Radiation therapy represents a common therapeutic approach for many forms of cancer, of which there are nearly 2 million new cases diagnosed each year.(24) As improvements in cancer therapy have increased the average length of cancer survival, a growing number of survivors are living with long-term sequelae of radiation therapy such as RIF. Shown to have a profound impact on long-term quality of life, RIF can lead to severe cosmetic and functional impairment.(25, 26) At the tissue and cellular levels, RIF manifests as epidermal thinning, eosinophilic homogenized sclerosis of dermal collagen, presence of scattered large and atypical fibroblasts, and fibrous thickening leading to luminal obliteration of deep vessels.(25–27) Dermal thickening paired with vascular damage results in an environment in which wound healing is impaired, leading to a uniquely challenging setting for surgical reconstruction.(25)
Despite the clinical significance and the rising incidence of RIF, the current array of therapeutic options remains restricted, particularly in the realm of topical treatments. Presently, patients have access to treatments such as physical therapy, fat grafting, and vitamin E. However, the limited and mixed evidence, coupled with logistical barriers and high costs, has hindered the widespread adoption of these options. Topical DFO has emerged as a treatment for RIF, and has previously been shown to attenuate cutaneous RIF in a murine model across biomechanical and histological measurements as well as improve perfusion to the skin.(12, 13)
To investigate the cellular mechanisms underlying the demonstrated efficacy of DFO, this study focused on the effect DFO treatment may have on ferroptosis, an iron-dependent mechanism of cellular death, which has recently been tied to IR-induced damage in a variety of tissue types.(28–31) Historically, DFO has also been known to restore vascularity by stabilizing HIF1α through chelation of iron, an integral co-factor necessary for prolyl hydroxylase domain-containing protein 2-mediated degradation of HIF1α. Stabilization of HIF1α leads to an increase in downstream angiogenic factors and recruitment of endothelial progenitor cells.(32, 33) In addition to this pathway, a previous study has suggested that DFO may act through additional cellular pathways to impart a therapeutic effect.(14)
This study is the first, to our knowledge, to demonstrate in vivo the occurrence of ferroptosis in skin following ionizing radiation injury. We observed that topical DFO treatment reduces a well-established marker of ferroptosis, 4-HNE, to levels comparable to that achieved by Fer-1, consistent with cutaneous inhibition of ferroptosis. In the case of Fer-1, this finding mirrors previous studies where IP injection has been proven effective at inhibiting ferroptosis in a variety of organ systems.(20, 34) DFO has also demonstrated this capability in vitro,(11) via IP injection,(35) and intraarticular injection,(36) though never before as a topical treatment.
Our findings show that DFO treatment resulted in tissue-level alteration measured by histology that indicate the prevention and/or alleviation of dermal architectural changes known to characterize RIF. Including reduced dermal thickness measured by H&E and reduced collagen density measured by MT staining, these results recapitulate some previous findings which have demonstrated the ability of topical DFO treatment to attenuate RIF across these parameters.(12) Notably, Fer-1 treatment was found to moderately improve these histological measures of RIF as well, though not as much as DFO treatment. This finding was mirrored in other outcome measures as well, including extracellular matrix ultrastructure analysis and biomechanical testing, with measured parameters more similar to that of normal skin, but not to the degree achieved by DFO.
Perfusion imaging with laser Doppler confirmed that topical DFO treatment minimizes hypoperfusion of the dermis characteristic of chronic RIF,(12–14) while Fer-1 treatment did not. Taken together, these results indicate that while topical DFO treatment may inhibit ferroptosis, this effect alone does not explain the full therapeutic action of the iron chelator. As discussed above, the ability of DFO to promote perfusion through HIF1α stabilization has been previously described and may account for other differences, as Fer-1 is not known to interact with the VEGF pathway and has not been demonstrated to support neovascularization.
Notably, the safety profile of Fer-1 is a topic of current investigation, as several studies have indicated that the drug may have therapeutic potential in a variety of clinical settings, such as acute kidney and lung injury as well as cardiovascular disease.(37–39) However, some studies have indicated concern for liver toxicity, induction of autophagy, and immunosuppression.(7, 40, 41)
While our findings in a murine model show promise, additional experimentation is required to assess the potential translation of topical DFO treatment into clinical practice. Delving deeper into the intracellular effects of DFO may offer additional insights to explain the observed differences in outcome measures between Fer-1 and DFO treatments. While Fer-1 acts as an antioxidant and inhibits ferrous iron and lipid hypdroperoxide-dependent peroxidation, DFO chelates iron directly. Paired with the delivery of DFO through a reverse micelle formulation which allows for penetrance of the stratum corneum and perhaps intracellular entry,(12) this difference in mechanism may also account for some of our results indicating that DFO more effectively rescues RIF of the skin and restores perfusion compared to Fer-1 alone.
As DFO is known to promote angiogenesis through the stabilization of HIF1α, a theoretical concern exists regarding the use of this agent in sites where oncologic pathology may present. However, no studies to our knowledge have demonstrated an increased risk for cancer growth, metastasis, or recurrence following local administration of DFO. Furthermore, iron is known to participate in critical cellular functions such as oxygen transport, metabolism, and cell growth, and evidence has suggested that DFO may thus impart an anti-tumor effect.(14, 42, 43) Some tumors, in fact, have demonstrated iron dependency making them vulnerable to iron chelation by agents such as DFO.(44)
Notably, the formation of fibrosis in murine skin differs from that of humans in clinically relevant ways. For example, murine skin is known to heal and fibrose more rapidly than human skin, and time points analyzed for chronic fibrosis in mice were based on previously published studies.(12, 45, 46) Mouse skin varies from humans morphologically, as well, containing layers of differing relative thickness and the addition of a layer of subdermal muscle called the panniculus carnosis.(47) For these reasons, further investigation of this topic in large animal models such as pigs, which offer a skin structure more similar to that of humans, would be of substantial translational value.