Radiation-induced cellular senescence in salivary glands
Peng, Julie
DOI:
10.33612/diss.103407924
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Peng, J. (2019). Radiation-induced cellular senescence in salivary glands. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.103407924
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Radiation-induced cellular
senescence in salivary glands
Xiaohong Peng
Peng, X.
Radiation-induced cellular senescence in salivary glands
PhD dissertation, University of Groningen, Groningen, The Netherlands
Printing of this thesis was financially supported by the Graduate School of
Medical Sciences, University of Groningen.
Cover:
Xiaohong Peng
Layout:
Xiaohong Peng, Peisen Su
Printed by:
Ridderprint, www.ridderprint.nl
ISBN electronic version: ISBN 978-94-6375-695-2
ISBN printed version: ISBN
Copyright © 2019, Peng. X, The Netherlands
All rights reserved. No part of this thesis may be reproduced, stored in a
retrieval system, or transmitted in any form or by any means, mechanically,
by photocopying, recording, or otherwise without prior written permission
of the author.
Radiation-induced cellular
senescence in salivary glands
PhD thesis
to obtain the degree of PhD at the
University of Groningen
on the authority of the
Rector Magnificus Prof. C. Wijmenga
and in accordance with
the decision by the College of Deans.
This thesis will be defended in public on
Wednesday 4 December 2019 at 11.00 hours
by
Xiaohong Peng
born on 02 March 1988
in Hubei, China
Promotor
Prof. R.P. Coppes
Co-promotor
Dr. L. Barazzuol
Assessment Committee
Prof. M.A.T.M. van Vugt
Prof. N. Cordes
Prof. M.A.G.G. Vooijs
Yi Wu
To my family
To my mother
Chapter 1
General introduction and thesis outline
9
Chapter 2
Role of glial-cell-derived neurotrophic factor in salivary
gland stem cell response to irradiation
Radiother Oncol. 2017; 124: 448-45441
Chapter 3
Cellular senescence contributes to radiation-induced
hyposalivation by affecting the stem and progenitor cell
niche
Manuscript submitted
65
Chapter 4
GDNF – a marker for radiation induced senescence in
salivary glands
Manuscript in preparation
93
Chapter 5
Differential cellular senescence response of salivary
gland organoids after low and high LET radiation
Manuscript in preparation as short communication
123
Chapter 6
Summary and future perspectives
139
Appendices Dutch summary
Acknowledgements
Curriculum vitae
List of publications
155
Chapter 1
General introduction and thesis outline
Radiation and cellular senescence
Cellular senescence is a multifaced process in which cells exit the cell cycle and irreversibly stop proliferation. Senescent cells undergo distinctive phenotypical alterations, including changes in morphology, chromatin, metabolism and secretome, the latter also known as senescence-associated secretory phenotype (SASP)[1-7].
Cellular senescence is considered to be a stress response which can be induced by a variety of intrinsic and extrinsic factors, such as DNA damage [8-10], telomere dysfunction [11], oncogene activation [12-14], mitochondrial dysfunction [15], reactive oxygen species [16], cell–cell fusion, as well as exposure to radiation [10] and cytotoxic drugs [3].
Senescent cells display a distinctive SASP, by which they secrete a combination of pro-inflammatory cytokines, chemokines, growth factors and extracellular matrix factors [17,18] that may result in a complex crosstalk with neighboring cells and surrounding tissues [3,17,19,20] interacting with multiple biological processes. These processes may have beneficial or detrimental effects depending on the specific composition of the SASP which can vary based on the cell type and the specific senescence triggering factor. Depending on the SASP content and other signaling cascade, such as those induced by ROS production [16], cell-cell interaction or exosome release [21,22], the roles of senescence are pleiotropic. Cellular senescence has an important role in physiological processes such as wound healing [23], tissue repair and regeneration [11,23-25], embryonic development [26,27]and aging [28]. However, cellular senescence also plays a deleterious role in processes such as tissue inflammation [12,29,30], fibrosis [31] and loss of tissue homeostasis [32-34].
