Cell-Surface Interactions in Pathogenesis

National Institute Of Dental & Craniofacial Research

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Abstract

Our goal in this project is to characterize new roles for cellular interactions in disease pathogenesis. A major current focus is on tumor cell invasion and metastasis, with a goal of identifying novel molecular regulators and mediators. A fundamental question in cancer biology is the relationship of the local microenvironment to cancer progression. We have been evaluating the roles of the extracellular matrix in cancer cell migration and invasion. The two major forms of matrix we examine in these interactions have been the 3D fibrillar extracellular matrix and the flat, sheet-like basement membrane. Recent models of cancer cell migration and invasion have used 3D extracellular matrix hydrogel systems. These model systems differ markedly in molecular composition and fibril organization, and they can produce conflicting findings. We directly compared five different 3D extracellular matrix systems for studying cancer cell migration and invasion using automated computer-based cell tracking. We chose HT-1080 fibrosarcoma and MDA-MB-231 breast carcinoma cell lines as examples of cancers of mesenchymal versus epithelial origin, respectively. The first three 3D model systems examined involved reconstituted 3D fibrillar gels consisting of two types of collagen hydrogel and tissue matrix gel (TMG). The last two model systems consisted of cell-derived extracellular matrices extracted from dense in vitro cultures of primary human or cancer-associated fibroblasts. HT-1080 cancer cells displayed rounded morphologies in all three reconstituted 3D matrices, but they became spindle-shaped in the two cell-derived matrices. MDA-MB-231 breast cancer cell morphologies were elongated in all matrices. Quantitative measures of key parameters of cell migration differed markedly between the different types of 3D matrix. For cells within the three reconstituted matrices, cells migrated most rapidly and furthest in TMG. However, comparing the three reconstituted matrices with the cell-derived matrices, cells migrated even more efficiently in the two cell-derived matrices. The most notable differences involved directionality (directional persistence) of migration, which was greatest in the two cell-derived matrices compared to the simpler hydrogel systems. These cell-derived matrixes had characteristic aligned fibrils that may promote rapid 1D cell migration along a matrix fibril. We conclude that measurements of cancer cell morphology, speed of translocation, and directional persistence of migration can differ dramatically depending on the type of 3D matrix system being employed. Consequently, studies of cancer cell migration and invasion will depend on the exact type of 3D matrix used. Conclusions about the mode of 3D cancer cell migration can differ depending on the type of matrix employed in the study, which should be chosen to most closely mimic the corresponding in vivo 3D environment. Tumor progression can be influenced by multiple external microenvironmental factors, including immune cells. A collaborative study involving a joint postdoctoral fellow in the laboratory of Dr. Alberto Baena at Oxford is continuing to evaluate the interplay between Drosophila tumors and immune cells. The roles of Drosophila macrophages appear to depend on regulation from caspases that is separate from their well-known roles in apoptosis. There is surprisingly widespread non-apoptotic caspase activation in Drosophila tumors engineered to mimic human tumors by activation of the EGF receptor and JAK/STAT signaling. This tumor-associated caspase activation provides partial suppression of JNK signaling and tumor expansion by limiting tumor cell proliferation and cell fate alteration in processes independent of classical apoptosis. An unexpected additional finding has been an effect of caspase activity on macrophage proliferation near the tumors associated with tumor expansion. Mycoplasma infections of cells can be problematic because they resist multiple antibiotics. We identified a novel approach to inhibiting mycoplasma infection. Treating mycoplasma-infected prostate cancer cells with hemin produced a striking >90% inhibition of infection. Hemin is FDA-approved for another disease and is a natural inducer of the cyto-protective enzyme heme oxygenase-1. The critical role of HO-1 was confirmed using specific siRNA suppression of hemin-induced HO-1 protein expression, which restored intracellular DNA levels of this mycoplasma. Proteomic analysis and transmission electron microscopy of hemin-treated cells confirmed complete absence of this mycoplasma from treated cells. Our study is the first to our knowledge suggesting the therapeutic potential of inducing HO-1 activation to enhance an innate cellular response for suppressing mycoplasma infections. Because we have extensive expertise in real-time imaging of cell behavior in vitro and in organ explants, we have an ongoing research collaboration involving a joint postdoctoral fellow with Dr. Ashok Kulkarni's laboratory to use the GCaMP6 mouse system for direct visualization of calcium signaling in pain-sensing neurons. Current research has focused on characterizing orofacial pain signaling and mechano-sensation in the mouse trigeminal ganglion with testing of the roles of Cdk5 in responses to a variety of stimuli. Quantification of calcium signaling responses has permitted detailed characterization of trigeminal neuronal responses during the sensing of acute and inflammatory pain, as well as the sensing of light touch and its pathological conversion to pain hypersensitivity analogous to allodynia. The ability to compare signaling in the same neurons during responses to different stimuli is providing an approach to be able to distinguish polymodal from more stimulus-specific responses of individual trigeminal neurons. Interestingly, experimental inflammation results in a notable increase in polymodal pain signaling. This system is also establishing evidence for the effects of peptide inhibition of Cdk5 activity on real-time trigeminal neuronal signaling. This approach can permit inhibition of pain signaling at a peripheral site rather than at a central (CNS) level, providing a possible alternative to opioids for suppressing orofacial pain. Our various primary and collaborative studies are providing new insights into the ways in which normal physiological cellular interactions are altered or subverted in disease pathogenesis.