Hall Ch 21 (Model Tumor Systems) Flashcards
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 ( ).
In practice, pure inbred strains of rats or mice are used and are maintained by
( ) mating, which also serves the function of reducing the variability
among animals to a minimum.
isogenic, brother–sister
The tumor from a donor animal is removed aseptically and, if possible, prepared into a ( ) suspension; this is accomplished by separating the cells with an enzyme such as trypsin and then forcing them through a fine wire mesh.
To effect a transplant, 10^4 to 10^6 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 is commonly used, but
sometimes, a special tumor requires a particular site, such as the brain.
single-cell
Some tumors cannot be handled in this way and must be propagated by transplanting a small piece of tumor rather than a known number of single cells; this is obviously less quantitative. Within days or weeks, depending on the type of tumor and the strain of animals, palpable tumors appear in the recipient animals that are uniform in size, histologic type, and so on. Hundreds to thousands of animals can be used, which makes it possible to design highly quantitative studies of tumor response to different radiations, fractionation regimens, sensitizers, and combinations of radiation and chemotherapeutic agents.
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 in this chapter. These assays take
into account both intrinsic cell sensitivity to ionizing radiation as well as the
influence of the microenvironment. Before we discuss the techniques used to
assess tumor response to radiation, we should briefly revisit how the type of cell
death and the tumor microenvironment can affect tumor response to therapy, in
particular the role of ( ) cell death.
apoptotic
It is generally thought that irradiated cells die in attempting the next or a
subsequent ( ). However, this is not the only form of cell death.
Programmed cell death, or ( ), also occurs in both normal tissues and
tumors, spontaneously, because of ( ), and can be triggered by changes in
the tumor ( ).
mitosis, apoptosis, irradiation, microenvironment
In Chapter 22, 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 ( ) resulting from ( ).
random cell death, apoptosis
Studies with transplanted mouse tumors, as well as human tumors growing
as xenografts in nude mice, have shown that the importance of ( ) as a
mechanism of cell death after x-irradiation varies substantially.
Apoptosis was most important in ( ), essentially ( ) in sarcomas, and intermediate and very variable in carcinomas.
apoptosis, lymphomas, absent
In a mouse lymphoma, for example, 50% to 60% of the cells may show signs of dying an ( ) death by 3 hours after ( ), whereas in a sarcoma, there may be so few apoptotic cells that the process is of ( ) significance.
If a tumor responds ( ) to a relatively low dose of radiation, it generally means that ( ) is involved because the process peaks at ( ) hours after irradiation.
apoptotic, irradiation, little, rapidly, apoptosis, 3 to 5
Susceptibility to the ( ) also may be an important factor determining ( ) because ( ) appears to be a prominent ( ) effect in radiosensitive mouse tumors and essentially absent in radioresistant tumors.
induction of apoptosis, radiosensitivity, programmed cell death, early
In particular, the transformation of mouse cells of different histologic origins with oncogenes in cell culture makes them particularly sensitive to DNA damage-induced apoptosis.
The concern with using cell lines that are highly susceptible to apoptosis is that cell culture will act as a ( ) over time to select for variants of these cells that have lost their ( ), thus changing their in vivo sensitivity to radiation and cytotoxic drugs used in cancer therapy.
selective pressure, apoptotic sensitivity
Changes in the ( ) can also influence the sensitivity of tumor
cells to therapy (Fig. 21.1).
Studies have shown that changes in tumor ( ) can induce apoptotic cell death in a subset of tumor cells that represent a therapeutically sensitive population.
Therefore, cells that are sensitive to ( ) will be more susceptible to killing by the environmentally restrictive conditions in experimental tumors and will be better able to be controlled by radiotherapy and chemotherapy.
microenvironment, oxygenation, pH, and growth factors, apoptosis
Cells with diminished ( ) potential, which represent most tumor cell lines maintained in culture, will have lost this rapid response to radiotherapy but can still die by a ( ).
Cells with diminished apoptotic sensitivity caused by genetic mutations in critical apoptotic pathways, such as the p53 tumor suppressor gene or the ( ) oncogene (see Chapter 18), are probably more reflective of human ( ) tumors that are treated clinically.
apoptotic, mitotic cell death, Bcl-2, solid
FIGURE 21.1 Regions of ( ) form within solid tumors as a result of the ( ) vasculature.
