Radiation-induced salivary gland damage/dysfunction in head and neck cancer: Nano-bioengineering strategies and artificial intelligence for prevention, therapy and reparation

Saliva is produced by and secreted from salivary glands. It is an extra-cellular fluid, 98% water, plus electrolytes, mucus, white blood cells, epithelial cells, enzymes, and anti-microbial agents. Saliva serves a critical role in the maintenance of oral, dental, and general health and well-being. Hence, alteration(s) in the amount/quantity and/or quality of secreted saliva may induce the development of several oro-dental variations, thereby the negatively-impacting overall quality of life. Diverse factors may affect the process of saliva production and quantity/quality of secretion, including medications, systemic or local pathologies and/or reversible/irreversible damage. Herein, chemo- and/or radio-therapy, particularly, in cases of head and neck cancer, for example, are well-documented to induce serious damage and dysfunction to the radio-sensitive salivary gland tissue, resulting in hypo-salivation, xerostomia (dry mouth) as well as numerous other adverse Intra-/extra-oral, medical and quality-of-life issues. Indeed, radio-therapy inevitably causes damage to the normal head and neck tissues including nerve structures (brain stem, spinal cord, and brachial plexus), mucous membranes, and swallowing muscles. Current commercially-available remedies as well as therapeutic interventions provide only temporary symptom relief, hence, do not address irreversible glandular damage. Further, despite salivary gland-sparing techniques and modified dosing strategies, long-term hypo-function remains a significant problem. Although a single governing mechanism of radiation-induced salivary gland tissue damage and dysfunction has not been yet elucidated, the potential for synergy in radio-protection (mainly, and possibly -reparation) via a combinatorial approach of mechanistically distinct strategies, has been suggested and explored over the years. This is, undoubtfully, in parallel to the ongoing efforts in improving the precision, safety, delivery, and efficacy of clinical radiotherapy protocols/outcomes, and in designing, developing, evaluating and optimizing (for translation) new artificial intelligence, technological and bio-pharmaceutical alternatives, topics covered in this review

Graphical abstract

It is well recognized that the incidence of cancer, the second leading cause of death, globally, is increasing; an ongoing major burden of disease and public health burden, Worldwide. While there were 14.1 million cancer cases reported in 2012, the World Health Organization (WHO) estimated about 1 in 6 deaths is due to cancer, with 9.6 million such deaths reported in 2018. In the United States, today, cancer is the second leading, after heart disease, cause of death among men and women, with over 1 million new cases diagnosed, annually [1]. Despite a reduction in tobacco consumption and significant modern advancements in medicine, the number of new cancer cases, per year, is projected to rise to 22.2 million by 2030 [1]. Cancers, often squamous cell carcinomas/neoplasms, that involve the oral cavity, nostrils, paranasal sinuses, naso-/oro-/hypo-pharynx, larynx, and the salivary glands, are common/collectively (despite their heterogeneity) termed head and neck cancers (HNC), which, together are responsible for nearly 200,000 deaths, a year, Worldwide [2]. In the United States alone, HNC represents 4% - 5% of all cancers, and in Europe, HNC is the 6th most frequent group of cancers [3]. Besides the alarming incidence and mortality rates, HNC suffers a relatively poor prognosis, overall, whether due to delays in diagnosis, staging, treatment, particulars of the tumor site, onset, type of symptoms, and/or efficacy of therapies, to mention a few. Such factors further contribute to permitting the progress and upstaging of the malignant tumor(s) which eventually result in enfeebled survival, despite the application of novel or advanced intensive therapeutic regimens. Briefly, treatment, often a multi-disciplinary case-specific approach, can employ chemo-/radio-/immune-therapy, surgery, or combinatorial strategies [4]. Herein, radiotherapy (RT), whether radical or prophylactic, remains a mainstay of HNC treatment, especially in light of modern improvements in precisely targeting and delivering the required radiation doses to the tumor, thereby allowing additional sparing of normal/healthy surrounding tissue(s), greatly reducing side or adverse effects of radiation, and consequently improving the quality of life (QoL) of patients as well as their families [5-8]. IMRT (intensity‐modulated radiotherapy), VMAT (volumetric modulated arc therapy), and particle (ion-based) therapy are perhaps fine examples of modern high-precision RT [7]. Indeed, it has been estimated that > 80% of HNC patients exhibit xerostomia and salivary gland hypofunction following RT [3,4], in which, as well, depending on the RT dose, delivery method, and employed salivary gland-sparing techniques, 64% - 91% of irradiated HNC patients, often suffer from chronic xerostomia [3,4,8].

