The Nature of Matter and Radiation

All matter, biological or otherwise, is composed of atoms. Atoms are made up of groups of electrons (i.e., small negatively charged particles) orbiting a nucleus consisting of protons (larger positively charged particles) and neutrons (uncharged particles having mass similar to that of a proton). The properties of an atom are generally defined by the number of protons in the nucleus. A matching number of electrons are held in orbit around the nucleus by electrostatic attraction. A specific, discrete set of possible electron orbits exists for each kind of atom, with each electron orbit corresponding to a specific energy. Moving an electron from one orbit to another requires adding or subtracting the energy difference between the two orbitals, and removing an electron from the atom entirely, that is, ionization, requires adding the full energy of the electron orbital.

The properties of a nucleus are defined by the number of protons and neutrons, with the number of protons defining the type of element and the number of protons and neutrons together defining the isotope. Analogously to the arrangement of electrons in an atom, protons and neutrons are arranged in discrete energy levels specific to a particular nucleus, and transitioning between energy levels requires adding or removing a comparable amount of energy. Of the 1400 known isotopes of the 92 naturally occurring elements, approximately 80% are unstable and spontaneously undergo a transition between energy levels, emitting energy in the process. The phenomenon of spontaneous energy release from a nucleus, called radioactivity, can take many forms, including combinations of the emission of γ-rays, the ejection of electrons, positrons, or α-particles, and the transmutation of one element to another.

The term “radiation” refers to energy emitted from a source that is transmitted through a material or space. This radiation can then deposit its energy by interacting with the matter through which it passes. Regardless of the source, most radiation used in radiation therapy involves electromagnetic interactions. This energy can take the form of packets of electromagnetic waves called photons or can be carried as the kinetic energy of freely propagating particulate radiation such as electrons, protons, or α-particles.

Photons are packets of oscillating electric and magnetic fields propagating through space at the speed of light (3 × 10¹⁰ cm/second). Photons are characterized by their wavelength, which is the distance traversed by a wave over the course of a single oscillation. The possible wavelengths of photons have no real limits, but common examples range in wavelength from AM radio (10³ m) to visible light (10⁻⁷m) to gamma rays (10⁻¹²m). Photons that have a shorter wavelength oscillate at a higher frequency (i.e., they have more oscillations per unit time) and are more energetic. Energy and frequency are related by Planck’s constant (4.135 × 10⁻¹⁵ eV-sec) and are generally expressed in units of electron volts (eVs), which are equivalent to the kinetic energy of a single electron accelerated over a potential of 1 volt. The energy of a photon determines its ability to penetrate matter. Visible light (~1 eV) can only interact with the surface of objects. Diagnostic (kilo eV [keV]) and therapeutic (mega eV [MeV]) photons can penetrate much more deeply, permitting therapeutic effects anywhere in the body. Photons at therapeutic energies can pass through many centimeters of tissue before experiencing any interactions.

Most particulate radiation consists of energetic charged particles. The electric fields around these particles cause them to interact with all the other charged particles in the surrounding medium. Charged particles are therefore much more efficient at depositing energy in matter, because the particles will continuously lose their energy as they attract or repel other charged particles along their path. Many of these interactions will cause ionization, which correlates with the amount of biological damage delivered. Heavy particles, such as protons and α-particles, ionize matter very efficiently, lose energy more efficiently, and have a higher linear energy transfer (LET), that is, amount of energy loss per length of the particle’s track (discussed later in this chapter) than do lighter particles such as electrons and positrons. Although most particulate radiation is charged, uncharged neutrons are also capable of depositing energy in a material. Unlike charged particles, neutrons can only interact with other nuclei. Generally, this interaction takes the form of a collision with a proton. The proton recoils with some fraction of the neutron’s initial energy. The positively charged proton then ionizes the surrounding particles, causing most of the biological damage.

