neoplastic transformation in the planarian: i. cocarcinogenesis and histopathology

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 240:211-227 (1986)

Neoplastic Transformation in the Planarian: I. Cocarcinogenesis and tiistopathology

FREDERICK HALL, MICHIO MORITA, AND JAY BOYD BEST Department o,f Physiology and Biophysics, Colorado State Uniuersity, Fort Collins, IColorado 80523

ABSTRACT Although several investigators have reported that exposure to mammalian carcinogens induces abnormal tumorlike growths and teratogenic remodeling in planarians, thiere is no general agreement that these, or comparable responses in any other invertebrates, model mammalian carcinogenesis. To investigate this question, freshwater planarians of the species Dugesia clorotocephala were exposed to culture water containing an initiator and a promoter, either alone or in combination. Cadmium, a potent carcinogen, was used as an initiator in the protocol. Treatment with sublethal concentrations of cadmium sulfate produced a benign, but persistent, tumor in a small per- centage of the planarians. The addition of 12-0-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester and well-known promoter, to the cadmium- containing solutions resulte'd in the induction of a progressive, potentially lethal, transplantable tumor in a large proportion of the treated flatworms. Light and electron microscopy revealed this particular tumor to be composed both of immature cells and of a single mature cell type: newly differentiated, but transformed, reticular cells. Further examination of the infiltrating tissue formations elucidated the profile of differentiation, from a population of mitotically active transformed stem cells through the transitional stages in the associated reticuloma. These results suggest that 1) the freshwater planarian displays the major phenomenology of mammalian cocarcinogenesis and that 2) the planarian reticuloma mlodels several important features of a neoplastic stem cell disease.

Spontaneous tumorlike growths and tera- tological malformations have been reported in several species of planarians (Goldsmith, '39, '41; Stephan, '62; Tar and Torok, '64; Lange, '661, and a variety of morbid cellular proliferations have been experimentally induced by surgery, faradic stimulation, elec- trocautery (Goldsmith, '40; Stephan and Schilt, ,661, metabolic antagonists (Hender- son and Eakin, '61), ionizing radiation (Mix and Sparks, '691, cytotoxic dyes (Seilern-As- pang and Kratochwil, '65), and polyaromatic hydrocarbons (Goldsmith, '37; Foster, '63, '69; Best and Morita, '82; Best, '83). There is, however, no general agreement th,at these, or comparable responses in any other invertebrates, represent a biological correlate of mammalian tumorigenesis. Indeed, some of these pathological planarian syndromes would preferably be classified as hyperpla- sias, dysplasias, or necroses, as well as teratogenic remodelings, rather than as true tumors associated with neoplastic transfor-

mation. Still, others appear to represent more promising invertebrate equivalents of vertebrate cancers.

The modern conception of mammalian carcinogenesis as a multistage process, involving initiation and promotion in the progression of neoplastic disease (Berenblum, '74; Slaga et al., '78; Hecker et al., '82; Greim et al., '84), was developed from experiments on mouse skin (Berenblum, '41; Berenblum and Shubik, '47a,b, '49) and expanded to include other tissues (Berenblum, '75) such as liver (Armuth and Berenblum, '72; Peraino et al., '78; Pitot et al., '78, '82), lung (Armuth and Berenblum, '72; Witschi and Lock, '781, bladder (Hicks et al., '78; Hicks, '821, and mam- mary (Armuth and Berenblum, '74; Day, '82) tumors. The classical division of chemical carcinogens into initiators and promoters has served as a useful organizing principle, pro- viding powerful analytical tools for the study of the mechanisms of tumorigenesis; and an appreciation of these operational definitions

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2 12 F. HALL ET AL.

has led to considerable advancements in cancer research. Unfortunately, specialists in invertebrate systems have yet to take full advantage of the insights available through the utilization of two-stage cocarcinogenic protocols. In planarians, it has been reported that the incidence of spontaneous tumors is influenced by nutritional status (Lange, ,661, and a controlling role of regeneration has been implicated in the augmentation of tumors in one case (Foster, '63) and in the nor- malization of tumors in another (Seilern- Aspang and Kratochwil, '65); however, there remains an absence of experimental evidence regarding the use of bona fide promoters in these animals and a resulting lack of information regarding the question of tumor promotion andor cocarcinogenesis. The present study was designed to determine whether or not the freshwater planarian displays the major phenomenology of mammalian carcinogenesis and to define the resulting histopathology in the appropriate terms and criteria established for mammalian tumorigenesis.

