In terms of fractionation, the decrease in dose rate can be considered as lowering the fraction size of the total radiation dose to be delivered in external beam HDR radiotherapy (Fig. 5.11). Referring to the LQ model (Sect. 5.5) for comparison of biological effectiveness of different radiation treatment schemes, low dose rate irradiation could be considered as super-fractionation (Fig. 5.11 and Box 5.7).

A line graph represents cell survival in Log 10 versus the total dose in gray. The plotted data, the dotted curve for continuous L D R, the solid curve for fractionated H D R, fraction number, and dose rate, gray per hour feature a downward trend.

Fig. 5.11
The dose rate effect seen as an extreme form of fractionation. Cell survival following fractionated HDR irradiation with increasing number of fractions (solid curves). With an infinite number of tiny fractions, and complete sublethal damage repair, the dose-squared β parameter of the LQ tends to zero, and only the dose-linear β parameter plays a role. Then, the Biologically Effective Dose (BED) is reached for a certain endpoint effect E. Similar sparing phenomenon with decreasing dose rate in continuous LDR exposure (dotted curves)


A line graph represents cell survival in Log 10 versus the total dose in gray. The plotted data, the dotted curve for continuous L D R, the solid curve for fractionated H D R, fraction number, and dose rate, gray per hour feature a downward trend.


At extremely low dose rate, i.e., irradiation with an infinitely large number of infinitesimally small dose fractions, the theoretical total dose required to produce an isoeffect is the Biological Effective Dose (BED) of the LQ model.


Thus, as exposure is elongated, the shoulder of the cell survival curve tends to become shallower, this is because the α parameter of the linear-quadratic model does not change significantly, while the β parameter tends towards 0.


This situation also implies, dependent on the fractionation sensitivities of irradiated tumor and normal tissues involved (i.e., their repair capacity characteristics expressed in their α/β values) as well as of their DNA repair kinetics (the half time for sublethal damage repair T1/2), an optimal therapeutic ratio situation. For the LQ model adaptation to correct for the dose rate effect and incomplete repair, additional parameters are introduced (e.g., Joiner and van der Kogel [16]). The dose rate effect of continuous low dose irradiation is discussed in view of the 6R’s of radiobiology in Sect. 5.4 below.


The repair process of radiation-induced DNA lesions has been explained in depth in Chap. 3. DNA DSB, if not repaired, are lethal to the cell. A DNA DSB can either be induced by single-track action or double-track action. A single-track X-ray lesion is independent of dose rate and linearly proportional to dose (the contribution of α in the LQ model). In double-track action, the two interactive single strand DNA lesions are produced by different tracks of X-ray photons, and the formation of double strand lesions is therefore dependent on the dose rate and is proportional to the radiation dose squared (the contribution of β in the LQ model). In fact, the protracted delivery of a given radiation dose reduces the effect of double-track action because time offered between lesions is long enough for repair to occur [28] (Fig. 5.12).

An illustration displays the X-ray penetrates the cancerous cell and goes through a single track action and double track action. A line graph below represents proportion of full effect versus dose per fraction. The linear and nonlinear are in a downward trend.

Fig. 5.12
Illustration single-track action and double-track action. In single-track action, the two interactive lesions are produced by a single track of ionization induced by an X-ray photon that subsequently produces a dose which is independent of dose rate and linearly proportional to dose. In double-track action, the two interactive lesions are produced by a different track of ionization induced by X-rays which subsequently produces a dose which is dependent on the dose rate (decreasing the dose rate reduces double-track action) and non-linearly proportional to the radiation dose squared


An illustration displays the X-ray penetrates the cancerous cell and goes through a single track action and double track action. A line graph below represents proportion of full effect versus dose per fraction. The linear and nonlinear are in a downward trend.


Figure 5.13 shows that lowering the dose rate has greater effect on cells or a tissue with a low α/β ratio, for example, 3 Gy than with a high α/β ratio of, for example, 10 Gy. At a low α/β ratio, the curves are spread out more, implying that late responding normal tissues are particularly spared relative to tumors when decreasing the dose rate.

Two line graphs represent effect versus dose in gray. The plotted data in each graph features a decreasing dose rate.

Fig. 5.13
The effect of lowering the dose rate on the survival of cells. (a) Cells characterized with an α/β ratio of 10 Gy (typical for a tumor or early responding normal tissue). (b) Cells with an α/β ratio of 3 Gy (typical for a late responding normal tissue). See text for details. (Figure from Shrieve and Loeffler [17], with copyright permission from Wolters Kluwer Health, Inc.)


Two line graphs represent effect versus dose in gray. The plotted data in each graph features a decreasing dose rate.


Also, for a tissue having an equivalent α/β ratio, larger sparing is obtained with decreasing tissue-specific half time (T1/2) for sublethal damage repair. Similarly, as with fractionated radiation, this can be attributed to incomplete repair between the “fractions” or during continuous exposure. Hence, at longer repair half time, low dose rate irradiation is causing more damage, and less discriminative between tissues with different α/β ratio.


It is well recognized that cells in the G2 or M phase of the cell cycle are more sensitive to radiation than cells in the G1, G0, or S cell-cycle phases (Sect. 5.4, Fig. 5.8). During continuous low dose rate irradiation, the process of redistribution would push initially relative radioresistant cells into a radiosensitive cell-cycle phase. This process is dependent on numerous cellular and tissue factors, and therefore difficult to predict.


Another phenomenon that might occur is the inverse dose rate effect, which represents a reversal of the typical pattern of the conventional sparing with decreasing dose rate. For the same radiation dose, radiation delivered at a certain specific lower dose rate increases the radiosensitivity of cells in comparison to radiation delivered at a higher dose rate. This is illustrated in Fig. 5.14.

A diagram displays the G 2 block to G 1 where G 1 leads to G 2 cells and then to the tumor, where it progresses to colon cancer, and then goes through the continuous low dose rate exposure of the tumor value.

Fig. 5.14
The inverse dose rate effect. When the dose rate delivered to HeLa cells is decreased from 1.54 to 0.37 Gy/h, the efficiency of cell killing increases, with damage generated similar to that from an acute exposure [35]. When cells are exposed to higher dose rates, they are kept in the phase of the cycle in which they are at the beginning of irradiation. However, use of lower dose rates may allow cells to continue cycling during irradiation. When cells are exposed to 0.37 Gy/h, cells tend to progress from other phases of the cell cycle and arrest in G2, which is a radiosensitive phase of the cycle. As a result, an enriched population of G2 cells is responsible for increasing the radiosensitivity of cells


A diagram displays the G 2 block to G 1 where G 1 leads to G 2 cells and then to the tumor, where it progresses to colon cancer, and then goes through the continuous low dose rate exposure of the tumor value.


Tumor cell repopulation during continuous low dose rate (LDR) exposure might negatively influence treatment outcome since a larger number of cells have to be sterilized if the repopulation rate outflows the duration of exposure, which might occur with fast repopulating tumor cells (e.g., cell doubling time of 24 h).


The impact of irradiation on the immune response has been shown to be dependent on the radiation dose (see Chap. 6), and the dose rate of exposure is likely to play a role [36]. The effect of low dose rate irradiation regarding reactivation of the immune response is however not well described.


Chronic low dose rate exposure will not cause oxygen depletion in initially well-oxygenated tumor cells. Initially, hypoxic cells might benefit from reoxygenation during long-term radiation exposure. However, as pointed out in Sect. 5.4, the kinetics of reoxygenation is very much dependent on the tumor type.


Whole-body irradiation (WBI) or total body irradiation (TBI) refers to the therapeutic protocol in which a patient’s total body is irradiated with γ/X-rays. WBI is used as part of the conditioning regimen for transplantation of bone marrow or hematopoietic stem cells for lymphoma, leukemia, or multiple myeloma and as a palliative regimen in selected cases of lymphoma and leukemia [37]. WBI implicates irradiation of the total body, with reduction of the dose to the lungs, to lessen the hazard of radiation-induced lung toxicity [38, 39]. Historically, in the fifth and sixth decade of the last century scientists trying to reverse early responding tissue effects of radiation, demonstrated experimentally that bone marrow engrafted with hematopoietic stem cells from a donor animal “could recapitulate the blood system” and thus showed that previously irradiated bone marrow could be rescued. This contributed to the development of therapeutic techniques involving bone marrow ablation followed by bone marrow engraftment with hematopoietic stem cells for the treatment of some marrow cancers, for example, leukemia or multiple myeloma [37]. This procedure is mainly used to eliminate residual cancer cells in the transplant recipient, and to further suppress or destroy the immune system; subsequently, it serves to prevent immunologic rejection of blood stem cells or transplanted donor bone marrow. Thus, the chances of engraftment are increased, and the bone marrow stromal cells of the patient are spared [38, 39].


Since a characteristic of WBI is that it can sterilize small numbers of widely spread cells that are sensitive to radiation, this makes it a treatment option for (residual) marrow disease. Biologically, leukemia is associated with a spectrum of intrinsic cellular radiosensitivity that ranges from notable radiosensitivity to significant radioresistance, which determine the extent of leukemic cell killing. The molecular biology responsible for the variety in radiosensitivity of leukemia is not entirely known, but increased apoptosis seems to require functional p53, c-myc, and Bcl2 genes. Therefore, it seems that radiosensitivity results from the apoptosis retention after activating p53, c-myc, and Bcl2 genes by radiation [40]. RT in conjunction with a wide range of treatment modalities such as (myeloablative) chemotherapy and the subsequent graft-versus-tumor effect are therefore required to obtain significant eradication of malignant clones [22, Chap. 16].


Bone marrow stem cells typically have D0 values ranging from 0.5 to 1.4 Gy. These cells are therefore intrinsically radiosensitive. Even though hematopoietic rescue (i.e., stem cells) could allow the delivery of high doses that eliminate the recipient’s marrow cells which in turn prepares the stem cell microenvironment for repopulation to occur, this procedure is associated with long-term or life-threatening consequences. Critical organs of concern in WBI are those described as late responding tissues. Fortunately, as effect on these tissues is dependent on total dose, dose rate and fractionation, appropriate scheduling of the treatment allows some protection. While a modest number of cancer cells being radiosensitive will be killed, complete cancer cell killing may not always be possible with radiation alone. Therefore, TBI often needs to be given together with chemotherapy. Moreover, incomplete bone marrow ablation may result in mixed chimerism of bone marrow after transplant [22, 40].


As a result of immunological mismatch between recipient and donor, rejection of donor stem cells may occur. In order to avoid this, TBI is used to prevent the recipient from rejecting donor stem cells.


Bone marrow transplantation results are influenced by the treatment schedule. Lymphoid cells repair a large amount of radiation-induced DNA damage during the time interval between fractions. Hence, the effectiveness of fractionated TBI is reduced significantly in comparison with single-dose TBI and results in more graft rejections. However, the fractionation effect is reversed for bone marrow stromal cells (“colony-forming unit fibroblasts”). The success of engraftment is based on the likelihood of sparing bone marrow stromal cells, and when treatment is delivered as single-dose TBI, the likelihood of damaging both bone marrow stromal cells and their progenitors increases. Importantly, the effectiveness of single-dose TBI is increased significantly in comparison with fractionated TBI, but at the cost of increased long-term toxicity [22].


