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Keywords:

  • radiotherapy;
  • hyposalivation;
  • xerostomia;
  • IMRT ;
  • gene transfer;
  • stem cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Radiation-induced hyposalivation
  5. IMRT
  6. Proton therapy
  7. Prevention of radiation damage with drugs
  8. Salivary gland transfer
  9. Gene transfer
  10. Stem cell therapy
  11. Conclusion
  12. Author contributions
  13. References

Radiation-induced hyposalivation is still a major problem after radiotherapy for head and neck cancer. Current and promising new thoughts to reduce or salvage radiation damage to salivary gland tissue are explored. The main cause underlying radiation-induced hyposalivation is a lack of functional saliva-producing acinar cells resulting from radiation-induced stem cell sterilization. Current methods to prevent that damage are radiation techniques to reduce radiation-injury to salivary gland tissue, surgical techniques to relocate salivary glands to a region receiving a lower cumulative radiation dose, and techniques to make salivary gland cells more resistant to radiation injury. These preventive techniques cannot be applied in all cases, also reduce tumor sensitivity, or do not result in a sufficient amelioration of the dryness-related complaints. Therefore, alternative methods on techniques to salvage salivary glands that are damaged by radiation are explored with promising results, such as stem cell therapies and gene transfer techniques to allow the radiation-injured salivary gland tissue to secrete water.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Radiation-induced hyposalivation
  5. IMRT
  6. Proton therapy
  7. Prevention of radiation damage with drugs
  8. Salivary gland transfer
  9. Gene transfer
  10. Stem cell therapy
  11. Conclusion
  12. Author contributions
  13. References

In 2012, in the USA, more than 40 000 new patients were diagnosed with head and neck cancer (American Cancer Society, 2012). Worldwide, head and neck cancer accounts for more than 550 000 cases annually (Jemal et al, 2011). The majority of these patients were treated with radiotherapy alone or in combination with chemotherapy, cetuximab and/or surgery. The 5-year survival rate of these patients is approximately 50% for non-metastatic locally advanced disease (Piccirillo et al, 2007). While radiotherapy significantly improves the patient's chances of survival, at the same time the exposure to ionizing radiation usually results in unavoidable co-irradiation of the normal tissues surrounding the tumor. The salivary glands are among these normal tissues as the ionizing beams have to pass the salivary glands to reach the tumor.

Protocols have been developed to minimize early and late loss of gland function following radiotherapy, but even when applying intensity-modulated radiation therapy (IMRT), the current evidence-based standard technique, commonly applied technique to irradiate head and neck cancer, still 40% of head and neck cancer patients experience a moderate or severe sensation of oral dryness (xerostomia) (Burlage et al, 2001; Malouf et al, 2003; Jellema et al, 2007; Jensen et al, 2010; Vissink et al, 2010; Beetz et al, 2013). In addition, induced by or related to the radiation injury to the salivary glands and consequential hyposalivation, many other post-treatment complications occur, such as hampered speech, increased risk on oral infections and dental caries, difficulties with swallowing and food mastication, impaired taste, and nocturnal oral discomfort. These symptoms can lead to a dramatic loss in quality of life for the patient and remains extremely difficult to manage (Jansma et al, 1992; Vissink et al, 2003a,b).

In this paper, first the pathophysiology underlying radiation damage to salivary tissue is be briefly reviewed. Next, currently explored and promising new thoughts to reduce or salvage radiation damage to salivary gland tissue are discussed.

Radiation-induced hyposalivation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Radiation-induced hyposalivation
  5. IMRT
  6. Proton therapy
  7. Prevention of radiation damage with drugs
  8. Salivary gland transfer
  9. Gene transfer
  10. Stem cell therapy
  11. Conclusion
  12. Author contributions
  13. References

The salivary glands of rodents and primates are composed basically of two saliva-producing cells types, namely mucous and serous acinar cells, of myoepithelial cells, which facilitate saliva expulsion, and of a ductal system which modifies saliva composition and through which saliva is secreted into the oral cavity (Figure 1). Saliva production is stimulated by intertwined cholinergic and adrenergic nerve fibers and indirectly by the blood vessels that supply the glands (Proctor and Carpenter, 2007).