To investigate the histological responses of planarians to mammalian cocarcinogens, subjects of the species Dugesia dorotoeephala were exposed to culture water containing an initiator and a promoter, either alone or in combination. Cadmium, long suspected on the basis of epidemiological evidence (Sun- derman, '78; Malcom, '79), has recently been classified as a positive mammalian carcinogen (Sax, '81). The cadmic ion, which i s associated with the production of chromosome anomalies in humans (Peereboom and Peer- eboom-Stegman, %l), implicated in the induction of neoplastic transformation in experimental animals (CRC, '74; IARC, '76; Takenaka et al., '83), and considered to be among the more potent of the recognized carcinogens (Gunn et al., '67; Sax, ,811, was selected as an initiator for these experiments. One of the most biologically active constitu- ents isolated from the prototypical promoter, croton oil (Berenblum, '41; Berenblum and Shubik, '47a,b; Van Duuren, '691, was found to be 12-0-tetradecanoylphorbol-13-acetate (TPA) (Hecker, '66, '68, '71; Van Duuren and Orris, '651, also referred to as phorbol myris- tate acetate (PMA). This 12J3-diester vari- ant of the tetracyclic parent alcohol, phorbol, was used as a promoter in these studies. Re- search has shown that TPA exhibits strong tumor-promoting action when employed either prior to (Roe, '59; Vesselinovitch, '58; Tannenbaum et al., '64) or after (Boutwell et

al., '82) the initiating stimulus. Whether the phorbol ester is applied repeatedly, as in the mouse skin model (Boutwell, '64; Van Du- uren and Sivak, ,681, or presented continu- ously, as in mammalian tissue culture systems (Han and Elkind, '82; Kennedy et al., '801, the promotional activity is enhanced by the persistence of administration; therefore, chronic exposures were employed in our cocarcinogenic protocols. The profound tumorigenic response of the planarian to cadmium and phorbol ester treatment is described in this and a following companion paper (Hall et al., '86).

MATERIALS AND METHODS

Asexual planarians of the species Dugesia dorotocephala were used as subjects for this study. They were selected for normal morphology and similar lengths (18-20 mm) from a laboratory colony that had been maintained in healthy condition, in aged tap water, and fed twice a week on raw beef liver.

Concentrated stock solutions of 12-0-tetradecanoylphorbol-13-acetate (TPA) (Sigma Chemical Co., St. Louis, MO) were prepared in acetone at 200 Fgirnl and were stored at -20°C. A weekly dilution to 0.1 pg/ml in aged tap water was carried out, and this solution was kept refrigerated at 5°C until final dilutions were made each day at room temperature. Stock solutions of cadmium sulfate (3CdS04:8H20, FW = 769.556) were prepared in aged tap water at a concentration of 120 mg/liter, and then were either adjusted to the final concentrations or were mixed with TPA prior to dilution.

Dose-response relationships, in terms of lethality, for the various solutions were determined by exposure of sets of ten planarians per glass specimen bowl to 50 ml of the test solution, which was changed every day. An appropriate amount of acetone was added to certain control groups. The optimum concentrations for chronic exposure were determined as the highest concentrations which produced negligible mortality during the treatment period; then larger populations (50 worms) were exposed to these test solutions (500 ml) in partially covered, porcelainized pans. Once established, the TPA dose-lethality curves served as a useful assay for the biological activity of the stored stock solutions, which was checked periodically.

The subjects were exposed to the various test solutions for a period of 2 weeks; then they were returned to aged tap water. They were observed closely for a period of 1 month,

PL,ANARIAN TUMORIGENESIS 213

during which time such toxicological indicators as the incidence of fissioning, behavioral activity, and overall appearance were monitored. In order to eliminate the possible promotional aspects of nutrition, the planarians were fasted during the entire 30-d,ay experimental period. After 30 days, the occurrence of disease was quantified; then, some tumor- bearing animals were selected for transplantation studies, others were prepared for histological examination, while the ]remainder became the subjects of continued observation under conditions of normal care and feeding.

The transplantation of tumorous tissues, as well as that of control tissues, was carried out under a dissecting microscope. Both do- nor and recipient planarians were placed on a slab of dental wax on top of crushed ice, with just enough water to keep them moist. The region of the tumor was dissected from the donor and surgically partitioned into several equisized pieces. A sliver of riuor blade was used as a scalpel, and a square incision (1 mm x 1 mm) was made on the dorsal surface of the recipient and a small amount of tissue was removed. The donor tissue was then placed into the incision and a ismall lens paper “bandage,” secured by surface tension, was applied to hold the transplanted tissue in place while the wound healed. After 24 hours in normal culture water at 20°C, the grafts had sufficiently sealed in and the bandage was no longer required. The recipients of such grafts were maintained in ordinary culture water for subsequent observation.

