Cancer is a group of diseases characterized by uncontrolled growth and spread of abnormal cells. If the spread is not controlled, it can result in death. Cancer is caused by both external factors (tobacco, infectious organisms, chemicals, and radiation) and internal factors (inherited mutations, hormones, immune conditions, and mutations that occur from metabolism. According to statistics of world health organization (2008), cancer is a leading cause of death worldwide, accounting for 7.6 million deaths (around 13% of all deaths). The main types of cancer are: lung (1.37 million deaths), stomach (736,000 deaths), liver (695,000 deaths), colorectal (608,000 deaths), breast (458,000 deaths), cervical cancer (275,000 deaths) . In 2012, about 577,190 Americans are expected to die of cancer, more than 1,500 people a day. Cancer is the second most common cause of death in the US, exceeded only by heart disease, accounting for nearly 1 of every 4 deaths .
Fig. 1.1 Proportion of frequent cancers in the world in men and women in 2008, the WHO .
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1.1.2 The fact of lung cancer carcinoma
As an estimation, there are about 226,160 new cases of lung cancer which are expected in 2012, accounting for about 14% of cancer diagnoses in the US. This type of cancer is increasing with aged-modality rate and accounts for more deaths than any other types of cancer in both men and women (28% of all cancer deaths) . There are many reasons that can cause of lung cancer but cigarette smoking is one of the most important risk factor that has involved. Exposure to other factors such as organic chemicals, radiation, air pollution or genetic susceptibility can play a contributing role to the cancer development. Non-small lung cancer is the most common type, accounting for 85% of lung cancer and another one is small lung accounting for about 14%. Currently, surgery, radiation therapy, chemotherapy, and targeted therapies such as bevacizumab (Avastin), erlotinib (Tarceva), and crizotinib (Xalkori) or combination therapies are being used for lung cancer treatments.
The conventional clinical methods still have limitations in both diagnosis and therapies such as non-specificity, side effect and almost fail to treat disease once cancer cells have been spreading to other organs (metastasis). The overall survival rate is low (16% for 5 years). The number will be higher if disease would have detected at earlier stage when tumor cell is still localized (52% survival for 5 years), however, only 15% cases are diagnosed at early stage . For that reason, it is vital to find out novel therapeutic strategies in which tumors can be earlier detected, feasibly controlled (localization and metastasis), less invasive as well as more tumor specific. And oncolytic therapies are one of such approaches which are being under developed.
1.2. Vaccinia virus
1.2.1 An overview of vaccinia virus
Vaccinia virus (VV) is enveloped virus belonging to the poxvirus family, a high pathogenicity and widely susceptible animal hosts . It has preserved the name vaccinia since Edward Jenner's time when it was first reported as an isolated material from milkmaid to vaccinate the smallpox disease in 1798. At that time it was known as cowpox virus. Until 1930s the cowpox vaccines used in 20th century for smallpox vaccination was determined as a distinct strain, named vaccinia. VV has the same genus with variola virus, a major strain causing smallpox pandemic which had been spread out over the world from 17th to mid-20th century with estimated deaths of about 300-500 million people. Until 1979, after the long vaccination campaign using attenuated VV , the WHO certified the eradication of smallpox in human . For that reason the viral structure and biology have been intensively investigated. Nowadays, despite not being used as a vaccine, VVs are still interesting tools for the transcription machinery studies or protein interactions . Moreover, with long-historical safely profile in human as a vaccine, broadly natural tumor tropism [7-8] and many other biological properties, VVs are considered potential candidates for oncolytic viral therapies [9-10].
The first vaccinia genome to be sequenced was vaccina Cophenhagen strain. The open reading frame (ORF) was determined by Hind III restriction map. VV is a large (about 191,636 kbp encodes for 2063 proteins) double stranded DNA containing virus with a complex enveloped virions . The virus replicates entirely in the cytoplasm of infected cells; thus, it must encode all of the enzymes and structural proteins necessary to transcribe the DNA genome.
