Differences in the growth-promoting effect of normal and peritumoral dermis on epidermis in vitro

DEVELOPMENTAL BIOLOGY 17, 679-691

Differences and

(1368)

in the Growth-Promoting Peritumoral PAULINA

Dermis REDLER'

on Epidermis

AND F,ucmra

ftrstitrlto de Owdogia Avcnida

Effect of Normal in Vitro

S. IXJSTIC;':

“A, H. Roffo,” Dcpartamento San Martin 5481, Buenos Airrs.

de Inr;c.vtifclcicitr, Arpuiina

INTRODUCTIOS

Embryonic epidermis is considered a favorable tissue to determine cxtraepithelial factors responsible for epithelial proliferation. Until now it has been known that for embryonic epithelium to grow and become differentiated in citru it has to be combined with other embryonic tissues of mesenchymal origin. In embryonic skin this finding was reported, among others, by McLaughlin ( 196la,b ), Jt’essells ( 1962, 1963), Rawles (1963), Sengel (1964), and Dodson ( 1967a,b ). Similar epidermal responses are feasible when epithelium is cultured in association with dermis belonging to both homologous and hetcrologous species (Mordoh and Lustig, 1966). It has been established in zjitx by Aigyris and Argyris (1962) that Ehrlich’s ascites tumor transplanted subcutaneously has a stimulating action on normal adjacent epidermis; these authors, however, do not state whether such action is due to the tumoral cells or to the stromal component. Others (c.g,, Rillingham et al., 1951; hlarchant and On. 1953; Orr, 1961; Van Scott and Reinertson, 1961; Clucksmann, 1963) have also studied in vioo the dermal role in the promotion of both normal and tumoral epidermal development. The present work demonstrates that certain types of adult tissue. such as peritumoral dermis, have a stimulating effect over epithelial development in vitro. This effect is much less cvideirt in normal oi inflammatorv adult dermis. Some characteristics of the dermal material ’ Fellow of the Consejo National de Investigacionrs Cientificas Argentina. ’ Career investigator of the Consejo Sacional de Investigwioncs T&nicas, Argentina. 679

! TPcnicas, Cientificas

J

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REDLER AN-D LUSTIC

responsible for epidermal cell proliferation entiation were also studied. MATERIALS

and morphological

differ-

AND METHODS

(a) Epidermis. This was obtained from the skin of the tarsometatarsal region of 11-12-day chick embryos. The skin was cut in fragments some 2-2.5 mm in diameter, and separation of epidermis from the underlying mesenchyme was undertaken after suspension in 0.25% disodium ethylenediaminetetraacetate in Ca’+- and Mg”- free Tyrod6s solution at room temperature (ZO-22°C) during 1 hour. The epidermis thus separated was then washed in equal parts of Tyrode’s and human serum. As demonstrated by Dodson (1967a) practically in every case the basement membrane remained on the dermis. (b) Dermis. Peritumoral,” inflammatory, cicatricial, and normal dermis from adult human skin was separated from the corresponding epidermis according to the method described in (a), but the separation usually took up to 3 hours. Care was taken to utilize strictly the densely hyalinized peritumoral stroma from early basal-cell epitheliomas and epidermoid carcinomas. In the case of nevi and papillomas the study comprised up to 3 mm of the corresponding underlying dermis. The age of the donors ranged from 27 to 86 years. Inflammatory dermis corresponded to chronic nonspecific inflammatory processes from donors 29 and 69 years old. Cicatricial dermis corresponded to an extensive traumatic scar which dated from fifteen years back, in a 23year-old donor. Normal dermis was obtained from donors with malignant and nonmalignant skin tumors, and with benign tumors of the breast and uterus. The ages of these donors ranged from 29 to 73 years. The dermal tissue was cut in pieces 2-2.5 mm in diameter and 1 mm thick and was used immediately or after having been kept in Tyrode’s plus human serum (2:l) at 4°C during a maximum of 48 hours. Some of the peritumoral dermal fragments were left untreated while others were submitted to one of the following procedures before use: 1. Freezing: This was carried out in an attempt to kill dermal cells ( Wessells, 1964; Cohen, 1965). In some cases peritumoral dermis was frozen solid during 1 hour at - 8°C in Tyrode’s, then thawed at room temperature; this freeze-thaw cycle was repeated twice. Other &gments of dermis were suspended in tubes containing small volumes of ‘Peritumoral of the tumor.