Selected specimens were fixed for 1 hour in a mixture of 1.5% glutaraldehyde and 0.5% formaldehyde adjusted to pH 7.3 with 0.1 M phosphate buffer solution and then postfixed for 1.5 hours in 1% buffered osmium tetrox- ide. After fixation, the specimens were dehy- drated through increasing concentrations of ethanol (from 50 to loo%), placed in propyl- ene oxide, and then embedded in Pcllybed 812 resin. Sections were cut with glass knives on a Sorvall Porter-Blum MT-2 ultramicrotome. Thick sections (1 pm) collected from each specimen block were stained with either toluidine blue or a modified Richardson stain and were examined with a light microscope to deline- ate the tumor regions. Thin sections (800 A) cut from selected regions were stained with uranyl acetate and lead citrate (Sato, ’68). Observations were made with a Philips electron microscope, EM-200, operating; a t 80 kV. The electron micrographs were taken at orig- inal magnifications of x 3,000 to x 7,000 and then enlarged photographically.

OBSERVATIONS

Exposure of planarians to increasing culture water concentrations of cadmium sulfate produced a variety of dose-dependent responses which were monitored. Several of these toxicological indicators, such as the suppression of fissioning and the incidence of head resorption, have been previously reported (Best and Morita, ’82, ’83). The per- centage of lethality in different concentrations of cadmium sulfate is presented as a function of time in Figure l a . The latency of lethality decreased and the rate of death increased with increasing concentration, pro- ducing 100% lethality within 3 days in 30 ppm, 6 days in 15 ppm, and 12 days in 7.5 ppm 3CdS04:8H20. The 14-day LC50 was determined to be 3.0 ppm and the LC25 for the same time period was 1.5 ppm. As a result of these findings, it was determined that concentrations less than or equal to 0.75 ppm cadmium sulfate would be suitable for chronic exposure. The dose-response relationship for TPA, displayed in Figure lb, revealed the remarkable sensitivity of the planarian system. Whereas concentrations from 0.1 ppm to 0.25 ppm TPA were found to be nontoxic to mammalian cell cultures (Han and Elkind, ,821, a concentration as low as 0.05 ppm TPA was found to be lethal to all exposed planarians within 5 days. From these results, 0.01 ppm TPA, a concentration which suppressed fissioning to a considerable extent, yet produced a negligible amount of mortality over a 5-day test period, was selected for utilization in the cocarcinogenesis experiments. As shown in Figure lc, the combination of cadmium sulfate and TPA produced an additional increase in the incidence of lethality, manifested in the post- treatment period. For the sake of clarity, and for reasons to be presented and discussed in detail, the following histopathological responses of the planarian to these chemical cocarcinogens will be referred to as tumors throughout the manuscript.

The tumorigenic response of the planarians upon 14 days of continuous exposure to cadmium sulfate proceeded in a dose-dependent manner, with concentrations of 0.3 ppm, 0.5 ppm, and 0.75 ppm 3CdS04:8H20 produc- ing tumors after 30 days at an incidence of 3.4%, 7.7%, and 14.7%, respectively. This particular type of tumor, referred to as a “type I” tumor, appeared externally as a small out- growth on the lateral margins of the “cervi- cal” area or, more commonly, in the post-

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F. HALL ET AL.

E . I-' + -

c E 50- 0

7a - - c e - a"

0 -

CdS04

LETHALITY AS A FUNCTION OF TIME

Cd = 0 3ppm CdS04

TPA = 0 01 pprn TPA

6 TPA Cd+TPA

l&Il Cd

I ' 2 ' 4 ' 6 ' i ' 10' 1 2 ' 14- l a Days of Exposure

CdS04 + TPA

LETHALITY AS A FUNCTION OF TIME

0.75 porn CdS04 0-01 ppm TPA

-0 .5 ppm CdSOq 0-01ppm TPA f 0 3 ppm CdSO4 0 01 ppm TPA

- 0 01 TPA "controls

Treatment Period

Ic - Fig. 1. a. Cumulative percent lethaiity of planarians

as a function of time in various aquatic concentrations of cadmium sulfate. b. Cumulative percent lethality of planarians as a function of time in various aquatic concentrations of 12-0-tetradecanoylphorbol-13-acetate (TPA). c. Cumulative percent lethality of planarians as a function of time in various concentrations of cadmium sulfate combined with 0.01 pg/ml TPA. d. Incidence of the tumorigenic responses of surviving planarians exposed to cadmium sulfate andor TPA, obtained as the

pharyngeal regions (Fig. 2b). Type I tumors first became recognizable at 3-4 weeks-that is, about 10 days after the animals were returned to normal culture water. Light microscopic examination of these tissues (Fig. 2d) revealed them to be composed of a heterogeneous population of cells, the proliferation of which had altered the normal histology (Fig.

