[CANCER RESEARCH 28, 1944-1951. October 1968]

In fact, it was not until the development of the air-turbineultracentrifuge (5) that an instrument capable of producingstratification in normal somatic and neoplastic cells becameavailable.

Guyer and Claus (12—14) were the first to study the physical nature of neoplastic cells by use of the air-turbine ultracentrifuge. They observed a difference between the displaceability of the contents of carcinomatous cells implanted intothe adrenal gland and that of the cortical adrenal cells, bothof which were centrifuged together at the same force (400,000x g) andfor the sameperiodof time (30mm). In general,they observed that the neoplastic cells showed less markeddisplacement of their components, both of the nucleus andcytoplasm, than did the normal cells, and from this evidencethey concluded that neoplastic cells possess “adecidedlygreater viscosity than that of normal tissue cells.―

On the other hand, Cowdry and Paletta (10), using thesame methods as Guyer and Claus (12), but studying theeffects on the nuclei only in several different types of malignant cells, observed them to stratify more readily than do thenuclei in their nonmalignant counterparts, and they concludedthat the nuclei of malignant cells possess a lower viscosity thando those of nonmalignant cells. Mateyko and Kopec (28) foundthat neoplastic cells of the frog kidney stratified more readilyand their contents redistributed more rapidly than they did incentrifuged normal cells.

Evidence cited above is conflicting concerning consistency ofnormal and malignant cells. It is known that certain cell typesseem to require more centrifugal force than others to effect astratification of their components; this may be due in part todifferences in density of the cellular components and to the difference in size of the cells (ci. 3) . We have not attempted tomake a direct comparison of the relative consistency of theascites cells with that of their progenitors ; instead, a comparison of the stratification occurring in the ascites cells with thatreported in published accounts of certain other ultracentrifuged somatic cells is discussed.

MATERIALS AND METHODS

A strain of Ehrlich ascites tumor cells was kindly suppliedus by Dr. T. C. Evans of the Radiation Research Laboratory,University of Iowa. The tumor was propagated in young, adultfemale Swiss mice. Five to seven days after inoculation, thecells were removed from the body cavity of the mouse andplaced in a test tube, where they were washed three times in

SUMMARY

Ehrlich ascites tumor cells were ultracentrifuged in a gumarabic solution, or as stacks of cells in 0.9% sodium chloridesolution for periods ranging from 20 to 30 mm. Those suspendedin gum arabic become greatly stretched and are sometimespulled into two parts. The nuclei are displaced centrifugallyand the relatively dense nucleolus (sometimes there are morethan one) is forced against the nuclear envelope and adjacentplasma membrane causing them to become greatly stretched.Sometimes the nucleolus is thrown through both the nuclearenvelope and plasma membrane becoming free of the cell. Theinterphase chromosomes are more dense than the nucleoplasm,and they are displaced centrifugally. However, when this occurs,it is revealed that the chromosomes adhere to the nuclearenvelope. The forces holding them to the inner membrane ofthe nuclear envelope are relatively strong, thus causing thedisplaced chromosomes to become greatly stretched, or evenbroken, before they are detached. When subjected to a comparable centrifugal force, the stratification in the ascites cellsis not so complete as has been reported for certain nonmalignant somatic cells.

INTRODUCTION

The study of the cancer cell has been approached from practically every point of view in an effort to understand thetransformation from the normal to the malignant state. Notwithstanding the enormous literature on this subject, “Classicaland modern investigations have failed to reveal a singlemorphological sign which is truly specific of cancer cells ; however, it is possible to enumerate certain phenomena which,when taken together, characterize the tumor cells― (31).

The centrifuge technic has been widely used in a study ofthe physical properties of noncancerous cells, particularly largecells such as oocytes, eggs, and protozoa (e.g., 8, 15—17,30).However, because of their relatively small size and high consistency, somatic cells from which most malignant cells arederived have been little studied by this method (ci. 25, 26).

1 Supported by Grants GM-5479, 4706, 09229,@ and HD-00699

from the NIH and Grant G-9879 from the National ScienceFoundation.