Radiation, given as part of cancer treatment, is one of the main triggering factors of cellular senescence in both normal and cancer cells. Both high single doses
1
hours with a total dose of 10 Gy [9,10,37,38] can induce senescence. Cellularsenescence may act as a double-edged sword in tumorigenesis. On one side senescence-associated cell cycle arrest may act as a tumor suppressor [2,3,39] counteracting tumor development. On the other hand, given the effects of SASP in stimulating pre-neoplastic cells, senescence can also accelerate tumor relapse and increase the risk of metastasis [40]. Interestingly, radiation can also induce senescence in normal tissues [9,10,37,38]. This can be caused by the unwanted radiation dose to the normal tissue delivered during radiotherapy for cancer treatment. Moreover, the senescent cell bystander effect (also known as senescence-induced senescence) through gap junction-mediated cell-cell communication, metabolic alterations [41] or cytokine release plays an important role in radiation-induced senescence [42,43]. Radiation-induced normal tissue effects and cancer tissue senescence may significantly influence both the treatment outcomeand the patient’s quality of life.
Molecular pathways involved in radiation-induced
senescence
Senescence is triggered by different events and can lead to multiple changes in the nucleus and cytoplasm resulting in changes in multiple signaling pathways. These signals crosstalk within and between cells upon changes in the micro-environment. Cell intrinsic signaling pathways, which are involved in the cell state switch to senescence, are listed below (Fig. 1).
Cell cycle arrest pathway
After radiation exposure, the recognition of DNA damage and the subsequent activation of the DNA damage response (DDR) trigger a downstream signaling cascade. In the nucleus, the key mediators, the Ser/Thr protein kinase Ataxia Telangiectasia Mutated (ATM) and Rad3-related (ATR), phosphorylate the sensors and effectors of the DDR, such as CHK1/2, extracellular signal regulated kinases
(ERK) and P53 [44-46]. These phosphorylated effectors trigger the activation of the cell cycle blockers, Cyclin-dependent kinase 2 (CDK2) inhibitor p21 (CDKN1A) [47-49] and CDK4/6 inhibitor p16 [50], both of which arrest the cell cycle in the G1 phase [13,14,51,52]. It has been shown that p21 and p16 play different roles in the process of senescence. P21 is more important in the early stages of senescence, whereas p16 plays an important role in the long term maintenance of cell cycle arrest [53-55]. Radiation-induced DNA damage induces CHK1, Ser345 phosphorylation and prevents CDK1 activation by CDC25-mediated dephosphorylation [56], which blocks the cell cycle in the G2 phase.
DDR-regulated SASP pathway
Senescence-associated DDR signaling, genomic fragments and mTOR dependent protein synthesis profoundly affect secretion by senescent cells [8,10][57,58]. Persistent DNA damage signaling can initiate cytokine secretion, such as IL-6 [10]. Rodier et al. [10] showed that radiation-induced cytokine responses need DDR proteins like ATM, NBS1 and CHK2 but not p53 and pRb, which are known as the cell cycle arrest enforcers. P53 deficient cells can initiate the cytokine response without cell cycle arrest. Thus, the DDR controls both the p53-dependent cell cycle arrest and the senescence associated inflammatory secretion. GATA Binding Protein 4 (GATA4), a senescence and SASP regulator, when stabilized by the DDR proteins, ATM and ATR, mediates suppression of autophagy, which in turn can activate nuclear factor kappa light chain enhancer of activated B cells (NF-κB) to initiate the SASP and facilitate senescence itself [8]. One of the most abundant SASP factors, IL6, can reinforce the senescence associated G1 cell cycle arrest in a secretion-independent manner [11]. The SASP can also either suppress or promote the neighboring cells in a paracrine manner [59].
