Pathogenesis of Desmoplasia. I. Immunofluorescence Identification
and Localization of Some Structural Proteins of Line 1 and Line 10
Guinea Pig Tumors and of Healing Wounds 1,2
Harold F. Dvorak,3,4 David M. Form,5 Eleanor J. Manseau,3 and Barbara D. Smith 5
Desmoplasia is a striking and characteristic feature of
many important animal and human tumors (1, 2).
However, the pathogenesis of tumor desmoplasia is
poorly understood. Prevailing opinion holds that the
collagen and other connective tissue structural proteins
that comprise desmoplasia are products of benign
fibroblasts that come to be associated with certain types
of tumors (2). Some investigators, however, have argued
that tumor cells themselves synthesize the desmoplastic
stroma (3-7). It is also possible that both tumor cells and
benign fibroblasts contribute to tumor connective tissue
synthesis.
Recently, a role for fibrin has been postulated in the
pathogenesis of tumor desmoplasia, on the basis of the
following evidence (8-16): 1) Fibrin is a regular component of human and animal tumor stroma; 2) at least in
animal tumors, where the kinetics of tumor growth can
be monitored closely, fibrin deposition is prominent
from the earliest stages after tumor implant and persists
throughout subsequent tumor growth; 3) tumors bring
about local fibrin deposition by secreting mediators that
a) increase vascular permeability, allowing extravasation
of plasma proteins including fibrinogen, b) effect the
coagulation of extravasated fibrinogen to form fibrin,
and c) modulate fibrin deposition by initiating fibrinolysis through plasminogen activator secretion; and 4)
newly deposited tumor stromal collagen is intimately
associated with fibrin deposits.
Fibronectin also may be reasonably expected to have a
role in tumor desmoplasia, although this specific ques-
tion has not been investigated extensively. Like fibrinogen, fibronectin circulates in the plasma, but a very
closely related molecule is also synthesized locally in a
variety of tissues (17-20). Cell surface fibronectin is
commonly altered in the course of malignant transformation, and fibronectin may, in turn, modulate the
biologic behavior of tumor and connective tissue cells
(17-20).
It is apparent from this analysis that many of the events
associated with tumor growth resemble at least superficially the events that take place in ordinary wound
healing (21-27). Indeed, the granulation tissue of healing
wounds includes fibrin as well as fibronectin and
collagen. In addition, both healing wounds and growing
tumors are characterized by the prominent ingrowth of
new blood vessels. These findings raise the possibility
that tumor desmoplasia and wound healing involve
similar pathogenetic mechanisms. For the further
investigation of this possibility, certain additional pieces
of information are required: knowledge of the types of
collagen and other structural proteins that comprise
tumor stroma and healing wounds; the anatomic relationships of the different types of structural proteins to
tumor cells, blood vessels, and fibroblasts; identification
of the cells responsible for synthesizing each type of
structural protein; and finally, the control mechanisms
that induce tumors and/or benign fibroblasts or other
cells to synthesize the various structural proteins.
In this paper we identify and localize by IF certain of
the structural proteins of 2 well-defined, solid-growing
ABBREVIATIONS USED: Fib=group of fibrinogen and fibrin-related
proteins that react with antibodies raised against fibrinogen; FITC=
fluorescein isothiocyanate; HBSS=Hanks' balanced salt solution;
HEET buffer=heparin (2,000 U/ml), hirudin (2,000 U/ml), €-aminocaproic acid (50 mg/ml), and aprotinin (1,400 mg/ml) in 0.15 M NaCI,
adjusted to pH 7.3; IF=immunofluorescence.
February 13, 1984; accepted July 3, 1984.
in part by Public Health Service grant CA-2847I from the
Division of Extramural Activities, National Cancer Institute; by a grant
from the National Foundation for Cancer Research; and by grant 1522
from The Council for Tobacco Research-USA, Inc.
3 Departments of Pathology, Beth Israel Hospital and Harvard
Medical School, and the Charles A. Dana Research Institute, Beth Israel
Hospital, Boston, Mass. 02215.
4 Address reprint requests to Dr. Dvorak at the Department of
Pathology, Beth Israel Hospital, Boston, Mass. 02215.
S Boston University School of Medicine and the Veterans Administration Outpatient Clinic, 17 Court St., Boston, Mass. 02108.
