RESUMO
BACKGROUND: Surgical management of pancreatic cancer depends on tumor resectability and staging. Lymph node (LN) metastases represent an important decision-making factor when it comes to surgical treatment. AIMS: To evaluate a new in vivo, endoscopic confocal microscopy (CM) system not requiring fluorescence markers, for detection and staging of pancreatic cancer in rats. METHODS: A confocal system consisting of a confocal scanning laser operating in reflection mode and a dedicated rigid Hopkins rod-lens endoscope were used for in vivo imaging in a rat model of pancreatic ductal adenocarcinoma. A double-blind study compared CM to standard histology in (1) the detection of tumors in rat bearing cancer (n = 11) and controls (n = 6), and (2) in the detection of local nodal involvement at 3 and 6 weeks after tumor induction. RESULTS: CM detected all pancreatic tumors with 100 % sensitivity and specificity and identified 15 metastatic LNs with an average adenocarcinoma nodule diameter of 2.3 mm (range from 1 to 4.2 mm) out of the 66 examined. CM demonstrated a sensitivity of 87.5 % and a specificity of 98 % in LN detection. The Spearman's rank correlation/rho calculator was of 0.87. CM demonstrated a negative predictive value of 96.1 % and a positive predictive value of 93.3 % in the detection of metastatic LNs. CONCLUSIONS: Interpretation of confocal images has a high concurrence rate with histopathology examination for primary tumor and lymphatic involvement detection making it a promising technique for in vivo real-time detection and staging of pancreatic cancer. Larger studies are warranted to confirm these preliminary results.
Assuntos
Adenocarcinoma/diagnóstico , Metástase Linfática/diagnóstico , Neoplasias Pancreáticas/diagnóstico , Adenocarcinoma/secundário , Animais , Método Duplo-Cego , Metástase Linfática/patologia , Microscopia Confocal , Estadiamento de Neoplasias , Neoplasias Pancreáticas/patologia , Distribuição Aleatória , Ratos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
UNLABELLED: Novel therapies employing oncolytic viruses have emerged as promising anticancer modalities. The cure of particularly aggressive malignancies requires induction of immunogenic cell death (ICD), coupling oncolysis with immune responses via calreticulin, ATP, and high-mobility group box protein B1 (HMGB1) release from dying tumor cells. The present study shows that in human pancreatic cancer cells (pancreatic ductal adenocarcinoma [PDAC] cells n=4), oncolytic parvovirus H-1 (H-1PV) activated multiple interconnected death pathways but failed to induce calreticulin exposure or ATP release. In contrast, H-1PV elevated extracellular HMGB1 levels by 4.0±0.5 times (58%±9% of total content; up to 100 ng/ml) in all infected cultures, whether nondying, necrotic, or apoptotic. An alternative secretory route allowed H-1PV to overcome the failure of gemcitabine to trigger HMGB1 release, without impeding cytotoxicity or other ICD activities of the standard PDAC medication. Such broad resistance of H-1PV-induced HMGB1 release to apoptotic blockage coincided with but was uncoupled from an autocrine interleukin-1ß (IL-1ß) loop. That and the pattern of viral determinants maintained in gemcitabine-treated cells suggested the activation of an inflammasome/caspase 1 (CASP1) platform alongside DNA detachment and/or nuclear exclusion of HMGB1 during early stages of the viral life cycle. We concluded that H-1PV infection of PDAC cells is signaled through secretion of the alarmin HMGB1 and, besides its own oncolytic effect, might convert drug-induced apoptosis into an ICD process. A transient arrest of cells in the cyclin A1-rich S phase would suffice to support compatibility of proliferation-dependent H-1PV with cytotoxic regimens. These properties warrant incorporation of the oncolytic virus H-1PV, which is not pathogenic in humans, into multimodal anticancer treatments. IMPORTANCE: The current therapeutic concepts targeting aggressive malignancies require an induction of immunogenic cell death characterized by exposure of calreticulin (CRT) as well as release of ATP and HMGB1 from dying cells. In pancreatic tumor cells (PDAC cells) infected with the oncolytic parvovirus H-1PV, only HMGB1 was released by all infected cells, whether nondying, necrotic, or succumbing to one of the programmed death pathways, including contraproductive apoptosis. Our data suggest that active secretion of HMGB1 from PDAC cells is a sentinel reaction emerging during early stages of the viral life cycle, irrespective of cell death, that is compatible with and complements cytotoxic regimens. Consistent induction of HMGB1 secretion raised the possibility that this reaction might be a general "alarming" phenomenon characteristic of H-1PV's interaction with the host cell; release of IL-1ß points to the possible involvement of a danger-sensing inflammasome platform. Both provide a basis for further virus-oriented studies.
