Expanding the therapeutic index of radiation therapy by normal tissue protection

Introduction

Cancer causes 8.2 million deaths each year globally. Today, most of the anti-cancer therapeutic improvements are achieved by combined treatment modalities, in which radiation therapy remains a cornerstone and is delivered with curative intent in 50% of cancer patients. During the past 20 years, ballistics and imaging improvements have enabled the individualized treatment of patients with a precise and conformal delivery of the dose to the tumor. These technological improvements have greatly enhanced the irradiation therapeutic index. Nevertheless, the level of dose that can be delivered, and accordingly, the possibility of achieving local control over the tumor, is still limited by the toxicity induced to normal (surrounding) tissues. In addition, the combined treatment protocols currently used against cancer are associated with an enhanced risk of toxicity. As the number of cancer survivors increase, preventing and reducing the treatment’s side effects is a priority. The present review provides a list of validated strategies as exhaustive as possible that have been validated to prevent, mitigate, or reverse radiation-induced toxicity in preclinical and clinical studies, together with several new options provided by novel types of radiation therapy.

Cancer causes 8.2 million deaths each year globally. Today, most of the anticancer therapeutic improvements are achieved by combined treatment modalities, in which radiation therapy remains a cornerstone and is delivered with curative intent in 50% of cancer patients. During the past twenty years, ballistics and imaging improvements have enabled the individualised treatment of patients with a precise and conformal delivery of the dose to the tumor. These technological improvements have greatly enhanced the irradiation therapeutic index. Nevertheless, the level of dose that can be delivered, and, accordingly, the possibility of achieving local control over the tumour, is still limited by the toxicity induced to normal (surrounding) tissues. In addition, the combined treatment protocols currently used against cancer are associated with an enhanced risk of toxicity. As the number of cancer survivors increase, preventing and reducing the treatment’s side effects is a priority. The present review provides a list of validated strategies as exhaustive as possible that have been validated to prevent, mitigate, or reverse radiation-induced toxicity in preclinical and clinical studies, together with several new options provided by novel types of radiation therapy.

Post-radiotherapeutic normal tissue injury

Typical side effects are systemic in the case of drug therapies, whereas radiation-induced normal tissue damages are local or locoregional and can be divided into early and late side-effects. Typically, in the clinic, early effects occur during the time-course of the treatment or within a few weeks of the completion of a fractionated radiotherapy schedule. These effects include skin erythema, dry or moist desquamation of the skin, mucositis, nausea, diarrhea, edema or headaches. Late effects are expressed after latent periods of months to years, and include radiation-induced fibrosis, atrophy, and vascular damage (Figure 1). Likewise, in pre-clinical models, radiation-induced normal tissue injury can be dichotomized into an acute inflammatory phase followed by a late chronic phase characterized by both chronic inflammation and fibrosis. The complications varies from undetectable to highly disabling levels for the patient, inducing a loss of function of the altered organ¹ depending upon extrinsic factors such as variations in the dose delivered, changes in the treatment volume or dose fractionation as well as intrinsic factors such as individual radiation sensitivity and the presence of comorbidity factors.^2–5

In order to reduce toxicities, it is important to understand the mechanisms underlying the radiation injury of normal tissues. While cell death is the primary and intended effect of ionizing radiation on tumor cells, the deleterious effects of irradiation on normal tissues comprise a cascade of molecular, cellular, and tissue events that spans for a long time after exposure,⁶ and has been compared to a “complex wound”.⁷ However, whereas wound repair has three distinct stages that include a clotting/coagulation phase, a restoration phase with fibroblast migration/proliferation, and a final remodeling phase where the normal tissue architecture is restored (Figure 1), this cascade of finely-tuned processes is disrupted post-radiation therapy. Tissue response to irradiation depends upon the intrinsic sensitivity of the various cellular compartments (direct cell death) that compose the organ and the complex crosstalk established in between all these compartments (indirect functional effects). For instance, rapid renewing compartments, like the epithelial layers and bone marrow, show an acute response to irradiation. This is due to a severe depletion of actively dividing upstream progenitor compartments, ultimately resulting in a loss of replenishment of downstream mature cell pools.^{8, 9}

