Designing a bio-inspired bio-mimetic in vitro system

for the optimisation of ex vivo studies of pancreatic cancer

Stella Totti1, Spyros I. Vernardis2, Lisiane Meira3, Pedro A. Pérez-Mancera4, Eithne

Costello4,5, William Greenhalf5, Daniel Palmer4, John Neoptolemos4,5, Athanasios Mantalaris2,

Eirini. G. Velliou1,*

1 Bioprocess and Biochemical Engineering Group (BioProChem), Department of Chemical and

Process Engineering, University of Surrey, Guildford, GU2 7XH, UK

2Biological Systems Engineering Laboratory (BSEL), Department of Chemical Engineering, Imperial

College London, SW7 2AZ, London, UK

3Department of Clinical and Experimental Medicine, University of Surrey, Guildford, GU2 7XH, UK

4Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Daulby Street,

Liverpool L693GA, UK

5NIHR Liverpool Pancreas Biomedical Research Unit, University of Liverpool, Daulby Street,

Liverpool L69 3GA, UK

*Corresponding author. Fax: 0044-(0)-1483686577

E-mail address: e.velliou@surrey.ac.uk

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Abstract

Pancreatic cancer is one of the most aggressive and lethal human malignancies. Drug

therapies and radiotherapy are used for treatment as adjuvants to surgery, but

outcomes remain disappointing. Advances in tissue engineering point that three-

dimensional cultures can reflect the in vivo tumour micro-environment and can

guarantee a physiological distribution of oxygen, nutrients and drugs, therefore, being

promising low cost tools for therapy development. In this work we review crucial

elements, i.e., structural and environmental, that should be considered for an accurate

design of an ex vivo platform for studies of pancreatic cancer. Furthermore, we

propose environmental stress response biomarkers as platform readouts for the

efficient control and further prediction of the pancreatic cancer response to the

environmental and treatment input.

Keywords

Pancreatic cancer, tissue engineering, 3D culture systems, stress biomarkers,

environmental stress, hypoxia, metabolic stress, metabolomics

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1. Introduction

Pancreatic cancer is the fourth leading cause of all cancer-related deaths in the United

States [1] fifth in the UK (see: http://www.cancerresearchuk.org) and eighth

worldwide [2]. Moreover, despite the fact that for most cancers the survival rate has been

increasing, the 5-year survival rate of pancreatic cancer remains persistently low at 3-6% [1]

(see: http://www.nhs.uk). This dismal outcome can be attributed to many factors such

as i) late stage diagnosis due to lack of early diagnostic biomarkers [3,4], ii) high

metastasis likelihood [5,6], iii) resistance to treatment [7]. Understanding the

pancreatic tumour growth/evolution as well as response to treatment is crucial in order

to improve treatment efficacy both for the benefit of society and individual patients.

Animal studies can be very informative, however they are expensive and complex to

reproduce. In vitro studies of cancer so far, are mainly conducted in traditional 2D

monolayer systems. Those systems can provide useful information, but usually they

lack predictability as they differ significantly from the in vivo tumour environment

[8,9]. Thus, in order to accurately understand cellular and tumour behaviour and

response to treatment, it is essential to simulate ex vivo, the in vivo tumour

environment. The latter, is the main aim of 3D tissue engineering constructs. These

3D systems better reflect the in vivo scenario in terms of structure, porosity, and

microenvironmental niches than the conventional two dimensional (2D) systems [9-

12]. Their spatial arrangement provides better cell-cell interactions while coating with

different extracellular matrix proteins (ECM) ensures better cell-matrix interactions

[13,14]. Co-cultivation with stromal cells, additionally to the above characteristics can

lead us one step closer to the accurate recapitulation of the in vivo tumour

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microenvironment and realistic stromal interactions [15]. Therefore, growing patient

derived samples outside of the patient body in appropriate 3D tissue engineered

platforms could lead to better understanding the tumour behaviour and response to

drug and irradiation therapies [16].

