Int. J. Cancer: 20, 87-92 (1977)

TUMOR NECROSIS AND CELL DETACHMENT Leonard WEISS Department of Experimental Pathology, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, N. Y. 14263, USA

Cell detachment from and around Walker 256 tumors grown in rats is quantitated by a standardized in vitro shaking procedure. The volume of cells detached from cancers of similar size is virtually the same in tumors growing in the liver, the spleen, in intramuscular and in subcutaneous sites. In cystic tumors, greater volumes are detached from the innermost regions of the walls adjacent t o necrotic material than from the outermost parts. More liver perenchymal cells are shaken free of liver adjacent to a tumor interface than from regions 0.5 and 1.O cm distant from it. Detachment of tumor and liver cells i s also enhanced by prior incubation of tissue samples with necrotic extracts. It i s suggested that the necrotic regions of tumors, and products derived from them, facilitate the detachment of tumor cells and cells composing the normal tissues surrounding them, thereby potentially promoting metastasis and invasion.

Although necrosis is a salient feature of many solid cancers having diameters exceeding several millimeters, the interactions between the necrotic and living parts of tumors and non-cancerous host tissues have been largely ignored. In this communication, cell detachment from and around Walker 256 tumors is quantitated with respect to interaction with necrotic tumor tissues and simple saline extracts prepared from them, with a view to identifying a necrosis-related, cell-release phenomenon. The possible roles of cell detachment in metastasis and invasion are discussed. MATERIAL A N D METHODS

Tumors

The present experiments were made with the Walker 256 " carcinosarcoma ", which was carried as an ascites tumor in adult, male Sprague-Dawley rats. Solid tumors were initiated by direct injections of the ascites cells into different sites, as described below. Necrotic material

Subcutaneous tumors of 3 to 5 cm diameter were bisected and their amorphous, necrotic centers were curetted out and gently rinsed in 0.85 % NaCl at 4" C. This material, which was sterile on culture, was pooled and homogenized with a Tekmar " Tissumizer " (Cincinatti, Ohio). An equal volume of saline was added to the homogenate, which was

centrifuged at 35,000 g for 1 h; the supernatant fluid was then passed through Nalgene 0.45 pm pore size filters (Sybron, Rochester, N.Y.) and stored at -20" c. The necrotic extracts (NE) contained 10 to 1 1 mg/ ml protein (Lowry et al., 195l), and their acid phosphatase levels ranged from 8.4 to 21.7 (mean = 12.4) mU/ml (Babson et al., 1966). Proteolytic activity at PH 7.2 was 29 to 4.3 NFU/ml, as determined by the '' Azocasein method (Markus and Werkheiser, 1964). Further chemical characterization of the NE must await the development of corresponding bioassays. Unless stated otherwise, in detachment experiments the controls for NE consisted of similar solvents containing similar protein concentrations of bovine serum albumin (Fraction V; Armor Pharmaceutical Co., Chicago, Ill.). "

Cell detachment from tissues

Cylinders were punched from tissues using a 13 gauge (ca 2 mm) trocar and cannula, and these were then divided into 2-mm lengths over a scale, under magnification. Two such cylinders were placed into each of a number of screw-capped, glass vials, 4.5 cm long and 1.3 cm diameter, containing 2 ml of cold HBSS. These operations, which took 20 to 30min, were all done in solutions at 4" C to minimize metabolic differences. The vials were clamped onto a reciprocating shaker, making 275 oscillations per minute, with an excursion of 4.5 cm, for 40 min, at room temperature (25" C). In some designated experiments, tissue cylinders were shaken for 10 min only. After shaking, 0.25 ml of 10% buffered formaldehyde was added to each vial which was mixed by gentle inversion. When present, the macroscopic remains of the cylinders rapidly settled to the bottoms of the vials, and the released cells and cell clusters in the supernatant fluid were counted in a Fuchs-Rosenthal chamber, and their diameters measured with an imagesplitting eyepiece (Vickers-A.E.I. ; England) at a final magnification of x300. It had previously been

Received: February March 30, 1977.

