MUC4 Mucin - MUC4 as Pancreatic Cancer Tumor Maker

1. Introduction

1.2 MUC4 as Pancreatic Cancer Tumor Maker

1.2.2 MUC4 Mucin

MUC4 is a high molecular weight transmembrane mucin that is expressed by various epithelial cells (trachea, lung, stomach, colon and cervix) in normal tissues [38]. MUC4 has two

subunits, MUC4α and MUC4β. The two subunits are non-covalently

link to each other.

Schematic representation of the modular structure of MUC4 is shown in Figure 1

. The mucin type subunit MUC4α is of 850 kDa and the

membrane-bound growth factor like subunit of 80 kDa [39].

Numerous studies have established association of MUC4 with the progression of cancer and metastasis. An aberrant expression of MUC4 is reported in precancerous lesions, indicating its early

involvement in the disease process with no detectable expression in the normal pancreas or chronic pancreatitis [40].

Figure 1 A: Schematic representation of the modular structure of MUC4.

B: Schematic representation of MUC4 protein. The representation is not drawn to scale.

[39]

The RT-PCR analysis of MUC genes expression in pancreatic adenocarcinoma and chronic pancreatitis are shown in Table 1 and Table 2 respectively. Overlapping symptomatological characteristics have been observed between pancreatic adenocarcinoma and chronic pancreatitis [8]. However from the Table 1 and Table 2 we can easily observe that, in pancreas aberrant expression of MUC4 gene exclusively occurs

during pancreatic adenocarcinoma making it noval biomarker for diagnosis of pancreatic cancer.

Table 1 Expression of MUC genes in pancreatic adenocarcinoma by RT-PCR analysis^a. Total RNA from 15 pancreatic tumor cell lines was isolated and subjected to semiquantitative RT-PCR^a

aData obtained from reference [40]. ^b D.S., differentiation stage; PD, poorly differentiated; MD, moderately differentiated; WD, well differentiated; ND, not determined. ^c_, no expression; +, low level; ++, moderate level; +++, high level; ++++, very high level; ND, not determined.^d Normal tissue samples were included as controls. S.I., small intestine; S.G., salivary gland

Expression level^c

Table 2 Expression of MUC genes in chronic pancreatitis by RT-PCR analysis^a. Total RNA from 10 chronic pancreatitis samples was isolated and subjected to semiquantitative RT-PCR.

Expression level^a

a Data obtained from reference [40].^b _, no expression; +, low level; ++, moderate level; +++, high level; ++++, very high level.

1.2.3 Role of MUC4 in Cancer Development

During cancer development controlled interaction between neighboring cells and the cell and the extracellular matrix are interrupted, whereas new interactions establish due to alterations in the cell surface proteins, extracellular matrix composition, and loss of cell polarity. A favorable environment is created by these molecular and cytoarchitectural changes at the tumor site which facilitate in tumor development [41]. The MUC4 is a multifunctional protein implicated in a variety of biological functions. Under normal conditions, MUC4 is localized at the apical surface of the epithelial cells.

Genetic/epigenetic changes, alternative splicing, and biochemical modifications may result in an aberrant expression of MUC4 [12]. In addition, MUC4 can change the role of other adhesion-associated signaling molecules via steric hindrance. Lose in polarity of tumor cell, facilitate MUC4 to find new interacting partner(s). Recent studies established

that MUC4 interacts with HER2/ ErbB2 and alter its expression [41]. Role of MUC4 in cancer development is systematically shown in Figure 2.

Figure 2 Role of MUC4 in cancer development^[12]

1.2.4 Monoclonal Antibodies Targeting MUC4

The overriding challenge in developing antibodies to specifically target mucin proteins is lies in their structure, 80% of their mass is composed of glycan chains. The Glycan chains cover greater part of the core protein epitopes. The main mucin-type antibodies available recognize glycan epitopes and therefore do not bind with a unique mucin. For instance, the DU-PAN-2 antibody is reactive with MUC1 and MUC4 [40]. Recently, a series of monoclonal antibodies (mAbs), 9H8-1F3, 13F12-2C9, 12B8-2D9, 8G7-1D1, 12C11-1G2, 11A8-1B7, and K2G6-1H6, directed against the TR region of MUC4 have been reported [38]. To generate these antibodies a 16-amino-acid sequence was chosen same as that of 16 amino acid residues repeated in tandem up to 400 times for the main MUC4 allele [42]. The mAb, 8G7 has been discovered to strongly react against the MUC4 peptide and with native MUC4 from human tissues or pancreatic cancer cells in Western blotting, immunohistochemistry, and confocal analysis [38].