Next to the actual architectural and micro-environmental characteristics of a tumour in

terms of structure, environmental conditions such as oxygen, glucose, nutrients,

temperature are of significant importance for the cancer kinetics. Fluctuations of those

environmental parameters naturally occur in patients with cancer and could alter

tumour proliferation and/or cause adaptation of the pancreatic tumour cells to

treatment [17-21].

The aim of this work is to review crucial elements, i.e., structural and environmental,

that should be considered for an accurate design of an ex vivo platform for studies of

pancreatic cancer. Furthermore, we propose environmental stress response

biomarkers as platform readouts for the efficient control and further prediction of the

pancreatic cancer response to the environmental and treatment input.

2. 3D tissue engineering constructs for in vitro studies of pancreatic cancer

A first challenging and promising step towards understanding the pancreatic cancer

evolution and further response to treatment is to recapitulate ex vivo the in vivo

environment. For decades, conventional two dimensional (2D) technology has been

used in cancer research. Generally, these 2D systems are simple to handle,

reproducible and cheap. Furthermore, they are responsive to drug therapies and

radiotherapy thus they are systematically used for treatment screening [9,22-25]. The

architecture of these systems enables homogenous distribution of nutrients and

oxygen, with no presence of gradients [9,23]. Thus, a significant amount of research

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on pancreatic cancer has been conducted in 2D [26-33]. However, experimental 2D

platforms cannot accurately recapitulate the 3D in vivo tumour microenvironment as

they do not capture aspects such as structure, porosity and three dimensional

extracellular matrix distribution [11,13,23,34-40]. Consequently, 2D-grown cells can

differ significantly in their morphology, cell-cell and cell-matrix interactions, as well

as in their response to fluctuations of environmental factors [15,41-44]. Furthermore,

several studies have revealed that cells change their original phenotype in 2D culture

conditions, therefore hindering predictability in vivo [45-47].

In contrast to 2D cultures, three dimensional (3D) tissue engineering constructs are

closer to the in vivo environment in terms of structure, architecture, porosity and

microenvironmental niches [48,49]. The features of a 3D system allow topologically

realistic cell-cell interactions and migration due to the spatial arrangement of these

constructs, as well as 3D cell-matrix interactions through coating with extracellular

matrix proteins [38,40,50,51] and co-cultivation with stromal cells [34]. The latter is

particularly important as it is known that stromal cells have a crucial role in cancer

progression and aggressiveness [52-54]. Especially in pancreatic cancer, presence of

intense extracellular matrix, i.e., dense desmoplasia, can contribute to the

development and progression of this malignancy [39,40,55,56] as well as to the

inhibition of apoptotic pathways, directly affecting the diffusion of the

chemotherapeutic drug reagents [57].

Additional to their architectural/structural characteristics, 3D tissue engineering

constructs provide a unique perspective for the realistic distribution of environmental

input (oxygen, nutrients & temperature gradients) and treatment input (irradiation,

drugs), in terms of mass transfer/diffusion as well as heat transfer and energy

deposition [58-61].

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All the above design parameters that differ between 2D and 3D culture systems, could

possibly lead to a different response of the cells to treatment, suggesting the 3D as

more accurate and predictive (Figure 1). There are several pancreatic cancer studies

that have been conducted in 3D systems. More specifically, the following 3D systems

have been reported for pancreatic cancer i) spheroids, ii) hydrogels, iii) synthetic

matrices.

2.1. Spheroids

Spheroids are the simplest and most widely used tissue engineering constructs, where

a small aggregate of cells grows in three dimensions without adhering to a solid

surface [62,63]. In vitro spheroid formation can be achieved by the forced floating

method [64], the hanging drop method [64,65] and agitation approaches [64] (Figure

1). Spheroids allow cells to interact with each other, with the (produced) extracellular

matrix and the microenvironment [23,66]. Matsuda et al. created 3D spheroid culture

systems for different pancreatic cell lines and captured differences in the expression

levels of cytoskeletal proteins between the developed spheroids and 2D conventional

monolayer cultures. [67]. Longati et al. captured differences in proliferation,

metabolism and chemoresistance between 2D cultures and 3D methylcellulose based

spheroid cell cultures, using various human pancreatic ductal adenocarcinoma

(PDAC) cell lines. Proliferation was lower in 3D compared to 2D, and lactate

accumulation increased in the spheroids. Additionally, higher production of collagen I

and fibronectin I and higher resistance to gemcitabine (GEM), i.e., commonly used

chemotherapeutic drug for pancreatic cancer treatment, was observed in 3D [68]. Wen

et al. developed a 3D spheroid based system using the forced floating method for

human pancreatic cell lines. Higher resistance of PANC-1 to the drugs gemcitabine