Hypoxic regions represent a gradient of oxygen concentrations, the highest being nearest the vessels and the lowest being the farthest away.
In the most ( ) regions, p53 is stabilized and induces apoptosis.
A selection pressure therefore exists to ( ) and allows the ( ) of cells.
These cells are also resistant to chemotherapy and radiotherapy, both of which require an efficient blood supply. (From Hammond EM, Giaccia AJ. Hypoxia-inducible factor-1 and p53: friends, acquaintances, or strangers? Clin Cancer Res. 2006;12:5007–5009, with permission.)
hypoxia, inefficient and disorganized, hypoxic, loose p53, clonal expansion
( ) measurement is possibly the simplest end point to use and
involves the daily measurement of each tumor to arrive at a ( ).
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.
Figure 21.2 illustrates the variation of tumor size with time for unirradiated controls and tumors given a single dose of x-rays. The untreated tumors grow rapidly at a relatively uniform rate; the radiation treatment causes a temporary shrinkage of the tumor, followed by regrowth.
Tumor growth, mean diameter
FIGURE 21.2 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.
( ) represents the time after irradiation that it takes for the tumor to regrow to the size at the time of irradiation.
Growth delay
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 ( ) 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.
TCON
Two different methods have been used to score the tumor response.
Barendsen and his colleagues have used ( ), illustrated in Figure 21.2,
as the time ( ) it was at the time of irradiation.
Clearly, this index of response is only suitable for tumors that ( ) significantly after irradiation.
growth delay, taken after irradiation for the tumor to regrow to the size, shrink
For tumors that do not shrink so obviously, a more convenient index of growth delay is the time ( ), compared with controls.
Either index of growth delay increases as a function of radiation dose. Figure 21.3A shows growth curves for a rat rhabdomyosarcoma irradiated with various doses of x-rays or fast neutrons. In Figure 21.3B, growth delay is expressed as a function of ( ).
taken for the irradiated tumor to grow to some specified size after exposure, radiation dose
FIGURE 21.3 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 of 300-kV x-rays. Curves 3 and 5 refer to tumors irradiated with 4 and 8 Gy 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 based on the cell survival curves. B indicates the observed ( ) for
x-rays. Note the good agreement between calculated and observed values of the
TCD90 for x-rays. (Adapted from Barendsen GW, Broerse JJ. Experimental
radiotherapy of a rat rhabdomyosarcoma with 15 MeV neutrons and 300 kV xrays: I. Effects of single exposures. Eur J Cancer. 1969;5:373–391, with
permission.)
TCD90
Tumor control provides data of most obvious relevance to radiotherapy. In
experiments of this kind, a large number of animals with tumors of uniform size
are divided into separate groups, and the tumors are irradiated locally with
graded doses.
The tumors subsequently are observed regularly for ( ) or local ( ).
The proportion of tumors that are locally controlled can be plotted as a function of dose, and data of this kind are amenable to a sophisticated statistical analysis to determine TCD50, the dose at ( ).
recurrence, control, which 50% of the tumors are locally controlled
This quantity (TCD50) is highly repeatable from one experiment to another in an inbred strain of animals. Suit and his colleagues, over a period of more than 30 years, have made an extensive study of the response to radiation of a mammary carcinoma in C3H mice.
Data from a typical experiment are presented in Figure 21.4. Tumors were
propagated by transplanting 4 × 10^4 cells into the outer portion of the mouse ear,
and irradiations were performed when the tumors had grown to a volume of
about 4 mm3.
A brass circular clamp was fitted across the base of the ear and maintained for at least a minute before the initiation of the irradiation so that the tumors were uniformly hypoxic.
Single-dose, 2-dose, and 10-dose experiments were performed, with a 24-hour interval between dose fractions. Tumor control results are shown in Figure 21.4.
The TCD50 for a single treatment is ( ).
This indicates that a marked and extensive repair of ( ) damage has taken place during a ( ) regimen.