refers to a distance

of up to approximately

2 mm from

the edge

GROWTH-PROMOTING

EFFECT

OF

PERITTJMORAL

DERMIS

681

Tyrode’s and immersed in an ethyl alcohol-water-dry ice mixture at - 25°C until freezing took place. This was followed by thawing at 20-22”C, and the procedure was repeated three times. 2. To test the stability to heat of the biological activity, samples of peritumofal dermis suspended in tubes containing Tyrode’s were immersed in a water bath at 60°C during 1 hour, and then washed in Tyrode’s. 3. Fragments of peritumoral connective tissue were exposed to collagenase (CLS, Worthington Biochemical Corporation), 1 mg/ml in Tyrode’s at pH 7 and 37°C during 1 ji hmlrs, and then washed XVera1 times in diluent alone. 4. Fragments of peritumoral stroma were incubated with 10 mg/ml Difco 1:250 trypsin or 0.5 mg/ml 2 X crystallized trypsin (Nutritional Hiochelnicals Corporation) in Tyrode’s at pH i.5 and 37°C during I!6 hours. Crystallized trypsin was used to rule out a possible effect due to proteolytic contaminants present in the crude preparation. Th(, tissue thus treated was then washed several times in diluent with human serum ( 1: 1) to remove and inactivate trypsin. 5. Peritumoral dermal material was incubated in Tyrode’s during lli hours at 37°C in order to control procedures 3 and 4. ( c ) Culture methods. Agar platforms were prepared in the following form: melted agar in distilled water was mixed with equal parts of medium 199 in 2 x concentration; to this mixtllre human serum was added. The resulting mixture was poured into watch glasses and the agar was allowed to solidify. The final composition of the culture medium was: 0.5% agar in medium 199 with 10% human serum. The watch glasses thus prepared were placed in dishes containing wet cotton in order to maintain a high degree of humiditv. The small derma1 fragments were covered with the embryonic epidermal fragments and placed on the agar surface, dermal side down. The tissues were cultured during 5-6 days at 37’C and then fixed in Bouin’s fluid. Serial paraffin sections were cut (7-9 /I. thick), mounted, and stained with hematoxylin and eosin. RESULTS

Association

of Normal

Adult

Dermis with Embryonic

Epidermis

Out of 58 cultured explants only 19 (33%) developed stratified layers. of healthy epithelial cells (Table 1) with a basal layer regularly

682

REDLER

AND

LUSTIG

arranged in which mitoses could be found. The epithelium of these explants was well keratinized. Some of the explants, however, contained areas in which the epidermal cells were not able to grow. Spiky projections of epithelial basal cells penetrated the dermal tissue in some of the cultures. Degeneration of the epidermis took place in the remaining explants in the form of necrotic cells and cellular debris, accompanied by lack of adhesion with the underlying dermis in which hardly any cells were evident (Fig. 1). Association

of Inflammatory

Dermis with Embryonic

Epidermis

Out of 12 explants only 2 (17%) revealed good epithelial (Table 1). Association of Peritumoral

Dermis with Embryonic

growth

Epidermis

All the peritumoral dermal tissues studied induced the same type of epidermal proliferation (Table 1). Out of 94 explants of peritumoral connective tissue without treatment, 63 (67%) showed growth of the associated epidermis: the basal cuboidal cells took on a columnar orientation, and their vitality was judged by the appearance of mitotic activity and the presence of harmoniously stratified cells undergoing gradual keratinization. In many cases as many as 8 layers were present. There was an intimate epithelial adhesion to the underlying dermis (Figs. 2 and 3). Some of the cultures of peritumoral stroma from malignant tumors showed about one mitosis per 100 epithelial basal cells; meanwhile none of the cultures of dermis from nonmalignant tumors reached this high mitotic index. In some of the cultures intrusive spurs of epithelial cells penetrated the dermal tissue. The epithelial growth was not uniformly arranged in all areas; indeed in some explants good epithelial growth was observed in the vicinity of necrotic epithelial cells. Histologically, tissue adjacent to the tumor resembled normal adult dermis rather than embryonic dermis and, excepting for the nevi, very few cells were present in the peritumoral stroma. When peritumoral dermis was submitted to freezing before association with the epidermis, the percentage of explants with viable epitheha1 cells was similar to that of untreated dermis; basal mitoses could be observed in the germinative layer. From 39 explants of frozen peritumoral dermis at -8°C and from 28 frozen at -2S’C, 30 (77%) and

1 1

:I 0 3 (Iti%,,)

4 1

664

REDLER

AND

TABLE

1 (Continued)

