Therapeutic Approaches for
Treatment of Gliomas
Sura
Zaki, Sha Jin
Sura Zaki, Sha Jin, Department of Biomedical
Engineering, College of Engineering, University of Arkansas, Fayetteville, AR
72701, the United States
Correspondence to: Sha Jin, Department of Biomedical
Engineering, College of Engineering, University of Arkansas, Fayetteville, AR
72701, the United States
Email: sjin@uark.edu
Telephone: +479-575-5298
Fax: +479-575-7696
Received: October 22, 2013
Revised: December 4, 2013
Accepted: December 9, 2013
Published online: March 18, 2014
ABSTRACT
Although tremendous research efforts have been made,
cancer remains one of the leading causes of death worldwide. Glioblastoma
multiforme (GBM) is the most aggressive primary malignant brain tumor due to
its highly heterogeneity and resistance to standard treatment approaches. Poor
survival rate in gliomas patients related to the presence of blood-brain
barrier (BBB) which represents potent obstacle against a wide range of drugs
including most anticancer agents. Many novel strategies have been developed to improve
glioma prognosis and treatment by designing delivery systems for targeting
therapies that specifically attack cancer cells without causing damage to
surrounding healthy brain tissue. These targeted therapies are often promising,
but with limited progress upon clinical applications. In this review, we
highlighted the recent novel techniques for malignant glioma treatment,
including cell encapsulation technique, gene therapy, nanotechnology, stem
cell-based therapy, immunotherapy, and targeted therapy.
© 2014 ACT. All rights reserved.
Key words: Glioma, Brain tumor,
Targeted treatment, Cell-based therapy, Gene therapy, Nanotechnology, Cancer
vaccine
Zaki S, Jin S. Therapeutic
Approaches for Treatment of Gliomas. Journal of Tumor 2014; 2(3): 99-107
Available from: URL: http://www.ghrnet.org/index.php/JT/article/view/
INTRODUCTION
Cancer is a fetal disease of
uncontrolled cell growth of genetically altered cells which can be developed in
almost all tissues. One of the most critical and challenging tumors is that of
the central nervous system (CNS) which arises in the tissues of the brain and
spinal cord. According to the World Health Organization, three major types of
brain tumors are characterized and classified into gliomas: astrocytomas,
oligodendrogliomas, and oligo- astrocytomas. Each year, more than 20 million
people are diagnosed with cancer and one million die from this disease despite
most recent available remedies[1]. Patients diagnosed with brain
tumors have the lowest survival rates within five years after diagnosis[2-4].
Astrocytoma is the most frequent type of brain tumors. It is developed in glial
cell type. It constitutes about 50% ~ 60% of primary brain tumors. And
glioblastoma multiforme (GBM), the grade IV astrocytoma, is the most common and
aggressive type of these primary malignant brain tumors[3,5,6]. The
invasive behavior, rapid and aggressive regression of GBM results from cellular
heterogeneity. Besides, GBM appears to be resistant to conventional treatment
like chemo and radiotherapies[2]. Glial tumor cells are highly
infiltrating. They can disperse within normal brain tissue leading to tumor
recurrence. Residual brain tumor cells resist conventional adjuvant remedies by
intrinsic factors like protection from alkylating agents, and extrinsic factors
like selective properties of the blood-brain barrier (BBB)[4]. These
anticancer techniques aimed at controlling tumor growth rather than trying to
cure it[1].
A unique challenge in brain
cancer treatment is the blood-brain barrier (BBB). BBB- associated selectivity
is controlled by the endothelium of capillary perfusion to the brain[7].
The concept of this unique membranous barrier that separates the blood and
brain was developed hundred years ago, after the evidence that most organs can
be stained by intravenously injected dye, excluding the brain as well as spinal
cord[8]. BBB is important for the roles of the central nervous
system (CNS)[9]. The protective and selective characteristics of the
BBB results from tight junctions between capillary endothelial cells formed
through cell adhesion molecules[8]. The BBB represents a powerful
hindrance against large number of drugs including most anticancer compounds,
peptides, and nucleic acids. Therefore, this barrier restricts the access of
effective remedies of many acute and life threatening diseases such as brain
cancer. The relative impermeability in order to constrain the access of
molecules and cells between blood and brain is giving a natural protection
against circulating toxic or infectious factors. However, to overcome and cross
the BBB, assertive research efforts have lately aimed to develop new approaches
to efficiently deliver drug targeting brain tumors[8]. In this
review, we review and discuss the conventional therapies as well as some new
strategies for brain tumors treatment.
CONVENTIONAL
THERAPIES IN GLIOMAS
Gliomas are normally resistant to
conventional therapies, including aggressive surgical removal of tumor mass,
chemotherapy and radiotherapy. Poor survival rate and inefficiency of
conventional treatment to eradicate gliomas is due to their highly invasive
nature since tumor cells disperse within normal brain parenchyma and typically
cause tumor recurrence at the surgical site[4,5,10]. The infiltrative
nature of glioma cells permits some cell migration into long distances from the
original tumor mass. Several factors result in GBM resistance to the therapies.
They are (1) poor ability to deliver anti-cancer drugs to the brain across BBB[11];
(2) because of their low molecular weight, chemotherapeutic agents do not
maintain efficient concentration within GBM due to short blood half-life[6];
(3) the expression of multidrug resistance genes is considered an important
factor in developing resistance[12]; and (4) continual repopulation
of gliomas from cancer stem cells allows prolonged cell survival[13].
Therefore, the cancer stem cells play a major role in tumor aggressiveness and
therapy resistance.
Surgery
Glioma surgery aims to establish a
tissue sample for a pathologic diagnosis, alleviate tumor mass and eliminate
the influence on the surrounding brain. Hence, surgery improves neurological
symptoms, provides possibility of achieving complete resection, and provides
time for other remedies such as irradiation and chemotherapy to be performed[14,15].
Several techniques are in use to improve glioma surgery. Some are designed to
conserve functional integrity including preoperative MRI diffusion tensor
imaging. Others are designed to maximize glioma removal such as intraoperative
MRI and fluorescence light microscopy[16]. Furthermore,
intraoperative electrostimulation mapping, which serves both aims, is
increasingly used by neurosurgeons to improve the advantage/risk ratio of
surgery[17]. Intraoperative stimulation mapping has been proven to
be a gold standard in neurosurgery in patients with glioma for the avoidance of
postoperative neurological worsening. Brain mapping techniques have been
utilized to provide clinical and radiological finding for preoperative
programming and enable the visualization of functional area and their proximity
to the tumor sites. Preoperative information determines the ability of not
causing injury in trajectory of tumor excising[14]. Intraoperative
stimulation mapping for cortical and subcortical mapping is a credible and
powerful approach to facilitate greater extent of surgical debulking with an
impact on survival and minimize associated morbidity profile even when gliomas
are located within or close adjacent functional pathways. Particularly, the
importance of mapping motor and language pathway is well accomplished for
secured resection of intrinsic tumors[18]. However, neuronavigation
is known to be associated with intraoperative brain shifts. To solve this
problem, intraoperative MRI has been introduced to continuously update imaging
data with compensation for the brain shift[14]. Historically, biopsy
has been utilized for tumor placed closed to or deep within eloquent territory
whereas stereotactic biopsy is adapted for deep- seated tumors. Thus, resection
is usually saved for superficial lesions. Moreover, craniotomy and resection
have been used for patient with considerable mass effect which leads to
enhanced intracranial pressure or permanent functional deterioration[18].