45.75 Gy, rising to 51.1 Gy for 2 fractions and to 84 Gy if the radiation is delivered in 10 equal fractions
sublethal
multifraction
FIGURE 21.4 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 ( ) in a ( ) mouse. The transplantation was made into the outer portion of the ear with 4 × 10^ 4 viable cells. The tumors were treated when they reached a diameter of 2 mm (i.e., a volume of about 4 mm3 ). (Adapted from Suit HD, Wette R. Radiation dose fractionation and tumor control probability. Radiat Res. 1966;29:267–281, with permission.)
spontaneous mammary carcinoma, C3H
The dilution assay technique was devised by Hewitt and Wilson, who used it to
produce the first in vivo survival curve in 1959. They used a lymphocytic
leukemia of spontaneous origin in mice.
A single-cell suspension can be prepared from the infiltrated liver of an animal with advanced disease and the tumor transplanted by injecting known numbers of cells into the peritoneal cavities of recipient mice, which subsequently develop leukemias. The leukemia can be transmitted, on average, by the injection of only two cells; this quantity—the number of ( )—is known as TD50. The dilution assay technique became the basis for obtaining an in vivo cell survival curve.
cells required to transmit the tumor to 50% of the animals
The procedure used, illustrated in Figure 21.5, is as follows. An animal containing the tumor may be irradiated to a given dose of radiation, for example, 10 Gy.
A single-cell suspension is then prepared from the infiltrated liver, the
cells are counted and diluted, and various numbers of these cells are injected
intraperitoneally into groups of recipient animals. It is then a matter of
observation and calculation to determine how many irradiated cells are required
to produce a tumor in half of the animals inoculated with that given number of
cells.
Suppose, for instance, that it takes 20 irradiated cells, on average, to
transmit the tumor; because it is known that only 2 clonogenic cells are needed
to transmit the tumor, it is a simple matter to decide that in the irradiated
population of cells, 2 of 20, or 10%, were clonogenic and survived the dose of
10 Gy.
That is, Surviving fraction = ( )
TD50 controls / TD50 irradiated
FIGURE 21.5 Schematic representation to show the general features of the
dilution assay technique. Various numbers of tumor cells from the donor animal
are injected into groups of recipients, and a determination is made of the ( ) (TD50).
The ratio of this quantity for control and irradiated donors is the ( )
fraction. (Adapted from Andrews JR, Berry RJ. Fast neutron irradiation and the
relationship of radiation dose and mammalian tumor cell reproductive capacity.
Radiat Res. 1962;16:76–81, with permission.)
number of cells required for a tumor to take in half of the animals of the group
surviving
If this process is repeated for several doses of radiation and the corresponding
surviving fractions are determined by this assay technique (Dilution Assay Technique), a survival curve for cells irradiated and assayed in vivo can be constructed.
This technique is a ( ), but it involves a ( ) as opposed
to a solid tumor. The cells, after reinoculation into the mouse, grow in the
peritoneal cavity in much the same way that the cells grow in a petri dish in the
in vitro technique; the mice are in fact being used as small portable incubators.
true in vivo system, leukemia
Since these pioneering efforts, the ( ) assay technique has been applied
by many different workers to measure survival curves for several ( ) and
solid tumors if the tumors can be removed and prepared into a single-cell
suspension; some collected results are shown in Figure 21.6.
dilution, leukemias
The survival curves (from the dose response curves in vivo using the dilution assay technique in figure 21.6) obtained have a D0 (A does of radiation that induces an average of one lethal event per cell leaves 37% of irradiated cells still viable) of about 4 Gy because the cells in the peritoneal cavity of the mouse are so numerous and so closely packed that they are deficient in ( ).
This technique, therefore, produces a ( ) survival curve. To obtain a
survival curve characteristic of aerated conditions, it is necessary either to
remove the cells from the donor animal and irradiate them in a petri dish in
which they are in contact with air or to inject ( ) into the
peritoneal cavity of the mouse before irradiation so that oxygen is available to
the tumor cells during the irradiation. If this is done, D0 is about ( ) Gy.
oxygen, “hypoxic”, hydrogen peroxide, 1.3 to 1.6
FIGURE 21.6 Dose–response curves in vivo, using the dilution assay technique,
for various murine tumors under ( ) and ( ) conditions. (Adapted
from Berry RJ. On the shape of x-ray dose-response curves for the reproductive
survival of mammalian cells. Br J Radiol. 1964;37:948–951, with permission.)
oxygenated, hypoxic
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 the recipient animals and
counting the number of ( ) colonies produced.
