Chapter 20 Model Tumor Systems

1. Chapter 20 Model Tumor Systems

2. TRANSPLANTABLE SOLID-TUMOR SYSTEMS IN EXPERIMENTAL ANIMALS A wide range of experimental tumors of various histologic types have been developed for radiobiologic studies. To produce a large number of virtually identical tumors, propagation by transplantation from one generation of animals to the next is used, which makes it mandatory that the animals be isologous.

3. The tumor from a donor animal is removed aseptically and, if possible, prepared into a single-cell suspension

4. To effect a transplant, 104 to 106 cells are inoculated subcutaneously into each of a large group of recipient animals of the same strain. The site of transplantation varies widely; the flank or back commonly is used, but sometimes a special tumor requires a particular site, such as the brain.

5. There are five commonly used techniques to assay the response of solid tumors to a treatment regimen: Tumor growth measurements. Tumor cure (TCD50) assay. Tumor cell survival determined in vivo by the dilution assay technique. Tumor cell survival assayed by the lung colony assays. Tumor cell survival using in vivo treatment followed by in vitro assay. Each of these methods is discussed briefly.

6. TUMOR GROWTH MEASUREMENTS Tumorgrowth measurement is possibly the simplest end point to use and involves the daily measurement of each tumor to arrive at a mean diameter. For tumor growth experiments, a large number of transplanted tumors are prepared as previously described. When they have grown to a specified size (e.g., a diameter of 8 to 10 mm in rats or 2 to 4 mm in mice), they are treated according to the plan of the particular experiment.

8. FIGURE 20.1 • The pattern of response of a tumor to a dose of x-rays. The size of the tumor, either the mean diameter or the volume, is plotted as a function of time after irradiation. Two different indices of tumor responses have been used by different investigators. Growth delay represents the time after irradiation that it takes for the tumor to regrow to the size at the time of irradiation. Alternatively, the index of radiation damage may be the time taken for the tumor to grow from a specified size A at the time of irradiation to some specified larger size B. Typically, this may be from 9 to 25 mm in diameter for rat tumors. This quantity is shown as TCON for unirradiated control animals and TX-RAY for tumors irradiated with a dose (D) of x-rays. Either index of tumor response may be plotted as a function of radiation dose.

10. FIGURE 20.2 • A: Volume changes of rhabdomyosarcomas in rats after irradiation. Curve 1 represents the growth of the unirradiated control tumors. Curves 2, 4, 6, and 7 refer to tumors irradiated with 10 to 40 Gy (1,000-4,000 rad) of 300-kV x-rays. Curves 3 and 5 refer to tumors irradiated with 4 and 8 Gy (400 and 800 rad) of 15-MeV d+ → T fast neutrons. B: Growth delay of rhabdomyosarcomas in rats as a function of dose of x-rays (curve 2) or fast neutrons (curve 1). A and C indicate the doses of neutrons and x-rays, respectively, required to “cure” 90% of the tumors, calculated on the basis of cell survival curves. B indicates the observed TCD90 for x-rays. Note the good agreement between calculated and observed values of the TCD90 for x-rays. (From Barendsen GW, Broerse JJ: Experimental radiotherapy of a rat rhabdomyosarcoma with 15 MeV neutrons and 300 kV x-rays: I. Effects of single exposures. Eur J Cancer 5:373-391, 1969, with permission.)

11. FIGURE 20.3 • Percentage of mouse mammary tumors locally controlled as a function of x-ray dose, for single exposures and for two different fractionation patterns. The tumors were isotransplants derived from a spontaneous mammary carcinoma in a C3H mouse. The transplantation was made into the outer portion of the ear with 4 × 104 viable cells. The tumors were treated when they reached a diameter of 2 mm (i.e., a volume of about 4 mm3). (From Suit H, Wette R: Radiation dose fractionation and tumor control probability. Radiat Res 29:267-281, 1966, with permission.)

12. Surviving fraction =TD50 controls/TD50 irradiated If this process is repeated for a number of doses of radiation, and the corresponding surviving fractions are determined by this assay technique, a survival curve for cells irradiated and assayed in vivo can be constructed. This technique is a true in vivo system, but it involves a leukemia as opposed to a solid tumor. The cells, after reinoculation into the mouse, grow in the peritoneal cavity in much the same way that

14. LUNG COLONY ASSAY Hill and Bush have devised a technique to assay the clonogenicity of the cells of a solid tumor irradiated in situ by injecting them into recipient animals and counting the number of lung colonies produced. The general principles of the method are illustrated in Figure 20.6.

16. The lung colony technique is not confined to the KHT sarcoma but has been used with other tumor cells. For example, the demonstration of the absence of repair of potentially lethal damage after neutron irradiation involved the use of the Lewis lung carcinoma, and the fraction of surviving cells was assayed by counting lung colonies.

17. IN VIVO/IN VITRO ASSAY A limited number of cell lines have been adapted so that they grow either as a transplantable tumor in an animal or as clones in a petri dish. These cells can be readily transferred from in vivo to in vitro and back. In one generation they may grow as a solid tumor in an animal, and in the next as a monolayer in a petri dish. The three most commonly used systems are a rhabdomyosarcoma in the rat (Hermens and Barendsen), a fibrosarcoma in the mouse (McNally), and the EMT6 mammary tumor, also in the mouse (Rockwell and Kallman). The steps involved in this method are illustrated in Figure 20.7. This method combines many of the advantages of the in vitro and in vivo techniques.

