Differential Response of Skin Epithelium to Growth-Promoting Effects of Subcutaneously Transplanted Tumor*

Differential Response of Skin Epithelium to Growth-promoting Effects of Subcutaneously Transplanted Tumor*

THOMASS.ARGYRISANDBERTIEF. ARGYRIS

(Department of Zoology, Syracuse University, Syracuse, New York)

SUMMARY Subcutaneously transplanted Ehrlich ascites tumor invaded the subcutis of the overlying skin and resulted in epidermal enlargement, consisting of cell hyperplasia and hypertrophy. The resting hair follicles, on the other hand, were completely un affected by the invading tumor. The growing tumor eventually invaded the dermis, resulting in a further increase in the thickness of the epidermis. However, the hair follicles, even though completely surrounded by the invading tumor, remained un affected. The cells of the resting hair follicles and those of the basal layer of the epidermis had the same developmental potentiality and were interchangeable. Therefore, the results indicate that the physiological condition of a tissue, or its competence, is important in determining a positive outcome in a growth-promoting interaction.

We report the results of an investigation which mor. Plucking initiated hair growth in the plucked demonstrates that Subcutaneously transplanted area only (13). Upon completion of the growth Ehrlich ascites tumor stimulates the epidermis of phase of the hair growth cycle the hair follicles the skin but does not affect the resting hair fol again entered the resting phase and remained licles. These results are important, because they there throughout the course of the experimental demonstrate that two tissuesâ€”namely, the epi period; if not, the animals were discarded. dermis and resting hair folliclesâ€”which are de- To study the effects of the Ehrlich ascites tumor velopmentally equivalent and are interchangeable on the skin, 69 mice were given inoculations sub- (12, 14), respond differently, presumably to the cutaneously of 0.1 ml. of Ehrlich ascites tumor. same growth-promoting influences. For details of the technic, precautions taken to maintain sterility, and source of tumor, see MATERIALS AND METHODS Argyris and Argyris (2, 3). Biopsy specimens of In total, 189 young adult C57BL mice of both the tumor and overlying skin were taken at 2, 4, 7, sexes were used in this investigation. All mice 10, 14, 21, and 28 days. Six hours prior to removal came from our own inbred colony or from the of the tumor and the overlying skin, the mice Jackson Memorial Laboratory, Bar Harbor, were given injections Subcutaneously of 0.1 mg. Maine. The mice were kept in an air-conditioned colchicine in 0.25 ml. of saline. As controls, six animal room, fed Purina Laboratory Chow and mice whose skin was in the resting phase were water ad libitum, and lettuce and cod liver oil- given injections of colchicine, as described above, soaked pellets, weekly. and biopsy specimens of the skin were taken 6 The hair follicles of mice undergo cycles of hours later. growth and rest (12, 14). To be certain that the To determine the effect of mild epidermal dam hair follicles would be in the resting phase through age, twenty mice with resting hair follicles were out the course of investigation, all mice were shaved. Biopsy specimens were taken at 2, 3, 4, 7, plucked 21 days previous to inoculation of the tu- and 9 days. These mice were pretreated with col * This investigation was supported by grants from Ruth chicine prior to removal of the biopsy specimens Estrin Goldberg Memorial Foundation and the National as described above. Science Foundation. All biopsy specimens were fixed in 10 per cent Received for publication July 24, 1961. formol-calcium, dehydrated and cleared in dioxan, 73