Ny&bms of

Source of dermal material

g. Incubated in Tyrode’s solution Nevi Epidermoid carcinomas

LUSTIG

Number of cultured explants

1 1

Cicatricial

dermis inflammations

dermis

Normal dermis Subjects with malignant or nonmalignant skin tumors Subjects with nonmalignant tumors of breast and uterus

-

10

6 (60%)

2

12

2 (17%)

1

20

12 (60%)

7

31

11

2

27

the harmonious

8 -

58 D In 16 of these explants longer maintained.

3 3

4 6 -

Inflammatory Nonspecific

Number of explants with mitotically active epithelial basal cells

stratification

19 (33%) of epithelial

layers was no

15 (54%) of the respective explants showed a good epidermal growth ( Figs. 4-7). When peritumoral connective tissue was submitted to heat treatment, a reduction was observed in its ability to stimulate epithelial proliferation; only 33% of 18 cultured explants developed stratified epithelial layers with healthy basal cells. In the majority pycnotic and vacuolated cells were observed, as well as loss of adhesion to the underlying dermis (Fig. 8). Trypsin treatment produced a remarkable decrease in the biological effect of peritumoral dermis. Data obtained with crude and crystalline FIG. 1. FIG. 2.

Epidermis cultured on normal adult dermis. X 375. Epidermis cultured in recombination with dermis adjacent to a basalcell epithelioma. X 375. FIG. 3. Epidermis cultured on dermis corresponding to a nevus. X 375. FIGS. 4 and 5. Epidermis cultured on frozen (--8’(Z)-thawed peritumoral dermis. X 375. FIG. 6. Epidermis cultured on frozen ( -25°C)-thawed peritumoral dermis. x 375.

GROWTH-PROMOTING

EFFECT

OF PERITUMORAL

DERMIS

685

686

REDLER

AND

LUSTIG

GROWTH-PROMOTISG

EFFECT

OF

PERITUMOHAL

DERMIS

687

trypsin were similar: from explants whose dermal components were incubated with crude trypsin (I2 explants) and crystalline trypsin (19 explants), 3 (25%) and 3 (16%), respectively, revealed good epiderma1 growth. In the remaining explants pycnosis was observed in the necrotic epithelial cells (Figs. 9 and 10). Exposure of peritumoral stroma to collagenase did not seem to interfere with epithelial growth, which was evident in 77% of 31 explants. However, columnar orientation of the basal cells, and the harmonious stratification of the epithelial layers on 16 of these explants was no longer maintained. The mitotic activity was poor (Figs. 11 and 12). Controls performed with peritumoral dermis in Tyrode’s solution rr;tainrd their biological effect and behaved like untreated peritumoral dermis. Out of 10 cultivated explants, the epidermis of 6 (60%) showed healthy basal cells and stratified epithelial layers.

Association

of Cicatricial Dermis with Embryonic Epiclcrmts

Out of 20 cultures 12 (60%) developed epidermal growth with a cslumnar basal layer with mitoses and normal stratification and keratiwas not uniformly arranged, the nization. This epidermal growth maximum number of epithelial layers being 6. Spiky projections of epithelial cells penetrated the dermal tissue in some of the cultures. Very few connective tissue cells were evident in these cultures ( Table 1) . As will be appreciated from Table 2 the epithelial response was not dependent on the sex of the donor. It should be noted that such iesponse was likewise unaffected by the age of the donor or the region from which the dermis originated. No appreciable differences werr: observed in the histological sections examined after 5-6 days culture. PIG. 7. Epidermis cultured on frozen ( -25’C)-thawed peritumoral dermis. x 375. Frc:. 8. Epidermis cultured on heated peritumoral dermis. 1~ 375. FIG:. 9. Epidermis cultured on peritumoral dermis incubated with crude trypsin. X 375. FIG. 10. Epidermis cultured on peritumoral dermis incubated with crystalline trypsin. X 375. FIGS. 11 and 12. Epidermis cultured on peritumoral dermis exposed to collagenase. X375. FIGS. l-12. All these photographs illustrate the results after 5-6 days of culture.

