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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: [email protected]
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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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