embedded in paraffin, and semi-serially sectioned at reasonable to consider the upper sheath of the 6 p. Sections were stained with toluidine blue, pH resting follicle functionally as a continuation of 3.5. the basal layer of the epidermis (9),2 a convention To relate the histological changes with tumor which we shall arbitrarily use. growth, about 60 mice were given inoculations of There is considerable mitotic activity in the 0.1 ml. of Ehrlich ascites tumor as described sebaceous glands but none in the dermis. Mitotic above. Tumors were dissected free and their wet activity is also absent in the panniculus carnosus, and dry weights determined at 2, 4, 7, 10, 14, 21, the muscle layer of the skin. Beneath the pannicu and 28 days. Similarly, 40 mice were given sub lus carnosus is a thin layer of loose areolar connec cutaneous inoculations of .05 ml. of tumor, and tive tissue, in which mitoses also are rare. the tumors were dissected out and their wet and Histological changes in Ehrlich ascites tumor and dry weights determined at 2, 7, 10, and 14 days. mouse skin following tumor inoculation.â€”Within 2 days after tumor inoculation the tumor showed RESULTS considerable growth (Table 1). It was composed of Histology of mouse skin.â€”A bare outline of the two areas: the outer area was deeply basophilic histological structure and mitotic pattern of mouse and had a number of layers of cells in which skin with resting hair follicles, pretreated with mitoses were frequent; the central area was under going necrosis, but mitoses might also be seen. TABLE1 The skin was not invaded and was normal, but WETANDDRYWEIGHTSOFEHRLICHASCITESTUMOR the subpannicular connective tissue was stimu (EAT)ATDIFFERENTDAYSAFTERSUBCUTANEOUS lated, showing a considerable amount of mitosis INOCULATION and metachromasia. Four to 6 days after tumor inoculation, the EAT*No.tumors7612151064WeightML. EAT*No.tumora895117WeightML. tumor did not show any significant increase in mass (Table 1); however, both outer and central mg.(Av.Â±S.E.)Wet58in tag.(Av.Â±S.E.)Wet19Â±434in DAT12467101421280.1 areas of the tumor appeared thicker. In ten cases the skin was normal except for a mild cellular in filtration in the subcutis. However, in three biopsy specimens the tumor had invaded the skin as far as the dermis. In these cases there was a +744 Â±.18.5+.113 definite enlargement of the epidermis, owing to an +859 +534 +437Â±738 +19Â±117 increase in cell number and size; but the resting +6114 +121+320+. hair follicles were unaffected (Fig. 2). +10108+4127 Tumor mass was increased by 10 days (Table 724 + 7Dry3Â±.66+.910+ 1 +7309 +251+60.05 1). Correspondingly, both the outer area and the + 36Dry12 central necrotic area were bigger. Most of the sections showed normal skin, with the exception of * Amount of fluid tumor inoculated subcutaneously. a definite cellular infiltration in the panniculus carnosus and subcutis. The underlying subpan colchicine, is presented for purposes of orientation. nicular connective tissue continued to be enor Details may be found in Chase (12) and Montagna mously thickened, had many mitotic figures, and (14). was metachromatic. The epidermis is one or two cell layers in thick In a few cases, the subcutis of the skin was in ness. The basal layer shows occasional mitotic vaded by tumor cells. As before, the epidermis, figures. The resting hair follicles were made up of but not the hair follicles, appeared to be stimu a single epithelial sheath, the external root sheath, lated. However, adjacent skin areas, where tumor a sebaceous gland, and a dermal papilla. The rest invasion had not occurred, did not show epidermal ing hair follicle sheath below the level of the outlet stimulation, even though the tumor beneath the of the sebaceous gland rarely shows mitotic activ skin had reached a considerable size. ity, but the upper part of the hair follicle, from the The situation at 14 and 21 days was essentially level of the sebaceous gland to the epidermal sur the same, except that the tumor was much larger face, has mitotic activity similar to the basal (Table 1), and the number of biopsy specimens layer of the epidermis. It also has the same diurnal showing tumor invasion and epidermal stimulation mitotic rhythm as the epidermis (9-11), and it had increased. In almost all cases tumor invasion responds similarly to the epidermis to a variety of 1T. S. Argyris, unpublished observations. damaging agents.1 Indeed, some suggest that it is 2H. B. Chase, personal communication.