image

Figure 1. Schematic representation of a generic primate salivary gland showing component cell types with likely stem and progenitor cell locations. The considered location of primitive stem cells, within larger excretory and striated ducts, and progenitor cells, within the striated and intercalated ducts, is shown. The stem cell pool supplies the progenitor cell pool, which in turn replenishes the population of functionally mature duct and acinar cell types. Both stem and progenitor cells have the capacity to self-renew and differentiate (from Pringle et al (2013), with permission)

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Saliva-producing acinar cells are largely postmitotic in nature and therefore according classical radiobiology theories not predicted to be very radiation sensitive (Vissink et al, 2010; Figure 2). By contrast, radiation to salivary gland tissues is followed by severe early (phases 1 and 2, 0–10 days and 10–60 days postradiotherapy, respectively) loss of salivary secretion, suggesting that salivary glands are more radiosensitive than anticipated (Coppes et al, 2001, 2002; Jensen et al, 2010; Vissink et al, 2010; Figure 3). Debate is still ongoing as to whether this observed early radiation-induced hyposalivation is attributable to apoptosis or to membrane damage-induced dysfunction of acinar cells (Abok et al, 1984; Stephens et al, 1989, 1991; Nagler et al, 1997; Zeilstra et al, 2000; Coppes et al, 2001; Konings et al, 2005a). Currently, treatments are explored to suppress radiation-induced apoptosis (Kojima et al, 2011b; Hai et al, 2012; Martin et al, 2012; see paragraph on preventive agents). The later phases of radiation-induced hyposalivation (phases 3 and 4, from 60 to 120 and 120 to 240 days after radiotherapy, respectively), wherein functionally mature acinar cells senesce and are not replenished with new ones, are currently suggested to be due to radiation-induced sterilization of salivary gland stem/progenitor cells (Denny et al, 1993, 1997; Roesink et al, 1999; Coppes et al, 2001; Man et al, 2001; Takahashi et al, 2001; Ihrler et al, 2002; Konings et al, 2005a, 2006; Lombaert et al, 2008a; Figure 1). Stem and progenitor cells are characterized by their self-renewal and differentiation capabilities, replenish damaged cells, and have been identified in many rodent and human tissues (Coppes and Stokman, 2011; Pringle et al, 2013). Thus, the number of remaining undamaged stem/progenitor cells will determine the regenerative capacity of the gland after irradiation. Recovery and compensatory responses in non-irradiated regions have been observed after radiation (Braam et al, 2005; Konings et al, 2006), suggesting compensatory responses of potential surviving stem/progenitors cells to regenerate salivary gland tissue (van Luijk et al, 2009).

image

Figure 2. On the basis of the slow turnover rates of their cells, salivary glands are expected to be late responding, but the changes in quantity and composition of saliva already occur shortly after radiotherapy (red circle). This resembles an immediacy of a radiation response (short latent interval) that is normally observed for cells with a higher labeling index (such as intestine). However, when one looks at the actual kill of acinar cells, the curve behaves just like any other late-responding tissue (gray circle). (Adapted from Stewart and Van der Kogel (2002) and Vissink et al (2010), with permission)

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image

Figure 3. Flow rate of 2% citric acid-stimulated parotid (single gland) and bilateral submandibular–sublingual (SM/SL) saliva as a function of time after start of radiotherapy (XRT: conventional radiotherapy: both parotid, submandibular, and sublingual glands located in the treatment portal, 2 Gy per day, 5 days per week, total dose 60–70 Gy; parotids/SM/SL IMRT: parotid/SM/SL sparing three-dimensional/IMRT, that is, bilateral [the majority] and unilateral radiotherapy [scattered radiation to contralateral gland]. For parotid IMRT data: 1.8–2.0 Gy per fraction, prescribed dose to primary target 64 Gy (range, 57.6–72 Gy); and for SM/SL IMRT data: 2 Gy per day, 70 Gy to gross disease planning target volume). Initial flow rates are set to 100% [modified after Jensen et al (2010) and Vissink et al (2010)]

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IMRT

  1. Top of page
  2. Abstract
  3. Introduction
  4. Radiation-induced hyposalivation
  5. IMRT
  6. Proton therapy
  7. Prevention of radiation damage with drugs
  8. Salivary gland transfer
  9. Gene transfer
  10. Stem cell therapy
  11. Conclusion
  12. Author contributions
  13. References