688

REDLER

AND

TABLE

LUSTIG

2

EFFECT OF SEX ON THE GROWTH-PROMOTING EFFECT OF UNTREATED PERITUMORAL, INFLAMMATORY, CICATRICIAL, AND NORMAL DERMIS

Type of dermis

Peritumoral Inflammatory Cicatricial Normal

%3X

Number of cultured explants

Number of explants with mitotically active basal cells

Male Female Male Female Male

5 9 2 -

50 44 12

32 (64%) 31 (70%) 2 (17%) -

Female Male Female

1 5 4

20 25 33

12 W%‘,) 9 (36%) 10 (30%)

DISCUSSION

The present paper confirms the possibility of in vitro development of epidermal growth when this tissue is associated with dermis of a different species (Mordoh and Lustig, 1966). During ontogeny mesenchymal tissue soon loses its ability to stimulate embryonic epidermal growth in vitro. This fact has been studied by Wessells ( 1963 ) in the skin of chick embryos and by us in mice (unpublished data), and such loss coincides with the appearance of abundant intercellular substance in the dermis which accompanies maturation of the mesenchymal cells. The reduced capacity to stimulate epithelial growth characteristic of human normal stroma is reversed in the peritumoral dermis without any relation to the donor’s age, and its action is similar to that of embryonic dermal tissue. Thus, as regards human peritumoral stroma, a disorder takes place in its epidermal growth control mechanisms. This disorder consists of the absence of inhibitors to epithelial growth present in normal adult tissue, or else of an enhancement factor(s) present in peritumoral stroma but and growthusually less evident in normal stroma. Growth-inhibiting stimulating substances have been found by several authors. Thus, working with normal adult connective tissue, Parshley ( 1965) was able to obtain in vitro inhibitors to the growth of some tumors, whereas Cohen (1965) extracted an epithelial growth factor from the submaxillary gland of adult mice. The high mitotic activity of basal cells in cultures with good epithe-

GROWTH-PROMOTING

EFFECT

OF

PbXITUMORAL

DERMIS

689

lial growth suggests that the number of layers is due to cellular proliferation; however, lateral cell movements have not been discarded. In experiments carried out with peritumoral dermis of 20-methylcholanthrene induced carcinomas in mice WC‘ were unable to reproduce the results obtained with peritumoral hrlman dermis (unpub lished data). In spite of having been submitted to freezing and thawing in order to destroy cellular integrity, peritumoral connective tissue induced a good epidermal proliferation, and only exceptionally was the columnarity of basal cells affected. These data show that the epidermal response is evoked even under conditions where continuous activitv of dermal cells seems not to be present. Trypsin sensitivity suggests that protein( s ) may play a dominant role in the biological effect, but in a complex system such as peritumoral dermis, indirect effects cannot be ruled out. It should also be borncb in mind that a heat-labile molecule(s) can participate in this effect. but negative results may also be due to tissue degeneration products provoked by heating process. Heat and trypsin treatments affect the stimulating activity of peritumoral dermis as well as that of embryonic dermis (Dodson, 1967). Whether the biological effect is mediated by similar molecules can only be determined by isolation and characterization of the activct substances. Peritumoral dermal material sensitive to collagenase, presumabl?, collagen, seems not to intervene greatly in the stimulation of epidermal growth but to play an important supporting role. A similar action ot collagenase has been observed in the embryonic submaxillary gland of mice (Grobstein and Cohen, 1965) where a change takes place in the adenomere morphology but not in the epithelial growth. The same authors have also reported that soluble tropocollagen does not replace salivary mesenchyme in its morphogenetic effect. Rutter et al. ( 1964) have found that collagen per se is unable to induce epithelial morphogenesis in the developing pancreas. In embryonic epidermis, Ivessells (1964) W;U able to observe that tropocollagen gels in defined nutrients failed to duplicate the effect of a complex medium. In view of the fact that the 20 explants from the cicatricial scar studied originated from only one donor, no definite conclusions can be arrived at in so far as concerns the unusual biological activity observed in this particular case.

696

REDLER

AND

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Some of the mesenchymal factors which have been found to influence epidermal development disappear at the end of embryonic life. Changes in connective factors in tumoral processes give rise to a new stimulant capacity of epithelial growth in vitro. However, as yet, we do not know whether such changes are the consequence of a tumoral process or whether they are the inducers of tumoral proliferation. SUMMARY

The response of embryonic epidermis on the basis of histologic transformation in tissue culture was studied when epithelium was associated with normal, peritumoral, inflammatory or cicatricial human dermis. The proliferation of embryonic epidermis was stimulated under the influence of peritumoral dermis; such action, however, was not so pronounced in the case of normal adult or inflammatory dermis. The promotor activity of peritumoral connective tissue was very similar to that observed when embryonic dermis and epidermis are combined in vitro. In order to determine the characteristics of such action, peritumoral dermis was submitted to different treatments; it was established that trypsin and heat reduced its stimulating property. However, this property proved resistant to freezing and thawing. When the dermis was incubated with collagenase, the harmonious stratification of the epithelial layers was no longer maintained. REFERENCES ARGYRIS, T. S., and hIGYRIS, B. F. (1962). Differential response of skin epithehum to growth-promoting effects of subcutaneously transplanted tumors. Cancer Res.