RT, in general, aims to realize localized destruction and control of the target tumor (-cells) and halt the reproductive potential, while minimizing toxicity onset. Specifically, high-energy radiation is deposited, causing DNA strands to break thereby damaging the cell genome either directly or indirectly (via free-radical production) and subsequently resulting in apoptosis, mitotic cell death, and tissue hypoxia, through different cascades and processes [5,7]. Depending on the radiation dose and tissue turnover, amongst other factors, RT can almost always be expected to result in a range of side effects, of which some are reversible, and others are irreversible (Figure 1). Indeed, HNC and oral squamous cell carcinoma (OSCC) patients receiving RT often experience pain, taste disturbances, difficulties in mastication and deglutition (swallowing) and suffer from mucositis, fungal infections, dental decay, alterations in speech, all of which are mainly due to or linked to salivary gland dysfunction which in turn results in hyposalivation and xerostomia [9-12]. Herein, xerostomia, a dry mouth sensation, is one of the main complications and complaints for HNC patients receiving RT, mainly as a sequela of the un-avoidable damage to the parotid and sub-mandibular glands (both produce over 80% of saliva) anatomically located with the radiation zone [8,12]. Inflammation, fibrosis, atrophy, and the reduced wound healing response, i.e. reparative and regenerative capacity of the glands, mainly due to lack of functional salivary gland stem/progenitor cells post-irradiation, render the inevitable radiotherapy-induced salivary gland damage and dysfunction, whether occurring early or late, a significant impediment to the QoL and survival of HNC and OSCC patients [10,13,14]. Hence, besides modern advancements in radiation engineering technologies, ample pharmacological and pharmaceutical solutions have been explored [14]. Accumulating knowledge in understanding the underlying signaling pathways, cellular and tissue responses, spatiotemporally, fuel the continuing efforts aimed to explore, develop and translate novel/innovative solutions to support the prevention (and treatment of) radiation‐induced side-effects and damage of salivary glands, the main focus of this review, designed to provide the clinical reader with a summary of relevant literature and recent innovative developments in salivary gland radioprotection and potential salivary gland repair, post-RT.

Saliva and salivary glands: pre-, during- and post-RT

Briefly, exocrine salivary glands are classified as either major (parotid, sub-mandibular, and sub-lingual) or minor (labial and buccal gland, glosso-palatine gland, and palatine and lingual) glands. Anatomically, all three major glands are highly vascularized, and innervated and are architecturally similar featuring a ductal structure with a secretory/excretory (saliva-producing acini surrounded by myoepithelial cells, myofibroblasts, immune cells, stromal cells, endothelial cells, and nerve fibers) opening into the oral cavity/mouth [15]. The glands differ in their type of acinar cells and as a result, in the type of produced saliva. While the parotid is composed of only serous acini thereby producing watery saliva, the sub-mandibular and sub-lingual glands contain a mix of serous and mucous (glycoprotein- rich) acini, thereby producing saliva of a different composition, a seromucous secretion. Secretion of saliva is stimulated by the sympathetic (proteins) and parasympathetic (serous/ions) branches of the autonomic nervous system [15,16]. Saliva is basically an oral lubricant fluid with multiple digestive functions critical for oro-dental health, QoL, and general well-being. It is composed of a complex mixture of water (99%), electrolytes (sodium, potassium, calcium, magnesium, etc ...), mucins, proteins, white blood cells, epithelial cells, immunoglobulins, anti-microbials/-bacterial and enzymes (1%) [16-18]. Hence, saliva is essential for moistening, chewing, swallowing, and chemically-digesting foods. It also facilitates speaking, aids the tongue in taste sensing, helps protect the oral mucosa (localized immunity/mucosal resistance), and plays a role in tissue re-mineralization. A healthy adult produces/secretes a daily average of 0.5 L - 1.5 L, at differential rates over the day, and at a near-neutral (buffer) pH of 6.7 [15-17,19-22]. Therefore, alterations in quantity (↓: hypo- or ↑: hyper-salivation) or quality of the secreted/produced saliva are associated with a variety of conditions and diseases and have been associated with some medications and therapies [23]. For instance, sialorrhea is a general term used for hyper-salivation (or drooling), often as a result of medication, systemic diseases, psychiatric disorders, and/or oral pathologies, amongst others [14]. It is also often linked to conditions such as Parkinson’s, epilepsy, amyotrophic lateral sclerosis or ALS, cerebral palsy, developmental disabilities, pregnancy, and/or drugs including clozapine [16,24] Common treatments for sialorrhea include surgical intervention, radiation of the salivary glands (to halt and diminish its function) and the use of oral anti-cholinergic drugs (to inhibit saliva production), however with known side or adverse effects. In recent years, numerous studies investigated the use of neuro-toxins, mainly botulinum neurotoxins or BoNTs, which basically are bacterial exotoxins that interfere with and block the exocytotic release of vesicular neuro-transmitters cholinergic neuromuscular activity in the target tissue, including commercially-available RimabotulinumtoxinB (RimaBoNT-B; FDA approval in 2000) and IncobotulinumtoxinA (IncoBoNT-A; FDA approval in 2010) in patients suffering sialorrhea, with attractively promising results [24,25]. On the other hand, salivary gland hypofunction (progressive loss of gland function) is commonly described or associated with the reduction of salivary flow and production, quantitatively. Frydrych [26] discussed salivary gland hypofunction etiology and classified causes into seven major areas; developmental, autoimmune/chronic inflammatory, endocrine, neurological/psychiatric, metabolic, infectious, and iatrogenic [26]. In a healthy individual, the un-stimulated “whole” salivary flow rate is averaged at 0.35 mL saliva per minute, with abnormalities indicated if the rate drops. For example, one of the most prevalent and studied diseases or disorders of the salivary gland is Sjögren’s syndrome (SS), a chronic auto-immune inflammatory reaction characterized by lymphocytic infiltration of the exocrine glands (mostly to the salivary or lacrimal glands), which generates a significant reduction in salivary flow rate - to below 0.1 mL whole saliva per minute secreted, un-stimulated [27]. It is perhaps noteworthy herein that the whole saliva indicates the collection of saliva (secreted from all salivary glands) present in the mouth. Other quantification techniques will require direct collection from the specific gland. Moreover, often it is reported in diagnosing SS that just the un-stimulated whole saliva flow rates are employed.

