RESUMO
The members of the picornavirus family include various viruses which, due to their impressive oncolytic activity, have the potential to be used for the treatment of cancer. However, the replication of these oncolytic viruses (OV) is not limited to tumor cells but can also take place in various normal tissues. To increase the safety of these OV, target sites (miR-TS) of microRNAs, which are expressed in normal tissues but are absent or only expressed at low levels in cancer cells, can be inserted into the viral genome. Here we describe how miR-TS can easily be inserted into the complementary DNA (cDNA) of coxsackievirus B3 (CVB3) RNA genome using the In-Fusion cloning technology. Here we provide the step-by-step protocol, how miR-TS containing recombinant CVB3 can be generated from these viral cDNA constructs, how the virus is amplified, purified and concentrated, and how the functionality of the miR-TS within the viral genome can be confirmed.
Assuntos
MicroRNAs , Vírus Oncolíticos , DNA Complementar , Enterovirus Humano B/genética , Genoma Viral , Células HeLa , Humanos , MicroRNAs/genética , Vírus Oncolíticos/genética , Replicação Viral/genéticaRESUMO
The coxsackievirus B3 strain PD-0 has been proposed as a new oncolytic virus for the treatment of colorectal carcinoma. Here, we generated a cDNA clone of PD-0 and analyzed the virus PD-H, newly generated from this cDNA, in xenografted and syngenic models of colorectal cancer. Replication and cytotoxic assays revealed that PD-H replicated and lysed colorectal carcinoma cell lines in vitro as well as PD-0. Intratumoral injection of PD-H into subcutaneous DLD-1 tumors in nude mice resulted in strong inhibition of tumor growth and significantly prolonged the survival of the animals, but virus-induced systemic infection was observed in one of the six animals. In a syngenic mouse model of subcutaneously growing Colon-26 tumors, intratumoral administration of PD-H led to a significant reduction of tumor growth, the prolongation of animal survival, the prevention of tumor-induced cachexia, and the elevation of CD3+ and dendritic cells in the tumor microenvironment. No virus-induced side effects were observed. After intraperitoneal application, PD-H induced weak pancreatitis and myocarditis in immunocompetent mice. By equipping the virus with target sites of miR-375, which is specifically expressed in the pancreas, organ infections were prevented. Moreover, employment of this virus in a syngenic mouse model of CT-26 peritoneal carcinomatosis resulted in a significant reduction in tumor growth and an increase in animal survival. The results demonstrate that the immune status of the host, the route of virus application, and the engineering of the virus with target sites of suitable microRNAs are crucial for the use of PD-H as an oncolytic virus.
Assuntos
Infecções por Coxsackievirus/imunologia , Enterovirus/fisiologia , Vírus Oncolíticos/fisiologia , Animais , Células CHO , Neoplasias Colorretais , Cricetulus , Enterovirus/classificação , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Nus , MicroRNAs , Miocardite , Neoplasias , Vírus Oncolíticos/classificaçãoRESUMO
It is now well accepted that many tumors undergo a process of clonal selection which means that tumor antigens arising at various stages of tumor progression are likely to be represented in just a subset of tumor cells. This process is thought to be driven by constant immunosurveillance which applies selective pressure by eliminating tumor cells expressing antigens that are recognized by T cells. It is becoming increasingly clear that the same selective pressure may also select for tumor cells that evade immune detection by acquiring deficiencies in their human leucocyte antigen (HLA) presentation pathways, allowing important tumor antigens to persist within cells undetected by the immune system. Deficiencies in antigen presentation pathway can arise by a variety of mechanisms, including genetic and epigenetic changes, and functional antigen presentation is a hard phenomenon to assess using our standard analytical techniques. Nevertheless, it is likely to have profound clinical significance and could well define whether an individual patient will respond to a particular type of therapy or not. In this review we consider the mechanisms by which HLA function may be lost in clinical disease, we assess the implications for current immunotherapy approaches using checkpoint inhibitors and examine the prognostic impact of HLA loss demonstrated in clinical trials so far. Finally, we propose strategies that might be explored for possible patient stratification.
Assuntos
Antígenos HLA/imunologia , Imunoterapia/métodos , Neoplasias/imunologia , Linfócitos T/imunologia , Animais , Humanos , CamundongosRESUMO
Arming of oncolytic viruses with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown as a viable approach to increase the antitumor efficacy in melanoma. However, melanoma cells may be partially or completely resistant to TRAIL or develop TRAIL resistance, thus counteracting the antitumor efficiency of TRAIL-armed oncolytic viruses. Recently, we found that TRAIL resistance in melanoma cells can be overcome by inhibition of antiapoptotic Bcl-2 protein myeloid cell leukemia 1 (Mcl-1). Here, we investigated whether the cytotoxicity of AdV-TRAIL, an oncolytic adenovirus, which expresses TRAIL after induction by doxycycline (Dox), can be improved in melanoma cells by silencing of Mcl-1. Two melanoma cell lines, the TRAIL-resistant MeWo and the TRAIL-sensitive Mel-HO were investigated. Treatment of both cell lines with AdV-TRAIL resulted in a decrease of cell viability, which was caused by an increase of apoptosis and necrosis. The proapoptotic effects were dependent on induction of TRAIL by Dox and were more pronounced in Mel-HO than in MeWo cells. SiRNA-mediated silencing of Mcl-1 resulted in a further significant decrease of cell viability and a further increase of apoptosis and necrosis in AdV-TRAIL-infected MeWo and Mel-HO cells. However, while in absolute terms, the effects were more pronounced in Mel-HO cells, in relative terms, they were stronger in MeWo cells. These results show that silencing of Mcl-1 represents a suitable approach to increase the cytotoxicity of a TRAIL-armed oncolytic adenovirus in melanoma cells. KEY MESSAGES: ⢠Cytotoxicity of TRAIL-expressing adenovirus can be enhanced by silencing of Mcl-1. ⢠The effect occurs in TRAIL-sensitive and TRAIL-resistant melanoma cells. ⢠Increase of apoptosis is the main mechanism induced by Mcl-1 silencing.