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cGAS-STING-regulated SASP pathway
DDR signaling drives only a subset of radiation-induced SASP factors. The Cyclic GMP-AMP synthase - stimulator of interferon genes (cGAS-STING) pathway mediates the other SASP factors including cytokines, chemokines and type I interferons (IFNs). Micronuclei [58] and cytoplasmic chromatin fragments (CCF) [11,60-62], arising from genome instability after exogenous DNA damage, can initiate a cGAS-STING-mediated SASP production. cGAS is a cytoplasmic double-stranded DNA sensor activated by double–stranded DNA damage. cGAS localized to micronuclei resulting from irradiation [58] gives rise to the production of a second messenger cGAMP which subsequently activates the STING [63]. STING subsequently upregulates interferon stimulated genes (ISGs) leading to type I IFNs (e.g. IFN-β and IFN-γ) and other inflammatory cytokines [58,64].
DNA damage and autophagy
Autophagy is essential to maintain cellular homeostasis by clearance of aged, damaged or misfolded proteins, which are recycled to sustain cellular metabolism [65-69]. Several studies showed that autophagy can be activated by DNA damage [70-72], in turn activation of autophagy contributes to DNA damage-induced senescence [70]. Recent studies showed that autophagy is needed for radiation-induced senescence in pituitary tumor-transforming 1 (PTTG1)-depleted cancer cells [73]. Autophagy can inhibit senescence by selective degrading GATA4 in a p62-dependent manner [8]. Conversely, radiation or oncogene-induced activation of ATM and ATR can block autophagic degradation of GATA4 which in turn activates the downstream NF-kB pathway contributing to SASP production [8]. This subsequently facilitates senescence via IL1 mediated cell cycle arrest [12] and paracrine senescence. In vivo data showed that GATA4 accumulates during mouse radiation-induced senescence, which could contribute to the senescence-associated inflammation. Moreover, it has been shown that autophagy promotes cellular senescence by facilitating the synthesis of SASP in a mTOR-dependent manner
within a cellular compartment called TOR-autophagy spatial coupling compartment (TASCC), to where the mTOR is recruited and accumulates during senescence for protein degradation and synthesis [57]. Degradation of the nuclear lamina by autophagy and subsequent formation of CCF can trigger SASP formation in a cGAS-STING-mediated manner [62,74].
Fig. 1. Molecular pathways controlling radiation-induced senescence. The DNA
damage response (DDR), evoked by radiation-induced DNA damage, leads to G1 cell cycle arrest via induction of CDK inhibitors p16 and p21 [14,75] and G2/M cell cycle arrest via CHK1-mediated CDC25 degradation [76]. Moreover, radiation induces autophagy which promotes radiation-induced senescence [73].The SASP is driven by the DDR through autophagy inhibition of GATA4 and subsequent NF-κB activation [8], as well as by cytoplasmic chromatin fragments (CCFs) [61,62], including microbial and self-DNA fragments, sensed by the cGAS-STING pathway which drives SASP by activating IFN-γ/β. SASP are produced in an m-TOR dependent manner [57]. Some cytokines, like IL6,
1
nuclear factor Κb; ERK, extracellular-signal-regulated kinase; CDK, Cyclin-dependent kinase; GATA4, GATA binding protein 4; IL6, Interleukin 6. cGAS, cGMP-AMP synthase; STING, stimulator of interferon genes; IFN-γ/β, Interferon gamma/beta; NK, Natural killer cells.
SASP
Senescent cells secrete a complex mixture of soluble and insoluble factors termed as senescence-associated secretory phenotype (SASP). The SASP consists of pro-inflammatory cytokines, chemokines, growth factors, proteases and extracellular matrix factors [77-80], which can have detrimental or beneficial functions depending on the cell type, the senescence inducing factor and the nature of the SASP (Fig. 2) [12,81,82]. Initiation and maintenance of SASP production requires ATM and its downstream NF-kB-dependent transcriptional program as the main regulators [10,79,82]. It has been demonstrated that radiation affects the surrounding normal tissue through SASP by AMP-activated protein kinase (AMPK) and NF-KB signaling pathways [41]. Recent data also showed that cytoplasmic chromatin fragments (CCFs) and its downstream cGAS-STING pathway also play a role in the maintenance of SASP production.