1195
1 Received
2 Supported
JNCI, VOL. 73, NO.5, NOVEMBER 1984
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ABSTRACT-The structural proteins of the scirrhous line 1 and the
medullary line 10 bile duct carcinomas, both syngeneic in strain 2
Sewall-Wright inbred guinea pigs, were studied. Tumor structural
proteins were compared with those of healing wounds. A provisional stromal matrix of cross-linked fibrin and fibronectin was
initially deposited in both tumors and wounds and was subsequently replaced by granulation tissue containing collagen types I
and III. The amounts of stromal fibrin-fibronectin and collagen
were characteristic of each tumor: Line 1 contained significantly
greater amounts than line 10. These differences were augmented
when line 1 tumor rejection was prevented with cyclosporine,
permitting time for stromal maturation. In tumors and wounds
laminin and collagen type IV were found only in basement
membranes. The findings suggest that 1) tumor desmoplasia is
analogous to wound healing, 2) both processes involve replacement
of an antecedent fibrin-fibronectin provisional matrix, 3) the extent
of fibrin-fibronectin is characteristic of each tumor, and 4) tumor
desmoplasia correlates with the amount of fibrin-fibronectin matrix
deposited.-JNCI 1984; 73:1195-1205.
1196
Dvorak, Form, Manseau and Smith
guinea pig tumors, the line 1 and line 10 hepatocarcinomas of the bile duct, and we compare these proteins
with the structural proteins deposited in healing skin
wounds.
MATERIALS AND METHODS
JNCI, VOL. 73, NO.5, NOVEMBER 1984
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Tumor celis, transplantation, and culture. -Ascites
variants of line 1 and line 10 bile duct carcinomas were
used in these experiments (8, 28, 29). Tumor cells
(1-3 X 107 ) were passaged in the peritoneal cavities of
syngeneic strain 2 Sewall-Wright inbred guinea pigs of
either sex at 7- to lO-day intervals and were recovered
from the peritoneal cavity by injection of 20 ml of HBSS.
Tumor cells were washed three times in HBSS and were
counted, and their viability was assessed with the use of
trypan blue. Viable cells (106 or 3XI06 in 0.1 ml HBSS)
were transplanted to the subcutaneous space of 300- to
350-g strain 2 guinea pigs for growth as solid tumors.
Tumor cell viability always exceeded 95%. Line 1 and line
10 solid tumors were grown in separate animals.
Tumor growth was monitored by daily palpation. At
intervals, animals were anesthetized with ether and were
exsanguinated, and tumors were promptly isolated and
divided into representative portions. In some instances
animals received an iv injection of 0.5 ml of an
anticoagulant-antifibrinolytic mixture (HEET buffer) 5
minutes before sacrifice. For IF, full-thickness tumor
slices were rinsed briefly in HEET buffer and snap-frozen
in O.GT. compound (Miles Laboratories, Inc., Naperville, Ill.) for preparation of frozen sections. Other tumor
slices were fixed in a mixture of paraformaldehydeglutaraldehyde for preparation of l-~m-thick, Eponembedded, light microscopic sections (8). A total of 32
separate tumors from 16 different animals was studied in
these experiments.
Effect of cyclosporine on line 1 tumor growth.-Line 1
tumors differ somewhat from typical scirrhous human
cancers in that their stroma is less mature; i.e., they
possess greater cellularity (mostly fibroblasts) and relatively less collagen. This fact may be attributed to the
relatively brief time available for connective tissue
formation in line I tumors prior to immunologic
rejection (8). To test this possibility, experiments were
undertaken to inhibit the immune response, with the
hope of preventing line 1 tumor rejection and thereby
providing a longer interval for maturation of the tumor's
connective tissue stroma. Tumor-bearing animals were
treated sc with 10 mg cyclosporine dissolved in 4.0 mg
absolute ethanol-81.0 mg Miglyol 812 (Kay-Fries Chemical Inc., Montvale, N.].) 5 days/week. Cyclosporine was
the gift of Dr. E. Wiscott, Sandoz Ltd., Basel, Switzerland.
A total of 6 separate tumors taken from 10 different
animals was studied by IF and in l-~m sections.
Wound healing model.-Standard skin wounds were
made with a disposable 4-mm biopsy punch to the level
of the panniculus carnosus in the shaved and depilated
flanks of 300- to 350-g strain 2 guinea pigs (26). Wounds
were left uncovered, and animals were sacrificed as in
tumor experiments at intervals of I, 3, 5, 7, 11, 15, and
21 days and the wound sites harvested and bisected for IF
and l-~m Epon section study. A total of 21 separate
wounds was studied in 6 different animals.