In contrast, in connective tissues where cellular turnover is low, radiation injury may be expressed months or even years after exposure if cell death occurs when the cellular division is attempted. The functional consequences are a result of non-lethal effects on different intra- and extracellular molecules and changes in gene expression in irradiated cells, synthesis of paracrine factors leading, e.g. to direct inactivation of anticoagulant molecules, activation of latent growth factors such as transforming growth factor, TGFβ₁, and activation of proteases. The various steps involved in this cascade can constitute valuable therapeutic targets to modify normal tissue injury and enhance the outcome of radiation therapy. In the following sections of this review, we will focus our attention on selected targets, explain their pathophysiological relevance and report the therapeutic strategies that have been used over the past decades. We also discuss how the selection of the timing of administration depends upon the pathway targeted and precludes the selection of the appropriate drug.

Inhibiting radiation-induced acute cell death and depletion

To prevent and inhibit radiation-induced toxicity, the early prevention of epithelial, endothelial, and stem cell deaths is one of the primary options that can be proposed. These interventions imply the administration of drugs delivered prophylactically (Table 1) including inhibitors of pro-apoptotic molecules, such as the transient blockade of p53 (Pifithrin);^{10, 11} the inhibition of p-53 upregulated modulator of apoptosis (Puma Inhibitors) or the inhibition of the ceramide pathway in endothelial cells.^{16, 17} Alternatively, stimulation of antiapoptotic molecules such as NF-ҡB by the flagellin-derivative, CBLB502^{18, 19} protects microvascular endothelial cells from radiation-induced death. Similarly, the enhancement of endothelial cell radiation resistance can be achieved by blocking the TSP1/CD47 pathway.²⁰ Other cytoprotective therapies such as keratinocyte growth factor, fibroblast growth factor and glucagon-like peptide-2 treatment^{15, 21,22} also have proven efficacy, and prevent the development of normal tissue toxicity. For instance, keratinocyte growth factor administration has been validated and shown to stimulate cell proliferation and promote epithelial cell survival along with the differentiation of oral mucosa, both in pre-clinical and clinical trials.^{23, 24} Furthermore, it displayed a beneficial off-target effect by decreasing reactiveoxygen species (ROS) levels and stimulating DNA repair.^{23, 24}

These strategies prevent the alteration of an organ’s structure and function, and/or enable the rapid restoration of the tissue, markedly when stem cells are protected and activated. Activation of deleterious cascades will be interrupted, avoiding the excessive release of inflammatory mediators and dramatic tissue injury. However, two major drawbacks are associated with these strategies: primarily, there is a high risk of tumor protection and secondly, the acute rescue of heavily damaged cells could have a delayed detrimental impact on normal tissue structure and function, including the occurrence of a secondary cancers.

Restoring the redox equilibrium in tissue after radiotherapy

Another important strategy that has been extensively explored to counteract radiation injury involves antioxidant molecules and scavengers aiming at restoring the redox equilibrium of the tissue, immediately after irradiation or at later time points (Table 2).

Direct interactions of ionizing radiation with biological matter induces excitations and ionizations resulting in the ejection of electrons from biomolecules. In addition, indirect interactions occur through ionization of the water and represent the major part of the radiation’s effects on the biological matter. Both effects lead to free radical formation. In addition to this rapid burst of free radicals that occurs immediately following radiation, persistent and prolonged increase in reactive oxygen species/reactive nitrogen species (ROS/RNS) is also observed after irradiation. While ROS/RNS in physiological conditions do perform useful functions such as cell proliferation and differentiation^{41, 42} and are involved in homeostatis processes such as wound healing,⁴³ when ROS production escalates beyond a certain threshold and becomes persistent, the antioxidant response is not sufficient to reset the system to the original level of redox homeostasis. These high levels of ROS/RNS result in pathological stress to tissues and cells^{2, 44} by acting as messenger molecules in cytoplasmic signalling pathways, and by direct effects on transcription.⁴⁵ These elevated concentrations of ROS/RNS not only cause DNA damage, but also alter proteins, lipids, carbohydrates, and complex molecules.

