Cell Tissue Kinet. (1976) 9, 547-552.

CELL KINETICS OF A CHONDROSARCOMA

ANNE DAWSON A N D N. F. KEMBER

Physics Department, St Bartholomew’s Hospital Medical College, London

(Received 15 March 1976; revision receiced 17 May 1976)

ABSTRACT

The cell kinetics of the transplantable DC-I1 mouse chondrosarcoma have been studied by the pulse labelled mitoses method. The analysis gave the following estimates for the phases of the cell cycle: GI, 10.5 hr; S, 9.5 hr; G2, 4 hr with an intermitotic time of 23.5 hr. Consideration of the overall growth of the tumour indicated that the growth fraction and cell loss factor both had values of about 0.5. The results are compared with cell kinetic data from sarcomas and other cartilage tissues.

The DC-I1 chondrosarcoma arose spontaneously in the ST/Eh strain of mouse, and was transplanted by Englebreth-Holm in Copenhagen. A brief history of the tumour and pre- liminary studies were reported by Swarm (1963) and Swarm et al. (1964) who investigated its histology and growth characteristics. They were able to grow the tumour in four strains of mouse, and in animals from 7 weeks to 12 months of age. The tumour did not differentiate or produce calcified tissue. Since we have made extensive studies of normal growth cartilage (Walker & Kember, 1972), it was felt that a study of an abnormal cartilage system would provide valuable comparative results.

METHODS

The tumour was received as deep frozen tissue in DMSO from the cell and tumour bank of Microbiological Associates Incorporated (Bethesda, Maryland, U.S.A.). The tissue was thawed, washed with Medium 199 and implanted through a small incision into BAlb/C mice. Subsequently, the transplantation was simplified, and a small (roughly 1 mm3) piece of non-vascular tissue from the edge of a chondrosarcoma was injected by trocar through a minimal skin incision into the axilla region. A record was kept of the transplant sequence, and the experiments reported here were carried out between the second and fourteenth transplant of the tumour.

For measurements of overall tumour growth, transplants were made from 2-3-week-old tumours into ten mice aged 9-1 1 weeks, and into six mice aged 4-6 weeks. The size of the tumours was measured at 3 4 day intervals from the time the tumour became palpable

Correspondence: Dr N. F. Kember, Physics Department, St Bartholomew’s Hospital Medical College, Charterhouse Square, London EClM 6BQ.

547 35

548 Anne Dawson and N. F. Kember until the animal was killed at about 5 weeks after implantation. Tumours were measured along three perpendicular axes through the skin with calipers.

For studies of histological development, a series of 10-week-old mice were implanted with tumour. At intervals from 7 to 42 days after implantation, a sequence of host animals was killed at 1 hr after an injection of 1 pCi/g body weight of tritiated thymidine (Radiochemical Centre, Amersham, 5 Ci/mmole specific activity). After death, the tumours were excised, fixed in formol saline and embedded in paraffin wax. Sections were cut at 5 pm for routine histology or for autoradiography.

Sections for autoradiography were dipped in Ilford K5 emulsion, exposed for 5 weeks and processed with Kodak D-19 developer and Johnson’s Fixsol. The sections were stained with haematoxylin and eosin after development. All labelled nuclei displayed at least ten grains and an arbitrary limit of five grains per nucleus was set for a cell to be classified as labelied. Background was one or two grains per nucleus.

For investigation of cell kinetic parameters using the percentage labelled mitoses technique, a series of mice was taken at 14-21 days after tumour implantation and injected with 1 pCi/g body weight of 3H-TdR. At intervals between 1 and 48 hr after injection, mice were killed and the tumours removed to formol saline for autoradiography. Sections from these tumours were lightly stained with Cole’s Haematoxylin after development. The percentage of mitotic figures labelled with 3H-TdR was calculated from observation of at least 100 mitotic figures in each tumour.

RESULTS Overall growth

Tumour volume was expressed as the product of the three axial measurements (including skin thickness), that is, as the volume of the cuboid containing the tumour. These values were pooled for the mice from the 4-6-week-old group, and for the 9-11-week-old group, and were plotted as tumour volume against time. Volume doubling times were read off the smoothed curves and were found to be about 95 hr for both groups of mice over the range 0-8-2.0 cm diameter (Dawson, 1975).

Macroscopic and histological appearance The 14-day tumour appears as a 1 cm diameter greyish-white translucent mass, often

nodular, and occasionally with pockets of blood in the centre. Large haemorrhagic spaces are present in the centres of older tumours (greater than 1 cm diameter). Within the chondroid tissue, cells usually occur in nest formation, with each cell nest containing more cells than in normal hyaline cartilage. At the edges of nodules, at tumour borders and often perpendicu- lar to blood vessels in areas of necrosis, cells take on a columnar arrangement that bears comparison with normal growth plate structure. No calcification has been detected with Von Kossa stain.

