the similarity in histologie appearance of some human cancer … · 2. histologie examination : a...

The Similarity in Histologie Appearance of Some Human"Cancer" and "Normal" Cell Strains in

Sponge-Matrix Tissue Culture*

JOSEPHLEIGHTON,!IRAKLINE, MORRISBELKIN,FRANCESLEGALLAIS,ANDHENRYC. ORR

(Laboratory of Pathology and Laboratory of Chemical Pharmacology, National Cancer Institute, Bethetda, Md.,%

and Department of Pathology, University of Pittsburgh, Pittsburgh, Pa.)

In 1951 Gey (7) succeeded in developing for thefirst time & strain of human cells (HeLa) thatcould be cultivated indefinitely in vitro. Thisstrain has since been used widely as a laboratorytool. In the few years following Gey's achieve

ment, other investigators have applied themselvesto establishing lines of human cells from normaland cancer tissue. Many of these efforts have beensuccessful. In the fall of 1955, twenty differentstrains of human cells were reported in answer toa questionnaire circulated by the Tissue CultureAssociation. Established strains of human cells arebeing used in a variety of areas in experimentalmedicine, including virology, cell nutrition, metabolism, genetics, and cancer research.

Cell strains have been of use to us in our studieson the biologic mechanisms that control the spreadof cancer. We have been involved with this problem at an experimental level since 1953, followingthe observation that cells of strain HeLa, derivedfrom an epidermoid carcinoma of the uterine cervix, invade and replace normal human connectivetissue in sponge-matrix tissue culture (11).

The principles of the technic are simple. A combined matrix of cellulose sponge and plasma clot isused to support the growth of the normal host tissue. A very small particulate inoculum of tumorcells in Gelfoam is placed on the cellulose spongeculture and held secure with a fresh plasma clot.Tumor cells can be seen growing from the inoculum into the normal connective tissue in the living

* Presented at the Seminar on the Biology of Cell Strains,annual meeting of the Tissue Culture Association, April 3,1956 (Anat. Ree., 124:492, 1956).

Supported in part by Research Grant C-2800 from theNational Cancer Institute.

t Present address: Department of Pathology, Universityof Pittsburgh School of Medicine, Pittsburgh 13, Pa.

ÃŽNational Institutes of Health, Public Health Service,U.S. Department of Health, Education and Welfare.

Received for publication December 7, 1956.

culture; the morphologic details of the invasionare studied in serial histologie sections. The site ofinoculation of the tumor, and hence some estimation of the extent of tumor outgrowth, can be determined in the histologie sections by the positionof the Gelfoam sponge, the microscopic appearance of which can be readily distinguished fromthat of the cellulose sponge.

Experiments on the spread of cancer in suchsystems have three primary separate variables:the normal tissue, the tumor tissue, and the conditions of cultivation. Impressive differences havebeen seen in the pattern of histologie organizationin cultures of host tissues alone, depending upontheir organ of origin. These differences are accompanied by varied patterns of interaction whentumor cells are added to cultures of the normaltissue (12).

The established cell lines that were studied werechosen simply on the basis of their immediateavailability. They are Gey's HeLa, Eagle's KB(4), and Osgood's Oregon J-96 (15), all of whicharose from human cancer tissue; Henle's Intestine4071 and Chang's Conjunctiva (2) from normalhuman tissue; and our Strain D-189 (13), a histo-logically malignant transformation of a normalhuman connective tissue. In this report we willcompare the histologie appearance of these six cellstrains growing in Gelfoam matrix.

MATERIALS AND METHODSMost laboratories using cell strains grow the

cells on the walls of glass bottles or tubes. Such aculture can be transferred to growth in Gelfoam2particle suspension as follows:

Preparation of Gelfoam particles.â€”A piece of

1G. Henle and F. Deinhardt. Reported in the Catalogueof Established Cell Strains, distributed by the Tissue CultureAssociation, 1956, and personal communication.

'"Gelfoam pack," The Upjohn Company, Kalamazoo,

Mich.