Unlike curative RT, palliative RT is used to control the symptoms of advanced, incurable cancer (the primary tumor or metastatic deposits) by slowing down tumor growth, controlling symptoms and causing cancer to regress [41]. WBI may be effective for palliation, especially for advanced leukemia or lymphoma, using rather low doses in the order of 0.1 Gy/fraction. In experiments with solid tumors, tumor cells with colony-forming abilities in both experiments of formation of artificial metastases and naturally developing metastases, these tumors could be suppressed with specific low doses [42]. It is assumed that either chronic TBI or low dose total body irradiation may stimulate the immune system to eliminate metastatic cancer cells. However, nowadays, TBI is only very rarely used for this indication. For further information, see tumor microenvironment changes and abscopal effect discussed in Sect. 5.15.


In WBI, the doses delivered for transplantation of bone marrow or stem cells are in the range of 10–12 Gy [43]. To reduce long-term complications in the recipient, this dose is typically divided into 2 Gy fractions [14, 44]. In the so-called reduced conditioning regimens, single fractions of 2 Gy or two fractions of 2 Gy are given. When WBI is split into multiple small fractions and spread over a period of time, outcomes are generally improved, and toxicity is diminished. While the former is due to the fact that the dose is still adequate to eradicate both any cells of residual malignant tissue and the recipient’s bone marrow, the latter is explained in Sect. 5.2 [45].


The dose rate in RT influences the effectiveness of the radiation exposure. An explanation of how a survival curve may become shallower at low dose rates was discussed in Sect. 5.5. In WBI, the pattern is different from localized RT because the effect of radiation dose depends on the tissue type. When low dose rate is used, the incidence of normal tissue toxicity is decreased in comparison with high dose rates [46–48]. However, changing the radiation dose rate from the low dose rate to high does not affect the probability of engraftment success [49, 50].


Functional parameters including reoxygenation, redistribution, repopulation, repair, and radiosensitivity are traditionally used to define radio-responsiveness.

The role of repopulation also proved to be a robust predictive marker as illustrated in head and neck cancer where increased tumor expression of EGFR indicates efficacy of accelerated radiation.

DNA, RNA, and proteins have recently been identified to define tumor RT responsiveness yet few of them have attained clinical validation and/or application.


Functional parameters including reoxygenation, redistribution, repopulation, repair, and radiosensitivity are traditionally used to define radio-responsiveness.


The role of repopulation also proved to be a robust predictive marker as illustrated in head and neck cancer where increased tumor expression of EGFR indicates efficacy of accelerated radiation.


DNA, RNA, and proteins have recently been identified to define tumor RT responsiveness yet few of them have attained clinical validation and/or application.


Radiation treatment has been improved greatly over the last two decades by integrating 3-D anatomy into planning systems, and developing of image-guided (IGRT), intensity-modulated (IMRT) and intensity modulated arc radiation therapy (IMAT) techniques, resulting in individualization of treatment. Radiation treatment portals and arcs are much more tailored to the anatomy of each patient’s tumor and normal tissue. Today medical professionals prescribe RT taking into account the type of primary tumor, its grade, stage, location, size, biological characteristics, and concomitant treatments. However, these clinical parameters do not give an accurate prediction of the effect of RT since wide variations in response occur between patients given the same treatment and having similar tumors [51–53]. Furthermore, diverse treatment options are now available. Moreover, several tumor RT sensitizing strategies using different drugs which can enhance the effects of RT, especially those that target the molecular pathways, are becoming available now. Treatments frequently employ combined approaches. One such avenue currently underway involves trials that combine chemotherapy/RT with immunotherapy.


RT can differentially affect tumor responses due to a variety of radiobiological factors, which are referred to as the 6R’s. Among them, hypoxia, proliferation, and radiosensitivity have proved to be fairly good predictive markers [51–34]. Beyond these well-known classical biomarkers (BMs), there are also a number of promising candidate molecular biomarkers currently being tested in preclinical and clinical studies such as genetic and epigenetic factors as discussed in Sect. 5.10. In this section, classical and modern BMs as well as their role in predictive assays will be discussed in addition to the available methods used to detect them.


RT constitutes approximately 60% of cancer treatment. If a clinical assay could successfully predict the RT response, it would have wide-ranging clinical implications. A broad group of old-fashioned radiobiological variables that affect RT outcome including tumor oxygen status, the degree of repopulation or proliferation rate, intrinsic radiosensitivity, and both individual and tumor radiosensitivity is shown in Fig. 5.15.

A spoke diagram of radiotherapy outcome includes hypoxia, redistribution, and repopulation.

Fig. 5.15
Review of classical biomarkers used to obtain information on relevant features of radiobiology


A spoke diagram of radiotherapy outcome includes hypoxia, redistribution, and repopulation.


Predictive factors in therapy may relate more directly to primary tumors and their local control. Metastatic disease may need to be considered separately even though it clearly plays a significant role in survival of the patient. Research to develop predictive assays for tumor RT response should generally measure local control and normal tissue effects [16, 51, 52].


Tumor vascular beds differ significantly from those in normal tissues in their structure and physiological characteristics. Tumor-related blood vessels are composed of single-layered endothelium, commonly containing gaps between the endothelium taken up by tumor cells, resulting in immature capillaries. A dysfunctional blood supply through the tumor reduces oxygen delivery, resulting in areas of tumor hypoxia, acidic intra-TME nutritional deprivation, and therewith the tumor response to IR For more information about predictive tests to assess Oxygen Effect to Tumor Hypoxia see Sect. 5.8.


Tumor repopulation is a key factor contributing to treatment failure after RT. Alternative fractionation schemes have been proposed as methods for modulating interfraction tumor repopulation. A phase III randomized trial in over 1000 patients with head and neck cancer showed significant improvement in regional control following accelerated and hyperfractionated RT as compared to conventional fractionated RT. Additional evidence, which has shown the importance of proliferation, demonstrated that higher doses are needed to control a tumor when overall time of treatment is prolonged. Clinical evidence that tumor repopulation is an important mechanism for treatment failure is notably apparent in a subset of patients. Therefore, evaluation of tumor repopulation has been a priority for developing predictive tests [52]. Table 5.5 shows several tests that can be performed in vitro and in vivo to measure tumor repopulation.Table 5.5
Description of biomarkers and laboratory assays for assessing repopulation

Test

Procedure

Method

Statements

The mitotic index

Using tissue sections to calculate mitosis ratios

Biopsy staining

A correlation exists between outcome results and the labeling index, but it is weak

DNA flow cytometry

Calculating the percentage of cells that are in the S phase of the cell cycle

Flow cytometry

The tumor potential doubling time, Tpot

Using bromodeoxyuridine as a stain in a tumor biopsy

Flow cytometry

Tpot is not a significant predictor for RT response

Endogenous markers (Ki67)

Proliferation-associated proteins can be detected using antibodies

Gene signature, biopsy studies, or plasma/serum

Molecular marker profiles (p-53, Bcl-2, Ki-67, or EGFR expression)

Measuring the level of these markers

Continuous hyperfractionated accelerated radiotherapy (CHART) is recommended in head and neck cancer patients who have high expression of EGFR in tumor or low organized patterns of Ki-67 and is negative for Bcl-2 or p53 expression


Various types of cell death, such as apoptosis and autophagy, which also result in a loss of colony-forming ability, contribute to tissue reactions caused by IR. Many publications proposed that cellular radiosensitivity could be measured by the clonogenic assay. A technical challenge of dispersing tumor cells ex vivo has interfered with its clinical application, however [52].


For more information about predictive tests to assess radiosensitivity, see Chap. 7.


Radiation responsiveness was traditionally defined using the 6R’s (see Sect. 5.4) but only three factors proved reliable as prognostic markers: hypoxia, repopulation, and radiosensitivity, and hence RT regimens have been modified according to fraction size, dose per fraction, and overall treatment time as depicted in Fig. 5.16. Individual assessments of these parameters could have predictive value since each of these parameters has a substantial effect on the outcome of the RT. Assays based on measurements of these parameters, however, had mixed success in developing predictive assays for many reasons. Firstly, the lack of success may be explained by the fact that few quantitative differences exist between human tumors and normal tissues, and their heterogeneity overlaps in many ways. Secondly, it was intended that the 6R’s can be used to understand emerging phenomena in radiation biology rather than predicting its outcomes.

A diagram of modern predictive assays includes proteins endpoint, D N A endpoint, and R N A endpoint.

Fig. 5.16
Review of modern biomarkers used to obtain information on relevant features of radiobiology


A diagram of modern predictive assays includes proteins endpoint, D N A endpoint, and R N A endpoint.


The first molecular techniques were applied to radiobiology about two decades ago and soon revealed the existence of proteins and genes that respond to and influence the cellular outcome of IR [53]. Radiation response of tumors is associated with a complex series of gene and protein alterations some which also are influenced by the underlying genomic alterations, for example, mutations. When cells experience IR-induced damage, it was early on observed that key proteins are induced [57, 58]. An example is the p53 protein which upon exposing cells to a photon beam is induced and control multiple pathways. For instance, a single fraction of 20 Gy X-rays was observed to induce key proteins such as MDM2 and CDKN1A in some cell lines, both which is regulated by p53 [58]. Furthermore, the response to radiation is influenced by polymorphisms in genes encoding proteins that participate in DNA damage repair, as well as by mutations affecting these genes. When cells experience radiation insult, multiple genes undergo a series of up and down regulations interacting through many pathways including p53-regulated genes such as p21 (CIP1/WAF1) and GADD45A. Also, the response to radiation is influenced by methylation, acetylation, ubiquitylation, phosphorylation, and sumoylation of genes and proteins which control the DNA damage repair. For instance, the presence of hyper-methylated promoters means that a gene is becoming actively transcribed; hyper-methylation of promoters attracts proteins that inhibit transcription and turn it off. Non-coding RNAs also contribute to radiation response. There are complementary forms of RNA, namely microRNA (miRNA) that are not translated into protein and play an important role in the initiation and progression, repopulation and programmed cell death (see Chap. 3). Therefore, this mechanism is reflected in terms of sensitivity or resistance to IR [16, 59]. Overall, gene and protein expression are altered in the tumor itself and by radiation which affects cellular outcomes and causes a heterogeneity of RT response in tumors. Accordingly, protein, DNA and RNA analysis can be used to obtain information on relevant features of radiobiology using several types of measurements such as genomics, transcriptomics, epigenomics, or proteomics, which analyze DNA, RNA, DNA–chromatin interactions and proteins, respectively, as described in Fig. 5.17 and Table 5.6.

A schematic diagram of the different types of biomarkers. These are the nucleus, chromosome, nucleosome, cell, proteomics, transcriptomics, R N A, chromatin, D N A, genomics, epigenomics, and phenomics.