1.3 Contrast Agent 1.3.1 Background

It had been known long back that atomic nuclei, which possess a spin angular momentum, will interact with magnetic field. In 1971, Raymond Damadian observed that certain mouse tumors display high relaxation times compared with normal tissue and this was the was the inception of imaging the human body [43]. MR signal is due to relaxation of water protons that are trying to realign with a static magnetic field following the application of radiofrequency (RF) pulse. In 1948, Bloch et al. reported the use of the paramagnetic ferric nitrate salt to enhance the relaxation rate of water proton. Three

decades later Lauterbur et al. applied Mn(II) salt to distinguish between different tissue based on the differential relaxation time and thus produce the first MR image [44].

Extensive application of MRI in clinical imaging and biomedical research has promoted the development of a new class of pharmacological product, called contrast agent. It acts as catalyst in shortening the longitudinal and transverse (i.e., T1 and T2) relaxation time of water protons in tissue in which the agent accumulates, consequently, enhances the image contrast between normal and diseased tissue and indicate the status of organ function or blood ﬂow. Currently, 30% of all MRI examinations worldwide are performed with contrast agents [45].

The efficiency of an MRI contrast agent can be quantified by its relaxivity, r1, which is defined as the increase of the longitudinal relaxation rate of the water protons per mM of the paramagnetic compound. Relaxivity can be expressed as function of two sets of parameters. surrounding and second set of parameters in equation is responsible for the dynamic part.

Most important interactions which influence the paramagnetic relaxation enhancement

are, Fermi contact (A_iso) and the dipolar (

↔

T ) hyperﬁne interactions (HFIs), i.e. the

magnetic interaction between the spins of nuclei and electrons. Coupling tensor,

↔

Q, i.e.

The interactions of nuclear quadrupoles with the electric field gradient (EFG) are

important for the description of relaxation of nuclei with a spin number larger than 1/2, for instance ¹⁷O nuclei [46]. The important dynamic parameters (second part of eq. 4) that influence the paramagnetic relaxation are the exchange rate between the bound water molecules and the bulk water, kex, the longitudinal and transverse electronic relaxation times, T1e and T2e, and the rotational correlation time τ R [46]. These parameters are schematically summarized in Figure 3.

Figure 3 Important interaction and dynamic parameters defining the efficiency of an MRI contrast agent.

1.3.2 Paramagnetic Agents

Metal ions with one or more unpaired electrones are paramagnetic, paramagnetism is due to the spin and orbital angular momentums of unpaired electrons, and therefore have a permanent magnetic moment. However, the magnetic moments of individual paramagnetic atoms in a material are only weakly coupled to each other, and room temperature thermal energy is sufficient to overcome these interactions to eliminate any net magnetic moment [47]. A small fraction of the atomic moments aligns parallel to the field in accord with the Boltzmann distribution when placed in a magnetic field. The paramagnetic interaction with the field is weak and disappears in the absence of an applied field [48]. In an aqueous solution of paramagnetic metal, there is a dipolar magnetic interaction between the electronic magnetic moment of the paramagnetic atom and much smaller magnetic moments of the protons in its vicinity belonging to water molecules. Random fluctuations in this dipolar magnetic interaction, mainly a result of molecular motions, reduce both the longitudinal (T1) and transverse (T2) relaxation of water proton [49]. Gadolinium (Gd(III)) and manganese (Mn(II)) are paramagnetic ions which have been successfully used in MR contrast agents. The Gd(III) ion is an ideal paramagnetic metal ion for MRI contrast agent. The Gd(III) is an ideal choice as a paramagnetic ion is for several reasons. First of all, this ion is characterized by a large magnetic moment due to the half-filled 4f shell (7 unpaired electrons!). Secondly, due to the symmetric S state this ion has a relatively long electron spin relaxation time which is one of the necessary requirements for efficient paramagnetic relaxation enhancement.

However Gd(III) cannot be used as contrast agents in their ionic form due to an undesirable biodistribution and the relatively high toxicity [50]. Therefore, ligands have

been designed that can form stable complexes with paramagnetic metal ions to form strong chelates which can remain stable in the body and thereby signiﬁcantly reducing toxicity. The Gd(III) coordinate strongly to diethylenetriamine backbones modified with carboxylic acids. In complexes with such ligands the ion is normally nine-coordinate, were introduced. Since the approval of [Gd(DTPA)(H2O)]²⁻in 1988, it is estimated that over 30 metric tons of gadolinium have been administered to millions of patients worldwide [45]. Mn(II) complexes have also been investigated for its potential application in MRI. However due to poor stability it is not used as widely as Gd(III) complexes. Consequently so far, MnDPDP (Teslascant) in which Mn(II) is coordinated by dipyridoxyl diphosphate, is the only clinically approved agent [51].