2 Recipient of a Career Development Award, GM-11,524, from

the National Institute of General Medical Sciences.Received January 15, 1968; accepted May 2, 1968.

CANCER RESEARCH VOL. 281944

Properties of the Ehrlich Ascites Tumor Cell as Determined by

Electron Microscopy, Ultracentrifugation, and Hydrostatic Pressure'

H. W. Beamsand R.G. Kessel2Department of Zoology, University of iowa, Iowa City, iowa 62240

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Physical Properties of Ehrlich Ascites Cells

0.9% sodium chloride solution. Some of the cells were placedin small plastic tubes containing 20 to 30% gum arabic dissolved in 0.9% sodium chloride solution, and others werepacked several layers deep within small tubes containing 0.9%sodium chloride solution. The tubes were then placed in anair-turbine ultracentrifuge (5) and centrifuged at approximately 300,000 x g for periods of 20 to 30 mm. Upon completion of the ultracentrifugation some of the cells were immediately examined under the phase-contrast microscope andphotographed. Others were fixed in Carnoy's, Bouin's, andChampy's solutions and stained in Heidenhain's hematoxylinor the Feulgen method. For electron microscope studies, thecells were fixed in cold, 3% phosphate-buffered glutaralydehydesolution (pH 7.3) for 1 to 2 hr and postfixed in 1% buffered(pH 7.3) osmium tetroxide (33). The cells were dehydratedrapidly in a series of cold ethanols and embedded in Epon 812(22) . Sections were stained in uranyl acetate (35), lead citrate(32), or in both uranyl acetate and lead citrate. The sectionswere studied in an RCA EMU 3D or 3G electron microscope.

Ascites cells were also exposed in 0.9% sodium chloride solution to a hydrostatic pressure of 9,000 lb/sq inch for periodsof 30 to 60 mm. The pressure was generated by a jack (modelP76, Blackhawk Mfg. Co., Milwaukee, Wisconsin) and delivered through a pressure hose to a specially constructedcylinder fitted with an automobile hydraulic brake wheelsleeve, which functioned both to separate the oil from thepressure chamber and to transmit the pressure from the jackto the experimental pressure chamber. Experiments were runat room temperature (22°C), and while a continuous recordof the temperature within the pressure chamber during anexperiment was not made, it was determined that the temperature within the chamber immediately upon its decompressiondid not vary over ±1°Cfrom that at the beginning of the experinient.

RESULTS

The structure of the Ehrlich ascites cell is so well known thata detailed description of it here seems superfluous. It will sufficeto point out that this cell possesses a relatively large nucleus,one or more nucleoli, and a prominent porous nuclear envelope(Fig. 1) . A well-developed Golgi apparatus consisting of lamellae and vesicles (GA ) , scattered filamentous mitochondria (M),and one or more lipid bodies (L) are usually present (Fig. 1).Numerous ribosomes, both free and attached to membranes ofthe endoplasmic reticulum, occur in the cytoplasm (Fig. 1, ER).A well developed centriole, bodies consisting of granules andvacuoles (lysosomes) , and bodies described as virus are oftenseen in electron micrographs of ascites cells (ci. 1) . Projectingfrom the cortex or ectoplasmic region of these cells are numerous microvilli. Cells exposed to a hydrostatic pressure of 9,000lb/sq inch for 30 mm often show fibrous rootlets extendingfrom the base of the microvilli to a position relatively deepwithin the cortex (Figs. 2, 3).

Effects of Ulfracentrifugation

When washed ascites tumor cells are suspended in a mediumof the same relative density or piled one upon the other in a

centrifuge tube, subjected to a centrifugal force of approximately 300,000 x g for 20 to 30 mm, and examined immediatelyunder the phase-contrast microscope, marked effects on thecells are observed, some of which are illustrated in Figs. 4 to8. They are often greatly stretched, even to the point of separating into two parts (Figs. 7, 8) . The nucleus is relativelydense and displaced centrifugally ; the lipid ( L) and vacuoles(V) are less dense and a@e displaced centripetally (Figs. 4—8).The lack of a sharp stratification of the mitochondria in manyof these cells (Fig. 4, M) suggests, but does not prove, thatthey possess a relatively high density as compared to certainnormal somatic cells in which the mitochondria are sharplystratified (ci. 3, 4, 6, 11).