Fig. 5.17
Schematic view of biomarkers. Proteins, DNA chromatin, DNA, or RNA that are analyzed by proteomics, genomics epigenomics, genomics, or transcriptomics, respectively
Table 5.6
Description of modern biomarkers and assays for analyses of RT response

Marker

Advantage

Assay

Protein

Expression profiling at the protein level can be a good candidate marker as proteins are responsible for the actual cellular functions.

Tissue microarrays for high-throughput immuno-histochemistry, luminex technology for multi-analyte immunobead-based profiling, mass spectrometry methods and antibody chip.

DNA

The DNA level can be a good candidate marker because everyone is genetically unique, cancer is caused by genetic changes, and is characterized by genomic instability. Variation in individual genes influences risk for cancer development. Variations in a patient’s response to treatment are ascribed to the heterogeneity of genetic changes in cancer.

In situ hybridization (ISH) for identifying a DNA sequence in tumor tissue, genetic variation sequence for analyses of duplications and deletions of large region DNA (copy number variation (CNV) and Single Nucleotide Polymorphisms (SNPs), epigenetic variation sequence for chromatin modification and DNA acetylation, ubiquitylation, phosphorylation, sumoylation, and methylation.

RNA

Low-density Taqman arrays for detecting small signatures after whole genome profiling, ISH and FISH for detecting RNA in sample in situ. Quantitative polymerase or real-time chain reaction for quantify and amplify a targeted DNA molecule. Gene expression array for deriving profiles, classifiers or signatures associated with prediction of treatment results or prognosis. Next-generation sequencing technologies for the study of gene expression profiles of different species of mRNA.


A schematic diagram of the different types of biomarkers. These are the nucleus, chromosome, nucleosome, cell, proteomics, transcriptomics, R N A, chromatin, D N A, genomics, epigenomics, and phenomics.


Hypoxia refers to conditions with low oxygen. The oxygen concentration of most normal tissue in the human body is around 5–7% and tissues with less than 3% oxygen are regarded as hypoxic. Hypoxic cells are known to be more resistant to radiation and chemotherapy. Hypoxia is also a potent microenvironmental factor promoting metastatic progression of cancer  [60].


Hypoxia in tumor cells can be of two types—acute hypoxia or chronic hypoxia. Acute hypoxia is a transient perfusion-limited hypoxia due to transiently occluded blood vessels [61]. Chronic hypoxia is a diffusion-limited hypoxia and can lead to necrosis [62].


Oxygen can generally diffuse approximately 150 μm at the arterial end of the capillary and less at the venous end (Fig. 5.18). Therefore, when the radius of the tumor is less than 160 μm, there is no central necrotic region. Between 160 and 200 μm, there may or may not be a hypoxic center. When the radius is more than 200 μm, the central portion consists of anoxic necrotic cells while the next layers consist of cells with different degrees of hypoxia and aerobic actively dividing cells at the outer layer [62]. The central portion becomes necrotic because the cells are deprived of both oxygen and nutrients.

A diagram of the components of the oxygen passing through the tumor in small amounts. These are the blood vessel, aerobic cells, hypoxic viable cells, and anoxic necrotic cells, all with increasing chemotherapy and radiation resistance.

Fig. 5.18
Diffusion of oxygen through tumor. As the distance from the blood supply increases, the oxygen levels available for the cells decreases. As the cells grow more hypoxic, they become more radioresistant


A diagram of the components of the oxygen passing through the tumor in small amounts. These are the blood vessel, aerobic cells, hypoxic viable cells, and anoxic necrotic cells, all with increasing chemotherapy and radiation resistance.


As hypoxic cells are more radioresistant than oxygenated cells (Chap. 3), irradiation of a tumor will predominantly kill the outer layer of cells leaving the hypoxic cells. One way to reduce this problem is to divide the radiation dose into many daily fractions, which will allow the hypoxic cells nearest to the oxygenated cells to be reoxygenated after the oxygenated cells have been killed [63]. In this section, we will give an overview of the most important mechanisms and pathways induced by hypoxia since these are potential targets in connection with treatments.


As described in Chap. 3, oxygen modifies the biological effects of low LET IR. For such radiation, DNA damage predominantly occurs through the indirect effect of radiation-induced water radicals. In the absence of oxygen, DNA radicals can become chemically restituted through donation of hydrogen atoms by SH-compounds (such as glutathione). However, if molecular oxygen is present, RO2· is produced which cannot be restored. Oxygen thus “fixes” the damage produced by free radicals (i.e., makes the radiation damage permanent). Therefore, cells are more sensitive to radiation in the presence of oxygen than in its absence.


The relative radiosensitivity of cells increases dramatically when the oxygen tension increases from 3 mmHg (0.4% O2) to about 30 mmHg (4% O2), which corresponds to the oxygen concentration in venous blood [64]. Beyond this, it reaches a plateau with no further effect of increasing the oxygen tension. In order to compete with restitution, molecular oxygen must be present during or within microseconds after the radiation exposure because the lifetime of the free radicals generated by the radiation is less than about 10−5 s.


The oxygen enhancement ratio (OER) is the ratio of doses under hypoxic to aerated conditions that produce the same biologic effect. In vitro studies have shown OER of X-rays to be around 3.5 for high doses and around 2.5 in low dose regions of the cell survival curves [65].


High LET radiations like alpha particles cause direct and complex damage to DNA with little possibility for restitution. Therefore, there is no enhanced effect with presence of oxygen (i.e., OER is 1). It is intermediate for neutrons and comparatively high for low LET radiations such as X-rays and gamma rays [66, 67]. OER decreases as LET increases. The OER falls slowly until about 60 keV/μm of LET, then falls rapidly and reaches 1, i.e., no oxygen effect, when LET reaches about 200 keV/μm [66]. Thus, one way to target hypoxia is to use high LET radiation (Box 5.9).


The oxygen enhancement ratio varies with:
Cell cycle phase

Type/quality of radiation

Radiation dose and dose rate


When a tumor grows, the existing vasculature will not be able to provide oxygen and nutrients to the more distant cells resulting in regions of hypoxia. During hypoxic conditions, the cells that survive are those that undergo adaptive responses, which are not only critical to survival but also promote malignancy and metastasis. The main purposes of the adaptive responses are to uphold ATP production and save energy as well as nutrients. ATP production is maintained by the formation of new blood vessels (angiogenesis), increased production of red blood cells (erythropoiesis), switch to anaerobic metabolism, and migration to a more favorable environment (metastasis). The processes to save energy include reduction of protein synthesis and recirculation of nutrients (autophagy). The signaling pathways regulating these processes are induced by upregulation of hypoxia-inducible factors (HIFs) and activation of the unfolded protein response (UPR).


HIF-1 was discovered in 1995. Its importance is emphasized by the award of the Nobel prize in Physiology or Medicine 2019 to William Kaelin Jr., Peter J. Ratcliffe and Gregg L. Semenza for their work in elucidating how HIF senses and adapts cellular response to oxygen availability. The HIF protein family consists of three different alpha subunits (HIF-1α, HIF-2α, HIF-3α), which bind to the same type of β-subunit. In this section, we will only discuss HIF-1, which is the only one which is ubiquitously expressed and the most well studied. In the presence of molecular oxygen, proline-hydroxylase enzymes (PHD) hydroxylate HIF-1α, i.e., PHDs add two OH-groups to proline residues on HIF-1α using oxygen as a cofactor. Hydroxylated HIF-1α is recognized by von-Hippel-Lindau (VHL)-ubiquitin ligase complexes, which then adds ubiquitin-groups that target HIF-1α for degradation. Without oxygen, HIF-1α is stabilized allowing it to bind to the β-subunit and activate gene transcription.


HIF-1 is known to induce transcription of more than 60 genes by binding to a common promoter called hypoxia response element (HRE). Some of these genes are involved in increasing oxygen supply, such as VEGF (which induces angiogenesis), erythropoietin (which stimulate red blood cell production), and iron transport to erythroid tissue. HRE-regulated genes are also involved in cell proliferation, such as insulin-like growth factor-2 (IGF-2) and transforming growth factor-α (TGF-α). HIF-1 also induces transcription of genes involved in switching of metabolic pathways to use glycolysis as a primary mechanism of ATP production. HIF thus regulate transcription of genes involved in glucose transport into the cell (i.e., GLUT1 and GLUT3), and in maintaining intracellular pH during increased lactate production such as carbonic anhydrase 9 (CAIX) and monocarboxylate transporter 4 (MCT4).


In addition to promoting hypoxia tolerance, HIF-1 also upregulates transcription of genes involved in several steps of metastasis: angiogenesis, epithelial-mesenchymal transition (EMT), cell motility, intra/extravasation, and the formation of a premetastatic niche facilitating colonization of metastatic tumor cells.


Stabilization of HIF and activation of its downstream pathways occur at relatively moderate levels of hypoxia (<about 2% O2) as compared to radiation resistance, which occurs below about 0.1% O2 [68].


Similar to IR resistance, activation of the unfolded protein response (UPR) occurs below about 0.2% O2 and contributes to hypoxia tolerance by reducing protein synthesis and inducing autophagy. UPR is activated by accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER). Such accumulation takes place under hypoxia because post-translational disulfide bond formation, which is necessary for correct protein folding, have been shown to be oxygen dependent [69]. There are three ER stress sensors in the ER membrane, PERK (protein kinase RNA-like endoplasmic reticulum kinase), IRE1a (inositol requiring kinase 1a), and ATF6 (activating transcription factor 6). All three are activated by the release of BiP (Binding immunoglobin Protein) from the ER membrane. Activated PERK phosphorylates eIF2α (Eukaryotic translation initiation factor 2 subunit α), which inhibits translation of protein except for some key proteins, which remain translated [70]. One of these is ATF4 (activating transcription factor 4), which induces transcription of genes required for autophagy, amino acid synthesis and import, redox balance, and angiogenesis [71]. However, if the stress becomes excessive, ATF4 can switch from pro-survival to pro-death and induce apoptosis [72]. Active IRE1α (Inositol-requiring transmembrane kinase/endoribonuclease 1α) reduces protein synthesis through the degradation of selected mRNAs. It also activates an inflammatory response and promotes autophagy as well as apoptosis. BIP release allows ATF6 translocation to Golgi, where cleavage of this protein results in release of transcriptionally active ATF6 (DNA-binding cytoplasmic domain of ATF6), which contributes to restoring ER homeostasis [70].


Hypoxia in tumor cells can be measured using oxygen probes or protein markers for hypoxia. Polarographic electrodes, in particular Eppendorf electrodes, have long been regarded as the gold standard for measuring tumor hypoxia. The limitations of these are that they must be inserted into the tumor damaging the tissue and that they consume oxygen resulting in a high signal-to-noise ratio for low oxygen levels [73]. Fiber-optic probes have also been developed [74], but the disadvantage of both is that this is an invasive method, which can only be used in accessible tumors. Another method is injection of pimonidazole to patients before biopsies or total resection of the tumor. Pimonidazole is a 2-nitroimidazole compound, which binds to thiol-containing proteins specifically in hypoxic cells, and it can be used in immunohistochemical analyses to quantitative assessment of tumor hypoxia [75].