Hypo-salivation, therefore, is salivary flow rate reduction, quantified, clinically via sialometry. Xerostomia, on the other hand, is the reported perception or sensation, subjectively, of oral dryness. Hypo-salivation may or may not be accompanied by xerostomia and vice versa. Dryness in the mouth can be a side-effect of medications or due to diseases such as HIV/AIDS, diabetes, hypertension, and/or other factors including smoking, dehydration, mouth breathing, aging, and/or head and neck irradiation [14,16,23,28,29]. Indeed, xerostomia is one of the most commonly reported (and to be inevitably expected) complications of RT (during and after RT) for HNC, and as mentioned earlier, mainly as a predictable consequence to the significant damage (and generated inflammatory immune response) caused to the salivary glands which are located and included within the RT-zone or field [30-32]. RT, besides impairing salivary gland function and salivary flow rate, impacts the quality of the secreted saliva, given the loss or atrophy of acinar and ductal cells and granules (and stem/stromal and progenitor cells) and the consequential morphological changes to salivary fluid quality (including pH and buffering capacity), thereby affecting the essential protective, functional, and overall physiologic processes (Figure 2). Such damage [32] and impact can appear as soon as one week after the first RT session (acute RT-induced damage is due to a disturbance in the involved signal transduction pathways on the cell membrane). A progressive decrease in salivary gland function is evident with more RT sessions (delayed or late RT-induced damage is due to apoptosis-driven parenchymal cell loss, inflammation, blood vessel dilation, function loss, nerve injury, and reduced parasympathetic nervous function, and fibrosis) rendering rescue, repair, and regeneration rather challenging [33-35]. As a result, the QoL of a large proportion of patients receiving RT is severely compromised [36,37], with thicker or more viscous saliva and xerostomia leading to reported complaints [38]. Indeed, RT-related biochemical and proteomic alterations where several key glycoproteins, proteins, and other molecules are affected have been identified [31,39]. For example, Jehmlich, et al. [40], discussed such variations post-RT, detected significant alterations in 48 proteins, and highlighted the development of oral mucositis as a result of salivary gland dysfunction. The psycho-social and emotional impact on QoL of HNC patients, especially the elderly [41], where they experience and suffer from a compromised ability to taste, chew and swallow foods extended to their forced switching of dietary preferences to soft and carbohydrate-rich foods, thereby resulting in serious nutritional deficiencies [38,40,41]. Hyposalivation and sequential xerostomia also affect speaking and communication abilities, and patients experience nocturnal oral discomfort, consequently, causing additional stress and leading to withdrawal from the needed every day or day-to-day societal and emotional interactions [42-44]. Furthermore, with the prolonged oral clearance of sugars, the oral mucosa becomes painfully dry, sticky, and more susceptible to infection, the progression of dental caries (tooth decay), gingival and periodontal disease and trauma, accentuating the importance of oro-dental hygiene and care, especially in the elderly patients [41]. Other sequelae include erosion and ulceration of mucosal tissues, oral candidiasis, dysgeusia and dysphagia. Therefore, it is common for HNC patients to suffer from depression, feelings of anguish, and anxiety after receipt of the RT protocol [14,37,45-49]. While the recovery of irradiated salivary glands at the cellular and molecular has been thus far shown to be limited, salivary recovery post-RT, from our clinical exposure and expertise, is a possible, yet lengthy (> 3 years), dire and capricious process; with underlying mechanisms not yet fully understood.

Radiation-induced damage prevention and potential repair/regeneration of salivary glands

Understanding the underlying mechanisms governing cellular and molecular control of salivary gland function is highly pertinent, during- and post-RT, to aid in developing suitable and effective therapies, whether preventive or reparative. To date, it is safe to state that available therapies continue to be symptomatic, and no definitive solution or approach has been shown to compensate for and/or recover the impairment of salivary glands and function. Lifestyle modifications, synthetic saliva, and/or use of salivary stimulants and sialagogues, suffer shortcomings and are not satisfactory to our patients, as they either only provide temporary (short-term) relief or might have other disquieting side effects. Hence, global attention has been diverted to seeking and developing alternative novel methods, tools, and therapies, to offer to HNC patients undergoing RT that can provide superior long-term efficacy. Herein, tissue engineering, regenerative medicine, pharmaceutics, and nanotechnology may contribute.