359

on May 17, 2021. © 1957 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

360 Cancer Research

Gelfoam pack measuring about 1X2 cm. isminced aseptically with scissors in 1-2 ml. offeeding mixture3 until it has been reduced to a

particle size of about 1 mm. in diameter. As theGelfoam first becomes moistened, the pH of themedium drops, and bubbles (presumably carbondioxide) are released. As the particles are minced,the pH of the brei goes up to 7.2 or higher.

Transfer of cells from glass surface to Gelfoam.â€”

The medium is removed from the culture tube, andmoistened Gelfoam particles are deposited overthe area of cell growth. The area is gently wipedwith the gelatin sponge particles, and 1 ml. ofmedium is added. The culture is then incubated asbefore.

Replenishing the nutrient medium.â€”The pH of

the culture usually drops from the initial level inthe medium, i.e., 7.6-7.8, to a level of 7.0 in 1 or

more days, depending on the cell population. It isdifficult to remove depleted medium with apipette, because the Gelfoam particles are suckedinto the pipette as well. This difficulty is avoided,however, by the addition of a slice of sterile cellulose sponge to the culture tube. The cellulosesponge is held against the glass wall with thepipette, and the depleted medium is aspiratedthrough it, thus preventing the Gelfoam particlesfrom entering the pipette. One to 4 ml. of nutrientare then added, depending upon the fall in pH thatoccurred during the previous feeding interval (12).

Subculture to other Gelfoam cultures.â€”One 2X2cm. square of Gelfoam pack is minced in 2 or 3 ml.of nutrient medium. The mixture is added to thedonor culture without prior removal of the partially depleted medium. The donor Gelfoam particlesare mixed thoroughly with the freshly minced Gel-foam. On the following day they are divided intoa few aliquots by pipetting them into sterile tubescontaining slices of cellulose sponge. Two ml. ofmedium are added to each tube, which is thenmaintained as was the original culture.

Microscopic observation.â€”

1. Living cultures: In the initial transfer of cellsfrom glass to Gelfoam, clumps of cells will be seenhere and there in contact with the refractile gelatinsponge. In the next day or 2, cells are seen migrating into the interstices and, as growth proceeds,coating the trabeculae of the sponge. Eventuallythe Gelfoam sponge particles are covered by densepopulations of cells.

2. Histologie examination : A portion of the gelatin sponge particles in a luxuriant donor culture

1A convenient feeding mixture that supports all the celllines described here consists of 40 per cent human serum and 1per cent beef embryo extract, diluted in Hanks' balanced salt

solution.

may be fixed for histologie study prior to the addition of freshly minced Gelfoam. A satisfactory histologie routine has been described previously (10).However, further improvements in technical detailhave since been made, and the procedure now being used is as follows:4

a) Fixation: Gelfoam particles are dropped into a vial containing about 10 ml. of fixative, e.g., Zenker's formalin, and

are agitated gently, then fixed for 2 hours.6) Washing and wet storage: Particles are washed in several

changes of tap water for a total of 2 hours, then transferred to80 per cent alcohol for at least 30 minutes. If storage beforeembedding is necessary, particles are kept in 80 per cent alcohol.

c) Preparation for sectioning:

Dehydration: 1) 95 per cent alcohol, 15 minutes2) 95 per cent alcohol, 15 minutes3) Absolute alcohol, 15 minutes4) Absolute alcohol, 15 minutes5) Absolute alcohol, 15 minutes

Clearing: 1) Toluol, 20 minutes2) Toluol, 20 minutes or longer, until par

ticles are translucent.

Embedding: The discrete fragments should not be placedin direct contact with one other; at least 1 mm. of spaceshould be allowed between fragments. Paraffin' with a meltingpoint of 56Â°-58Â°C., should be used, and the oven kept at 65"

C. while tissue is being infiltrated.

1) Paraffin, 30 minutes2) Paraffin, 30 minutes3) Paraffin, 30 minutes

d) Sectioning: The knife must be kept in excellent condition. Stropping may be required several times during thepreparation of serial sections from a single culture. The knifeand paraffin block should be chilled thoroughly just beforesectioning.