Assuntos
Antineoplásicos/metabolismo , Morte Celular , Desoxicitidina/análogos & derivados , Células Epiteliais/fisiologia , Vírus Oncolíticos/crescimento & desenvolvimento , Parvovirus/crescimento & desenvolvimento , Linhagem Celular Tumoral , Desoxicitidina/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Proteína HMGB1/metabolismo , Humanos , Transdução de Sinais , GencitabinaRESUMO
Hypoxia and dysfunctional tumor vessels represent a prominent feature of pancreatic cancer, being, at least in part, responsible for chemotherapy resistance and immune suppression in these tumors. We tested whether the increase of oxygen delivery induced in vivo by myo-inositol trispyrophosphate (ITPP) can reverse hypoxia, control tumor growth and improve chemotherapy response. Tumor size, metastatic development (microcomputed tomography scan follow-up) and the survival of rats and nude or NOD.SCID mice, (bearing syngenic rat and MiaPaCa2- or patient-derived pancreatic tumors), were determined on ITPP and/or gemcitabine treatment. Partial oxygen pressure, expression of angiogenic factors and tumor histology were evaluated. Infiltration and oxidative status of immune cells, as well as chemotherapy penetration in tumors, were determined by fluorescence-activated cell sorting, fluorometry, nitric oxide release assays, Western blot and confocal microscopy. Weekly intravenous ITPP application resulted in the inhibition of metastasis development and restricted primary tumor growth, showing a superior effect on the rats' survival compared with gemcitabine. ITPP treatment restored tumor normoxia and caused a reduction in hypoxia inducible factor-1α levels, with subsequent VEGF and Lox downregulation, resulting in improved vessel structure and decreased desmoplasia. The latter effects translated into elevated immune cells influx and improved susceptibility to gemcitabine treatment. Growth of human pancreatic tumor xenografts was strongly inhibited by administration of ITPP. ITPP exploits a two-stage mechanism causing rapid, early and sustainable late stage normoxia. This is due to the angiogenic factor modulation and vascular normalization, leading to enhanced chemotherapy delivery and synergistic life prolongation, on combination with low doses of gemcitabine.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Desoxicitidina/análogos & derivados , Hipóxia/tratamento farmacológico , Fosfatos de Inositol/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/uso terapêutico , Sinergismo Farmacológico , Imunofluorescência , Humanos , Hipóxia/metabolismo , Hipóxia/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neovascularização Patológica , Oxigênio/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos Lew , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , GencitabinaRESUMO
Myo-inositol trispyrophosphate (ITPP), a synthetic allosteric effector of hemoglobin, increases the regulated oxygen-releasing capacity of red blood cells (RBCs), leading to suppression of hypoxia-inducible factor 1α (HIF-1α) and to down-regulation of hypoxia-inducible genes such as vascular endothelial growth factor (VEGF). As a consequence, tumor growth is markedly affected. The effect of weekly intravenous injection of ITPP on an orthotopic, syngenic rat hepatocellular carcinoma (HCC) model was compared to that for untreated animals and animals subjected to conventional Doxorubicin chemotherapy. The longitudinal examination of HCC was performed by microCT imaging, and the cellular and molecular changes were evaluated by histology and Western blotting analysis of HIF-1α, VEGF, and caspase-3 gene expression in the tumor and in the surrounding liver. Hematologic impact was evaluated by blood cell-count measurement and determination of P50 (oxygen partial pressure for a 50 % oxygen saturation of hemoglobin). The HCC evaluation by microCT revealed a high potency of ITPP for tumor growth inhibition, thus allowing long-term survival and even cure of almost all the treated animals. The P50 value of hemoglobin in RBCs underwent a shift of 30 % following ITPP injection. Under these conditions, HIF-1α activity was strongly decreased, VEGF expression was down-regulated, and apoptosis was induced in HCC and surrounding liver cells, as indicated by Caspase-3 expression. ITPP did not affect hematologic parameters during treatment. The observations of in vivo tumor eradication suggest a significant clinical potential for ITPP in cancer therapy.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fosfatos de Inositol/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Animais , Apoptose , Carcinoma Hepatocelular/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/patologia , Masculino , Oxigênio/metabolismo , Ratos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND & AIMS: Surgical management of pancreatic cancer depends on tumor resectability and staging. This study evaluated a new in vivo technique, fiberoptic confocal fluorescence microscopy (FCFM), for detection and staging of pancreatic tumors in rats. METHODS: FCFM was used with a protease-activated fluorescent marker (ProSense; VisEn Medical Inc, Woburn, MA) for in vivo imaging of solid organs (1.8-microm resolution) in a rat model of pancreatic ductal adenocarcinoma. A preliminary study described the FCFM rendering of normal and pathologic tissues. Subsequently, 2 double-blind studies compared FCFM to standard histology in (1) detection of tumors in rat models of cancer and controls and (2) detection of nodal involvement (splenic, celiac, mesenteric, and colic) 4, 5, and 6 weeks after tumor induction vs controls. RESULTS: Tumor cells displayed a fluorescent ductal pattern compared with non-fluorescent normal pancreas or normal follicular pattern of lymph nodes (LNs). FCFM detected all the pancreatic tumors (1.7-mm mean diameter) and identified 23 LNs that contained metastases of 99 LNs examined. Standard histologic analyses resulted in 1 false-negative result in tumor detection and 2 false negatives in LN detection, whereas FCFM produced no false-negative results. Additional serial sectioning confirmed all tumors and 16 metastatic LNs; FCFM had a negative predictive value of 100% and a positive predictive value of 69.6%. CONCLUSIONS: Real-time "virtual biopsy" using FCFM detects tumors and LN metastases with 100% sensitivity and 92.2% specificity in rats, making it a reliable technique for detection and staging of pancreatic cancer.