At acute time point post-irradiation, early hypoperfusion occurs due to vascular changes, endothelial cell damages^{16, 46} and escalated oxygen consumption, a consequence of increased cellular metabolism. It generates tissue hypoxia, which further exacerbates the injury.⁴⁷ Then, as time passes, the system may still reach an equilibrium associated with higher ROS concentrations called chronic oxidative stress,⁴⁸ which does not really involve a loss of homeostasis but rather a chronic shift in the level of homeostasis. The high level of ROS/RNS are immediate activators of the fibrogenic signals including transcriptional activation of one of the most potent fibrogenic growth factor TGFβ₁,^{49, 50} that subsequently upregulates collagen synthesis and perpetuates self-induction and autocrine induction of another potent fibrogenic growth factor, connective tissue growth factor (CTGF).⁵¹ Activation of inflammation mediators has also been described in the case of a high ROS level status, leading to deleterious chronic inflammation in the irradiated tissue. These observations support the fact that radiation-induced late effects are partially propelled by a chronic oxidative stress⁴⁸ induced at late stages by the redox imbalance that occurs in the tissue as a result of intrinsic hypoxia⁵² which further enhance the redox imbalance.

Given the central and persistent role played by the loss of redox equilibrium in the tissue response to irradiation, targeting redox imbalance, ROS/RNS, and hypoxia is an obvious therapeutic option with applications during any of the steps of the cascade as shown in Table 2. Treatment with hyperbaric oxygen (HBO)⁵³ and antioxidant therapy^54–56 were both successfully used despite their apparent antagonistic mechanism of action. HBO induces transient tissue hyperoxia (typically ~2 h day^-1) that should not overcome natural antioxidant defenses,⁵⁷ but may help to remobilize tissue remodelling by activating signaling molecules in transduction cascades (see the review)⁵⁸ and stimulate angiogenesis.⁵⁹ In addition, the results from recent clinical trial show no benefice of HBO on lymphoedema.^{60, 61} Antioxidant therapies are based on a different mode of action and aim at scavenging ROS. Initial studies with amifostine^25–27 and bovine liposomal Cu/Zn superoxide dismutase showed antifibrotic efficacy associated with TGFβ inhibition.⁶² More recent trials investigated the benefits of tocol isoforms (Vitamin E analogs) such as high-dose alpha-tocopherol combined with pentoxifylline and clodronate,^{36, 37} and γ-tocotrienol (GT3).³⁹ In addition to their antioxidant action, both strategies have displayed off-target benefits with protective endothelial activity^{63, 64} and miRNA regulation.⁶⁵ Interestingly, the efficacy of GT3 is enhanced when combined with pentoxifylline.⁶⁶ Lastly, hypoxia-regulating molecules such as 2-methoxyestradiol have been shown to downregulate HIF1α-mediated Smad activation and inhibit radiation-induced lung fibrosis in mice.⁶⁷

Targeting inflammatory and fibrogenic signals induced by radiotherapy

The third type of strategy is based upon the understanding of the pathophysiological processes (cellular and molecular) governing normal tissue toxicity. This knowledge has provided us with tools to improve the therapeutic ratio of radiation therapy, and biology-driven efforts have enabled the development of translatable therapeutic approaches to prevent, mitigate, and even reverse radiation injury based upon the targeting of signalling pathways. The relevance of these various signalling pathways to the pathogenesis and maintenance of radiation injury has been extensively and recently reviewed in several articles.^{40, 51,68,69} Therefore, we will focus on recent results that highlight the relevance of immune cells in response to irradiation. Elucidating the impact of radiotherapy on the immune compartment and subsequent immunomodulation is nowadays one of the most promising strategies for improving anticancer treatment,⁷⁰ and recent studies suggest that it may also enhance the differential effect of radiotherapy.