1.3.3 Superparamagnetic Agents 1.3.3.1 Superparamagnetism

In general, macroscopic ferromagnetic materials are divided up into domains of parallel magnetic moments for minimization of their energy. Within a magnetic domain, the magnetic moments orient in one direction, while the alignment of spins in neighboring domains is usually antiparallel. The oppositely aligned magnetic domains are separated

from each other by a domain wall (Bloch wall). As the particle size decreases below some critical value, the formation of domain walls become energetically unfavorable and the ferromagnetic particle can support only a single domain structure. The critical diameter for a magnetic particle to reach the single domain limit is equal to [52]

36 ₂ saturation magnetization. Magnetic particles of nanometer size are usually in a single domain structure [53]

The amount of energy required to reverse the magnetization of a single domain particle, over the energy barrier from one stable magnetic conﬁguration to the other is proportional to KV/k_BT where V is the particle volume, k_B is Boltzmann’s constant and T is temperature [54]. In zero magnetic fields, the energy barrier ∆E has to be overcome to rotate the magnetization of a single-domain particle as shown in Figure 4. Because the height of the barrier, ∆E = KV, is proportional to the particle volume V, ∆E may become comparable to the thermal energy (kBT, where kB shows the Boltzmann’s constant) when the particle size decreases. If the thermal energy is large enough to overcome the anisotropy energy so that the energy barrier can no longer pin the magnetization to the time scale of observation, the magnetization is no longer stable due to thermal fluctuations and the particle is said to be superparamagnetic (SPM) [55].

Figure 4 Energy barriers from one stable magnetic configuration to the other

1.3.3.2 SPIO Nanoparticle in Molecular Imaging

Molecular imaging, in general, refers to the study of cellular and molecular events through noninvasive investigation. In MRI, molecular imaging depends on induced changes in proton relaxivity of in vivo water molecules on the molecular and cellular level. In last one decade, biocompatible iron oxide particles conjugated with targeting moiety for targeted molecular imaging applications has been extensively investigated.

Monoclonal antibodies labeled with superparamagnetic nanoparticles are expected to be good tumor-specific contrast agents because of their high specificity against some cancers. Superparamagnetic iron oxide nanoparticles (SPION) are currently used for clinical imaging of liver tumors and prostate, breast and colon cancers as well as for the delineation of brain tumor volumes and boundaries.

1.3.3.3 Recent Advancement in Superparamagnetic Agents

Magnetic nanoparticle probes are emerging as a next generation contrast medical imaging. Super paramagnetic iron oxide (SPIO) particles were suggested as potential liver speciﬁc MR contrast agents as early as the mid 1980s [56]. Recent research shows SPIO particles have potentially to generate higher contrast enhancement in MRI than conventional paramagnetic Gd-based contrast agents. Numerous chemical methods have been reported for synthesize of SPIO nanoparticles for medical imaging applications:

microemulsions [57], sol-gel syntheses [58], sonochemical reactions [59], hydrothermal reactions [60], hydrolysis and thermolysis of precursors [61], ﬂow injection syntheses [62], and electrospray syntheses [63]. Aqueous co-precipitation process in the presence of the coating material has been frequently employed for the synthesis of SPIO and USPIO.

The main advantage of this process is that a large amount of nanoparticles can be synthesized. However, the control of particle size distribution is limited, because only kinetic factors are controlling the growth of the crystal [64-66]. The saturation magnetizations (Ms) Values of nanoparticles obtained by these methods are in the range of 30–50 emu/g, which is lower than the 90 emu/g reported for their bulk form. The low M_s value is due to incorporation of impurities hampering the crystal structure and surface effect [67]. Monodisperse magnetite nanoparticles has been synthesized at high-temperature by decomposition reaction of iron(III) acetylacetonate with 1,2-hexadecanediol in the presence of oleic acid and oleylamine to. The particle diameter can be tuned from 4 to 20 nm by varying the reaction temperature [68]. However, use of hydrophobic oleic acid and oleylamine surfactants in the process results in a hydrophobic coating on the particle surface, amphiphilic polymer or surface surfactant exchange have been utilized to overcome this problem [69]. In order to meet the demand of excellent

magnetic property for the applications such as, molecular imaging, metal doped iron oxide nanoparticles have drawn much attention due to their enhanced magnetic properties. Spinel metal ferrites with a composition of MFe2O, where M is +2 cation of Mn, Fe, Co or Ni, have been fabricated by various methods to tune speciﬁc magnetic properties. Comprehensive study conducted by Lee et al. on 12 nm ferrite nanoparticles (MnFe2O4, Fe3O4, CoFe2O4, NiFe2O4) for MRI application shows that 12 nm MnFe2O4

NPs have the highest mass magnetization value of 110 (emu per mass of magnetic atoms) among MnFe2O4, Fe3O4, CoFe2O4, NiFe2O4 as shown in Figure 5. Furthermore their results also suggest that MnFe2O nanoparticles are nontoxic in vitro and possess higher magnetic susceptibility than magnetite nanoparticles, suggesting that they may be used as an ultrasensitive MR imaging probe [70].