Assuntos
Adenocarcinoma/secundário , Microscopia Confocal , Microscopia de Fluorescência , Estadiamento de Neoplasias/métodos , Fibras Ópticas , Neoplasias Pancreáticas/patologia , Adenocarcinoma/cirurgia , Animais , Estudos de Viabilidade , Corantes Fluorescentes , Masculino , Neoplasias Pancreáticas/cirurgia , Ratos , Ratos Endogâmicos Lew , Reprodutibilidade dos TestesRESUMO
UNLABELLED: Pancreatic carcinoma is a gastrointestinal malignancy with poor prognosis. Treatment with gemcitabine, the most potent chemotherapeutic against this cancer up to date, is not curative, and resistance may appear. Complementary treatment with an oncolytic virus, such as the rat parvovirus H-1PV, which is infectious but nonpathogenic in humans, emerges as an innovative option. PURPOSE: To prove that combining gemcitabine and H-1PV in a model of pancreatic carcinoma may reduce the dosage of the toxic drug and/or improve the overall anticancer effect. EXPERIMENTAL DESIGN: Pancreatic tumors were implanted orthotopically in Lewis rats or subcutaneously in nude mice and treated with gemcitabine, H-1PV, or both according to different regimens. Tumor size was monitored by micro-computed tomography, whereas bone marrow, liver, and kidney functions were monitored by measuring clinically relevant markers. Human pancreatic cell lines and gemcitabine-resistant derivatives were tested in vitro for sensitivity to H-1PV infection with or without gemcitabine. RESULTS: In vitro studies proved that combining gemcitabine with H-1PV resulted in synergistic cytotoxic effects and achieved an up to 15-fold reduction in the 50% effective concentration of the drug, with drug-resistant cells remaining sensitive to virus killing. Toxicologic screening showed that H-1PV had an excellent safety profile when applied alone or in combination with gemcitabine. The benefits of applying H-1PV as a second-line treatment after gemcitabine included reduction of tumor growth, prolonged survival of the animals, and absence of metastases on CT-scans. CONCLUSION: In addition to their potential use as monotherapy for pancreatic cancer, parvoviruses can be best combined with gemcitabine in a two-step protocol.
Assuntos
Carcinoma/tratamento farmacológico , Desoxicitidina/análogos & derivados , Vírus Oncolíticos/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Parvovirus/metabolismo , Animais , Antimetabólitos Antineoplásicos/farmacologia , Carcinoma/terapia , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Humanos , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias Pancreáticas/terapia , Ratos , Ratos Endogâmicos Lew , GencitabinaRESUMO
The incidence of lymphomas developing in both immunocompetent and immunosuppressed patients continues to steadily increase worldwide. Current chemotherapy and immunotherapy approaches have several limitations, such as severe side toxicity and selection of resistant cell variants. Autonomous parvoviruses (PVs), in particular the rat parvovirus H-1PV, have emerged as promising anticancer agents. Although it is apathogenic in humans, H-1PV has been shown to infect and suppress various rat and human tumors in animal models. In this study, we demonstrate the capacity of H-1PV for efficiently killing, through necrosis, cell cultures originating from Burkitt's lymphoma (BL), while sparing normal B lymphocytes. The cytotoxic effect was generally accompanied by a productive H-1PV infection. Remarkably, parvovirus-based monotherapy efficiently suppressed established BL at an advanced stage in a severe combined immunodeficient (SCID) mouse model of the disease. The data show for the first time that an oncolytic parvovirus deserves further consideration as a potential tool for the treatment of some non-Hodgkin B-cell lymphomas, including those resistant to apoptosis induction by rituximab.