1, 1977 and in revised form

88

WEISS

demonstrated that the described formaldehyde fixation did not affect these two numerical determinations. An estimate of the volume of tissue liberated by shaking was obtained from the product of the cube of the mean " particle " radius and the mean number of such cell units. Volume functions of this type were used for purposes of comparison. Small pieces of tissue were fixed in formalin and subsequently examined in 4-pm stained sections. Aliquots of liberated cells were centrifuged onto slides (Cytospin : Shandon-Elliott, London, England), and morphologically characterized after reaction with Wright's stain.

TABLE I TUMOR CELLS RELEASED FROM 8(*3)-DAY WALKER 256 TUMORS A T DIFFERENT SITES ___

~~

~~~

Numbers

Tumor size

released

(cm)

( x 1,000) +SE (n)

ca.>1.0

IM

ca.>1.0

Liver

~

214t32.8 (87) 204+13.0 (51)

Spleen 218*13.1 (31) SC 163+9.4 (79)

ca.>1.0

ca. 1

~~

Comparative Unit diameter volumes (wn)+sE (n) released (cf SC)

48*0.65 (714) 5010.86 (408) 4611.31 (204) 5410.81 (612)

1 .o I .o 0.9

I .o

RESULTS

Types of cells detached Liver. Differential counts made on liver sections revealed a mean of 72% parenchymal cells (range: 67-83 %); the centrifuged deposits of detached cells showed a mean of 88% (range: 82-92%). Although these data suggest a preferential release of parenchymal cells on shaking, the difference may simply reflect differences in visualization in tissue sections and centrifuged deposits. On morphologic criteria, over 80% of the identified, liberated liver cells appeared intact. A small number of acellular strands were present, which appeared similar to the nucleoprotein material released from dead cells. Examination of sections of the tumors growing in liver usually revealed a band of necrotic material approximately 0. I mm thick, between the healthy tumor and the liver. This material was removed from cylinders prior to shaking. Tumors. Differential counts made on sections of tumors revealed that approximately 85 % (range: 70 to 89%) of the cells present were identifiable cancer cells; the centrifuged deposits consisted of approximately 75 % (range: 76 to 86%) cancer cells. It is not possible to give accurate estimates of the viability of the liberated cancer cells, although over 70% were morphologically intact, and cultures of tumor cells have been established from them. In addition, care was taken to exclude necrotic material from the whole cylinders before cutting them into sections prior to shaking. Parenthetically, it is this problem of viability assessment which has directed extensions of this work into utilization of cell cultures. The detachment of Walker 256 tumor cells or normal liver cells was measured as follows: Eflect of organ site The release of cell units from cylinders taken from tumors of approximately 1 cm diameter, growing in

various sites 5 to 11 days after direct injection of lo7 cells in 0.2 ml of Hanks' balanced saline (HBSS), was studied to determine the effect of anatomic site on release. Approximately six counts of units released and 100 diameter measurements were made on the material shaken free from each tumor. The results summarized in Table I show that when the volume functions released from different sites are compared, they differ but little from those released from subcutaneous tumors. It was not possible to obtain discrete 1 -cm tumors in the lungs or kidneys, Efect of site within tumor

Cystic, subcutaneous Walker 256 tumors were selected, with necrotic centers of approximately 3 cm mean diameter and with wall thicknesses of 2 to 5 mm of " healthy " cancerous tissue. The release of cells from the inner and outer 1-mm lengths of cylinders taken radially through the cyst walls is shown in Table 11. In the two representative examples shown, three and six times greater volumes of tumor were released from the inner, juxtanecrotic regions of the cyst walls than from their outer parts. In four more tumors studied, the ratios all fell between these values. TABLE I 1

TUMOR CELLS RELEASED FROM INNER AND OUTER PARTS OF WALLS OF SUBCUTANEOUS, CYSTIC WALKER 256 TUMORS

Tumor diameter cm

&-:'

