines, across various microenvironments, and over time.</p> <p>Conclusion</p> <p>The spatial nature of the NEA makes its outcome susceptible to the global influence of many cellular parameters at once (e.g., motility, protease secretion, cell-cell adhesion). We propose the NEA as a mid-throughput technique for screening and simultaneous examination of factors contributing to cancer cell invasion, particularly suitable for parameterizing and validating Cancer Systems Biology approaches such as mathematical modeling.</p

Soluble fibrin inhibits monocyte adherence and cytotoxicity against tumor cells: implications for cancer metastasis

BACKGROUND: Soluble fibrin (sFn) is a marker for disseminated intravascular coagulation and may have prognostic significance, especially in metastasis. However, a role for sFn in the etiology of metastatic cancer growth has not been extensively studied. We have reported that sFn cross-linked platelet binding to tumor cells via the major platelet fibrin receptor αIIbβ3, and tumor cell CD54 (ICAM-1), which is the receptor for two of the leukocyte β2 integrins (α(L)β2 and a(M)β2). We hypothesized that sFn may also affect leukocyte adherence, recognition, and killing of tumor cells. Furthermore, in a rat experimental metastasis model sFn pre-treatment of tumor cells enhanced metastasis by over 60% compared to untreated cells. Other studies have shown that fibrin(ogen) binds to the monocyte integrin α(M)β2. This study therefore sought to investigate the effect of sFn on β2 integrin mediated monocyte adherence and killing of tumor cells. METHODS: The role of sFn in monocyte adherence and cytotoxicity against tumor cells was initially studied using static microplate adherence and cytotoxicity assays, and under physiologically relevant flow conditions in a microscope perfusion incubator system. Blocking studies were performed using monoclonal antibodies specific for β2 integrins and CD54, and specific peptides which inhibit sFn binding to these receptors. RESULTS: Enhancement of monocyte/tumor cell adherence was observed when only one cell type was bound to sFn, but profound inhibition was observed when sFn was bound to both monocytes and tumor cells. This effect was also reflected in the pattern of monocyte cytotoxicity. Studies using monoclonal blocking antibodies and specific blocking peptides (which did not affect normal coagulation) showed that the predominant mechanism of fibrin inhibition is via its binding to α(M)β2 on monocytes, and to CD54 on both leukocytes and tumor cells. CONCLUSION: sFn inhibits monocyte adherence and cytotoxicity of tumor cells by blocking α(L)β2 and α(M)β2 binding to tumor cell CD54. These results demonstrate that sFn is immunosuppressive and may be directly involved in the etiology of metastasis. Use of specific peptides also inhibited this effect without affecting coagulation, suggesting their possible use as novel therapeutic agents in cancer metastasis

Lysophosphatidic Acid Upregulates Laminin-332 Expression during A431 Cell Colony Dispersal

Lysophosphatidic acid (LPA) is a bioactive phospholipid that affects various biological functions, such as cell proliferation, migration, survival, wound healing, and tumor invasion through LPA receptors. Previously, we reported that LPA induces A431 colony dispersal, accompanied by disruption of cell-cell contacts and cell migration. However, it remains unclear how LPA affects cell migration and gene expression during A431 colony dispersal. In this paper, we performed cDNA microarray analysis to investigate this question by comparing gene expression between untreated and LPA-treated A431 cells. Interestingly, these results revealed that LPA treatment upregulates several TGF-β1 target genes, including laminin-332 (Ln-332) components (α3, β3, and γ2 chains). Western blot analysis also showed that LPA increased phosphorylation of Smad2, an event that is carried out by TGF-β1 interactions. Among the genes upregulated, we further addressed the role of Ln-332. Real-time PCR analysis confirmed the transcriptional upregulation of all α3, β3, and γ2 chains of Ln-332 by LPA, corresponding to the protein level increases revealed by western blot. Further, the addition of anti-Ln-332 antibody prevented LPA-treated A431 colonies from dispersing. Taken together, our results suggest that LPA-induced Ln-332 plays a significant role in migration of individual cells from A431 colonies

A novel circular invasion assay mimics <it>in vivo </it>invasive behavior of cancer cell lines and distinguishes single-cell motility <it>in vitro</it>

Abstract Background Classical in vitro wound-healing assays and other techniques designed to study cell migration and invasion have been used for many years to elucidate the various mechanisms associated with metastasis. However, many of these methods are limited in their ability to achieve reproducible, quantitative results that translate well in vivo. Such techniques are also commonly unable to elucidate single-cell motility mechanisms, an important factor to be considered when studying dissemination. Therefore, we developed and applied a novel in vitro circular invasion assay (CIA) in order to bridge the translational gap between in vitro and in vivo findings, and to distinguish between different modes of invasion. Method Our method is a modified version of a standard circular wound-healing assay with an added matrix barrier component (Matrigel™), which better mimics those physiological conditions present in vivo. We examined 3 cancer cell lines (MCF-7, SCOV-3, and MDA-MB-231), each with a different established degree of aggressiveness, to test our assay's ability to detect diverse levels of invasiveness. Percent wound closure (or invasion) was measured using time-lapse microscopy and advanced image analysis techniques. We also applied the CIA technique to DLD-1 cells in the presence of lysophosphatidic acid (LPA), a bioactive lipid that was recently shown to stimulate cancer cell colony dispersal into single migratory cells, in order to validate our method's ability to detect collective and individual motility. Results CIA method was found to be highly reproducible, with negligible levels of variance measured. It successfully detected the anticipated low, moderate, and high levels of invasion that correspond to in vivo findings for cell lines tested. It also captured that DLD-1 cells exhibit individual migration upon LPA stimulation, and collective behavior in its absence. Conclusion Given its ability to both determine pseudo-realistic invasive cell behavior in vitro and capture subtle differences in cell motility, we propose that our CIA method may shed some light on the cellular mechanisms underlying cancer invasion and deserves inclusion in further studies. The broad implication of this work is the development of a reproducible, quantifiable, high-resolution method that can be applied to various models, to include an unlimited number of parameters and/or agents that may influence invasion.</p