Electron micrographs also reveal in some of the ultracentrifuged cells that the mitochondria are not completely displacedfrom all parts of the cells, but they appear a little more concentrated in the centrifugal and centripetal halves than elsewhere (Figs. 9, 10) . It may be that the mitochondria in theascites cell, like those in the Arbacia egg ( 19) , differ slightlyin density, a condition which would account for their concentration in the two ends of the ultracentrifuged cells. The Golgicomplex in ultracentrifuged cells is usually found in the centripetal half of the cell (Fig. 9, GA).

The Nucleus

A marked effect of the high centrifugal force is noted on thenucleus. Generally, it is displaced centrifugally and becomesgreatly stretched (Figs. 4—10). The densest component in thenucleus is the nucleolus (there are sometimes more than one);it is always forced to the centrifugal end of the nucleus, whereit may cause the nuclear envelope and adjacent plasma membrane to become greatly stretched, even to the point of breaking, thus allowing the nucleolus to become detached from thecell (Figs. 5, 6).

Of special interest is the effect of the centrifugal force onthe interphase chromosomes. The chromatin is more dense thanthe nucleoplasm and is displaced centrifugally (Figs. 6—10,DCII) . When this occurs, it is observed that some, if not all, ofthe interphase chromosomes are “attached―to the nuclear envelope, and they adhere so tightly to it that they become greatlystretched (Figs. 4, 6—8,SCH) . There seems little doubt thatthe filaments adhering to the nuclear envelope represent chromosomes, as they readily stain with the Feulgen method. Thebuoyancy of the centripetally displaced nucleoplasm preventsthe collapse of the nuclear envelope at the centripetal end ofthe cell (Figs. 9, 10 NE) . Although not clearly illustrated inthe figures, the nuclear envelope at the centripetal end of thenucleus often shows small indentations, presumably caused bythe displacement pull of the centrifugal force on the “attached―chromosomes.

The adherence of the interphase chromosomes to the innermembrane of the nuclear envelope is not a special characteristicof neoplastic cells, as it has been observed in certain other typesof nonmalignant cells. It has been suggested that the chromosomes may be anchored to the nuclear envelope at chromocentres (29) ; however, we were unable to obtain evidence inthe ascites cells for a specialized structure or body in the chromosome where it is attached to the nuclear envelope.

OCTOBER 1968 1945

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H. W. Beams and R. G. Ke8seL

Other evidence bearing on the consistency of tumor cells hasbeen noted by Lewis (20). After comparing several differenttypes of sarcoma cells in tissue culture with their normal prototype cells, he concluded that the characteristic differencesbetween them was that the cytoplasm of the malignant cellsis more dense, suggesting a relatively high viscosity. Physiologicstudies seem to indicate that the surface properties of tumorcells differ from those of normal cells and that they are moreeasily separated from one another than are normal cells (e.g.,Ref. 2) . However, a study involving the use of the microdissection technic revealed no difference in consistency and structure between the normal and malignant cell (7) . Good generaldiscussion of the cytology of the cancer cell may be found inarticles by Cowdry (9) and Oberling and Bernhard (31).

A condition which has been observed by a few investigatorsbut which is not of general knowledge is the fact that in certaintypes of cells the interphase chromosomes are “attached―oradhere securely at one or more positions on their surface to theinner membrane of the nuclear envelope. The nature and significance of this association is not clear. Are the chromosomesalways “attached―to the inner nuclear membrane in a givenposition on their surface, such as the chromocenter (29), ormay they adhere to it at any position? In any case, the chromosomes are so tightly bound to the membrane that they areoften broken by the centrifugal force before becoming detached(4) . Obviously, as the cell enters prophase, the chromosomesautomatically shorten, thicken, become detached from the innernuclear membrane and writhe about “likeeels in a box―(34).

