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Protocol no. 25

LASER DIFFRACTION MEASUREMENT OF TUMOUR SPHEROIDS

Tumour cell lines cultured as aggregates can be utilised for in vitro radiosensitivity and/or chemosensitivity tests. Chemical effects are monitored by studying the changes in spheroid diameter measured by laser diffraction.

CONTACT

Dr J.E. Dyson Dept. of Radiobiology Tunbridge Building Cookridge Hospital Leeds, LS16 6QB UK Tel: England - 0532 673411 ext. 219 Dr Dyson is happy to discuss the operation of this procedure by telephone, and invites interested scientists to visit the laboratory for a demonstration.

RATIONALE

The study of in vivo tumour therapy is hampered by the homogeneous nature of the monolayer of the cell lines. The heterogeneity of several of the most important factors that influence the response of a tumour is absent in a monolayer culture, and limits its applicability to the in vivo situation. These factors have led to a growing interest in cell aggregates which mimic the heterogeneous situation encountered in vivo more closely because they develop a necrotic centre surrounded by a layer of hypoxic cells, due to the presence of nutrient and oxygen gradients. Thus, the cellular heterogeneity which may affect tumour cells in vivo is present to a considerable degree in multicellular aggregates.

BASIC PROCEDURE

Multicellular aggregates are formed from monolayer cultures of tumour cell lines and grown as suspensions in spinner flasks. Aliquots are removed for testing when required and spheroids of a defined size are obtained using a cascade system of filters. In addition, latex calibration beads and plastic microcarrier beads are used to provide regular-shaped spheres with similar light scattering properties to cells. Samples of beads and spheroids are added to the particle sizer measuring chamber and 200 sequential scans are performed. Aliquots are also added to a calibrated microscope graticule and the crossed diameters measured. Chemicals under test should be added directly to spheroid cultures for an appropriate exposure period. An iterative least squares calculation is carried out by a computer interfaced with the particle sizer which fits particle size distribution to light scattering distribution. From the plots/tabulation obtained the mean diameter of the spheroids can be determined.