Tissue engineering and reparative/regenerative medicine: current regimens and strategies

Several tissues and organs are highly sensitive to irradiation, such as the skin, esophagus, and bone marrow. However, the salivary glands are intricately radiosensitive, given their highly-differentiated cell content marked with a very low or slow proliferative rate [50]. This can help explain why the salivary glands, in specific, are somewhat unique in their early- and delayed effects post-RT, when compared to other tissues and organs. Nonetheless, salivary gland dysfunction and/or hypofunction have been shown, in some cases, to be reversible. Such treatment intervention is multi-factorial and highly dependent on original causality, for example, in cases of alcohol abuse and dehydration or hypothyroidism. RT-induced salivary gland damage and dysfunction is a far more challenging scenario. Auto-immune/chronic inflammatory diseases, such as SS or systemic lupus erythematosus also result in irreversible damage to the salivary glands [26]. Today, as mentioned earlier, only palliative and efficacy-limited regimens are commercially-available [47]. Tables 1-4 highlight a selection of various radio-protection strategies, at different stages of development, pre-clinically (in vitro and in in vivo testing) and clinical (human clinical trials). Briefly, a database search was performed in PubMed-indexed articles using a multi-search of the following keywords: “Salivary Glands AND Radioprotection [Title/Abstract]”, “Salivary Glands AND Radioprotection [MeSH]”, “Salivary AND Glands AND Radioprotection [Title/Abstract]”, “Salivary Gland AND Radioprotection [Title/Abstract]”, “Salivary AND Gland AND Radioprotection [ALL FIELDS]”, “Salivary Glands AND Radioprotection [ALL FIELDS] and “Salivary Gland AND Radioprotection [ALL FIELDS]”. Eligibility and inclusion criteria included English articles reporting radio-protection data from in vitro, in vivo, and/or clinical settings/trials. Articles dated back to 1978 up to the search end date of October 16th of 2020 were analyzed. Reviews, communications, or articles with preliminary results were not included in our analysis (Figure 3). Herein, our purpose is to screen the available literature and assess the level of development of new strategies, regimens and/or innovative solutions, to provide a usable prior-Art formatted report. Hence, not all included articles, which are tabulated for the reader, were aimed to be presented and dissected to be discussed in detail. This review attempts to provide an overview of the current understanding, status, and prospect of salivary gland radioprotection systems, with a look onto potential reparative and regenerative keys; where we, amongst other clinicians and researchers, do aspire for a superior, safe, efficacious, and long-term innovative solution that reverses RT-induced damage to the salivary glands of our HNC patients. Moreover, we opted to avoid concluding our overview with calls for additional research or validation; given that vital tissue engineering strategies employing the design, characterization, and optimization of novel biomaterials (and three-dimensional or 3-D printing), that can also be housing/incorporating release-controlled nanoparticles or nanocapsules that also are designed to encapsulate distinct mesenchymal stem cells, induced pluripotent stem cells (iPSCs), growth factors or cytokines and/or pharmaceutical agents or drugs, currently investigated at different levels of development are limitless in distinctions and details.

Palliative care for RT-induced salivary gland dysfunction: Current and commercially-available palliative options for HNC patients undergoing RT include chewing gum (sugar-free), saliva substitutes, oral and topical lubricants, malic and ascorbic acid, saliva stimulants and sialogogues such as pilocarpine (Salagen, for example) and cevimeline (Evoxac, for example). As mentioned above, none have proven to restore normal QoL and patient satisfaction, mainly due to their limited efficacy and effectiveness [30,42]. On top, adverse side effects are common, and such options are often costly to patients, requiring multiple daily uses over long periods of time. In parallel, patients, especially the elderly, institutionalized and frail, need to go through education and training to acquire new eating and lifestyle habits; learn to prevent or avoid impaired swallowing and potential choking, and improve their oral and dental hygiene practices and tools to prevent (or halt the progression of) dental and oral mucosal diseases, infections, and tooth loss. Other palliative care options including acupuncture and electro-stimulation (enhancement of salivary reflexes) are currently undergoing investigation [30,51].

The only Food and Drug Administration/FDA–approved radio-protective and anti-xerostomia drug for clinical use (adjuvant setting) is Amifostine; an organic thiophosphate, cryoprotective agent and free radical scavenger administered subcutaneously or most often intravenously upon reconstitution with normal saline prior to or simultaneously with RT to then accumulate within the salivary glands, has been extensively studied since its development, initially under the nuclear warfare program [14,52]. Today, while it continues to benefit some patients, prophylactically, via minimizing the effects of xerostomia and taste loss, it is often associated with severe side effects including a rapid decrease in blood pressure (hypotension), nausea, and emesis or vomiting. A recent analysis of several clinical trials associated Amifostine with low-quality and mixed evidence in preventing dry mouth complaints in patients receiving RT to the head and neck region, in the short- to medium-terms (up to three months post-RT) and has questioned its potential in tumor cell protection; thereby further narrowing its clinical safety and efficacy window, especially in light of its high cost [52,53]. Essentially, its use in radiation-induced xerostomia has already been cautioned in the year 2008 by the American Society of Clinical Oncology [54] and so, its controversial and debatable safety and use in all cancer cases linger.