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and 5-fluorouracil was observed in the spheroids as compared to classical 2D

monolayers [69]. As can be seen from the above findings, pancreatic cell lines behave

differently in spheroids as compared to 2D cultures and generally they display an

increased resistance to treatment in the spheroids, making the latter a very attractive

system for in vitro studies. However, spheroids have some disadvantages as their

spatial characteristics result in very high gradients of nutrients/oxygen [9,23,70].

Moreover, it is difficult to control the shape and the morphology of the formed

spheroids [9,11] . A summary of the advantages and disadvantages of spheroids for

pancreatic cancer screening are shown on Table 1.

2.2. Hydrogels

Hydrogels are water swollen and cross-linked polymeric networks produced by a

polymeric reaction of one or more monomers [71,72]. Due to their ability to simulate

the native tissues in terms of architectural and spatial characteristics, their

biocompatibility and their hydrate structure that allows nutrient and oxygen diffusion,

hydrogels are attractive materials for 3D ex vivo cancer models [13,73-75] (Figure 1).

Hydrogels can be formed from purely non-natural molecules such as poly(ethylene

glycol) (PEG) [76] and poly(vinyl alcohol) (PVA) [77]. Sempere et al. developed a

hydrogel, composed of 3% collagen IV/laminin-rich gelatinous medium (Matrigel®)

and soft agar, to study the effect of transforming growth factor (TGFβ) on the growth

of human pancreatic cancer cell lines. Although TGFβ had an inhibitory effect in soft

agar, TGFβ had a growth stimulatory effect in the 3D culture [78]. Raza et al.

developed a hydrogel system for PANC-1 using 4-arm poly(ethylene glycol)-tetra-

norbornene (PEG4NB) and four different cross-linkers. Morphological differences

were observed in the hydrogel as compared to 2D. Specifically, the pancreatic cells

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formed clusters within the hydrogels within 4 days of cultivation. Moreover, matrix

characteristics such as stiffness and the type of cross-linkers used, influenced the cell

growth as well as the structure of the hydrogels. In particular, softer matrices lead to

enhanced metabolic activity of cell populations and cyst like formations. Furthermore,

matrices cross linked with the MMP (Matrix Metalloproteinase) supported cell growth

& enhanced metabolic activity compared to a dithiothreitol linker to form the

hydrogel and resulted in formation of cyst-like cell structures [79]. Ki et al. developed

a hydrogel system for pancreatic cell lines and evaluated the effect of hydrogel matrix

stiffness and epidermal growth factor receptor (EGFR) on cell proliferation within the

different hydrogel matrices. Higher metabolic activity was observed in softer matrices

as compared to the medium and stiff ones. Moreover, treatment with EGFR inhibitor

reduced the cell viability on stiff hydrogels but had no effect on soft hydrogels nor in

2D cultures [80]. Ki et al. developed a semi-synthetic in vitro microenvironment

mimicking pancreatic desmoplasia. More specifically, a hydrogel was developed and

coated with collagen type I. Increased cell proliferation and drug resistance to

gemcitabine was observed in the hydrogel matrix compared to the 2D system [81].

Boj et al. developed a 3D organoid matrigel system for pancreatic cancer from

primary pancreatic ductal adenocarcinoma cells that can accurately recapitulate the

histology and the disease stage –specific characteristics of the tissue from which they

were derived (normal and neoplastic human tissue). This organoid system managed to

retain both the tumour and stromal production [82,83].

However, despite their great advantages in comparison to classical 2D cultures,

hydrogels cannot provide a consistent in vitro tumour model, due to the lack of

uniform spatial distribution of cells within them [9,84]. Additionally, hydrogels

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present weak mechanical strength, provoking difficulties in handling them [13,85]

(Table 1).