Chemotherapy
Chemotherapy has gained a
significant function as primary treatment for progressive pediatric low grade
glioma (LGG). Several promising studies have demonstrated that the safety and
efficiency of using chemotherapy as an alternative therapy for LGG due to the
concerns about toxicity of radiotherapy in young children. Radiotherapy is
known for its late effects on neurocognitive, endocrine function and growth. It
may also enhance malignant transformation and recurrence. Therefore, there is
an intensive interest regarding repeated chemotherapy for progressive LGG,
especially in young children to delay or replace radiotherapy[19,20].
Chemotherapy has been mostly used as a single agent approach, by either
alkylating or platinum- based chemotherapies[19]. Recently, combined
therapies over prolonged periods were applied to address the biology of tumors[19,21,22],
such as receiving carboplatin and vincristine (CV) or thioguanine,
procarbazine, lomustine, and vincristine (TPCV)[14]. It was found by
meta- analyses that adjuvant chemotherapy could be beneficial, and this was
indicated by a significant increase in the survival rate for particular time
intervals. Patient with anaplastic astrocytomas can take the advantage more
than GBM patients[21]. The best outcomes of adjuvant chemotherapy
are achieved with a nitrosurea based- regimens either carmustine (BCNU) or a
combination of procarbazine and lomustin (CCNU) besides vincristine, known as
PCV-3 therapy[15].
In chemotherapy,
temozolomide (TMZ) represents a class of second generation imidazotetrazine
prodrugs[2]. TMZ undergoes spontaneous hydrolysis under physiologic
pH into highly reactive methylating agent, methyl- triazenyl imidazole
carboxamide (MTIC)[15,23]. TMZ acts as an alkylating agent with antitumor
activity against malignant glioma[21]. In vitro, TMZ has
shown schedule- dependent activity against broad spectrum of tumor types such
as sarcoma, lymphoma, and melanoma[22]. TMZ gains its antitumor
activity in CNS tumors due to its small molecular weight and lipophilic
characters which enables it to cross the BBB[24]. It also has shown
distribution to all tissues, relatively low toxicity. It displays 100%
bioavailability within 1-2 hr when taken orally and has been reported to pose
antineoplastic activity in relapsed high grade glioma (HGG) and mycosis
fungoides. This mild- to moderate- cytotoxicity which is predictable and easy
to be managed has made TMZ a valuable potential in treating and improving the
quality of life of patient with glioma[22]. On the other hand, the
enzyme methylguanine methyl transferase (MGMT) is responsible for repairing
mechanism such as chlorethylation or methylation damage stimulated by
nitroseurea and TMZ. Therefore, characterization of molecular markers such as
MGMT has shown to be predictive marker for treatment response since the
expression of MGMT can be silenced by methylation of the CpG island in the
promoter region[14,25]. Therefore, suppressed MGMT gene results in
lacking the full ability to repair the chemotherapy- induced DNA damage and
consequently tumor reduction[26]. This was associated with better
survival in all patient with GBM, especially those who are treated by RT plus
TMZ[25]. Although both TMZ and dacarbazine (DTIC) are structurally
and functionally related, and both are hydrolyzed to the same active compound
MTIC, TMZ has a major potential benefits over DTIC in that TMZ is able to
penetrate the CNS thus affects CNS tumor metastases. Furthermore, DTIC is only
metabolized in the liver whereas TMZ does not require hepatic metabolism for
activation due to its ability to cross CNS[22]. Compared to other
chemotherapeutic agents such as nitrosureas, platinum compound, and
procarbazine, TMZ does not lead to cross- linking of DNA strands. Therefore, it
has less cytotoxicity to the hematopoietic progenitor cells in the bone marrow
than the other compounds[26].
Chemotherapy is also used as
an alternative choice of treatment if tumor continues progression after surgery
or radiation[19]. In 2007, See and Gilbert demonstrated the
significance of using chemotherapy combined with RT. According to a study
conducted on randomized patients with GBM, there was a considerable increase in
survival rate of 26.5% at 2 years compared to 10% in the combined RT with MTZ
group and the RT alone group respectively[25]. Depends on the
outcomes of clinical studies, TMZ has been approved by European Union for the
treatment of patient with recurrent GBM after standard therapy[22]. Moreover,
TMZ received approval from FDA for the treatment of refractory and relapsed
anaplastic astrocytoma (AA) in adult[24]. Furthermore, understanding
the mechanisms of tumor angiogenesis have had an immense impact on targeting
recurrent GBM by applying bevacizumab (BV)[27]. BV, also called
Avestin, is a humanized monoclonal that was accelerated to be approved from FDA
in May 2009. It acts as an antiangiogenic agent by targeting the formation of
new blood vessels or angiogenesis which contribute to a considerable
development in the treatment of this fatal disease[28]. GBM are
highly vascularized tumors that highly utilize vascular endothelial growth
factor (VEGF) for new vascular supply which is required for further tumor
expansion and aggressiveness[29]. By targeting the tumor
neovascularization, it is possible to bypass the poor drugs penetration across
the BBB to reach their target. Moreover, it was evident experimentally and
clinically that antiangiogenic agents can minimize vasogenic edema and
patients’ demand for corticosteroids, which is considerably associated with
morbidity[27]. Addition of BV to RT/TMZ first line therapy is
endurable without obvious unexpected toxicities, except for potentially
increased frequency of arterial and venous thromboembolism. Increased median
progression- free survival (PFS), but prolongation of overall survival is still
unclear. Therefore, further clinical studies are required to determine patients
who will benefit from the incorporation of BV into upfront treatment of GBM[29].
Radiotherapy
Radiotherapy is always applied in
the treatment of malignant glioma. The addition of radiotherapy and surgical
resection is more beneficial compared to surgical therapy alone[14,15].
Also, postoperative radiotherapy combined with TMZ has considered being the
mainstay of care for patient with newly diagnosed GBM, according to the outcome
gained from a large European- Canadian phase III trial. This phase III trial
indicates prolong median survival of one year after applying radiotherapy alone
to 14.6 months after applying combined therapy RT plus TMZ with survival rate
of 1.9% versus 9.8%, respectively[30]. Radiation therapy is
recommended when the complete surgical resection is not possible with tolerable
morbidity[31], and when symptomatic patients display progressive
relapsed glioma[30]. External beam radiation is advantageous for
selected patients. However, it only occasionally results in prolonged survival[21].
This so called ”limited-field” approach of irradiation is applied to the
majority of gliomas (greater than 90%) because the relapse at the primary site,
the junction of the primary site, and the surrounding brain[15]. Due
to this recurrence pattern, brachytherapy, stereotactic radiosurgery (SRS) or
stereotactic radiotherapy (SRT) techniques were applied. These techniques
included the delivery of high dose of radiation by the tumor volume and
minimizing the radiation dose to the surrounding normal brain tissues[21].
A woman diagnosed with optic glioma was treated with radiotherapy which has
contributed to the improvement of ten years PFS, and overall survival. Modern
RT techniques were used that permits high specificity in delivering radiation
therapy including fractionated stereotactic radiotherapy (FSRT) combined with
image guided radiation therapy (IGRT). Therefore, these modern techniques are
more beneficial than classical ones because it provides the opportunity to
reduce the amount of normal tissue within the high dose volume[31].