Compared with conventional radiotherapy, IMRT allows a more accurate and specific delivery of radiation dose to the tumor and allows for the possibility of better sparing of surrounding tissues, for example major salivary glands (Jensen et al, 2010; Vissink et al, 2010; Nutting et al, 2011; Figure 3). Recently, the Quantitative Analyses of Normal Tissue Effects in the Clinic (QUANTEC) Group proposed practical guidelines to reduce the toxicity risk based on dose constraints to be used in IMRT treatment planning (Deasy et al, 2010). More specifically, the QUANTEC Group concluded that severe xerostomia, defined as long-term stimulated salivary flow <25% of baseline, can be reduced if at least one parotid gland is spared with a mean dose of less than 20 Gy or if both glands are spared with a mean dose of less than <25 Gy (Deasy et al, 2010). Recently, Nutting et al (2011) showed in a randomized controlled trial that parotid sparing IMRT indeed significantly reduces the risk of severe hyposalivation as compared to 3D-CRT. Thus, IMRT is currently recommended as the standard radiation delivery technique in head and neck cancer to limit the cumulated radiation dose to the parotid gland. The problem, however, is that the QUANTEC criteria cannot be met in many patients. Beetz et al (2013) showed that the QUANTEC criteria worked well to prevent moderate-to-severe xerostomia in the long term, but that these criteria could only be met in a minority of patients and thus even after IMRT, still about 40% of patients complain of xerostomia.

The sparing effect of IMRT on normal tissue might be affected by the subtolerance doses that regions adjacent to a region that are exposed to the high-dose receive, the so-called bath-and-shower effect. Indeed, in a rat model, it was shown that such dose bath to parotid gland region resulted in enhanced function loss (van Luijk et al, 2009). This bath-and-shower effect may explain the less-than-expected sparing of salivary gland function after IMRT. In other words, as modern IMRT can control the dose in sufficient detail to allow sparing of specific subvolumes of a salivary gland, knowledge on mechanisms and the role of dose to these substructures would facilitate specific sparing of the most critical substructures of the gland, potentially reducing radiotherapy-induced hyposalivation. It is presumed that identifying these critical substructures is essential to make IMRT more effective to reduce radiation damage to salivary glands. As a first step, in a series of preclinical studies, it was found that inclusion of the area containing the main excretory ducts in the radiation field aggravated parotid gland dysfunction (Konings et al, 2005b, 2006; van Luijk et al, 2009). Interestingly, it has been suggested that these ducts contain the salivary gland stem cells (Pringle et al, 2013). Therefore, these ducts may represent a critical structure that needs to be spared using IMRT to reduce postradiation hyposalivation.

Next to this, blood vessels will need to be salvaged to allow for less functional damage to salivary glands. It was shown in mice that injury to the adjacent microvasculature may play an important role in radiation damage to salivary glands (Cotrim et al, 2007). In miniature pigs, single-dose irradiation resulted in significant reduction of microvascular density and local flow rate of blood in parotid glands, which indicated marked damage to microvascular endothelial cells (Xu et al, 2010). Furthermore, it was shown in mice that postirradiation mobilization of bone marrow stem cells facilitates regeneration of the submandibular gland and ameliorates vascular damage (Lombaert et al, 2008b). The latter was presumed to be partly due to bone marrow-derived endothelial progenitor cells differentiating into vascular cells and the direct stimulation of proliferation of existing blood vessel cells. Next, Sumita et al (2011) showed that bone marrow transplant indeed resulted in increased level of tissue regenerative activity such as blood vessel formation and cell proliferation in irradiated salivary glands in mice leading to a higher salivary output.

Proton therapy

  1. Top of page
  2. Abstract
  3. Introduction
  4. Radiation-induced hyposalivation
  5. IMRT
  6. Proton therapy
  7. Prevention of radiation damage with drugs
  8. Salivary gland transfer
  9. Gene transfer
  10. Stem cell therapy
  11. Conclusion
  12. Author contributions
  13. References

Another method to improve sparing of salivary gland tissue is the use of radiation techniques using charged particles, for example protons instead of the currently used photons. The physical properties of protons allow a superior dose distribution as compared to current photon radiotherapy, thereby minimizing the dose to normal tissues and significantly reducing acute and late side effects (van de Water et al, 2011a; Figure 4). Using risk models for xerostomia, a reduction in the risk of side effects of up to 70% is expected (van de Water et al, 2011b). In addition, it has been shown that with reduced spot-size intensity-modulated proton therapy improved dose sparing of the salivary glands can be obtained (van de Water et al, 2012), which is expected to result in a further reduction in severe xerostomia (Langendijk et al, 2013). Interestingly, applying reduced spot intensity-modulated proton therapy will enable an even better sparing of the main excretory ductal area, allowing improved postirradiation regeneration, and could result in a further reduction in the radiation-related loss of salivary gland function.