A noninvasive alternative method is hypoxia PET imaging. 2-Nitroimidazoles have been developed as radiosensitizers due to their ability to undergo up to six electron reductions. However, they can also be used as radiotracers carrying a positron emitter. The reduction requires the activity of reductases that are only present in viable hypoxic cells resulting in the radiolabeled molecules being accumulated in hypoxic regions visible to PET imaging. Examples of nitroimidazole or nitroimidazole-derivative tracers are 18F-Fluoromisonidazole (18F-FMISO), 18F-Fluoroazomycin-Arabinofuranoside (18F-FAZA), 18F-labeled fluoroerythronitroimidazole (18F-FETNIM), 18F-fluoroetanidazole (18F-HX4), and 18F-2-(2-Nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide (18F-EF5) [73, 76]. Other redox-sensitive compounds include Copper(II) diacetyl-bis (N4-methylthiosemicarbazone) (60Cu-ATSM) and 123I-iodoazomycinarabinoside (123I-IAZA) [77]. Of these, 18F-MISO is the most extensively used in both preclinical and clinical studies. F-MISO is highly lipophilic, which means it can easily diffuse across cell membranes but also that the clearance of the tracer from normoxic tissue is very slow. This results in a low tumor to blood ratio and the need to wait for typically 2 h between tracer injection and imaging. A general problem in PET imaging is the low spatial resolution.


Magnetic resonance imaging (MRI) is non-ionizing and has a better spatial resolution than PET. Different MRI techniques have been evaluated for quantification of the blood oxygenation level in tissue. Blood oxygen level-dependent MRI (BOLD-MRI) uses the paramagnetic quality of deoxyhemoglobin in contrast to oxygenated hemoglobin. This can be measured as an increase in T2*-relaxation rate. A similar technique, tissue oxygen level-dependent MRI (TOLD-MRI) uses an increase in T1 relaxation caused by the presence of dissolved oxygen. A third technique is dynamic contrast-enhanced MRI (DCE-MRI) in which a paramagnetic contrast agent such as gadolinium is used and imaged over time [78].


HIF and its target proteins such as CA9 and GLUT1 are upregulated under hypoxia (see above). Accordingly, they have been studied as endogenous markers for hypoxia and seem to co-localize with 2-nitroimidazoles. However, their expression can also be regulated by other factors than oxygen, which reduces their reliability in quantifying tumor hypoxia.


Tumor hypoxia is a major barrier for effectively treating cancer patients. Various approaches have been investigated both in vitro and in vivo to overcome hypoxia in tumor cells. The main clinical strategies to mitigate treatment resistance due to tumor hypoxia are either to improve oxygenation, mimic the effect of oxygen, use drugs that are cytotoxic only in hypoxic cells, apply dose escalation to hypoxic areas, or inhibit pathways that are important for cell survival under hypoxia (Table 5.7).Table 5.7
A summary of different approaches to improve treatment of hypoxic tumors

Approaches

Rationale

High LET radiotherapy

Direct damage to DNA, less dependent on oxygen levels

Hyperbaric oxygen

Improve tumor oxygenation

Blood transfusion

Improve oxygen supply

Perfluorocarbons

Artificial blood substances to improve oxygen supply

Nicotinamide

Vitamin B3 analogue to overcome acute hypoxia

Carbogen

95% oxygen + 5% carbon dioxide to overcome chronic hypoxia

Hypoxic cell radiosensitizers

Compounds that mimic oxygen and can radio-sensitize tumors

Bioreductive drugs

Reduces to cytotoxic form during hypoxic conditions

Hypoxia-targeted radiotherapy

Dose escalation/adaptation in hypoxic sub-volumes of tumor

Dichloroacetate

Inhibits pyruvate dehydrogenase kinase 1, alter tumor metabolism, enhances efficacy of hypoxic cytotoxins

Electron transport chain inhibitors

Decreased oxygen consumption in mitochondria and increased radiosensitivity


The simplest approach to reduce tumor hypoxia is to let the patient breathe hyperbaric oxygen (HBO) (2–4 atmosphere 100% oxygen). However, this may cause vasoconstriction. To avoid this, a mixture of 5% carbon dioxide and 95% oxygen called carbogen was introduced. Carbogen breathing reduces chronic diffusion limited hypoxia. For acute hypoxia, nicotinamide can be used to mitigate transient fluctuations in tumor blood flow [79]. In the ARCON strategy, a combination of carbogen and nicotinamide was used in association with accelerated hyperfractionated RT to reduce tumor proliferation during treatment and spare normal tissues [80].


Hypoxic cell radiosensitizers are chemical or pharmacologic agents that can increase the radiosensitivity of cells, which are deficient in oxygen. These agents are oxygen substitutes that diffuse into poorly vascularized areas of tumors and penetrate to reach hypoxic cells within the tissue. Therefore, when administered with radiation these agents cause fixation of the free radical damage caused by IR and thereby increases its lethal effect. A radiosensitizer should have an increased effect on tumors relative to normal cells to achieve a therapeutic window/gain. An ideal hypoxic cell radiosensitizer should be chemically stable; have low metabolic breakdown; be highly soluble in water and lipids; act at all phases of cell cycle; be active at low doses of radiation; and cause low toxicity to normal cells [81].


The commonly used hypoxic cell radiosensitizers belong to the nitroimidazole group of drugs. In the nitroimidazole ring structure, a nitro group can be present in any of positions 2–5. Based on the position of the nitro group, drugs are named 2-nitroimidazole to 5-nitroimidazole. The presence of the nitro group in position 2 increases electron affinity and IR sensitization capacity [82]. Therefore, 2-nitroimidazole is a more efficient hypoxic cell radiosensitizer. Three widely used nitroimidazole compounds in clinical trials and their characteristics are summarized in Table 5.8.Table 5.8
Nitroimidazole group hypoxic cell radiosensitizers (adapted from [13])

Compound name

Position of nitro group

Properties

Toxicity

Misonidazole

2-Nitroimidazole

Lipophilic, crosses blood–brain barrier

Neurotoxic—dose limiting

Etanidazole

2-Nitroimidazole

Hydrophilic, shorter half-life, does not cross blood–brain barrier

Less neurotoxic

Nimorazole

5-Nitroimidazole

Less active

Less toxic


The enhancement ratio is the ratio of X-ray doses needed to control 50% of the tumors in the absence and presence of the drug. It was found to be around 1.82 for misonidazole in a mouse mammary tumor model for a single-dose treatment but for multifraction regimens, it is usually less [83].


The Danish Head and Neck Cancer 5 (DAHANCA 5) study which comprised 422 patients with pharyngeal and supraglottic carcinomas found a clear benefit of using nimorazole as a radiosensitizer. Thus, no increase in late radiation toxicity and a highly significant benefit in 5-year local control rates and disease-free survival rates. The use of nimorazole as a radiosensitizer in head and neck cancer is standard practice in Denmark [55].


Bioreductive drugs are drugs that are reduced by cells to form active cytotoxic agents only under hypoxic conditions while being non-reduced in an oxic cell. Thus, the differential action on hypoxic cells (i.e., tumor cells) compared to oxic cells, i.e., normal cells) gives a therapeutic window/gain. Unlike the direct action of hypoxic cell radiosensitizers, which increases the radiosensitivity of cells, bioreductive drugs act synergistic with radiation to improve tumor cell kill [84].


The hypoxic cell cytotoxicity ratio (HCR) is the dose of a drug required to kill a certain fraction of oxic cells compared to the dose of drug required to kill an equal fraction of hypoxic cells. HCR varies for different classes of bioreductive drugs [85]. The main classes of bioreductive compounds are fused ring benzoquinones (e.g., mitomycin C), organic N-oxides (e.g., tirapazamine), and nitroheterocyclic compounds (e.g., RSU-1089), among which tirapazamine is the widely investigated drug.


As described above, imaging techniques such as PET and MRI can be used to identify hypoxic regions within the tumor. They are used in RT treatment planning to define radioresistant sub-volumes that can be targeted with boost dose or dose escalation. Targeting hypoxic sub-volumes in tumors by adaptive RT/dose painting can lead to improved tumor control. There is early clinical evidence from a phase 2 randomized trial that dose escalation to hypoxic volumes using 18F-FMISO PET is feasible without increased normal tissue toxicity in head and neck cancers [86]. The utility of hypoxia-directed RT dose painting is being studied in other cancer subsites such as lung, pancreas, and cervix cancer [87–89].


Drugs that target specific molecules to improve radiosensitivity in hypoxic tumors are being investigated in preclinical and clinical studies. Examples are dichloroacetate, electron transport chain inhibitors, and other pathway inhibitors.


Dichloroacetate (DCA) is a specific inhibitor of the pyruvate dehydrogenase kinase (PDK), which has been shown to increase ROS production in hypoxic cancer cells but not in aerobic cells leading to radiosensitization of hypoxic tumor cells [90]. Electron transport chain inhibitors have been developed to reduce oxygen consumption and thereby improve tumor oxygenation. One such is an anti-diabetic drug, Metformin, which has been shown to reduce oxygen consumption through inhibition of mitochondrial complex I and thus improve tumor oxygenation and response to RT [91]. Other approaches include inhibition of pathways that are important for cell survival under hypoxia (e.g., HIF-1α, CAIX, MCT4, VEGF, and lactate dehydrogenase-A (LDHA) [92].


Tumor hypoxia can be acute (perfusion-limited) or chronic (diffusion-limited).

Tumor hypoxia leads to resistance to RT and chemotherapy.

Hypoxic tumor cells undergo adaptive responses that are critical to survival but also promote malignancy and metastasis.

Strategies to either increase oxygenation or interfere with the hypoxic response pathways can improve the outcome of RT.

Drugs that either mimic the effect of oxygen or are toxic under hypoxic conditions can be used to increase the effect of RT.


Hypoxic tumor cells undergo adaptive responses that are critical to survival but also promote malignancy and metastasis.


Strategies to either increase oxygenation or interfere with the hypoxic response pathways can improve the outcome of RT.


Drugs that either mimic the effect of oxygen or are toxic under hypoxic conditions can be used to increase the effect of RT.


RT is an effective cancer treatment, but a large portion of patients subsequently experience radioresistance and recurrence of their cancers.

Mechanisms of radioresistance in cancer treatment are related to several factors: Increased DNA damage repair capacity, cell-cycle redistribution, cancer stem cell resilience, signaling pathways, epithelial-mesenchymal transition (EMT), and tumor metabolism.


RT is an effective cancer treatment, but a large portion of patients subsequently experience radioresistance and recurrence of their cancers.


Mechanisms of radioresistance in cancer treatment are related to several factors: Increased DNA damage repair capacity, cell-cycle redistribution, cancer stem cell resilience, signaling pathways, epithelial-mesenchymal transition (EMT), and tumor metabolism.