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1962 American Association for Cancer Research. ARGYRISANDARGYRISâ€”GrowthPromotionof Skin by Tumor 75 continued to be limited to the subcutis, although damage. To test this possibility the effects of in some cases the tumor had begun to invade the shaving have been studied. dermis (Fig. 3). Shaving produced epidermal enlargement; but, By 28 days the tumor had further increased in in addition, hair follicle stimulation also occurred size (Table 1) and in some cases had resulted in even though histological examination showed that skin ulcÃ©ration.Marked tumor invasion of the skin shaving had not resulted in any open wounds or was prominent. In many cases the tumor had com damage to the hair follicles. Moreover, the resting pletely invaded the subcutis and dermis and might hair follicles had their club hairs intact as late as reach the epidermal-dermal junction (Fig. 4). In 9 days after shaving. This indicates that dislodge- such cases the epidermal stimulation was usually ment of club hairs, which can be sufficient stimu maximal. The epidermis might contain five to lus for hair growth, had probably not occurred, eight cell layers of enlarged basophilic cells. The since club hairs which have been dislodged usually hair follicles, however, remained completely de fall out within 5-6 days.2 Finally, epidermal en void of mitotic activity even though they might be largement was transient. It began at about 2-3 not only completely surrounded by the tumor but days and was gone by 9 days. actually in the process of degeneration. In the cases where the tumor had resulted in ulcÃ©ration, DISCUSSION microscopic examination showed the epidermis We may conclude that the Ehrlich ascites tu gone and only remnants of the hair follicles left. mor, subcutaneously inoculated, can result in the The epidermis immediately next to the ulcer was enlargement of the epidermis of the overlying skin. not stimulated but actually showed atrophie de This epidermal enlargement is a function of both generation, a result which was opposite to that cell hypertrophy and hyperplasia. Apparently seen in epidermis adjacent to all other kinds of epidermal stimulation occurs over considerable wounds studied (5, 6, 8). Progressing further away distances, since the epidermis is enlarged when from the ulcer, but still in the area of tumor in tumor invasion is limited to the subcutis, which is vasion, the epidermis showed marked stimulation. at least 100-200 p from the epidermal-dermal As before, in areas where tumor invasion had not junction. On the other hand, the resting hair occurred, the epidermis was not stimulated (Fig. follicles are completely unaffected, as far as any 1). increases in cell size or number are concerned. This We point out in passing that the sebaceous refractoriness is most obvious when the tumor has glands did not show obvious qualitative changes, invaded the dermis and completely surrounds the even in areas of complete dermal invasion (Fig. 4). hair follicles. Only in areas of ulcÃ©rationdidthey show degenera It may be asked whether the epidermal stimula tive changes. tion is due to nonspecific damage, since it is well Histological changes in skin damaged at the time known that many forms of mild damage can of tumor inoculation.â€”The above results show a stimulate skin epithelium (7, 10, 12). This seems strong correlation between skin invasion and epi unlikely, because in many cases only a small part dermal stimulation. If stimulation of the epidermis of the skin shows epidermal stimulation, and we is dependent on tumor invasion, then we should be would have to hypothesize that wherever we have able to artificially speed up stimulation by enhanc epidermal stimulation, by coincidence that point ing invasion. This has been done in nine mice by on the skin has been damaged. We feel this im damaging the panniculus carnosus with the needle probable, especially since a close inspection was of the syringe just prior to tumor inoculation. It is always made of the overlying skin. Our results well known that damage can enhance tumor in also show that mild damage due to shaving results vasion (17). In all cases tumor invasion occurred in epidermal stimulation and, in addition, growth within 4 days, and concomitantly the epidermis of the resting hair follicles. Similarly, small bites showed definite enlargement. By 7 days the tumor due to fighting or mites, small enough to be in had invaded the dermis up to the level of the epi visible to the unaided eye, also result in hair dermis, a result usually not observed in un growth stimulation (12).3 Finally in skin wounds, damaged skin of mice until at least 21 days after epidermal stimulation, irrespective of wound size tumor inoculation. or depth, is never more than 1 mm. (10). Tumor Effects of damage on mouse skin.â€”Since it is stimulation may extend, however, as much as 8 well known that handling or mild surface damage mm. over the surface of the skin. Thus, we feel of mouse skin stimulates the epidermis (10, 12), that even mild damage produces different and the question arises whether the epidermal enlarge additional effects from those of the tumor, thereby ment we observed was due to nonspecific surface 1T. S. Argyris, unpublished results.