DRAWBACKS OF
CONVENTIONAL THERAPIES
Cancer patients receiving
chemotherapy suffer from unwanted side effects. Many chemotherapy drugs induce
mutation that causes abnormal changes in the DNA. They also cause skin
irritations, hair loss, weakness, anemia, and loss of appetite. Chemotherapy is
toxic, and it should only be utilized when it is verified to be effective. Therefore,
side effects occur when the chemotherapy damages normal and healthy cells which
maintain the body's function and image[32]. Radiotherapy has similar
side effects as chemotherapy and a number of studies have shown that radiation
can kill oligodendrocytes, stem cells populating the subventricular zone, and
progenitor cells of the dentate gyrus of the hippocampus in rodent models[33].
Therefore, there is an urgent need to develop novel therapeutic approaches that
would both specifically targeting tumor cells and repairing brain damaged
tissues[33]. We describe some approaches in brain tumor treatment in
the following sections.
MANIPULATING
TUMOR ACIDIFICATION
Controlling of the extra and/or
intracellular pH of tumors may have significant prospects as anticancer remedy[34].
Acidification of extracellular space plays an important role in cancer
invasiveness or what is called acid-mediated invasion[35,36]. Otto
Heinrich Warburg, the Nobel Prize winner, found that malignant tumors unlike
normal cells use glycolytic metabolic pathway, wasteful glycolytic conversion
of glucose into lactate, even in the presence of sufficient oxygen tension,
this phenomenon is called aerobic glycolysis, the Warburg effect[5]
which is less efficient than oxidative phosphorylation to generate energy (ATP)[37],
or what is called mitochondrial respiration[34]. Therefore cancer
cells are able to survive and proliferate in the acidic microenvironment that
developed as a result of (a) tumor production of lactic acid by anaerobic
glycolysis in tumor sites which are hypoxic, and (b) aerobic glycolysis or
Warburg effect[34,35]. Tumor cells keep their high proliferating
rate in this hostile microenvironment by continuous removal of accumulating
protons produced as a result of high lactic acid production to avoid
acidification of intracellular pH, so glycolytically produced acid must be
expelled by tumor cells via a number of proton transporter[37], such
as V-ATPase[36], the Na+/H+ exchanger (NHE)[1], the
carbonic anhydrases 9[37]. These hyper active proton pumps
contribute to disturbance of pH gradients that enhance cancer phenotype[1].
Therefore, blockage or inhibition of the membrane ion pumps tends to reduce
intracellular pH. Few drugs have been used clinically as proton pump inhibitors
(PPIs) like omeprazole, esomeprazole. For example, PPIs can covalently inhibit
the V-ATPase when activated by mildly acidic environment[34]. The
anticancer role of PPIs was first identified in human B-cell tumors. These
inhibitors enhance pro- apoptotic action in different B-cell tumor- derived
cell lines. PPIs can be tumor- specific agents as they are activated in the
acidic extracellular pH, which is a characteristic of cancer cells[1].
Another strategy is to suppress cancer metastasis achieved by oral
administration of high doses of systemic buffer such as sodium bicarbonate[35]
or trisodium citrate or even special diet of low to moderate protein content
and full of potassium enriched juices, fruits, and vegetables. These substances
are able to inhibit cancer aggressiveness by reducing extracellular
acidification[34].
CELL
ENCAPSULATION TECHNOLOGY AS A THERAPEUTIC APPROACH FOR BRAIN TUMORS
In 1964, T.M.S. Chang suggested the
concept of using ultrathin polymer membrane microcapsules for the
immunoisolation of implanted cells and introduced the term 'artificial cells'
to define the idea of bioencapsulaion[38]. Cell encapsulation
system, by which viable cells entrapped within a non-degradable, selectively
permeable membrane to be isolated from host cells and immune response, shows
promising method for long-term delivery of therapeutic factors including
powerful cancer remedies[39]. Encapsulation technique can be
classified as either macro or microencapsulation[33,40,41].
Macroencapsulation approach
In the 1940s, macroencapsulation
was developed by Algire who implanted cells into diffusion champers and it
works as arteriovenous (AV) shunts which are connected to blood vessels
enabling blood flow through the lumen of these synthetic blood vessels. Drug-
containing cells are immobilized on the surface of the AV shunts and they are
within the diffusion area of influence of the blood vessel surrounded by a
membrane[40,41]. Macroencapsulation devices can be removed easily in
case of undesired side effects or graft failure. However, it is unfavorable
application because of their physical properties related to their large size,
especially their surface-to-volume ratio; it also results in membrane breakage
and limited cell loading capacity[42]. In addition, due to the large
sizes of macroencapsulation devices, they trigger an inflammatory response in
host tissue, and this leads to limited use of this technique as therapeutic
release system[41].
Microencapsulation approach
Microencapsulation has many
considerable advantages in comparison to macroencapsulation[40],
including small size, larger surface-to-volume ratio. It can be synthesized
easily from different types of stable and biocompatible polymers. Furthermore,
it can be easily implanted and retrieved[42]. In this aspect,
genetically modified cells that produce recombinant proteins may affect the
tumor microenvironment. The encapsulated cells that are protected from immune
rejection have been implanted to target tumor cells. Genetically engineered
cells can be immobilized in polymers. Currently, various polymers have been
utilized for immunoisolation. Alginate had the lowest cytotoxic effect and the
optimal cell attachment properties[40]. Cells embedded in alginate
beads meet all required considerations which include the capsule should be
semi-permeable to nutrients, oxygen, and the manufactured drug, while
antibodies and immune cells are excluded. Also, biocompatibility is considered
between the encapsulated cells and the implantation site. Moreover, the capsule
should be mechanically and chemically stable[38]. Biodistribution is
another index for the success of therapeutic applications. For cancer therapy,
implanted cells which can be encapsulated for immunoisolation may comprise (1)
Cells that secrete chemotherapeutic drugs targeted tumor tissues[41],
or cells that code for enzymes that are locally activated a non-toxic prodrug
to a cytotoxic drug at the tumor site[10]. (2) Cells uploaded with
anti-angiogenic factors such as angiostatin and endostatin[33,40,41],
and also anti-vascular endothelial growth factor (VEGF) which inhibits the
overexpressed VEGF, a prominent growth factor which function an important role
in tumor progress[43]. (3) Cells that release cytokines that trigger
host's immune response against tumors such as TNF-α[10,41].
GENE
THERAPEUTIC APPROACHES
The use of gene delivery for brain
cancer therapy is another promising strategy as it involves in situ delivery of
the required genes that drive the therapy to tumor cells. Hence, it is
important to design a vector that delivers a specific gene to tumor tissues
effectively[2,44]. Some strategies of these vectors have been
developed to deliver therapeutic transgenes for cancer treatment including
prodrug- activating genes, conditional cytotoxic approach[3], or
'suicide' genes that enhance local cytotoxicity of anticancer drugs[45].
For instance, tumor cells were transduced with herpes simplex virus thymidine
kinase (HSV-tk) gene which activates systemically delivered ganciclovir (GCV)[10,44].