Figure 5 Magnetism-engineered iron oxide (MEIO) nanoparticles. TEM images of MnFe2O4 (MnMEIO), Fe3O4 (MEIO), CoFe2O4 (CoMEIO) and NiFe2O4 (NiMEIO).

Scale bar, 50 nm^[70]

Recently, Seo et al. reported synthesis of bimetallic FeCo core of 7 nm and 4 nm with a single-graphitic shell through chemical vapor deposition (CVD), a systematic graphical

design shown in Figure 6. The Ms of the 7 nm and 4 nm nanocrystals were 215 emu/g and 162 emu/g, respectively with excellent r1 and r2 relaxivities [24].

Figure 6 Systematic diagram of a FeCo/GC nanocrystal ^[24]

The direct use of SPION as in vivo MRI contrast agents results in biofouling of the particles in blood plasma and formation of aggregates due to high surface energy that triggered by the “opsonization” process resulting in fast clearance of SPIO nanoparticles [71]. Therefore, it is essential to engineer the surface of the SPION to minimize biofouling and aggregation of the particles in physiological conditions for long periods.

Poly (ethylene glycol) (PEG) is widely used polymer for nanoparticle coating. Kohler et al. developed bifunctional PEG silanes capable of forming self-assembled monolayers (SAMs), furthermore terminal functional group (amine or carboxyl) extending out from the nanoparticle surface provide sites for conjugation of functional ligand.

1.3.4 Carbon Based Materials for Future MRI Contrast Agent

In last two decades a wide range of all-carbon nanostructures have been discovered for instance, fullerenes (1985), carbon nanotubes (1991), graphene (2004). Since the discovery of carbon nanotube (CNT) in 1991 [72], it has been used extensively used for various technological applications [73-74]. Chemically modifiable outer surface have been used as diagnostic and therapeutic agents in medicine [75-76]. Single-walled carbon nanotubes (SWNTs), fabricated from single sheets of graphene have been extensively investigated forms of carbon nanotubes for biological and medical and other technological application owing to its intriguing physical properties such as quantum electronic transport [77], a tunable band gap [78], extremely high mobility [79], high elasticity [80] and electromechanical modulation [81].

Although there has been doubt as to whether the ferromagnetic features of some of the graphitic materials was due to magnetic impurities such as iron. Recent study shows occurrence of intrinsic magnetism in carbon-based materials possessing the sp² network [82]. Carbon-based magnetic materials would bring a new prospective to technologies relying on magnetism for instance MR imaging. Surprisingly, the first organic ferromagnet, the γ-phase p-nitrophenyl nitronyl nitroxide (p-NPNN) was discovered only in 1991 [83]. Such materials may have low density, be transparent or environment-friendly.

Graphene, a two-dimensional honeycomb lattice of sp-bonded carbon atoms, has shown a wealth of exceptional properties. Since the discovery of the first isolated graphene in 2004, it has attracted major interest, because of its high charge mobility and crystal quality [84]. Xie et al. and Yu et al. have reported synthesis of carbon nanoscrolls

[85] and carbon nanotube [86] from monolayer graphene respectively. Schematic representation of CNS formation is shown in Figure 7.

Figure 7 Schematic representation of the formation of CNS. Step1: surface strain is induced in graphene after it is immersed in IPA solution. Step2: the edge of graphene is lifted up with the help of the surface strain and the intercalation of IPA solution. Step3:

the initial bending of the graphene is energetically unfavorable and might be caused by perturbations. Once the graphene gets selfstacked, the scrolling process will be easier.

Step4: the graphene continued to roll up until a CNS is formed.^[85]

Richerd et al. have reported noncovalent functionalization of carbon nanotube with amphiphilic Gd(III) chelates, a systematic scheme shown in Figure 8. The study shows T2 frequencies at any GdL concentration are remarkably lower than that of pure water, and they are practically independent of both the frequency and the Gd(III) concentration (8.5-13.8 ms for MWNT/GdL versus 2500 ms for water). Interestingly, the transverse relaxation times, T2 values of suspensions containing MWNT and the amphiphilic ligand L at various concentrations without any Gd(III) are in the same range as those measured in the solutions containing Gd(III) [87].