The SASP can reinforce the cell cycle arrest of its own senescent cells by an autocrine loop. This autocrine function can suppress the tumor growth and trigger the neighboring cells to undergo senescence in a paracrine manner, termed as paracrine senescence [16,30,83]. Paracrine senescence is triggered by SASP components through a mechanism that generates ROS and DNA damage [16,30,83]. This is reminiscent of the bystander effect of radiation by which irradiated cells influence non-irradiated neighboring cells through cellular gap junctions and by oxygen radicals and cytokines shown to be released in culture media [16,84]. Bystander responses have been observed in several cell types, like lymphocytes, fibroblasts, endothelial cells and tumor cells [85-87]. Increasing evidence indicates that SASP mediates the bystander effect of radiation by inducing senescence and affecting the microenvironment [16,88].
Therefore, with potent autocrine and paracrine activities, SASP may act as an important mediator of the pathophysiological functions of senescent cells, such as the induction of tissue inflammation and fibrosis, attraction of immune cells, the reinforcement of senescence itself and the induction of paracrine senescence. Acting as a double-edged sword in tumor, SASP can induce malignant phenotypes in senescent cells and their nearby cells (Fig. 2).
Fig. 2. The detrimental and beneficial effects of the SASP in the pathophysiological function of senescent cells. Scheme summarizing some of the SASP functions.
1
Therapeutic interventions for radiation-induced senescence
Several studies have shown that cellular senescence might be involved in the progression of several diseases, like pulmonary fibrosis [31], cancer [89,90], neurodegenerative diseases [91,92] and atherosclerosis [93], making senescent cells an attractive therapeutic target. Thus, to reduce the possible detrimental effects of senescent cells, identification and inhibition of SASP or selective elimination of senescent cells have been suggested as possible therapeutic strategies (Fig. 3).Modulation of SASP
Since SASP plays an important role in the physiological function and pathological progression of age-related diseases and treatment-related side effects, it is of great interest to explore its regulation and subsequent effects. The modulation of the SASP, for example, by inhibiting the function of NF-KB [94], the major driver of SASP, or by neutralizing some of SASP members, was proposed as a potential therapeutic option. Indeed, inhibition of SASP signaling, by knock down of IL-6R, IL6 or CXCR2 (a receptor for IL-8), has been shown to prevent senescence [12,81,95]. Moreover, mTOR inhibitors (RAD001 and rapamycin) or Janus kinase 1 (JAK1) and JAK2 inhibitors have been shown to reduce some SASP components, to enhance physical function and to increase the lifespan of mice [96-99]. The complex nature of SASP requires a context-dependent therapeutic strategy to specifically modulate SASP without unwanted and unpredictable toxicities. However, most of the SASP inhibitors are unspecific for senescent cells, potentially leading to side effects on the long-term [100]. More studies need to be performed to better understand which specific SASP factors have to be modulated to improve patients’ outcome considering the different functions of these factors in different diseases.
Semi-genetic and pharmacological elimination of senescent cells
The selective removal of senescent cells, the source of detrimental factors, has been suggested to be an attractive therapeutic approach for age-associated diseases. Semi-genetic clearance of p16-expressing senescent cells [101] was shown to delay age-associated pathologies, such as tau-mediated disease and the aged hematopoietic system [32,102,103]. Given that resistance to apoptosis is the main feature of senescent cells, pharmacological therapies targeting apoptosis related proteins in senescent cells have become a major focus. Several compounds have been discovered so far like ABT-263, ABT-737, dasatinib and quercetin, Alvespimycin ( 17-DMAG), FOXO4-DRI and cell-penetrating peptides (CPPs) (Table 1). ABT-263, a specific inhibitor of the BCL-2 family of anti-apoptotic proteins, was shown to mitigate radiation injury of senescent hematopoietic and muscle stem cells. The combination of Quercetin (Q, a polyphenol found in fruits and vegetables) and Dasatinib (D, a Src kinase family inhibitor with various functions [94]) can decrease senescent cells in a variety of tissues [104-108]. A single dose of D+Q treatment can improve limb exercise capacity impaired by 10 Gy irradiation in mice [105]. The FOXO4-DRI peptide can selectively promote p53 nuclear exclusion and cause apoptosis of senescent cells by disturbing the FOXO4-p53 interaction [109]. To limit off-target toxicity, antisenescence compounds should not only have the ability to selectively target senescent over normal cells, but also specifically target the deleterious senescent cells. CCPs can specifically redirect protein-protein interactions and they can be designed as antisenescence compounds selectively impairing the crucial signaling pathways important for senescent cell viability [101].