Assuntos
Linfoma/terapia , Terapia Viral Oncolítica/métodos , Parvovirus/fisiologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Linfoma de Células B/terapia , Camundongos , Camundongos SCID , Necrose/virologia , Parvovirus/genética , Ratos , Replicação Viral/genéticaRESUMO
Currently, gemcitabine is approved as the first-line therapy for patients with locally advanced or metastatic pancreatic cancer. Unfortunately, because of pre-existing or acquired chemoresistance of most of the tumor cells, gemcitabine has failed to significantly improve the outcome for pancreatic carcinoma patients. The present study explored the possibility of sensitizing pancreatic cancer to gemcitabine chemotherapy by combining the chemotherapy with the proapoptotic genes Bax and TNF-related apoptosis-inducing ligand (TRAIL). We designed two tetracycline-inducible recombinant adenoviruses using the human telomerase reverse transcriptase (hTERT) promoter for transcriptional apoptogene targeting. Our data showed that treatment with the adenoviral systems resulted in high-level expression of Bax and TRAIL genes directly related to apoptosis induction, leading to a significant sensitization of resistant pancreatic tumor cells. Furthermore, treatment with Bax and TRAIL adenoviruses plus a suboptimal dose of gemcitabine resulted in significant tumor regression and prolongation of the experimental animal';s life, in contrast to the weak retardation in tumor growth observed when gemcitabine alone was used. Additionally, using an orthotopic tumor model, we showed the usefulness of a non-invasive whole-body optical imaging for real-time evaluation of therapeutic efficacy. Together, these findings suggest that hTERT-targeted proapoptotic gene expression in combination with gemcitabine may be a potential therapeutic strategy for treatment of pancreatic adenocarcinoma.
Assuntos
Desoxicitidina/análogos & derivados , Terapia Genética/métodos , Neoplasias Pancreáticas/terapia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Telomerase/genética , Proteína X Associada a bcl-2/genética , Adenoviridae/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/fisiologia , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Modelos Animais de Doenças , Feminino , Vetores Genéticos/genética , Humanos , Camundongos , Modelos Genéticos , Regiões Promotoras Genéticas/genética , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Transcrição Gênica , Proteína X Associada a bcl-2/fisiologia , GencitabinaRESUMO
Oncolytic viruses represent novel tools for cancer treatment. Besides specifically killing cancer cells (oncolysis), these agents also provide danger signals, prompting the immune system to eliminate virus-infected tumours. As a consequence of oncolytic events, the innate and adaptive immune systems gain access to tumour antigens, which result in cross-priming and vaccination effects. Here the aim was to see whether we could enhance this adjuvant capacity by incorporating immunostimulatory CpG motifs into the single-stranded genome of an oncolytic parvovirus (H-1PV). We engineered 2 CpG-enriched H-1PV variants (JabCG1 and JabCG2), preserving both the replication competence and the oncolytic features of the parental virus. In keeping with their increased CpG content, the JabCG1 and JabCG2 genomes proved in vitro to be more potent triggers of TLR-9-mediated signalling than wild-type H-1PV DNA. Antitumour activity was evaluated in a rat model of MH3924A hepatoma lung metastases, where an infection with parental or modified viruses served as an ex vivo adjuvant to a subcutaneously administered autologous cell vaccine. In this setup, which excludes direct oncolytic effects on metastases, the JabCG2 vector displayed enhanced immunogenicity, inducing markers of cellular immunity (IFN gamma) and dendritic cell activation (CD80, CD86) in mediastinal (tumour-draining) lymph nodes. This led to a significantly reduced metastatic rate (50%) as compared to other vaccination schedules (H-1PV-, JabCG1-, JabGC- or mock-treated cells). The data provide proof of principle that increasing the number of immunostimulatory CpG motifs within oncolytic viruses makes it possible to improve their overall anticancer effect by inducing antitumour vaccination.
Assuntos
Ilhas de CpG , Parvovirus/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , DNA Viral , Terapia Viral Oncolítica , Parvovirus/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
AIM: Retrospective studies have suggested a protective effect of regional anesthesia against recurrence after cancer surgery. But confirmation of the in vivo antitumor effects is lacking. We examined the in vitro antitumor effects of lidocaine on various breast cancer cell lines and then assessed these properties in vivo at clinically relevant concentrations. MATERIALS AND METHODS: In vitro experiments: normal breast epithelial cells (NBEC) MCF-10A and three tumor breast epithelial cells (TBEC) lines (MCF-7 luminal A, MDA-MB-231 triple-negative and SKBr3 HER2 positive) were exposed to increasing concentrations of lidocaine. Cell viability, migration and anchorage-independent growth were assessed by MTT, wound healing, and soft-agar growth assays. In vivo experiments: 6-week-old severe combined immunodeficient mice were injected intraperitoneally with MDA-MB-231 cells and were treated with intraperitoneal lidocaine or phosphate-buffered saline. The mice were euthanized when they reached experimental endpoints or sacrificed to determine peritoneal carcinomatosis index and global tumor volumes. RESULTS: Lidocaine reduced the viability of all the cell lines, inhibited migration of TBEC compared to the NBEC, and compromised the anchorage-independent growth of the triple-negative cells. Intraperitoneal lidocaine improved survival of mice with MDA-MB-231 peritoneal carcinomatosis using doses that are consistent with the current clinical settings for analgesia. CONCLUSION: In agreement with the notion that local anesthesia may be beneficial for cancer therapy, lidocaine has a protective effect against breast cancer cells in experimental studies. However, the beneficial impact of local anesthetics on breast cancer needs to be strengthened by additional preclinical and clinical trials.
Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Lidocaína/farmacologia , Lidocaína/uso terapêutico , Anestésicos Locais/farmacologia , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Camundongos SCID , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Peritoneais/patologiaRESUMO
The aim of this study was to demonstrate the potential of electrogene therapy with the bacterial purine nucleoside phosphorylase gene (ePNP), on pancreatic carcinoma (PC) large tumors. The in vivo electroporation (EP) conditions and efficacy were investigated on both subcutaneous xenografts of human PC cells in immunocompromised mice and orthotopic intrapancreatic grafts of rat PC cells in syngenic rats. After intratumoral injection of naked plasmid DNA, EP was performed using a two-needle array with 25-msec pulses and either a 300 V/cm field strength for subcutaneous or a 500 V/cm field strength for orthotopic PC, parameters providing the best electrotransfer as reflected by the measurements of both luciferase activity and ePNP mRNA. As expected, tumors developed sensitivity to prodrug treatment (6-methylpurine deoxyribose or fludarabine phosphate). We observed both significant inhibition of tumor growth and extended survival of treated mice. In fact, after prodrug treatment, PC growth in the subcutaneous model was delayed by 50-70% for ePNP-expressing tumors. In an orthotopic pancreatic tumor model, the animal survival was significantly prolonged after ePNP electrogene transfer followed by fludarabine treatment, with one animal out of 10 being tumor-free 6 months thereafter. The current study demonstrates for the first time on PC the in vivo feasibility of electrogene transfer and its therapeutic efficiency using the suicide gene/prodrug system, ePNP/fludarabine. These findings suggest that electrogene therapy strategy must be considered for pancreatic cancer treatment, particularly at advanced stages of the disease.
Assuntos
Adenocarcinoma/terapia , Antineoplásicos/farmacologia , Genes Transgênicos Suicidas , Terapia Genética/métodos , Neoplasias Pancreáticas/terapia , Purina-Núcleosídeo Fosforilase/genética , Vidarabina/análogos & derivados , Animais , Linhagem Celular Tumoral , Eletroquimioterapia , Eletroporação , Técnicas de Transferência de Genes , Genes Bacterianos , Humanos , Neoplasias Experimentais/terapia , Pró-Fármacos/farmacologia , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos Lew , Vidarabina/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Oncolytic viruses have emerged as a novel class of potent anticancer agents offering an improvement on chemo- and radiotherapy in terms of tumor targeting and reduction of side-effects. Among these agents, autonomous parvoviruses have attracted the attention of researchers for their ability to preferentially replicate in and kill transformed cells, and to suppress tumors in the absence of adverse reactions in various animal models. We have previously shown that lethally irradiated autologous tumor cells can support parvovirus H-1PV production and serve as carriers to deliver progeny H-1PV into the vicinity of lung metastases in a rat tumor model, resulting in H-1PV infection of and multiplication in metastatic cells. It is known that irradiated autologous (neoplastic) cells can also act as a therapeutic vaccine against the original tumor. Yet the ability of these cells to suppress metastases in the above model was found to be much increased as a result of their H-1PV infection. This prompted us to determine whether H-1PV boosted the tumor-suppressing capacity of the autologous vaccine by increasing its immunogenic potential and/or by making it a factory of oncolytic viruses able to reach and destroy the metastases. Both effects could be dissociated in the presence of neutralising antibodies which either prevent the progeny viruses from spreading to metastatic cells, or deplete the CD8 effector cells from the immune system. This strategy revealed that the H-1PV infection of tumor cells enhanced their ability to trigger an immune response for which uninfected tumor cells could be the targets, thereby amplifying and taking over from the direct viral oncolytic activity. This dual oncolytic/vaccinal effect of H-1PV holds out promises of clinical applications to cancer therapy.
