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University of Groningen

Radiation-induced cellular senescence in salivary glands

Peng, Julie

DOI:

10.33612/diss.103407924

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from

it. Please check the document version below.

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Publisher's PDF, also known as Version of record

Publication date:

2019

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):

Peng, J. (2019). Radiation-induced cellular senescence in salivary glands. Rijksuniversiteit Groningen.

https://doi.org/10.33612/diss.103407924

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Chapter 1

General introduction and thesis outline

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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].

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1

hours with a total dose of 10 Gy [9,10,37,38] can induce senescence. Cellular

senescence 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 outcome

and 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

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(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

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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

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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].

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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.

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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.

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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].

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Fig. 3. Therapeutic interventions for cellular senescence. Senolytics selectively induce

death of senescent cells, while SASP inhibitors work by interfering with SASP signaling pathways.

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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

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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

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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.

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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.

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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

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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

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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

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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 p16

INK4a

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.

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