3.5 3

" Cell "-release ( x 1,000) *SE (n)

Inner

Outer

~ m m ~rnm

Mean size pmksa (n) Inner Outer ~ m m ~ m m

2 to 202-1-65 77&12 77k3.5 7413.1 3.5 (9) (9) (99) (99) 3 to 9012636*8.2 11013.3 8313.5 4 (9) (10) (100) (100)

Volume ratio

Inner! Outer

3:1 6:l

89

NECROSIS AND CELL DETACHMENT TABLE 111 SUBCUTANEOUS WALKER 256 CARCINOMA. TUMOR CYLINDERS INCUBATED WITH FOR 20 MIN AT 37°C THEN SHAKEN FOR 10MIN Experimental (E) Tumors

No. released ( x 1,000) +SE

NE

"

Control (C)

Clump diameter (pm)

(n)

"

&SE

(n)

No. released ( x 1 ,OOO) *SE

(n)

Clump diameter (am) +SE

ratio E'C

(n)

ca. 1 cm ca. 1 crn ca. 1 cm ca. 1 cm

171 +56 (17) 359*38 (18) 680180 (12) 812+80 (12)

573~2.0(100) 533Z1.9 (100) 4511.4 (100) 4911.4 (100)

245~t16(17) 389126 (18) 464134 (12) 4681t37 (12)

2310.3 (100) 26h0.5 (100) 2410.3 (100) 27k1.7 (100)

1011 811 911 1111

3 4 cm > 4 cm

376k.56 (10) 330146 (10)

23k1.0 (199) 2410.9 (203)

477161 (10) 458164 (10)

21 rt0.6 (197) 20h0.5 (199)

I .O/I 1.311

Effects of necrotic extract on tumor-cell release

Effects of necrotic extract on liver-cell release

The subcutaneous tumors from which material was obtained fell into two groups. The first were roughly spherical, with diameters of approximately 1 cm, and contained minimal, friable necrotic cores. Those of the second group were roughly egg-shaped with diameters of approximately 4 cm and 6 cm in the short and long axes respectively, and were largely necrotic, with healthy cancerous " rims " varying from 2 to 4 mm in depth. Cylinders obtained from the peripheral regions of the tumors were first incubated with 1/10 dilutions of necrotic extracts in HBSS for 20 rnin at 37" C, and then shaken for 40 rnin at room temperature (24" C). The results of six separate experiments with three separate batches of necrotic extract are summarized in Table 111. In the case of the 1-cm diameter tumors, it is seen that an 8 to 11 times greater volume of tumor was released after exposure to necrotic material than in the appropriate controls. In the case of the largely necrotic tumors, it is seen that pretreatment with necrotic extract produced no significant increase in the volume of tumor released by shaking, compared with controls.

Standard tissue cylinders were obtained from the livers of normal rats. These were incubated for 20 rnin at 37", in either 10% necrotic extract in HBSS, or in BSA control solutions. The cylinders were shaken for either 10 or 40 min. The results of four separate experiments given in Table V show that after 10 min shaking, a three times greater volume of cells was released from liver cylinders pretreated with necrotic extract than from the control series; after 40 rnin shaking, the ratios of extract-treated to controls increased to 18:l and 50:l.

Effects of tumor on surrounding '' normal

"

tissues .

Following the direct injection of Walker ascites cells (lo7cells in 0.2 ml HBSS) into the liver, tumors of approximately 1 cm diameter grew within 10 days. The tumors and the surrounding liver were bisected, and cylinders of liver were removed at its junction with the tumor and at 0.5 and 1.0 cm from this interface. The results of 10 separate experiments, together with release data from two normal livers, are given in Table IV. It is shown that a greater volume of parenchymal cells is released from tumor-bearing than from normal livers. In addition, the closer the normal liver samples are to the tumor interface, the more readily are cells detached from them.

TABLE IV RELEASE OF '' NORMAL" LIVER PARENCHYMAL CELLS SURROUNDING 256 TUMORS AS FUNCTION OF DISTANCE FROM TUMOR INTERFACE

Distance from tumor edge

0 cm

0.5 cm 1 .O cni

Normal liver

Numbers released ( x 1,000) ~ S (n) E

Clump size @m) +SE (n)

324116 (65) 256i-20 (36) 2243Z17 (1 9)

42&0.6

350116 (84)

25+0.2 (400)

Volume ratio (cf. normal)

4.41I

(510)

43 &0.6 (306) 38k0.5 (204)

3.711 2.311 1 .o

DISCUSSION

The techniques used here to quantitate cell detachment are a refinement of those used by McCutcheon et al. (1948) and Weiss and Holyoke (1969). The observations reported above, as " Types

90

WEISS TABLE V

RELEASE OF PARENCHYMAL CELLS FROM NORMAL