Cellular kinases convert GCV- monophosphate into triphosphate form, which leads
to DNA polymerase inhibition and binding to the DNA of proliferated cells. This
results in inability of cell proliferation, promotes cell apoptosis, and cell
death of transduced cells[4]. Alternatively, genes that code for
immunostimulatory molecules to initiate antitumor immune responses[44,45]
and to enhancing the immunologic memory against tumors[3] are
employed for tumor therapy as well. For instance, cytokines play an important
role as immunoregulators, and they show positive effects in experimental animal
tumor models. It is significant to use gene therapy since the direct administration
of cytokines is restricted by their possible cytotoxic effect and their short
half life time. This approach has been applied successfully in experimental
brain cancer patterns[10]. The best studied cytokines include IL-2,
IL-4, IL-12, granulocyte-macrophage colony stimulating factor (GM-CSF)[44],
IFN-α, IFN-β, IFN-γ, and TNF-α. In addition to
stimulate an immune response, these molecules can promote programmed cell death
and also act as anti-angiogenic agents[10]. Another strategy for
gene therapy is to use RNA interference (RNAi) to block glioma pro-survival
pathways and to silence oncogenic genes[3,10]. RNAi technique
utilizes antisense oligonucleotides to inhibit gene expression at translational
level by binding specifically to certain mRNA sequences[3]. For
example, inhibition of overexpressed epidermal growth factor receptor (EGFR)
which is responsible for the propagation of 90% of GBM and inhibition of
transforming growth factor β II (TGFβ2) expression by RNAi,
can lead to suppression of glioma growth[11,44]. Furthermore, genes
that code for anti-angiogenic factors can be another therapeutic gene. Endostatin,
as angiogenic molecule, is one of the most important factors required for brain
tumor development and progression[44]. Endostatin was delivered via
viral vector which injected intra-arterially and promoted survival time by more
than 47% in brain cancer rats[10].
Gene transfer can be
accomplished by the utilization of various vectors which can be divided into
three major groups: viral, non-viral, and cell-based vectors[4]. Retroviruses,
adenoviruses, and herpes simplex are the best and widely used vectors against
brain cancers[10,44]. Viral vectors can be subdivided into
replication-deficient[10], replication-competent or oncolytic viruses[44],
and oncolytic viruses associated with a therapeutic gene[45]. Bacteria
have shown an important role as efficient vehicles of cellular-based gene
delivery, especially to hypoxic areas of tumor, and also to produce a potent
immune response[44]. Recently, the use of neural stem cells for the
delivery of prodrug- converting enzyme has been developed for a new cell-based
delivery carrier[10]. Non-viral vectors involve naked DNA and
liposomes which enter the target cancer cells by endocytosis[44].
APPLICATION OF
NANOTECHNOLOGY TO BRAIN CANCER TREATMENT
Nanoparticles (NPs) can be defined
as solid particles at nano scale. Their size ranges from 10-1000 nm.
Nanotechnology-based cancer therapies have shown wide application in medicine
such as screening, diagnosis, and treatment of cancer. Therapeutic agents are
dissolved, restrained, encapsulated, or bounded to the matrix of nanoparticles[46].
Due to their small size, NPs can easily interact with biomolecules located on
the cell surface or inside the cells. Furthermore, small size of NPs enables
them to penetrate cancerous tissues deeply in specific manner, resulting in
enhanced tissue-specific drug delivery[47]. Hence, nanoparticles
offer broad spectrum applications in the treatment of tumors. They can either
function as drug delivery system or promote cytotoxicity to cancer cells[3].
It is important to use nanomaterials-based therapy technique since it has
several significant advantages including the improvement of poorly
water-soluble drugs, prolonged circulation half-time by minimizing
immunogenicity[48], sustained or controlled release rather than
frequent administration of drug[47], and preferential accumulation
of NPs at the site of disease as a result of passive targeting via enhanced
permeability and retention effect
by passing through fenestrations of tumor's blood vessels which are more
permeable than normal ones because of their deformity[6,48,49]. Moreover,
NPs concentrate within tumor mass via active targeting specific surface
ligands. Although NP-mediated drug delivery can reduce systemic side effects[48],
a drug delivery NP must be stable in the circulation for adequate period of
time, may be for days, to reach their desired targets[6]. After
their parenteral administration, NPs are easily recognized by plasma proteins
called opsonins. These opsonins include reticulo-endothelial system (RES) cells
which bind onto surface of NPs through a process called opsonization that
function as a bridge between NPs and phagocytes. Delivering this drug-carrier
system to phagocytic cells leads to its rapid clearance from blood and hence
alters the drug biodistribution profile[6,46]. For above reasons, it
is essential to develop NPs for medical applications by surface coating them
with hydrophilic molecules such as polyethylene glycol (PEG), polyethylene
oxide, polysorbate 80, and poloxamine[6,46-49]. Studies show that
PEG is designed to enhance pharmacokinetics and biodistribution of
NPs-associated drugs for lowering clearance rate and reducing uptake by
opsonins[48,49]. PEG- surface coated NPs or stealth-shielding on the
surface of these drug carrier systems are referred to as PEGylation, a process
of either covalent or non-covalent binding or adsorption of PEG onto NPs[6].
Two major classes of NPs are
used in clinical trials include liposomes and polymer-drug conjugates. Other NP
modalities like dendrimer, nanoemulsions, inorganic[47,48], gold,
and ceramic NPs have also utilized as therapeutic carrier systems[48].
Gold nanoparticle-mediated hyperthermia is based on the systemically
administration of the gold nanoparticles and their accumulation in tumor
tissue. The tumor is then exposed to a heated environment through external
inducer like near infrared (NIR) laser light, radiowaves, or magnetic field. Thus,
NIR- absorbing gold nanoparticles like gold-silica nanoshells, gold nanocages,
and gold nanorods can kill tumor tissues by heating both in vitro and in vivo. This
type of cancer therapy has demonstrated an immense impact in cancer treatment[3,49].
Importantly, the amount of scattered light is proportional to the absorbed size
of tumor site. Moreover, magnetic targeted hyperthermia relay on their specific
inorganic characteristics by using metallic nanoparticles which turns
electromagnetic energy into heat[49,50]. Direct cytotoxicity can be
enhanced by targeted delivery of chemotherapeutic agents specifically to GBM. Madhankumar
et al. illustrated that specific delivery can be occurred by the specific
binding of liposome-based anticancer agents with IL-13Rα2 receptors which
overexpressed by GBM but not by normal counterparts. IL-13-conjugated liposome
is an efficient and promising approach for direct killing of cancerous lesions[51].
Liposomes have been utilized as therapeutic drugs carriers due to their good
biocompatibility, easy preparation, low toxicity, and commercial availability[6].
This combination between nanoparticle and anticancer drug can reduce systemic
cytotoxicity and side effects[51].
STEM
CELL-BASED THERAPIES
In the 1960s, stem cells were
discovered by Drs. Ernest McCulloch, James Till, and professors at the
University of Toronto and the Ontario Cancer Institute. These cells have unique
properties such as the ability to divide and self-renew over long period of
time; they are unspecialized; and they can differentiate into more than
multiple types of cells, a feature known as plasticity[52]. It has
been anticipated that over 100 million Americans can get benefits from using
stem cell in the targeting therapy including cancer treatment[53]. Among
organs that harboring adult stem cells, neural stem cell and neural progenitor
cell populations (NSC/NPC) of the central nerve system are found from brain
early development to adult stage. These cells are responsible for the
maintenance of neurogenesis and gliogenesis in the developing and adult brain[54].
In general, there are three sources of human stem cells that can be used for neurological
disorders treatment: NSCs, embryonic stem cells (ESCs), and bone marrow[53].