2.3. Synthetic polymeric scaffolds

Polymeric scaffolds are a promising approach for ex vivo modelling of pancreatic

cancer. Their architectural characteristics provide a realistic ex vivo 3D spatial

arrangement of the cells in the scaffold (Figure 1). Moreover, the possibility of

controlling accurately the porosity of such structures allows mass and energy transport

phenomena such as diffusion of oxygen, nutrients, drugs as well as heat and

irradiation (radiotherapy) to occur to a more similar manner to the actual in vivo

behaviour [35,86-89]. Additionally, polymeric scaffolds, depending on their

fabrication, can have strong mechanical properties [9,12,35,70]. Furthermore, features

of the tumour microenvironment such as the presence of extracellular matrix proteins

and co-culture of stromal cells can take place in order to imitate the in vivo

progression of cancer [43,49]. Therefore, cellular proliferation, signalling, migration,

differentiation and additional bio-physical-chemical and mechanical phenomena that

occur in the tumour microenvironment in vivo can take place in those polymeric

systems, rendering them strong candidates for 3D culture platforms. A synthetic

scaffolding system consisting of poly(vinyl alcohol) – gelatine (PVA/G) sponges was

constructed by Funel et al. Cell growth of primary pancreatic cells was further studied

in the polymeric scaffolds. The results indicated viable cells that adhered within the

sponge, with enhanced metabolism in the 3D model compared to the traditional 2D

culture, resulting in a growth ratio (3D/2D) of 1.38 [90]. Wang et al. indicated that

polyglyconate/gelatine electrospun scaffolds provided a favourable microenvironment

for pancreatic cancer stem cells derived from patients, ensuring increased proliferation

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capacity in comparison to the 2D system for a time-spam of 7 days [91]. Additionally,

He et al. created a disk-like polymeric scaffolding system based on poly(glycolide-co-

trimethylene carbonate) and gelatine (PGA-TMC)/G. Pancreatic cancer stem cells

were seeded in the scaffold and proliferated for up to 7 days. This polymeric scaffold

model demonstrated better neoplastic formation and accelerated tumour evolution as

compared to the 2D system. [92]. Ricci et al. developed three different biocompatible

scaffold types based on two polymers (poly(ethylene oxide

terephthalate)/poly(butylene terephthalate) and poly(vinyl alcohol)/gelatine) and two

polymeric formulations (fibre mesh, sponge like). The type of polymer and the

formulation technique alter the internal architecture, therefore, affecting the cell

growth and morphology as well as the tumour-specific MMPs synthesis of PDAC

[48]. Totti et al. developed a highly porous (90% porosity) polyurethane (PU) based

scaffold which successfully supported the long term (up to 5 weeks) cultivation of

pancreatic cancer cell lines [93].

Polymeric scaffolds are sometimes complex to produce as compared to other 3D

systems [9] and present difficulties in retrieving cells after the culture formation [64]

(Table 1). However, their overall properties as compared to the other 3D systems

make them very strong tissue engineering candidates. Namely, the possibility of an

accurate monitoring of porosity (size, type, distribution), and the ability to recapitulate

and actual 3D structure with controlled size makes them ideal for ex vivo drug

screening. Furthermore, their strong mechanical strength enable the construction of

robust perfusion systems that could allow vascularisation mimicry.

Overall, from all the above studies, it is clear that there are significant differences in

the biological behaviour as well as the response/resistance to treatment of pancreatic

cancer cells in 3D tissue engineering constructs compared to 2D culture systems, with

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3D tissue engineering constructs featuring more accurately elements of the tumour

microenvironment (Table 2).

It is therefore essential to move towards the development of efficient 3D systems as

in vitro models for studying, understanding and eventually predicting the pancreatic

tumour evolution and response to different types of treatment.