A ntibodies and IF procedures. -Sheep antibodies (lgG)
to collagen types I, III, and IV and rabbit antibodies to
laminin and fibronectin were the gifts of Dr. George
Martin, National Institutes of Health, Bethesda, Md. The
specificity of these antibodies was tested in our laboratory
on standard 4-~m-thick cryostat sections of frozen normal
guinea pig skin with the use of a two-stage method in
which the second antibody consisted of FITC-conjugated
F(ab'h fragments of goat anti-sheep or sheep anti-rabbit
IgG. A checkerboard technique was employed to select
optimal dilutions of both primary and secondary antibodies. As expected, antibodies to coUagen types I and III
stained normal guinea pig dermal collagen but not other
connective tissue structures. Antibodies to collagen type
IV and to laminin did not stain dermal collagen but did
stain blood vessel basement membranes, epidermal and
hair follicle basement membranes, and the sheaths
enveloping piloerector muscles of the skin and the
perimysium surrounding the muscle fibers of the
panniculus carnosus. Antibodies to fibronectin stained in
a pattern similar to that of collagen type IV, except that
epidermal basement membrane staining was weak and
perimysial staining absent. Substitution of any of the
above antibodies with appropriate dilutions of normal
rabbit or sheep sera evoked no specific staining.
In addition to the above, FITC-conjugated rabbit
antibodies to fibrinogen and to fibronectin were purchased from Cappel Laboratories, Cochranville, Pa., and
we raised an antibody to guinea pig fibrinogen by
immunization of rabbits with highly purified guinea pig
fibrinogen (30). Antibodies to fibrinogen are known to
react with fibrin and with certain fibrinogen and fibrin
breakdown products, with a range of specificities we have
referred to collectively as "anti-Fib" (31). To characterize
anti-Fib-reactive material better in tissue sections, we
subjected frozen tumor sections to extraction in freshly
prepared 3 M urea or in 2% acetic acid or in 2%
monochloroacetic acid for 30-60 minutes at room
temperature or at 37°C prior to staining. Any of these
treatments is known to solubilize naturally occurring
fibrinogen and fibrin species except for fibrin crosslinked by activated factor XIII (31,32). Thus the anti-Fib
staining observed in urea- or acid-extracted sections
might be expected to represent only cross-linked fibrin;
we established this fact by staining frozen sections of
cross-linked and non-cross-linked fibrin gels with or
without prior extraction (Dvorak HF: Unpublished
data).
Antisera to fibrinogen often include antibodies to
fibronectin. Therefore, anti-Fib antibodies were routinely
absorbed with Sepharose beads conjugated with purified
human fibronectin to ensure specificity. These absorbed
antibodies strongly stained fibrin clots but not WI-38 cell
mono layers that contained abundant fibronectin. Conversely, antisera to fibronectin may contain antibodies
reactive with fibrinogen. For removal of these antibodies,
anti-fibronectin antibodies were absorbed with Sepha-
Desmoplasia and Wound Healing I
rose-conjugated human fibrinogen that had been specifically freed of fibronectin contamination. Other details of
the IF procedure have been published (12, 26).
RESULTS
Growth Pattern of Line 1 Tumors
T ABLE I.-IF evaluation of structural proteins in line 1 and
line 10 tumors early (4 days) and late (?11 days) after
transplant into the subcutaneous space of syngeneic
strain 2 guinea pigs a
Interval after transplant
Early
Structural proteins
Line 1
Late
Line 10
Line 1 b
Line 10
1-2+
2-3+
4+
4+
2+
1-2+
Stroma
c
Fibrin
Fibronectin
Collagen types I and III
4+
2+
±
1+
Trace
1+
Tumor cell basement membranes
Laminin
Fibronectin
Collagen type IV
1+
1+
1+
Trace
Trace
Trace
2+
2+
2+
Blood vessel basement membranes
Laminin
Collagen type IV
4+
4+
4+
4+
±
±
±
d
4+
4+
4+
4+
a Staining was scored on a semiquantitative scale of 0-4+. ±
and 1+, scattered focal deposits; 2+ and 3+, moderate to abundant deposits; 4+, extensive confluent deposits.
bldentical line 1 tumor results were obtained in cyclosporineimmunosuppressed guinea pigs studied as late as 36 days after
transplant.
C Cross-linked fibrin as determined by resistance to extraction
with urea or acid.
dFibronectin associated with blood vessel basement membranes
could not be evaluated because of extensive extravascular fibronectin deposits.
