influence of cultivation, trypsinization and aggregation on the transplantability of melanoma b16...

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Int. J. Cancer: 11, 663-675 (1973) INFLUENCE OF CULTIVATION, TRYPSINIZATION AND AGGREGATION ON THE TRANSPLANTABILITY OF MELANOMA B16 CELLS1 Bjorn HAGMAR and Klas NORRBY Department I of Pathology, University of Goteborg and Department II of Pathology, University of Linkoping, Sweden To elucidate the usefulness of tumour cells grown in vitro f o r transplantation studies, Melanoma B16 was grown in monolayer culture. Three types of suspensions were prepared from the cell cultures: two virtually monocellular suspensions, one produced by mechanical procedures and Versene ( C V ) , the other with the addition of trypsini- zation ( C T V ) ; a third suspension containing cell aggregates ( C V A ) was produced by centrifuging a C V suspension. For comparison, a well-dissociated suspension of solid tumor origin was prepared by a trypsin- Versene procedure ( S T V ) . AN suspensions were characterized with regard to dissociation grade, cell volumes and cellular electro- phoretic mobility and were transplanted in graded cell doses subcutaneously, intraperi- toneally and intravenously. While inoculurn-doses needed for progressive growth were lower IP than SC, the transplantabilities were similar in these sites for all suspensions, indicating similar viabilities and absence of major dilution errors. The growth patterns for I V infused cells exhibited significant diferences between the suspensions, C V cells gave rise to lung tumours almost exclusively, while CVA animals, in addition to a large total lung tumour volume, had some extrapulmonary takes. C T V had the greatest number of extrapulmonary tumours, but in combination with small lung tumours, S T V cell recipients were intermediate with respect to pulmonary as well as extrapulmonary turnours. These observations were collated with data regarding aggregation, cell volumes and electrophoretic mobility. It was concluded that (1) cultivated tumour cells can have similar transplantation characteristics to the tumour of origin: (2) such cells may be useful in turnour dissemination studies; (3) trypsinization as well as the presence of cell aggregates aflects the growth pattern of I V infused tumour cells by yielding an increased extrapulmonary tumour crop. In tumour transplantation studies it is often desirable to use highly viable tumour-cell sus- pensions of a defined, high-dispersion grade (Norrby et al., 1966; Boeryd et al., 1971b; Knutson et al., 1971). A little-exploited possi- bility of obtaining such suspensions is to use tumour cells grown in vitro. If no major biological alterations occur in vitro, such suspensions might offer some advantages over other alternatives, e.g. ascites tumours and enzymatic suspensions of solid turnours, viz: (1) availability in suspen- sions of tumours which do not lend themselves to ascites transformation or enzymatic digestion; (2) a more uniform cell population, without contaminating cell types; (3) higher growth fraction which makes it easier to label the population with radioactive DNA-precursors ; (4) means of " synchronizing " cells; (5) less harmful and less varying procedures for cell separation (compared to solid tumours). Received: December 27, 1972, and in revised form March 12, 1973. This work was supported by grants from the Swedish Cancer Society. 663

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Int. J. Cancer: 11, 663-675 (1973)

INFLUENCE OF CULTIVATION, TRYPSINIZATION AND AGGREGATION ON THE TRANSPLANTABILITY

OF MELANOMA B16 CELLS1

Bjorn HAGMAR and Klas NORRBY Department I of Pathology, University of Goteborg and Department II of Pathology,

University of Linkoping, Sweden

To elucidate the usefulness of tumour cells grown in vitro for transplantation studies, Melanoma B16 was grown in monolayer culture. Three types of suspensions were prepared from the cell cultures: two virtually monocellular suspensions, one produced by mechanical procedures and Versene ( C V ) , the other with the addition of trypsini- zation ( C T V ) ; a third suspension containing cell aggregates ( C V A ) was produced by centrifuging a C V suspension. For comparison, a well-dissociated suspension of solid tumor origin was prepared by a trypsin- Versene procedure ( S T V ) . AN suspensions were characterized with regard to dissociation grade, cell volumes and cellular electro- phoretic mobility and were transplanted in graded cell doses subcutaneously, intraperi- toneally and intravenously. While inoculurn-doses needed for progressive growth were lower IP than SC, the transplantabilities were similar in these sites for all suspensions, indicating similar viabilities and absence of major dilution errors. The growth patterns for I V infused cells exhibited significant diferences between the suspensions, C V cells gave rise to lung tumours almost exclusively, while CVA animals, in addition to a large total lung tumour volume, had some extrapulmonary takes. CTV had the greatest number of extrapulmonary tumours, but in combination with small lung tumours, S T V cell recipients were intermediate with respect to pulmonary as well as extrapulmonary turnours. These observations were collated with data regarding aggregation, cell volumes and electrophoretic mobility. It was concluded that (1) cultivated tumour cells can have similar transplantation characteristics to the tumour of origin: ( 2 ) such cells may be useful in turnour dissemination studies; (3) trypsinization as well as the presence of cell aggregates aflects the growth pattern of I V infused tumour cells by yielding an increased extrapulmonary tumour crop.

