AMERICAN JOURNAL OF CLINICAL PATHOLOGY Vol. 34, No. 2, August, I960, pp. 125-130 Printed i?i U.S.A. THE RESPONSE OF CONNECTIVE TISSUE ASSOCIATED WITH TUMORS OF T H E SKIN RICHARD D. MOORE, M.D., JOHN STEVENSON, B.A., AND MELVIN D. SCHOENBERG, P H . D . Institute of Pathology, Western Reserve University, Cleveland, Ohio The connective tissue adjacent to experimental and spontaneous neoplasms of the skin is frequently altered. Gliicksmann4 and On 13 have described the histologic changes that result from the application of coal tar carcinogens to the dermis. Gersh and Catchpole3 suggested that depolynierization of mucopolysaccharides of the ground substance may have a role in the invasion of connective tissue by tumors. Simpson16 has indicated that mucolytic enzymes may be elaborated by undifferentiated epithelial cells, and suggested that these enzymes may act as a spreading factor to promote invasion by malignant tumors through degradation of the ground substance. Fanger and Barker2 described variations in the metachromatic response of the dermis associated with a variety of tumors. They suggested that the alterations in the connective tissue are a proliferative response of the connective tissue to the tumor. The study described in this paper deals with the reaction of connective tissue to benign and malignant lesions of human skin. MATERIALS AND METHODS Thirty-one specimens of basal cell carcinoma of the skin, 22 of squamous cell carcinoma, 12 of seborrheic keratosis, and 12 of verruca vulgaris were used in this study. The specimens were obtained during Received, December 30, 1959; accepted for publication March 30, 1960. Dr. Moore is Associate Professor of Pathology; Mr. Stevenson is a medical student, Western Reserve Univ.; and Dr. Schoenberg is Assistant Professor of Experimental Pathology and Research Fellow, American Cancer Society, Cuyahoga County, Ohio Unit. This work was supported in part by a grant from the National Institutes of Health, United States Public Health Service, Department of Health, Education, and Welfare. surgical procedures and at autopsy, and were fixed in 10 per cent neutral buffered formalin. Normal skin was present in these specimens and 8 additional-samples of normal human skin were used aVco'iitrols. The sites of the lesions and s.a&iplqgffjf normal skin were random, but .,\j£rai>-considerable overlap of. anatomic locaiskm. The' tissue »was embed'ded in. paraffin and sectioned -in the usual manner. All of the specimens were treated by the following procedures. Staining with hematoxylin and eosin was used for the study of the histologic features. The metachromatic staining reaction for the acidic polysaccharides of the ground substance was performed with twice recrystallized toluidine blue (National Aniline Division, Allied Chemical Company), before and after hyaluronidase digestion. Dye solutions of 1.5 X 10 -4 M were prepared in various buffers10 from pH 2.5 to 5.0 in increments of 0.5 pH units at ionic strength 0.0025 M. The sections were dialyzed for 2 hr. in the appropriate buffer and then stained with toluidine blue for 20 min. Digestion of the tissue with testicular hyaluronidase (Nutritional Biochemicals Corporation) was accomplished in 0.12 M NaCl at pH 5.8 for 24 hr. at 37 C , and at a concentration of 150,000 turbidity reducing units (TRU) per liter.12 The enzymetreated sections were washed in 0.12 M NaCl and distilled water prior to dialysis. RESULTS An alteration of the connective tissue adjacent to and surrounding the invasive portions of the basal cell carcinomas was observed microscopically in 23 of 31 specimens. The remainder of the specimens did not have any histologically apparent alteration in the connective tissue adjacent to the tumor. The fibroblasts in affected areas 125 126 MOO KB ET varied in number and maturity in each of the specimens. The mature forms were associated with numerous bundles of collagen (Figs. 1 to 3). Twelve of 22 specimens of squamous cell carcinoma had areas of fibroblastic proliferation adjacent to the tumor. Study of the specimens of seborrheic keratosis, verruca vulgaris, and normal skin did not reveal a--reaction by the connective tissue. AL. Vol. U Lymphocytes, plasma cells, macrophages, and polymorphonuclear leukocytes were frequently seen in the dermis adjacent to the lesions. There was a metachromatic reaction of the connective tissue adjacent to the tumor in those examples of basal cell carcinoma that had a fibroblastic response associated with them. The metachromatic reaction was first apparent at pH 4.0, and the extent of FIG. 1 (upper left). Basal cell-carcinoma and adjacent connective tissue. There is a proliferation of fibroblasts and a few_small collagen fibers. This type of connective tissue has a strong metachromatic reaction. Hematoxylin and eosin. X 100. FIG. 2 (upper right). Basal cell carcinoma and adjacent connective tissue. The fibroblasts are not as numerous and the collagen fibers are more prominent. This type of connective tissue has less of a metachromatic response than that of Figure 1. Hematoxylin and eosin. X 100. FIG. 3 (lower left). Basal cell carcinoma and adjacent connective tissue. There are very few fibroblasts and the collagen bundles are large. This type of connective tissue