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3. Towards a biomimetic distribution and control of the environmental (stress)

input in pancreatic cancer tissue engineering platforms

The formation of desmoplasia around the pancreatic tumour coupled with an

avascular tumour microenvironment leads to hypoperfusion of nutrients as well as

hypoxia [26,94,95]. Therefore, environmental parameters should be monitored in 3D

experiments, especially since within a tissue engineering construct those parameters

could fluctuate as a result of diffusional limitations and/or increased cell density at

different locations of the 3D [43]. Furthermore, additionally to cell survival, easily

readable quantitative measurements, i.e., in situ as well as in the cell culture medium

surrounding the 3D systems should be considered for the detection of

stressed/unstressed regions within the 3D culture systems that could be linked to

resistance to treatment (Table 3). Such environmental stress monitoring would lead to

a more efficient control of the input of the environmental fluctuations on the cell

evolution (Figure 1). In particular, a correlation of metabolic (biomarker) information

and the overall cell survival (cell growth/inactivation kinetics) under different levels

of environmental (stress) stimuli in 3D could be highly beneficial as it would enable a

more accurate control and further prediction of the disease progression (in vitro). The

following section summarizes important environmental parameters and suggests

biomarker candidates that could enable efficient control of such parameters in 3D

systems.

3.1.The Impact of Oxygen Stress on Pancreatic Cancer Evolution

Low oxygen levels (hypoxia) as well as fluctuations on the levels of oxygen, naturally

occur within a solid tumour [96]. These hypoxic regions are correlated with altered

metabolism and resistance of a tumour to drug therapies and irradiation [96,97]. For

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example, Shibamoto et al. showed that four different pancreatic cell lines displayed

resistance to radiotherapy under hypoxic conditions as compared to normoxic

conditions [98]. On the contrary, Mizumoto et al. showed enhanced cytotoxicity of

the drug PR-350 under hypoxia. More specifically, under hypoxia the cytotoxicity of

PR-350 was significantly enhanced almost 5% [99].Yokoi and Fidler observed that

pancreatic cells are more resistant to chemotherapy under hypoxic conditions. In

particular, they showed that under hypoxic conditions, the highly metastatic

pancreatic cell line L3.6pl was resistant to apoptosis triggered by the drug

gemcitabine [17]. Cheng et al. observed that pancreatic cells grown in hypoxic

conditions were resistant to chemotherapy in comparison with cells grown in

normoxic conditions [100]. Furthermore, Maftouh et al. showed that pancreatic

tumour spheroids under hypoxia exhibited increased chemoresistance to gemcitabine

than the corresponding spheroids in a normoxic environment [101].

Oxidative stress biomarkers

Oxygen homeostasis and hypoxia signaling is mediated by the hypoxia-inducible

factors (HIFs) [102]. HIF-1α, the master regulator of O2 homeostasis, is

overexpressed in 88 % of pancreatic cancer tissues, versus in only 16% of healthy

pancreas [94,103]. Hypoxia mediated HIF-1 induction in cancer correlates with

resistance to apoptosis, tumour size and cell proliferation, invasion and metastasis

[102,104,105]. For example, Kizaka-Kindoh et al. indicated that killing/deactivation

of HIF-active pancreatic cells, impaired the tumour evolution [106]. Additionally,

Schwartz et al. showed that the in vitro and in vivo use of HIF-1α inhibitors led to a

decrease in pancreatic cancer cell survival after fractionated radiation [103]. Another

study by Kang et al. showed that HIF-1 degradation triggers pancreatic tumour cell

death [107].

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Collectively, the above studies indicate hypoxia is a direct cause of therapeutic

resistance. Both radiotherapy and chemotherapy are significantly influenced by

hypoxia, therefore, it is very important to include oxygen input as a parameter on the

design as well as the readout (by HIF status monitoring) of the pancreatic cancer in

vitro platform (Figure 1 & Table 4).