1
Fig. 3. Therapeutic interventions for cellular senescence. Senolytics selectively induce
death of senescent cells, while SASP inhibitors work by interfering with SASP signaling pathways.
Table 1. List of compounds used as antisenescence drugs.
Compounds Targets Type of target Functions Reference
Rapamycin mTOR SASP
Reduce production of
SASP
[110] RAD001 mTOR SASP [40][97] Metformin NF-KB SASP [111]
Anakinra IL-receptor SASP [78]
5Z-7-oxozeaenol Transforming growth factor-β1-activated kinase-1 (TAK1) SASP [112]
ABT-263 BCL-2 family Anti-apoptotic proteins
Induce senescent cells to die
[102,103] ABT-737 BCL-XL/BCL-2 Anti-apoptotic proteins [113,114] A1331852 and
A1155463 BCL-XL Anti-apoptotic proteins [115] FOXO4-DRI interfering peptide FOXO4-P53 [109] Quercetin Lipoprotein lipase (LPL), Glycolysis Antioxidant enzymes [104]
Dasatinib tyrosine kinases Pan-receptor Receptor tyrosine kinases [104] Alvespimycin (17-DMAG) Heat-shock protein 90 (HSP90) Chaperone subfamily PI3K/AKT anti-apoptotic pathway [116] Cell penetrating
peptides (CPPs) Protein-protein interactions
Endogenous interaction parters [101] MAP kinase-activated protein kinase 2 （MAPKAPK2）
ZEP36L1 m-RNA binding protein
Target SASP mRNA
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Glial-cell-line-derived neurotrophic factor in response to
radiation
Introduction to GDNF and its functions
Glial-cell-line-derived neurotrophic factor (GDNF) was first identified as a survival factor in midbrain dopaminergic neurons [111,118], which degenerate in Parkinson’s disease [119]. It has also been shown that GDNF is produced in spinal motor neurons [120], noradrenergic neurons, astrocytes, oligodendrocytes and Schwann cells [120] acting as a trophic factor. Neurotrophic factors regulate many critical aspects of the development of neurons, such as neurite branching, synaptogenesis and electrophysiological maturation. Exogenously applied GDNF promotes the growth of dopaminergic neurons both in vitro and in vivo [119,121]. However, the side effects and effectiveness of GDNF treatment on Parkinson’s disease patients varies between trials [122-125]. GDNF hypermorphic mice revealed that GDNF plays an important role in the development of the postnatal nigrostriatal system and regulates the structure and function of the nigrostriatal dopaminergic system [126]. In addition, GDNF plays an important role in many peripheral neurons, for instance, sympathetic, parasympathetic, sensory and enteric neurons [127-129]. The production of GDNF from oligodendrocytes, Schwann cells and astrocytes showed neuroprotective function to neighboring neuronal populations through anterograde transport in dorsal root ganglia and motor neurons [130,131]. In addition to those neuroprotective effects [132-134], GDNF plays a role in mitigating astrocyte cell death and minimizing microglia activation [135,136].