RT is one of the most efficient therapeutic regimens for various tumors. The main factor in determining the therapeutic effect is the tumor radiation response that is correlated to intrinsic or acquired radioresistance after the treatment [93].  Intrinsic radioresistance relies on inherent characteristics of the tumor (for instance, the presence of cancer stem cells (CSCs). The acquired radioresistance is a process of tumor adaptation to the RT-induced changes and develop resistance to the IR. This complex process involves multiple genes, factors, and mechanisms [94, 95]. Radioresistance leads to poor prognosis in cancer patients, and it represents the main reason for RT failure, which can ultimately lead to tumor recurrence and/or progression/metastases [95]. Radioresistance of tumors is influenced by several factors, including increased DNA damage repair capacity, cell-cycle redistribution, CSCs resilience, signaling pathways, epithelial-mesenchymal transition (EMT), and tumor metabolism (Fig. 5.19).

A spoke diagram of treatment failure includes cancer stem cells, D N A repair, cell cycle redistribution, extracellular signaling pathways, epithelial-mesenchymal transition, and tumor metabolism.

Fig. 5.19
Description of the tumor biological responses to radiation and the mechanisms for its resistance


A spoke diagram of treatment failure includes cancer stem cells, D N A repair, cell cycle redistribution, extracellular signaling pathways, epithelial-mesenchymal transition, and tumor metabolism.


RT kills cancer cells mostly by inducing DNA damage. Radiation-induced DNA lesions are multiple including base and sugar modifications, cross-links, single-stranded breaks (SSBs), and double-stranded breaks (DSBs) [96] (see also Chap. 3). DNA DSBs are responsible for most of IR-induced cell killing [97]. To respond to DNA damage, cells initiate signaling pathways named DNA damage response (DDR) signaling. The capacity of tumor cells to trigger a DDR following radiation, through initiation of DNA repair and cell-cycle checkpoints, promotes radiation resistance and tumor cell survival. In radiobiology, it is considered that cells with a high capacity to trigger DSBs repair will be radioresistant, while cells with frail repair ability will be more sensitive in response to radiation [93].


Activation of DDR and proper repair of DNA lesions are of paramount importance for cellular integrity, survival, and genomic stability [98]. There are four main DNA repair pathways in tumor cells: DNA DSB repair, base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR) [99]. BER and SSB repair (SSBR) are important for repairing altered bases and SSBs. Besides DSBs caused directly by IR unrepaired damaged bases and SSBs, can generate DNA DSBs when they face a replication fork. Non-homologous end joining (NHEJ) and homologous recombination (HR) two principal cellular DSB repair pathways (see Chap. 3).


Defects in DNA repair factors are a characteristic of many cancers which promote genomic instability and mutation in cells [98, 100–102]. Deficiency of DDR and DNA repair genes caused diseases such as Xeroderma pigmentosum, Ataxia Telangiectasia (ataxia telangiectasia mutated (ATM)), Nijmegen breakage Syndrome (Nibrin (NBS1)), Werner syndrome (WRN), Fanconi anemia (FA), associated breast cancer syndrome (BRCA1 and BRCA2), lead to cancer susceptible disease syndromes (reviewed in [98, 102] and in Chap. 7). Cancers present germ line mutations, somatic mutations or distinctive expression of DDR components that lead to the impairment of DDR and altered checkpoints. In addition, DNA replication stress due to oncogene stimulation also causes initiation of DDR and tumor progression.


The same DNA repair deregulation that commenced carcinogenesis is associated with cancer progression, aggressiveness [102]. There are situations in which the tumor is preserved after the therapy, becoming even more aggressive. It is known that IR can activate stress and adaptive response to DNA lesions causing cancer progression, metastatic behavior [103]. Several dysfunctional DDR signaling genes are found in cancer and correlated with radioresistance (Table 5.9).Table 5.9
Examples of dysfunctional DNA damage response signaling genes found in cancer and linked with cancer radioresistance and progression

Molecule

Function(s)

Type of defects

Reference

ATM

DDR—DSBs
Cell-cycle redistribution

Mutations; increased copy number and autophosphorylation, overexpression

Squatrito et al. [104], Zhang et al. [105]

DNA PKcs

NHEJ

Overexpression

Kotula et al.  [106]

Ligase IV/XRCC complex

NHEJ

Polymorphism in LIG4 gene

Srivastava et al. [107]

RAD51

HRR

Overexpression

Chen and Kuo [96] Bhattacharya and Asaithamby [98]

RPA

HRR

Overexpression

Liu et al. [93]

BRCA1 and BRCA2

HRR transcriptional regulation of many DNA repair genes

Loss-of-function mutation; BRCA1 overexpression

Kan and Zhang [108]

HSP90

Regulates HRR
Regulates protein related to DDR signaling (ATM, NBS1, ATR)

Overexpression

Kinzel et al. [109]

APE1 (apyrimidinic endonuclease 1)

BER

Overexpression

Kiwerska and Szyfter [102], Madhusudan [110]

DNA polymerase β (pol β)

BER

Overexpression; missense mutations in pol β gene coding for variant proteins

Bhattacharya and Asaithamby [98]

PARP

PARP 1 and PARP 2-SSB, BER

Overexpression

Li et al. [111]

Ku 70/80

NHEJ

Overexpression

Baptistella et al. [112]

MSH6/MSH2

MMR

Reduced expression

Uraki et al. [113]

ERCC1

NER

Overexpression
Polymorphism

Kiwerska and Szyfter [102], Peng et al. [114]

XRCC1

Excision repair

Polymorphism

Li et al. [115], Liu et al. [93]


Activation of cell-cycle checkpoints is important downstream IR-induced DDR signaling. Several molecules in the cell-cycle checkpoints regulate and block cell-cycle progression to allow for DNA repair and prohibit premature entering into mitosis with unrepaired DNA lesions. The same machinery responsible for DNA damage recognition, ATM and ATR, activates the cell-cycle checkpoints kinases CHK1 and CHK2 to regulate the checkpoints and repair DNA damage. Two distinct signaling cascades, the ATM-Chk2 and ATR-Chk1 axes respond to DSBs damages, respectively, to ssDNA. Activated Chk1 and Chk2 inactivate CDC25C by phosphorylation. In the absence of CDC25C phosphatase activity, WEE1 kinase impedes cyclin-dependent kinase 1 and 2 (CDK1/2) activity triggering cell-cycle arrest in the G2/M phase and promoting DNA damage repair (reviewed in [93, 95, 111]). Molecules in the cell-cycle checkpoints were also found to be linked with the development of radioresistance (e.g., ATM, p53, p21, Chk2, Cdc2).


A considerable amount of research has been done on the modulation of cellular signaling pathways in response to IR and many of them are involved in radioresistance, including extracellular and intracellular signaling cascades. This subsection focuses specifically on the major oncogenic signaling pathways such as Ras and phosphatidylinositol-3′-kinase pathways (PI3K)/AKT that contribute to radioresistance (Fig. 5.20).

A spoke diagram of resistance to radiation includes the suppressors L K B 1, Ras pathway, suppressor P T E N, and P I 3 K, A K T pathway.

Fig. 5.20
Review of tumor suppressors and the molecular signal pathways that contribute to radioresistance


A spoke diagram of resistance to radiation includes the suppressors L K B 1, Ras pathway, suppressor P T E N, and P I 3 K, A K T pathway.


Radiation causes many behavioral changes in the extracellular domain of EGFR as well as to its kinase domain, including truncations and common mutations of an extracellular domain of EGFR [16, 116, 117]. Photon radiation can cause EGFR aberrations that over-activate downstream pro-oncogenic signaling pathways, including the RAS-RAF-MEK-ERK MAPK and AKT-PI3K-mTOR pathways that modulate cell processes, including cell resistance to RT, angiogenesis, migration, invasion, and apoptosis [118–120].


When EGFR is activated by photon radiation, this activation, triggers the RAS-RAF-MEK-ERK MAPK pathways. Radiation resistance is associated with this pathway targets due to its pro-survival nature, and mutated RAS has been associated with resistance to photon-irradiation in cancer cells [121–124].


Also, when EGFR is activated by photon radiation, this triggers the AKT-PI3K-mTOR pathways and activated EGFR induces the phosphorylation of PI3K and downstream AKT phosphorylation [124]. Radiation resistance is also linked to activation of this target with tumor cells being protected by decreased autophagy and apoptosis, as well as increased DNA repair capacity [125].


Overall resistance to photon irradiation appears to be modulated by the transmembrane protein EGFR but also repression of tumor suppressors. In this circumstance, the tumor suppressors LKB1 and PTEN has been reported to correlate to resistance.


EMT is a series of changes involving epithelial cells that phenotypically transform into mesenchymal cells [126]. To acquire local invasiveness, the first step involves a profound phenotypic shift within the primary tumor of cancer cells. The progression of cancer is dependent on tumor cells undergoing the EMT and invading blood vessels [127, 128]. This results in invasion and distant metastasis, the main cause of cancer-related death. Normal epithelial cell layers cannot accommodate the motility and invasiveness of malignant carcinoma cells, yet this epithelial organization plan continues to be recognized in many primary carcinomas. Despite their overall topology differing quite a bit from that of comparable normal epithelia, these tumors contain well-organized sheets of epithelial cells. Although it’s not confirmed, cancer cells require a partial or complete EMT to become invasive as in the following steps [129]:1.
Factors, including growth factor (GF), initiate EMT by promoting E-cadherin replacement by N-cadherin.
 2.
Due to its ability to allow these tumor cells to form homotypic interactions with a variety of types of mesenchymal cells, N-cadherin increases the affinity of cancer cells for stromal cells composed of fibroblasts, which support tumor cell invasion.
 3.
N-cadherin forms a weaker intermolecular band than E-cadherin, so molecules whose intermolecular bands are weaker favor motility.


Factors, including growth factor (GF), initiate EMT by promoting E-cadherin replacement by N-cadherin.


Due to its ability to allow these tumor cells to form homotypic interactions with a variety of types of mesenchymal cells, N-cadherin increases the affinity of cancer cells for stromal cells composed of fibroblasts, which support tumor cell invasion.


N-cadherin forms a weaker intermolecular band than E-cadherin, so molecules whose intermolecular bands are weaker favor motility.


When normal cells transform into cancerous cells, E-cadherin turns into N-cadherin and such cancer cells can escape their keratinocyte neighbors. Thus, the switch from E- to N-cadherin expression promotes invasion of the stroma. N-cadherin allows cancer cells to form homotypic interactions with mesenchymal cells, including endothelium, fibroblasts, and others in the stroma of the organ (Fig. 5.21).

A diagram of the epithelial to mesenchymal transition. Some of the steps are Epithelial, stable defined polarity, E M 1, E M 2, E M 3, meta-stable, Apico-Basal polarity loss, gain of front-back polarity, mesenchymal, stable, front-back polarity, and metastasis, cancer cell in blood stream.

Fig. 5.21
An overview of a typical EMT program that causes cadherin shifts in the cell and invasion of cancers


A diagram of the epithelial to mesenchymal transition. Some of the steps are Epithelial, stable defined polarity, E M 1, E M 2, E M 3, meta-stable, Apico-Basal polarity loss, gain of front-back polarity, mesenchymal, stable, front-back polarity, and metastasis, cancer cell in blood stream.