3.2. Metabolic stress- Nutrient deprivation

As previously stated, hypoperfusion is a common characteristic of pancreatic cancer

due to the desmoplasia which surrounds the tumour. Therefore, it may lead to not only

oxygen but also nutrient limitations in the tumour area [108]. Environmental stress

can affect normal tissue cells and turn them into other types, after accumulation of

genomic and epigenomic alterations (Figure 2). The metabolism of proliferative cells

(cancer cells) tends to be more glycolytic and dependent less on oxidative

phosphorylation than a normal/healthy cell (Figure 2) even when oxygen is sufficient

(Warburg effect). This kind of metabolism covers the needs of proliferative cells in

macromolecules’ building blocks (monomers), energy and establishes a redox

potential balance [109]. Nevertheless, pancreatic tumours show an inherited ability to

survive under starvation conditions. A study by Izuishi et al. showed that different

pancreatic cell lines displayed remarkable survival under nutrient starvation

conditions. More specifically, the cells maintained 50 % of their viability up to 48 h in

nutrient deprived cultured medium [21]. Similarly, Kim et al. observed that among

different cancer cell lines (lung carcinoma, colorectal carcinoma), pancreatic cells

displayed remarkable tolerance to extreme nutrient deprivation. [20]. However,

several studies showed a beneficial effect of metabolic stress on treatment of

pancreatic cancer. For example, Lu et al. reported that Kigamicin D, a potential

cytotoxic compound, resulted in enhanced cytotoxic effect in glucose deprived culture

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medium, compared to nutrient rich conditions [110]. Similarly, Momose et al.

investigated the effect of various potential cytotoxic compounds on the survival of

PANC-1. The presence of those agents led to enhanced cytotoxicity under nutrient

deprived conditions compared with nutrient abundant conditions [111]. Ueda et al.

observed increased cytotoxicity of the drug Grandifloracin in PANC-1 under nutrient

deprivation [112].

From all the above studies it is clear that the cocktail of nutrients plays a key role on

the pancreatic cancer response to drugs, therefore, the nutrient level and content is a

necessary input when screening pancreatic cancer ex vivo (Figure 1).

Metabolic stress biomarkers

The relevance of metabolic stress on the response of pancreatic cancer to treatment

underscores the potential clinical value of candidate biomarkers of metabolic stress.

Indeed, several studies identify biomarkers that are expressed under metabolic stress

conditions. Guo et al. reported that PANC-1cells under starvation have higher

expression of LC3-II protein levels. LC3-II is the autophagy –associated form of the

microtubule-associated protein 1 light chain 3 [113-115]. Treatment with the

autophagy inhibitor chloroquine under nutrient deprived conditions significantly

increased LC3-II expression and inhibited cell growth [116]. Moreover Ueda et al.

presented that combination of nutrient deprivation and treatment with the natural

compound Grandifloracin increased LC3-II expression up to 22 fold in comparison to

nutrient abundance and resulted to reduction of cell viability [112]. Similarly,

Hashimoto et al. showed that nutrient deprivation increased LC3-II levels by almost

70% in PANC-1 cells compared to nutrient rich conditions. Additionally, combination

treatment of PANC-1 and BxPC-3 cells with common chemotherapeutic drugs and

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chloroquine led to an increase in LC3-II protein expression which was coupled with

enhanced cytotoxicity for both cell lines [27].

Overall, as stated above, LC3-II is one of the key proteins in the molecular

mechanism of autophagy, which is a general environmental cell stress response

mechanism to metabolic stress (nutrient deprivation) [20,112,116], but also to

oxidative stress [117-119] and treatment (chemotherapy and/or radiotherapy)

[28,31,120-122]. This molecular mechanism (autophagy) is highly elevated in

pancreatic cancer [123,124]. Therefore, the LC3-II protein could be an interesting

biomarker candidate to be quantified and monitored in 3D tissue engineering systems,

especially due to the occurrence of oxygen/nutrient/drug gradients in such systems

(Figure 1, Table 4).