Interactions of Radiation and Matter

In order of roughly increasing energy, photons can interact with:

Photoelectric Effect

Photons having sufficient energy to ionize an atomic electron can undergo the photoelectric effect (Fig. 27-1). In this process, the photon energy is entirely absorbed. Some energy is lost to breaking the electron binding energy, and the rest is carried away as kinetic energy of the ejected electron. The probability of a photoelectric interaction scales with the cube of the atomic number (Z) and the inverse cube of the photon energy (E), making the photoelectric effect very sensitive to material type and much more prevalent for lower photon energies. The photoelectric effect is the dominant photon interaction in tissue below 30 keV.

The Generation of Therapeutic Radiation

To be useful in radiation therapy, radiation must be generated in a manner in which it can be directed at the targeted tissues. Radiation for cancer therapy is predominantly generated through two means: linacs and radioactive sources.

Linear Accelerators

The most common modality used in radiation oncology is external beam radiation therapy. Although a small number of radiation therapy facilities generate external beams using radioactive sources such as cobalt-60, the vast majority of therapeutic electromagnetic radiation is generated in a linac. A linac is a device that accelerates charged particles (electrons) to velocities near the speed of light using oscillating electric fields to push the electrons through a series of accelerating cavities. A schematic of a linac is shown in Figure 27-2, A. Electrons are accelerated to energies typically between 4 and 18 MeV. Electric and magnetic fields focus and steer the high-energy electrons such that they strike a thin metal target that stops the electron beam, with some fraction of the electron energy converted to a spray of photons through the bremsstrahlung process. The bremsstrahlung photons, called x-rays, move approximately in the same direction as the electrons and have an energy spectrum, ranging from a few 10’s of keV up to the maximum energy of the initial electrons. The resulting photon beam then passes through a series of filters and beam-shaping elements that flatten and define the edges of the beam.

The dose from a photon beam is related to its intensity, defined as the number of photons per unit area. Two major effects serve to decrease the intensity of a photon beam as it passes through tissue. First, as with any photon source, the beam intensity decreases with increasing distance from the source, just as is the case for a light bulb. In addition, beam intensity decreases as photons are attenuated from the beam via various scattering and absorption effects. This process leads to a characteristic decrease in intensity versus depth that varies based on photon energy. Although the photon intensity begins decreasing immediately upon entering a material, the energy released through the photon interactions is spread over a few centimeters as the electrons scattered by the photons gradually lose their energy as they pass through the material. The resulting dose distribution is characterized by a region of rapid increase near the surface, a leveling off of dose at a depth of 1 to 3 cm, and a gradual dose falloff as depth increases. The plot of dose versus depth is called a percent depth dose curve, as shown in Figure 27-3. Because higher energy photons are more penetrating, higher energy beams will attenuate more slowly, leading to a more gradual decrease in dose with depth.

Linacs designed to produce photon beams can also be configured to produce therapeutic electron beams. Removing the photon-generating target and replacing it with a comparatively thinner electron scattering foil allows the transmission of the initial electron beam, but not without scattering the initially narrow beam into a broader distribution. Multiple filters and beam-shaping elements, as shown in Figure 27-2, B, produce an even distribution of customized shape at the surface of the patient. Electron beams lose their energy through different types of interactions than photons, leading to a different pattern of dose versus depth for electron beams. Rather than periodically removing photons from the beam through attenuation, electrons lose their energy gradually and at a relatively constant rate until the entire kinetic energy of the electron is expended and the particles simply stop. Ideally, this phenomenon would lead to a region of constant dose with increasing depth until the depth of full energy loss is reached, at which point the dose would drop abruptly to zero, with the depth of the dose drop-off being dependent on the initial electron energy. In practice, scattering and redirection of electrons within the beam lead to a mildly peaked dose plateau region and a somewhat more gradual dose falloff, as shown in Figure 27-3.