1.2.2 Vaccinia virus molecular biology
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Fig. 1.2 The overview of vaccinia virus structure and replication cycle. (A) Virion structure includes several membranes and core, containing double strand DNA, enzymes, factors, v.v. for the initial replication of virus in host cells. (B) life cycle happens entirely in cytoplasm and occurs in three phases; early, intermediate and late, accompanying with different forms of virions . Mature vaccinia virions exist in four forms which differ in number of membrane surrounding and location of viral particles. Intracellular mature virus (IMV) is a first assembled and simplest infection form of virus with a membrane, which is remained inside of infected cells following virus maturation. Intracellular enveloped virus (IEV) is essentially IMV with two additional surrounding layers which is warped in Golgi apparatus . Cell-associated enveloped virus (CEV) is derived from IEV which outer IEV membrane is fused with plasma membrane. CEV retains attached to the outer surface of cell. And extracellular enveloped virus (EEV) is CEV which has been released from cell surface. Therefore, EEV is mainly responsible for the spread of virus during infection .
Initial step for virus entry is attachment of virus to cell plasma membrane. The detail mechanism of action is still not well understood. It can occur via direct fusion at plasma membrane or low-pH dependent endosomal pathways . Three viral membrane proteins: A27 , D8  and H3L  were showed to involve the attachment of virus to glycoaminoglycans (GAGs) at cell surface. But virus can also use GAG independent pathway to enter the cell . Soon after the entry, core was released from virion and virus uses packaging materials to start an early phase, followed by intermediate and late phases as illustrated in fig. 1.2. Life cycle of virus happens entirely in cytoplasm of host cell hence will not interfere or integrate to the host structural genome. Virus proteins which are necessary for functions of certain phages will be synthesized at different stages of infectious cycle. Gene expression during VV infection occurs in tightly regulated by temporal cascade featuring sequential synthesis of early, intermediate and late gene classes which are distinguished by special transcriptional promoters and its enclosed factors.
About half of VV genes belong to early class . The VV early class mRNA appears in minutes (20 min) and reaches a peak at about 100 min after virus' entry into the cells . Viral mRNAs are synthesized inside the core and are extruded outside its surface by packaged initial materials along with DNA genome which is necessary for early transcription and translation . They include early transcription factors such as 80 kDa and 72 kDa encoded by A7L and B6L correspondingly  which bind to promoters with assistance of RNA polymerase to activate the transcription processes [24-26]. Subsequently, the synthesis of early proteins is made. They include proteins needed for core wall uncoating reactions, viral DNA replicational and transcriptional factors to activate intermediate genes.
Early viral DNA releasings are those templates for intermediate-gene transcriptions which then are used to translate into transcription factors that at the end will be used for the transcription of late- phase genes. The intermediate genes are believed to be few in number . The late genes are detectable about 140 min after infection , mainly encoded for viral structures, virion enzymes as well as essential proteins such as early initial proteins which must be incorporated into virus particles during assembly. Once all necessary materials are synthesized, the assembled process is initiated to first form immature virions. These viral particles mature with brick shape called IMVs. IMVs acquire a second, double membrane from Golgi apparatus to form IEVs. Next step, IEVs release to outside of the infected cell and may contain one more layer of cell plasma membrane named EEV or CEV (if it is still retained on cell plasma membrane). While there is no evidence for the participant of host proteins in early phase of virus infection, the situation is different in intermediate and late phases. Virus appears to be borrowed from host cell proteins for mRNA synthesis which have been reported elsewhere [27-28]. Soon after infection, VVs develop multiple mechanisms to interfere the host gene expression such as inhibition of host mRNA synthesis , induction of actin and tubulin mRNA degradation. Eventually, in about 2-4 hours, host translation is drastically impaired in order to temporally maximize the expression of viral genetic information [30-31].