No. released ( x 1,000)

10 m 10 m 40 m 40 m

~

S

(n) E

88f4.4 (10) 147110.9 (9) 327127 (10) 250k4.9 (10)

Clump diameter (rm) +SE

EXTRACT

Controls (C)

Experimental (E)

Shaken for

LIVER+ NECROTIC

(n)

31h1.2 (137) 32f1.4 (100) 46f1.8 (200) 51.33Z2.9 (100)

of cells detached” show that within the noted qualifications of technique, no obvious differences were detectable with respect to morphology and inferred viability, between the liver cells released in the various experiments, or between cancer cells released in the different experiments with tumor cylinders. The present results show that following standardized shaking, the volume of cancer cells released from tumor specimens varies considerably, depending on the region of the tumor from which the specimens are taken. The data given in Table I1 show that greater volumes are released from “ healthy ” regions of a tumor adjacent to central necrotic areas than from regions located a few millimeters further out, at the tumor periphery. Interestingly enough, these variations in detachment paterns within individual tumors are greater than those detected between the peripheral regions of tumors growing in four different anatomic sites (Table I). The facilitated release of constituent cancer cells from tumor cylinders is also observed after they are shaken in the presence of saline extracts of necrotic material (Table 111). These observations support the view that material diffusing from central necrotic regions of these tumors can facilitate the detachment of live tumor cells from them. It was also observed that a greater volume of parenchymal cells was released by shaking cylinders taken from livers surrounding growing cancers than from livers in non-tumor-bearing animals. In addition, the volume decreased with increasing distance of the liver cylinders from the tumor edge (Table IV). This distance-dependence strongly suggests that material diffusing out from tumors facilitates the mutual detachment of the normal cells surrounding them, which in turn indicates a weakening of the intercellular region. This weakening does not represent a complete dissolution of the intercellular region, because the cylinders of liver d o not fall apart spontaneously but require the application of mechanically generated force.

No. released ( X 1,000) SE (n)

Clump diameter @m)

60f3.5 (9) 2033~13.3(10) 16319.0 (10) 40k4.8 (10)

19h0.9 (127) 201-1.0 (100) 2 3 h l . l (200) 25.6h1.6 (100)

+SE

Voiume ratio E’C

(n)

311 311 18/1 5011

Neither the respective relative contributions to cell detachment of the viable and the necrotic regions of the tumor tissues nor those of the “ normal ” tissues surrounding them can be determined from the present data. Thus, although it is seen (Table V) that necrotic extracts alone can facilitate the detachment of normal liver cells, and it would be surprising if this material played no role in vivo, it is well known that healthy tumor cells in vivo (Sylven and Bois ,1960; Sylven, 1968) and in vitro (Wu, 1959) release into their environment enzymes which can facilitate cell detachment (Weiss, 1965). However, regardless of the relative contributions of living cells and tissues to cell detachment, the experimental data clearly indicate that material originating from dead and/or dying cells within tumors is active in this respect. Other work (Weiss, 1965; Weiss and Holyoke, 1969) indicates that lysosomal activation may also facilitate cell detachment, through a process of sub-lethal autolysis at the cell periphery (Weiss, 1967). Thus, many tissue and/or cell interactions, including therapy, which result in cell damage and/or death and are characterized by lysosomal activation, are expected to facilitate homotypic or heterotypic cell detachment. Although preliminary assays indicate that the extracts of necrotic material are rich in lysosomal enzymes, it is not known whether in fact the observed facilitation of cell detachment was totally explicable in terms of the direct action of these and/or other enzymes. In addition, it must not be assumed that the detachment-activity of the necrotic extract is in whole or part derived from malignant cells or from surrounding normal tissues, although the experimental data (Tables I1 and IV) indicate that in intact tumors this activity appears to be diffusing out from their necrotic centers, rather than diffusing in. In the present context, it is emphasized that solid cancers consist of heterogenous cell populations, only some of which are malignant cells. For example, Alexander (1976) has stressed the considerable macrophage content of different tumors, and these and other cellular components of tumors can release enzymes affecting cell detachment (Weiss,