Preventive and interventional care for RT-induced salivary gland dysfunction: The main objective of any planned and/or prescribed preventive option should be the relief of symptoms and complications associated with hypo-salivation and xerostomia in HNC patients scheduled to receive RT, in order to prevent deterioration in their QoL thereby enhancing their battle with/against underlying cancer, its treatment and consequences thereof [55]. As discussed earlier, despite advancements in irradiation techniques and regimens including IMRT, only palliative, and prophylactic options are currently available (commercially), all of which do suffer substantial shortcomings [56,57]. One might even consider IMPT or intensity-modulated proton therapy, used to deliver a much-reduced irradiation dose and subsequently less toxic than IMRT, thereby alleviating much of the typical side effects of RT; however, IMPT is recognized to be more expensive and lacks accessibility or availability [43,56,57].

Contemporary alternative strategies and solutions

1. Surgical intervention: To prevent RT-induced hyposalivation, sub-mandibular gland preservation, and protection from irradiation via surgical relocation to the sub-mental space, thereby away or out of the irradiation zone, has been explored, with positive results. It is perhaps worth mentioning herein that the sub-mandibular salivary gland supplies up to 90% of the unstimulated saliva formation/secretion. However, such highly-invasive interventional procedures are peculiar and require exquisite surgical manipulation skills and settings. Further, surgical transfer of salivary glands is not indicated or possible for cancers of the oral cavity or patients undergoing (systemic) chemotherapy. In addition, for the gland to either retain or restore functionality, the connection of the gland to the main duct must be maintained or restored, respectively [58]; altogether rendering it a very limit-ed/-ing option. In terms of innovative approaches, Rao, et al. [59] recently described the use of a synthetic hydrogel (TraceIT; composed of water and iodinated cross-linked polyethylene glycol), injected via an 18-gauge needle, to serve as a minimally-invasive “spacer” (previously demonstrated in the treatment of prostate cancer), and displace or relocate the sub-mandibular gland in order to protect it from irradiation toxicity and be able to deliver a reduced irradiation dose; however, the experimental model used herein comprised of four refrigerated cadaveric specimens and no further in vivo or clinical studies evaluated the usability, malleability, safety, and efficacy, amongst other central factors or parameters, in clinical organ spacing.

2. Tissue engineering and regenerative medicine: Clearly, better approaches need to be explored and developed, driving the search elsewhere, into the multi-disciplinary areas of tissue engineering and regenerative medicine, in order to combine with and improve current options or to innovate and translate new alternative solutions, for wound healing. This is especially true, in light of accumulating knowledge and understanding of the underlying mechanisms governing radiation-induced salivary gland damage and dysfunction [47]. Indeed, from inducing DNA damage (via a. the generation of ROS/reactive oxygen species or b. the breakage of the DNA double-strand) to mutations to cell death (by apoptosis or necrosis, depending on cell type, injury, and cellular responses), to the loss of salivary progenitors, to the accruing evidence regarding the regenerative capacity (slow yet existent) of salivary glands following RT-induced injury, more evidently upon the administration of stimuli (exogenous delivery of stem cells and/or growth factors, for example), altogether re-emphasize the potential of such complex yet innovative approaches in finding a better clinical alternative solution. In a recent clinical study, Ho, et al. [60] evaluated the effects of a commercially-available slowly-dissolving adhering disc/tablet formulation (OraCoat XyliMelts) on the oro-dental health, enamel remineralization, bio-film formation, saliva presence, pH and buffering in 5 patients diagnosed with xerostomia (criteria: un-stimulated whole saliva flow rate below 0.2 mL per minute and a stimulated saliva flow rate of less than 0.5 mL in 5 minutes). They also assessed the patient self-reported comfort with the mint-flavored, xylitol-releasing tablets. Subjects were instructed to use the disc as often as needed for dry mouth symptom relief. In the end, a mean of 4+1 discs each day and 2 discs each night, were used. Overall, desirable effects of the product on symptomatic alleviation and management of xerostomia were reported. The authors reported effective local palliation, reduced dental sensitivity, improved salivary production and buffering capacity, reduced plaque formation, and alleviated xerostomia symptoms, without the need to use any systemic sialagogue medications throughout the 21 days of the study [60]. Yet, this is a pilot study, limited to involving a small number of participants.

3. Biomaterials and cell therapy: One of the fundamental roles for the maintenance of the body of any living organism is regeneration, which enables the repair and restoration of lost or damaged tissue [47,61]. Adult stem/stromal and progenitor cells have been identified in many tissues and are known to have a key role in the regeneration and repair, initiated or activated either by the excessive loss of differentiated cells (pool) or via (niche) environmental cues. In the presence of functional biomaterials such as the previously-described injectable hydrogel spacer [59] and a feasible agent-delivery tablet or disc [60], would loading, encapsulating, or incorporating putative salivary progenitor or stem/stromal cells, for example, a distinct type of stimuli, yield better results? Supplying salivary gland progenitor and stem/stromal cells, via a proper release-controlled dose-responsive carrier, might be able to re-establish the disrupted salivary stem/progenitor cell pool and niche, restore glandular tissue homeostasis, reverse hypo-salivation, and perhaps control xerostomia, a hypothesis we are currently examining in our laboratory, employing natural and synthetic polymers, liposomes, solid lipid nanoparticles, and core-shell nanocapsules, and further supplemented by other pharmaceutical agents. Modern medicine and biomedical research aim to control and enhance radioprotective as well as regenerative and reparative capabilities through the utilization of cells (cell lineages or primary cells); growing surface control using bio-scaffolds and/or manipulating growth factor/cytokine concentrations [47,62]; strategies designed to stimulate residual cells to regenerate acini and other parenchymal elements (ductal ligation) and infiltrate growth factor doses to boost salivary gland repair post-RT [63].