TPA LETHALITY AS A FUNCTION OF TIME

I 1 2 3 4 5

Days of Exposure

Ib

CdS04 + TPA

TUMORIGENIC RESPONSE TO COCARCINOGENS

1 Id

mean i SD of experiments performed in triplicate, with a total of 90 animals per test group. Those type I tumors induced by cadmium sulfate alone were considered to be benign, while those type I1 produced by cadmium sulfate plus TPA, or by TPA alone, were considered to be malignant. (The difference in the mean incidence of malignant type I1 tumors between the TPA and the cadrnium-sulfate-plus-TPA groups was highly significant: t = 10.35, df = 4, P < ,001.)

2a,c); however, the various cell types and the relative frequencies of these were not mark- edly abnormal. These tumors did not appear to increase dramatically in size, or to spread, but they did remain prominent for several months after the chemical treatment was discontinued. The appearance of these outgrowths well after the exposure to cadmium

PLANARIAN TUMORIGENESIS 2 15

Fig. 2. a. Photograph of a control plan,srian of the asexual strain of the species Dugesia dorotocephala displaying normal morphology. Horizontal bar = 1 mm ( x 5). b. Photograph of a cadmium-treated planarian displaying a small type I tumor on the lateral aspect of the postpharyngeal region. This benign type of tumor persisted long after the chemical treatment, but did not appear to spread or increase in size. Horizontal bar = 1 mm ( X 5). c. Light micrograph of the lateral edge of a

transverse section representing normal histology. Tolui- dine blue stain. Horizontal bar = 50 pm ( x 350). d. Light micrograph of a transverse section through the benign type I tumor. The tissue is composed of a heterogeneous population of cells, yet the histology is somewhat dis- torted. Note the vertical muscle (arrow) that is displaced by the cellular proliferation. Toluidine blue stain. Hori- zontal bar = 50 p ( X 350).

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sulfate was terminated, and the persistence of these outgrowths for months, in the absence of the initiating stimulus, in an animal which is capable of regenerating an entire head within 2 weeks, suggest that a perma- nent alteration in morphogenesis has taken place, and that the term benign neoplasia rather than repair hyperplasia might be associated with these localized tumors.

The addition of the promoter, TPA, to the cadmium-containing solutions resulted in the induction of a tumorous condition that was not seen in the planarians that were exposed to cadmium sulfate alone. Approximately 10- 14 days after the 2-week cocarcinogen treatment, infiltrating “type 11” tumors were observed in a large proportion (76.2%) of the exposed animals. These tumors originated invariably as a swelling in the postpharyngeal region (Fig. 3a), grew rapidly, distorting the general morphology (Fig. 3b), and spread widely (Fig. 3c), leading eventually to the death of the planarian. Irreparable ulcerations began to appear in the epidermis dur-

ing the terminal stages of the disease; and all but a few of the subjects, in which the pernicious disease was observed, died within the next 2-3 months. Thus, the progressive, infiltrating type I1 tumors were considered to be malignant. Due to the combined toxici- ties of cadmium and the phorbol ester, increased lethality was observed within the latent period in the higher cadmium concentrations (0.75 ppm and 0.5 ppm 3CdS04: 8Hz0; Fig. lc), and although the type I1 tumorous disease was observed in a large per- centage (> 50%) of the survivors, the overall yield was reduced; therefore, solutions containing 0.3 ppm 3CdS04:8HzO (which corre- sponds to 0.13 ppm Cd) and 0.01 ppm TPA were considered to be optimal for the gener- ation of these type I1 tumors.

Treatment with the phorbol ester alone, in the absence of the cadmic ion, resulted in the induction of malignant type I1 tumors in considerable proportions (17.2%), although at a much reduced incidence from that observed with joint exposure to both cadmium and

Fig. 3. Photographs of planarians treated with chem- tually spreads throughout the animal (Fig. 3c). ical cocarcinogens (cadmium and TPA) representing dif- Irreparable ulcerations, such as those seen in Figure 3c, ferent stages of the resulting malignant disease. This appear during the terminal stage and death inevitably particular type I1 tumor originates in the postpharyn- follows. Horizontal bar = 1 mm (3a: x 5,3b: x 6,3c: x 5). geal region (Fig. 3a), grows rapidly (Fig. 3b), and even-

PLANARIAN TUMORIGENESIS 217

TPA. Figure Id illustrates the coniparative incidence of types I and I1 tumors induced by the different chemical treatments, revealing an interaction of cadmium and TPA that is not additive, but synergistic in effect.

