Ecotoxicol Environ Saf. 2017 Aug;142:544-554. doi: 10.1016/j.ecoenv.2017.04.044.

Planarians as models of cadmium-induced neoplasia provide measurable benchmarks for mechanistic studies.

Voura EB1, Montalvo MJ2, Dela Roca KT2, Fisher JM3, Defamie V4, Narala SR4, Khokha R4, Mulligan ME2, Evans CA2.

1 School of Science, Technology and Health Studies, Morrisville State College, 80 Eaton Street, Morrisville, New York 13408, USA. Electronic address: vouraeb@morrisville.edu.
2 Department of Math and Science, Dominican College, 470 Western Highway South, Orangeburg, New York 10962, USA.
3 Colgate University, 13 Oak Drive, Hamilton, New York 13346, USA.
4 Ontario Cancer Institute, University Health Network, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada.

Abstract

Bioassays of planarian neoplasia highlight the potential of these organisms as useful standards to assess whether environmental toxins such as cadmium promote tumorigenesis. These studies complement other investigations into the exceptional healing and regeneration of planarians – processes that are driven by a population of active stem cells, or neoblasts, which are likely transformed during planarian tumor growth. Our goal was to determine if planarian tumorigenesis assays are amenable to mechanistic studies of cadmium carcinogenesis. To that end we demonstrate, by examining both counts of cell populations by size, and instances of mitosis, that the activity of the stem cell population can be monitored. We also provide evidence that specific biomodulators can affect the potential of planarian neoplastic growth, in that an inhibitor of metalloproteinases effectively blocked the development of the lesions. From these results, we infer that neoblast activity does respond to cadmium-induced tumor growth, and that metalloproteinases are required for the progression of cancer in the planarian.

KEYWORDS:

Cadmium; Metalloproteinase; Neoblast; Planarian; Stem cells; Tumorigenesis

PMID: 28482323

 

Supplement:

Planarians have been studied for decades because of their unparalleled healing and regenerative abilities. And it was hypothesized early on that these organisms must contain a population of cells that are capable of developing into all the tissues necessary for their homeostasis. Of course, with time, scientists came to understand that these cells are stem cells, or neoblasts, and that the planarian has a rich supply of these supporting its physiology. It was also observed that planarians undergo tumorigenesis when subjected to various toxins. As a result of their sensitivity to carcinogens, planarians were suggested as potential models to assess environmental contaminants for their possible carcinogenic activity. But rodent models readily supplanted studies of planarian tumor growth, owing to their greater similarity to human physiology. However, with the advent of advanced molecular biological techniques, planarians have come again to the forefront as research models in many laboratories focused on understanding regeneration. And in the over twenty years since planarians were suggested as a tumor model, the cancer field also progressed significantly on many fronts. Within that work, we were interested the concept of the cancer stem cell, as well as the intriguing suggestion that tumors are reminiscent of uncontrolled or chronic wound healing.

In searching for a model to understand the behavior of stem cells during tumorigenesis in a tissue environment providing a context that is naturally conducive to wound healing and regeneration we aimed to explore the potential of the planarian system for our work. In our article we outlined a straightforward approach to reliably grow cadmium-induced tumors in the planarian Dugesia tigrina. In characterizing the role of the stem cells in the system we examined their mitotic activity, and, with the knowledge that neoblasts are the smallest cells in the planarian, monitored the general population of planarian cells by size with exposure to cadmium. Then, to show that our planarian tumor model is amenable to mechanistic studies, we demonstrated that metalloproteinases are important for the process. In short, we developed a useful assay system using the planarian to study tumorigenesis and stem cell activity, and, to our knowledge, provided the first clue to the mechanism involved in planarian tumor growth.

Cadmium is classified as a human carcinogen, but the underlying mechanism responsible for the tumor promoting effects of cadmium has been elusive. We believe our assay provides a practical animal model to work out a unifying mechanism behind cadmium-induced carcinogenesis, and that it can provide insight into how stem cells respond to tumorigenesis. We are currently focusing our efforts to better understand the role of metalloproteinases in planarian physiology. In addition, we are examining ways to study the movement of neoblasts during planarian tumorigenesis. If these cells migrate to the site of tumorigenesis metalloproteinases might be necessary. These recruited stem cells could be triggered to augment tumor growth, or they may promote the healing and regenerative mechanisms that are likely activated by tumorigenesis.

By staining for F-actin, we have circumstantial evidence of tissue remodeling around the lesions. Planarians have an extensive, seemingly ‘transcellular’ cytoskeletal system critical for the formation of the muscle layer ‘net’, which is dramatically exemplified by the stunning F-actin distribution of the organism (Fig 1A-C; meshwork) in the subepidermal muscle layer1. This tissue layer is necessary to both maintain the structure of the planarian, and to coordinate changes during healing and regeneration 2-5. Any regenerative activity will require the rearrangement of this network to support the initial wound healing of the organism. Indeed, others have observed that stem cells of the muscle layer are key to regeneration5-7. Our exploratory experiments looking at the distribution of F-actin around tumor lesions provides evidence of cellular activity affecting the regular actin network of the surrounding tissue including thickened fibers, fiber clusters and an irregular pattern that does not match the rest of the network in the exposed tissue (Fig 1D and E). These studies also indicate that cell staining and likely in situ hybridization studies can be used to study the regions surrounding the tumors. These data can then be compared with published data on planarian regeneration and wound healing to provide further insight into the mechanisms affecting the physiology of the tissues surrounding tumorigenesis in the planarian.

 

Figure: Regular, ‘transcellular’, cubic F-actin distribution in a healthy worm (A). Higher magnification view of the healthy planarian F-actin network is shown in (B) and (C). An image through a small tumor in (D) shows a distribution of F-actin locally – not affecting the regular network outside the tumor. An image showing the edge of a large tumor (arrow; tumor proper lost) in (E) shows an additional redistribution of actin outside the tumor. These images were taken using a Zeiss AxioVert epifluorescence microscope equipped with a Zeiss LSM 510 Meta confocal attachment and software. The optical slice thickness for all images was 10 mm and a Zeiss PlanApo 20x lens having an NA of 0.8. Scale bars = 20 mm.

 

References:

  1. Cebriá, F., Vispo, M., Newmark, P.A., Bueno, D. & Romero, R. Myocyte differentiation and body wall muscle regeneration in the planarian Girardia tirgrina. Dev. Genes Evol.207, 306-316 (1997).
  2. Cebriá, F. & Romero, R. Body-wall muscle restoration dynamics are different in dorsal and ventral blastemas during planarian anterior regeneration. Belg. J. Zool.131, 5-9 (2001).
  3. Pascolini, R., Lorvik, S., Maci, R. & Camatini, M. Immunoelectron microscopic localization of actin in migrating cells during planarian wound healing. Tissue Cell20, 157-163 (1988).
  4. Pascolini, R., Panara, F., DiRosa, I., Fagotti, A. & Lorvik, S. Characterization and fine-structural localization of actin- and fibronectin-like proteins in planaria (Dugesia lugubris s.l.). Cell Tissue Res.267, 499-506 (1992).
  5. Cebriá, F. Planarian body wall muscle: Regeneration and function beyond a simple skeletal muscle support. Front. Cell Dev. Bio. 4, (2016).
  6. Witchley, J.N., Mayer, M., Wagner, D.E., Owen, J.H. & Reddien, P.W. Muscle cells provide instructions for planarian regeneration. Cell Rep.4, 633-641 (2013).
  7. Dingwall, C.B. & King, R.W. A muscle-derived matrix metalloproteinase regulates stem cell proliferation in planarians. Dev. Dyn.245, 963-970 (2016).