4. Growth factor and cytokine therapy: Somatomedin C is a hormone, similar to insulin in molecular structure, and actually is better known as IGF-1 or insulin-like growth factor 1 [64]. While a statement as “increased insulin-like growth factor signaling induces cell proliferation, survival and cancer progression” is true, it is traditional and partial, to a great extent. Today we understand that the issue is much more complex. For instance, IGF regulates cellular senescence which is known to halt the proliferation of aged and stressed cells and does play a key role in cancer development. Actually, there is accruing evidence that, over time, IGF not only regulates but also induces premature cellular senescence (tumor suppressor protein p53-dependant, in terms of acetylation, stabilization, and activation) [65]. Hence, despite the understandably-alarming, at first and for some, suggestion to exogenously administer/supply cytokines and growth factors to sites of cancer, recent years have indeed witnessed a note-worthy increase in the study of growth factors as cytoprotectants including their use as radioprotectors for salivary glands, and to reduce RT-induced symptoms, such as oral mucositis. To date, various growth factors have emerged as potential radioprotectors, including neurotrophic factors [66,67], epidermal growth factor (EGF), fibroblast growth factor (FGF) [68,69], keratinocyte growth factor (KGF) [70,71] and the afore-mentioned insulin-like growth factor-1 or IGF-1 [72-74]. Meyer, et al. [73], for example, investigated and determined the radioprotectant and therapeutic effect of IGF-1, in a murine model. They found that IGF-1 is mediated by the activation and maintenance of a histone deacetylase, specifically the Sirtuin 1 (SirT-1). Pre-treatment with IGF-1 enabled the repair of double-stranded breaks in the DNA of parotid salivary gland cells within the first hours post-irradiation, thereby allowing for optimal DNA repair (i.e. IGF-1 promotes DNA repair in irradiated parotid salivary glands via the maintenance and activation of SirT-1) to fulfill the cell cycle checkpoints. However, hours later and as early as 8h, RT-induced apoptotic cells were detected [73]. Such observations lead to further study of the signaling cross-talk between IGF-1 and SirT-1, thereby identifying several activators, stabilizers, and inhibitors, including the afore-mentioned inhibition of the p53-mediated apoptosis and the phosphoinositide 3-kinase (PI3K) – protein kinase B (Akt) pathway [65], in-depth study-worthy topics, beyond the scope of this concise review. To date, studies, collectively indicate that cytokines can be radioprotective, and anti-apoptotic and suggest/promote that the exogenous and localized (via a release-controlled delivery system, preferably directly injectable) utilization of growth factors do stimulate endogenous stem cell populations/niche and will eventually contribute to the desired and/or pursued clinical solution suitable for preventing RT-induced damage, diminishing salivary hypo-function, and restoring salivary gland function in RT- HNC cases.

5. Gene transfer therapy: The utilization of gene transfer, DNA transmission, and cell transduction to produce high levels of transgenic protein in order to correct cellular dysfunction and/or induce a new cellular function, post-RT, is a wide area of investigation and development. Baum, et al. [75], utilized an adenoviral technique to transfer the Aquaporin-1 (AQP1) gene into the sub-mandibular gland, reporting an increase in salivary flow when compared to control viruses into a rat or mini-pig models [75,76]. Yet, key shortcomings continue to exist for non-viral as well as viral vectors [61], rendering translation for routine clinical use difficult. Likewise, the therapeutic potential of genetic modification and application of small-interfering RNAs or siRNA for the purpose of target gene silencing are intensively investigated, progressing from pre-clinical testing in animal models to ongoing clinical trials for cancer, lung disease, and liver damage in human subjects. Thus far, highly limited in salivary gland tissues and accompanied by significant safety concerns [50]. For example, AQP-1 gene transfer into the salivary glands via adenoviral vectors to treat disorders such as SS, yielded strong immune responses, mainly due to the limited or low efficiency of intra-cellular siRNA delivery [77,78]. Herein, similar to growth factors, cell therapy, and pharmaceutical agent administration, the availability of a reproducible, scalable, safe and effective, release-controlled carrier/vehicle suitable for therapeutic siRNA delivery, directly into the salivary gland, ensuring sufficient residency/retention, is a challenge.