Assuntos
Vacinas Anticâncer , Parvovirus H-1/fisiologia , Neoplasias Pulmonares/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos/fisiologia , Animais , Anticorpos Antivirais/farmacologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Parvovirus H-1/efeitos dos fármacos , Parvovirus H-1/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Vírus Oncolíticos/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The 3Rs guideline is the gold standard for ethics in animal experimentation. Two of those rules, namely refinement and reduction, require further improvement. The objective of this study was to define pathways to better compliance with these prerequisites. Two methods which move us in this direction are: (1) using small animal imaging techniques for pancreatic ductal adenocarcinoma (PDAC) follow-up and (2) reduction of the number of control animals included in a study of PDAC progression under treatment. MATERIALS AND METHODS: Firstly, we used MicroCT scan to diagnose events showing PDAC progression prior to any clinical symptoms to thereby define more humane endpoints identifiable before any painful phenomenon is observed. Secondly, in order to test the hypothesis of using a reference control group in all preclinical studies of a new treatment of PDAC, we investigated the stability of the results obtained with the control groups in three successive identical studies comparing placebo and gemcitabine in tumor-bearing Lewis rats. RESULTS: Two imaging endpoints were found. The first was the observation of a liver metastasis assessing PDAC diffusion and, earlier than liver metastasis, the presence of bands of fluid along the flanks, with more or less a medial displacement of bowel and solid viscera, reflecting a peritoneal ascites. Results of the longitudinal follow-up of rats in the gemcitabine study revealed heterogeneity in the survival rate in the three control groups, as opposed to the survival rate in the three treated groups which did not differ statistically. As a result, the significance of improved survival with chemotherapy varied greatly according to the control group used for the comparison, ranging from no impact to a highly significant effect. CONCLUSION: The early detection by the means of animal imaging of one or more signs indicating the onset of a critical step in the development of the disease (e.g., ascites or/and metastasis) allows the researcher to prevent the occurrence of animal pain, thereby ensuring better animal welfare. However, using a single standard control group in an effort to use fewer animals for a given model runs such a significant risk of false results that it mars the entire study. Although reducing the number of animals in a study remains the gold standard of our experimental practice, in this case it would come at the price of a loss of validity of the results.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Carcinoma Ductal Pancreático/tratamento farmacológico , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Antimetabólitos Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/diagnóstico por imagem , Carcinoma Ductal Pancreático/mortalidade , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais , Seguimentos , Estudos Longitudinais , Masculino , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/mortalidade , Ratos Endogâmicos Lew , Taxa de Sobrevida , Microtomografia por Raio-X , GencitabinaRESUMO
INTRODUCTION: Toolan's H-1 parvovirus (H-1PV) exerts a cytotoxic/oncolytic effect, predominantly mediated by its non-structural protein (NS1). This rat parvovirus is harmless, unlike other parvoviruses, and its antitumor potential may be useful to clinicians as its oncolytic action appears to be true in numerous non-digestive and digestive cancers. AREAS COVERED: After a brief review of parvovirus genus and biology, we summarize the proposed mechanisms to explain the cytotoxicity of H-1PV to tumors which results in dysregulation of cell transcription, cell-cycle arrest, termination of cell replication, activation of cellular stress response and induction of cell death. Viral oncolysis induces a strong tumor-specific immune response leading to the recognition and elimination of minimal residual disease. As the action of H-1PV is not limited to the digestive tract, we initially analyse studies performed in non-digestive cancers such as glioma (as the virus is able to cross the blood brain barrier), and then focused more particularly on the results in digestive cancers. EXPERT OPINION: Based on the results of studies showing little H-1PV toxicity to living bodies, we advocate for the use of the parvovirus in cancers such as melanoma, glioma and pancreatic ductal adenocarcinoma in addition to conventional chemotherapy.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias do Sistema Digestório/terapia , Parvovirus H-1 , Terapia Viral Oncolítica/métodos , Animais , Morte Celular , Parvovirus H-1/fisiologia , HumanosRESUMO
OBJECTIVES: Our aim was to establish and characterize a novel pancreatic ductal adenocarcinoma cell line from a patient in whom the origin of the invasive carcinoma could be traced back to the intraductal papillary mucinous neoplasm (IPMN) precursor lesion. METHODS: The primary patient-derived tumor was propagated in immunocompromised mice for 2 generations and used to establish a continuous in vitro culture termed ASAN-PaCa. Transplantation to fertilized chicken eggs confirmed the tumorigenic potential in vivo. Molecular analyses included karyotyping, next-generation genomic sequencing, expression analysis of marker proteins, and mucin-profiling. RESULTS: The analysis of marker proteins confirmed the epithelial nature of the established cell line, and revealed that the expression of the mucin MUC1 was higher than that of MUC2 and MUC5AC. ASAN-PaCa cells showed rapid in vitro and in vivo growth and multiple chromosomal aberrations. They harbored mutations in KRAS (Q61H), TP53 (Y220C), and RNF43 (I47V and L418M) but lacked either IPMN-specific GNAS or presumed pancreatic ductal adenocarcinoma-driving mutations in KRAS (codons 12/13), SMAD, and CDKN2A genes. CONCLUSIONS: ASAN-PaCa cell line represents a novel preclinical model of pancreatic adenocarcinoma arising in the background of IPMN, and offers an opportunity to study how further introduction of known driver mutations might contribute to pancreatic carcinogenesis.