enabling us to rule out surface damage as the tissue acts as a barrier to diffusible growth- cause of epidermal stimulation. promoting substances released by the tumor. We Neither the size of the inoculum nor large have previously suggested that muscle or connec amounts of tumor growth are important in de tive tissue may act as a barrier to the stimulation termining epidermal stimulation. Inoculation of of the mammary gland ducts by transplanted one-half the amount of Ehrlich ascites tumor re Ehrlich ascites tumor (4). The possibility that tu sults in sufficient tumor growth (Table 1). Tumor mor invasion is critical, because it simply damages growth per se beyond a certain minimal amount is the connective tissue of the skin, which in turn not critical, because epidermal stimulation can be releases growth-promoting substances which observed as early as 3-4 days when little tumor stimulate the epidermis, we believe is mitigated growth has occurred. On the other hand, the by the fact that Bullough and Laurence (10, 11) tumor may grow for as long as 28 days and achieve have shown that damage to the connective tissue a large size without any epidermal stimulation. of the skin does not result in epidermal stimula Similarly, increases in blood supply, connective tion. It is possible, however, that the damage to tissue proliferation, inflammation, or massive tu connective tissue produced by the invading tumor mor necrosis are not directly responsible for epi releases different substances than those released dermal stimulation. All these factors are present by mechanical damage. within a few days after tumor inoculation, and The resting hair follicles and the basal layer of they increase in magnitude with increased tumor the epidermis have been shown to be experimental growth. Yet, just like tumor growth, there ap ly interchangeableâ€”i.e., the cells of the hair fol pears to be no direct correlation between them and licles can be used to make epidermis, and vice epidermal stimulation. A lack of correlation be versa (12, 14). In other words, these tissues are tween these variables and growth promotion is developmentally equivalent. Therefore, a pro seen also in the stimulation of the mammary gland found difference in the physiological condition duct epithelium by the Ehrlich ascites tumor in must exist between the epidermis and resting hair ovariectomized, normal, pregnant, and lactating follicles, since only the epidermis responds to the C57 mice (3, 15), and in the stimulation of hair growth-promoting effects of the Ehrlich ascites growth in wound-healing (7). tumor. This focuses our attention on the fact that Pressure which has also been associated with the role of the receiver, or its competence, is im growth promotion (1, 16) cannot be responsible portant for a positive outcome in any growth- for epidermal stimulation, even though there is promoting interaction. The question arises why considerable distention of the skin as the tumor the epidermis is competent to respond and the grows. We have had cases in which the tumor has hair follicles not. We have no answers to this ques grown for 30 days, without skin invasion, resulting tion at the moment, but we offer the following in a considerable pressure on the skin, yet epi speculations. dermal stimulation has not been seen. Conversely, One obvious difference between the epidermis we have obtained perfectly good stimulation and the resting hair follicles is the fact that the within 4 days after tumor inoculation. epidermis is an actively dividing tissue, whereas It appears to us that the most critical factor the resting hair follicles are not. Perhaps the reason determining epidermal stimulation is invasion of that the epidermis can respond is because some of the skin by the tumor cells. Indeed, very little its cells are already dividing, whereas the resting invasion is necessary for epidermal stimulation to hair follicles rarely, if ever, contain mitoses (12). occur. We speculate that tumor invasion of the If this is the case, then the growth-promoting skin is necessary because the panniculus carnosus message(s), presumably liberated by the tumor, is and/or the hyperplastic sub-pannicular connective sufficient only for stimulating tissues in which

FIG. 1.â€”Sectionof skin 21 days after the subcutaneous in follicles. The tumor has invaded the subcutis of the skin but not oculation of Ehrlich ascites tumor. The skin is normal. Arrow the dermis. XI68. points to some of the growing tumor cells that are just begin FIG. 3.â€”Section of skin 21 days after tumor inoculation. ning to invade, but have not entered in sufficient numbers to Tumor beginning to invade dermis. Note enlarged epidermis. cause stimulation of the overlying epidermis. X168. X230. FIG. 2.â€”Section of skin 7 days after tumor inoculation. FIG. 4.â€”Section of skin 28 days after tumor inoculation. Note the large number of mitotic figures in the surface epider Tumor has completely invaded the dermis. Note the marked mis and in the part of the epidermis which dips toward the epidermal enlargement and intact sebaceous glands. X168. sebaceous gland. Note also the absence of mitoses in the hair