Light microscopic examination of the af- flicted subjects outlined the histopathological nature of the malignant type I1 condition: large, distinct tumor formations were observed in the parenchyma, outside of the intestinal regions (Fig. 4a). These tumor masses displayed a uniformity of cellular staining and appearance. An abundance of mitotic figures were observed within the cells of the tumor areas (Fig. 4b). The infiltrating character of the disease was examined, at this level, in a variety of sections in which it became apparent that tumor cells were spreading into otherwise normal tissues (Fig. 4c). At the electron microscopic level, these tumors were found to be composed both of immature cells and of a single mature cell type; yet the mature cell type was observed within a wide spectrum of its differentiation. Certain areas within a given type 11 tumor were found to be occupied by cells which were similar to neoblasts in their phenotype: exhibiting spherical nuclei, prominent nucleoli, nuclear satellite material, numerous free ribosomes, and a high nuclear-to-cytoplasmic ratio. Surrounding or adjacent to these assembies of neoblastlike cells (Fig. 5) were found newly differentiating cells that were intermediate in composition to the mature tumor cell-which although grossly abnormal, can be identified as a derivative of the reticular cell. Normally, reticular cells are migratory, phagocytic cells that can be distinguished from other parenchymal cells by their irregular (ameboid) profile and their voluminous cytoplasm, which contains an abundance of glycogen granules, mitochondria, and lysosomes, as well as a considerable amount of granular endoplasmic reticulum and, quite often, phagosomes. Based on ultrastructural similarities, the type I1 tumorous derivatives of the reticular cell line are henceforth referred to as reticuloma cells. Although the mature tumor cells display the basic characteristics of reticular cells (such as glycogen granules, mitochondria, and lysosomes) the quantity of granular endoplasmic reticulum is greatly reduced, and phagosomes, common in normal reticular cells, are not observed. Such deficits tend to indicate an altered or compromised function. These reticuloma cells display extremely ir-

regular nuclei, an abnormally large number of mitochondria, and extensive cytoplasmic interdigitations with adjacent cells, assemblies of which compose the major portion of the tumor areas.

The sequence of type I1 tumor cell differentiation was determined originating from mitotically active assemblies of neoblastlike stem cells (Fig. 6), the development progresses through nonmitotic intermediate stages to the mature reticuloma cells and their characteristic tissue formations (Fig. 7). Nuclear satellite material, which appears solely in the perinuclear cytoplasm of neoblasts and then diminishes with differentiation, can be used as marker for elucidating the sequence of cellular maturation (Morita et al., ’69; Morita and Best, ’74, ’84a,b). Nor- mally, mature reticular cells do not exhibit the “dense lumps’’ of nuclear satellite material that are indicative of recent cytodiffer- entiation. In the case of the malignant type I1 tumors, an abundance of nuclear satellite material is found in the undifferentiated cells and in the early stages of differentiation (Figs. 5,6), which diminishes to vestiges (Fig. 8a,b) as the cytoplasm becomes more extensive and the nucleus becomes increasingly irregular-resulting, ultimately, in the production of the mature, but nonfunctional, reticuloma cell (Fig. 8c). All stages of this sequence of cellular differentiation were observed within the malignant tumors and, be- sides a stray reticular cell which displayed normal morphology (as seen in Fig. 6), these were the only cells found in the tumor areas. The observed restriction of differentiation to a single type of abnormal mature cell indicates that the neoblastlike precursor cells found within the tumor areas are, indeed, functionally transformed stem cells.

Surgical transplantation studies were un- dertaken in which small pieces of tumorous reticuloma tissues were implanted into the bodies of otherwise healthy planarians. Out of a total of 50 type I1 tumor transplantations, 48 planarians survived the operation, and eight of the surviving recipients, or 16.7%, developed the tumorous disease during the following month. It should be noted that only those recipients of tumor pieces containing a mitotically competent transformed stem cell would be expected to develop the disease, since the more mature reticuloma cells are nonmitotic. Light and electron microscopy confirmed that the transplanted disease represented the same

2 18 F. HALL ET AL.

Fig. 4. a. Light micrograph of a transverse section of (arrow) can be seen in the central portion of the tumor. a tumor-bearing planarian. The type I1 tumorous masses, Modified Richardson stain. Horizontal bar = 50 p outlined by the arrows, are located peripheral to the ( X 415). c. Light micrograph of a horizontal section of intestinal areas (I). Modified Richardson stain. Horizon- the planarian type I1 tumor. The arrows point out the tal bar = 50 pm ( x 104). b. Higher magnification of the tumorous streams of cells infiltrating the mesenchymal above transverse section shows the tumor (T) in contrast region (Me) outside of the intestinal area (I). Modified to the normal mesenchymal (Me) region. A mitotic cell Richardson stain. Horizontal bar = 50 p ( x 262).