In tumour transplantation studies it is often desirable to use highly viable tumour-cell sus- pensions of a defined, high-dispersion grade (Norrby et al., 1966; Boeryd et al., 1971b; Knutson et al., 1971). A little-exploited possi- bility of obtaining such suspensions is to use tumour cells grown in vitro. If no major biological alterations occur in vitro, such suspensions might offer some advantages over other alternatives, e.g. ascites tumours and enzymatic suspensions

of solid turnours, viz: (1) availability in suspen- sions of tumours which do not lend themselves to ascites transformation or enzymatic digestion; (2) a more uniform cell population, without contaminating cell types; (3) higher growth fraction which makes it easier to label the population with radioactive DNA-precursors ; (4) means of " synchronizing " cells; ( 5 ) less harmful and less varying procedures for cell separation (compared to solid tumours).

Received: December 27, 1972, and in revised form March 12, 1973. This work was supported by grants from the Swedish Cancer Society.

663

HAGMAR AND NORRBY

Since enzymatic treatment is not required to dissociate cells cultivated in vitro, such cells may also be used for elucidating what enzymatic treatment per se does to the transplantability of tumour cells at different anatomical sites. For enzymes, apart from having possible direct cell effects, may also affect the relationship between the cells and the host in several ways. In the case of intravascularly injected cells, their surface characteristics may also to some extent determine the distribution of the cells in the body (Wilkins, 1967a, b ; Hagmar, 1972). Since trypsinized cells are often used in experimental metastasis studies, it would be relevant to determine whether and to what extent trypsinization itself determines the metastatic pattern. The same is true for the dispersion grade, i.e. whether the presence of cell aggregates affects metastatic growth (cf Watanabe, 1954).

Such questions prompted the present study with syngeneic Melanoma B16, used in its solid, serially transplanted form (S) and as a monolayer- cultivated cell line (C). C-cells were suspended with and without trypsinization, and in one type of C-suspension aggregates were induced. The suspensions were characterized with regard to dispersion grade, cell size and cellular electro- phoretic mobility. The transplantability was tested subcutaneously (SC), intraperitoneally (IP) and intravenously (IV).

MATERIAL A N D METHODS

The Melanoma B16 turnour was purchased in 1966 from the Jackson Laboratory (Bar Harbor, Maine, USA), with litter-mate pairs of the inbred C57Bl/J6 mouse strain. It has since been trans- planted subcutaneously in such mice. The cell suspensions used in the transplantation experi- ments emanated from a tumour in generation 126.

A cell line was derived from explants of the tumour in generation 115, and maintained in Minimum Essential Medium, MEM (Eagle, 1959) with 10% non-heat-inactivated newborn calf serum. The cultivation technique has previously been described in detail (Norrby, 1970).

Cultivated cells frozen in liquid nitrogen were thawed and subcultivated twice before use in experiments. One hundred and fifty thousand &50,000 cells were seeded per plastic Petri dish (50 mm diameter; Falcon Plastics, Los Angeles,

USA). Each culture received 5 ml growth medium, mentioned above, and the medium was changed 48 h after subcultivation. The cells were dissociated and used when they were multiplying exponentially, about 72 h after subcultivation.

Suspensions of cell cultures To obtain cell suspensions of maximal degree

of dissociation the following steps were taken: A. Two ways of dissociating the cells: (V)

Versene (ethylene diamine- tetra-acetic acid) 0.02% (w/v) in salt solution (Paul, 1959) free from Ca++ and Mg++ and buffered to about PH 7.4; (TV) Trypsin Difco 1:250 0.25%- Versene 0.02% (w/v) in the same buffer as in (V). This trypsin will convert 250 times its own weight of casein to proteoses, peptones and amino acids, under the N.F. assay for pancreatin; it contains small amounts of other pancreatic enzymes such as chymotrypsin, amylases, lipases, elastases and carboxypeptidases.

The dissociating procedure consisted of the following steps: (a) one rinse of the culture dishes with V or TV; (b) addition of 2 ml of V or TV; (c) after 2.5, 5 , 10, 15 or 20 min the cells were dissociated by pipetting (vide infra); (d) the suspensions were centrifuged at 4" C for 5 min at approx. 80xg; (e) the supernatant was dis- carded and the cells were resuspended in nutrient medium containing 10% serum; the suspensions thus dissociated were designated CV and CTV. For transplantation experiments syngeneic serum was used.

The dissociating procedure with V regularly yielded more cells than with TV; the cells were more easily damaged by TV in terms of cell loss due to cell rupture. Exposure of the cultures during 2.5 and 5 min to either V or TV resulted in maximal cell yields; longer exposure resulted in cell loss due to rupture.