The general principles of the method are illustrated in Figure 21.7.
The tumor used in these studies was the ( ), which is a transplantable tumor that arose originally in a C3H mouse and which has been propagated serially through many generations.
Tumors are irradiated in situ, after which they are removed and made into a
preparation of single cells by a combined trypsinization and mechanical
procedure.
A known number of cells then are mixed with a large number of
heavily irradiated tumor cells and injected intravenously into recipient mice.
About 3 weeks later, these mice are sacrificed, and the colonies formed in the
( ) are readily countable. The number of lung colonies is a measure of the
number of surviving ( ) cells in the injected suspension.
lung, KHT sarcoma, lungs, clonogenic
FIGURE 21.7 The lung colony assay system. The tumor is irradiated in situ,
after which it is excised and made into a ( ). A known
number of cells are then injected intravenously into the recipient animals. About
3 weeks later, the recipient animals are sacrificed and the colonies that have
formed in the ( ) are counted.
The number of lung colonies is a measure of the number of ( ) cells in the injected suspension. (Adapted from Hill RP, Bush RS. The effect of continuous or fractionated irradiation on a murine sarcoma. Br J Radiol. 1973;46:167–174, with permission.)
single-cell suspension, lungs, surviving clonogenic
The lung colony technique is not confined to the ( ) but has been
used with other tumor cells. For example, the demonstration of the absence of
repair of potentially lethal damage after ( ) irradiation involved the use of
the ( ), and the fraction of surviving cells was assayed by
counting lung colonies.
KHT sarcoma, neutron, Lewis lung carcinoma
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 ( ) in the rat (Hermens and Barendsen), a ( ) in the mouse (McNally), and the ( ) tumor in the mouse (Rockwell and Kallman).
rhabdomyosarcoma, fibrosarcoma, EMT6 mammary
The steps involved in this method (In vivo/in vitro assay) are illustrated in Figure 21.8. This method combines many of the advantages of the in vitro and in vivo techniques. The
tumors are treated in vivo in a natural environment so that the cellular response is
modified by the various factors that are important in determining gross tumor
response.
After treatment, each tumor is removed and prepared into a single-cell
suspension, and the cell concentration is counted in a hemocytometer or
electronic cell counter.
Known numbers of cells can then be transferred to petri dishes containing fresh growth medium, and the proportion of clonogenic cells can be determined by counting colonies 10 days later. The speed, accuracy, and relative economy of the in vitro system replace the expense and inconvenience of the recipient animals in the dilution assay technique.
FIGURE 21.8 The principle of the in ( ) system using the rhabdomyosarcoma in the rat. The solid tumor in the animal can be removed and the tumor cells assayed for colony formation in petri dishes. This cell line can be transferred ( ) between the ( ). (Adapted from a drawing courtesy of Drs. G. W. Barendsen and J. J. Broerse.)
vivo/in vitro assay, back and forth, animal and the petri dish
A xenograft is a transplant from ( ). In the cancer field, this usually refers to a human tumor transplanted into a laboratory animal. If the recipient animal has a ( ) immune system, a xenograft should ( ) grow, but there are two main ways in which growth has been achieved.
First, animal strains have been developed that are ( ) deficient.
one species to another, normal, not, congenitally immune
Best known are nude mice, which, in addition to being hairless, also lack a ( ). Many human tumors grow ( ). More recently, there have been nude rats and severe combined immune-deficient (SCID) mice, which suffer from the severe combined immunodeficiency syndrome and are deficient in both B cell and T cell immunity.
Second, it is possible to severely immunosuppress mice by the use of ( ) or a combination of both to the point at which they accept human tumor grafts. It is important to recognize that neither type of host completely fails to reject the human tumor cells: Rejection processes are still present, and these complicate the interpretation of in situ tumor therapeutic studies.
thymus, under the skin of nude mice, radiation or drugs