In vitro and in vivo, EMT is induced by a variety of factors, including radiation, ROS, hypoxia, and transforming growth factor [130–132]. Radiation causes many changes in a cell, which are directly affiliated with epithelial cancer. These include aberrant multicellular organization, genomic instability, dysregulation of differentiation, and phenotypic transition, i.e., EMT. Multiple evidence suggests that Notch1 signaling is essential for EMT [133]. Unlike normal tissue, when cancerous tissues experience radiation insult, the irradiated cells show overexpression of an active Notch-1 protein in both cytoplasm and nucleus. This results in decreased expression of E-cadherin and increased expression of vimentin and fibronectin, which consequently alleviate radiation-induced EMT as reported both in in vitro and in vivo models [134, 135]. Furthermore, low dose IR promotes NF-κB p65 signaling in cervical cancer cells. The transcription factor NF-κB regulates tumorigenesis as well as apoptosis and is essential for the initiation and maintenance of EMT. This signaling subsequently induces EMT that downregulates of expression of the epithelial markers E-cadherin and Cytokeratin 18 (CK-18), which is a hepatocyte-secreted protein that is released into the blood during apoptosis and necrosis while it upregulates vimentin and the mesenchymal markers N-cadherin. Therefore, this leads to improving the progression of cancer cells [136]. Additionally, a variety of evidence indicates that Granulocyte Colony-Stimulating Factor (G-CSF) (formerly believed to promote granulocytes) is strongly associated with lung cancer progression [137]. Moreover, it has been reported that IR activates Granulocyte Colony-Stimulating Factor Receptor (G-CSFR)/JAK/STAT3 signaling pathways via secreted G-CSF, resulting in EMT in non-small cell lung cancer cells (NSCLCs) [137]. The matrix metalloproteinase (MMP) family proteins are essential for EMT, as well. The γ-IR treatment of nodular NSCLC cells was associated with increased invasion via EMT induction, which is characterized by a highly active MMPs system [138]. Following irradiation, an EMT program is induced, causing breast cancer cells to develop a resistance and progression behavior. The proto-oncogene tyrosine-protein kinase (SRC), being a non-receptor kinase, integrates diverse signal transduction pathways that contribute to a variety of cellular processes including survival, proliferation, angiogenesis, differentiation, migration, and angiogenesis. Upon irradiation, SRC is activated, which promotes EMT through AKT, p38, and PI3K activation. Similarly, fractionated IR inhibits PTEN-related expression, which is associated with activation of the Drosophila embryonic protein SNAI1 (Snail) protein accumulation and Akt/GSK-3β signaling and provoking EMT [139]. As a result of radiation, GSK-3β is phosphorylated at serine 9 residue, and silencing the tank-binding kinase-1 (TBK1) tones down the phosphorylation, indicating that TBK1 inhibition is associated with activating GSK-3β. Indeed, GSK-3β functions in several key signaling pathways that can contribute to EMT, such as the Wnt, TGF-β, Hedgehog, Notch, and PI3K pathways. Upon phosphorylation at its N-terminus, GSK-3β can be inactivated and Snail is stabilized and succumbs to EMT. In this way, it is suggested that TBK1 inhibition has the potential to reduce radiation-induced Zinc Finger E-Box Binding Homeobox 1 (ZEB1) expression via activation of GSK-3β. This hypothesis is supported by the observation that pre-treatment of cells with the GSK-3β inhibitor SB216763 increases TBK1 inhibition, increases E-cadherin expression, and reduces ZEB1. In conclusion, it was seen that TBK1 signaling is influenced by GSK-3β in radiation-induced EMT [140]. RT can cause both lessening of tumor cells and killing of them as it can also lead to damage to normal tissues, radioresistance, invasion, and distant metastases. Several studies have suggested that EMT generates malignant characteristics such as a propensity to recurrence, resistance to treatment, and dissemination of metastatic cells. Radiation is generally considered a key initiating factor for EMT, as it activates signal pathways that initiate the process.


Radiation resistance has in multiple studies been linked to tumor metabolism alterations as a result of radiation-induced changes in mitochondrial metabolism or glycolysis [141, 142]. The development of radioresistance is closely related to glucose- as well as mitochondrial metabolism. The radiosensitivity of the tumors can be explained by radiation-induced metabolic changes in glucose and mitochondrial functions or by tumor-induced metabolic changes (Table 5.10).Table 5.10
Glucose and mitochondrial metabolism induced by the tumor itself that contribute to tumor resistance and progression

Entity

Type

Cause

Effect

Outcome

Nicotinamide adenine dinucleotide phosphate (NADPH)

Glucose metabolism

The decrease in OxPhoslevels in mitochondria and the pentose phosphate pathway (PPP)

Increasing tumor dependence on glycolysis and reducing intracellular ROS levels

Tumor proliferation

An organic compound (dinitrophenol)

Glucose metabolism

Mitochondrial respiratory modulators

Increase in the glycolytic index

Rejoining of DNA strand breaks that leads to reducing the probability of cancer cells damage by radiation

Proteins (GLUT1)

Glucose metabolism

GLUT family in cell membrane

Upregulated under hypoxic circumstances

Poor prognosis, radioresistance, oncogene activation and inhibition of tumor suppressors, stimulation of hypoxia, and the regulation of different signaling pathways, including PI3K/AKT and MAPK

Lactic acid

Glucose metabolism

Products of glycolysis

Accumulation in tumor tissues

Promoting recurrence, tumor metastasis and radioresistance

Fibroblasts in tumors release hyaluronic acid in response to lactic acid

Neovascularization, cell clustering, migration, and VEGF secretion

Lactate dehydrogenase (LDHA)

Glucose metabolism

Distribute in human tissues

Enzyme helps make lactic acid from pyruvate

Overexpression indicates hypoxic conditions, which can be associated with radioresistance, lower survival rate, local recurrence, and distant metastases

Soluble adenylate cyclase (sAC)

Glucose metabolism

Activation of BRAF/ERK1/2 signaling

Release of LDHA

The anti-irradiation effects and cell proliferation.

HIF-1β

Glucose metabolism

Activated in hypoxic environments

The adaptive cellular responses to hypoxia, important mediator of the conversion of OxPhos to glycolysis in the metabolism of carbohydrates convert ADP and phosphoenolpyruvate into ATP and pyruvate

Radioresistance by removing ROS from the cells and activating cell autophagy

Pyruvate kinase (PK)

Glucose metabolism

Enzyme involved in glycolysis

Convert ADP and phosphoenolpyruvate into ATP and pyruvate

PK expression correlates with radioresistance

Hexokinase 2 (HK2)

Glucose metabolism

A critical role in glucose metabolism

Upregulation of this enzyme can induce glycolysis

HK induces glycolysis, making it essential for the progression and maintenance of tumors

Antigen 2 (NG2)

Mitochondrial metabolism

A glial cell antigen

By interacting with serine protease OMI/HtrA2 of mitochondria, protect oligodendrocyte precursor cells from oxidative stress

Development of radioresistance

Protein 3A (ATAD3A)

Mitochondrial metabolism

Cancer patients

Activating ATAD3A leads to the expressions of histone H3, H2AX and ATM increase

Inhibit the IR-induced apoptosis in glioblastoma cells

Matrix metalloproteinase

Mitochondrial metabolism

The extracellular protein

Radiation resistance is induced by molecules or signaling pathways that increase MMP or inhibit its decrease

Radiation resistance


After the tumor cells have been exposed to radiation for an extended period of time, these cells experience alteration in the glucose metabolic pathway. As an important kinase, AKT regulates multiple biological processes, including cellular metabolism. As described in the previous section AKT is phosphorylated in cells after IR exposure to radiation which results in radioresistance of tumor cells through the AKT-mediated alteration of cellular glucose metabolism. A disruption in glucose metabolism also occurs after radiation exposure of tumor cells, resulting in an accumulation of lactic acid. This is the primary product of glycolysis and contributes to the development of malignant tumors. Moreover, glycolysis may cause tumor metastasis, recurrences, and resistance to RT.


Radiation exposure of tumor cells likewise disrupts mitochondrial metabolism. In the post-RT setting, manganese superoxide dismutase (MnSOD) activity increases significantly in pancreatic cancer cells irradiation. MnSOD is the enzyme which catalyzes the formation of superoxide anion radicals, fighting the effects of oxidative stress ROS-induced damage. As a consequence, G2 checkpoint block is activated and cells are protected against mitochondrial oxidative stress by mitochondrial antioxidant systems, promoting radioresistance development. Radiation exposure to Burkitt lymphoma similarly disrupts mitochondrial metabolism by inducing the proteins (mitochondrial proteomes) that prompt radioresistance. Radiation of tumor cells targets Mitochondrial MAPK phosphatase (MKP1). Radiation increases MKP1 expression in breast cancer cells, where it translocates into the mitochondria and inhibits apoptosis by phosphorylating the c-Jun N-terminal kinase (JNK).


Palliative RT may be used to slow down tumor growth, prevent or treat symptoms, and improve quality of life.

Common applications include prevention of bleeding and pain and treatment of metastases.

Brain and spinal cord metastases are often treated to relieve neurological symptoms.

Palliative RT is often hypofractionated with potentially unique radiobiology.

The radiobiological mechanisms of palliative RT are currently not well understood.


Palliative RT may be used to slow down tumor growth, prevent or treat symptoms, and improve quality of life.


Common applications include prevention of bleeding and pain and treatment of metastases.


Brain and spinal cord metastases are often treated to relieve neurological symptoms.


The radiobiological mechanisms of palliative RT are currently not well understood.


While radical RT aims to eliminate cancer cells with the intention of cure, palliative RT is used to slow down tumor growth, to prevent or treat symptoms and improve quality of life. It is estimated that more than 50% of RT patients receive palliative RT. This is particularly relevant in populations in less well-resourced countries where the burden of advanced disease is high, and palliation is commonplace (Box 5.11).


Typically, palliative RT is used to treat or prevent bleeding [143–146] or obstruction [147]. In addition, palliative RT is commonly used to treat pain, particularly bone pain, as well as brain and spinal cord metastases to address neurological symptoms [148]. Sites frequently treated palliatively include bone, brain, pelvis, and lung.


Despite the widespread use of RT with palliative intent, the radiobiology of palliative RT is not well understood. Radiation can reduce tumor burden by targeting proliferating tumor cells, and thus reduce tumor growth and result in relief of obstructions and pressure. However, mechanistic effects of how RT can stop bleeding and pain are still somewhat obscure. It has been speculated that cytokine modulation may alter pain levels and targeting of vascular components may prevent bleeding [149]. Palliative regimens are also typically shorter and frequently involve hypofractionated approaches or single (7–8 Gy) fraction schedules, which may induce differential biological effects to those induced by typical conventionally fractionated radical treatments. Enhanced vascular targeting and immune modulation have been implicated [150].