REFERENCES

1. Adams, R., and Prince, M. An Electron Microscope Study ofthe Morphology and Distribution of the Intracytoplaemic“Virus-Like―Particles of Ehrlich Ascites Tumor Celia. J.Biophys. Biochem. Cytol., 8: 161—170,1957.

2. Ambrose, E. J. The Surface Properties of Tumor Cells. In:E. J. Ambrose and F. J. C. Roe (eds.), The Biology of Cancer,pp. 65-77. London : D. Van Nostrand Co., Inc., 1966.

3. Beams, H. W. The Effects of Ultracentrifugal Force on theCell with Special Reference to Division. Ann. N. Y. Acad. Sci.,51: 1349—1364,1951.

4. Beams, H. W. Interphase Chromosome-Nuclear Envelope Relationship in Certain Somatic Cells. Anat. Record, 160: 459,1968.

5. Beams, J. W., Weed, A. J., and Pickels, E. G. The Tjltracentrifuge. Science, 78: 338—340,1933.

6. Bessis, M. The Blood Cells and Their Formation. In: J.Brachet and A. E. Mirsky (eds.), The Cell, Vol. 5, pp. 163-.217. New York : Academic Press, 1961.

7. Chambers, R., and Ludford, R. J. Microdissection Studies onMalignant and Non-malignant Tissue Cells. Arch f. Zellforsch.,12: 555—569,1932.

8. Conklin, E. G. The Effects of Centrifugal Force on the Organization and Development of the Eggs of the Fresh Water Pu!monates. J. Exptl. Zool., 9: 417-454, 1910.

9. Cowdry, E. V. Cancer Cells, pp. 1-667. Philadelphia: W. B.Saunders Co., 1955.

10. Cowdry, E. V., and Paletta, F. X. Alterations in Nuclear Viacosity During Experimental Carcinogenesis Determined byIfltracentrifugation. Am. J. Pathol., 17: 335—337,1941.

Ascites cells are not killed by ultracentrifugation, as evidencedby the fact that they seem to grow as rapidly as noncentrifugedascites cells when transferred to the body cavity of mice (cf.11, 12). Mateyko and Kopac (26) report that frog and ratliver cells exposed to a 40% gum arabic solution for 2.5 hrshow slight shrinkage. No morphologic evidence was revealedin this study, even in the electron micrographs, to suggest thatthe gum arabic had produced a marked distortion of the ascitescells, but admittedly it is not an ideal medium on which tocushion cells in the ultracentrifuge.

Hydrostatic Pressure

As shown here, the Ehrlich ascites cells seem to be moreresistant to the action of hydrostatic pressure than are certainnormal cells (23) . Exposure to a hydrostatic pressure of 9,000lb/sq inch for a period of 30 mm produced little effect on thesecells. Longer exposure (1 hr) showed effects illustrated in Fig.11. Here the mitochondria are vacuolated and appear dense,and the endoplasmic reticulum is less definite, but still recognizable. Most, but not all, of the microvilli have disappearedfrom the surface.

DISCUSSION

Investigators who have studied the biochemistry of theEhrlich ascites tumor cells are in agreement that they are muchmore difficult to break than most nonmalignant somatic cells.In fact, it has been necessary to devise special methods to clisrupt these cells, such as grinding with sand (21), because “...homogenization in sucrose solution as commonly employed fornormal tissue, gave completely unsatisfactory results― (18, cf.36) . The fact that these cells are so difficult to homogenize canbe interpreted to mean that the plasma membrane and/or thecytoplasm is more firm and substantial than it is in many othertypes of nonmalignant cells. As reported here, ultracentrifugedascites cells are readily stretched, but less sharply stratifiedthan many other types of somatic cells such as certain leukocytes (6) . This condition may be interpreted to mean that thecytoplasmic hyaloplasm has a relatively higher consistency orthat the cytoplasmic components are nearly of the same densityas the hyaloplasm. However, it should be emphasized that fromour studies there is no direct evidence that this condition isassociated with the state of malignancy, nor does it suggest thatall types of malignant cells would react to ultracentrifugationin a similar way. In fact, the results of Guyer and Claus (13,14) and Cowdry and Paletta (10) suggest that different typesof neoplastic cells may react to ultracentrifugation in differentways, some showing little stratification, presumably because oftheir high consistency (viscosity) ; others, especially their nudci, stratify readily, indicating a relatively low consistency.The most extensive study on the physical properties of tumorcells is that of Mateyko and Kopac (24—28). They have usedboth the technics of microdissection and high speed centrifugation to investigate human ascites cells (24), human gynecologictumors, both benign and malignant (27), and frog adenocarcinoma (28) . Some variation in consistency was found to existamong different types of tumor cells, but more often than notthey seemed to be of a lower consistency than most nontumorcells.