NECROSIS AND CELL DETACHMENT

19776). Thus, macrophages continuously secrete lysozyme, and following various stimuli may also secrete lysosomal acid hydrolases and/or neutral proteases (Davies and Allison, 1976). Once the diameters of many “ solid ” tumors exceed a few millimeters and/or following therapy, regions of necrosis commonly occur. Although cell death has often been considered as a major cause of cell “ loss ” in analyses of tumor kinetics (Steel, 1967, 1968; Iversen, 1967), the effects of tumor necrosis on the living cells composing and surrounding tumors have been largely overlooked (e.g. Cooper et al., 1975), with the notable exception of Revesz (1958). In this communication the interactions between necrotic material and normal and cancer cells have been assessed in terms of one parameter only, namely cell detachment, which is one of a number of factors thought to play a role in metastasis and invasion (Weiss, 1976, 1977a). Inherent to the basic concept of metastasis is the detachment of malignant cells from primary cancers to form non-contiguous secondary deposits (Willis, 1952; Weiss, 1967). While only a very small proportion of those cancer cells shed into the bloodstream for example, give rise to metastases (Fidler, 1976), and while the complexity of the metastatic process precludes direct and simplistic numerical correlations between it and cell detachment (Weiss, 1977b), by definition, metastasis cannot occur unless malignant cells are detached from primary tumors, and it is reasonably expected from the results of many animal inoculation studies that, within limits, the greater the number of malignant cells arriving at a site, the greater the chance of a metastasis developing there. The mechanism whereby cancer cells invade tissue is also not well understood at present (Strauli and Weiss, 1977). However, whatever mechanisms of invasion of the tissues by cancer are operative, it is to be expected that invasion will take place along the path of least mechanical resistance. This pathway may be along natural tissue planes or cavities, or through “solid” tissues; the latter may occur between cells and/or through them. The intercellular pathway would be favored by conditions weakening the material between cells, which in turn would

91

facilitate their separation from each other, as measured in the present experiments. Thus, from the restricted viewpoint of cell detachment, and regardless of mechanisms, it is expected that interactions between necrotic material and adjacent cancerous tissues will mobilize malignant cells, facilitating invasion and metastasis. In addition, the weakening of intercellular planes in surrounding normal tissues is expected to facilitate invasive processes into and through them. In contrast to work emphasizing the correlation between tumor growthrate and metastasis (Glucksmann, 1948; Slack et al., 1969), the present work raises the possibility that some parts of the metastatic process may be correlated with tumor death. However, growth of solid cancers, particularly rapid growth, is often associated with necrosis, therefore discrimination between the separate role of one or other of these opposite processes is somewhat artificial, and the old term “ necrobiosis ” (Virchow, 1860), used at the tissue rather than the cellular level, appears more appropriate in this situation. Some 280 different morphologic types of human tumor are recognized, and the biologic behavior of many of these types varies both between and within these different histologic groups. It is hardly necessary to emphasize that the present results were obtained on an allogeneic system, the Walker 256 tumor grown in rats. Thus, while it seems likely that the results and interpretation are of general applicability, this is by no means certain. In this respect it is noteworthy that patterns of necrosis vary with different types of cancer, from the central necrosis observed here and in many other solid tumors, to the peripheral necrosis described by Rubin and Casarett (1966) in some lymphoid cancers. ACKNOWLEDGEMENTS