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1.2.3 Vaccinia virus as an oncolytic agent
There are numerous inherited biological properties that make VV suitable for development as oncolytic agents. As a member of poxvirus family, VV has been reported with broad spectrum of host range in which virus can use multiple host entrance mechanisms [18-19, 32]. VV is one of the safest viruses which have been intensively studied about molecular biology and phathogenesis, as a result of being used as a vaccine for eradiation of smallpox disease in human. Besides, it owns natural tumor tropism which can selectively infect, replicate and destroy tumor cells while does no harm to normal cells [7, 33]. VV owns a large double DNA genome (~200 kbp) encoded almost all needed enzymes and factors for virus replication and entry in cytoplasm, and therefore physically independence on host-genome modification. In addition, this virus is able to carry multiple large transgenes, up to 25 kbp which allows a variety of genes to be added and engineered without affecting viral replication . Recently, there are many genetically engineered VVs created based on wide type VV backbones [10, 35-36]. Those new generations showed significantly improved tumor selectivity and efficacy in both cell culture and animal studies of various cancer cell types as well as some potential candidates are being tested in different tumor models in clinical trials (reviewed in ).
188.8.131.52 First vaccinia virus generations
The term "oncolytic virus" means that virus is able to selectively infect, replicate within and eventually destroy the cancer cells once a number of viral particles are well accumulated. These properties arrive from either inherence or genetic engineering. The first vaccinia generations mostly exploit natural tumor tropism to target the cancer cells. In 1922, VVs have been used to infect and inhibit the growth of many tumor kinds in mice, rats and rabbits. Concomitantly, at the same year, Salmon and Baix tried to use the virus for treatment of breast and enocarcinoma in human. However, no tumor regression outcome has been reported . The years later, more virus strains have been isolated. They showed potential therapeutic spectrum on different cancer cells such as leukemia 9471, sarcoma, human cervix carcinoma or in combination with x-ray and so on . VVs displayed ability to replicate in tumor cells preferentially. However, non-specific effects such as high virus replication in normal organs or high toxicity are those limitations with first vaccinia virus' generations .
184.108.40.206 Second vaccinia virus generations
In order to improve tumor specificity, the 2nd virus generations were genetically manipulated to inactivate some critical genes necessary for the virus replication in normal cells but expendable in cancer cells. Thymidine kinase (TK), 19 kDa vaccinia virus protein , is a key function enzyme for DNA replication by making the nucleotide pools. A hallmark of cancer cells is unlimitedly dividing therefore abundant nucleotide pools in tumor can be served as the sources for viral DNA replication and thereby propagation. Deletion of TK showed tremendous attenuated virus virulence in normal tissue such as 7000 folds lower in lung, 3000 folds lower in liver, and 250 folds lower than ovary after 4 days i.p. injection of viruses while still strongly active in tumor , resulting in the increase of survival rate . VV also expresses vaccinia growth factor (VGF) that binds to epidermal growth factor receptor (EGFR) to promote cell proliferation through Ras signaling pathway , creating material sources for virus replication. Therefore, the deletion of both TK and VGF in cancer cells with activating Ras pathway can result in higher therapeutic index than either mutants with single deletion [31, 42]. Vaccinia hemagglutinin (HA) has specific affinity with membrane structure proteins which is believed to work as a bridge for virus binding . The complete tumor regression was also achieved when Zhang et al. used triplet inactivation of HA, TK and F14.4L from original vaccinia LIVP strain, named GLV-1h68, to treat breast carcinoma xenografts while poor or non-detected replication of virus on normal tissues compared to wild type strains . It has been known that virus induces many adaptation processes in infected cells which are similar to the hallmarks of malignant cells, including anti-apoptosis , induction of interferon  and immune evasion. So it means that virus with many virulent genes which are inactivated in cancer cells are already adapted for viral replication. Therefore their deletions result in viral strains that are attenuated in normal tissue but not in tumors . Beside the deletion of viral virulent genes, the insertion of biomarker genes such as fluorescent proteins (green fluorescent protein (GFP) [33, 46], red fluorescent protein (RFP)), luminescent proteins (luciferase-GFP fusion protein ) or product converting enzymes (ï¢-galactosidase, ï¢-glucoronidase ) make it feasible for real time visualization and quantification of virus actions in cancerous tissues. Because of their advantages, we can understand why almost recent vaccinia constructs carrying live biomarkers were applicable [35, 39, 45, 47]. Lately, JX495, an engineered vaccinia virus expressing GFP, ï¢-glucoronidase and glanulocyte-macrophage colony stimulating factor (GM-CSF), beautifully showed for the first time positive expression of their marker genes in tumor tissues of treated patients .