1946 CANCER RESEARCH VOL.28

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11. Dornfeld, E. Structural and Functional Reconstruction ofUltracentrifuged Rat Adrenal Cells in Autoplastic Grafts.Anat. Record, 69: 229—241,1937.

12. Guyer, M. F., and Claus, P. E. Growth of Cancerous and ofEmbryonic Tissues Stratified in the Ultracentrifuge. Proc. Soc.Exptl. Biol. Med., 35: 468-473, 1936.

13. Guyer, M. F., and Claus, P. E. Relative Viscosities of TumorCells as Determined by the Ultracentrifuge. Anat. Record, 73:17—27,1939.

14. Guyer, M. F., and Claus, P. E. Increased Viscosity of Cells ofInduced Tumors. Cancer Res., 2: 16—18,1942.

15. Harvey, E. B. Development of the Parts of Sea Urchin EggsSeparated by Centrifugal Force. Biol. Bull., 64: 125—148,1933.

16. Harvey, E. N., and Marsland, D. A. The Tension at the Surface of Amoeba dubia with Direct Observations on the Movement of Cytoplasmic Particles at High Centrifugal Speeds. J.Cellular Comp. Physiol., 2: 75-78, 1932.

17. Heilbrunn, L. V. The Dynamics of Living Protoplasm, pp. 1-327. New York : Academic Press, 1956.

18. Hudack, E. D., and Baker, N. Methods of Isolation of Nuclei.Exptl. Cell Rca., 2@: 327-337, 1961.

19. Lansing, A. I., Hillier, J., and Rosenthal, T. B. ElectronMicroscopy of Marine Egg Inclusions. Biol. Bull. 103: 294,1952.

20. Lewis, W. H. Some Cultural and Cytological Characteristicsof Normal and Malignant Cells In Vitro. Arch. f. Zellforsch.,23: 5-26, 1939.

21. Lindberg, 0., Ljunggren, M., Ernater, L., and Revesz, L. Isolation and some Enzymic Properties of Ehrlich Ascites TumorMitochondria. Exptl. Cell Res., 4: 243—245,1953.

22. Luft, J. H. Improvements in Epoxy Resin Embedding Methods. J. Biophys. Biochem. Cytol., 9: 409-414, 1961.

23. Marsland, D. The Action of Hydrostatic Pressure on Cell Division. Ann. N. Y. Acad. Sci., 61 : 1327—1335,1951.

24. Mateyko, G. M., and Kopac, M. J. Cytophysical Studies onHuman Ascites Cells of Malignant Ovarian Origin. Acta UnioIntern. Contra Cancrum, 16: 91—109,1960.

25. Mateyko, G. M., and Kopac, M. J. Cytophysical Studies onLiving Normal and Neoplastic Cells. I. Separation into IsolatedPopulations. Ann. N. Y. Acad. Sci., 106: 185-218, 1963.

26. Mateyko, G. M., and Kopac, M. J. Cytophysical Studies onLiving Normal and Neoplastic Cells. II. Isopyknotic Cushioning During High-speed Centrifugation. Ann. N. Y. Acad. Sci.,106: 219-285, 1963.

27. Mateyko, G. M., and Kopac, M. J. Physical Properties ofHuman Gynecological Tumors : Micrurgical and Centrifugation Studies on Living Cells. Progr. Exptl. Tumor Res., 4 : 27-.56, 1964.

