RESUMO
Cancer is one of the most common reasons for death in dogs. One promising approach is oncolytic virotherapy. We assessed the oncolytic effect of genetically modified vaccinia viruses in canine cancer cells, in freshly excised tumour biopsies, and in mice harbouring canine tumour xenografts. Tumour transduction efficacy was assessed using virus expressing luciferase or fluorescent marker genes and oncolysis was quantified by a colorimetric cell viability assay. Oncolytic efficacy in vivo was evaluated in a nude mouse xenograft model. Vaccinia virus was shown to infect most tested canine cancer cell lines and primary surgical tumour tissues. Virus infection significantly reduced tumour growth in the xenograft model. Oncolytic vaccinia virus has antitumour effects against canine cancer cells and experimental tumours and is able to replicate in freshly excised patient tumour tissue. Our results suggest that oncolytic vaccinia virus may offer an effective treatment option for otherwise incurable canine tumours.
Assuntos
Doenças do Cão/terapia , Neoplasias/veterinária , Terapia Viral Oncolítica , Vaccinia virus/fisiologia , Animais , Biópsia , Linhagem Celular Tumoral , Doenças do Cão/patologia , Cães , Camundongos Nus , Neoplasias/patologia , Neoplasias/virologia , Neoplasias Experimentais/terapia , Vírus OncolíticosRESUMO
In our earlier studies, Semliki Forest virus vector VA7 completely eliminated type I interferon (IFN-I)-unresponsive human U87-luc glioma xenografts, whereas interferon-responsive mouse gliomas proved refractory. Here, we describe in two clones of CT26 murine colon carcinoma, opposed patterns of IFN-I responsiveness and sensitivity to VA7. Both CT26WT and CT26LacZ clones secreted biologically active interferon in vitro upon virus infection but only CT26WT cells were protected. Focal infection of CT26WT cultures was self-limiting but could be rescued using IFN-I pathway inhibitor Ruxolitinib or antibody against IFNß. Whole transcriptome sequencing (RNA-Seq) and protein expression analysis revealed that CT26WT cells constitutively expressed 56 different genes associated with pattern recognition and IFN-I signaling pathways, spanning two reported anti-RNA virus gene signatures and 22 genes with reported anti-alphaviral activity. Whereas CT26WT tumors were strictly virus-resistant in vivo, infection of CT26LacZ tumors resulted in complete tumor eradication in both immunocompetent and severe combined immune deficient mice. In double-flank transplantation experiments, CT26WT tumors grew despite successful eradication of CT26LacZ tumors from the contralateral flank. Tumor growth progressed uninhibited also when CT26LacZ inoculums contained only a small fraction of CT26WT cells, demonstrating dominance of IFN responsiveness when heterogeneous tumors are targeted with interferon-sensitive oncolytic viruses.
Assuntos
Neoplasias do Colo/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos/genética , Vírus da Floresta de Semliki/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Efeito Espectador , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Vetores Genéticos , Proteínas de Fluorescência Verde/biossíntese , Interferon Tipo I/farmacologia , Interferon Tipo I/uso terapêutico , Interferon beta/metabolismo , Camundongos Endogâmicos BALB C , Necrose , Transplante de Neoplasias , Fator de Transcrição STAT1/metabolismo , Transfecção , Resultado do TratamentoRESUMO
Oncolytic vaccinia virus is an attractive platform for immunotherapy. Oncolysis releases tumor antigens and provides co-stimulatory danger signals. However, arming the virus can improve efficacy further. CD40 ligand (CD40L, CD154) can induce apoptosis of tumor cells and it also triggers several immune mechanisms. One of these is a T-helper type 1 (Th1) response that leads to activation of cytotoxic T-cells and reduction of immune suppression. Therefore, we constructed an oncolytic vaccinia virus expressing hCD40L (vvdd-hCD40L-tdTomato), which in addition features a cDNA expressing the tdTomato fluorochrome for detection of virus, potentially important for biosafety evaluation. We show effective expression of functional CD40L both in vitro and in vivo. In a xenograft model of bladder carcinoma sensitive to CD40L treatment, we show that growth of tumors was significantly inhibited by the oncolysis and apoptosis following both intravenous and intratumoral administration. In a CD40-negative model, CD40L expression did not add potency to vaccinia oncolysis. Tumors treated with vvdd-mCD40L-tdtomato showed enhanced efficacy in a syngenic mouse model and induced recruitment of antigen-presenting cells and lymphocytes at the tumor site. In summary, oncolytic vaccinia virus coding for CD40L mediates multiple antitumor effects including oncolysis, apoptosis and induction of Th1 type T-cell responses.
Assuntos
Antineoplásicos/farmacologia , Ligante de CD40/genética , Vetores Genéticos/administração & dosagem , Terapia Viral Oncolítica/métodos , Células Th1/imunologia , Neoplasias da Bexiga Urinária/terapia , Animais , Apoptose/efeitos dos fármacos , Ligante de CD40/imunologia , Linhagem Celular Tumoral , Genes Reporter , Vetores Genéticos/uso terapêutico , Humanos , Camundongos , Neoplasias Experimentais , Vírus Oncolíticos/genética , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
At present, it is not possible to reliably identify patients who will benefit from oncolytic virus treatments. Conventional modalities such as computed tomography (CT), which measure tumor size, are unreliable owing to inflammation-induced tumor swelling. We hypothesized that magnetic resonance imaging (MRI) and spectroscopy (MRS) might be useful in this regard. However, little previous data exist and neither oncolytic adenovirus nor immunocompetent models have been assessed by MRS. Here, we provide evidence that in T2-weighted MRI a hypointense core area, consistent with coagulative necrosis, develops in immunocompetent Syrian hamster carcinomas that respond to oncolytic adenovirus treatment. The same phenomenon was observed in a neuroblastoma patient while he responded to the treatment. With relapse at a later stage, however, the tumor of this patient became moderately hyperintense. We found that MRS of taurine, choline and unsaturated fatty acids can be useful early indicators of response and provide detailed information about tumor growth and degeneration. In hamsters, calprotectin-positive inflammatory cells (heterophils and macrophages) were found in abundance; particularly surrounding necrotic areas in carcinomas and T cells were significantly increased in sarcomas, when these had been treated with a granulocyte-macrophage colony-stimulating factor-producing virus, suggesting a possible link between oncolysis, necrosis (seen as a hypointense core in MRI) and/or immune response. Our study indicates that both MRI and MRS could be useful in the estimation of oncolytic adenovirus efficacy at early time points after treatment.