OBSERVATIONS"CANCER" CELL LINES

Stock cultures of three tumor cell lines, HeLafrom an epidermoid carcinoma of the uterine cervix, KB from an oral carcinoma, and J-96 from amonocytic leukemia, have been maintained formore than a year in Gelfoam particle suspension.As cultures became densely populated, Gelfoamparticles covered with tumor cells sometimes adhered to the glass walls of the roller tubes and gaverise to sheets of epithelial-like cells. The sheetsformed by each of the tumor cell lines could not bedistinguished from one another.

From time to time, particles of Gelfoam coveredby luxuriant growth were serially sectioned. Manycultures of each of the cell lines have been exam-

4We wish to acknowledge, with thanks, the assistance ofMr. Joseph Albrecht and Mr. Willie Morgan of the Laboratoryof Pathology, National Cancer Institute, in perfecting thehistologie routine with which excellent sections of sponge-matrix tissue cultures can now be prepared.

'Bioloid Embedding Paraffin, Will Corporation, Baltimore 3, Md.

"**



LEIGHTONet al.â€”Similarity of "Cancer" and "Normal" Cell Strains 361

ined; the number of distinct Gelfoam particlesfrom each line is in the hundreds. It was impossible to identify any of the cell lines specifically.

With low magnification a pattern of radial symmetry could be recognized in sections through thecenter of the largest particles. The Gelfoam wascovered by a thick crust of compact cells. Most ofthe interior of the gelatin sponge was very sparselypopulated (Fig. 1).

With higher magnification, as the growth wasexamined from the surface to the depths of thesponge, an effect of the gradient of progressivelydepleted essential metabolites and of increasingproducts of cell metabolism could be seen. On thesurface of the cellular zone, in direct contact withthe medium, the cells were often almost contiguousand free of degenerating forms. In the deeper layers of the crust, the cells were less compact, andviable ones and some in all stages of degenerationand necrosis were intermingled in a random pattern. Dividing figures were seen wherever livingcells were found and were not concentrated in anyparticular layer. No identifiable organoid arrangement was observed at any level of the metabolicgradient of this system.

Various fixatives and stains were used in an effort to distinguish one cell line from another.Bouin's solution and alcoholic formalin fixationwere used as well as Zenker's formalin. In additionto hematoxylin and eosin, Regaud's iron hematox-

ylin, methyl green pyronine, Feulgen, Giemsa,and the Periodic acid-Schiff reaction with andwithout diastase digestion were all used. None ofthese was of help in the specific identification ofany one of the tumor cell lines.

The cells in each of the lines had the appearanceof malignancy (Figs. 2-4). They were hyperchro-matic, and there was considerable variation in cellsize. Some were enormous, with multiple nuclei orsingle, large nuclei containing a very large nucle-olus. The nuclei were all relatively large, and thecytoplasm was scanty. The chromatin in manynuclei was coarse, nuclear membranes staineddarkly, and single large or multiple nucleoli werecommon. Many kinds of abnormal divisions wereseen, and bizarre multipolar figures were common.Large amounts of glycogen were seen in the cytoplasm of some of the cells in each of the lines, butits distribution was varied and did not provide abasis for separating the cell lines from one another.

"NORMAL"CELL LINESCultures of Henle's Intestine 407 and Chang's

Conjunctiva were transferred from growth on aglass surface to Gelfoam sponge. The flat epitheli-al-like growth seen on the glass could not be dis

tinguished from the three "cancer" cell lines de

scribed above.Histologie sections of several luxuriant cultures

of each of the normal cell lines were prepared(Figs. 5 and 6). Both of the "normal" lines had allthe cytologie features of malignancy seen in the"cancer" cell lines, and the two groups could not be

separated when presented as unknowns to severalpathologists. All the sections were read as malignant.

"CANCER" CELL LINE THAT AROSEin

Vitro FROMNORMALTISSUEWe have observed the transformation of a

strain of normal human connective tissue into ahistologically malignant strain (D-189) in cellulosesponge-matrix tissue culture (Fig. 7). The originalexpiant was from the foreskin of a four-day-oldinfant. The altered cells (unlike their normal ancestors) grew well on glass but had less of a tendency to form unbroken sheets than did the five celllines already described.