B. Pipetting in order to dissociate the cells in the culture dish was standardized, 25 ins and outs being used. Extensive pipetting resulted in great cell losses (in sister cultures) whereas fewer pipettings often resulted in an insufficient break-up of aggregates. C. To dissolve ajilamentous gel-like substance-

probably deoxyribonucleoprotein emanating from ruptured nuclei (cf Norrby et a/., 1966)- entrapping cells, DNase (2 x crystallized; Nutri- tional Biochemical Corporation, Cleveland, Ohio, USA) was used in concentrations >60 pg/ml.

664

TRANSPLANTABILITY OF MELANOMA s16 CELLS

D. Filtration of cells was effected by vacuum aspiration through sintered glass filters (obtained from James A. Jobling & Co., Ltd., Sutherland, Scotland) of various pore sizes. The filtration was made necessary by the occurrence of fairly large cell aggregates in some STV suspensions. G2 filters with a stated pore size of 60-90pm were found most suitable; filters with finer pores retained a large proportion of cells. In order to treat the suspensions of cultivated cells similarly to the enzymatically produced cell suspensions from the solid tumour (vide infra) all suspensions were passed through G2 filters and subjected once again to centrifugation and resuspension. This procedure did not noticeably alter the degree of dissociation in the CV or CTV suspensions.

Each step in the procedure was monitored by two independent methods for estimating the presence of cell aggregates (cf Norman and Norrby, 1971). A formaldehyde-fixed sample (Norrby, 1970) of the suspensions was filtered onto a Millipore filter with pore size 5.0+1.2 pm for subsequent staining according to Papanicolaou and microscopic examination (at approximately 300 to 500x magnification) of numbers and sizes of aggregates. Another sample of the same suspension was analyzed in an electronic particle counter (Celloscope 302, AB L. Ljungberg & Co., Stockholm, Sweden). Information on the effect of the fixative on the resistivity of the suspension, on instrument error, and the representativeness of the counted samples has been published else- where (Hagmar and Norrby, 1970; Norrby, 1970).

Aggregation was induced in samples of CV suspension by centrifugation at approximately 100 xg for 10 min. The aggregates in CVA suspensions could not be wholly broken up by pipetting. The aggregate-containing suspension was designated CVA (where A stands for aggre- gation).

On the Millipore filters thousands of particles were microscopically examined in each sample. The number of aggregates (per 1,000 particles) in those suspensions actually used for the transplan- tation experiments is shown in Table I and the particle size distribution in Figure 1 . Cells in late mitosis-anaphase and telophase-were included among two cell aggregates.

Suspension from solid tumour

A procedure was used for enzymatic produc- tion of cell suspension employing trypsin Difco

TABLE I

PRESENCE OF SINGLE CELLS AND CELL AGGREGATES OF VARIOUS SIZES PER 1,OOO

PARTICLES

Number of aggregates containing Type of Number of the following cell numbers

suspension single cells 6- 16- 10 IS 20

~~ ~

CTV 916 73 1 1 cv 894 89 17 CVA 466 170 86 17 22 79 56 44 STV 886 88 14 7 5

1 :250 (0.25 % w/v) and DNase for approximately 90 min, and resuspension of the end product in chemically defined medium containing extra amounts of DNase and serum (Norrby et al., 1966; Knutson et al., 1971). Many of the suspen- sions thus produced from the solid Melanoma B16 tumour contained a small number of rela- tively large aggregates, consisting of up to approximately 40 cells. Considerably fewer and smaller aggregates were obtained by introducing further modifications : an additional treatment with trypsin-Versene, as employed for the CTV cells; the cells in the end product were sedimented by centrifugation (80 xg, 5 min), resuspended and agitated by repeated pipetting in TV-solution (vide supra) at 37" C.

After this exposure the cells were centrifuged once more (80 xg, 5 min) and resuspended in ordinary Parker 199 containing 60 pg/ml DNase and 5 % serum. Syngeneic serum was used for transplantation experiments. Any large persisting aggregates were eliminated by passing the suspension through a sintered Jobling G2 glass filter. Although the transplantation experiments required an additional centrifugation to obtain an adequate cell concentration (vide infra), the suspension actually used for transplantation contained only a few small aggregates (Table I and Fig. 1).

By the above procedures the cells used in vivo consequently were washed twice after trypsiniza- tion by centrifugation and resuspension in serum-containing medium (MEM or Parker). AS final medium for injection Parker (95 %)-syn- geneic serum (5 %) was used throughout (Boeryd et al., 1971~).

665

HAGMAR A N D NORRBY

v(mean volume, p m 3 ) 2S.E. : 1618+

, 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

30 CVA

I5 c t S.E. : 3660 2 63

10 i i i i 1 ik 18 2’0 122 24 26 1 zi 3’0 3’2 34 3k 3’8

Diameter in pm

1767 5060 10192

Volume in pm3

FIGURE I Relative size distribution of Melanoma B16 tumour cell suspensions used

for transplantation. 5,OOOrt 1,000 particles per suspension were analysed in a Celloscope 302.