Delivery of Therapeutic Radiation

The cytotoxic properties of ionizing radiation provide an opportunity for tumor control but also require that care be exercised to limit the exposure of healthy tissue to radiation. For external beam radiation therapy, linacs are typically mounted on rotating gantries (Fig. 27-4) that allow beams to pass through the patient and the target from a variety of directions. By placing the area to be treated at or near the center of rotation, multiple beams can be made to overlap in the region of the tumor, delivering a high dose to the overlap area and a comparatively low dose to other areas. For treatment based on implanted radioactive sources, a procedure known as brachytherapy, proper dose delivery consists of designing and delivering a three-dimensional (3D) distribution of radioactive seeds within the volume to be treated, creating a high-dose region that decreases rapidly beyond the treatment volume. Both of the aforementioned treatment modalities require a 3D understanding of the patient anatomy and require:

The Molecular Biology of Cellular Radiation Responses

Much more has been learned about the radiation response of cells at the molecular level since the molecular biology and biotechnology revolutions beginning in the early 1980s. In fact, many of the radiobiological phenomena carefully characterized decades ago but necessarily couched in operational terms (e.g., clonogenic survival, sublethal damage recovery, and the cell cycle age response) now have solid molecular underpinnings. Of particular interest to radiation biologists is the molecular basis of radiation sensitivity, the pertinent aspects of which will be summarized here. An important ramification of our ever-growing understanding of the molecular workings of cells is the promise of being able to identify and target specific genes, proteins, or pathways for therapeutic gain, such as to render tumors cells more radiosensitive or normal cells more radioresistant.

A cell’s (normal or tumor) radiation response represents a complex interplay between intrinsic properties and extrinsic factors imposed by the cell’s microenvironment. Properties intrinsic to the cell include, for example, its capacity to recognize and repair DNA damage, its position in the cell cycle, and its response to being damaged, that is, to either adapt and grow or to die. However, extrinsic, environmental factors also can contribute to cellular radiosensitivity. Examples include the availability of nutrients and oxygen, the ability to eliminate waste, and the presence or absence of cytokines, growth factors, or other signaling molecules that instruct the cell how to respond to its radiation injury.

Normal mammalian cells have in common key molecular signaling pathways that regulate growth, death, and differentiation, many of which are activated in response to radiation exposure. Many of these pathways also exist in tumor cells but are typically dysregulated—that is to say, hyperactive or hypoactive—secondary to the activation of oncogenes and/or the inactivation of tumor suppressor genes. Not surprisingly, the activity (or lack thereof) of these pathways can influence both cellular radiosensitivity and other radiation responses of intact tissues.

The first activated oncogene identified to confer radiation resistance was NRAS (see references 11 and 12). Ras proteins, products of RAS family member genes, are small guanosine triphosphatases activated by upstream receptor tyrosine kinases. They transduce signals to a complex downstream cascade of protein kinases that have the net effect of promoting cell survival (for example, by the suppression of apoptotic cell death pathways) and unlimited proliferation (secondary to, for example, the downregulation of antiproliferative proteins), two of cancer’s hallmark phenotypes. When the ras protein is mutated, however, as is the case in approximately 30% of all human cancers, these pathways become overactive and no longer responsive to the normal regulatory mechanisms that antagonize the ras pathway and reign in excessive proliferation. This situation is exacerbated in many tumors by the concurrent loss of function of tumor suppressor proteins.

Ras became, accordingly, a desirable target for molecular cancer drug development.¹³ The general approach to targeting ras focused on its need for posttranslational modification through prenylation to become activated.¹⁴ Despite encouraging preclinical results with a class of prenylation-inhibiting agents known as farnesyl transferase inhibitors—drugs that, when used alone or combined with radiation, produced sensitization of tumor cells with constitutively active ras pathways—these drugs did not meet expectations in clinical trials because of alternative prenylation pathways.¹³

Subsequent drug development focused on inhibiting proteins upstream of ras, in particular, members of the human epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases that transduce growth signals through ras and other signaling proteins.15,16 These transmembrane glycoproteins are activated through binding of ligands belonging to the EGF family of peptide growth factors.