image

Figure 4. The benefit of protons compared with photons. The green curve represents the dose distribution of irradiation the target area (tumor) when applying photons, showing a rather high cumulative dose in the normal tissues in front of the target area and also a radiation load to the normal tissue located behind the target area. When applying protons (blue curve), both the cumulative dose to the normal tissues in front of and behind the target area is reduced

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Prevention of radiation damage with drugs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Radiation-induced hyposalivation
  5. IMRT
  6. Proton therapy
  7. Prevention of radiation damage with drugs
  8. Salivary gland transfer
  9. Gene transfer
  10. Stem cell therapy
  11. Conclusion
  12. Author contributions
  13. References

A number of drugs have been used to reduce the sensitivity of salivary glands to radiation among which are radical scavengers (amifostine, tempol), sialogogues (pilocarpine), suppressants of apoptosis (insulin growth factor 1, keratinocyte growth factor), and agents enhancing survival and proliferation of progenitor cells and expansion of ductal and acinar cells (pilocarpine, insulin growth factor 1, keratinocyte growth factor) (Jensen et al, 2010; Vissink et al, 2010). More recently, roscovitine, a cyclin-dependent kinase inhibitor that acts to transiently inhibit cell cycle progression and allow for DNA repair in damaged tissues, has been shown to have potential to preserve normal salivary function following targeted head and neck irradiation (Martin et al, 2012). Roscovotine induces transient G2/M cell cycle arrest allowing for suppression of apoptosis. Also, basic fibroblast growth factor was shown to prevent salivary gland dysfunction after irradiation. Again, this effect was attributed to the inhibition of radiation-induced apoptosis in secretory salivary gland tissue and the paracrine effect for this factor on these tissues in the salivary glands that received this treatment (Kojima et al, 2011b). It also was proposed that concurrent transient activation of the Wnt/b-catenin pathway could prevent radiation-induced salivary gland dysfunction (Hai et al, 2012). Other authors have shown, however, that Wnt/ß-catenin signaling may lead to radioresistance of cancer stem cells too (Wang et al, 2013). This is also a concern of many of the other substances tested to prevent radiation damage.

Salivary gland transfer

  1. Top of page
  2. Abstract
  3. Introduction
  4. Radiation-induced hyposalivation
  5. IMRT
  6. Proton therapy
  7. Prevention of radiation damage with drugs
  8. Salivary gland transfer
  9. Gene transfer
  10. Stem cell therapy
  11. Conclusion
  12. Author contributions
  13. References

Salivary gland transfer has the potential to prevent radiation-induced hyposalivation and xerostomia as not-irradiating a salivary gland is the best prophylaxis against radiation injury to salivary gland tissue. Recently, a number of studies has been published showing the potential benefit of relocating the submandibular salivary gland to a position minimizing the radiation dose to this tissue (Jha et al, 2009, 2012; Liu et al, 2011; Okoturo et al, 2012; Rieger et al, 2012; Zhang et al, 2012; Sood et al, 2014). The salivary gland transfer method of Jha et al (2000), which is performed in patients with clinically negative cervical lymph nodes, is performed by mobilizing the submandibular gland, cutting and ligating the facial artery and vein, and preserving the retrograde blood flow to the submandibular gland through the distal facial vessels. The mylohyoid muscle is cut to allow for repositioning of the submandibular duct and submandibular ganglion. The mobilized gland is then repositioned in the submental space with the distal facial vessels as a pedicle under the anterior belly of the digastric. Importantly, suspicious nodes in the level I zone (submental and submandibular) are sent for frozen section biopsy and when affected by cancer, the salivary gland transfer is abandoned, and a formal neck dissection is performed. The gland is anchored in place with absorbable sutures, and the posterior and inferior borders are marked with titanium miniclips to ease identifying the gland during radiotherapy planning. Skilled surgeons are needed to perform this approach. Even more important is a proper selection of patients in whom this approach is a reasonable option. Eligible patients are patients with a negative neck and in whom it is possible to plan the radiation schedule such that the translocated salivary gland is exposed to no or a radiation dose far below the tolerance level of that gland.