STV designates an enzymatically produced cell suspension from the solid tumour, finally exposed to trypsin (0.25 %, Difco I :250) and Versene (0.02 %). CTV is a suspension of cultivated cells dissociated with trypsin (0.25 %, Difco I :250) and Versene (0.02%). CV is a suspension of cultivated cells dissociated by Versene (0.002%) only. CVA is a sample of the CV suspension where aggre- gates were induced by centrifugation

CTV, CV and CVA cells are considerably larger than the STV cells, whose suspension contains smaller cells and aggregates, than the CV and CVA suspensions. In comparison with the CV suspension the distribution of particles is wider in the CVA suspension which contains a considerable number of aggregates (Table I) and more particles in size levels above 21.3 ,urn in dia- meter (5060 pm3). The aggregation reduces the total number of particles and shifts their sizes mainly from the modal and the paramodal sizes toward larger size levels. The differences in mean particle size are significant between all suspensions (p.<O.OOi ; Student’s-test.

Three arbitrary particle volumes, corresponding to three particle diameters (15.0, 21.3, 26.9 pm) are shown at the bottom of the Figure.

Cell electrophoresis T h e lneasurements were made in a cylindrical

cell apparatus (Bangham et al., 1958) a t 25 f0.5” C with a voltage gradient of 3.45 V per c m between grey sintered platinum electrodes. Twenty cells were measured in both directions before a n d

after reversal of the current. T h e determinations were made in R ~ k e r Or Parker (95 %)-serum (5 %)

The results are shown in Table 11. Untryp- sinized cultivated cells (CV) had t h e highest mobility, i.e. the greatest negative surface charge

666

TRANSPLANTABILITY OF MELANOMA ~ 1 6 CELLS

TABLE 11 RESULTS ELECTROPHORETIC MOBILITY OF CTV, CV

A N D STV CELLS IN PARKER 199 (P) OR PARKER The incidence and time of appearance of palpable SC tumours are plotted in Figure 2. (95%) SERUM (5%) (PS) MEDIUM Figures 3 and 4 depict the time and incidence of spontaneous death from IP and IV cells.

The tumour incidence, as well as the time of Electrophoretic

wm/v/crn ( f SD) Cell type Suspension mobility medium

CTV P 1.02 (10.15) CTV PS 1.09 (+0.11)

CV P 1.33 (10.19) cv PS 1.30 (i0.16) STV P 1.06 (10.12) STV PS 1.15 (+0.20)

density. Trypsinized cells, whether from cells grown in vitro (CTV) or from the solid tumour (STV) had similar but lower mobilities. No statistical differences (Student’s t-test) were obtained between cells in medium with and without serum.

Transplantation studies

The transplantability of four different cell suspensions was tested viz. STV (Solid-Trypsin- Versene), CTV (Cultivated-Trypsin-Versene), CV (Cultivated-Versene) and CVA (Cultivated- Versene- Aggregates).

The cell numbers were estimated from haema- cytometer counts, made in triplicate to minimize sampling errors. All injections were of 0.1 ml from agitated suspensions kept on ice. From dilution series in Parker (95%) serum (5%) of each type of suspension 5 x104, lo4, lo3 and 102 cells were injected SC or IP into groups of four animals (20, 2?). Intravenously, i.e. in the exposed jugular vein, 1.8 x lo5, lo5, 5 x lo4, lo4 and lo3 cells were given to groups of six animals

The hosts were observed daily for 4 months. Animals with SC transplants were observed for palpable tumours. The time of spontaneous death was noted for animals with IP and IV transplants. Autopsies were carried out without delay and the gross tumour extent was recorded. Lungs and livers from mice injected IV, as well as any doubtful tumour, were examined histologically. The organs were prepared and sectioned accord- ing to Boeryd et al. (1965). Before paraffin embedding, the lungs were weighed to quantify the tumour mass (Hagmar and Boeryd, 1969).

(30, 353.

appearance of tumours or spontaneous death, show a positive correlation with the cell dose given for all suspensions in all transplantation sites. As will be described, however, there were differences in take frequencies between the transplantation sites.

Subcutaneously (Fig. 2) all suspensions of 5 x lo4 cells grew in all animals, while lo4 cells grew in a varying number of animals up to 4/4 for STV. Occasional animals became positive with still lower cell doses from all the C suspen- sions.

The STV tumours appeared somewhat earlier than the C tumours.

Intrapcritoneally (Fig. 3) 5 x lo4 and lo4 cells took in all animals. The STV and CVA suspen- sions took in 4/4 animals with lo3 cells, and all groups developed one or more positive animals also with the lo2 cell dose.