Wnt/β-catenin pathway: radio-protective role and bio-effect in RT-induced salivary gland damage

In existing irradiation studies and radio-protection literature, numerous cellular signaling pathways and cell-cycle alteration mechanisms have been explored. Of those, the Wnt/β-catenin signaling pathway seems to receive the utmost attention, recently, towards preventing the damage caused by irradiation [86]. Briefly, this canonical Wingless–Int (Wnt) pathway leads to the accumulation and translocation of co-activator β-catenin, a multi-functional protein involved in cell-cell adhesion, gene transcription, and physiologic homeostasis (adult), into the nucleus, via a series of molecular events initiated through the binding of specific Wnt proteins to the frizzled receptors on the cell surface. The pathway plays a critical role in cell regulating cell migration and determining cell fate, and mutations have been linked to human birth defects, cancer, and other disorders and diseases [79-83]. Activating the canonical Wnt/β-catenin signaling pathway is complex. It depends on a family of glycoproteins involved in cell-to-cell communication. To simplify, the interaction of ß-catenin with the cell adhesion molecule, e-cadherin, is involved in phenotypes: adhesion, mobility, and proliferation [80,81]. In absence of a Wnt ligand, β-catenin is degraded by the “destruction complex”. Several proteins are involved within this complex whereby Axin acts as a scaffold protein facilitating the interaction of Glycogen Synthase Kinase 3β (GSK-3β), Adenomatous Polyposis Coli (APC), and Casein Kinase 1α (CK1α), for β-catenin phosphorylation [82, 83]. Then, phosphorylated β-catenin is recognized by the β-transducin-repeat-containing protein (β-TrCP) and goes through the ubiquitin-proteasome degradation pathway. When the Wnt ligand activates Wnt signaling through the plasmatic membrane receptor frizzled with other lipoprotein receptors, the cytoplasmic protein disheveled (Dvl) is recruited and thereby activated. Herein, the activation of Dvl disrupts the “destruction complex” by dissociation of the GSK-3β from the Axin and inhibits the GSK-3β. As a result, β-catenin phosphorylation is also inhibited, allowing stabilization and translocation of β-catenin into the nucleus. Nuclear β-catenin then binds to a transcription factor-T cell factor and a lymphoid-enhancing factor (Tcf/Lef) and finally activates a response, i.e. changes in gene expression [79,84,85]. The Wnt signaling pathway cross-talks with other signaling pathways and can be modulated by several activators and inhibitors. For example, the utilization of growth factors, to activate or inhibit, has been extensively studied, further adding to the complexity given the wide range of involved genes [86]. Cross-talk between signaling pathways is possible via the common regulatory protein GSK-3β. For example, when the epidermal growth factor (EGF) is recognized by its native receptor (EGF-R), this complex activates the aforementioned phosphoinositide 3-kinase (PI3K) which facilitates the activation of AKT kinase regulator. Herein, the activation of AKT results in the inhibition of GSK-3β by phosphorylation [87-89] and ultimately leads to the translocation of β-catenin into the nucleus. On the other hand, the fibroblast growth factor (FGF) is also able to cross-talk with GSK-3β (common pathway with EGF) and the activation of its native receptor (FGF-R) is followed by PI3K which then results in the inhibition of GSK-3β via AKT activation [84,90]. Herein, FGF-R activation also involves MapK activation which inhibits GSK-3β through the p90 ribosomal protein s6 kinase (p90rsk) in an AKT-independent manner [91-93]. Therefore, activating the Wnt signaling pathway (Figure 4) through the utilization of cytoplasmic regulatory proteins (from other signaling pathways) is potentially able to promote β-catenin stabilization, its translocation to the nucleus, and the activation of survival genes [94]. Such understanding and revelations can lead to producing a plausible and innovative alternative strategy for the activation of native repair systems that may allow and promote the survival of the cells during and after RT. Possibly, can be even extended to explore the plausibility of prevention. To the best of my knowledge, Hakim, et al. [95] conducted one of the first/earliest clinical studies connecting signaling pathways (Wnt/β-catenin and TGF-β) with salivary gland irradiation damage. They reported an alteration in the expression pattern of Wnt1 in viable irradiated acinar cells of xerostomia patients, suggesting a possible therapeutic effect of the Wnt pathway in controlling RT-induced salivary gland damage and dysfunction [95], in accordance with previous in vitro studies [79]. Following this line of research, Hai, et al. [96] carried out a study analyzing the transient activation of the Wnt/β-catenin signaling pathway to prevent irradiation damage to the salivary glands. They reported, using a murine model, that activating the Wnt/β-catenin pathway through the transient activation of Wnt1 in the basal epithelium helped to prevent chronic salivary dysfunction generated by local irradiation, specifically via suppressing apoptosis and preserving or rescuing the life of salivary stem/progenitor cells. Salivation in experimental mice when compared to controls (animals receiving only RT) was increased/higher [79,96]. However, the radioprotective effect of Wnt/β-catenin activation seems, thus far, to only occur within a limited time lapse. Activating the signaling path 3 days before or 3 days after irradiation yielded dissimilar effects on the tissues [96]. Indeed, in another approach, the activation and modulation of a cell signaling pathway(s) using a cocktail (more than one) of activators has been suggested, with the Wnt signaling pathway (and its components) as a therapeutic target(s). Thula, et al. [69] evaluated the effect of EGF and bFGF (basic FGF) in salivary gland explants, reporting promising results regarding gland radioprotection [69]. Overall, taking the studied findings into account, it can be proposed that a Wnt/β-catenin signaling pathway activator might be a good candidate to be developed as a potential preventive and therapeutic strategy against RT-induced salivary gland damage in HNC patients. Herein, as was and is the present scenario with distinctive cells, proteins, cytokines, genes, growth factors, and drugs, a suitable/ideal delivery vehicle is once more, deemed vital.