220.127.116.11 Third vaccinia virus generations
In 3rd generations, VVs were equipped with more powerful "tools" to specifically find and destroy the tumor cells. With huge advantages of carrying multiple large transgenes, many strategies have been designed to efficiently use VVs in cancer treatment. Recently, Chen and Szalay had a detailed review on those strategies .
In brief, vaccinia viruses express immunostimulatory cytokines such as interleukins (IL-2, IL-6, IL-12), interferons (IFN-ï§, IFN-ï¢ ) and alpha tumor necrosis factor (TNF-alpha). They are important mediators in immune responses against tumors by promoting immune cells such as B cells, T cells, or monocytes. The expression of some IFNs also has protective function of normal cells from virus infection but not in cancer cells. It has been showed that the combination of virus with cytokines enhanced tumor selectivity and antitumor effects in many tumor models [45, 50-52]. However, increased cytokine-associated toxicity has been reported elsewhere, so the control of timing and concentration of administration must be taken into consideration to achieve the successful therapy .
Monoclonal antibody is also a strategy that has been mentioned a century ago. Multiple mechanisms have been exploited to inhibit tumor growth beside oncolysis. They include induction of apoptosis, alternation of tumor intracellular signaling, and inhibition of growth factor receptor . Tumor-antigen specific monoclonal antibody (TA specific mAb) may exert their effects through Fc-based mechanisms such as antibody dependent cell-mediated cytotoxicity and complement dependent cytotoxicity, subsequently activate cell-mediated immunity such as nature killer cells, macrophages, monocytes or neutrophills to produce cytokines [55-56]. Avastin is one of the most successful immunotherapeutic proteins, which has been approved by the US Food and Drug Administration for treatment of metastatic colorectal cancer and most forms of metastatic non-small cell lung cancer. This monoclonal antibody is supposed to inhibit the growing of blood vessel in tumor (angiogenesis) which is a phenomenon characteristic found in most tumors and metastasis. Exploiting the same strategy, Fentzen el al. had genetically engineered vaccinia viruses expressing anti vascular endothelial growth factor (VEGF) antibody (GLAF-1), and they showed significantly enhanced tumor therapeutic efficacy compared to control via the massive reduction of tumor vascularization .
Apoptosis is a common way used by tumor cells to avoid cell-death programming which all normal cells have to pass through. Apoptotic cells display a number of characteristic features such as DNA fragmentation, chromatin condensation, mitochondrial dysfunction, and plasma membrane alterations ultimately leading to the death of the cell . P53 is a tumor suppressor protein that in humans is encoded by the TP53 gene. P53 is important in living organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer. In most human tumors, P53 have been defected either by PT53 mutation or inactivation of the P53 signal transduction. Therefore restoration of the activation of P53 could be beneficial to inhibit cancer growth. Vaccinia virus expressing wild-type P53 (rVV-p53) showed tumor growth inhibition of different human and rat glioma cell lines underwent apoptosis  or in combination with radiotherapy .