All suspensions began to kill hosts at approxi- mately the same time, the times for spontaneous deaths being more concentrated for STV than for C groups.

Intravenously (Fig. 4) lo3 cells only gave takes in a few, randomly distributed animals. Ten times more cells, however, gave fatal tumours in 616 CVA and STV animals, and 416 CTV and CV animals. As in the SC and IP groups the different suspensions thus showed similar trans- plantabilities IV.

All suspensions were also similar in giving higher take-frequencies IP, i.e. growing from smaller cell inocula, than SC or IV. This point emerges from Table 111, where the incidences of tumours are shown for the dose interval 1O3-5x1O4, which was common for all three transplantation sites. When takes from all suspensions are combined, there is a highly significant preferance for tumour growth IP over SC (p <0.005) and IV (p <0.005) with Chi-square analysis. These two latter loci did not differ in tumour incidence. There was a similar differ- ence between SC and IP transplantability (p<0.0005) for all doses (102-5 x lo4 cells) combined.

667

HAGMAR AND NORRBY

rnll.

I

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mice surp.n,ion CI" , p - 1 . ~ 0 4 L . ~ ~ l

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..... .i.. ... ~ . _ , , I

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. I :-..I .......................... L I.. I____..

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0 I 4 18 2 2 26 10 I4 18 4 2 46 I 0 I 4 D n 7 i

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101

FtGURE 2 Incidence and time of appearance of palpable

tumours after transplantation of different cell doses stibeutaneously .

...............

. . , 10 14 1 8 32 16 4 0 4 1 48 I2 56 ? O 7 1 78 8) Bb 90 D v r i

. . . . . . . . . . . . . . . . = . . . . . . . . . . . I

I . , 10 1 4 2 8 32 l b 40 44 48 5 2 5 6 lb 74 7 8 81 8b V O O w r

. . . . . . . . . . . . . .:. . . . . . . . . . . . 70 74 1 8 12 36 40 44 48 51 56 70 74 7 8 82 86 90 Dnyl

FIGURE 3 The incidence and time of spontaneous death after

transplantation of different cell doses intruperi- toneally.

668

TRANSPLANTABILITY OF MELANOMA B l 6 CELLS

1

0 1 1 , , , , , , , , , , 1 ' , , , I 20 2 4 28 3 1 36 4 0 44 4E 52 56 60 64 80 84 88 Do",

TABLE 111

INCIDENCE OF TUMOUR TAKES IN THE SUBCUTANEOUS (SC), INTRAPERITONEAL (IP),

A N D INTRAVENOUS (IV) INJECTION SITES FOR

(SEE TEXT) THE DOSE INTERVAL 1OS-5 X 10' CELLS

Incidence of turnours

sc IP IV

Type Of from cells injected suspension

CTV 8/12 11/12 12/18 cv 5/12 9/12 11/18 CVA 8/12 12/12 1311 8 STV 9/12 12/12 14/18

5u,panr,on C" , * '"'cc t

~ ____ ~ _ _ _ _ ____

Total incidence 30148 44/48 50172

, - . .

Even though the IV tumorigenicities were similar for the various suspensions, the distribu- tion of the resulting tumours was nevertheless characteristically dissimilar (Table IV). The lung tumours were recorded as organ weight, since it was confirmed histologically that metastases alone had enlarged the organ.

In the livers only a few microscopical metas- tases were found: STV 1 animal given 1.8 x lo5 cells, CTV 1 with lo5, CTV 1 with 5 x lo4, CV 2 with 1.8 x lo5 and CV 1 with lo5 cells.

The lung weights, i.e. the tumour masses, were largest and similar in the CV and CVA groups, smallest in the CTV group. The differences between the two former and the latter were significant (p t0.001) with Student's t-test. The STV group was intermediate, differing from CTV (p <0.05) but also from CV (p <0.005) and CVA (p <O.OOOl). The metastases were visible to the naked eye in all groups, large and con- fluent in the CV and CVA groups, small and scattered in the CTV group. Characteristically, the S-metastases were unpigmented, while the C-metastases more often than not were pigmented (Fig. 5 and 6).

The lung tumours were smallest in CTV animals, but the number and incidence of gross metastases in extrapulmonary organs were greater in this group than in any other (p <0.01- 0.001). The CVA and STV suspensions were similar in these respects, but compared to them the CV suspension gave a still smaller incidence of extrapulmonary tumours (p <0.05).

The survival times of the different groups were dose-dependent, but similar for those receiving

669

HAGMAK AND NORRBY

FIGURE 5 FIGURE 6 Tumour growth (" metastasis ") in the lung from Tumour growth (" metastasis ") in the lung from

STV cells injected intravenously; specimen from a CTV cells injected intravenously; specimen from a spontaneously dead animal. The tumour is profusely spontaneously dead animal. Note the presence of infiltrating the lung parenchyma. Note the absence heavily pigmented tumour cells. V. Gieson stain, of pigment. V. Gieson stain, approximately x 600. approximately x 600.

any of the C-suspensions. This is true whether all animals are included, or when calculated only on those animals which received 2 5 x lo4 cells (to avoid influences on the mean survival time by varying take-frequencies in animals receiving smaller cell doses). However calculated, the mean survival times were shorter in the STV groups than in any other.