Figure 8 Carbon nanotubes noncovalently functionalized by amphiphilic Gd3+

chelates^[87]

Recently, Ananta et al. have report that raw HiPco SWNTs (r-SWNTs), purified SWNTs (p-SWNTs), and US-tubes show inherently high performance T₂-weighted MRI contrast agents by virtue of their superparamagnetic character, with the US-tubes being the most efficacious of the materials by far. The high-efficacy contrast performance is due to contributions from both the iron catalyst nanoparticles (originating from the synthesis of SWNT materials) and the carbon SWNT material itself. Table 3 shows relaxation time of Aqueous SWNT solution along with clinically-used SPIO (Ferumoxtran) T₂ agent measured at 1.41 T and 37 °C [88].

Table 3 Relaxation Times of Aqueous SWNT Solutions (dispersed using pluronic surfactant)^a

aData were collected with a Bruker Minispec Mq-60 spectrometer at 1.41 T and 37 °C.

For comparison, a clinically-used SPIO T2 agent (Ferumoxtran) is also included . ^b Based on the Fe concentration. ^c Based on the SWNT concentration ^[88]

Wang et al. has reported synthesis of soluble carbon nanotubes by the sonication of graphene oxide nanosheets. Systematic diagram of synthesis procedure is depicted in Figure 9.

Figure 9 systematic illustration of the mechanism for transforming GO nanosheets into carbon nanoparticles and nanotubes following their ultrasonication in acid. (I) Oxidative cutting of graphene oxide produces PAH molecules in concentrated HNO₃. In the dehydrating acidic medium, the polyaromatic fragments fuse and nucleate into (II) carbon nanoparticles or (III) nanotubes via acid-catalyzed intramolecular or intermolecular dehydration reactions.^[89]

sample [Fe] mM T1(ms) T2(ms) r1 (mM-1 s-1)b r2 (mM-1 s-1)^b r2/r1 T2(ms ·mg-1)^c

r-SWNT 0.449 34.6 63.6 4.6

p-SWNT 0.167 242.2 46.5 22.8 126.8 5.6 5.1

US-tube 0.075 1000 67.7 8.9 192.5 21.6 31.7

Ferumoxtran-10 9.9 65 6.6

The saturation magnetization of CNT synthesized by Wang et al. increased from 25 µemu cm^-2 to a maximum of 100 µemu cm^-2 upon annealing to 400 °C and gradually dropped to 41 µemu cm^-2 on annealing at higher temperatures [89].

Chapter 2

Experimental Section

2.1 Instruments and Regents 2.1.1 Instruments

1. Particles diameter analyzer (Dynamic Light Scattering): English Malvern Instruments Company, Model: ZetaSizer 3000 HAS

2. NMR relaxometer (NMS-120 Minispec, Bruker) 3. CO2 incubator (Japan SANYO, Model: MCO-20AIC)

4. MR scanner (3.0 T) (Sigma; GE Medical Systems, Milwaukee, WI) 5. MPCVD (ASTeX type microwave plasma CVD)

6. XRD

7. Field-emission scanning electron microscopy (FESEM, JEOL JSM-6700F) 8. Transmission electron microscope (Philips TEM)

9. Raman microscopy (LABRAMHR).

2.1.2 Reagents

1. Zymed Laboratories:

Mouse anti- MUC4, clone 1G8 2. HyClone

RPMI-1640 medium 3. Malliockrodt Methanol 99.7 % 4. TEDIA

Dimethyl sulfoxideDMSO99.9 %

5. GIBCO:

Dulbecco's modified Eagle's medium

2.1.3 Cell Culture Medium

Culture Medium for BxPC-3

RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 90%; fetal bovine serum, 10%

Culture Medium for PANC-1

90% Dulbecco's modified Eagle's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose + 10% fetal bovine serum

2.2 Experimental Methods

2.2.1 Immobilization of 8G7 mAb on SPIO-mPEG-NH

One hundred microliters of the SPIO-mPEG-NH₂ at a concentration of 4 mg Fe/mL was added to 400 lL of 8G7 mAb (ZYMED Laboratories, UK), using 1-hydroxybenzotriazole and (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexaﬂuorophosphate as catalysts, and the mixture was stirred for 24 h at room temperature. The solution was separated from unbound 8G7 mAb by dialysis.

2.2.2 Measurement of Particle Size

The hydrodynamic diameter of the SPIO-mPEG-NH2 particles was measured using a

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