Tumor microenvironments (TME) can recourse the tumor origination, expansion, invasion, metastasis, and its response to therapies like RT and chemotherapy.

RT immunomodulates the TME to induce a local anti-tumor immune response leading to tumor regression.


Tumor microenvironments (TME) can recourse the tumor origination, expansion, invasion, metastasis, and its response to therapies like RT and chemotherapy.


RT immunomodulates the TME to induce a local anti-tumor immune response leading to tumor regression.


The tumor microenvironment (TME) is an “ecological niche” to facilitate the promotion and the succession of cancer cells. TME intricacy is coupled with tumor expansion, metastasis, and reaction to therapy. Potent alteration stirring in the TME is the basis of tumor cell variant selection, which may endorse genomic instability [31]. Cancer is an exceptionally assorted disease as the cells within the tumor have diverse mutations at different locations within the primary tumor and the metastatic site. Tumor growth and development are not only by actions relating tumor cells, but also by the milieu they inhabit, the TME. In 1889, Stephen Paget introduced “seed and soil” proposition which recommended that metastasis does not occur due to accidental events, but some tumor cells (the “seeds”) nurture ideally in particular organs (the “soil”) and metastases only emerges when the appropriate seed and soil combination takes place [151] (Box 5.12).


Tumors are normally greatly heterogeneous and intricate in genetics. Different cell types, such as adipocytes, fibroblasts, immune cells, endothelial cells, and neuroendocrine (NE) cells, possess unique purpose in TME (Table 5.11). Acellular constituents of the TME like the extracellular matrix (ECM), extracellular vesicles (EVs), and cytokines remain adjacent to these cells. Physical and chemical uniqueness of the TME that are considered as vital the microenvironmental components comprise of the high interstitial pressure, hypoxia, fibrosis, and low pH. Further, communications involving cells and stromal elements also play an important role in cancer evolution and development [152].Table 5.11
Functions of various components of the tumor microenvironment (adapted from [152])

TME components

Function

Neutrophils

Augmentation of angiogenesis and metastasis; associated with poor diagnosis.

Tumor-associated macrophages (TAMs)

Promote deterioration of the extracellular matrix; support the increase of inflammatory cytokines (TNF-β); augment of angiogenesis and remodeling.

CD8+ cytotoxic T cells (CTL)

Provoke apoptosis, necrosis, and growth arrest by secreting INF-γ and other cytotoxic cytokines; set up an anti-tumor environment.

Regulatory T cells (Tregs)

Exude cytokines such as IL-10, TGF-β; set up an immunosuppressive environment; associated with poor diagnosis.

Myeloid-derived suppressor cells (MDSCs)

Linked with tumor development and neoangiogenesis; repress T cells and NK cells; differentiating into TAMs in a hypoxic environment.

Mesenchymal stem cells (MSCs)

Differentiating into mesenchymal tissues such as bone, cartilage, and fat tissues, vasculogenic mimicry; form the premetastatic niche; promoting cancer initiation and malignancy.

Endothelial cells

Constitutes of tumor blood vessels; secreting angiocrine factors such as adhesion molecules; intercommunicating with tumor cells via secreting EVs including CD106, CD49a.

Adipocytes

Regulating the balance of systematic energy and metabolism; secreting exosomes, cytokines, chemokines, and hormones; promoting cancer progression.

Neuroendocrine cells (NE cells)

Promoting proliferative signaling; secreting neurotransmitters, including CgA, chromophilic, and vasoactive polypeptide; regulating NK cell migration and toxicity ability.

Vascular network

Providing oxygen, clearing carbon dioxide, and metabolizing wastes; providing nutrition support for cancer cells; promoting angiogenesis and metastasis.

Lymph vessels

Helping immune cells to avoid immunity and dissemination; providing a physical link between lymph nodes and tumor.

Extracellular matrix (ECM)

Forming the complex macromolecular network; controlling cancer invasion and metastasis, angiogenesis; contribution to growth and proliferation signaling, inhibiting cancer cell apoptosis.

Extracellular vesicles (EVs)

Carrying biologically active molecules such as proteins, miRNAs, and lncRNAs from donor cell to recipient cell; regulating key signaling pathways, proliferation, drug resistance, and stemness; reprogramming stromal cells to create a niche for survival.

Cancer-associated fibroblasts (CAFs)

Sustaining proliferative signaling; activating angiogenesis and metastasis; tumor-promoting inflammation; evading immune destruction; reprogramming cellular metabolism; promoting genome instability and mutation.


IR can modify exchanges among tumor cells and their microenvironment. The TME does not only persuade tumor cell proliferation, invasion and metastasis of cancer cells, angiogenesis, modulation of immune cell infiltration and the immune response, but also has a brunt on the therapeutic response [151]. The effects of radiation on the TME diverge with dose and fractionation plan. Radiation stimulates the cellular and DNA damage that can lead to both production as well as release of tumor-associated neo-antigens and secretion of cytokines from tumor and the stromal cells. These proceedings can budge the equilibrium towards an immuno-reactive microenvironment, as contrasting to immunosuppressive microenvironment, causing the recruitment and activation of cytotoxic T cells. Figure 5.22 gives an overview as to how radiation affects the TME.

A diagram of the effects of radiation on the tumor microenvironment involves the cancer cell, dying cancer cell, N K cell, C D 8 plus T cell, T subscript reg cell, D C, activated D C, T A M, C A F, M D S C, endothelial cell, and ionizing radiation.

Fig. 5.22
Overview of effects of radiation on the tumor microenvironment. (Figure adapted from Barker et al. [153], with permission)


A diagram of the effects of radiation on the tumor microenvironment involves the cancer cell, dying cancer cell, N K cell, C D 8 plus T cell, T subscript reg cell, D C, activated D C, T A M, C A F, M D S C, endothelial cell, and ionizing radiation.


The damage induced by IR affects many cells in the TME. The sensitivity of tumor cells is higher to radiation and their death commences the inflammatory signaling cascade. Also, the levels of intercellular adhesion molecule 1 (ICAM) and vascular cell adhesion molecule 1 (VCAM) expression go high as that of the cells of the innate immune system. Interestingly, the upregulation of the integrins present on the endothelial cells is associated with better survival as it acts as a system for radioresistance. Vascular diminution heightened the effect of hypoxia inducing the HIF-1α signaling. It also leads to the pro-angiogenic and pro-vasculogenic stimuli through VEGF and chemokine (C-X-C motif) ligand 12 (CXCL12). Following radiation cancer-associated fibroblast (CAF) activation stimulated the altered growth factor secretion and secret several ECM and cytokine modulators. Also, the transforming growth factor-β (TGF-B) signaling pathway is not only complex but also pleiotropic. It directly affects the tumor cells and CAFs, driving the HIF-1 signaling and reducing the activation of T cells and the dendritic cells (DC). In the immune compartment, due to high levels of mTOR there is not only an increase in the accessibility of tumor cell antigen but also a boost in the processing of the antigen in combination with damage-associated molecular pattern (DAMP) related Toll-like receptor (TLR) responses. Also, there is an increased pro-inflammatory cytokine signaling to activate the DCs and as a consequence T cells. The activated DCs move to the proximal lymph nodes. In the TME, such signaling is frequently obstructed by high Treg cytotoxic T lymphocyte antigen 4 (CTLA-4) suppression of co-stimulation. Radiation upregulates the NKG2D signals on the tumor cells that enforce direct cytotoxic effects by the CD8+ T cells and NK cells. However, there are other tumor cells which getaway with the PD-L1 signaling but the MDSC derived IL-10 immunosuppression remains intact.


Hence, depending on the organ, there are organ-specific interstitial cells such as the presence of astrocytes in the central nervous system or the osteoblasts in bone tissues. Such cells are collectively called as the stroma of the tumor and along with other essential factors like the surrounding pH, oxygen levels, extracellular matrix make up the microenvironment of the tumor [139].


The tumor cells are known to interact with the stroma or the adjacent milieu. The tumor stroma comprises of a range of diverse cells, and extracellular matrix (ECM), that are known to advocate in limiting the host immune reaction against tumor cells. The tumor stroma puts forward a versatile complex network that maintains the tumor propagation by secreting immunosuppressive cytokine, diverse cellular processes and metabolic alterations [154]. Stromal cells involving the endothelial cells and adipocytes can modify the radiosensitivity by their roles in angiogenesis and vasculogenesis, and their secreted adipokines, respectively. The extracellular matrix can control radiation sensitivity by manipulating the oxygen proximity and managing the equilibrium and bioavailability of growth factors and cytokines [155]. Even though several RT-mediated stromal transformations like the renewal of or polarization towards tumor-suppressing immunity are valuable, RT can operate as a double-edged sword in tumors.


Chronic inflammation is one of the radiation effects on the stroma which acts as a chief driver of fibrosis during which there is occurrence of persistent immune responses in addition to tissue remodeling and repair processes. Some of the radiation-induced alteration on the TME is by altering the way CAFs manage their collagen assembly. In addition, CAFs that receive RT-undergo variation and modification in terms of their variety, secretome, and phenotype. Moreover, RT augments the stimulation of the proliferating machinery linking the RAS and mitogen-activated protein kinase (MAPK) signaling pathway; the invasion pathway that is associated with tumor evolution, resistance, and metastasis. When the stroma receives RT, there is augmentation in the invasiveness of the tumor because of the augmented hepatocyte growth factor (HGF)/c-Met (HGF receptor) signaling and MAPK activity. This aids the improvement in the tumor movability which can prove to be fatal. RT can also stimulate the stromal traits that can potentially impart resistance to therapy. Studies are still ongoing in understanding as to how fractionated RT alters the TMEs stromal machinery and how all the modifications can affect after the cancer cells response to RT [155].


An important feature of solid tumors is the vascular network that develops from the vasculogenesis, angiogenesis, and fibroblasts. A characteristic of these vessels is that they lack their supporting pericytes or basement membrane, which makes them vulnerable to radiation [156–158]. When tumor tissue is exposed to radiation, blood vessels at the level of the microvessel network are firstly destructed and the degree of this destruction depends on the radiation dose. The vessel’s thickness increases, which raises the risk of atherosclerotic changes and intimal proliferation, since the destructed microvessel network reduces distance between functional vessels and vascular density, resulting in hypoperfusion. Irradiation not only modulates the vessel structure, but it also has long-term effects, including medial necrosis and fibrosis, but these effects are affected by radiation dose. When a radiation dose is high, for instance, blood flow is permanently reduced; necrosis and fibrosis are produced, and the tumor is revascularized by vasculogenesis, which is a disorganized process of vessel growth in comparison to angiogenesis. Secondly, radiation to tumor tissue induces the chemokine receptor CXCR4 which induces bone marrow of matrix metalloprotease 9 that is responsible for radiation vasculogenesis and subsequent post irradiation [159, 160].