3.3. Temperature stress

Increased temperature could affect cell proliferation and consequently influence the

response/resistance to treatment. For that reason, there are clinical studies that

investigate the synergistic effect of hyperthermia and chemotherapy and/or

radiotherapy [125,126]. Ohguri et al. notified that chemoradiotherapy combined with

regional hyperthermia applied to 29 patients with advanced pancreatic carcinoma led

to better survival than the chemoradiotherapy alone [125]. Similarly, Assogna et al.

reported the beneficial effect of applied hyperthermia on the adverse effects of

chemotherapy for 25 pancreatic cancer patients. Apart from the clinical studies, in

vitro studies also examined the role of temperature stress on pancreatic cancers’

resistance/ sensitivity to treatment [126]. For instance, Liao et al. reported that when

different pancreatic cell lines were exposed to heat stress at 45oC they overexpressed

the BAG-3 (Bcl-2 Associated Athanogene 3) protein, a modulator of cellular anti-

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apoptotic activity [127]. Furthermore, Mocan et al. studied the growth of pancreatic

cancer cells, as influenced by hyperthermia via laser treatment, in a nano-biosystem.

The cell viability was significantly reduced after thermal treatment [128]. Similarly,

Guo et al. indicated that hyperthermia increased the gemcitabine sensitivity in

pancreatic cell lines [129]{Guo, 2015 #273;Guo, 2015 #273}.

Temperature stress biomarkers

Cells respond to heat by inducing the synthesis of a group of proteins called the heat

shock proteins or hsps [130]. HSP27 belongs to the family of heat shock proteins that

basically act as molecular chaperones in cells exposed to different types of stress,

such as heat shock and/ or irradiation, oxidative stress, chemotherapeutic drugs

[130 ,131-133]. Schäfer et al. indicated that the application of hyperthermia (at

41.8oC), increased the over-expression of heat shock protein 27 (HSP27) and the

sensitivity of pancreatic cell lines to the chemotherapeutic agent gemcitabine [134].

Similar findings on HSP27 were reported by Guo et al. [129]. In contrast, heat shock

protein HSP 27 overexpression has also been reported to increase gemcitabine

resistance in pancreatic cancer cell lines [18,19,135]. Overall, these studies strongly

indicate that temperature stress affects the response of pancreatic cells to treatment

and it is very important to monitor the quantitative evolution of the HSP27 as readout

of the platform (Figure 1 &Table 4).

4. Monitoring environmental stress using metabolomics: a great potential for

novel pancreatic cancer biomarker detection in 3D cultures

Further to the previously listed biomarker candidates for monitoring the

environmental stress response in 3D systems new biomarker molecules can be

detected through metabolomics. Metabolomics is the high-throughput analysis of all

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the measurable free metabolite pools [136]. It offers a quantitative and holistic insight

of the metabolome of a particular biological system. There is already a significant

interest of the scientific community in the application of metabolomics analysis in

deciphering aspects of cancer metabolism, as it offers a metabolic snapshot of the

cancerous state. Metabolites are a very big group of compounds (41993 now

described at the Human Metabolome Database [137,138]) and it is highly likely that

some of them are correlated with specific diseases. Additionally, metabolic profiles,

which consist of the relative concentrations of many metabolites simultaneously, can

be proven very sensitive and accurate as biomarkers as they reflect the total metabolic

condition of a sample and can include subtle differences which cannot be easily

detected by classic single metabolite quantitative methods. Moreover, they do not

require the full knowledge of the metabolic network and metabolomics samples’

collection is not necessarily invasive, as samples can be collected from body fluids

such as plasma, saliva, tears, sweat and urine [139].

Several metabolomics analyses have already been applied for the study of pancreatic

cancer. Most of them concern of human samples and not pancreatic cells, but plasma

[140,141], saliva [142], urine [143],serum [144-147], tumour tissues [148] and all the

common metabolomics platforms, such as GC-MS, LC-MS, CE-MS and NMR have

been applied. Specifically, Berger et al. applied lipidomics profiling and managed to

discriminate normal plasma profiles and profiles collected from pancreatic cancer

patients [140]. Based on the same idea, Urayama et al. found differences on the

metabolic profiles of pancreatic cancer plasma samples vs. healthy plasma samples

with lactate characteristically increased in pancreatic cancer plasma samples [141].