Salivary gland transfer is not without complications. The most common complications are ipsilateral facial edema (13.6%), neck numbness (6.8%), bleeding/hematoma formation 4.5%, wound infection (4.5%), shoulder weakness (4.5%), ipsilateral neck numbness (4.5%), hypoglossal nerve injury (2.5%), lingual nerve injury (2.5%), gland positional movement (2.5%), and cerebral embolism/cerebrovascular accident (2.5%) (Sood et al, 2014). The procedure was shown to be oncologically safe (Okoturo et al, 2012; Sood et al, 2014).

Preservation of the secretion of the (sero) mucous glands is thought to be the most valuable option to reduce oral dryness as these glands contribute to both the resting and stimulated salivary secretion. Therefore, prevention radiation-induced loss of the submandibular salivary glands, the major contributor to (sero) mucous secretion, is considered essential. Indeed, salivary gland transfer was shown to be very effective in eligible patients to reduce both the sensation of oral dryness and increasing (un)stimulated salivary flow (Jha et al, 2009; Liu et al, 2011; Jha et al, 2012; Rieger et al, 2012; Zhang et al, 2012; Sood et al, 2014; Figure 5). In the 177 patients at mean follow-up of 22.7 months reviewed by Sood et al (2014), xerostomia was prevented in 82.7% (95% CI, 76.6–87.7%) of patients. Unstimulated and stimulated salivary flow rates rose to 88% and 76% of baseline values, respectively, compared with subjects in whom it was attempted to reduce radiation damage to salivary gland tissue by peri- and postradiotherapy administration of pilocarpine (Scarantino et al, 2006; Burlage et al, 2008; Sood et al, 2014).

image

Figure 5. Salivary gland transfer results in a better preservation of salivary gland function postradiatherapy. SGT: whole saliva flow in patients subjected to salivary gland transfer followed by conventional/IMRT; control: whole saliva flow in conventionally/IMRT treated patients (modified after Sood et al, 2014)

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Gene transfer

  1. Top of page
  2. Abstract
  3. Introduction
  4. Radiation-induced hyposalivation
  5. IMRT
  6. Proton therapy
  7. Prevention of radiation damage with drugs
  8. Salivary gland transfer
  9. Gene transfer
  10. Stem cell therapy
  11. Conclusion
  12. Author contributions
  13. References

Gene therapy is considered a therapeutic option for radiation-induced salivary hypofunction in patients that are suffering from radiation-induced salivary gland dysfunction. This method might be applicable in future for selected patient cohorts. Transfer of genes into cells can be accomplished using viral and non-viral vectors. Currently, viral vectors provide the most efficient means for gene transfer, but they raise safety concerns including, in particular, the risk of insertional mutagenesis and the possibility that their manufacture or use will generate replication-competent viruses. Additionally, viral vectors can elicit immune responses, which can be marked and prevent the repeated use of the product (Samuni and Baum, 2011).

The delivery of DNA via non-viral vectors poses less of a safety risk, but is inefficient in transducing mammalian cells. Methods for non-viral gene delivery use physical and chemical approaches to improve efficacy and cell specificity. For salivary glands, non-viral vectors are seldom employed, while Ad5 or serotype-2 adeno-associated viral (AAV2) vectors are most often used (Samuni and Baum, 2011). Currently, research is directed toward nanoparticle delivery of siRNAs targeting a proapoptotic gene (Arany et al, 2012, 2013) and adenoviral delivery of Tousled kinase (Palaniyandi et al, 2011). Both methods have been suggested as effective means to provide radioprotection to salivary glands.

As animal experiments with adenoviral-mediated aquaporin-1 cDNA (AdhAQP1) transfer were promising (Wang et al, 2000; Li et al, 2004; Shan et al, 2005; Voutetakis et al, 2008), this viral vector was administered to a single previously irradiated parotid gland in 11 subjects (Baum et al, 2012). The design was an open label, single-dose, dose-escalation study (AdhAQP1 vector; four dose titers from 4.8 × 107 to 5.8 × 109 vector particles per gland). All subjects tolerated vector delivery and study procedures well over the 42-day study period reported. No serious adverse events or dose-limiting toxicities occurred. Objective responses were seen in six of these 11 subjects, none of these subjects had received the highest dose. Five of these six subjects also experienced subjective improvement in xerostomia, while four of five non-responders did not perceive amelioration or worsening of their oral dryness. In one non-responder, oral dryness improved, which was not accompanied by increased parotid flow (Figure 6). Remarkably, the positive responses in responders did not follow a time course predicted from previous AdhAQP1 vector studies with mice, rats, miniature pigs, and macaques (Wang et al, 2000; Li et al, 2004; Shan et al, 2005; Voutetakis et al, 2008). Generally, those studies showed peak transgene expression times 24–72 h after vector delivery, with biological responses occurring shortly thereafter. Among the six responders, the time at which peak elevations in parotid salivary flow occurred was quite variable and much later than in animal studies (Baum et al, 2012; Figure 6). It is yet unknown how long the increase in salivary flow will last in human.