GDNF has several roles outside the central nervous system. It functions as a regulator of kidney morphogenesis and spermatogonial differentiation. In the embryonic kidney, GDNF is expressed by the metanephric mesenchyme and binds to its receptor RET, which is expressed by the adjacent tips of the branching
ureteric bud, and promotes the ureteric budding [137-139]. Indeed, knock out of GDNF or its receptors lead to mouse death at birth due to severe hypodysplasia and lack of kidney development [126,137]. GDNF is expressed by the sertoli cells in the testis and controls spermatogonial stem cell differentiation and self-renewal in a paracrine manner [140]. The role of GDNF varies with the dosage level. When GDNF is low, the spermatogonial stem cells undergo differentiation, while high levels of GDNF promote spermatogonial stem cell self-renewal instead of differentiation. It has been shown that overexpression of GDNF in the testis can lead to mouse infertility and seminomatous germ cell tumors [141]. Moreover, GDNF promotes salivary gland stem cell self-renewal capacity in vitro and increases the number of salivary gland stem cells [142,143] (Fig. 4).
Fig. 4. GDNF functions. Scheme summarizing some of the functions of GDNF.
Abbreviations: GDNF, glial cell-derived neurotrophic factor; GFRα1, GDNF receptor alpha-1; RET, receptor tyrosine kinase.
1
GDNF receptors and molecular signaling
GDNF belongs to the transforming growth factor-β (TGF-β) superfamily. GDNF binds to glycosyl-phosphatidylinositol-anchored coreceptor (GFRα1) to form a homodimer, then activates the receptor tyrosine kinase (RET) through transphosphorylation of specific tyrosine residues in their tyrosine kinase domains and downstream intracellular signaling [127] (Fig. 4). RET activates the PI3K/AKT, MEK/ERK, p38 activated MAPK and JNK pathways. Through the downstream targets, the GDNF/RET signaling pathway is involved in neuronal and glial cell survival and differentiation [144-146], kidney morphogenesis [147] and spermatogenesis [148].
Interestingly, GDNF can also signal independently from RET through GFRα1. In RET-deficient cell lines and primary neurons, GDNF triggers RET independent Src and Met activation. It has been demonstrated that NCAM functions as an alternative signaling receptor for GDNF [149]. GDNF-GFRα1-NCAM signaling activates the Src-like kinase Fyn and the focal adhesion kinase FAK [149]. Through GDNF-NCAM pathway, GDNF contribute to Schwann cell migration and promote axonal growth.
GDNF and radiation
It has been shown that GDNF was highly expressed and localized in the secretory ducts of irradiated murine and human salivary glands [142,143]. Moreover, RNA sequencing data showed that GDNF consistently increased in radiation-induced senescence in three cell lines (fibroblasts, keratinocytes and melanocytes) and also in senescent astrocytes induced by oxidative stress [150]. Therefore, GDNF may act as a core transcriptome signature of senescence. However, there is no universal marker of senescence, whether GDNF can act as a specific hallmark of radiation-induced senescence still needs to be further investigated.
Senescence and different radiation types
Linear energy transfer (LET) is the average amount of energy that an ionizing particle transfers to the material is traversing per unit of distance. Heavy ions (e.g. carbon ions) have higher LET values compared to low LET photons and protons. Importantly, high LET radiation are biologically more effective per Gray and in addition they display an improved dose distribution when compared to photons. These characteristics make high LET radiation a promising radiation modality in cancer treatment.
Indeed, high LET particle therapy can be used to more accurately and effectively target the tumor while reducing the volume of co-irradiated normal tissue [151,152]. This is due to the depth-dose distribution characterized by what is known as the “Bragg peak”. In clinical practice high LET radiation can be modulated to cover the whole tumor in a “spread-out Bragg peak”, which exhibits a higher LET than the plateau entrance dose, thereby minimizing the entrance and exit dose, and thus sparing the surrounding normal healthy tissue.
Preclinical studies with high LET radiation focused more on mechanisms involved in the differential induction of biological responses, such as clonogenic survival [153], induction and repair of DNA damage [154], and apoptosis [155]. The increased level of cell killing (higher relative biological effect, RBE) together with more precise tumour targeting and sparing of normal tissue support the idea that high LET radiation can achieve better tumor control while limiting normal tissue side effects compared to photons, largely used in conventional radiotherapy.