No changes were found in tumours from the second to the fourteenth transplant generation, and in general these findings agree with Swarm’s (1963) description of the tumour.

Cell labelling studies The percentage of nuclei which were labelled at 1 hr after tritiated thymidine injection was

measured at 100-200 sites within active areas in each tumour, each site containing 50-100 cells. No variation was found in the average value of 18 i- 3 % in tumours from 7 to 42 days

Cell kinetics of a chondrosarcoma 549 after implantation. In some sites the percentage was as high as 40 %, but such local variation is also found in normal growth cartilage.

In one tumour a detailed study was undertaken in an attempt to relate local differences in labelling percentage to the gross structure of the tumour, i.e. to proximity of blood vessels or areas of necrosis. No correlations between labelling percentages and proximity of histological features were observed, although it is unlikely that variations could be detected in a two- dimensional slice through an unstructured tumour. Full details of this study are given in Dawson (1975).

Percentage of labelled mitoses The results of this study are presented graphically in Fig. 1. The data were sent to Dr G. G.

Steel at the Institute of Cancer Research, for analysis by a computer curve fitting program (Steel & Hanes, 1971). This program fits log-normal distributions for the phases of the cell cycle to the experimental points. The data derived from the computer program are presented in Table 1, where comparable data for other cartilage tissues are also given. The log-normal distribution for the cell cycle time of the chondrosarcoma cells is plotted out in the inset to Fig. 1.

' " L J U 0 4 8 12 16

Cycle time (hr)

Hours after triiiaied thymidine injection

FIG. 1. Experimental points (x) and computer fitted line showing the percentage of labelled mitoses recorded at intervals after an injection of tritiated thymidine. The inset shows the log-normal distribution of cycle times fitted by the computer program to the data.

TABLE 1. Cell kinetic parameters for three cartilage cell types

G2 S GI T C

Chondrosarcoma DGII 4.0 f 1.2 9.5 rt 1.5 10.5 It 6-5 2 4 0 Neonate rat vertebra* 4.6 f 2.0 11-6 f 2.2 6.8 2 2-5 22.0 Growth plate cartilage 3.2 f 3-5 6.5 & 0.28 44.5 2 38.9 55 f 40

from 6 week male rat?

* Results of Dixon (1971). t Results of Walker (1972).

550 Anne Dawson and N . F. Kember Growth fraction and cell loss factor

The results obtained above enable estimates to be made of the growth fraction of the chondrosarcoma cell population at 14-21 days after implantation. Since the median inter- mitotic time (T,) is 24 hr, the proliferating cells would double in number every 24 hr, provided that no cells were lost from this population.

The method of Steel (1968) estimates the potential doubling time of the whole population of tumour cells, when the synthetic period T,, the labelling index LI, and a correction factor 1, which depends on the position of S within the cell cycle, and the age distribution of proliferat- ing cells, are known. With the values Ts and G2 given in Table 1, a value for il of 0-9 is given by Steel.

In this study we have not measured the labelling index directly, but have measured the percentage of cells labelled at 1 hr after injection. The difference between these two par- ameters for a T, value of 9-5 hr is likely to be small compared with the experimental errors in the measured labelling percentage of 18 & 3%. So, substituting our values for T, and LI, together with 1 in the formula: doubling time = ATJLI, the potential doubling time of the whole population is 47 hr.

The difference between the potential doubling times for the proliferating cells and for the whole population is likely to depend on the growth fraction of the tumour.

The theoretical labelling index for the proliferating cells can be calculated from the formula: LIP = T,/T,. Hence, LIP = 36%. Now, since the growth fraction, that is, the proportion of cells in the proliferative compartment, is given by the ratio LI/LIp, for this chondrosarcoma, the growth fraction is 50%.

The value of 47 hr for the potential population doubling time of the whole tumour popula- tion is still considerably shorter than the doubling time of 95 hr obtained from the measure- ment of overall growth rate. This discrepancy may well be due to the loss of cells from the tumour, and the presence of necrotic centres in larger tumours is evidence that this occurs.

Steel (1968) defines a ‘cell loss factor’, Cp, as the rate of loss of cells expressed as a fraction of the rate at which cells are added to the tumour by mitosis, i.e. 4 = rate of cell loss (KJ rate of cell production (Kp) and it can be shown that Cp = 1 - T,/T,,, where To is the observed doubling time. From our results, Cp = 1 - 47/93 = 0.5. Therefore, the rate of cell loss in the chondrosarcoma at 14-21 days after implantation is about one half of the rate of cell production.