NSCs are characterized by their unique migratory ability and targeting glioma[3],
in addition to self-renewal capacity and multipotency[53,55]. In the
adult brain, neurogenesis is primarily occurring in two precise regions: the
dentate gyrus of the hippocampus and the subventricular zone (SVZ) of the
cerebral cortex. NSCs localize in a specific microenvironment known as the stem
cell niche[54]. The complexities of treatment of the brain tumors is
due to the unique neuroanatomical location of the injuries next to special
neurovascular structures[56] and number of lesion region(s) which
can be either focal or multifocal[57], as well as cellular
heterogeneity that includes cells expressing neural/progenitor stem cells,
astroglial, and neuronal markers[3,54]. Furthermore, the highly
diffuse invasiveness of tumor cells and their resistance to conventional
therapies result in rapid and aggressive recurrence[56,58]. While as
NSC selectively migrates to tumor site, it is considered as the best candidate
for the treatment of brain tumors either for injured tissue replacement or as
drug delivery system in vivo[12,56,59]. Because of the
selective migratory potential, NSC can be engrafted into injured CNS within
distinct areas. After NSCs implantation, the migrated cells are incorporated
into the local neural site of injury and accompanied by their regular gene
expression. Recent evidence has indicated that the implanted exogenous NSCs may
affect the surrounding niche by enhancing protection and renewal of host neural
pathways[56]. Damaged tissue repairing can be promoted by
recruitment of endogenous stem cells or lineage-committed precursor cells. Experimental
stimulation of some lesions was accompanied by maximizing proliferation of stem
cells of the subventicular zone and homing of newly formed cells into the site
of injury[59]. Moreover, the recruitment of endogenous stem cells
and their ability of self-renewal are associated with the formation of new
neurons and oligodendrocytes that become normally integrated into the CNS
parenchyma[59]. Transplantation of unmodified NSCs leads to extend
survival of animals with experimentally induced tumors[56]. The
preferential migration and homing of stem cells to brain tumor sites is
enhanced by factors secreted by gliomas and cells in the surrounding niche, and
this can play a role in delivering anti-tumor agents to gliomas[12].
In addition, NSCs that are genetically modified to produce IL-4 enable them to
promote tumor remission. Using NSC treatment, the life time was prolonged in
glioma mice[59].
On the other hand,
genetically modified NSCs are considered to be a desirable option for
delivering anti-cancer remedies because of NSC’s glioma-tropism[12].
Such modified cells can display either direct cytolytic molecules like
TNF-related apoptosis inducing ligand (TRAIL)[3] or suicide genes
which regulate the enzymatic conversion
of non-toxic prodrug into activated oncolytic agents[3,55]. A variety of prodrug/enzyme systems have been
developed by using NSCs as an enzyme delivery vehicle for brain tumors, such as
cytosine deaminase and HSV- thymidine kinase[3,12]. There are other
two types of stem cells except NSC have been also used in the treatment of
brain tumors[12]. Mesenchymal and embryonic stem cells (MSCs and
ESCs) have been suggested for cell transplantation. MSCs derived from bone morrow
have attractive properties such as providing degree of immune tolerance. This
may help reduce host rejection to transplanted cells. ESCs have also been used
to produce neural precursors by their pluripotent potential with a degree of
immune privilege[53]. The route of cell administration depends on
the site and number of CNS lesions[57]. The concept of intranasal
delivery of NSCs may be a convenient and noninvasive alternative approach for
stem cell-based therapies since the direct intracerebral administration is an
invasive approach with low engraftment efficacy[58].
IMMUNOTHERAPY
FOR TREATMENT OF MALIGNANT BRAIN TUMORS
The immune system
The immune system is designed to
function in the specific recognition and to eliminate foreign pathogen from the
body within minutes of infection. The patterns of response can be classified
into innate response and adaptive response. The innate immunity comprises
monocytes and tissue macrophages such as microglia, granulocytes, natural
killer cells (NK), and antigen presenting cells such as dendritic cells (DC). These
cells are capable of initiating rapid and nonspecific immune response as a
result of responding to signals from damaged tissues or infection through
recognition of surface pathogen-associated molecular patterns (PAMP) such as
mannose and lipopolysaccharides as well as heat-shock proteins derived from
tumor cells. Inflammatory mediators and cytokines are involved in the immune
response[60]. On the other hand, adaptive immune response including
highly specific, lymphocyte-directed response which developed through four
distinct stages: recognition and activation, clonal expansion, and effector
function, and memory. This type of response is either humeral (antibody-
mediated immune response) secreted by differentiated B-cells (antibody
producing plasma cells) or cell-mediated immune response achieved by CD4+
helper T-cells or CD8+ cytotoxic T-cells (CTL)[61,62].
Immune privilege in the brain
The brain is considered an
immune-privileged organ to which the immune system has limited access due to
the lack of lymphatic draining system and its separation from circulation by
BBB[60]. The concept of immune privilege in brain has been supported
by the successful engraftment of allogeneic tissue grafts placed within the
brain of experimental models[63]. In addition, many autopsy studies
have revealed the absence of naïve T-cells in the brain due to their inability
to pass through BBB, suggesting the lack of major machinery important to
initiate an immune response in the brain. Nevertheless, there is growing
evidence that supports the presence of multiple immune pathways in the brain[60].
First, local microglial cells, which can process and present tumor antigens,
function as major antigen presenting cells (APCs) in the brain. These cells
express phenotypic and functional features of both DC and macrophage[64-66].
Second, activated lymphocytes can infiltrate into a tumor and initiate
anti-brain tumor immune response[63,67]. These tumor-infiltrative
lymphocytes (TILs) are associated with long survival in patient with GBM. Finally,
in vitro expansion of TILs revealed the existence of tumor antigen-specific
lymphocyte, indicating that an effective adaptive immune response can be
occurred[66].
Tumor mediated immune suppression
Patients with GBM display a profound
immune suppression induced by gliomas production of immune inhibitory factors. Such
immune suppression is mediated by cytokines and growth factors such as
interleukin-10 (IL-10), transforming growth factor-β (TGF- β), prostaglandin- E2[3],
macrophage chemoattractive protein (MCP-1), IL-6[65], as well as
VEGF. Those inhibitory factors can cause lymphopenia, downregulated lymphocyte
protein expression, and impaired antibody yield[64]. Therefore, it
is important to develop effective strategies that specifically combat these
suppressive effects and to find effective approaches to deliver the immunologic
effector molecules to the brain tumors[3,64]. Immunotherapy can be
divided into active immunotherapy which imply the use of tumor vaccines in
which the patients can be immunized by tumor cells themselves or characterized
tumor antigens, and passive immunotherapy in which the tumor-specific effector
cells are prepared in vitro and transferred to the patients either
systemically or intracranially[64,68].
Immunotherapy
The immune system has various levels
of control to ensure the appropriate balance between immune stimulation and
immune suppression. Over two decades the basics of this regulation have become
comprehensible. The knowledge may provide opportunities for a precise immune
response against tumors[65]. For example, tumor- associated antigens
expressed by gliomas and their ability to artificially stimulate class I and II
MHC- restricted antigen presentation have contributed to manipulate the immune
response as a target of immunotherapy[67]. T-cells play an important
role in tumor vaccine therapy because of their ability to specifically activate
and proliferate against tumor cells and also generate a memory mechanism[62].