Cetuximab, a human-murine chimeric immunoglobulin G1 monoclonal antibody raised against the EGFR, is among the more successful molecularly targeted drugs, yielding improved outcomes in squamous cell head and neck cancers when combined with radiation therapy.¹⁷ Details of its mechanism of action and clinical use are discussed later in this chapter. Other classes of drugs that target EGFR include the small molecule tyrosine kinase inhibitors gefitinib and erlotinib, which inhibit EGFR phosphorylation and its downstream cascade by blocking the intracellular catalytic domain of the receptor.¹⁸ These drugs have shown some efficacy in selected patients with treatment-refractory, advanced non–small cell lung cancer (NSCLC).¹⁹

A second molecular target of interest is vascular endothelial growth factor (VEGF) and/or its cell surface receptor. VEGF is the most potent of the proangiogenic endothelial cell proliferation stimulators and chemoattractants and plays a pivotal role in promoting tumor survival by stimulating the growth of new blood vessels derived from the host vasculature. This phenotype of sustained angiogenesis is a hallmark property of cancer. Several isoforms of VEGF bind to the corresponding receptors VEGFR1, VEGFR2, and VEGFR3 (encoded, respectively, by the genes FLT1, KDR, and FLT4).²⁰

Bevacizumab, a recombinant humanized monoclonal antibody, is the first molecularly targeted antiangiogenic drug to gain approval for clinical use by the U.S. Food and Drug Administration. This antibody binds to VEGF, with the net effect of eliminating signaling to vascular endothelial cells to initiate angiogenesis.21,22 The clinical use of bevacizumab in combination with radiation therapy is discussed later in this chapter.

Cell Survival and Tissue Dose-Response Curves

Tumor control is achieved only when essentially all clonogenic cells (the putative “tumor stem cells”) are killed or otherwise rendered unable to sustain tumor growth indefinitely. To estimate the likelihood of cure, it is necessary to know, at least to a first approximation, how radiosensitive or resistant these cells are—that is, some measure of cell killing efficiency per unit radiation dose.

Death as a permanent, irreversible cessation of vital functions is not the same as what constitutes “death” to the radiation biologist or oncologist. For proliferating cells, including those maintained in vitro and the stem cells of both normal tissues and tumors in vivo, cell death in the radiobiological sense refers to a loss of reproductive integrity or clonogenicity, that is, an inability to sustain proliferation indefinitely. It is important to note that the term “clonogenic death,” as first described more than 50 years ago, is operationally defined and today serves as a catch-all term encompassing various mechanistic ways that cells die, all of which culminate in a cell losing its ability to divide indefinitely. These modes of cell death include mitotic catastrophe (the most common form of cell death after radiation exposure), apoptosis, autophagy, necrosis, senescence, and, strictly speaking, differentiation as well, to the extent that differentiated cells lose their ability to divide.²³ Another noteworthy feature of clonogenic death is that it does not necessarily preclude the possibility that a cell may remain physically intact, metabolically active, continue its tissue-specific functions, and even divide a limited number of times after irradiation.

The first report of a quantitative measure of intrinsic radiosensitivity for a human cell line (HeLa, derived from a cervical carcinoma) was published by Puck and Marcus in 1956.²⁴ For different doses of x-rays, the reproductive integrity of HeLa cells was measured by their ability to form macroscopic colonies of at least 50 cells (corresponding to approximately six successful postirradiation cell divisions) on Petri dishes. The HeLa cell survival curve, in which the log of the surviving fraction of cells was plotted as a function of the radiation dose, was characterized by a roughly exponential dose response at intermediate to high doses and a bending, “shoulder” region at low doses where cell killing was less effective. This phenomenon is illustrated graphically in the upper panel of Figure 27-7. Radiation survival curves for hundreds of cell types derived from mammalian tumors and normal tissues have been generated in the years since the pioneering work of Puck and Marcus,²⁴ and most are qualitatively similar to the original HeLa survival curve (see lower panel of Fig. 27-7).