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Figure 6. Summary of clinical response data. Clinical responses following vector delivery as measured by (a) absolute parotid salivary flow rate from the targeted gland and (b) the proportional increase in peak parotid salivary flow shown as the percent of baseline. Individual changes in parotid salivary flow are shown (c) for absolute salivary flow rates and (d) for proportional changes compared with baseline. Coding for individual subjects is shown as indicated in the Inset in c. All subjects shown in black were considered non-responders (<50% increase in salivary flow rate). All subjects shown in colors were considered responders (at least 50% increase in parotid salivary flow rate following AdhAQP1 administration). The days indicated to the right of each peak data point correspond to the days on which that peak parotid flow rate was observed. Visual analog scale (VAS) results from all subjects, at baseline and peak time of parotid salivary flow, are shown for both the amount of saliva perceived (e) (rate how much saliva is in your mouth) and dryness of their mouth (f) (rate the dryness in your mouth). Note that lower VAS results indicate an improvement in symptoms. The colors and symbols used to identify individual subjects are identical to those shown in c (from Baum et al (2012), with permission)

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Stem cell therapy

  1. Top of page
  2. Abstract
  3. Introduction
  4. Radiation-induced hyposalivation
  5. IMRT
  6. Proton therapy
  7. Prevention of radiation damage with drugs
  8. Salivary gland transfer
  9. Gene transfer
  10. Stem cell therapy
  11. Conclusion
  12. Author contributions
  13. References

Another potential method to salvage salivary glands that are damaged by radiation is stem cell therapy. Preclinical studies have shown that stem/progenitor cell transplantation not only rescues hyposalivation (Lombaert et al, 2008a), but also restores tissue homeostasis of the irradiated gland, necessary for long-term maintenance of adult tissue (Nanduri et al, 2013).

Stem/progenitor cells might be harvested from salivary glands before start of radiotherapy and be returned to the salivary complex after radiotherapy has been completed. These salivary stem/progenitor cells might then repopulate the damaged salivary glands. A proper place to deliver the stem/progenitor cells is probably the ductal complex as ducts usually better survive radiation treatment than acini. Alternatively, adipose-derived stromal cells (Kojima et al, 2011a), bone marrow-derived clonal mesenchymal stem cells (Lim et al, 2013), and human amniotic epithelial cells (Zhang et al, 2013) have been applied in rodents with some success to salvage radiation damage to salivary gland tissue.

Adult salivary gland stem/progenitor cells

As mentioned earlier, proliferating cells in the salivary gland are localized mainly in the excretory and intercalated ducts (Ball, 1974; Denny et al, 1993, 1997; Man et al, 2001; Takahashi et al, 2001; Ihrler et al, 2002; Pringle et al, 2013; Figure 1). Furthermore, ligating the major excretory duct of the salivary glands, creating a dysfunctional/apoptotic acinar cell environment, results in the proliferation of intercalated and excretory duct cells (Denny et al, 1993; Man et al, 1995; Takahashi et al, 2004a,b; Osailan et al, 2006a,b; Cotroneo et al, 2008, 2010; Katsumata et al, 2009). As a result, the initial functional ablation in ligated glands can be rescued after deligation through proliferation and suggested differentiation of these ductal cells, and saliva flow will return rather rapidly to pretreatment levels (Man et al, 2001; Takahashi et al, 2004a; Katsumata et al, 2009; Osailan et al, 2006a). Although acinar cells themselves seem to display a limited degree of proliferative ability, it is unlikely that rescue of function following the total ablation of acinar cell function in ligation experiments is due to acinar cell proliferation only (Man et al, 2001; Takahashi et al, 2004a,b; Osailan et al, 2006a; Cotroneo et al, 2008; Katsumata et al, 2009; Cotroneo et al, 2010). Therefore, it is presumed that cells capable of proliferation and differentiation reside within the ducts of the salivary glands and may represent a potent salivary gland stem/progenitor cell population (Pringle et al, 2013). It also was shown that these putative stem/progenitor cells are responsive to growth factor-mediated stimulation, whereby radiation-induced hyposalivation was rescued. However, this is dependent on the number of surviving stem cells as reducing the number of surviving putative stem/progenitor cells by increasing the radiation dose prevented rescuing salivary gland function (Lombaert et al, 2006, 2008a,b). These data emphasized the importance of a functional residual salivary gland stem/progenitor cell population for hyposalivation recovery. Therefore, the next step in developing this approach to salvage salivary glands is characterization of the most potent stem/progenitor cell population and being able to expand these cells in vitro.