Fig. 5 . Electron micrograph montage of a planarian type I1 tumor induced by joint treatment with chemical cocarcinogens (cadmium and TPA). A population of neoblastlike tumor cells (RT) displaying a high iiuclear (N)- to-cytoplasmic ratio, several nucleoli (Ne), End nuclear satellite material (NSM): characteristics which are rep- resentative of the early stages of cellular differentiation.

Mitochondria (m) and glycogen granules (g) can also be seen at this magnification. The gradual differentiation to a large, interdigitating reticuloma cell WM), with its distinctively irregular nucleus (N), is found to take place within these type I1 tumors. The central electron-dense complex is mucus. Horizontal bar = 5 p ( X 5,200).

220 F. HALL ET AL.

Fig. 6. Electron micrograph montage Of a cocarcinogen-induced type I1 tumor area revealing a cell undergoing mitosis N) in a field of newly differentiating, presumably transformed, stem cells CRT). An abundance of glycogen granules (g) and mitochondria (m) is ob-

served in these tumor cells, as well as nuclear satellite material (NSM). An apparently normal reticular cell CRC) can be seen near the top of the plate. Horizontal bar = 5 p ( x 5,200).


Fig. 7. Electron micrograph montage of a cocarcinogen-induced type I1 tumor area representing the tissue formations characteristic of the later stages of tumor cell differentiation. These large tumor cells (RM), with their extensive interdigitations, exhibit an abundance of mi-

tochondria (m), a distinctively irregular nucleus, and a comparatively electron-dense cytoplasm. Newly differentiating cells (RT) and some lipid deposits (L) can also be seen. Horizontal bar = 5 p (x 4,700).

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Fig. 8. Electron micrographs showing the intermedi- nucleus (N) becomes increasingly irregular (c). The abun- ate stages of cellular differentiation which take place dance of glycogen granules (g) and mitochondria (m), as within these malignant type I1 tumors. The cell in (a) well as the presence of lysosomes fLy) characterize these displays a nucleolus (Ne) and nuclear satellite material tumor cells as derivatives of reticular cells and the can- (NSM) indicative of the relative youthfulness of the cell. cerous type I1 disease as malignant reticuloma. Horizon- The nuclear satellite material persists @), as telltale tal bars = 2 p (a, x 7,700; b, x 9,100; c, x 6,700). vestiges, as the cell grows dramatically in size and the


cellular and histopathological qualities as the donor tissues. Continued observation confirmed the relentless progression to lethality. Although several (out of 25) control transplantation subjects failed to survive the operation, no such controls were found to exhibit any signs of the malignant, disease. A notable problem that is encountered in planarian grafting studies is that of auto- tomy (Dawe, '66); that is, the uncanny ability of the recipient planarians to reject, by fissioning, large areas of tissue disturbance. Whether induced by the actual surgery or by the foreign tissues, this type of wholesale tissue rejection was observed in both the tumor transplantations and the conti~ols. The selection for recipient planarians that had recently fissioned (exhibiting blunt tails), and the transplantation of tissues just posterior to the pharynx, managed to reduce the incidence of postoperative fissioning to about 50% and produced the aforementioned success rate. The fissioned tails frorn tumor transplanted recipients also displayed the malignant disease in four out of 26 cases, or 15.4%, which indicates that some of the tumorous tissues were obviously "autectom- ized." In any event, the transplantation studies serve to confirm the metastatic spread of the cadmium- and phorbol-ester- induced malignant reticuloma disease.

In addition to the transplantation studies, the regenerative capabilities of the type II tumor-bearing planarians were examined. Upon first recognition of the reticuloma disease, the subjects were cut in half by transverse section and were then observed for several months under normal colony conditions. The anterior portions, which iincluded the head but not the visible tumor, regener- ated the missing portions effectively to produce normal-appearing flatworms in eight of the ten cases studied, while the posterior portions, which included the tumor, displayed little or no regenerative capacity and either died or remained moribund, displaying the terminal stages of the syndrome. Nor did simple decapitation improve the survivabil- ity of those planarians stricken with the malignant disease. Although no definitive conclusions can be made from these prelimi- nary findings, it appears that the malignant reticuloma is inherently pathological and cannot be normalized by the morphogenic stimulus of regeneration.