Mathematical models were developed to fit the cell survival data, with survival curve theory originating in a consideration of the physics of energy deposition in matter by ionizing radiation. An assumption inherent to target theory was that a biological response (cell killing in this case) resulted from critical “targets” receiving random “hits”²⁵ in a probabilistic manner. Further, for cell survival curves with shoulders, each target was envisioned as requiring more than one hit to elicit the response, that is, that “sublethal” damage had to accumulate first before the cell would be killed. One mathematical expression derived from target theory that provided a fairly good fit to survival data was:

< ?xml:namespace prefix = "mml" />S=1−(1−e−D/D0)n

In this equation, S is the fraction of cells that survive a given dose (D), D₀ is the dose increment that reduces the cell surviving fraction to 37% (1/e) of some initial value on the exponential portion of the curve, and n, the extrapolation number, is the back extrapolation of the exponential portion of the survival curve to zero dose.

Over time, it became apparent that some features of this model were inadequate,²⁶ not the least of which was that its basis was the probabilistic nature of energy deposition in matter by ionizing radiation and not anything biologically based. For example, target theory was not concerned per se with which biomolecules in the cell were the purported “targets” of radiation damage, what the nature of the damage was in a molecular sense, or how the cell responded to it. (Of course, it was not lost on radiation biologists of the day that at least one cellular target was likely to be the DNA contained in chromosomes.²⁴)

A different and more biology-based interpretation of the dose response for radiation-induced cell killing was proposed by both Kellerer and Rossi²⁷ and Chadwick and Leenhouts.²⁸ The linear-quadratic (LQ) or “alpha-beta” equation,

S=e−(αD+βD2)

was shown to fit cell survival data quite well, particularly in the low-dose region of the curve where the target theory model often failed.²⁶ In this expression, S is again the fractional cell survival following a dose (D), α is the rate of cell kill by a single-hit process, and β is the rate of cell kill by a two-hit mechanism. Implicit in the LQ model was that (borrowing the language of target theory for comparative purposes only) DNA (or a chromosome) was the target, and the hits corresponded to irreparable or misrepaired lesions produced by either one or two radiation tracks traversing the cell nucleus (Fig. 27-8).

A comparison of the features and parameters of the target theory and LQ survival curve models is shown in Figure 27-9.

To bridge the gap between the radiation responses of single cells grown in culture and tissues or tumors in a laboratory animal or human patient, several ingenious methods were developed to measure, or at least estimate, the radiation sensitivity of intact normal tissues and tumors in vivo.31–31 Some of these assays used the reproductive integrity of cells as an end point, similar in principle to the in vitro survival curve assay, but in which the animal essentially served as its own Petri dish. One classic example of an in vivo clonogenic assay is the spleen colony assay of Till and McCulloch,³⁰ which originally was developed as a model system for the study of bone marrow transplantation. These authors determined that lethally irradiated mice could be “rescued” by a bone marrow transplant and that the transplanted, viable bone marrow stem cells were noted to form discrete nodules or colonies in the spleens of irradiated animals. By extension, then, a mouse’s spleen could be used as a pseudo Petri dish, with the number of clonogenic bone marrow colonies countable as a function of the radiation dose that the donated bone marrow received prior to transplantation. Assays such as this showed that the radiosensitivity of individual cells (tumor cells or normal cells) was largely unchanged whether the cells were irradiated in relative isolation in Petri dishes or as parts of a more complex tissue containing many different, interacting cell types in 3D contact.

Unfortunately, in vivo clonogenic assays necessarily involved the sacrifice of the animal and thus obviously are not applicable for clinical use. Further, such assays are labor intensive and involve long waiting periods before results are obtained, adding to their impracticality. A second type of in vivo radiosensitivity assay is nonclonogenic, using a tissue structural or functional end point as a surrogate for cell survival. Data derived from nonclonogenic assays and plotted as a function of radiation dose are properly called dose-response curves rather than cell-survival curves, because cell survival is not the end point being assessed. Regardless, cell-survival curves and dose-response curves are often analyzed and interpreted similarly; for example, a mathematical model is commonly used to fit the data and survival curve parameters are calculated.