Evaluation of survival time and tumour distribution was somewhat complicated, a t least for the STV group, by tumour growth o n the neck in some animals at the site of injection in the jugular vein (Table IV). In such animals the recorded parameters might have been affected, e.g. by ( I ) loss of tumour cells a t injection, (2) secondary spread from tumours growing around the vein, and (3) shortened survival

owing to the neck tumours. However, animals with and without neck tumours had similar lung weights in all groups and similar numbers of extrapulmonary tumours. In the whole series animals with neck tumours had a shorter mean survival time (33 days) than those without (39 days). In the only group (STV) where neck tumours could have affected the statistical evaluation, however, the survival time was only marginally shorter for animals with tumours a t the injection site (29 versus 31 days).

DISCUSSION

The cell cultures (C-cells) were dissociated in a phase of exponential multiplication with the growth fraction (Mendelsohn, 1960) essentially

670

TRANSPLANTABILITY OF MELANOMA B I 6 CELLS

TABLE IV

SURVIVAL TIMES AND TUMOUR PARAMETERS AFTER INTRAVENOUS CELL INJECTION

Mean survival Extrapulmonary Tumour

Type of suspension Tuniour time of Mean lung weights metastases growth at and cell number per dose incidence positive g*S.D. injection

animals Incidence Total No. site (days)

CTV 1.8 x 105 616 35 0.27 (+0.03) 516 13 I I6

10 216 I 05 516 37 0.28 (10.07) 415

33 0.26 (+0.11) 516 5 x 104 616

65 0.27 (10.14) 212 5 012 I 0 3 216

4 115

52 0.50 ( t0.53) 214 2 1 14

5/23

I 04 416

All dose levels 23/30 40 0.31 (*0.23) 18/23 34

CV 1 . 8 ~ 1 0 ~ 616 31 0.95 (+0.32) 116 1 216 I 0 5 616 36 0.54 (+0.30) 116 1 216

0 216 5 x 104 616 104 416

0 o/ 1 2 8/23

I 03 116 0 214

36 0.77 (k0.46) 016

65 0.72 (t - ) 01 1 39 0.80 (+0.41) 2/23

54 1.06 (+0.55) 014

A l l dose levels 23/30

2 316 7 016 3 316 0 016 1 01 1

CVA 1 . 8 ~ 1 0 ” 616 30 0.67 (t0.31) 216 I05 616 36 1.07 (10.28) 316

104 616 42 1.03 (+0.15) 016 103 116 83 0.80 (_ t - ) 111

5 x lo4 616 39 0.96 (+0.46) 316

6/25 All dose levels 25/30 38 0.93 (*0.33) 9/25 13

25 0.33 (t0.08) 416 7 416 8 516 0 416

STV i . 8 ~ 105 616

5x104 616 I 0 4 616 103 216

10’ 616 27 0.45 (*0.28) 416

3 316 0 012

18 16/26

32 0.56 (10.35) 016 33 0.37 (*0.18) 216 41 1.14 (10.41) 012

10126 All dose levels 26/30 30 0.48 ( i0.31)

unity (Norrby, unpublished). All cell cycle phases should be present and probably distributed approximately according to the general model of Steel and Hanes (1971). We lack relevant data on the duration of the cell cycle phases in either C- or S-cells but we know that mitosis probably is under-represented in the STV suspensions (cf Boeryd et a/., 1965). The growth fraction in the solid tumour is as yet unknown.

These basic kinetic data are important in the cell size analysis, since the size distribution is determined by the true variability in size of cells as well as by their position in the cell cycle, the increase in volume being 2-3 times from early telophase to metaphase (Terasima and Tolmach, 1963; Prescott, 1964; Cone, 1969). When sizing cells electronically, which probably is more precise than optical measuring (Anderson and Petersen, 1967), size has been reported to equal volume xshape factor (Grover et a/., 1972); also

differences in deformability of the cells are of importance. Undoubtedly the cultivated cells were considerably larger than the STV cells (Figure I), which agrees with Moore et al. (1968).

The transplantability of tumour cells has been variously affected by in vitro cultivation in previous studies (Surveys : Foley and Drolet, 1964; Hu and Lesney, 1964; Moore et al., 1968; Jacobs, 1970). In the present study with Melanoma B16 an early cell line in v i m (C tumour) had in most respects similar trans- plantation characteristics in syngeneic animals as enzymatically dissociated cells from the solid, serially transplanted (S) tumour. The C tumour only had a longer latency before appearing as tumours SC and IV, indicating an adaptation period in vivo before growth. TP, however, the latent periods were similar for the C and S forms.