28. Mateyko, G. M., and Kopac, M. J. Studies on the Cytophysiology of Frog Renal Adenocarcinoma. Ann. N. Y. Acad. Sci.,156: 22—58,1965.

29. McLeish, J. The Action of Maleic Hydrazide in Vicia. Heredity 6: Suppl, 125-147, 1952.

30. Morgan, T. H. Cytological Studies of Centrifuged Eggs. J.Exptl. Zool., 9: 593-655, 1910.

3!. Oberling, Ch., and Bernhard, W. The Morphology of the Cancer Cells. In : J. Brachet and A. E. Mireky (eds.), The Cell,Vol. 5, pp. 405-496. New York : Academic Press, 1961.

32. Reynolds, E. S. The Use of Lead Citrate at High pH as anElectron-Opaque Stain in Electron Microscopy. J. Cell. Biol.,17: 208-212, 1963.

33. Sabatini, D. D., Bensch, K. G., and Barrnett, R. J. Preservationof Uitrastructure and Enzymatic Activity of Aldehyde Fixation. J. Histochem. Cytochem., 10: 652, 1962.

34. Strangways, T. S. P. Observations on the Changes Seen inLiving Cells During Growth and Division. Proc. Roy. Soc.,London,5cr.B,94:137—141,1922.

35. Watson, M. L. Staining of Tissue Sections for Electron Microscopy with Heavy Metals. J. Biophys. Biochem. Cytol., 4:475—478,1958.

36. Woernley, D. L., and Carruthers, C. A Versatile Method ofIsolating Cell Nuclei Based on Employment of a High Viacosity Suspending Medium. Arch. Biochem. Biophys., 67 : 493-495,1957.

Fig. 1. Electron micrograph of ascites cell showing nucleus (N), microvilli (MV), Golgi complex (GA), mitochondria (M), endoplasmic reticulum (ER), and lipid body (L). x 10,000.

Figs. 2, 3. Electron micrographs of ascites cells exposed to a hydrostatic pressure of 9,000 lb/sq inch for 30 mm. Rootlets (arrows)extending from the base of microvilli are especially prominent. Fig. 2, X 10,000; Fig. 3, x 13,500.

Fig. 4. Ascites cell centrifuged at 300,000 X g for 20 min. Note elongation of cell with the nucleus displaced centrifugally and thelipid (L) and vacuoles (V) centripetally. A clear stratification of the mitochondria (M) is not evident. Observe stretched chromosomes (SCH) adhering to the nuclear envelope. Direction of the centrifugal force is toward upper left in figure. Phase-contrast photomicrograph. x 4000.

Figs. 5, 6. Ultracentrifuged cells showing displacement of nucleolar material (NU) to the centrifugal end of the nucleus. Here thecentrifugal force acting on the nucleoli has caused both the nuclear envelope and plasma membrane to become greatly stretched. Notestretched (SCH) and displaced (DCH) chromosomes in Fig. 6. Centrifugal force directed toward left in both figures. Phase-contrastphotomicrograph. x 4000.

Figs. 7, 8. Ijltracentrifuged cells that have become stretched and are in the process of separating into two components. Nuclei aredisplaced centrifugally, vacuoles (V) and lipid bodies (L) centripetally. Some of the chromosomes adhere to the nuclear envelopeat the centripetal end (SCH), while others appear to be completely displaced centrifugally. Direction of centrifugal force is towardbottom of figures. x 4000.

Figs. 9, 10. Electron micrographs showing stretched chromosomes (SCH) adhering to the nuclear envelope (NE). Mitochondria appear at M. Microvilli (MV) and Goigi apparatus (GA). Centrifugal force directed toward bottom of figures. Fig. 9, x 20,000 ; Fig.10, x 17,000.

Fig. 11. Portion of ascites cell exposed to hydrostatic pressure (9,000 lb/sq inch) for one hr. The mitochondria (M) appear dense andvacuolated; the endoplasmic reticulum (ER) is less clearly defined than in the control cells. Electron micrograph. x 32,000.

1947OCTOBER 1968

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