However, radiation-induced normal tissue effects are not only caused by cell death but can also depend on many other biological processes such as cell differentiation, cellular senescence, extracellular matrix deposition, epithelial to mesenchymal transition, proliferation of specific cells, and other responses [156-160]. Therefore, a better understanding of normal tissue responses such as those related to
1
Salivary glands and radiation-induced side effects
The salivary glands are exocrine glands that produce saliva through the acinar compartment (formed by mucous, serous and seromucous acini) which is secreted through a system of ducts (intercalated, striated and finally excretory ducts). The three pairs of major salivary glands (parotid, submandibular and sublingual glands) can produce 90% of the saliva in humans and rodents [161]. Multiple causes, including Sjögren’s syndrome, uncontrolled diabetes, age-related senescence [162] and radiotherapy for head and neck cancer patients [163], contribute to the loss of saliva production. Radiation-induced hyposalivation and resulting xerostomia is a severe side effect of radiotherapy for head and neck cancer patients, which severely influences patients’ quality of life. Multiple events could be involved in radiation-induced hyposalivation, such as the damage to salivary gland epithelial cells, impairment of microvessels and parasympathetic innervation [163,164]. After radiation, cells will undergo cell death, cellular senescence or DNA repair depending on the severity of the DNA-damage. Cell death, loss of stem/progenitor cell function and cellular senescence [165-168], may play a role in the development of this severe radiation-induced side effect.
Use of organoids in radiation research
Organoids, mini-organs derived from organ specific stem cells, can be used for drug screening [169-171], disease diagnosis [170,171], disease modeling [172,173], and radiation-induced side effects [153] (Table 1.). Moreover, organoids can be derived both from animal and human organs. Human organoids can offer the unprecedented opportunity to study patient and organ specific drug and/or radiation interactions holding the possibility to further develop personalized medicine. When compared to 2D cultured cells, 3D organoid models resemble more closely the in vivo tissue response to drugs and radiation [174,175]. For instance, 2D cells are more sensitive than 3D organoids to radiation [174]. In addition, 3D organoids offer a more complex environment, for example, 3D cultured cells have different
cell-cell and cell-matrix interactions which may play a role in radio-resistance compared with 2D cultured cells. Therefore, organoids offer the opportunity to study tissue specific responses to radiation including radiation-induced side effects. Recently, using 3D cultured salivary gland stem cell derived organoids, our group developed a novel organoid model to study normal tissue responses to radiation treatment with both photons and particles [153].
Table 1. Organoid models have been developed from certain healthy tissues or cancer types. These organoids may be used for several purposes including the
study of radiation-induced side effects.
Healthy tissue organoid
model Cancer tissue organoid model organoid model Potential use of
Kidney [176] Breast cancer [177] Disease diagnosis [171] Lung [99][178] Prostate cancer [179] Drug screening [169]
Liver [180] Glioblastoma [179] Gene-modified therapy [171]
Pancreas [181] Pancreatic cancer [182] Radiation induced side effects [153]
Salivary gland [183] Colon/Colorectal cancer[184]
Brain [185] Gastrointestinal cancers [186] Small intestine [180] Liver cancer [187] Gastric epithelium [188] Bladder cancer [189]
Mammary [190] Esophagus [191]
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Concluding remarks
While the development of high precision radiotherapy techniques might reduce the normal tissue side effects, there is still much to be explored about the fundamental mechanisms underlying the response of normal tissue to radiation. Many questions still remain unanswered, for instance, what is the role of radiation-induced cellular senescence in normal tissue side effects? Do different types of radiation induce different levels of senescence? What is the effect on normal tissue when senolytics are used to eliminate radiation-induced senescent cells? The development of 3D organoid models to study radiation-induced side effects [153,192] on tissue resembling structures in vitro can be used to investigate mechanisms and even allow testing of modulators of damage such as the use of GDNF and senolytics.
Aim and outline of the thesis
The main goal of this thesis is to characterize and modulate radiation-induced senescence in the salivary gland and its relation to tissue regeneration.
Although GDNF pretreatment can increase mouse salivary gland stem/progenitor cell (SGSC) number and rescue saliva production in irradiated salivary glands, the role of GDNF in the modulation of SGSC response to irradiation remains elusive. In Chapter 2, a GDNF hypermorphic mouse model was used to study whether GDNF may act as a radio-protector on SGSCs by enhancing post-irradiation regeneration.