Closing expert remarks

Technology promise in translational tissue engineering and nanomedicine: The interplay between tissue engineering, regenerative medicine, biomaterials, bio-nanotechnology, and nanomedicine continues to be the hallmark of current scientific research Worldwide, promising to change every aspect of human life via creating revolutionary materials of biological origin for use in the diagnosis and treatment of devastating human diseases; a multi-disciplinary approach to innovative and translational solutions, suitable for scale-up, safe, efficacious and cost-effective routine clinical use [97-99]. Whether conventional small-molecule agents or emerging protein and/or peptide-based macromolecular biopharmaceutics, the therapeutic effect is of vital significance. Controlled or at least predictable delivery is also substantially necessary. An intense effort is invested into engineering such complex bio-systems capable to achieve optimum cell-material interactions while keeping intact the material’s bulk properties. One of the core interests of nanobiotechnology, for example, this decade has been drug/gene/cell bio-functional delivery, driving the design and development of bio-inspired, intelligent, or “smart” nano-systems [97,98,100]. It can be stated that a competitive and superiorly successful delivery system should offer: therapeutic outcome enhancement, patient compliance improvement, and overall cost reduction of therapy. For HNC cases suffering RT-induced salivary gland damage and dysfunction, an attractive delivery system, for clinical ease-of-use, can perhaps entail a directly injectable formulation, sterilizable, capable of efficiently holding a dose-responsive bio-load, maintaining its bio-activity over time, and “predictably” control its pharmaco-kinetic release profile.

Oxidative stress and ROS in RT-induced salivary gland damage and dysfunction: Although the mechanism of RT-induced salivary gland injury has not yet been determined, as previously mentioned throughout this review, recent studies have shown that RT-induced salivary gland damage and dysfunction are associated with the mechanisms of Aquaporin and activated water channel (AQP1 and AQP5) [141], calcium signaling (G-protein-coupled receptors, Ca2+ and Ca2+i) [142], micro-vascular injury (reduction in micro-vascular density or MVD: IR-induced DNA damage initiates ceramide generation via the activation of mitochondrial ceramide synthase and the de novo synthesis of ceramide) [143], cellular senescence (p16 INK4a/Rb-dependent pathway induced by growth/differentiation factor-15) [144] and apoptosis (phosphorylated PKCδ, caspase-3 and activating/inducing p53 expression and transcription) [145], which are closely related to oxidative stress. Indeed, RT significantly increases the levels of reactive oxygen species or ROS, thereby causing oxidative stress in the salivary glands; critical issues to consider for prevention and treatment strategies.

Artificial intelligence in HNC: For implementing a better diagnostic capability, standardizing reporting, quantifying scoring, and automating the work-flow as well as effective communication between surgeons, radiologists, pharmacists, and pathologists (146-148), for example, computational methods to detect patterns, make therapeutic (response to treatment) and prognostic predictions (i.e. machine learning or ML) using algorithms, big medical imaging datasets and digital image processing (including simple light microscopy) applying multi-factor analysis, conventional logistic regression and Cox analyses [146-149], have been increasingly advancing and attracting multi- and inter-/intra-disciplinary collaborations in recent years [146, 149]. Indeed, such can be combined with traditional strategies and approaches to further evolve and improve the accuracy or precision of detecting/diagnosing head and neck pre-cancerous and cancerous lesions [146,148,149]. Further, retrospective and radiomic (ML texture analysis of images) data would be a valuable tool to help predict prognosis or the course of HNC and provide treatment [146,147,149]. Today, the R&D&I trend (Figure 5) seems to focus more on exceeding the capabilities of human judgment [149] via designing novel data fusion ‘deep learning’ algorithms [148-150] that essentially would merge radiology, histology, and molecular measurements [149], from large-scale multi-centric prospective studies, for far more precise clinical predictions [150].

Conflict of interest

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding and acknowledgment

This SPECIAL ISSUE work was supported by operating grants provided to the HAiDAR R&D&I LAB/BioMAT’X (Laboratorio de Biomaterials, Farmacéuticos y Bioingeniería de Tejidos Cráneo Máxilo-Facial), member of CiiB (Centro de Investigación e Innovación Biomédica), Faculties of Medicine and Dentistry, Universidad de los Andes, Santiago de Chile, through the ANID-NAM (Agencia Nacional de Investigación y Desarrollo, Chile and National Academy of Medicine, USA) Grant código # NAM21I0022 (2020-2022), CORFO Crea y Valida I+D+i Grant código # 21CVC2-183649 (2021-2023), CORFO Crea y Valida — Proyecto de I+D+i Colaborativo - Reactívate” Grant código # 22CVC2-218196 (2022-2024), and FONDEF Concurso IDEA de I+D, ANID, Grant código # ID22I10215 (2022-2024). The author wishes to acknowledge the exceptional F-ODO students behind inspiring this piece: Yr3 (Andrea Bustos, Ismael Valenzuela, and Zabdiel Faundez), Yr4 (Alondra Beniscelli) and Yr6 (Ignacio Fernández). Finally, thanks to Ms. Lisa P., Publishing Manager-Journals at Heighten Science Publications Inc, USA, and colleagues for their incessant help and support.

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