For C cells the transplantabilities at all sites were similar whether the cells were brought into

67 1

HAGMAR AND NORRBY

suspension by mechanical means and Versene (suspensions CV and CVA) or by such procedures combined with trypsinization (CTV). Similarly, d itrerences in aggregability between Versene- dissociated cells (CV vc’rsus CVA) were not reflected in any altered transplantability SC or I P.

Cultivated melanoma B16 cells thus seem to preserve their transplantability when denuded of trypsin-sensitive surface material (as revealed by electrophoretic mobility) and administered singly rather than in groups. These findings must be borne in mind when interpreting the IV tram- plantation test. For if tumour-cell viabilities, defined as SC and IP transplantability, were similar for all suspensions used, differences in tumour patterns from TV infused cells must rather be ascribed to other factors, as discussed below.

I t is not clear why the melanoma cells had a higher transplantability IP than SC and IV. With regard to the difference in transplantability between SC and IP our results disagree with findings obtained in an earlier study with Melanoma B16 and with an MC-induced mouse sarcoma (Boeryd et a/., 1971b). On that occasion the trypsin t DNase treated suspensions from the B16 tumour, in generation 70, turned out to be practically monocellular and the cells were not exposed to Versene nor did they pass a glass filter. With other tumours Vaage et a/. (1971) found a preference for the IP site, while for instance Wexler et a/. (1969) noted a higher take-frequency IV than intramuscularly. Probably the ’‘ favourite soil ” differs among tumours, reflecting the interaction between the tumour cells and their immediate neighbourhood. Presumably t h e transplantability may vary even with the age and the treatment of the tumour.

If the IP site is poorly defended immunologi- cally (McCarthy, 1968), this may be of some importance provided the animals raise any significant immunological defence against this tumour. We have not yet assessed the antigeni- cities of the S and C forms of B16 cells, but indirect evidence (e .g . low inoculation dose, no tumour regression, absence of splenomegaly in tumour-bearing animals) does not suggest major antigenic discrepancies between the syngeneic hosts and either cell type. This and the similar transplantabilities might indicate a similar anti- genicity for in v i m grown cells as for their in vivo derived origin, although the former cells

may have acquired new surface antigens through genetic changes or antigen adsorption from the culture medium (Hamburger et al., 1963; Foley and Drolet, 1964; Franks, 1968; Moore et a/., 1968).

When injected IV, the presence of cell aggre- gates (CVA versus CV) surprisingly promoted the development of extrapulmonary tumours rather than inhibiting it. The reverse would have indicated an arrest of cell aggregates in lung capillaries. As i t is, one must assume that not only single cells but also cell aggregates passed through the lung vessels, presumably via arterio- venous shunts (Lawrence et a/., 1953). The individual cells of an aggregate are probably better protected against proliferation controlling and/or noxious influences, than single cells which are sparsely lodged in different organs, and thus provide their own favourable micro- milieu (Watanabe, 1954; Weiss, 1967). Tumours arising from more than one cell will of course also be larger than metastases arising from single cells which may not have attained recordable sizes at the termination of the experiment. However, considering the frequent occurrence of gross extrapulmonary metastases from the well- dispersed STV and CTV suspensions, this explanation seems less likely. In any case, the dispersion grade is obviously important and should be defined when experimental metastasis distribution is studied (cf Norrby ef a/., 1966).

Otherwise the most dramatic change in IV tumour distribution was constituted by the numerous extrapulmonary tumours in animals receiving trypsinized C-cells (CTV) compared to animals receiving CV and CVA suspensions. The concomitant decrease of CTV lung tumours indicates shunting of tumour cells and thereby metastases beyond the lungs. If this is not the case, one must postulate a growth restraint on CTV cells lodged on the lungs, but not on cells lodged elsewhere.

Assuming that a greater initial distribution of CTV cells from the lungs to other organs did occur, the underlying mechanism is still not clear. The results may be compared with a similar shift of metastases caused by polyanion-treatment of cells (Hagmar and Norrby, 1970; Hagmar, 1972), where it was surmised that altered surface properties and notably an increased negative surface charge density of the tumour cells would promote their transpulmonary passage. Trypsi-

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TRANSPLANTABILITY OF MELANOMA B I6 CELLS

nized B16 cells (CTV) had, however, even in the presence of serum (Wilkins, 1967b), a less negative surface potential than untrypsinized (CV) cells. So the mechanism surmised for polyanions, i.e. increased cellular electronega- tivity/decreased endothelial adhesiveness, cannot have been operative here.