JNCI. VOL. 73. NO.5. NOVEMBER 1981
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Line I tumors appeared within 1-2 days of transplant
as gelatinous papules, composed of an abundant fibrin
gel enveloping much smaller, irregularly spaced clumps
of tumor cells. Beginning at 2-3 days vessel sprouts and
accompanying fibroblasts began to penetrate the fibrin
gel from without. Thereafter, collagen was laid down
and tumor cell clumps became scattered in a cellular
connective tissue matrix. After day 8 lymphocytes and
other inflammatory cells appeared in increasing numbers,
and tumors were rejected by days 13-15. Tumors were
harvested for IF studies at early (4 days) and late (11 days
or later in immunosuppressed animals) intervals, corresponding to times when fibrin-fibronectin matrix or
desmoplasia was expected to predominate.
Line 1 tumors, 4 days after transplant.-IF results are
summarized in table 1. At 4 days line 1 tumor cell clumps
were enveloped in an extensive fibrin gel (fig. IA). The
fibrin staining pattern was indistinguishable when
sections were extracted with 3 M urea or with 2% acetic
acid or 2% monochloroacetic acid prior to antibody
staining, indicating that the fibrillar material observed
was largely cross-linked fibrin. Since these extractions
were effective and had no deleterious effect on IF
histology, they should be applicable as a general method
for distinguishing cross-linked fibrin from other fibrinogen-fibrin species in tissue sections.
Fibrin deposition in tumors, as observed by IF, could
be affected by tissue handling. If anticoagulant-antifibrinolytic buffer was not injected iv and if excised
tissues were not rinsed in HEET buffer prior to freezing
for IF, overall fibrin content was sometimes irregularly
reduced, particularly if removal of tumors from guinea
pig carcasses was delayed. Thus, if the above precautions
are not taken, tumor fibrin deposition may be underestimated.
Fibronectin staining (fig. IB) was observed in the same
distribution as that of fibrin. Occasional tumor cell
clumps were outlined with faint, granular staining when
they were reacted to antibodies specific for laminin
(fig. IC). No other staining was observed except at the
tumor periphery, where fibroblasts and new vessels were
beginning to invade the fibrin gel. Here there was strong
staining for antigens found in normal blood vessel
basement membranes [i.e., laminin (figs. IC, ID),
fibronectin, and collagen type IV] as well as staining of
small amounts of newly formed stroma with collagen
types I and III (fig. IE).
Eleven-day line 1 tumors and 15- to 36-day line 1
tumors in cyclosporine-immunosuppressed animals.Study of line I tumors beyond 11 days was complicated
by immunologic rejection. To circumvent this problem,
we immunosuppressed line I tumor-bearing guinea pigs
with cyclosporine. In such animals line 1 tumors grew
initially as in untreated animals but, in contrast to their
behavior in normal guinea pigs, continued to grow
progressively at least through day 36 and metastasized to
regional lymph nodes. Histologic examination revealed
that line I tumors in cyclosporine-treated animals
differed from II-day line I tumors in untreated animals
in two respects: sparse lymphocytes (consistent with
immunologic suppression) and greater maturity of the
connective tissue stroma (relatively fewer fibroblasts,
more collagen). Thus the stroma of line 1 tumors
growing in cyclosporine-treated guinea pigs 15-36 days
after transplant closely resembled the stroma of typical
scirrhous human cancers (fig. 2A).
The IF results were similar for II-day line I tumors
in untreated animals and for 15- to 36-day line I tumors
in cyclosporine-treated animals (table I). Antibodies
directed against fibrinogen intensely stained the desmoplastic stroma, but fibrin deposits were less extensive
than at 4 days (fig. 2B). Fibronectin deposits (figs. 2C, 2D)
were intense and were localized in two distinct distributions: I) in the tumor stroma along with fibrin and
collagen and 2) in basement membrane-like structures
occurring around and between some tumor clumps.
Fibronectin in tumor blood vessels could not be defined
because the entire tumor stroma was extensively stained
with anti-fibronectin antibodies.
Antibodies directed against laminin (figs. 2E, 2F) and
collagen type IV also stained in a patchy and irregular
1197
1198
Dvorak, Form, Manseau and Smith
manner basement membrane-like structures between
clumps of tumor cells. Not all tumor cells or tumor cell
clumps exhibited such staining, and the intensity of
staining varied from one area of the tumor to another.
These same antibodies also consistently and intensely
stained the new blood vessels that infiltrated the connective tissue of the tumor stroma, serving as excellent
markers for these vessels. Antibodies directed against
collagen types I and III (figs. 2G, 2H) stained the
connective tissue of the tumor stroma but did not stain
either blood vessels or tumor basement membranes.