does not have a metachromatic response. Hematoxylin and eosin. X 100. FIG. 4 (lower right). Squamous cell carcinoma and adjacent connective tissue. Numerous fibroblasts and few small collagen fibers. This type of connective tissue manifested a strong metachromatic reaction. Hematoxylin and eosin. X 200. Aug. 1960 CONNECTIVE TISSUE ANM) TUMORS OK SKIN such reaction varied with the maturity of the population of fibroblasts and the extent of formation of fibers. In instances with numerous immature cells that had large nuclei and abundant cytoplasm, and little or no associated collagen in the extracellular regions (Fig. 1), the metachromatic reaction was intense. When the fibroblasts were numerous and had a moderate amount of cytoplasm and a fairly large nucleus, but associated with small collagen fibers (Fig. 2), the metachromatic reaction was still observable, but less intense. When the cells were few in number, and of the mature spindle-shaped variety, as well as associated with large bundles of collagen, there was no metachromatic reaction (Fig. 3). The metachromatic reaction observed in the specimens of squamous cell carcinoma was confined to the areas of fibroblastic activity and had the same relation to the maturity of the cells and bundles of collagen as that observed in basal cell carcinoma (Figs. 4 and 5). It did not parallel the degree of invasion by the tumor. There was no metachromatic reaction in the connective tissue associated with the lesions of seborrheic keratosis and verruca vulgaris. The metachromatic response of normal skin was confined to the vicinity of skin appendages.11 As reported by others,2 mast cells were present in all of the specimens, and were metachromatic from pH 2.5 to 5.0. In basal cell carcinomas, the mast cells were more numerous in the immediate vicinity of the tumors. In some instances, 50 of these cells were present per 12.5 by 40 field. The number of mast cells associated with any one tumor was not related to the metachromatic response of the connective tissue or to the presence of a metachromatic reaction. The population of mast cells in squamous cell carcinomas, seborrheic keratosis, and verruca vulgaris was similar to that of normal skin. They averaged 1 to 6 per 12.5 by 40 field. No metachromasy was observed in the connective tissue after treatment of the sections with hyaluronidase. Digestion with testicular hyaluronidase did not affect the metachromatic reaction of the mast cells. 127 DISCUSSION In the connective tissue adjacent to basal cell and squamous cell carcinoma of the skin, there is frequently a proliferation of fibroblasts and an alteration of fibers that resemble the repair process in skin. The maturity of the fibroblasts and the extent of formation of fibers varied within a single specimen, and from specimen to specimen, similar to the stages of maturation of connective tissue in healing wounds of skin. The intensity of the reaction of the connective tissue was not a function of the aggressiveness of the tumor. The more invasive squamous cell carcinoma revealed less change than the less invasive basal cell carcinoma. The nonmalignant lesions of seborrheic keratosis and verruca vulgaris did not have an associated connective tissue proliferation. In conjunction with the reaction of the connective tissue, there was a metachromatic reaction in the ground substance of the connective tissue, and the intensity of the response paralleled the maturity of the connective tissue. This is histologically and histochemically analogous to the metachromatic response observed in various stages of a healing wound.17 The association of the metachromatic reaction with 128 MOORE ET immature fibroblasts and the extent of formation of fibers is consistent with the findings in previous studies. These have revealed the relation of polysaccharides and formation of fibers to the fibroblast in the development and repair of connective tissue and in tissue cultures of fibroblasts.5' 8,i5,i7 j n n o r m a ] skin, a metachromatic reaction can not be elicited, although the mucopolysaccharides are present. In the lesions of seborrheic keratosis and verruca vulgaris, there was no evidence of a metachromatic response. This is similar to normal skin. It is of interest to consider the nature of the material in the ground substance that participates in the metachromatic reaction. Theoretically and experimentally, it has been demonstrated that the metachromatic phenomenon is the result of the orientation of several, consecutive, planar dye molecules in such a manner as to form an array comparable to a stack of coins.14 In general, the substrates for this reaction are molecules that have regularly spaced functional groups for interaction with the dye. It has been shown that at least 4 such groupings are necessary.19 In the instance of connective tissue, the metachromatic reaction of the ground substance is dependent upon the successive carboxyl and sulfate groups in hyaluronic acid and the chondroitin sulfates. The availability of these groups for interaction with the dye determines whether or not a metachromatic reaction will occur. Hyaluronic acid and chondroitin sulfates B and C have been isolated from the skin,9 but chondroitin sulfate A has not been found.7 In