Assuntos
Adenocarcinoma , Neoplasias Pancreáticas , Animais , Linhagem Celular , Humanos , Camundongos , Mucina-2RESUMO
Peritoneal dissemination is a common end-stage complication of pancreatic cancer for which novel therapeutic modalities are actively investigated, as there is no current effective therapy. Thus, we evaluated, in a mouse model of pancreatic peritoneal carcinomatosis, the therapeutic potential of a novel nonviral gene therapy approach consisting of bis-guanidinium-tren-cholesterol (BGTC)-mediated lipofection of a combined suicide gene system. Human BxPC-3 pancreatic cells secreting the carcinoembryonic antigen (CEA) tumor marker were injected into the peritoneal cavity of nude mice. After 8 days, intraperitoneal (i.p.) lipofection was performed using BGTC/DOPE cationic liposomes complexed with plasmids encoding the two prodrug-activating enzymes Herpes Simplex Virus thymidine kinase and Escherichia coli cytosine deaminase, the latter being expressed from a bicistronic cassette also encoding E. coli uracil phosphoribosyltransferase. Administration of the lipoplexes was followed by treatment with the corresponding prodrugs ganciclovir and 5-fluorocytosine. The results presented herein demonstrate that BGTC/DOPE liposomes can efficiently mediate gene transfection into peritoneal tumor nodules. Indeed, HSV-TK mRNA was detected in tumor nodule tissues by semiquantitative reverse transcription-polymerase chain reaction analysis. In addition, green fluorescent protein (GFP) fluorescence and X-gal staining were observed in the peritoneal tumor foci following lipofection of the corresponding EGFP and LacZ reporter genes. These expression analyses also showed that transgene expression lasted for about 2 weeks and was preferential for the tumor nodules, this tumor preference being in good agreement with the absence of obvious treatment-related toxicity. Most importantly, mice receiving the full treatment scheme (BGTC liposomes, suicide genes and prodrugs) had significantly lower serum CEA levels than those of the various control groups, a finding indicating that peritoneal carcinomatosis progression was strongly reduced in these mice. In conclusion, our results demonstrate the therapeutic efficiency of BGTC-mediated i.p. lipofection of a combined suicide gene system in a mouse peritoneal carcinomatosis model and suggest that BGTC-based prodrug-activating gene therapy approaches may constitute a potential treatment modality for patients with peritoneal carcinomatosis and minimal residual disease.
Assuntos
Colesterol/análogos & derivados , Genes Transgênicos Suicidas/genética , Terapia Genética/métodos , Guanidinas , Lipossomos/administração & dosagem , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Animais , Biópsia , Antígeno Carcinoembrionário/análise , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citosina Desaminase/genética , Citosina Desaminase/metabolismo , Progressão da Doença , Escherichia coli , Feminino , Flucitosina/farmacologia , Flucitosina/uso terapêutico , Ganciclovir/farmacologia , Ganciclovir/uso terapêutico , Humanos , Lipossomos/química , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Cavidade Peritoneal/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Simplexvirus/enzimologia , Simplexvirus/genética , Timidina Quinase/genética , Timidina Quinase/metabolismo , TransfecçãoRESUMO
Hepatocellular carcinoma (HCC) is one of the most common cancer related deaths worldwide. One of the main challenges in cancer treatment is drug delivery to target cancer cells specifically. Preclinical evaluation of intratumoral drugs in orthotopic liver cancer mouse models is difficult, as percutaneous injection hardly can be precisely performed manually. In the present study we have characterized a hepatoma model developing a single tumor nodule by implantation of Hep55.1C cells in the liver of syngeneic C57BL/6J mice. Tumor evolution was followed up by µCT imaging, and at the histological and molecular levels. This orthotopic, poorly differentiated mouse HCC model expressing fibrosis, inflammation and cancer markers was used to assess the efficacy of drugs. We took advantage of the high precision of a previously developed robotized system for automated, image-guided intratumoral needle insertion, to administer every week in the tumor of the Hep55.1C mouse model. A significant tumor growth inhibition was observed using our robotized system, whereas manual intraperitoneal administration had no effect, by comparison to untreated control mice.
Assuntos
Carcinoma Hepatocelular/patologia , Avaliação de Medicamentos/instrumentação , Neoplasias Hepáticas/patologia , Robótica , Microtomografia por Raio-X , Animais , Carcinoma Hepatocelular/diagnóstico por imagem , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The rat parvovirus H-1PV has oncolytic and tumour-suppressive properties potentially exploitable in cancer therapy. This possibility is being explored and results are encouraging, but it is necessary to improve the oncotoxicity of the virus. Here we show that this can be achieved by co-treating cancer cells with H-1PV and histone deacetylase inhibitors (HDACIs) such as valproic acid (VPA). We demonstrate that these agents act synergistically to kill a range of human cervical carcinoma and pancreatic carcinoma cell lines by inducing oxidative stress, DNA damage and apoptosis. Strikingly, in rat and mouse xenograft models, H-1PV/VPA co-treatment strongly inhibits tumour growth promoting complete tumour remission in all co-treated animals. At the molecular level, we found acetylation of the parvovirus nonstructural protein NS1 at residues K85 and K257 to modulate NS1-mediated transcription and cytotoxicity, both of which are enhanced by VPA treatment. These results warrant clinical evaluation of H-1PV/VPA co-treatment against cervical and pancreatic ductal carcinomas.