Densely populated Gelfoam particle cultureswere examined in histologie section. All the cytologie features characteristic of malignancy thatwere seen in the previous two "normal" and thethree "cancer" cell lines were also found here (Fig.8). Cultures of D-189 could sometimes be distinguished from the other lines by the presence of agreater percentage of very large cells with enormous nucleoli. In addition, D-189 generallyshowed less of a tendency to form a coating on thesurface of the Gelfoam particle than did the otherlines. Instead, cells migrated, in general, into thesubstance of the Gelfoam to a greater depth thanseen in the other cell lines.

DISCUSSIONWhen primary expiants of human cancer or of

normal adult or fetal human tissues are grown insponge-matrix tissue culture, a varied picture isseen in the outgrowth as well as in the area of theexplant. The histologie picture of the culture canusually be related to the microscopic appearanceof the tissue of origin. For example, tumors ofglandular origin such as breast, thyroid, or pancreas may grow as nests of glandular cells in afibrous stroma. Epidermoid carcinoma of the lungproduces keratin pearls. Cultures of malignantmelanoma may grow as nests of polyhedral tumorcells in a stroma of spindle-shaped cells in whichpigment-laden macrophages abound. Many human tissues grow with vigor for 2-6 weeks andthen, whether subcultured or not, lose their pro-liferative and metabolic vigor. Histologie sectionsmade after this decline often show the neoplastic



Cancer Research

epithelial components to be scanty. Where identification can be certain, as in the case of keratinpearls in epidennoid carcinoma, such componentsare fully differentiated and probably irreversiblymature.6

The established cell strains appear to be of a different nature altogether, and their relationship tocancer in man is obscure. In contrast to the organized growth pattern seen in cultures of primaryexpiants, the histologie appearance of the established cell strains lacks tissue organization and isbest characterized morphologically as anaplasticcancer. Even a cell line from fetal intestine growswithout an organo id pattern and resembles linesderived from epidennoid carcinoma or from connective tissue of infant's foreskin.

The similarity of established cell lines derivedfrom normal and cancerous tissues, which we havedescribed here, has been observed by other investigators who have used biochemical and biologic,as well as morphologic, technics (1, 3, 5, 6, 14).Fennell (6) has used the approach of clinical ex-foliative cytology. He examined smears of "normal" and of "cancer" cell lines after staining with

the Papanicolaou method and found that smearsfrom both kinds of cell lines fulfilled the criteriarequired for a diagnosis of cancer. In addition, heobserved in each of the smears that the appearanceof the cells varied and that some cells could not beclassified as malignant.

In seeking an explanation for this variationwithin an established cell line, one wonders whether the variations may be a part of the normal lifecycle of the cell or whether these variants indicatethe presence of different clones in a population ofcells. If the latter alternative is the case, as is suggested by the work of Puck (16), it would be ofinterest to compare the biologic behavior of malignant-appearing and nonmalignant-appearingclones isolated from a cell strain in terms of theirfunctional resemblance to cancer.

Berman and Stulberg (I)7 observed that celllines obtained from both cancerous and noncancer-ous tissue were of similar appearance, and all exhibited some of the morphologic features usuallyassociated with malignancy. Both kinds of celllines invaded the mesoderm of the chick embryo.We take the invasion observed here, as well as thatobserved by others (14), as evidence of malignancy.

Eagle (3, 5) has compared the nutritional re-â€¢¿�We have'reported on the growth patterns of primary ex-

plants of four human tumors in sponge-matrix tissue culture(9). A larger series providing a broader basis for generalizationis being studied in both cellulose and Gelfoam matrices.

7Personal communication.

quirements and the response to chemotherapeuticagents of several "normal" and "malignant" cell

lines. He was impressed by their resemblance toone another and suggested that rapid growth is thecommon factor responsible for the metabolicsimilarity.