Rodent salivary gland stem/progenitor cells

Numerous studies have demonstrated that in vitro culture of processed salivary gland tissue is possible (Kishi et al, 2006; David et al, 2008; Pringle et al, 2013). Among others a non-adherent method for culturing potential murine salivary gland stem/progenitor cells was developed (Lombaert et al, 2008a; Nanduri et al, 2011; Pringle et al, 2011). With this method, after mechanical and enzymatic digestion, aggregates of cells cultured in suspension (salispheres), increased in size over time in culture, and contained proliferating cells (Lombaert et al, 2008a; Pringle et al, 2011). These salispheres were also found to express the adult stem cell marker proteins CD117, CD24, CD29, CD49f, Sca-1, Mushashi-1, CD44, CD90, and CD34, which are also present in ducts of naïve salivary glands (Lombaert et al, 2008a; Nanduri et al, 2011; Banh et al, 2011; Figure 1). Furthermore, it was observed that CD117 expression in 3-day cultured salispheres (>0.6%) was markedly higher than that immediately following salisphere isolation (<0.01%), suggesting that salisphere culture represents a form of lineage selection. On the basis of these observations, it was hypothesized that particularly ductal-like cells are promising candidates for enrichment for stem cells prior to therapeutic use (Feng and Coppes, 2008; Lombaert et al, 2008a; Banh et al, 2011; Nanduri et al, 2011; Pringle et al, 2013).

The first evidence of ductal-like stem/progenitor cells functionality in vivo was reported from studies in which donor cells isolated from salisphere cultures were transplanted back into irradiated recipient murine glands (Feng and Coppes, 2008; Lombaert et al, 2008a; Nanduri et al, 2011). Functional integration and differentiation led to recovery of salivary gland function of 70% of the transplanted animals and was achieved with as few as 100–300 c-Kit+ stem/progenitor cells from primary salispheres (Lombaert et al, 2008a). Furthermore, it was shown that stem/progenitor cell transplantation not only rescues hyposalivation, but also restores tissue homeostasis of the irradiated gland, necessary for long-term maintenance of adult tissue (Nanduri et al, 2013).

Human salivary gland stem/progenitor cells

Preliminary data have suggested that salisphere-based culture principles and employment of protein markers can be utilized in the study of human stem/progenitor cells (Feng and Coppes, 2008; Lombaert et al, 2008a; Feng et al, 2009; Banh et al, 2011). In this respect, human salisphere differentiation into three-dimensional organoid structures containing acinar and ductal-like regions was encouraging in terms of the potential differentiation capabilities of these cells to continue this search (Feng et al, 2009). Furthermore, it was shown that human salivary gland cells grown in monolayers also express a panel of stem cell-associated marker proteins (CD44, CD49f, CD24/CD49f, CD90, CD104, p75NGFR) (Pringle et al, 2013). Co-localization of two such markers, CD49f and CD90, in the periductal region of human salivary gland tissue was suggested to be evidence for a ductal location of hSSPCs (Sato et al, 2011; Palmon et al, 2012). Cells derived from human salispheres probably will also express multiple stem cell markers, but it is still necessary to show that these cells can self-renew and differentiate in vitro and in vivo (Pringle et al, 2013). Certainly, the search for the most potent stem cell containing populations has to continue.