My thanks are due to Ms. J. Holmes, Ms. D. Lombard0 and Mr. D. Graham for their assistance, and to Dr. T. Chu and Ms. J. Ciszkowski for the enzyme determinations. This work was partially supported by Grand No. PDT-14 from the American Cancer Society Inc., and Core Grant No. CA-I7609 from the National Institutes of Health.

NECROSE TUMORALE ET DETACHEMENT DES CELLULES Les cellules qui se detachent des tumeurs 256 de Walker inoculks ii des rats ou des regions adjacentes ont Bte mesur6es au moyen d’une technique normalis& d’agitation in vitro. Le volume des cellules qui se detachent de tumeurs de taille equivalente est pratiquement le m%medans les tumeurs du foie et de la rate ou de localisations intramusculaires et sous-cutanbes. Dam les tumeurs kystiques, il y a davantage de cellules qui se detachent des regions internes des parois adjacentes au materiel nkrotique que de cellules qui se d6tachent des parties exterieures. Les cellules parenchymateuses du foie qui se dbtachent d’une region adjacente a une interface tumorale sont plus nombreuses que celles qui proviennent d’une region distante de 0.5 ou 1 cm. Le detachement des cellules tumorales et hepatiques est Bgalement favorid par une incubation prtalable des sp6cimens de tissu avec des extraits nkrotiques. L’auteur suggkre que les regions necrotiques des tumeurs et les produits qui en proviennent facilitent le dttachement des cellules tumorales et des cellules composant les tissus normaux qui les entourent, favorisant ainsi potentiellement les metastases et l’envahissement.

92

WElSS REFERENCES

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SLACK,N. H., BLUMENSON, L. E., and BROSS,1. D. J., Therapeutic implications from a mathematical model characterizing the course of breast cancer. Cancer, 24, 960971 (1969). STEEL,G. G., Cell loss as a factor in the growth rate of human tumours. Europ. J . Cancer, 3, 381-387 (1967). STEEL,G . G., Cell loss from experimental tumours. Cell Tissue Kinet., 1, 193-207 (1968). STRAULI,P., and WEISS, L., Cell locomotion and tumor penetration. (Workshop Report). Europ. J. Cancer, 13, 1-12 (1977). SYLVEN, B., Lysosonial activity in the interstitial fluid of solid mouse tumor transplants. Europ. J . Cancer, 4,463-474 (1968). SYLVEN,B., and Bois, I., Protein content and enzymatic assays of interstitial fluid from some normal tissues and transplanted mouse tumors. Cancer Res., 20, 831-836 (1960). ViRcnow, R., Cellular pathology as based upon physiological and pathological histology. Translated from 2nd Ed. by F. Chance, p. 318, Churchill, London (1860). WEISS,L., Studies on cellular adhesion in tissue culture. VIII. Some effects of antisera on cell detachment. Exp. Cell Res., 37, 540-551 (1965). WEISS,L., The cell periphery, metastasis, and other contact phenomena, pp. 92-98, N. Holland, Amsterdam (1967). WEISS,L. (ed.), Fundamentalaspects of metastasis, N. Holland, Amsterdam (1976). WEISS,L., A pathobiologic overview of metastasis. Seminars in oncology, 4, 5-17 (1977~). WEBS, L., Cell detachment and metastasis. Gann (in press) (1 9776). WEISS,L., and HOLYOKE,E. D., Some effects of hypervitaminosis A o n metastasis of spontaneous breast cancer in mice. J. nut. Cancer Inst., 43, 1045-1054 (1969). WILLIS,R. A., The spread of tumours in the human body, Butterworth Co., London (1952). Wu, R., Leakage of enzymes from ascites tumor cells. Cancer Res., 19, 1217-1222 (1959).

Tumor necrosis and cell detachment.

Int. J. Cancer: 20, 87-92 (1977) TUMOR NECROSIS AND CELL DETACHMENT Leonard WEISS Department of Experimental Pathology, Roswell Park Memorial Institu...
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