the study described in this paper, the metachromatic reaction in untreated tissues was observed at pli 4.0 and above. These findings are consistent with a carboxylated, rather than a sulfated polysaccharide. If it were the latter, metachromasy should have been detectable at considerably lower levels of pH, inasmuch as the sulfate groups are completely dissociated even at pli 2.5. The dissociation of the carboxyl group is suppressed when the pH is less than 3.8. It can be demonstrated that chondroitin sulfate C is metachromatic, even at pH AL. Vol. U 2.0, in solution and also in tissue that is rich in this polysaccharide. The sensitivity of the metachromatic material to testicular hyaluronidase does not differentiate hyaluronic acid and chondroitin sulfate A or C.9 Chondroitin sulfate A, however, has not been isolated from skin, and chondroitin sulfate B, which has been observed, is not sensitive to testicular hyaluronidase.6 Whereas the effect of testicular hyaluronidase does not distinguish between chondroitin sulfate C and hyaluronic acid, the metachromatic reaction suggests that the substrate is probably a carboxylated polysaccharide. This does not preclude the possibility that the substrate is a nonsulfated or incompletely sulfated form of chondroitin sulfate C. It has been demonstrated, by means of the use of S3504, that the sulfation process is predominantly extracellular, and that it occurs after the carbohydrate chain has been synthesized.18 In mature connective tissue of the skin, a metachromatic reaction can not be demonstrated. In this connection, it should be realized that the procedure is dependent upon electrostatic interaction between the dye and the functional groups. It is certainly possible that the carboxyl and sulfate groups, or both, may interact with other substances more strongly than with the dye. Several authors 1 , 3 ' 1 0 have proposed that invasion of connective tissue by neoplasms is the result of a depolymerization of the mucopolysaccharides of the connective tissue. The alteration of the metachromatic reaction does not provide support for this hypothesis. Metachromasy is not a function of molecular weight of a substrate, but only requires 4 functional groups.19 The depolymerization hypothesis would be tenable only if unmasking of the reactive groups occurred at the same time. Inasmuch as it is well known that immature connective tissue contains fibroblasts that produce large amounts of mucopolysaccharide, this view should be favored over the depolymerization hypothesis. The presence of mast cells in significant numbers adjacent to basal cell carcinoma, and the relatively normal population of these cells in squamous cell carcinoma and cer- Aug. 1960 129 CONNECTIVE TISSUE AND TUMORS OF SKIN tain benign lesions of the skin has been noted previously.2 In this study, there was no correlation between the metachromatic properties of the connective tissue and the presence or absence of mast cells. It becomes attractive to consider the proliferative reaction of the connective tissue surrounding neoplasms of the skin as a result of mechanical stress induced by the tumor. The elaboration of mucopolysaccharides and formation of fibers is a natural consequence of the proliferation of the fibroblasts. The differences that were observed would be dependent upon the degree of distortion and the chronicity of the process. On the basis of the observations described in this paper, there are no grounds for inferring that the observed alteration in the connective tissue adjacent to these neoplasms of skin are related either to the aggressiveness of the neoplasm or to the capacity of the host to resist it. SUMMARY The connective tissue of normal skin and that associated with basal cell carcinoma, squamous cell carcinoma, seborrheic keratosis, and verruca vulgaris was studied by means of histochemical methods. The connective tissue adjacent to basal cell and squamous cell carcinomas was frequently altered. There was an increase in the number and the immaturity of fibroblasts, which was more conspicuous in the specimens removed from basal cell carcinomas. The acidic polysaccharides of the ground substance were also more prominent, and this change was associated with the increased numbers of fibroblasts. The similarity of these changes to the developmental cycle and repair process of connective tissue is discussed. SUMMAKIO IN INTERLINGUA Methodos histochimic esseva utilisate in un studio del tissu conjunctive de pelle normal e de illo associate con carcinoma de cellulas basal, carcinoma de cellulas squamose, ceratosis, seborrheic, e verruca vulgar. Le tissu conjunctive in sitos adjacente a carcinomas de cellulas basal e de cellulas squamose esseva frequentemente alterate. Esseva notate un augmento del numero e del grado de immaturitate de fibroblastos. Isto esseva plus conspicue in le specimens obtenite ab carcinomas de cellulas basal. Le polysaccharides acidic del substantia basal esseva etiam plus prominente, e iste alteration esseva associate con le augmentate numero de fibroblastos. Es discutite le similaritate de iste alterationes con le cyclo disveloppamental e le processo de reparo in tissu conjunctive. REFERENCES 1. 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