22, 73-77. BILLINGHAM, R. F., ORR, J. W., and WOODHOUSE, D. L. ( 1951). Transplantation of skin components during chemical carcinogenesis with 20-methylcholanthrene. Bait. J. Cancer 5, 417432. COHEN, S. (1965). The stimulation of epidermal proliferation by a specific protein (EGF). Develop. Bid. 12, 394407. DODSON, J. W. (1967a). The differentiation of epidermis. I. The interrelationship of epidermis and dermis in embryonic chicken skin. J. EmbryoI. Exptl. Morphol. 17, 83-105. DODSON, J, W. (196713). II. Alternative pathways of differentiation of embryonic chicken epidermis in organ culture, J. Embryol. Exptl. M~~phol. 17, 107-117. GL~CKSMANN, A. ( 1963). Epithelial tissue of the skin during carcinogenesis. Natl. Cancer Inst. Monograph 10, 509-529.

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EFFECT

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T)ER1\IIS

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Gl.O.XI’LI~, C., aid CoII~h, J. ( 1965). Collugcnast~:eflcct 011the niorphogenesis of embryonic salivary epithelium in citro. Sciencx 150, 626-628. MAHCHANT, J., and Onn, J. \‘V. ( 1953). Further attempts to analyze the roles ol epidermis and deeper tissues in experimental chemical carcinogenesis by tran\plantation and other methocls. Brit. J. Cancer 7, 329-341. \IcLouc~~~x, C. B. (1961a). The importance of mesmchymal factors in thus differentiation of chick epidermis. I. The differentiation in culture of the isolated epidermis of the embryonic chick and its I-rsponse to t3wbs vitamin A. J. Emhry~l. Exptl. Morphol. 9, 370-383. JICLOUGIILIK, C. B. ( 1961b). II. hlodification of el~idern~al diflerentiation by co!)tact with different types of mesenchymc. 1. Evhryol. Erpfl. Jlorphoi. 9, 385499. MOHDOH, P. R., and Lusrrc, E. S. (1966). Dermo-epiclernlal interactions in tissrlc, culture between heterologous species. Exptl. Cell Res. 42, 384-386. ORH, J. \V. (1961). Stromal changes during carcinogenrsis. 1,~ “Biological Iutrractions in Normal and h’eoplastic Growth” (hl. J. Brmnan ant1 \I’. I,. Simpson, eds. ), pp. 321-329. Little, Brown, Boston, Massachusetts. I'AIWILEY, hl. S. (1965). Effect of inhibitors from adult connective tissue> 011 growth of a series of human tumors in citro. Cawxv Rcs. 25, 387-401. HAWLES, hl. E. (1963). Tissue interactions in scale and feather development as studied in dermal-epidermal secombinations. .[. E1h!/01. Rz.ptl. 12lor)7llol. 11, 765-789. REDLER, P., and LUSTIG, E. S. (1967). Unpublished RCTTE:R, W':. J., WESSELLS, N. K., and GROBSTEIK,

data. C. ( I964 ) Control of specific synthesis in the developing pancreas. N&l. Cancer Itlst. Afotqruph 13, 51-65. SEXEL, 1’. ( 1964). The determinism of the differentiation of the skin and the cutaneous appendages of the chick embryo. 111“The Epidermis” (\$‘. hlontagrra and W. I’. Lobitz, eds.), pp. 1%34. A ca d emit Press, Ne\v York. \'AN SCOTT, E. J., and REINERTSOS, R. P. (I961 ). Th e modulating influence of stromal environment on epithelial cells studietl in hrrman autotransplantl;. J. Inwst. Dermafol. 36, 109-132. WESSELLS, N. K. ( 1962). Tissue interactions tlrnirl,q skin histodifferentiatiorr. Dewlop. Biol. 4, 87-107. \I’ESSELLS, IV. K. ( 1963). Effects of extra epithelial factors on the incorporation of thymidine by embryonic epidermis. Exptl. Cell Res. 30, .36-ri,j. U'ESSELLS, N. K. (1964). Substrate and nutrient effects upon epidermal basal cell orientation and proliferation. Proc. Nutl. Acud. Sci. [J.S. 52, 252-259,