The importance of myeloid cells in the radiation-induced response has been proposed and the role of macrophage reprogramming by radiotherapy has been demonstrated.^71–73 Macrophage phenotypes are highly dependent upon the microenvironment and recent publications have revealed their complexity.⁷⁴ In fibrotic tissue, macrophages do display immunosuppressive properties, secrete large amounts of the fibrogenic mediator TGF-β1⁷⁵ that activates the Smad pathway, and stimulate downstream fibrogenic genes such as CTGF and PAI-1.⁷⁶ The macrophages isolated from bronchoalveolar fluid from patients undergoing thoracic irradiation spontaneously released platelet-derived growth factor, another important fibrogenic growth factor.⁷⁷ Several older studies have suggested possible benefits of macrophage depletion using clodronate liposomes.⁷⁸ The reduction of the number of macrophages by clodronate in wounded tissue indeed reduced excessive scar formation and delayed cutaneous wound healing.⁷⁹ Froom and colleagues⁸⁰ showed that the oral administration of clodronate (bisphosphonate) significantly reduced bone marrow fibrosis, and in the early 2000’s Delanian and Lefaix successfully administered clodronate in combination with a pentoxifylline–vitamin E treatment, and showed improved efficacy in the treatment of radiation-induced fibronecrosis.^{81, 82}

More recent data validate and refine this strategy bringing molecular highlights and a biological rationale for macrophage targeting in the management of radiation-induced normal tissue complications.^{83, 84} Recently, the P Huber group showed that blocking CTGF with a specific antibody (FG-3019) was able to attenuate radiation-induced pulmonary remodeling and reverse fibrosis. Interestingly, they showed that this treatment was associated with the abrogation of M2-like macrophages influx.⁸³ We extended these findings and recently characterized the contribution of pulmonary macrophages to radiation-induced pulmonary fibrosis.⁸⁴ We showed that the populations of pulmonary macrophages are heterogeneous and their contribution to fibrosis is complex. A differential phenotype for alveolar and interstitial macrophages was indeed shown along with a specific fibrogenic contribution of interstitial macrophages but not alveolar macrophages. Ultimately, selective targeting of interstitial macrophages with CSF1R mAb was shown to display antifibrotic action.

Lastly, an overview of the drugs that have been used to prevent and mitigate radiation damages as well as the drugs that have been successfully used to reverse radiation-induced complications, fibrosis in particular, are provided in Tables 3 and 4. The impressive list of compounds shows the vitality of the research in this field with an impressive rate of translational/clinical studies (Table 4) using curative strategies. Three parameters have probably fostered this progress: first, the irreversibility of fibrosis was challenged by many cellular and experimental studies; second, curative strategies are clinically interesting as they do not interfere with anticancer treatments through possible tumor protection; third, they can be delivered to the targeted population of patients that need it.

Use of novel RT approaches to protect normal tissues

Aside from novel biological interventions, improvements in physics have been critical for protecting normal tissue, enhancing differential effects, and making progress on tumor control. Novel technologies based upon sophisticated instrumentation used in conjunction with imaging, such as stereotactic body radiotherapy, have helped to protect normal tissue by reducing the irradiated volume and more accurately targeting the tumor. More complex and expensive technologies, such as proton and carbon ion therapies, take advantage of their specific pattern of energy deposition in the biological matter and are already implemented in clinical practice. Other novel ideas and approaches have also been recently proposed such as dose rate escalation.^{108, 109} This last approach should be translatable into clinical application soon, which illustrates the broad range of opportunities that exist in radiation therapy, and highlight the need for interdisciplinary working teams composed of biologists, physicists, and physicians.

Conclusion

The selective protection of normal tissue function using modern targeted radiotherapy is a powerful approach to improve cure rates and simultaneously enhance the quality of life of long-term cancer survivors. Nowadays, high precision radiation therapy induces a drop in the rate of complications. In parallel, advancements in radiobiology have deciphered the complexity of the biological response induced by tissue exposure to ionizing radiation, and enabled the identification of therapeutic targets. These processes include profound microenvironment remodeling with alteration of the vascularization, perfusion/hypoxia, inflammation, modulation of immune compartments and stromal remodeling. Therefore, currently, well-selected combination strategies that target distinct pathogenic pathways induced by irradiation at specific time points of the pathogenic process can be proposed, and the next challenge will be to develop rational radiotherapy–drug combinations to maximize the therapeutic impact. The management of RT complication has also reach the era of personalized medicine and in many centers in Europe (France, UK, CH for instance), patients presenting with complications are managed in the frame of multidisciplinary consultations to adapt the best therapeutic answer for each specific situation. Simultaneously, novel radiotherapy approaches such as the ultra-high dose rate, Flash RT, have been developed, offering the potential to radically change the way radiation therapy is employed and delivered over the next few years.