IR kills cancer cells directly (atomic ionization) and indirectly (radiolysis of water) [16, 20, 158]. IR also induces a host immune response to tumor cells and facilitates tumor recognition and healing. These effects occur in a variety of ways, including immune response signaling. When cells are dying, they translocate calreticulin (ERp57) and their endoplasmic reticulum (ER) protein complex to the cell membrane which functions as a “eat me” signal. Secondly, upon IR of a tumor, inflammatory molecules, such as ATP and high mobility group protein B1 (HMGB1), are released, promoting a T cell and DC response. Signaling of this type uses three types of signals in anti-tumor immune responses, which are:1.
DCs take captive of released danger signals and process them.
 2.
Danger signals are presented into T cells by MHC molecules. As a result, naïve tumor-specific T cells are activated and become effector T cells.
 3.
Finally, macrophages and DCs may be recognized by cytotoxic T cells, and they destroy remaining cells of tumor tissues through their circulation through the bloodstream [161–165].


Danger signals are presented into T cells by MHC molecules. As a result, naïve tumor-specific T cells are activated and become effector T cells.


Finally, macrophages and DCs may be recognized by cytotoxic T cells, and they destroy remaining cells of tumor tissues through their circulation through the bloodstream [161–165].


However, not all irradiated cells sustain damage, and some survive. These IR surviving cells may expose surface molecules like antigen-presenting molecules of major histocompatibility complex class I (MHCI), death receptor Fas, and ICAMs which lead to an improvement in the recognition and killing of tumors by immune cells that are reactivated by tumor antigens.


To cure tumors, radiation needs to cause cell death. There are five different types of cell death depending on the pathways in the response system of DNA damage, namely, apoptosis, senescence, autophagy, mitotic catastrophe, and necrosis [13, 157, 158]. Via apoptosis pathways macrophages may be triggered to clean out these dying cells which causes polarized M1 macrophages thereby driving an anti-tumor immune response and indirectly killing of tumors. However, this process may also contribute to carcinogenesis and inflammation [163].


The most well-known tumors in declining order of radiosensitivity as follows: (1) lymphoma, (2) embryonal tumors, (3) cellular anaplastic tumors, (4) basal cell carcinoma, (5) adenoma and adenocarcinoma, (6) desmoplastic tumors, such as squamous carcinoma, (7) fibroblastic sarcoma; osteosarcoma; neurosarcoma. Tumors composed of fast-growing cells like the embryonal tumors are sensitive to radiotherapy.


Lymphoid cells are predominantly vulnerable to radiation. On the other hand, the radioresistant category comprises of the neurosarcomas, gliomas, and melanomas. Tumor sensitivity to radiation is affected by the variation on the radiosensitivity exhibited by the subgroups of major cancer forms. There are numerous factors affecting the cancer radiosensitivity and at times they are even obscure. The most important factor that plays the most vital role in influencing the tumor behavior under RT is the general condition of the patient. Also, there is an explicit association between radiosensitivity and the grade of malignancy which might not be uniform always. Hence, all these factors should be carefully evaluated before imparting radiotherapy and predicting its outcomes.


One of the most prominent factors imparting the tumor radiosensitivity and which was recognized very early on was the high metabolism of the tumor cells. This is similar to the fast growth and also understood as the tumor growth rate imparting radiosensitivity. Augmented or uneven vascularity also goes with fast growth. These factors combined provide the rapidly growing tumor cells with sensitivity to radiation. Especially when tumors are bulky hypersensitivity can be encountered in the treatment of such tumors. Therefore, to avoid accidents like bulky necrosis or interstitial hemorrhage appropriate precaution should be taken. Other factors like the large quantity of autolytic cell ferments and susceptibility of mitotic nuclei are also crucial in imparting tumor sensitivity to rapidly growing tumor cells. It is seen that the tumors exhibiting the embryonic trait in the origin cells provide the complete group of embryonal tumors a very high radiosensitivity. Hence, their response to radiation treatment is probably quicker and complete compared to other tumors. Therefore, such tumors should be identified as especially favorable.


While radiation effects classically result from direct damage to cells and tissues within the target, effects outside the field also known as abscopal effects have been observed in both tumors and normal tissue. For example, irradiation often leads to systemic fatigue [166], and bilateral pneumonitis sometimes occurs after unilateral lung irradiation [167]. Occasionally, metastatic tumors have been observed to regress dramatically outside the primary irradiation field, which is a manifestation of the systemic anti-tumor effect [168].


An illustrative example is the case of a man diagnosed with ulcerated malignant melanoma in his left arm [169]. The arm was resected, and no further treatment was indicated. Twenty months later, the patient had a painful skin lesion on the head and an asymptomatic metastatic lesion in the lung. The skin lesion was irradiated with 3 Gy in 10 sessions, after which the lesion and pain disappeared. Unexpectedly, the lung lesion also disappeared although it had never been treated. Reports of cases of abscopal effects in melanoma are relatively common. Another example is the case of a woman with squamous carcinoma of the anal canal with metastases to pelvic lymph nodes, liver, and bone. After palliative RT limited to the pelvis with sensitizing chemotherapy, complete regressions were observed 4 months after treatment not only in the primary tumor but also in the bone and liver metastases, an effect that persisted 4 years later [170]. Figure 5.23 gives an overview as to how radiation induces Abscopal Effects.

A diagram of a patient with the tumor metastasized to the head and lungs after 12 months display inset images of tumor antigens, cell death, tumor neoantigens, and danger signals that forwards to an anti-tumor immune response.

Fig. 5.23
The abscopal effect of RT. On the left side of this schematic case, the patient suffers from a tumor in the shoulder (e.g., a melanoma) that has metastasized to the lung. The treatment protocol consisted of surgery of the primary tumor and the metastatic nodules were treated with a combination of RT and immune checkpoint blockade, which promotes abscopal anti-tumor immunity. On the right side of this schematic case, RT triggers the release of tumor antigens, neoantigens, and DAMPs, which are recognized and processed by macrophages and DCs. Subsequently, these antigen-presenting cells present these tumor antigens to T cells in a draining lymph node of the tumor. The antigen-presenting cells are then taken up by cytotoxic T cells, which subsequently destroy the remaining cells of the cancerous tissue


A diagram of a patient with the tumor metastasized to the head and lungs after 12 months display inset images of tumor antigens, cell death, tumor neoantigens, and danger signals that forwards to an anti-tumor immune response.


The previous clinical cases showing unexpected regression of tumors outside the irradiated field are examples of a phenomenon first described by Mole as the “abscopal effect” [171]. The word abscopal comes from the Latin ab (position away from) and Scopus (target). However, there is now clear evidence that the abscopal effects associated with RT are due to systemic anti-tumor immune responses triggered by the RT. However, despite millions of patients receiving RT, abscopal effects are too unpredictable to be a therapeutic target, as only a limited number of cases have been identified. In a recent study, only 46 cases were identified between 1969 and 2014 [172]. With the advent of immune checkpoint blockade therapy, clinical interest has soared. Since the introduction of the anti-CTLA-4 antibody ipilimumab into clinical practice in 2011, abscopal effects have been observed in several patients who received RT while taking ipilimumab [173, 174]. Other immune checkpoint therapies have also shown promise. There are reports of abscopal effects following treatment with RT and anti-PD1 therapies such as pembrolizumab or nivolumab [175]. In this section, we will cover the biological events in cancer cells that precede the occurrence of radiation-induced abscopal effects.


Radiation can alter the immunogenicity of tumors by promoting tumor antigens, neoantigens, and danger signals.


Tumor antigens result from protein degradation due to damage to cellular proteins caused by radiation-induced free radicals. Radicals such ROS are generated during radiation-induced radiolysis of water. RT then causes cellular damage that is independent of DNA and leads to an increase in the pool of peptides, i.e., tumor antigens that bind to the major histocompatibility complex (MHC) [176].


Tumor neoantigens are tumor-specific antigens resulting from the expression of a large number of mutations which may be a result of tumor genetic instability but also due to severe DNA damage. The degrees of DNA damage can strongly influence the immunogenicity of the tumor. In this context, RT leads to DNA SSBs or DSBs or other DNA alterations (see Chap. 3) [177]. DSBs are the most lethal DNA lesions if not repaired. Repair depends on the type of radiation causing the damage. Low LET radiation causes simple DSBs, which are repaired by NHEJ pathway. High LET radiation causes clustered DNA lesions (or multiple damaged sites) that are usually repaired by HR. Non-DSB lesions at clustered DNA damage sites are corrected with Base Excision Repair. If the DNA repair mechanisms are not sufficient to repair the damage, or if the RT itself causes defects in the DNA repair mechanisms, mutations occur that can lead to the expression of tumor neoantigens. These neoantigens are then processed by tumor cells and presented by MHCs [178].


Damage signals properly known as Damage-Associated Molecular Patterns (DAMPs) are endogenous molecular signals released by irradiated cells after damage or when dying [176]. These signals enable enhanced recognition and killing of the tumor by macrophages and DCs resulting in antigen presentation to T cells [161, 164]. Some of the most frequently found radiation-induced DAMPs in tumor cells are:
Fragmented double-stranded DNA and micronuclei. These self-DNA fragments and micronuclei appear as cytosolic DNA in tumor cells, which are later recognized by immune cells.

High mobility group box 1 (HMGB1) is a nuclear protein associated with chromatin that is released extracellularly by dying tumor cells, normally necrotic cells [179]. HMGB1 binds to Toll-like receptor 4 (TLR4) expressed by DCs and activates them.

Nucleotide release by apoptotic cells function as a chemotactic signal for phagocytic myeloid cells including DCs by stimulating the Purinergic receptor 2 (P2RY2) [180].

Extracellular ATP can also function through the activation of Purinergic receptor 7 (P2RX7) to initiate inflammasome activation and subsequent Interleukin 1β production. This mechanism was shown to be required for the induction of tumor antigen-specific CD8+ T cells following challenge with dying tumor cells [181].

Calreticulin is an ER protein that gets translocated to the cellular membrane after RT. Calreticulin is essential for efficient uptake of dying tumor cells by antigen-presenting cells [182].

Chemokines are a frequent part of DAMPs. Chemokines, such as C-X-C motif (CXC) ligand 16, 10, and 19 (CXCL16, CXCL10, and CXCL9), are known for being released after radiation exposure; they trigger differentiation of immune cells, and stimulate immune modulators such as Interferons, Tumor necrosis factor α, and Interleukin 1β [162, 183–185].


Fragmented double-stranded DNA and micronuclei. These self-DNA fragments and micronuclei appear as cytosolic DNA in tumor cells, which are later recognized by immune cells.


High mobility group box 1 (HMGB1) is a nuclear protein associated with chromatin that is released extracellularly by dying tumor cells, normally necrotic cells [179]. HMGB1 binds to Toll-like receptor 4 (TLR4) expressed by DCs and activates them.


Nucleotide release by apoptotic cells function as a chemotactic signal for phagocytic myeloid cells including DCs by stimulating the Purinergic receptor 2 (P2RY2) [180].


Extracellular ATP can also function through the activation of Purinergic receptor 7 (P2RX7) to initiate inflammasome activation and subsequent Interleukin 1β production. This mechanism was shown to be required for the induction of tumor antigen-specific CD8+ T cells following challenge with dying tumor cells [181].