Assuntos
Adenoviridae/genética , Apoptose , Inativação Gênica , Terapia Genética , Melanoma/terapia , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Terapia Viral Oncolítica , Vírus Oncolíticos/genética , Neoplasias Cutâneas/terapia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Adenoviridae/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/virologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Necrose , Vírus Oncolíticos/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/virologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
Oncolytic virotherapy represents one of the most advanced strategies to treat otherwise untreatable types of cancer. Despite encouraging developments in recent years, the limited fraction of patients responding to therapy has demonstrated the need to search for new suitable viruses. Coxsackievirus B3 (CVB3) is a promising novel candidate with particularly valuable features. Its entry receptor, the coxsackievirus and adenovirus receptor (CAR), and heparan sulfate, which is used for cellular entry by some CVB3 variants, are highly expressed on various cancer types. Consequently, CVB3 has broad anti-tumor activity, as shown in various xenograft and syngeneic mouse tumor models. In addition to direct tumor cell killing the virus induces a strong immune response against the tumor, which contributes to a substantial increase in the efficiency of the treatment. The toxicity of oncolytic CVB3 in healthy tissues is variable and depends on the virus strain. It can be abrogated by genetic engineering the virus with target sites of microRNAs. In this review, we present an overview of the current status of the development of CVB3 as an oncolytic virus and outline which steps still need to be accomplished to develop CVB3 as a therapeutic agent for clinical use in cancer treatment.
Assuntos
Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Vírus Oncolíticos/fisiologia , Animais , Linhagem Celular Tumoral , Ensaios Clínicos como Assunto , Infecções por Coxsackievirus/virologia , Engenharia Genética , Humanos , Camundongos , MicroRNAs , Receptores Virais/metabolismo , Internalização do VírusRESUMO
Dysregulation of HLA (human leukocyte antigen) function is increasingly recognized as a common escape mechanism for cancers subject to the pressures exerted by immunosurveillance or immunotherapeutic interventions. Oncolytic viruses have the potential to counter this resistance by upregulating HLA expression or encouraging an HLA-independent immunological responses. However, to achieve the best therapeutic outcomes, a prospective understanding of the HLA phenotype of cancer patients is required to match them to the characteristics of different oncolytic strategies. Here, we consider the spectrum of immune competence observed in clinical disease and discuss how it can be best addressed using this novel and powerful treatment approach.
RESUMO
Coxsackievirus B3 (CVB3) has strong oncolytic activity in colorectal carcinoma but it also infects the pancreas and the heart. To improve the safety of the virus, here we investigated whether pancreas and cardiac toxicity can be prevented by insertion of target sites (TS), which are complementary to miR-375 and miR-1 into the viral genome. Although miR-375 and miR-1 are abundantly expressed in the pancreas and in the heart, respectively, their expression levels are low in colorectal carcinomas, which allows the carcinomas to be selectively attacked. To investigate the importance of the microRNAs, two viruses were engineered, H3N-375TS containing only miR-375TS and H3N-375/1TS containing miR-375TS and miR-1TS. In vitro, both viruses replicated in and lysed colorectal carcinoma cells, similar to a nontargeted control virus H3N-39TS, whereas they were strongly attenuated in cell lines transiently or endogenously expressing the corresponding microRNAs. In vivo, the control virus H3N-39TS induced strong infection of the pancreas and the heart, which led to fatal disease within 4 days after a single intratumoral virus injection in mice xenografted with colorectal DLD-1 cell tumors. In contrast, three intratumoral injections of H3N-375TS or H3N-375/1TS failed to induce virus-induced sickness. In the animals, both viruses were completely ablated from the pancreas and H3N-375/1TS was also ablated from the heart, whereas the cardiac titers of H3N-375TS were strongly reduced. Long-term investigations of the DLD-1 tumor model confirmed lack of virus-induced adverse effects in H3N-375TS- and H3N-375/1TS-treated mice. There was no mortality, and the pancreas and the heart were free of pathological alterations. Regarding the therapeutic efficiency, the treated animals showed high and long-lasting H3N-375TS and H3N-375/1TS persistence in the tumor and significantly slower tumor growth. These data demonstrate that miR-375- and miR-1-mediated virus detargeting from the pancreas and heart is a highly effective strategy to prevent toxicity of oncolytic CVB3.