Growth Pattern of Line 10 Tumors
Pattern of Healing Skin Wounds
The punch wound defe€.t quickly filled with clotted
blood, which became organized over a period of days by
JNCI, VOL. 73, NO.5, NOVEMBER 1984
Healing wounds
Structural proteins
1-3
5-7
11-21
days
days
days
1-2+
3+
4+
±-o
3+-±
4+
Normal
skin
Stroma
Fibrin
Fibronectin
Collagen types I and III
4+
4+
0
0
Trace
4+
Epidermal basement membranes
Laminin
Fibronectin
Collagen type IV
Fibrin
±
3+
±
3+
4+
1-2+
4+
1-2+
4+
4+
Trace
Trace
4+
4+
1+-0
0
Blood vessel basement membranes
Laminin
F ibronectin b
Collagen type IV
4+
4+
4+
?
?
?
4+
4+
4+
4+
4+
4+
·Scoring as in table 1; arrow indicates change over the
indicated time interval.
h Fibronectin associated with blood vessels of healing wounds
could not be evaluated because of extensive extravascular fibronectin deposits.
ingrowth of granulation tissue-fibroblasts and new
blood vessels-from the wound base (22, 26, 27). In
parallel, tongues of epidermis grew centripetally from the
wound periphery, dissecting between desiccated clot
(scab) above and underlying granulation tissue; in this
fashion the wound defect was bridged by 7-9 days.
Thereafter, the granulation tissue matured, as fibroblasts
and blood vessels decreased and collagen matrix increased. IF results are summarized in table 2.
One- and 3-day wounds.-Intense fibrin (fig. 3A) and
fibronectin (fig. 3F) staining characterized the blood clotfilled punch biopsy site and extended patchily for as
much as several millimeters away from the wound edge
into otherwise normal dermis (figs. 3B, 3G). Normal
epidermal basement membrane stained strongly with
antibodies to laminin (fig. 4A) and collagen type IV,
stained weakly to anti-fibronectin antibodies (fig. 31), and
not at all with anti-fibrin antibodies. In contrast, the
basement membrane zone of epidermis migrating
centripetally to cover the wound gap was characterized by
strong fibrin and fibronectin staining [fig. 3H; (26)] and
by weak staining with antibodies to laminin and
collagen type IV.
Five- and 7-day wounds. -Fibrin staining persisted in
the granulation tissue that filled the punch defe~t (fig~.
3C, 3D) and in the basement membrane of the epIdermIS
that had spread inward from the edges to cover the
wound defect by 7 days (fig. 3C). Basement membrane
staining for fibronectin was reduced, but antibodies to
fibronectin intensely stained the granulation tissue
stroma throughout the thickness of the healing wound
(fig, 3J), in striking contrast to the minimal staining of
normal dermis (fig. 31).
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The highly malignant line 10 tumors maintained a
relatively constant histologic pattern at all. stag~s of
growth (8). A fibrin investment, though mvanably
present, was always small compared to that of line 1
tumors and never accounted for more than 5-10% of the
tumor mass. Similarly, line 10 tumors induced little
desmoplasia. Line 10 tumors provoked a modest
lymphocyte and basophil response after day 8 but
nonetheless grew progressively to kill the host.
Line 10 tumors, 4 days after transplant.-Intense fibrin
and fibronectin staining was observed in the scanty line
10 tumor stroma (fig. IF) but in substantially smaller
amounts than in line 1 tumors at any stage of growth
(table 1). As in the case of line 1 tumors, fibrin in line 10
tumors was unaffected by urea or acid extraction,
suggesting that it also consists largely of cross-linked
fibrin. Antibodies to fibronectin, laminin, and collagen
type IV stained individual tumor cell surfaces foca~l~ or
outlined small clumps of tumor cells; however, stammg
was faint, patchy, and generally less extensive than in
line 1 tumors. As in line 1 tumors, areas of beginning
angiogenesis were characterized by prominent laminin
and collagen type IV staining of blood vessel basement
membranes. Only minimal stromal collagen was detected.
Eleven-day line 10 tumors. -As with the staining in
line 1 tumors, patchy, intense staining of fibrin (fig. 21)
and fibronectin (fig. 2J) persisted about tumor cell
clumps and in the granulation tissue at the tumor
periphery (table 1). Antibodies to f~bronectin .als?
revealed speckles of staining that outhned some mdIvidual tumor cells and cell clumps (fig. 2J); whereas only
a minority of tumor cells was so outlined, such staining
was considerably more extensive than at day 4.