Major histocompatibility complex (MHC) class I-tumorigenic peptide complex
which is able to activate CD8+ T-cells has been utilized in the
preparation of tumor vaccines[61,69]. Moreover, CD4+
T-cells are involved in the elimination of tumors that are resistant to CD8+
mediated response. CD4+ cells cooperated with NK cells to perform
this effector function. This observation of anti-tumor activity by CD4+
cells is independent on the expression of MHC molecules on tumor cells,
suggesting the possibility to recognize poorly MHC-expressing tumor cells by
designing approaches to stimulate CD4+ T-cells response against
tumor antigens[65]. Thus, an attractive strategy is to use DCs to
stimulate an immune response against tumors by loading of tumor antigens on the
autologous DCs which are isolated from the patient by leukapheresis followed by
re-administration of these cells into the patient. During the incubation
period, autologous DCs are activated by a mixture of recombinant cytokines or
immunostimulatory molecules[65].
Furthermore, it is possible
to utilize irradiated autologous whole cancer cells[68]. Gliomas not
only display a variety of tumor antigens, but also are able to present these
antigens to T cells. During in vitro and in vivo stimuli, such as
exposure to IFN-γ, it was found that
gliomas are able to express low levels of class I MHC. Hence, glioma showed
ability to present tumor- associated antigens to CTLs by the class I MHC
pathway[67]. Moreover,
phase I studies of subcutaneous vaccinations using synthetic peptides against
glioma- specific antigen epitopes have been conducted. The ultimate goals of
vaccination were safety and to obtain CD8+ T-cells sensitization
against targeted glioma- specific antigen. It was found that patient with newly
diagnosed high- risk LGG
demonstrated better vaccine- responsiveness than recurrent patient
represented by a positive tendency for IFN-γ ELISPOT responses as well as median PFS[70]. Another approach
involves ex-vivo activation and proliferation of tumor-specific CTLs by
peptide-pulsed DCs and to stimulate potent CTLs immune response in vitro
by using mRNA- transfected DCs[3]. Transfection of DCs with mRNA
encoding certain tumor proteins is simple and efficient[71]. Furthermore,
specific targeting of overexpressed determinant in GBM such as epidermal growth
factor variant III (EGFRvIII) by DC vaccines. The overexpression of those
surface receptors which are commonly mutated in malignancies results in
uncontrolled cell growth[3]. Moreover, monoclonal antibodies can be
directed towards the gliomas by targeting EGFRvIII[11]. For
instance, Nimotuzumab and Cetuximab/Erbitux are the two antibodies used
currently[66]. Another cytotoxic approach involves the blocking of
early angiogenesis by targeting of tumor endothelial cells which display
distinct surface markers that are not found on normal tissues. Anti-angiogenic
activity can be achieved by the adoptive transfer of T-cells that specifically
recognize tumor endothelial cells and leads to destruction of tumors by cutting
off the blood supply to the growing tumor[64]. Anti-angiogenic
activity can also be reached by means of anti-VEGF pathways such as using
Avastin antibody that competitively binds to VEGF receptors and thus inhibits
the cytokine binding and simulation of these receptors[66].
TARGETED
THERAPY
The long-term usage of anti-cancer
agents leads to lethal destruction for normal cells in addition to the cancerous
cells due to their systemic cytotoxicity and limited specificity. Therefore,
development of novel anti-cancer drugs with minimized toxicity and targeted
delivery approaches for the treatment of GBM are highly desired. Accomplishment
of targeted therapy requires preferential binding to tumor cells[6].
To overcome the drawbacks of chemotherapy, many strategies can be used such as
enzyme-prodrug therapy which confers a highly localized drug at the site of
choice. Well-characterized enzyme-prodrug combination involves the use of
herpes simplex viral vector-thymidine kinase/ganciclover (HSV-TK/GCV). It
achieves in situ delivery of effector enzyme by encapsulation[41]. Inhibiting
angiogenesis in glioma is another targeted treatment. This approach is based on
the fact that glioma stem cells are regulated by producing VEGF and glioma
tissues receive nutrient and oxygen via vascular niche. VEGF and
stromal-derived factor-1 are two angiogenic agents that stimulate angiogenesis.
Hence, utilizing anti-angiogenic therapy is believed to have the potential to
overcome GBM resistance to conventional therapy[54]. Besides, VEGF
contributes to BBB destruction in glioma, results in increase in infiltration
of interstitial fluid and the formation of intracerebral edema. This formed
edema reduces the delivery efficacy of medication to glioma cells. Moreover,
hypoxia plays a role in the sensitivity of gliomas to chemotherapy and
radiotherapy. Hence, it was proposed to use angiogenic inhibitors to reduce
brain edema, and to promote drug delivery and increase radio-sensitization. Anti-cancer
treatment can be achieved by using single inhibitor or a combination of
inhibitors and also a combination of inhibitors with chemotherapeutic agents[6].
Taken together, many efforts
have been made for brain tumor treatment even though GBM is still incurable. Clinical
trials have been conducted based on targeted drug delivery strategy and can be
found elsewhere[29,72-74]. As a direction for further development of
effective treatment of brain tumors, eliminating cancer stem cells from the
tumor is likely the key in order to control recurrence of brain tumors. CD133
antigen has been considered as a putative stem cell marker in malignant brain
tumor[75]. Thus, targeting this cancer stem cell marker could impact
the survival of GBM patients. Mammalian target of rapamycin (mTOR) is another
GBM cancer stem cell marker that can be used in a targeted therapy[76].
Overall, identifying biomarkers for targeted drug delivery which can overcome
BBB would improve patient response to therapy and enhance survival of brain
tumor patients.
CONFLICT OF INTERESTS
There are no conflicts of interest
with regard to the present study.
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Peer reviewers: Kanno Hiroshi, Professor, Department of Neurosurgery,
Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku,
Yokohama, 236-0004, Japan; Jian Yi Li, MD, PhD, Assistant Professor and
Co-Director of Neuropathology, Department of Pathology and Lab Medicine,
Director of Neuropathology at the Brain Tumor Institute, North Shore-Long
Island Jewish Health System, Hofstra North Shore-LIJ School of Medicine, 6 Ohio
Drive, Suite 202, Lake Success, NY 11042, the United States.
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eaquam apercid qui dipsum re volupis et qui doluptatus, que laboruntur sum re
consere moloreptatur aut velendipsant ipsam sinverepudam vit poris elibus aut
aciisin rerit autas eum a quam aute santuris rerchillis sit que od mo ipsa ipsae
et fugiand istiunt ipsam aut aut ullignit excerum quae magnis ea dolo quidus
nis aut que sit optae pediam errum in prae sae. Ut re laborem laut veri venimag
natur? Et harumet eum atus veniam idel ipsanda dellam, quos dolest, oditatur
abore cum fugit, toresequam exerfer uptatur?
Luptat min cumquam, sum,
qui omnisqui custio. Bis ulles conecta eatquis aut aut maximint officipsum qui
dolore, quation conestibus volo consedit es amusam quat as es ad millabo. Nam,
enihit quia pra venis accab illo cor assi dolum dolorer natisse quameniet
velecta non re es enissequam asition poritasim ipsam et fugiti doluptia conecus
aut magnam, opti se eic te consedi blam ad es assequam, non est, nuscipic
tendignat rat fugias verferentiat landania sumet od ut velendent.
Untiis alic totas eum
voluptat aut ationsequi beatem non con es nobit repera sam, si berio. Nam des
ipsa sae sunt fugitatiist ut laut quae et volecto ipsaped explati istiatempore
pratio. Itatecti sa dic tem. Vel eatem. Nem nust endia none di torpos
recepernam quaspeditia demperios in praecae ctibus doluptat.