DISCUSSION The study of tumorigenesis in simplified,

yet biologically relevant, model systems is

an area of considerable scientific interest. The goals and pitfalls inherent in the devel- oping field of comparative oncology have been reviewed on several occasions (Scharrer and Lochhead, '50; Dawe, '66, '69; Harshbarger, '67; Sparks, '69; Best, '83). Freshwater planarians are endowed with a number of physiological, histological, and biochemical attributes which have historically attracted interest from the fields of neuroscience and developmental biology, as well as environ- mental toxicology. Studies of the tumorigenic responses of planarians to mammalian carcinogens exhibit, also, a long history; yet, documents only limited success. The consid- eration that a general failure to take into account the promotional aspects required for neoplastic transformation has retarded the development of this field prompted these cur- rent investigations. The overall results of our experiments indicate that this is, perhaps, the case.

The tumorigenic response of the planarian to cadmium sulfate was found to result from a localized proliferation of a heterogeneous population of cells whose distribution dis- torted the normal histology, but did not me- tastasize. Now, a hyperplastic response to a recognized carcinogen is, by itself, interest- ing in regard to the well-known clinical observation that neoplastic transformation often occurs at a site of previous hyperplasia. Yet the development of the type I tumor after cessation of the chemical treatment and the time scale of the tumor's persistence after the stimulus was withdrawn suggest the ap- propriateness of the term benign neoplasia, although we acknowledge that the precise terminology is, in this case, somewhat debat- able. There is little doubt, however, that the addition of the promoter, TPA, to the cadmium-containing solutions produced a pernicious type of tumor that qualifies for the appellation: malignancy. The uncontrolled proliferation and extensive infiltration of the type I1 tumor, which progresses to lethality unabated by regeneration, with metastatic spread confirmed by transplantation, signify the malignant quality of the disease. The incidence of tumorigenesis upon exposure to the dual cocarcinogens, when compared to either the cadmium or the TPA alone, was found not to be additive, but revealed a remarkable synergism. The latency of tumorigenesis, which was predicted to be approximately 3 months on the basis of previous studies involving polyaromatic hydrocarbons (Foster, '69; Best and Morita, '82), was reduced to less than 1 month by the cocarcinogenic protocol.

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Ideally, a tumor promoter, by itself, would produce little or no incidence of tumors. But, in actuality, all known promoters display tumorigenic activity to some extent (Van Du- uren, '691, and TPA in particular has been regarded as a weak carcinogenic initiator (Boutwell et al., '82). In mammalian tissue cultures also, TPA was found to be a weak initiator as well as a strong promoter (Han and Elkind, '82). Therefore, the observed incidence of tumorigenesis in the planarians exposed solely to TPA is not surprising. What is noteworthy, in this context, is the type of cancers that the promoter induces in mammalian systems; that is, systemic administration of the unesterified parent compound, phorbol, in the mouse results in the induction of lymphoid leukemias in a large proportion of the treated animals (Berenblum and Lonai, '70). The leukemogenic action of phorbol was confirmed in the rat (Armuth and Berenblum, ,741, but whereas the lymphoid tumors of the mouse were determined to be predominantly nonthymic, those of the rat appeared to involve the thymus in every case. This specific leukemogenic action of phorbol becomes increasingly relevant when one con- siders the type of cells involved in the malignant planarian disease.

Thorough examination of the tissue formations and the cellular architecture of the malignant type I1 tumors revealed that the pathology involved the massive proliferation of a single type of differentiated cell: a tumorous derivative of the reticular cell. The normal reticular cell is known to be mobilized to the wound area as a first line of defense following surgical injury (Morita and Best, '74) and is demonstrably capable of phagocy- tizing damaged or degenerating cells (Morita and Best, '84a). Since the tumorous disease involved a derivative of this particular type of cell, which participates in a form of cell- mediated immunity, the question naturally arises as to whether the cellular accumula- tions develop, as an immune response, from migratory reticular cells in a planarian ver- sion of inflammation. This is definitely not the case. The malignant tumors are composed not of normal mature reticular cells migrating into these areas as a form of immune surveillance, nor of normal reticular cells proliferating in response to some kind of antigenic stimulation, but of abnormal reticuloma cells whose presence defines the disease. The malignant reticuloma cells exhibit a characteristic pathological morphology, several ultrastructural abnormalities, and

are apparently, as determined by the dimin- ished granular endoplasmic reticulum and the complete lack of phagosomes, nonfunctional. Further, the mature reticuloma cells, with their extensive cytoplasmic interdigitations, appear to be relatively immobile. These reticuloma cells are found to differentiate from mitotically active stem cells which proliferate within the tumor areas. The predicted incidence of mitotic figures in this species of planarian was determined from previous studies to be one in approximately 30,000 cells (Best et al., '681, and although precise quantification was not possible in the present study, due to the selective nature of the electron microscopy, the comparative incidence of mitotic cells appeared to be dramatically increased in the reticuloma areas.