It seems unlikely that these common tendenciesin morphology, in metabolism, and in biologic behavior are all artefacts of in vitro life. We view theresemblance as real and suggest that "normal" celllines are indistinguishable from "cancer" cell lines

because the former are, in fact, a kind of cancer.Many of the ideas advanced to explain the causeof cancer in the patient or in animals can be applied equally well to explain the development ofcancer in tissue culture. Anoxia has been proposedas a common factor in the cause of cancer. Directevidence for the role of anoxia is presented byGoldblatt and Cameron (8), and circumstantialevidence by others (17). The technics employed inculturing cells, in almost all laboratories, necessarily expose large groups of cells to chronic anoxia.The solubility of oxygen in mixtures of balancedsalt solution and serum is small, and circulatingred blood cells are not present in vitro to makemore oxygen available for the tissues. In areas ofthe culture chamber where the cell population isdense, the portion of dissolved oxygen availablefor each cell must indeed be meager.

In efforts to obtain an easily grown, uniformcell line, unrelenting factors of selection for rapidgrowth are constantly present. Subcultures areusually prepared from the most rapidly growingcultures. Cells are encouraged to abandon functionsnot related to rapid growth, and those that fail todo so are gradually lost in successive subcultures.It is not surprising, then, that cell strains arisingfrom many kinds of tissue, fed on the same kindsof medium, under the same conditions, and selected through many subculture generations for rapidgrowth, may eventually possess many biologicproperties in common, including microscopicstructure and the ability to parasitize normal tissue under appropriate conditions (i.e., malignancy) .

Upon examination of these cell lines, either inGelfoam alone or in combination with normal tissues, some pathologists have called such unorganized (anaplastic) arrangements of tumor cells "un-differentiated carcinoma." The use of this term is

unfortunate, since it leads to an inference thatmay not be correct. Undifferentiation is a state inwhich the potentiality exists for differentiation inseveral directions. As differentiation progressesmultiple potentiality is lost, and the cell realizesspecific, limited functions. Perhaps the capacity



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All the figures, with the exception of Figure 7, representmicroscopic sections of cultures in Gelfoam particle suspension.Figure 7 shows a microscopic section of a culture in cellulosesponge matrix.

FIG. 1.â€”Aspherical particle of Gelfoam with a thick crustof strain HeLa cells. The cells on the surface, in contact withthe medium, are compact and are free of degenerating forms.In the deeper layer, the cells are less compact; viable and de

generating cells are mixed. Hematoxylin & eosin. X96.Fio. a.â€”Gey'sstrain HeLa, derived from epidermoid carci

noma of the cervix. Regaud's iron hematoxylin. X420.FIG. 3.â€”Eagle'sstrain KB, derived from an oral carcinoma.

Regaud's iron hematoxylin. X4Â¿0.FIG. 4.â€”Osgood's strain Oregon ,1-96, derived from the

peripheral blood of a patient with monocytic leukemia.Regaud's iron hematoxylin. X420.

FIG. 5.â€”Henle's Intestine 407, derived from normal humanfetal intestine. Regaud's iron hematoxylin. X420.

FIG. 6.â€”Chang's Conjunctiva, derived from the normalconjunctiva of a child. Regaud's iron hematoxylin. X420.

FIG. 7.â€”Strain D, derived from normal infant's foreskin.

The cell line was 5 months old when this subculture was pre

pared and appeared to be a normal (i.e., nonmalignant) connective tissue. Regaud's iron hematoxylin. X420.FIG. 8.â€”Strain D-189, an offspring of Strain D, that has a

malignant appearance such as seen in Figures 1-6. Regaud's

iron hematoxylin. X420.



- - -



LEIGHTONet al.â€”Similarity of "Cancer" and "Normal" Cell Strains 363

for indefinite rapid growth here is itself as much aspecialized, differentiated function as is the production of melanin, keratin, or mucus by otherspecialized cells.

Finally, various agents, chemical substances aswell as viruses, have been implicated as causalfactors in the origin of cancer. Perhaps normalcells denied the homeostatic immune mechanismsof the host, and compelled to divide as fast as possible under conditions of oxygen deprivation, areespecially vulnerable to neoplastic alteration induced by viruses brought in with the original ex-plant or with the biologic materials in the medium,or by other carcinogenic factors.