DISCUSSION

This chondrosarcoma is a primitive proliferation system beyond the homeostatic control of the host mouse, and its internal structure, before necrotic breakdown occurs, is similar to that of undifferentiated embryonic cartilage tissue. The gross and microscopic structure was described by Swarm (1963), and we have no evidence to suggest that the structure has changed during subsequent passages, although we have occasionally seen a corded arrangement of cells in advanced tumours that was not recorded by Swarm.

The cell cycle parameters of this chondrosarcoma lie within the range of values measured for other sarcomas (Steel, 1972). Steel lists eight sarcomas which have been transplanted frequently, and the DC-I1 tumour has an intermitotic time that is, at 24 hr, very close to the mean for the group. However, the volume doubling time of 95 hr is nearly twice the mean doubling time of 50 hr for these sarcomas. The explanation is that the chondrosarcoma has

Cell kinetics of a chondrosarcoma 55 1 a low growth fraction combined with a higher cell loss factor than all the other sarcomas. The values of growth fraction and cell loss factor for this tumour are, however, based on a simple model of the growth of the tumour that does not take into account the full complexity of the relationship between cell division, necrosis and overall growth. The relative volumes of cells and matrix vary from area to area within the tumour. This variation has not been quantified. Further, as the tumour advances in age, the necrotic region in the centre appears to increase in volume. If some of the new cell growth is directed inwards as space becomes available within the tumour, the apparent overall growth rate will be reduced.

Our chief interest in the tumour was to make a comparison of its cell cycle parameters with those of other chondroid tissues that have been studied (Table 1). The three tissues have similar durations for the S and G2 phases of their cell cycles, and the variation occurs in the lengths and degrees of variability of the GI phases. Here it is evident that the chondrosarcoma tissue is closer in growth characteristics to the growth cartilage of the neonate rat, than to that of the 6-week rat. The cartilage of theneonate and the tumour tissue also show a similar lack of organization into columns, while the chondrosarcoma cells show further immaturity in their lack of progression towards calcification. Even where blood vessels penetrate the tissue, the tumour breaks down rather than form centres of ossification. Whether the vascular invasion is a primary effect, or occurs secondarily to hypoxia and cell death, we cannot say. Perhaps in the corded older tumours mentioned above, it might be possible to correlate cell labelling and cell death with the appearance of blood vessels in the structured tumour and thereby to answer the question, but this study has still to be carried out.

The intermitotic time of this chondrosarcoma is close to that of the neonate cartilage (Table I), but it is double the time observed in the faster growing sarcomas (Steel, 1972). The hypothesis might, therefore, be offered that all chondroid tissues have a similar lower limit to their cell cycle time. This limit could be fixed either by the need to produce a minimum volume of extracellular cartilage matrix before further cell division can occur, or by some common internal control. In an attempt to look for stimulatory or inhibitory effects of a tumour on the growth cartilages of the host animal no such effect was detected (Dawson, 1975). Thus, if chalones or other growth effectors are produced within the chondrosarcoma they must either be tumour specific or be released in too low a concentration to affect other chondroid tissues in the mouse.

ACKNOWLEDGMENT

We wish to thank the Joint Research Board of St Bartholomew’s Hospital who supported A.E.M.D. during this project.

REFERENCES

DAWSON, A.E.M. (1975) Cell kinetic studies of growth cartilage and of a chondrosarcoma in rodents. Ph.D.

DIXON, B. (1971) Cartilage cell proliferation in the tail-vertebrae of new-born rats. Cell Tissue Kinet. 4,21. STEEL, G.G. (1968) Cell loss from experimental tumours. Cell Tissue Kinet. 1, 193. STEEL, G.G. (1972) The cell cycle in tumours. Celf Tissue Kinet. 5,87. STEEL, G.G. (1973) The measurement of the intermitotic period. In: The Cell Cycle in Development and

STEEL, G.G. & HANFS, S. (1971) The technique of labelled mitoses: analysis by automatic curve fitting. Cell

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552 Anne Dawson and N . I;. Kernber SWARM, R.L. (1963) Transplantation of a murine chondrosarcoma in mice of different inbred strains. J. nut.

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WALKER, K.V.R. & KEMBER, N.F. (1972) Cell kinetics of growth cartilage in the rat tibia. I. Measurements in young male rats. Cell Tissue Kinet. 5,401.