Staining with antibodies to laminin (fig. 2K) and to
collagen type IV also revealed speckling around a
minority of tumor cells as well as intense blood vessel
staining. Again, line 10 tumor basement membrane
staining was less extensive than it was for line 1 tumor
cells and involved a much smaller proportion of tumor
cells and cell nests. Antibodies to collagen types I and III
(fig. 2L) stained the scanty stroma between tumor cell
clumps, but they did not stain tumor cell surfaces.
2.-IF evaluation of structural proteins in healing
skin wounds and in normal skin of strain 2 guinea pigs·
TABLE
Desmoplasia and Wound Healing I
DISCUSSION
The data presented here describe several of the
structural proteins of growing solid line I and line 10
tumors and of healing skin wounds and define their
distribution in relation to epithelial elements and other
structures. In both tumors and wounds a provisional
stromal matrix rich in cross-linked fibrin and fibronectin
was deposited initially, which was subsequently invaded
by fibroblasts and new blood vessels with deposition of
collagen types I and III. The amount of fibrin-fibronectin
matrix and the amount of subsequent desmoplastic
collagen were characteristic of each tumor and remained
constant with repeated tumor passages. In every experiment both the provisional matrix of fibrin and fibronectin that enveloped early line I tumors and the
desmoplastic stroma of collagen types I and III that
developed subsequently greatly exceeded those about line
10 tumors. Taken together, our findings are consistent
with the following statements: I) Tumor desmoplasia is
analogous to wound healing; 2) both processes involve
the gradual replacement of a fibrin-fibronectin provisional matrix with granulation tissue; 3) the extent of
tumor desmoplasia correlates with the amount of fibrinfibronectin matrix deposited earlier; and 4) the amount of
fibrin-fibronectin matrix, like the amount of desmoplasia, is characteristic for each tumor. Recent data
suggest that the consistently different patterns of fibrin
deposition in line I and line 10 tumors are attributable to
differences in tumor-associated fibrinolysis. Thus line 1
and line 10 tumors deposit fibrin at equivalent rates, but
line 10 tumors turn over fibrin more rapidly, apparently
by secreting more plasminogen activator than line 1
tumors (33).
Despite the obvious similarities, certain differences
remain with regard to fibrin and fibronectin deposition
and dissolution in tumors and wounds (d. tables 1 and
2). Substantial fibrin deposits were found about both
tumors (about line 1 always> line 10) from the earliest
intervals, and these persisted for as long as tumors were
followed. 6 By contrast, fibrin deposits in punch biopsy
wounds were prominent only at early intervals and
declined progressively thereafter so that by day II only
small amounts remained (table 2). However, fibronectin
deposition followed different kinetics. Moderate fibronectin was present in the relatively acellular stroma of
both tumors (again line 1 > line 10) at 4 days after
transplant and increased substantially at later intervals
when fibroblasts and new blood vessels contributed
prominently to the stroma and very likely also to
fibronectin synthesis (24-27). In healing wounds, however, stromal fibronectin and fibrin were maximal at
early intervals and declined thereafter, fibronectin more
slowly than fibrin, so that little of either remained at 21
days. Thus healing in wounds involved not only a laying
down but also a subsequent programmed dissolution of
the fibrin-fibronectin provisional matrix, the entire
process being complete within 2-3 weeks. By contrast, the
fibrin-fibronectin component of line 1 and line 10
transplanted tumors persisted much longer, more than 1
month and perhaps indefinitely. In agreement with these
observations, fibrin and fibronectin deposits have been
found in random sections of many human tumors,
including breast cancer and Hodgkin's disease (l0, 12,
14). We conclude that the fibrin-fibronectin provisional
matrix persists for a longer time in tumor desmoplasia
than in wound healing, perhaps identifying an important
distinction between these processes. It remains uncertain
to what extent the persistence of fibrin-fibronectin in
tumors results from incomplete dissolution of provisional
matrix or, alternatively, from continuing laying down of
new provisional matrix. We have found, however, that
the blood vessels of both line I (in cyclosporine-treated
guinea pigs) and line 10 tumors remain hyperpermeable
for at least I month after transplantation, suggesting that
continuing extravasation of plasma fibrinogen and
fibronectin contributes new provisional matrix for at
6 All fibrin deposited in tumors or wounds is ultimately derived from
circulating plasma fibrinogen. Tumors effect fibrin deposition by
secreting mediators that render the adjacent microvasculature hyper·
permeable (13) and that coagulate extravasated fibrinogen (14, 16). In
wounds fibrin is derived from fibrinogen that escapes from cut blood
vessels and possibly from the leaky new vessels of granulation tissue.