Um aut eos rehenem lab
inctem remperi vendit aut exerchil etusapiendis dolora dellabo rioribusam es
aliat.
Parum, cum aut quis
dolum ullesequi aut assimus rerepudaest quos explabo. Otatus eiciendi sunti as
evelibea con rehendis nonsecturem dest, quid quibus vere, temperro blam fugit,
cones que ipicitio exeriae rchilli busdae sunt.
Soluptio ommolorem con
prae. Agnim quae volecto to veliti doluptaque eatiistio. Da seditia
digendictate pre velluptatur alibusa vellabor aut qui blaborum et vendis si que
sus abo. Mus audam debit laccum nossus.
Optaspe lluptatiorem et
aut enihict iostiunt veritat iuntendebit harum illuptae consende pre vende
essunt abor rehendae eum fugiam volupta spidella dolupta soluptae molorep ersped
qui tem veliquaessit fuga. Elest poreptaque nihillibus remposapero tecus con es
audant harciat ut et volore cus, aut pliquatassit occulparume nam, omnia
voluptate ma doloreribus aut officiaspit pore pro odis maximpo riscipi
tatinvenimi, ut laccae aut aciis recture hendaes trupiendunte ea quiatur ad et
aliti iunturepel etuscia cus doloribust ab iusandam quoste pa dis quatius solo
iunt lacessi ut que nia conectate vere culloreria aut quia dolectatis es am
estiam consequi cus es as et harupta dolupta tiores debit ex esto eum volore
non porectatia doloratus estionem idendi doluptibusam aut est, explamus, aut
fuga. Giae molorias sitatum untis sinvenetur re sunt discit, ommo voluptus
evelitiunt porese exerent que optiorum erum, sedi ut restiora sum voloreium, si
de nusandiasim que volent et mo quam acipsunt raturia consecae vernate porrovit
qui beres eius volut eate odit doluptatur? Quia pratat labor samusda velignatem
voloriores magnatates exerupid enis non prat vit volupis simente eataquis et
vel ipsae. Itatest emquibus eum, solo volesseque sed quamus in corum am re
sequo eatent.
Lis sum estintiis dis
eium sequis denderc ipictur remporu metur?
Simoste pre, torumqu
asimint urento toreptate voluptatium il intio odi blabo. Et am et ommolentus
dis repudit utatio odi occusa cusam qui dolorum et adi consequi cullend anitio
vellaudae vent aut aut ipsaerios ipsunt, optatem porendenitas sunt aditas aut
ma sit aut et optatquam quatur sit ius.
Faccatem et que
porporio. Quis archic tem reperum nobis dolecusa vid molenim uscium quunten
tendestium voluptas debit, ea conseque nis poressint in remporum si atur magnis
et es aut aut omnia doluptur? Quisitiae. Vit voloribus everum dolupta dolut et
odioreperia vid eos et quos earum as estiatur aut magni dicaepr eiusantes
soluptatet pore parcia porerro vitata cone laboria as dit aut aliquiam eos
exerum si offictorro occus estemol oreptur? Ab isciis conserro odis quid que
vent imod modis que expligent et endam quos eriatem fuga. Untiaepudi dolorum
lacea et, comnis restius apidestrum quam rerios doloribus as et que et doloria
que non placcum quis eum dolest fugia pro molum m faciendam rentota tinctorest
et occatur, intium fugiae experiae non nos ea consendae nobis estem eosaper
spienec umquid moloreperi nihit volorem. Ed molore esequat urector empores
etures volupti ncillab oruptatatium es nobit molupta tiberum nullorp
orehenimusam et es ero in porrum, ute aut que volestio. Que sae debis dipieni endelis
erem audae nonsequas entiis ut aut alignatem que consedi volupti il is dolorest
hil ipsam dem nonsent volorerios et, quam fugias moluptatet volorep taspici
tianis ipsaect otatur? Aditam vendandis sime oditest est, tem aliquibus sitem
fugitasit, id eici quation serferat fugit vera ipit viduciis con nonsend
esendis ullab ipsum et volesequasi re, voluptatur, optatem doluptatur simo
dolupta tiberitibus magnia quas ipis aceptae volorem quiaepero exceperovit
estibus sequam reptatur, sanditi asimporempel ilique et enis simaion coriones
mincitionsed quiam con nus exceatur solut et fugiam, omnihicim fuga. Osamusa
ectibus eatem del et arcides nis aliqui dionsequi aut eos sitio blam nestis
dolorib usdant.
Facera coristis
ape volor reptaspid min eates arianime net pa qui cus non repudamenis et et
elest, cuptatur ratiani hilitempor sit et que sim quame placcum que delitatis
alia nem lita ero corpos magnimusamet velibus, sequam faccust otaersp eribusa
ectate poreic tem sanda volora quas enia a iduciatur?
Aximi, nonseque
cupta nimolen iendeniame dolorit, occae earum ea quatest est aut accum faccumq
uostia int.
Cium vero im
velestrum que dus modicte velicia im none nihitis ea dolupist faciden dipsum
dolupictet poribus, seque prerios a corae. Xerum quia accum aut quatur?
At magnient od
es corro velescil eos dolorem exerfero veribus.
Borem rest
ratiumquam eatur? Parumet, optam quasperi ut asped quam, sit, optatis erum
quassitis sequossin pra nihilis re destis volorum quae comniendi consequis
nectis dolupit, quidebit ut dolupta pori id ent experum quibereptat.
Vellatur, cor
aut aliquiate lam harumendem eossi ipsunto tendem ut moluptatem. Itatus ipsunt,
ipsam ad eum iducias doluptae ipidelitatur mo illupti abor sit volorep udicia a
evenima num vel entius reium, eum nobis pel mod quide excerov itaspid mi,
seritatur alitatur modiorerum aut eliqui conest, et ere et acepell accumenda
dent accumende nus etur? Quiduciet reste dolo te volor aspitis earios aut
hitatur magnis comnia pore, ipidita veliquis et quiatur rerum quis que id
quatiis si alitet fugitam aditin re cusapic tem faccusdaniet landistia denderum
estiis qui antinci lluptas rerfersperis seque preperepel id molum illabo. Riae
voluptae diostores doluptaquas sapedi re nusdae peria eles untio ent, odia
deliamus, imus ipsam laborernam ut eaquo totati receaque laccus et lacest, sunt
et experum ipsandunt quos molo is eossit dolor molupid que volore nist aut ulpa
perovit velenis venecus iur? Uptae sim aut quo earum aut faccus, cores im ea
sinit dessitis experrum natiistin nit aruptate vellaccus, num imiliciis simo
qui a quia qui con nempere in nulliciis pe expe et fugia comniani blabo. Ut
moluptatis excerruntium quiaestem fuga. Hendias alis reiciae ea consequam et
voloreh enetus precatu reperatiur audis aut moles aut quis et quaeped ma
voluptae modis necest, cum aborent orpossum re restia ni re, et odi aut deni
vollaboriti siniend andebis sequodisque omnis dolupta nobis dolupiscium
explitatur amus ditinctia nullitaquo bearum et rehenim enimil etus apernam
laudaec umquia nos volescianda volecus molupti aperroribus quiam numquae quam
que omnim vene reptatusda simil ipicab int.