CRITICAL ASSESSMENT

The term multicellular spheroids was introduced when cell aggregates of V79-171 hamster lung cells were investigated in liquid media stirred in spinner flasks. The aggregates formed were perfectly spherical in shape and hence were called "multicellular spheroids". These cellular structures resembled nodular structures observed in C3H mouse mammary carcinomas and in several types of human tumours. In addition, similar survival curves were found when radiation experiments were performed, with either aggregates or solid tumours and, therefore, the use of multicellular spheroids was considered as an in vitro tumour model for mimicking basic biological properties of cancer cells in vivo (the early avascular stage of tumour growth). Tumour cells grown under conditions that result in them growing as multicellular spheroids mimic the in vivo situation more closely. Tumour cell monolayers, on the other hand, are essentially homogeneous in respect to nutritional state, oxygen tension, proliferative state, pH and elimination of waste products. In addition their cell-to-cell interactions are minimised, losing the heterogeneity associated with a tumour in vivo. Common characteristics of solid tumours and multicellular spheroids Both models have similar matrix components, such as glycosaminoglycans or collagen. In contrast spheroid cells grown as monolayers have reduced or no capacity to synthesis extracellular matrix material. Thus a degree of structural and functional differentiation in primary tumour may be retained in spheroids. In addition, there are large spatial variations in cellular proliferation. Solid tumours contain proliferating cells in areas which are well supplied with oxygen and nutrients, for example adjacent to microvessels. In less oxygenated areas, cell cycle times increase due to the diffusion-limited nutritive supply and restricted removal of wastes (proliferation gradients). This decrease in proliferative activity of tumour cells may result in the occurrence of quiescent cells. Proliferation gradients have also been well documented in multicellular aggregates. It has been shown by several investigators that the proliferating cells in spheroids are localised within superficial layers with a thickness of 50-100mm. As the depth of the spheroids increases the cell cycle times are prolonged and cells pass into a non-proliferating state. These quiescent cells in spheroids are considered to match Go-phase cells in vivo more closely than corresponding plateau-phase cells in monolayer culture. Three phases of spheroidal growth can generally be distinguished. Firstly, when all the spheroid cells are proliferating as characterised by exponential growth up to spheroid diameters of 50-200mm. Secondly, a change in cell cycle distribution with an increasing accumulation of non-proliferating cells in central regions of the spheroids. In addition, mitotic cells are sequestered from peripheral parts of the spheroids into the culture medium. Thus, there is a progressive reduction of the proliferating fraction of cells and a linear increase in the spheroid diameter with time during the second growth phase. During the third growth phase, a considerable retardation in volume expansion can be observed, with spheroids eventually attaining a maximum diameter of 1-4mm depending on the cell type and culture conditions used. Spheroids of this size can be maintained in culture for several weeks with virtually no increase in volume regardless of how often culture media are replenished. Growth saturation appears to be a general feature of macroscopic tumour expansion as well, although the life-span of the host is usually terminated by the disease before this growth phase can be attained. Cell lines The cell lines employed possess different growth characteristics and thus form distinct spheroid cultures. The colorectal HCCO cell line produces rather loose spheroids whereas the cervix 754 cell line produces firm spheroids. Additionally, the cell lines form spheroids of a varied cell diameter (see Boothby et al., 1989) but the size ranges from 250-1000mm. The spheroids develop a necrotic centre surrounded by a layer of hypoxic cells due to the presence of nutrient and oxygen gradients. Spheroid cultures Cultures can be initiated from either a single cell or by inducing cell aggregation first with subsequent growth of the aggregates. Cultures grown up from a single cell are mainly used as screens to test the ability of cells for colony formation in semi-solid agar (considered as an indicator of malignancy). Although, in general, both normal and malignant cells may form aggregates, non-malignant cells show little growth in spheroid culture. However, established cell lines (e.g. sublines of V79 and 3T3) may be readily grown as spheroids. In general, the cells in the aggregates stay loosely attached to each other for a given time during which they can be easily dissociated by mechanical forces. This initial phase of aggregation, which possibly represents the phase of intercellular recognition is followed by stabilisation of the cell aggregates through the development of junctional complexes between the cells, such as gap junctions or desmosomes. The spinner flask is suited for growing large populations of spheroids under controlled external conditions with regard to O2, nutrient supply and pH. It has the convenience of allowing the removal of representative samples of spheroids from the flask at given time intervals, enabling the assessment of changes in various biological parameters as a function of spheroid growth, such as variations in number of cells per spheroid, or in the thickness of the viable rim with the time in culture. The liquid overlay technique (cultures in liquid media kept in agar-coated Petri dishes) enables the study of the characteristics of individual spheroids. In this case the number of spheroids which can be cultured is restricted when the external supply conditions are well-defined. During growth in spinner flasks, medium has to be renewed periodically, and removed routinely from the spinner flasks in such a way that the cellular concentration remains at a constant level for the duration of spheroid culturing. Ideally, culture media should be exchanged continuously using feedback systems for controlling relevant parameters, such as pH of O2 tension in the medium. Although such systems are available they have not been applied to spheroid growth up until now. Laser diffraction particle sizer This machine consists of a power helium neon laser (5mW), a stainless steel sample chamber with optically flat windows mounted on a magnetic stirrer, and a detector consisting of 31 concentric annular rings to analyse the light scattering of the sample. The components are mounted linearly on an optical bench. With appropriate optics the long-bench model is capable of measuring particles within the range 0.5 to 1880mm. The diffractor is interfaced with a computer (in this case an Olivetti model M24 computer, with dedicated software). It may be programmed to carry out repeated scans of the same sample (up to 32,767). Particles move into and out of the laser beam and change their orientation within the beam due to the magnetic stirrer, and essentially all particles in the sample are measured when an adequate number of scans are carried out. Measurements have been made by the authors to determine the effect of the number of scans on reproducibility of measurement values for mean particle diameter and standard error of the mean (±SE) by using plastic beads. The mean and standard deviation was found to reach a plateau at 500 scans and above (see table in Results). Multicellular spheroids are not necessarily regularly spheroid or have such a narrow range of diameters as PVC beads, and therefore a higher number of scans (2000) are routinely used. It takes 2 minutes for the machine to perform 2000 scans, calculate and tabulate the spheroid diameter frequency and statistical values, together with a cumulative undersize plot of the logarithm of spheroid diameter. Comparison of the particle sizer with microscopic measurements In initial investigations, a direct comparison was made between mean spheroid diameters determined by measurement under the microscope and by measurement by the particle sizer. The correlation coefficient between the two procedures calculated from the value is 0.996 (see Boothby et al., 1989). Growth curves for the cells lines can be determined by samples taken from the spheroid suspensions in three separate spinner flasks for each cell line and measured under the microscope and by the particle sizer. As a spheroid moves within the laser beam it will be measured in several different orientations; its contribution to the size-frequency plot will, therefore, be the average of the diameters measured in the different orientations. This is in contrast to the crossed diameters measured in a single orientation under the microscope. This may lead to a slightly larger size being measured by the particle sizer. The standard errors for the measurements by the particle sizer were much reduced compared to those resulting from microscopic measurement due to the increased number of spheroids measured and the repeated measurements. (20 for microscopic, 400 for particle sizer) Microscope-image analysis can be very time-consuming when there are large numbers of samples to be measured. Stirring To ensure the spheroids are maintained in suspension and that all the spheroids move through the laser beam, relatively vigorous stirring is required. However, this may result in loss of cells from the spheroid surface during the time of measurement, resulting in a slightly reduced spheroid size. This can be assessed by studying cell lines which form different sized spheroids with varied growth characteristics. Several samples of the same spheroid culture can be taken and the effects of stirring measured and averaged out (Boothby et al., 1989). Stirring has been found to have only a marginal effect on the mean spheroid diameter within the time required to introduce the sample and carry out the scans (5 minutes). A correction factor may be necessary for cell lines that form loosely packed spheroids. Treatment of the spheroids Low concentrations of some cytotoxic drugs cause increased cell shedding, or fragmentation of the spheroids without appreciable effect on cell viability, which can lead to the incorrect interpretation of growth delay curves. The particle sizer measures all cells and spheroids in the suspension and, therefore, any such effects are readily apparent when a frequency vs. size plot is performed.

TEST STATUS

In-house

CHEMICALS TESTED

Methotrexate

REFERENCES

  1. Boothby, C.D; Daniel, J.; Adam, S. & Dyson, J.E.D. (1989) Use of a laser diffraction particle sizer for the measurement of mean diameter of multicellular tumour spheroids. In Vitro Cellular and Developmental Biology, 25 (10), 946-950.
  2. Mueller-Klieser, W. (1987) Multicellular spheroids: A review on cellular aggregates in cancer research. J. Cancer Res. Clin. Oncol., 113, 101-122.
  3. Sutherland, R.M. & Durand, R.E. (1976) Radiation response of multicellular spheroids - an in vitro tumour model. Curr. Top. Radiat. Res., 11, 87-139.
  4. Wibe, E., Berg, J.P., Tveit, K.M., Nesland, J.M. & Lunde, S. (1984) Multicellular spheroids grown directly from human tumour material. Int. J. Cancer., 34, 21-26.
  5. Wigle, J., Freyer, J.P. & Sutherland, R.M. (1983) Use of a sedimentation column to obtain uniformly sized populations of multicell spheroids. In vitro, 19, 361-366.

IP-25 © August 1991