Like fibrin, fibronectin (17-20) may arise in tissue from extravasation of
plasma fibronectin; however, fibronectin may also be deposited as the
result of local synthesis by fibroblasts and endothelial cells, for
example.
JNCI, VOL 73, NO.5. NOVEMBER 19H1
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Antibodies to laminin clearly delineated the epidermal
basement membrane and extensive new blood vessels
present in the granulation tissue (figs. 4B, 4C); these
vessels, however, appeared to have a thinner basement
membrane than comparably sized vessels in the adjacent
normal dermis (fig. 4A), but staining intensity was
equivalent. Similar staining was observed with antibodies
to collagen type IV (fig. 4E). Antibodies to collagen type I
(fig. 4F) and collagen type III intensely stained the
granulation tissue of the wound as well as the surrounding normal dermis.
Eleven-, 15-, and 21-day wounds.-Abnormal fibrin,
but not fibronectin, staining persisted in the epidermal
basement membrane of the healing wound through day
15 (fig. 3E). The patchy fibrin staining of the underlying
granulation tissue and adjacent normal dermis declined
progressively, so that by day 11 it was confined to a few
flecks of fibrin staining in the superficial dermis of the
healed wound (fig. 3E). Abundant fibronectin deposits
remained in the granulation tissue (figs. 3K, 3L) and
declined gradually as these deposits were reworked into
normal-appearing dermis. Nonetheless, trace residual
fibronectin staining remained in the wound dermis even
at day 21. The basement membranes of wound blood
vessels continued to stain being thinner structures than
vessels of the surrounding normal dermis, as delineated
by antibodies to laminin (fig. 4D) and to collagen type
IV. However, vessel frequency was reduced from the
greatl y increased levels noted at earlier intervals, coming
to approach the frequency of normal dermis.
1199
1200
Dvorak, Form, Manseau and Smith
46).
It is of interest that the intensity and amount of
epithelial basement membrane staining with laminin
and collagen type IV correlated inversely with the degree
of malignancy. Healing epidermis exhibited a continuous
and prominent basement membrane at all times. In
contrast, line 1 tumors exhibited basement membrane
structural protein deposition about a minority of tumor
cell clumps, whereas the highly malignant line 10 tumors
exhibited only rudimentary tumor cell surface-associated
deposits of laminin and collagen type IV. We cannot say
whether these differences represent differences in rates of
basement membrane component synthesis or degradation
(or both), but these differences are consistent with the
results of Liotta et al. (47) and with the view that loss of
basement membrane is a measure of malignancy and
invasiveness (48).
The composition of wound epidermal basement membrane changed with healing (26; table 2). Thus fibrin and
fibronectin were strongly represented at early intervals.
However, laminin and collagen type IV, prominent
components of normal epidermal basement membrane,
were reduced in amount or were absent early; they
reappeared fully only after healing was advanced, fibrin
had disappeared, fibronectin was reduced, and epidermal
cell migration was complete. No comparable evaluation
was possible in tumors where basement membrane
deposition was slower to develop and remained incomplete.
Taken together, our findings support the hypothesis
that tumor desmoplasia and wound healing share similar
pathogenetic mechanisms. In the accompanying paper
(28) we test this hypothesis further, examining the
capacity of certain individual cell types that comprise
tumors and wounds to synthesize stromal proteins.
JNCI. VOL. 73, NO.5, NOVEMBER 1984
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FIGURE I.-IF photomicrographs of line I (lA-IE) and line 10 (IF) harvested at 4 days after transplant to the subcutaneous space of syngeneic strain 2
guinea pigs. IA) Abundant fibrillar pattern of fibrin staining in the stroma enveloping line I tumor nests (T). X 97. IB) Similar field stained with
anti-fibronectin. X 97. IC and ID) Antibodies to laminin focally stain the surfaces of individual line I tumor cells in a granular pattern (arrows in
I C) and strongly stain the basement membranes of newly formed tumor blood vessels (I C, I D). I C: X 243; I D: X 97. IE) Staining of newly formed
coHagen type III in granulation tissue developing at the junction of a line I tumor (T) with subcutaneous muscle (M). X 97. I F) Fibrin staining of
stroma around line 10 tumor (T) nests. X 97
Desmoplasia and Wound Healing I
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FIGURE 2.-Photomicrographs of line 1 (2A-2H) and line 10 (2I-2L) harvested at late intervals (2) 11 days) after transplant to the subcutaneous space.