Elentiur as eum
faceri doluptatem voluptas mo dolores nonsequati doloreh enderunt eaquia
nulleni anietur re, volum nist, ut harit, qui ipsum cus dolo dolorem quia nis
aut aut ut odit molorro magnihil ipsamet odit ut pere litae nosa cus magniminia
deremodi diorem quias pernamusamus ad modipsa que por accatem is dolum quia
cusae volorer chiciur?
Volore nobitae
nestiorepro cusdae verionsed ut qui con resed que et es ipsum quatet ea
doluptati doloria doluptius et ilia corpore iciuscimpedi volupta tiistibusam
sitaquam vendae corite sit plaborem fuga. Nemos ra erspiduci im dolupta
tecuscit ipitae od quassun tenimi, idenissus, tem abor aditaeperit ma is quis
plis vernam quate suntotas et quia con rae sit, solupit, aut excessinciis sim
natia nonsequibus dios voluptatur a ad ullorrume evelit ad maionsequo etur sum
aniam quam, consequam quatis etur maxim qui optur, sum vid mi, veriosam autem
aut re magnam, quam nonsed quis et, explit, to et ut volumqui aditaepudis ipsam
abor molupic tem susa quodit, omnim fugita veris nobit earitibus ium, quunt,
quibust, idelige ntione earum el imiliquia dolor aut quamet molorem oluptus
doluptatur aut ad eos digent.
Sum que reperep
roreped molestrum quod quam vide essi consequ odicia porion expe volut vollente
nostemque dolorestrum landunt.
Eculpa que vit
rest, optation reria ab in periti ande natem dendel et quunt explaboribus
ipsuntotae. Itati officitatur aut que viduciis consequunt molorite nobit
liaepel itiae. Itat et pratur?
Omnimpore
dolupit ut ea consequae. Acimporis est, ium vendunt re, sandam, atur?
Omnimin velenis
sequo bearum dem faccus eos doloreserum int fuga. Itae laut illestrum inverum
rem cum, tem volupic to estia as commolu ptaerrum quo ium faccatendam quia quam
laboremqui ut lanita is as nat qui iliquam enis eosa sinimincium est, volorem
vel idus ra exceat dolupta eprovitaquas sum, te quis es es expedist, que
conesto reperaepel modit est esenihil es aris sum explign ihiciur? Qui optam
accus doluptur, volupta tempora voluptat.
Descipic te
nonecti blaborum quasper roremqu aection secuscium aut odio vitaten imagnat ut
ius, sit rernate mporest quo omnim velestis et volluptibus.
Se niet
entur adictor erovidel inverup tatusam quiatureped quiatet quist, te num
recullu ptatet aceaque nos archici pitiorum aut es quos raerspercium consequam
facepta conse verrorerit lautatatem qui sapid es qui dollaborit offictur
acepero videst dolorro debis as sit plibus inventi usdae. Dolupti umquiae praepudam erchictur re doluptis el id mos mo comnis
eiuntur sanda comnis inciate mpossed mos sae. Luptati squiandus voluptatus
maior modi blaudaerciat ute nobis illes asit endi que laborit pro con ea dolum
si optatium et lant odici aut la nis voluptatem. Turiatur aut as es aspersp
elignaturem essit erae nusam rati dundunt oriorum volor si sequis de nesed
magnam, suntorr oviduciet laborem olupta sit et, ium reperia dellendio beatiunt
laturitatis aut faccust quo ipsunt offictis moluptatem quasi quundanda aut aperovit
fugit que sitatisincto volore sincidita nosanto torio molupta doluptus eos esti
ut hitem voluptat aut aut audam, optat.
Porrovit ad
mint fuga. Ut pre, alitem voloreperum rem que rehent exceruptate quid quis eat.
Rita dolorem
faceper uptiisqui aut et et fugitin eatia corum faces sunt.
Epresci
opturitis sandi raest, tem qui ipsandi que exerum illaboresti num in raecte
volut dolupti quos endaect emoluptas maximus sae occum ipis as doluptur re, est
illacepuda int, tem laborrum iduntur?
Aque magnitat
eum voluptatur aliquid esequatiist, non reruptiament la dis dolecto moluptae
nis quidus doluptaquam, santi restio blabore rioremo luptas eium ut volum non
pliquiate velenisquunt quam, quamet omnimax imenihic to idebit voloreped ma
volor alignatur, que doluptatur rem quibus.
Labor maionecum
conse sus aliquis maionemporro ommolor emoste dolorem nihici occum et
voluptatur rem volendanit qui voluptiis maximinus dolupicae a si re quid quos
sunt ad quassi num eatur asinveliquos id mod que dis ut occum que sit, vellace
ssimusam quatemolore pa venis doluptat assuntur?
Inume pre
dellupt aectetusam, offici quia que consectendae prere nus, seque cusam dipsust
eum fuga. Sit et qui utat officiatur a simusam veni ipsamus di anto te dollend
itateca borecte molupta taquis ea pore omnimus perrum iliquis sitas milias
denda que nos non nihilique que pliquo occumquia aut volorru ptatur alitat ut
quame ratur aut ut imusdae maxime estem demporr ovitataspe re natur soluptas
dolesequam ium unt que voluptae et ipsanim etustiaerios moluptaturia que
coreprores illupta quiatia teculpario cum nos esendi dolupta simpos maxim quae
quam fuga. Ignis ex evenimo luptatiumquo excest, am doloriore plic tem quae
volestore conserum m eat. Busae nihilib usamus erem remolup tianda ipis ut
hillorumeni comnimporis et, secero vento id et lab iminus volum ne ditemolo
odioratum venditem aut laborroribus delibus et ea sum fugitiumquam remque
magnis audio magniet, con repellab id qui blabo. Eribeaquas nest labor autat et quossit esequam nimin naturi demolentium voluptat.
Ehendae event quo cum ium ium ipsapictis assites aperrovit quiam sincimi
llabore rumque consequisque eictat quidem aut ut lab ipsum, voloritam ea
comnimporro et ant.
Hil maximin
imolestrume magnis voluptur?
Ovitatibus. Tur,
omnis minvelibust ipsum que lam, si dolorem re, solendi voloresequia versperum
qui del mi, ium unditatius volorro inctem est et hilia ducimporia antisci ut
acerrum quam ad quid quam es accusdam nustiunti nobit, to cus, is et et volupta
conem faccat.
Occulparcia aut
volent.
Simpore con
consequam volecab oriorestia nam, as experatecus esed ma eost qui utessit,
earum rerionet la que porunt labor aliquo modis ut isquat.
Os et dolupta
ecepudit quam conseque num soloriae vel millati aectibus a poraeculpa
doluptatem dolorera non est, volo oditae cum aut exerum nonsern atemque plia
volorit atatiori con nihit restet que solor reribus dent.
Pudisit ea quistotatet periora
ectionsequis dipsani ut dic test esequi unti iscia por magnam, sa voleseque num
faccusc ieture laccum quat doloreius suntibus, oditatiume doles rerae nonsentum
experup turenduci ni sit quaspid eatisque eost, erumque saerepudi verum ipis
ame offictas eariaspicte di odio berfernaturi occae volutat eniminc taerum ut
aligentenda pariam qui dolupti asperunt, nem nobit faceprore, sitatas inctas
dellitiis duci nos dolut ide aruptaepudis modit quis moluptatios mollate
dolores idellit optas molorporrum dolenis porem lant ut aut laborrorest et eius
accaecepero eremquate nienimilit unt ditio et omnis denihilist dolendelita
veniet lam
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