Sugimoto et al. used saliva samples to compare oral, breast and pancreatic cancer and

managed to identify 57 metabolites that could be used for early detection purposes

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[142]. Napoli et al. used urine samples to discriminate the metabolic profiles of males

with PDAC and healthy ones [143]. Tesiram et al. applied NMR to serum samples

and detected significant differences in lactate, taurine, cholines and fatty acids

between normal samples and samples collected from patients with pancreatic cancer

[146], while Kobayashi et al. claim to have reached a diagnostic-level of sensitivity

with the same kind of samples [147]. In a study with a total number of 99 samples,

Bathe et al. compared the serum metabolic profiles of benign hepatobiliary diseased

and pancreatic cancer individuals [145]. They were able to distinguish the two types

of profiles while there are important differences in glutamate, glucose, creatine and

glutamine concentrations.

Pancreatic cancer cell lines were also compared with pancreatic ductal epithelial cells

revealing that normal ductal epithelial cell line (H6C7) displayed metabolic profiles

that were significantly distinct from three pancreatic cancer cell lines (MIA PaCa-2,

PANC-1, AsPC-1).[149].

However, all the above studies have been conducted with cell lines in 2D systems.

Metabolomic analysis in 3D pancreatic cancer culture systems, simulating in vivo

conditions, could be translated into sensitive, dynamic and accurate monitoring of the

cancerous system ex vivo. 3D systems monitoring with the use of metabolomics

analysis offers a quick and robust snapshot of the metabolic physiology which reflects

even to subtle changes of the cell physiology, something that cannot be achieved with

other omics analyses. More interestingly, the ability to combine intracellular

(fingerprint) and extracellular (footprint) metabolomics data could give the

opportunity to achieve a good level of knowledge on the biological system and watch

the dynamic transitions of the cells towards different physiological conditions, as the

population of cancer cells within a 3D matrix is not homogenous.

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Overall, quantitative knowledge on the relation of the production/evolution as well as

the potential distribution of one or more biomarkers within a tissue engineering

construct as influenced by (micro-) environmental conditions as well as treatment

would be a significant step towards enhancement of predictability of treatment

response of pancreatic cancer [43,150-152].

6. Conclusions

For decades, pancreatic cancer remains a highly lethal disease. Despite the useful

information retrieved from animal studies they still lack translatability and they are

expensive and not always reproducible. Furthermore, the classically used 2D cultures

for in vitro drug and/or irradiation screening have been unable to efficiently predict

the in vivo response to treatment. Clinical trials have shown that combinatory drug

therapy may be beneficial for pancreatic cancer, as summarised in Table 5 [153,154].

The development of in vitro tumour models which would capture elements of the in

vivo pancreatic tissue are a very promising, low cost, accurate approach for

accelerating drug development and application of novel therapies from bench to bed.

Advances in tissue engineering have enabled the construction of in vitro platforms

that recapitulate mammalian tissue aspects such as structure, porosity, extracellular

matrix mimicry, and cell-cell interactions. Next to the ex vivo architectural tissue

mimicry, monitoring the impact of the environmental stress adaptation through the

quantitative control of the production and/or distribution of stress biomarkers within

the in vitro tissue (in situ analysis) could allow a more accurate correlation of the

pancreatic cancer cell survival with the cellular environmental (stress) metabolic

response, therefore, enhancing treatment predictability.

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Acknowledgements

This work was supported by the Department of Chemical and Process Engineering of

the University of Surrey as well as an Impact Acceleration Grant (IAA-KN9149C) of

the University of Surrey, an IAA-EPSRC Grant (RN0281J) and the Royal Society.

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Figures Legends

Figure 1: Designing a bio-mimetic bio-inspired in vitro platform for pancreatic cancer

screening

Figure 2: Metabolism of healthy and cancer cells. Fluctuations of the micro-

environment and time evolution may accumulate mutations in normal tissue cells,

leading them to a cancer stem cell (CSC) phenotype or to a malignant cell. Normal

and cancer cell metabolism are characterized by activities of metabolic pathways.

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Tables Legends

Table 1: Advantages and disadvantages of current systems for in vitro studies for

pancreatic cancer

Table 2: 3D culture systems for pancreatic cancer screening

Table 3: Effect of environmental stress factors on the response of pancreatic cancer to

treatment

Table 4: Environmental stress response biomarkers candidates for pancreatic cancer

Table 5: Recent clinical trials of combinatory drug therapies for pancreatic cancer

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