Assuntos
Neoplasias Colorretais , MicroRNAs , Animais , Cardiotoxicidade , Neoplasias Colorretais/genética , Neoplasias Colorretais/terapia , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/genética , PâncreasRESUMO
Coxsackievirus B3 (CVB3) has potential as a new oncolytic agent for the treatment of cancer but can induce severe pancreatitis. Here, we inserted target sequences of the microRNA miR-375 (miR-375TS) into the 5' terminus of the polyprotein encoding sequence or into the 3'UTR of the CVB3 strain rCVB3.1 to prevent viral replication in the pancreas. In pancreatic EndoC-ßH1 cells expressing miR-375 endogenously, replication of the 5'-miR-375TS virus and that of the 3'-miR-375TS virus was reduced by 4 × 103 -fold and 3.9 × 104 -fold, respectively, compared to the parental rCVB3.1. In colorectal carcinoma cells, replication and cytotoxicity of both viruses were slightly reduced compared to rCVB3.1, but less pronounced for the 3'-miR-375TS virus. Thus, CVB3 with miR-375TS in the 3'UTR of the viral genome may be suitable to avoid pancreatic toxicity.
Assuntos
Enterovirus Humano B/genética , Engenharia Genética , MicroRNAs/genética , Pâncreas/citologia , Regiões 3' não Traduzidas/genética , Sequência de Bases , Linhagem Celular Tumoral , Células HEK293 , Humanos , Pâncreas/virologiaRESUMO
Coxsackievirus B3 (CVB3), a single-stranded RNA virus of the picornavirus family, has been described as a novel oncolytic virus. However, the CVB3 strain used induced hepatitis and myocarditis in vivo. It was hypothesized that oncolytic activity and safety of CVB3 depends on the virus strain and its specific receptor tropism. Different laboratory strains of CVB3 (Nancy, 31-1-93, and H3), which use the coxsackievirus and adenovirus receptor (CAR), and the strain PD, which uses N- and 6-O-sulfated heparan sulfate (HS) for entry into the cells, were investigated for their potential to lyse tumor cells and for their safety profile. The investigations were carried out in colorectal carcinoma. In vitro investigations showed variable infection efficiency and lysis of colorectal carcinoma cell lines by the CVB3 strains. The most efficient strain was PD, which was the only one that could lyse all investigated colorectal carcinoma cell lines. Lytic activity of CAR-dependent CVB3 did not correlate with CAR expression on cells, whereas there was a clear correlation between lytic activity of PD and its ability to bind to HS at the cell surface of colorectal carcinoma cells. Intratumoral injection of Nancy, 31-1-93, or PD into subcutaneous colorectal DLD1 cell tumors in BALB/c nude mice resulted in strong inhibition of tumor growth. The effect was seen in the injected tumor, as well as in a non-injected, contralateral tumor. However, all animals treated with 31-1-93 and Nancy developed systemic infection and died or were moribund and sacrificed within 8 days post virus injection. In contrast, five of the six animals treated with PD showed no signs of a systemic viral infection, and PD was not detected in any organ. The data demonstrate the potential of PD as a new oncolytic virus and HS-binding of PD as a key feature of oncolytic activity and improved safety.
Assuntos
Neoplasias Colorretais/terapia , Neoplasias Colorretais/virologia , Enterovirus Humano B/metabolismo , Heparitina Sulfato/metabolismo , Vírus Oncolíticos/patogenicidade , Animais , Antígenos CD55/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Citotoxicidade Imunológica , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Especificidade de Órgãos , Receptores Virais/metabolismo , Carga Viral , VirulênciaRESUMO
PURPOSE: Hemorrhagic cystitis (HC) is the most common urotoxic side effect of cyclophosphamide (CP). The aim of this study was to compare the classical efficacy of mesna (2-mercaptoethane sulfonate sodium) with three different doses of resveratrol (RES) on cyclophosphamide-induced HC in rats. METHODS: Forty-six male Sprague-Dawley rats were divided into six groups. Group 1 served as a negative control (sham). Five groups received a single dose of cyclophosphamide (150 mg/kg) intraperitoneally at the same time. Groups 2, 3, 4, 5, and 6 received only CP, CP + 20 mg/kg RES, CP + 40 mg/kg RES, CP + 80 mg/kg RES, and CP + classical protocol of three doses of mesna (30 mg/kg three times), respectively. Antioxidants, cytokines, and malondialdehyde levels were measured in all groups. In addition, histopathological alterations in tissues were examined. RESULTS: CP administration induced severe HC with marked edema, hemorrhage, and inflammation in group 2. RES 20 mg/kg showed meaningful protection against bladder damage compared to the control group. It was seen that RES 40 mg/kg gave weaker protection but RES 80 mg/kg was not found to be effective. CONCLUSION: In conclusion, marked bladder protection was found in 20 and 40 mg/kg RES applications compared to the control group, but this protection was weaker than with mesna.