Vol. 34, No. 2, August, I960, pp. 125-130
Printed i?i U.S.A.
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
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.
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
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.
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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
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
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.
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
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
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
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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
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.
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.
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.
1. B U N T I N G , H . : T h e distribution of acid mucopolysaccharides in mammalian tissues as
revealed by histochemical methods. A n n .
New York Acad. S c , 52: 977-982, 1950.
2. F A N O E R , H . , AND B A R K E R , B . E . : Histochemi-
cal studies of some keratotic and proliferating skin lesions. I . Metachromasia.
A. M . A. Arch. P a t h . , 64: 143-147, 1957.
3. G E R S H , I., AND CATCHTOLE, H . R . : T h e organ-
ization of t h e ground substance and basement membrane and its significance in tissue injury, disease and growth. Am. J .
Anat., 85:457-521, 1949.
4. GLUCKSMANN, A . : T h e histogenesis of benzpyrene-induced epidermal tumors in t h e
mouse. Cancer Res., 5: 3S5-400, 1945.
5. G R O S S F E L D , H . J M E Y E R , K . , AND GODMAN, G.
C : Differentiation of fibroblasts in tissue
culture, as determined by mucopolysaccharide production. Proc. Soc. Exper. Biol.
& Med., 88: 31-35, 1955.
6. H O F F M A N , P . , L I N K E R , A., AND M E Y E R ,
T h e acid mucopolysaccharides of connective tissue. I I . F u r t h e r experiments on
chondroitin sulfate B . Arch. Biochem., 69:
435-440, 1957.
7. L O E W I , G., AND M E Y E R , K . : T h e acid muco-
polysaccharides of embryonic skin. Biochim. et biophys. acta, 27: 453-456, 195S.
S. MALINSKY, J . : Histochemical demonstration
of carbohydrates in intervertebral discs of
human embryos. Histology of intervertebral discs. Acta histochem., 3 : 297-307,
9. M E Y E R , K . , D A V I D S O N , E . , L I N K E R , A., AND
P . : T h e acid
mucopolysaccharides of connective tissue. Biochim. e t
biophys. acta, 2 1 : 506-518, 1956.
10. M I L L E R , G. L., AND C O L D E R , R . H . : Buffers of
p H 2 t o 12 for use in electrophoresis.
Biochem., 29: 420-423, 1950.
11. M O N T A G N A ,
MELARAGNO, H . R . : Histology and cytochemistry of human skin. I . Metachromasia
in t h e mons pubis. J . N a t . Cancer Inst., 12:
591-597, 1951.
12. M O O R E ,
Studies on connective tissue. IV. T h e mast
cell and its relation to t h e ground substance
polysaccharides of t h e human umbilical
cord. L a b . Invest., 7: 418-425, 1958.
13. O R R , J . W . : Changes antecedent t o tumour
formation during t h e t r e a t m e n t of mouse
skin with carcinogenic hydrocarbons. J .
P a t h . & Bact., 46: 495-515, 1938.
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AND H A M E R M A N , D . : M e t a -
17. T A Y L O R , H . E . , AND S A U N D E R S , A. M . : T h e
chromasia. Chemical theory and histochemical use. J . Histochem., 4:159-189, 1956.
association of metachromatic ground substance with fibroblastic activity in granulation tissue. Am. J . P a t h . , 33: 525-537, 1957.
14. SCHUBERT, M . ,
M . D . , AND M o O R E ,
R. D.:
Studies on connective tissue. I I I . Enzymatic
studies on t h e formation and nature of t h e
carbohydrate intermediate of t h e connective tissue polysaccharides in t h e human
umbilical cord. A. M. A. Arch. P a t h . , 65:
115-124, 1958.
16. SIMPSON, VV. L . : Mucolytic enzymes a n d invasion by carcinomas. Ann. N e w York
Acad. Sc.', 52: 1125-1132, 1950.
18. U P T O N , A. C , AND O D E L L , T . T . , J R . : Utiliza-
tion of S 36 -labeled sulfate in scorbutic guinea
pigs. Uptake in healing wounds, megakaryocytes, and blood platelets. A. M. A. Arch.
P a t h . , 62: 194-199, 1956.
19. W A L T O N , K . W., AND R I C K E T T S , C.
vestigation of t h e histochemical basis of
metachromasia. Brit. J . Exper. Path., 35:
227-240, 1954.