The human stem/progenitor cell hierarchy might to a certain extent mirror what is observed in the mouse system (Feng et al, 2009; Pringle et al, 2013). Moreover, due to the relatively long-turnover time of salivary gland tissue, a cocktail of stem and progenitor cells will most likely provide optimal (fast and sustained) salivary gland recovery. In that scenario, short-term recovery may result from the progenitors within the graft and long-term sustained improvement from the stem cells. A definitive minimal stem/progenitor cell number required for salivary gland rescue is still unknown and is likely to differ depending on, for instance, patient age and extent of irradiation (Feng et al, 2009; Pringle et al, 2013). Furthermore, due to the lobular nature of the salivary glands, the exact localization of injected stem/progenitor cells cannot be guaranteed. Unpublished data from our group using rats suggest that retrograde injection of stem/progenitor cell solutions directly into the opening of the submandibular or parotid glands might be used to control transplantation direction and efficacy, while other studies suggest that echo guidance may also be useful to overcome this problem (Jongerius et al, 2003; Passineau et al, 2010).

Aside from the delivery of the cells, various facets of the culture systems must be further optimized before this technique can be applied in human. In salisphere culture, for example, the majority of culture components are now compliant to current Good Manufacturing Practice (GMP) regulations. Moreover, GMP-compliant selection of human stem/progenitor cells from monolayer or salisphere cultures should be achievable using MACS and cGMP-approved antibodies (Palmon et al, 2012). Next to this, human stem/progenitor cells will need to be cultured briefly and then undergo cryopreservation until the desired time point during the 7- to 10-week period of radiotherapy. Cryopreservation is already possible using GMP-approved reagents, and the preserved function of CD24+ CD49f+ putative rat stem/progenitor cells frozen for 3 years has been documented (Neumann et al, 2012). Once thawed, these stem/progenitor cells demonstrated equal and in some cases better proliferative ability and expression of differentiation markers compared to their non-cryopreserved counterparts (Neumann et al, 2012). Parallel safety experiments using human stem/progenitor cells remain to be performed, to provide the equivalent functional guarantee for patients awaiting transplantation.

Regretfully, most of the patients are of old age and have been suggested to respond even more dramatically to the deleterious effects of radiation on the salivary glands (Beetz et al, 2012). Moreover, we observed a reduction in salisphere-forming capability of cells from salivary glands of mice of old age (Feng et al, 2009). These issues challenge the application of this technique in elderly. These challenges, in combination with the fact that often only a small salivary gland biopsy may be obtained prior to the radiotherapy, make it essential to expand the number of stem/progenitor cells before transplantation. It is therefore of eminent importance to find protocols that safely permit this. Current in vitro culture, self-renewal and differentiation assays for stem/progenitor cells open new possibilities for the screening of novel factors and genes that may be useful tools for stem/progenitor cell amplification. Applying adult stem cell therapy in a clinical situation may dramatically improve the quality of life of patients after anticancer treatments.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Radiation-induced hyposalivation
  5. IMRT
  6. Proton therapy
  7. Prevention of radiation damage with drugs
  8. Salivary gland transfer
  9. Gene transfer
  10. Stem cell therapy
  11. Conclusion
  12. Author contributions
  13. References

In conclusion, the best option is to preserve the stem cell compartment to allow for the best repair of radiation damage to salivary gland tissue by intensity modulating techniques applying either photons or protons. Alternatively, relocation of the submandibular gland outside the radiation treatment portal can be considered in eligible patients. When the damage is beyond repair, there is a need for methods to salvage the radiation damage that has occurred. These methods are still in development and include (i) stem cell transplant techniques to restore the self-renewal potential of salivary gland tissue and (ii) restoring the water secretion capacity of the salivary glands by inducing water channels in the radiation damaged salivary glands by gene transfer. While stem cell transplant techniques to salvage radiation-induced salivary glands are still in the preclinical stage, a phase I trial has been performed with regard to gene transfer, which has shown some promising results.

Author contributions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Radiation-induced hyposalivation
  5. IMRT
  6. Proton therapy
  7. Prevention of radiation damage with drugs
  8. Salivary gland transfer
  9. Gene transfer
  10. Stem cell therapy
  11. Conclusion
  12. Author contributions
  13. References

Arjan Vissink compiled first draft of the paper and was responsible for all subsequent edits. Peter van Luijk and Hans Langendijk en Rob Coppes carefully reviewed all section of the paper and added valuable additions/comments/suggestions to all sections and the sections of their expertise in particular. All approved the final version of the paper.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Radiation-induced hyposalivation
  5. IMRT
  6. Proton therapy
  7. Prevention of radiation damage with drugs
  8. Salivary gland transfer
  9. Gene transfer
  10. Stem cell therapy
  11. Conclusion
  12. Author contributions
  13. References