The loss by trypsinization of some 20% of the cell surface charge indicates the dissociation of peptide-bound material carrying exposed, ionized negative surface groups. For with our washing technique and in the presence of serum, adsorp- tion of trypsin onto the cells seems unlikely (Weiss and Kapes, 1966; Poste, 1971). The loss of surface material, obviously not affecting the cellular viability, was further substantiated by a slight or moderate reduction of cell volumes as also found by Weiss (1958). A smaller mean cell volume of course may have favoured the lung passage. Our results show, however, that a reduction of this order of the cell volumes is of minor, if any, importance for the metastatic distribution. This is clear since STV gave fewer extrapulmonary tumours than CTV and since CVA gave more extrapulmonary tumours than cv.

Trypsinization has previously been shown to produce structural and functional alterations of the cell surface (for review, see Boeryd et al., 1968). If, for instance, such changes affect cellular deformability and/or mobility, they may also affect the retention of cells in vessels (cf Weiss, 1967). Another, less substantiated, hypo-

thesis would be that trypsin might affect more or less specific surface structures, determining where the cells will be retained in the body, in the same way as enzyme treatment may alter the “ homing ” of lymphocytes in lymphoid organs (Woodruff and Gesner, 1968, 1969). In any case, it must be concluded that trypsinization rather drastically altered the B16 metastasis pattern. So enzyme treatment, as frequently used in experimental metastasis studies, must also be regarded as a co-determinant of the resulting tumour crop.

Although there are potential drawbacks in using cultivated cells, e.g. altered transplantability (Moore et al., 1968; Jacobs, 1970) or antigenicity (Franks, 1958; Weiler, 1959; Hamburger et al., 1963; Moore et al., 1968; Kollmorgen et al., 1972), the results of the present study indicate that cells cultivated in vitro will be useful in future studies related to the spread of malignan- cies. For, summing up, the investigation on our Melanoma B16 tumour demonstrates that: (1) cultivated tumour cells can have similar transplantation characteristics to the tumour of origin; (2) the transplantability is higher in the intraperitoneal site than in the subcutaneous or intravenous sites, regardless of degree of cellular dispersion and of trypsin treatment; (3) cellular aggregation, like trypsinization, alters the pattern of tumour distribution following intravenous transfusion of tumour cells, since both presence of cell aggregates and trypsinization greatly increased the occurrence of “ metastases ” in extrapulmonary organs.

INFLUENCE DE LA CULTURE, D E LA TRYPSINISATION ET DE L’AGGLOMERATION SUR LA TRANSPLANTABILITE

DES CELLULES DE MELANOME B16

Afin de dkterminer l’utilitk des cellules tumorales cultivkes in vitro pour les ktudes de transplantation, les auteurs ont cultivk un mklanome B16 en couche unique. Trois types de suspensions ont ktk prtparts a partir des cultures cellulaires: deux scrspensions virtuellement monocellulairrs, I’une produite par des prockdts mtcaniques et du Versdne ( C V ) , I’autre avec en plus une trypsinisation (CTV) ; m e troisi2me suspension contenant des agrtgats cellulaires (CVA) a ktk produite par centrifugation d’une suspension CV. Pour la comparaison, une suspension bien dissocike provenant d’une tumeur solide a Ptk prkparke par un proctdt trypsine- Verstne ( S T V ) . Toutes les suspensions ont e‘tk carac- tkriskes en ce qui concerne le degrk de dissociation, Ies volumes cellulaires et la mobilith tlectrophorktique des cellules; elles ont ttk transplantkes en doses cellulaires graduies par voie sous-cutanke, intrapkritonkale et intraveineusr. Si les doses-inoculum ntcessaires pour une croissance progressive ktaient plus faibles par voie intraptritonkale que par voie sous-cutanke, lu transplantabilitk Ptait similaire pour toutes les suspensions, ce qui

673

HAGMAR AND NORRBY

dinote une viahilitk analogue et I’absence de grosses erreurs de dihtion. Les schhmas de croissance des cellules infushes par voie intraveineuse sont trBs diflhrmts selon les suspensions. Les cellules C V ont presque exclusivement donnh naissance a des tumeurs pulmonaires, alors que pour les CVA, on notait, outre un volume total klevh de tumeurs pulmonaires, quelques prises extra-pulmonaires. Les CTV donnaient le plus grand nombre de tumeurs extrapulmonaires, mais elles htaient comhinkes de petites tumeurs pulmonaires; les rtcrpteurs de cellules STV se classaient a un niveau intermkdiaire pour I P S tumeurs pulmonaires uussi bien qu’extrapulmonaires. Ces observations ont Ptt com- parkes avec les donnies concernant l’agglomkration, les volumes cellulaires et la mobilitk tlectrophorktique. Les auteurs en concluent que I ) les cellules tumorales en culture peuvent avoir des caractkristiques de transplantation analogues a celles de la tumeur initiale; 2 ) ces cellules peuvent Ptre utiles pour les dudes sur la disskmination tumorale; 3 ) la trypsinisation et la prhsence d’agrhgats cellulaires afectent la croissance des cellules tumorales infushes par voie intraveineuse en produisant davantage de tumeurs extrapulmonaires.

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