It is generally believed that the immune system evolved not only as a means of defense against infectious microorganisms, but as an internal surveillance for cellular discontinu- ity, and that cancer, therefore, represents a malfunction of this immune surveillance system. Based on the essential relationship between the lymphoid system, immunity, and malignancy, and on the (mis)conception that the lymphoid system and reactive immunity came into existence for the first time in the primitive fishes, it has been boldly stated that none of the "lumps and bumps or cellular ac- cumulations among the invertebrates have features that are associated with the malignant tumors of mammals" (Good and Finstad, '69). Others have, less narrowly, viewed the tissue rejection phenomena described in planaria and other invertebrates, together with the systems of powerfully phagocytic cells, as a fundamental form of cell-mediated immunity (Cooper, '77). Further, it has been postu- lated that an "ancestral stem cell" emerged and gave rise to granulocytes, lymphocytes, and macrophages entirely within Inverte- brata (Cooper, '77). The planarian reticular cell system, described and referred to throughout this manuscript, is known to be mobilized as a first line of defense following injury (Morita and Best, '74), is demonstrably capable of phagocytosis (Morita and Best, '84a), and is now reportedly subject to neoplastic transformation: all features one would expect of such an ancestral stem cell. The planarian reticular cell is also considered to function as a nutrient carrier (Morita and Best, '84a) and may well represent a primal predecessor of the entire hemocytic (hence, hematopoietic) lines that developed within the phylogenetically higher animals.


Judging from the research of others (Lange, ’66; Foster, ’691, as well as from our own experiments with polyaromatic liydrocar- bons (Best and Morita, ’821, we predicted that the only cells in the asexual planarian capable of mitosis, and therefore capable of undergoing the promotional events required for transformation into malignancy, would be neoblasts. Accordingly, we anticipated, and previously expected, that all planarian “cancers” would be neoblastomas. This conception, though partly correct, must be reex- amined in view of new evidence: .4lthough an abundance of the planarian type I1 (malignant) tumor cells was found to be morpholog- ically indistinguishable from neobllasts, the only line of differentiation observed was that of the reticuloma cell. This functional restriction of totipotency was confirmed by the transplantation studies to involve more than morphological “field effects,” which suggests that these neoblastlike cancer cells are indeed transformed. What is novel here is the indication that these planarian caiicer cells have become committed prior to the loss of mitotic competency. The transformed (committed) stem cell, detected in the case of the planarian malignant reticuloma, parallels the pathogenesis of a variety of mammalian neoplastic stem cell diseases. Inasmuch as the identification and characteriza.tion of a particular cancer cell, as distinct from a plu- ripotent neoblast, is unique in the reported literature, the ultrastructure and limited differentiation of this reticuloma cell are described in additional detail in the following companion paper (Hall et a1.,’86).

In summary, two distinct types of tumors were induced in planarians by the differen- tial action of mammalian chemical cocarcinogens. The question of the appropriate terminology for these cocarcinogen-induced planarian tumors was addressed at the functional, histopathological, and ultrastructural levels. On the basis of these observations, the type I tumors produced by the action of cadmium sulfate alone that persisted but did not spread were considered to be benign, while the type I1 tumors that were induced by cadmium plus TPA, or by TPA alone (to a lesser extent), exhibited a pathogenesis and a cellular character which bears direct compari- sion with the malignant tumors of phyleti- cally higher animals. The type 11 tumors were determined to result from the progressive infiltration of a single line of highly abnormal cells: the reticuloma cell line. Ma- lignant reticuloma cells were found to differ-

entiate from mitotically active, presumably transformed, stem cells which proliferate within the infiltrating tissue formations. The metastatic spread of the malignant reticuloma disease was confirmed by transplantation into otherwise healthy animals that subsequently exhibited the identical histopathology and the same progression to lethality. As a result of these studies, it became evident that the freshwater planarian, Du- gesia dorotocephala, displays the major phenomenology of mammalian cocarcinogenesis. The histopathological and cellular characteristics of the malignant reticuloma model several important features of a neoplastic stem cell disease. Further investigations into the sequence and timing of the cocarcinogenic treatment are thus encouraged, and additional studies utilizing a variety of other recognized initiators, as well as different promoters, are called for as further investigations into the tumorigenic response of planarians to mammalian cocarcinogens. Ex- plorations into the physiological qualities of the postpharyngeal region that predispose this area to neoplastic transformation could also be potentially important.

ACKNOWLEDGMENTS

This work was supported in part by science and education grants from the US. Depart- ment of Agriculture to the Colarado State University Experiment Station.

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neoplastic transformation in the planarian: i. cocarcinogenesis and histopathology

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