In recent years, various tumor antigens (tumor-associated or tumor-specific antigens) have been discovered. They are the attractive targets for specific immunotherapy. Due to ability to escape the recognition and destruction by the host immune system of cancer cells, a strategy in which tumor antigens is expressed by oncolytic virus will exert synergic action of both direct tumor destruction and activation of immune cells. Attenuated VV encoded oncofetal antigen (5T4), is a trans-membrane glycoprotein over-expressed by a wide spectrum of cancers, including colorectal, ovarian, and gastric, but with a limited normal tissue expression, showed significant tumor retardation compared with mice vaccinated with controls in syngenenic models of melanoma and colorectal tumors . Some other virus-expressing tumor specific antigens were also shown increase of therapeutic benefit such as 47-LDA-Fcï§ , prostate-specific antigen . Moreover, virus itself or debris from lysed tumor cells is able to initiate immune responses. Immune response to viral infection is a double-edged sword for virus therapy. On the one hand, it can eliminate viral particles and therefore decreasing anti-tumor efficacy. But on the other hand, the local infiltration of immune cells help recognize and attack tumor cells . GLV-1h68 treated various cancer cell types (breast , pancreatic , prostate , canine carcinoma , squamous carcinoma ), which showed massive infiltration of immune cells with cytokine and chemokine productions in tumors, in parallel with virus replication and eventually lead to inhibition of tumor growth.
Product-converting enzyme-encoded viruses help overcome the limitations of conventional chemotherapy. With conventional chemotherapy, drugs were unspecifically delivered to whole body under the active forms therefore side effects are major concerns to decide the use of this method. In drug combined virus-mediated expressing enzyme, since an inactive form (non-toxic) of drug Which was administrated will only be activated inside the tumor where needed enzyme is provided by virus, the side toxicity is minimized. VV carrying cytosine deaminase (CD) showed virus-specific targeting in liver metastasis after virus administration in both immunocompetent and athymic nude mice models. Concomitantly, treatment with 5-fluorocytosine (5-FC) drug lead to significant tumor response and subsequent survival benefit with cure rate up to 30% of established liver metastasis .
18.104.22.168 Combination therapies
Combination with radiotherapy
Combination of oncolytic virus with radiation therapy continues to grow as the relationship between these two therapies is better understood. Through either radiation-mediated enhancement of viral oncolysis or virus-mediated sensitization of cells to radiation therapy, the combination of these two treatments has resulted in synergistic antitumor effects in numerous preclinical models . Using U-87 glioma xenograft models (subcutaneous and orthotopic) pre-treated with radiation showed preferential virus replication in tumor and correlated with tumor regression and survival rate when compared to mono-therapies . In another study, VV expressing P53 combination with radiation exhibited lower tumor incidence and tumor progression on C6 glioma tumor model .
Combination with chemotherapies
Chemotherapy is a conventional therapeutic option to treat cancer by preventing rapid dividing cells basically. One of the advantages of chemotherapy over other cancer treatments is effectiveness. However, with chemotherapy, the drugs are introduced to entire body, any other cells in the body that have the ability to quick multiply will also be killed by the drugs such as bone marrow, reproductive cells or hair follicles. Consequently, chemotherapy will associate to high level of cytotoxicity and significant side effects. On the other hand, oncolytic viruses have higher tumor specificity due to body immune barriers . The synergistic actions , therefore, are thought to be beneficial in combination of oncolytic virus and chemotherapy, whether the mechanism of action of those two therapies are quite different . In preclinical modes, the enhanced tumor therapeutics have been reported elsewhere. In human and rat glioma models, EGFP-expressing vaccinia virus (vvDD-EGFP) combined with rapamycin or cyclophosphamide promoted virus replication in tumor and further prolonged animal survival rate . Another VV, GLV-1h68, showed enhanced tumor regression in pancreatic tumor model in combination with cisplatin or gemcitabine . One derivative of GLV-1h68, GLV-1h90 encoded hyper-IL6, reduced chemotherapy-associated side effects of mitomycin C in a prostate model, at the end significantly improved anti tumor efficacy .
Although numerous encouraging results from in vitro and in vivo studies have been shown, the transfer from preclinical to clinical setting still faced with many limitations. This fact could be explained by the complex interactions between tumor and its microenvironment, the virus and the host immune response . During the evolution, human body has developed many barrier defenses against virus infections. Especially for VV in this case, which had been used as vaccine for smallpox eradication campaign with estimated a hundred million people who still have some degree of immunity to the VV. This has imposed a significant barrier to achieve maximum therapeutic efficacy of oncolytic viruses.