Two examples of nonclonogenic assays, one for normal tissues and one for tumors, are worth mentioning, especially because aspects of both are used routinely in the clinic, if not in the exact same way as the laboratory assay. One of the first nonclonogenic methods developed to assess normal tissue radioresponse was the skin reaction assay.³² (Pigs were used in the original studies because their skin is similar to that of humans in several key respects, although rodents have also been used.) An ordinate scoring system was used to quantify the severity of the skin reaction; for example, a skin score of “1” might correspond to mild erythema, whereas a score of “4” might correspond to confluent moist desquamation over more than half of the irradiated area. Then, when comparing different time, dose, and fractionation schedules, any combination that resulted in the same skin reaction score was assumed to correspond to an equivalent amount of cell killing. In this way, and by collecting information for different severities of skin reactions, a dose-response curve could be generated. A common nonclonogenic assay of tumor response to radiotherapy is the regrowth delay assay.³³ In this assay, the tumor’s dimensions (or volume) are measured periodically as a function of time after irradiation, with the degree of tumor shrinkage assumed to be a reflection of the fraction of clonogenic tumor cells killed. Dose-response curves are generated by plotting the amount of growth delay (in days) as a function of radiation dose.

Modifiers of Radiation Sensitivity

As discussed previously, factors both intrinsic and extrinsic to the cell can alter its radiosensitivity. In addition to the intrinsic, genetic determinants of radiation sensitivity, other physical and chemical modifiers can also play important roles. The type of radiation used for treatment can be considered a physical modifier of radiosensitivity to the extent that high LET types of radiation (e.g., neutrons and heavy ions) are more biologically effective for a given unit of dose than low LET types (e.g., x-rays and electrons). Representative dose-response curves for radiations with different LETs are shown in Figure 27-10. In light of these differences in biological potency, the term relative biological effectiveness (RBE) has been coined to compare and contrast two radiation beams of different LET. RBE is defined as the ratio of doses of a known type of low LET radiation (historically, 250 kVp x-rays were the standard, but others can also be used) to that of a higher LET radiation to yield the same biological end point. RBE is highly variable and depends on several irradiation parameters, including the type of radiation, total dose, dose rate, dose fractionation pattern, and the biological effect being assessed. An example of how RBE values are obtained from cell survival curves is shown in Figure 27-11.

Chemical modifiers of radiation response are also important, and perhaps the “chemical” of greatest significance in this regard is molecular oxygen, a potent radiosensitizer. Mechanistically speaking, the reason that well-oxygenated cells are more sensitive to radiation than cells relatively lacking in oxygen is that oxygen readily participates in the free radical reactions that occur in the micro- to milliseconds after irradiation and has the net effect of enhancing the radiation damage to cellular macromolecules. Poorly oxygenated cells experience less enhancement of radiation injury and are therefore more radiation resistant. This phenomenon is termed the oxygen effect. The relative resistance of poorly oxygenated cells compared with well-oxygenated ones can be expressed in terms of an oxygen enhancement ratio (OER). The OER is the ratio of doses to produce the same biological effect under low versus normal conditions of oxygenation. The OER typically ranges between 2.5 and 3.0 for large single doses of x-rays or γ-rays and 1.5 to 2.0 when multiple small dose fractions are used.³⁴ Further, the oxygen effect is reduced or eliminated as the LET of the type of radiation increases; OERs of 1.5 to 2.0 are obtained for radiations of intermediate LET, and an OER of 1.0 (i.e., no oxygen effect) is obtained for high LET radiations. Representative radiation survival curves generated in the presence or relative absence of oxygen are shown in Figure 27-12.