19. A xenograft is a transplant from one species to another. In the cancer field, this usually refers to a human tumor growth transplanted in a laboratory animal. If the recipient animal has a normal immune system, a xenograft should not grow, but there are two main ways in which growth has been achieved. First, animal strains have been developed that are congenitally immune deficient. Best known are nude mice, which in addition to being hairless also lack a thymus. Many human tumors grow under the skin of nude mice.

20. Second, it is possible to severely immune-suppress mice by the use of radiation or drugs or a combination of both, to the point at which they accept human tumor grafts.

21. Third, although the histologic characteristics of the human source tumors are usually well maintained by xenografts, the stromal tissue is of mouse origin. Consequently, xenografts of human tumor cells are not much more valid than murine tumors for any studies in which the vascular supply plays an important role.

23. SPHEROIDS: AN IN VITRO MODEL TUMOR SYSTEM Mammalian cells in culture may be grown either as a monolayer attached to a glass or plastic surface or in suspension, in which case they are prevented from settling out and attaching to the surface of the culture vessel by continual gentle stirring. Most cells in suspension, or in “spinner culture,” as it often is called, remain as single cells; at each mitosis the progeny cells separate, and although the cell concentration increases with time, it continues to consist of individual, separate cells.

24. The spheroid system has been applied to a number of problems in radiobiology and in the study of pharmacologic agents, such as radiosensitizers or chemotherapeutic agents. A major problem in the application of these drugs to human tumors is the presence of resistant cells that are resting or noncycling, often located away from blood vessels. Drugs are required to diffuse in effective concentration to these cells through layers of growing, actively dividing cells, which may inactivate the drug through their metabolism. The spheroid system mimics many of these tumor characteristics and provides a rapid, useful, and economic method for screening sensitizers and chemotherapeutic agents because it is intermediate in complexity between single-cell in vitro culture and tumors in experimental animals.

25. SPHEROIDS OF HUMAN TUMOR CELLS Many types of human tumor cells can be cultured as spheroids, with a wide spectrum of morphologic appearances and growth rates. In general, cells from disaggregated surgical specimens form spheroids if cultured in liquid suspension above a nonadhesive surface, which can be a thin layer of agar or agarose gel or the bottom of a culture dish not prepared for cell culture. Only if the spheroid is formed and grown to a certain size can it be transferred to a spinner culture vessel and grown in the same way as P.358 spheroids of established rodent cell lines

26. Morphologic studies of spheroids of human tumor cells show that they maintain many characteristics of the original tumor specimens taken from the patient and of the cells if grown as a xenograft in nude mice. Radiobiologic studies show that in addition to maintaining histologic characteristics of individual tumors, spheroids of human cells preserve characteristic radiosensitivity, because dose-response curves for spheroids are virtually identical to those for cells growing as xenografts in nude mice.

27. COMPARISON OF THE VARIOUS MODEL TUMOR SYSTEMS In all transplantable systems described, the tumor is treated in situ, with all of the realism and complexities of the in vivo milieu, such as cell-to cell contact and the presence of hypoxic cells, factors that cannot be fully simulated in a petri dish. The tumor cure (TCD50) and growth delay systems share the additional advantage that they are left in situ undisturbed after treatment. In the other techniques, the tumor must be removed, minced, and prepared into a single-cell suspension by the use of an enzyme, such as trypsin, before survival is assessed. Although this step does not appear to affect the assessment of the effects of radiation, it can result in artifacts in the case of other agents, such as chemotherapeutic drugs or hyperthermia, in which the cell membrane may be involved in the cellular response. The procedure of breaking up the tumor and partially dissolving the cell membrane with a digestive enzyme may influence results. For this reason, in the testing and evaluation of a new drug, one tumor system involving the determination of growth delay or TCD50 is always included. By the same token, these same systems are very expensive because they require a large number of animals for the amount of information produced. The determination of TCD50 is perhaps ideal for producing data relevant to clinical radiotherapy. It is certainly the most expensive; to produce a single TCD50 value for one of the lines in Figure 20.3, six to eight groups of up to ten animals must be kept and observed for weeks. The same information can be obtained in 10 days with one or two mice and six petri dishes using the in vivo/in vitro technique.

29. APOPTOSIS IN TUMORS It is generally thought that irradiated cells die in attempting the next or a subsequent mitosis. This is not the only form of cell death, however. Programmed cell death, or apoptosis, also occurs in both normal tissues and tumors, spontaneously and as a result of irradiation. In Chapter 21, it is pointed out that tumors grow much more slowly than would be predicted from the cell-cycle time of the individual cells and the fraction of cells actively dividing. One of the reasons for this “cell loss,” as it is called, is random cell death resulting from apoptosis. Studies with transplanted mouse tumors, as well as human tumors growing as xenografts in nude mice, have shown that the importance of apoptosis as a mechanism of cell death after x-irradiation varies substantially. Apoptosis was most important in lymphomas, essentially absent in sarcomas, and intermediate and very variable in carcinomas.