Unless otherwise indicated. line I-bearing animals were immunosuppressed with cyclosporine. 2A) Scirrhous pattern of line 1 tumor growth at 36
days. Tumor cells appear in small clumps amid abundant. dense collagenous stroma and without significant inflammatory cell infiltrate. I-JoLm
thick; Giemsa; Epon section; X 205. The remaining figures are IF photomicrographs. 2B) Persistent fibrin staining in line 1 tumor stroma at 15
days. Tumor cells (T) do not stain. X 155. 2C) Intense fibronectin staining of tumor stroma at 15 days. X 155. 2D) Fibronectin staining of stroma as
well as linear staining between tumor (T) cell nests in a basement membrane-like pattern at 15 days. X 243. 2E) Laminin staining of basement
membranes enveloping tumor cell clumps and interdigitating between individual tumor cells at 22 days. X 243. 2F) Linear pattern of laminin
staining of line 1 tumor basement membrane. as well as granular staining between individual tumor cells at 11 days in an untreated animal. X 389.
2G) Collagen type III staining of line I tumor stroma at 15 days. X 243. 2H) Collagen type I staining of line 1 tumor stroma. X 243. IF staining of
II-day line 10 tumors: 21) Persistent fibrin staining in line 10 tumor stroma. X 155. 2J) Fibronectin staining of II-day tumor stroma (extreme right)
as well as granular or linear staining between tumor (T) cell clumps. X 155. 2K) Intense laminin staining of blood vessels as well as granular and
finely linear deposits between tumor cells. X 243. 2L) Collagen type III staining of tumor stroma. Tumor (T) cells do not stain. X 155.
JNCI, VOL. 73. NO.5, NOVEMBER 1984
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Desmoplasia and Wound Healing I 1205
FIGURE 4.-IF photomicrographs of healing skin punch biopsy wounds, continued. 4A) Laminin distribution in normal skin, illustrating staining
of epidermal (E) basement membrane and dermal blood vessels below. X 97. 4B, 4C) Laminin staining of 7-day wound, illustrating normal
staining pattern of epidermal basement membrane and extensive new blood vessel formation. Vessels are considerably more numerous than in
normal dermis, and individual vessels display a thinner laminin-staining layer than normal skin blood vessels (ef. with fig. 4A). X 97. 4D)
Laminin staining of an II-day wound. Vessel frequency is reduced to near-normal levels. E, epidermis. X 97. 4E) Collagen type IV staining of
epidermal (E) basement membrane and blood vessels of a 7-day wound mimics that of laminin staining. X 97. 4F) Collagen type I staining of
granulation tissue (right) adjacent to normal dermis (left) at edge of a 7-day wound. Note differing patterns of organization. X 97
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FIGURE 3.-IF photomicrographs of healing skin punch biopsy wounds. 3A) Fibrin in base of I-day wound at junction with underlying
(nonstaining) panniculus carnosus (PC). X 97. 3B) Extensive fibrin deposition extends 1-2 mm from wound edge into normal dermis at day l. X
97. 3C) Intense fibrin staining of basement membrane of spreading epidermis (E) that has covered the wound defect at 7 days. Fibrin deposits are
also scattered in the underlying granulation tissue. X 97. 3D) Higher magnification of residual fibrin deposits in granulation tissue of 7-day
wound. X155. 3E) Eleven-day wound illustrating residual fibrin deposits in basement membrane of new epidermis (E) and fainter deposits
scattered in the underlying granulation tissue. X 97. 3F) Fibronectin staining of wound bed at day I in a field analogous to that of fig. 3A. PC.
panniculus carnosus. X 97. 3G) Extensive fibronectin staining in otherwise normal dermis 1-2 mm from edge of a I-day wound in a pattern
similar to that of fibrin illustrated in fig. 3B. X 97. 3H) Bright fibronectin staining of basement membrane of epidermis (E) at edge of a 3-day
wound. Basement membrane of new epidermis covering the wound defect also stained brightly for fibronectin. Farther removed from the wound
normal epidermal basement membrane does not stain for fibronectin. X 155. 31) Fibronectin staining pattern of normal guinea pig skin. Dermal
blood vessels stain strongly, but staining of epidermal (E) basement membrane is weak or negative. X 97. 3J) Bright fibronectin staining of
granulation tissue of 7-day wound. The wound defect has been bridged with hyperplastic epidermis (E) whose basement membrane stains for
fibronectin. X 97. 3K) Fibronectin staining of II-day healing wound. Epidermal (E) basement membrane no longer stains, but extensive
fibronectin staining persists in underlying granulation tissue. X 97. 3L) II-day wound, illustrated here at higher magnification, is dearly
differentiated from normal dermis (fig. 31), which is fibronectin-negative. X 243