Hypoxic cell radiosensitizers, exogenous chemicals that mimic the radiation damage–enhancing effects of oxygen, have been used clinically in an attempt to combat the relative radiation resistance of tumors that contain a clonogenic fraction of poorly oxygenated, hypoxic cells. A second approach to the problem posed by tumor hypoxia is to combine radiotherapy with a drug that, rather than sensitizing hypoxic cells, kills them outright. These drugs are called “bioreductive” because their toxic effects only occur secondary to the reductive metabolism common in cells relatively lacking in oxygen.35,36 These drugs are discussed further later in this chapter.

With few exceptions, clinical trials over several decades involving such hypoxia-directed therapies have been unsuccessful, in no small part because there was no way to preselect patients whose tumors contained sizeable hypoxic fractions and therefore might be expected to reap the most benefit from the use of a hypoxic cell radiosensitizer. In fact, there was no way to directly measure hypoxia in human tumors at all until the late 1980s.

One of the first studies demonstrating an association between directly measured oxygen tension in tumors and clinical outcome was published in 1988 by Gatenby et al.³⁷ An oxygen-sensing electrode was inserted into patients’ tumors (advanced squamous cell carcinomas of the head and neck), and multiple readings of partial pressure of O₂ were taken at different depths along the probe’s track; the arithmetic mean pO₂ value for each tumor positively correlated with local control rate, as did the tumor volume–weighted pO₂ value. A high tumor oxygen tension was associated with a high complete response rate and vice versa. Several years later, comparable oxygen electrode results for uterine cervix cancers were obtained by Höckel et al.³⁸ They made the important discovery that tumor hypoxia was a negative prognostic indicator in general, regardless of whether the patient had received radiation therapy, suggesting that radiation resistance was only one aspect of the hypoxia problem. A large meta-analysis of the relationship between tumor oxygenation status measured with oxygen electrodes and clinical outcome was published more recently for advanced head and neck tumors,³⁹ with comparable findings of improved overall survival in patients whose tumors were less hypoxic.

Another method of directly identifying and quantifying hypoxic cells in tumors is through the use of “hypoxia markers.” Both exogenous and endogenous markers for tumor hypoxia are available and can be studied in relation to each other, the tumor vasculature, or other “markable” features of the tumor microenvironment (e.g., the proliferation status of tumor cells). Exogenous markers consist of injectable drugs or chemicals that are bioreducible only under hypoxic conditions, causing them to bind to cellular proteins. These bound metabolites can be marked radioactively or with antibodies and can be visualized using several different techniques, such as autoradiography⁴⁰ or fluorescence immunohistochemistry.⁴¹ Two exogenous markers studied extensively in both preclinical cell and animal systems, as well as in human patients, are pimonidazole hydrochloride^42,43 and EF-5.⁴⁴ Both are of the chemical class known as nitroimidazoles that were originally studied as possible radiosensitizers of hypoxic cells (e.g., see references 45 and 46, as well as the discussion later in this chapter) but were serendipitously found to also have the property of bioreduction and binding to cellular macromolecules under hypoxic conditions. This facilitated their use as hypoxia markers. Endogenous markers, on the other hand, consist of genes and proteins that are upregulated as part of the cellular stress/adaptive response to hypoxic conditions and whose expression levels can, with caveats, be used as surrogates for tissue oxygenation status.^47,48 Commonly studied endogenous hypoxia markers include the hypoxia-inducible factor 1–alpha (HIF-1α, a transcription factor),⁴⁸ the enzyme carbonic anhydrase IX (CA-9 or CAIX),⁴⁹ glucose transporter–1 (GLUT1),^52–52 and osteopontin.⁵³

Figure 27-13 illustrates the potential of deriving “geographic” information about the extent and location of hypoxia in six different human tumors, using the exogenous marker pimonidazole hydrochloride. Simultaneously marking other tumor features, the location of blood vessels and proliferating cells in these examples, provides additional information about the tumor microenvironment.