Assuntos
Carcinoma/terapia , Vírus Oncolíticos/fisiologia , Parvovirus/fisiologia , Ácido Valproico/farmacologia , Animais , Apoptose/efeitos dos fármacos , Carcinoma/tratamento farmacológico , Carcinoma/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Células HeLa , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Estresse Oxidativo/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Parvovirus/metabolismo , Ratos , Ratos Nus , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Ácido Valproico/uso terapêuticoRESUMO
Animal experimentation is a prerequisite for preclinical evaluation of treatments such as chemotherapy. It's strictly regulated with the purpose of reducing the number of experimental animal as well as their pain. Small animal imaging should provide a painless longitudinal follow up of tumor progression on a single animal. The aim of the study is to validate small animal imaging by microscanner (µscan) in longitudinal follow up of a hepatocellular carcinoma (HCC) and to demonstrate its interest for in vivo evaluation of tumor response to different therapeutics. An HCC model achieved by orthotopic graft of the MH3924A cell line in ACI rats was followed using a Imtek/Siemens microscanner (µscan) with contrast agents (Fenestra(®) LC/VC). The procedures giving the optimal enhancement of the liver as well as a reliable determination of tumor volumes by µscan were validated. Three protocols for therapeutic assessment through µscan longitudinal follow up were performed. Each consisted in three groups testing a chemotherapy (gemcitabine, gemcitabine-oxaliplatine or sorafenib) versus two control groups (placebo and doxorubicine). Comparison was done on tumor volumes, median and actual survivals. There was a significant correlation between tumor volumes measured by µscan and autopsy. Treatment by sorafenib, at the contrary of gemcitabine alone or with oxaliplatine, resulted in a significant reduction in tumor volumes and prolongation of actuarial survival. These results are consistent with available clinical data for these diverse therapeutics. In conclusion, small animal imaging with µscan is a non-invasive, reliable, and reproducible method for preclinical evaluation of antitumor agents.
Assuntos
Carcinoma Hepatocelular/diagnóstico por imagem , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/tratamento farmacológico , Microtomografia por Raio-X/métodos , Animais , Antineoplásicos/uso terapêutico , Benzenossulfonatos/administração & dosagem , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Meios de Contraste/administração & dosagem , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Modelos Animais de Doenças , Progressão da Doença , Doxorrubicina/administração & dosagem , Monitoramento de Medicamentos/métodos , Neoplasias Hepáticas/patologia , Masculino , Niacinamida/análogos & derivados , Compostos Organoplatínicos/administração & dosagem , Oxaliplatina , Compostos de Fenilureia , Piridinas/administração & dosagem , Ratos , Ratos Endogâmicos ACI , Sorafenibe , Análise de Sobrevida , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Oncolytic viruses with their capacity to specifically replicate in and kill tumor cells emerged as a novel class of cancer therapeutics. Rat oncolytic parvovirus (H-1PV) was used to treat different types of cancer in preclinical settings and was lately successfully combined with standard gemcitabine chemotherapy in treating pancreatic ductal adenocarcinoma (PDAC) in rats. Our previous work showed that the immune system and particularly the release of interferon-gamma (IFNγ) seem to mediate the anticancer effect of H-1PV in that model. Therefore, we reasoned that the therapeutic properties of H-1PV can be boosted with IFNγ for the treatment of late incurable stages of PDAC like peritoneal carcinomatosis. Rats bearing established orthotopic pancreatic carcinomas with peritoneal metastases were treated with a single intratumoral (i.t.) or intraperitoneal (i.p.) injection of 5 x 108 plaque forming units of H-1PV with or without concomitant IFNγ application. Intratumoral injection proved to be more effective than the intraperitoneal route in controlling the growth of both the primary pancreatic tumors and peritoneal carcinomatosis, accompanied by migration of virus from primary to metastatic deposits. Concomitant i.p. treatment of H-1PV with recIFNγ resulted in improved therapeutic effect yielding an extended animal survival, compared with i.p. treatment with H-1PV alone. IFNγ application enhanced the H-1PV-induced peritoneal macrophage and splenocyte responses against tumor cells while causing a significant reduction in the titers of H1-PV-neutralising antibodies in ascitic fluid. Thus, IFNγ co-application together with H-1PV might be considered as a novel therapeutic option to improve the survival of PDAC patients with peritoneal carcinomatosis.