Radiation-induced loss of salivary gland function has been suggested to be driven by cellular senescence. Therefore, Chapter 3 aims to develop a radiation-induced senescence system by using maturated salivary gland organoids as a tool for investigation of the role of senescence in radiation-induced salivary gland damage. Furthermore, this model is used for testing the effects of senolytics on the elimination of senescent cells in vitro using the self-renewal potential of salivary
gland stem cell derived organoids and subsequently the in vivo effect of senolytics on radiation-induced salivary gland dysfunction.
Interestingly, GDNF is highly upregulated in irradiated glands with severe morphological damage and impaired saliva secretion [143]. Moreover, GDNF has been found to be a core transcriptome signature of radiation-induced senescence in different cell lines [150]. Therefore, using the radiation-induced senescence model described in Chapter 3, Chapter 4 aims to assess the role of GDNF in radiation-induced senescence in salivary glands.
Various mechanisms have been proposed to be involved in the development of normal tissue damage and may be differentially regulated depending on the radiation type and its linear energy transfer (LET). Hence, Chapter 5 explored the difference in induction of cellular senescence between low (photons) and high LET (carbon ions) radiation in salivary gland tissue resembling organoids derived from p16-3MR mice. These mice carry a 3MR (trimodality reporter) protein under the control of the promoter for p16INK4a which can be used to identify senescence and selectively kill senescent cells [23,32,193]. Differences in senescence induction between photons and carbon ions could be assessed by comparing survival, luminescence reporting p16 expression as a measure of senescence and SASP gene expression.
In Chapter 6, we summarize and discuss the data obtained in our experimental chapters and provide future perspectives.
1
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Chapter 2
Role of glial-cell-derived neurotrophic factor in
salivary gland stem cell response to irradiation
Xiaohong Peng
1, Kärt Varendi
2, Martti Maimets
1,3, Jaan-Olle Andressoo
2,4,
Rob P. Coppes
1 1Departments of Cell Biology and Radiation Oncology; University of Groningen,University Medical Centrum Groningen, The Netherlands. 2Institute of Biotechnology, University of Helsinki, Finland. 3BRIC-Biotech Research and Innovation Centre, Copenhagen, Denmark, 4Institute of Biosciences and Medical Technology - BioMediTech, University of Tampere, Finland.
Abstract
Background and purpose: Recently, stem cell therapy has been proposed to allow
regeneration of radiation damaged salivary glands. It has been suggested that glial-cell-derived neurotrophic factor (GDNF) promotes survival of mouse salivary gland stem cells (mSGSCs). The purpose of this study was to investigate the role of GDNF in the modulation of mSGSC response to irradiation and subsequent salivary gland regeneration.
Methods: Salivary gland sphere derived cells of Gdnf hypermorphic (Gdnfwt/hyper) and wild type mice (Gdnfwt/wt) were irradiated (IR) with γ-rays at 0, 1, 2, 4 and 8 Gy. mSGSC survival and stemness were assessed by calculating surviving fraction measured as post-IR sphere forming potential and population doublings. Flow cytometry was used to determine the CD24hi/CD29hi stem cell (SC) population. QPCR and immunofluorescence was used to detect GDNF expression.
Results: The IR survival responses of mSGSCs were similar albeit resulted in
larger spheres and an increased cell number in the Gdnfwt/hyper compared to Gdnfwt/wt group. Indeed, mSGSC of Gdnfwt/hyper mice showed high sphere forming efficiency upon replating. Interestingly, GDNF expression co-localized with receptor tyrosine kinase (RET) and was upregulated after IR in vitro and in vivo, but normalized in vivo after mSGSC transplantation.
Conclusion: GDNF does not protect mSGSCs against irradiation but seems to
promote mSGSCs proliferation through the GDNF-RET signaling pathway. Post-transplantation stimulation of GDNF/RET pathway may enhance the regenerative potential of mSGSCs.