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1962 American Association for Cancer Research. ARGYRISANDARGYRISâ€”GrowthPromotionof Skin by Tumor 77 some cells already are dividing. This speculation Substances. Symposia of the Society for Experimental Biology #11, pp. 235-54. Cambridge, England: Cambridge is supported by the fact that the Ehrlich ascites Univ. Press, 1957. tumor implanted in the mouse kidney does not 2. ARGYRIS,B. F., and ABOYRIS,T. S. Mammary Duct stimulate adjacent kidney cells to divide.4 Mouse Stimulation by Subcutaneous Ehrlich Ascites Tumor kidney cells are also essentially "differentiated" Transplants. Cancer Research, 20:325-28, 1960. 3. . Mechanism of Mammary Duct Stimulation by cells which normally show little mitotic activity Tumor Transplants. Ibid., 21:75-81, 1961. (16). However, it is probably more than the mere 4. . Effect of Ehrlich Ascites Tumor on Mammary absence of mitosis in a tissue which places it in a Glands of Mice of Low and High Mammary-Cancer class unable to respond to the tumor, since mam Strains. J. Nat. Cancer Inst., 26:25-35, 1961. mary gland ducts of ovariectomized mice are also 5. ARGYRIS,T.S. The Relationship between the Hair Growth Cycle and the Response of Mouse Skin to X-Irradiation. essentially devoid of mitosis, but they are stimu Am. J. Anat., 94:439-71, 1954. lated by the growing Ehrlich ascites tumor (2). In 6. . The Effects of Wounds on Adjacent Growing or this case it may be argued that, although the Resting Hair Follicles in Mice. A.M.A. Arch. Pathol., mammary gland ducts in ovariectomized mice are 61:31-36, 1956. devoid of mitosis, the machinery for cell division 7. ARGYRIS,T. S., and ARGYRIS,B. F. Stimulation of Hair Growth during Skin Regeneration. Develop. Biol., 1:269- has been previously synthesized. On the other 80, 1959. hand, the fact that a tissue shows mitosis does not 8. ARGYRIS,T.S., and BELL,E. The Phsyiological Activity of guarantee it will respond to the tumor-growth- the Skin and Its Response to Ultra Sound. Anat. Ree., promoting influences. Recent work of Simmons 125:105-19, 1959. (15) has suggested that, in mice, ducts of lactat- 9. BULLOUGH,W.S. Age and Mitotic Activity in the Male Mouse Mus musculua L. J. Exp. Biol., 26:261-86, 1949. ing mammary glands exhibit mitoses, but they 10. BULLOUGH,W.S., and LAURENCE,E. B. The Control of are not stimulated by transplanted Ehrlich ascites Epidermal Activity in the Mouse. Proc. Roy. Soc., s.B, tumor. If these speculations are correct, then the 161:517-36, 1960. growth-promoting influences released by the Ehr 11. . The Control of Mitotic Activity in Mouse Skin lich ascites tumor must be different from those Dennis and Hypodermis. Exp. Cell. Research, 21:394-405, 1960. observed after damage, since wound healing stimu la. CHASE,H. B. Growth of the Hair. Physiol. Rev., 34:113- lates not only the epidermis but also growth of 26, 1954. resting hair follicles (7), and damage to the kidney 13. CHASE,H. B.; RAUCH,H.; and SMITH,V.Critical Stapes in results in an intense mitotic proliferation around Hair Development and Pigmentation in the Mouse. the damaged area.4 Physiol. Zool., 24:1-10, 1951. 14. MONTAGNA,W.The Structure and Function of Skin. New York: Academic Press, Inc., 1956. ACKNOWLEDGMENTS 15. SIMMONS,J. E. Physiological Modification of Mammary The capable assistance of Miss Mary Ellen Trimble is Gland Response to Growth Stimulation by Ehrlich Ascites gratefully acknowledged. Tumor Transplants. Thesis. Syracuse, New York: Syracuse University, 1961. REFERENCES 16. SWANN,M. M. The Control of Cell Division: A Review.II. Special Mechanisms. Cancer Research, 18:1118-60, 1958. 1. ABERCHOMBIE,M.Localized Formation of New Tissue in an Adult Mammal. In: The Biological Action of Growth 17. WALLACE,A.C., and HERSHFIELD,E.S. The Experimental Implantation of Tumor Cells in the Urinary Tract. Brit. 4T. S. Argyris and B. F. Argyris, unpublished results. J. Cancer, 12:622-30, 1958.

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1962 American Association for Cancer Research. Differential Response of Skin Epithelium to Growth-promoting Effects of Subcutaneously Transplanted Tumor

Thomas S. Argyris and Bertie F. Argyris

Cancer Res 1962;22:73-77.

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