Experiments are in progress in which the biologic behavior of the "cancer" and "normal" cell

lines and primary expiants from clinical cancer arebeing compared by adding them to cultures ofhistologically nonmalignant adult and embryonichuman tissues.

SUMMARYThree established "cancer" cell strains that

arose from human tumor tissue, Gey's HeLa,Eagle's KB, and Osgood's J-96, were grown in

Gelfoam particle suspension. In histologie sectionsthese were indistinguishable from one another.Each had the same malignant features, i.e., absence of architectural patterns indicative of thetissue of origin, great variation in size of cells, hy-perchromatism, high nuclear-cytoplasmic ratio,prominent and often multiple nucleoli, and manymitoses including numerous tripolar and otherabnormal forms. On the basis of their microscopicappearance all three could be reasonably classifiedas anaplastic carcinoma (or sarcoma).

Two "normal" human cell lines, Henle's Intestine 407 and Chang's Conjunctiva, were indistin

guishable on histologie examination from the three"cancer" strains mentioned above.

Strain D-189 had the same malignant featuresseen in both the "cancer" and the "normal" cell

lines. It arose in vitro in our laboratory as a morphologic malignant transformation from normalhuman connective tissue.

We suggest that when cells have been kept invitro long enough for their growth to have becomestabilized to the extent that they can be consid-eredjestablished cell strains, they may bear littlerelationship to their tissue of origin. In addition,cell lines with the morphologic characteristics ofcancer may well be malignant, whether originallyfrom normal or tumor tissue.

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(Detroit) of Human Epithelial-like Cells. Proc. Soc. Exper.Biol. & M.-.I., 92:730-35, 1956.

2. CHANO,R. 8. Continuous Subcultivation of Epithelial-likeCells from Normal Human Tissues. Proc. Soc. Exper. Biol.& Med., 87:440, 1954.

3. EAOLE, H. Nutritional Needs of Mammalian Cells inTissue Culture. Science, 122:501-4, 1955.

4. . Propagation in a Fluid Medium of a HumanEpidermoid Carcinoma, Strain KB. Proc. Soc. Exper.Biol. & Med., 89:362-64, 1955.

5. EAGLE, H., and FOLGT, G. The Cytotoxic Activity ofCarcinolytic Agents in Tissue Culture. Proc. Am. Assoc.Cancer Research, 2:107,1956.

6. FENNELL,R. H. Exfoliative Cytology as a Tool in TissueCulture. Anat. Ree., 124:486, 1956.

7. GET, G. O.; COFFMAN,W. D.; and KUBICEK,M.T. TissueCulture Studies of the Proliferative Capacity of CervicalCarcinoma and Normal Epithelium. Cancer Research,12:264-65, 1952.

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9. LEIGHTON,J. Studies on Human Cancer Using SpongeMatrix Tissue Culture. I. The Growth Patterns of aMalignant Melanoma, Adenocarcinoma of the ParotidGland, Papillary Adenocarcinoma of the Thyroid Gland,Adenocarcinoma of the Pancreas, and Epidermoid Carcinoma of the Uterine Cervix (Gey's Strain HeLa). TexasRep. Biol. & Med., 12:847-64, 1954.

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11. LEIGHTON,J., and KLINE, I. Studies on Human CancerUsing Sponge Matrix Tissue Culture. II. Invasion ofConnective Tissue by Carcinoma (Strain HeLa). TexasRep. Biol. & Med. 12:865-73,1954.

12. LEIGHTON,J.; KLINE,I.; BELKIN,M.; and TETENBAUM,Z.Studies on Human Cancer Using Sponge-Matrix TissueCulture. III. The Invasive Properties of a Carcinoma(Strain HeLa) as Influenced by Temperature Variations,by Conditioned Media, and in Contact with RapidlyGrowing Chick Embryonic Tissue. J. Nat. Cancer Inst.,16:1353-73, 1956.

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1957;17:359-363. Cancer Res Joseph Leighton, Ira Kline, Morris Belkin, et al. Culture''Cancer'' and ''Normal'' Cell Strains in Sponge-Matrix Tissue The Similarity in Histologic Appearance of Some Human

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