Efficient colonization and therapy of human hepatocellular carcinoma (HCC) using the oncolytic vaccinia virus strain GLV-1h68

Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In this study, we analyzed for the first time the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 in two human hepatocellular carcinoma cell lines HuH7 and PLC/PRF/5 (PLC) in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 efficiently colonized, replicated in, and did lyse these cancer cells in culture. Experiments with HuH7 and PLC xenografts have revealed that a single intravenous injection (i.v.) of mice with GLV-1h68 resulted in a significant reduction of primary tumor sizes compared to uninjected controls. In addition, replication of GLV-1h68 in tumor cells led to strong inflammatory and oncolytic effects resulting in intense infiltration of MHC class II-positive cells like neutrophils, macrophages, B cells and dendritic cells and in up-regulation of 13 pro-inflammatory cytokines. Furthermore, GLV-1h68 infection of PLC tumors inhibited the formation of hemorrhagic structures which occur naturally in PLC tumors. Interestingly, we found a strongly reduced vascular density in infected PLC tumors only, but not in the non-hemorrhagic HuH7 tumor model. These data demonstrate that the GLV-1h68 vaccinia virus may have an enormous potential for treatment of human hepatocellular carcinoma in man.     

Imaging characteristics, tissue distribution, and spread of a novel oncolytic vaccinia virus carrying the human sodium iodide symporter

Introduction

Oncolytic viruses show promise for treating cancer. However, to assess therapy and potential toxicity, a noninvasive imaging modality is needed. This study aims to determine the in vivo biodistribution, and imaging and timing characteristics of a vaccinia virus, GLV-1h153, encoding the human sodium iodide symporter (hNIS.

Methods

GLV-1h153 was modified from GLV-1h68 to encode the hNIS gene. Timing of cellular uptake of radioiodide ¹³¹I in human pancreatic carcinoma cells PANC-1 was assessed using radiouptake assays. Viral biodistribution was determined in nude mice bearing PANC-1 xenografts, and infection in tumors confirmed histologically and optically via Green Fluorescent Protein (GFP) and bioluminescence. Timing characteristics of enhanced radiouptake in xenografts were assessed via ¹²⁴I-positron emission tomography (PET). Detection of systemic administration of virus was investigated with both ¹²⁴I-PET and 99m-technecium gamma-scintigraphy.

Results

GLV-1h153 successfully facilitated time-dependent intracellular uptake of ¹³¹I in PANC-1 cells with a maximum uptake at 24 hours postinfection (P<0.05). In vivo, biodistribution profiles revealed persistence of virus in tumors 5 weeks postinjection at 10⁹ plaque-forming unit (PFU)/gm tissue, with the virus mainly cleared from all other major organs. Tumor infection by GLV-1h153 was confirmed via optical imaging and histology. GLV-1h153 facilitated imaging virus replication in tumors via PET even at 8 hours post radiotracer injection, with a mean %ID/gm of 3.82±0.46 (P<0.05) 2 days after intratumoral administration of virus, confirmed via tissue radiouptake assays. One week post systemic administration, GLV-1h153-infected tumors were detected via ¹²⁴I-PET and 99m-technecium-scintigraphy.

Conclusion

GLV-1h153 is a promising oncolytic agent against pancreatic cancer with a promising biosafety profile. GLV-1h153 facilitated time-dependent hNIS-specific radiouptake in pancreatic cancer cells, facilitating detection by PET with both intratumoral and systemic administration. Therefore, GLV-1h153 is a promising candidate for the noninvasive imaging of virotherapy and warrants further study into longterm monitoring of virotherapy and potential radiocombination therapies with this treatment and imaging modality.     

Virotherapy of Canine Tumors with Oncolytic Vaccinia Virus GLV-1h109 Expressing an Anti-VEGF Single-Chain Antibody

Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for cancer therapy. We have previously reported that oncolytic vaccinia virus strains expressing an anti-VEGF (Vascular Endothelial Growth Factor) single-chain antibody (scAb) GLAF-1 exhibited significant therapeutic efficacy for treatment of human tumor xenografts. Here, we describe the use of oncolytic vaccinia virus GLV-1h109 encoding GLAF-1 for canine cancer therapy. In this study we analyzed the virus-mediated delivery and production of scAb GLAF-1 and the oncolytic and immunological effects of the GLV-1h109 vaccinia virus strain against canine soft tissue sarcoma and canine prostate carcinoma in xenograft models. Cell culture data demonstrated that the GLV-1h109 virus efficiently infect, replicate in and destroy both tested canine cancer cell lines. In addition, successful expression of GLAF-1 was demonstrated in virus-infected canine cancer cells and the antibody specifically recognized canine VEGF. In two different xenograft models, the systemic administration of the GLV-1h109 virus was found to be safe and led to anti-tumor and immunological effects resulting in the significant reduction of tumor growth in comparison to untreated control mice. Furthermore, tumor-specific virus infection led to a continued production of functional scAb GLAF-1, resulting in inhibition of angiogenesis. Overall, the GLV-1h109-mediated cancer therapy and production of immunotherapeutic anti-VEGF scAb may open the way for combination therapy concept i.e. vaccinia virus mediated oncolysis and intratumoral production of therapeutic drugs in canine cancer patients.     

Preclinical evaluation of oncolytic vaccinia virus for therapy of canine soft tissue sarcoma

Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for canine cancer therapy. In this study we describe the establishment of an in vivo model of canine soft tissue sarcoma (CSTS) using the new isolated cell line STSA-1 and the analysis of the virus-mediated oncolytic and immunological effects of two different Lister VACV LIVP1.1.1 and GLV-1h68 strains against CSTS. Cell culture data demonstrated that both tested VACV strains efficiently infected and destroyed cells of the canine soft tissue sarcoma line STSA-1. In addition, in our new canine sarcoma tumor xenograft mouse model, systemic administration of LIVP1.1.1 or GLV-1h68 viruses led to significant inhibition of tumor growth compared to control mice. Furthermore, LIVP1.1.1 mediated therapy resulted in almost complete tumor regression and resulted in long-term survival of sarcoma-bearing mice. The replication of the tested VACV strains in tumor tissues led to strong oncolytic effects accompanied by an intense intratumoral infiltration of host immune cells, mainly neutrophils. These findings suggest that the direct viral oncolysis of tumor cells and the virus-dependent activation of tumor-associated host immune cells could be crucial parts of anti-tumor mechanism in STSA-1 xenografts. In summary, the data showed that both tested vaccinia virus strains and especially LIVP1.1.1 have great potential for effective treatment of CSTS.     

FOXP3+ Lymphocyte Density in Pancreatic Cancer Correlates with Lymph Node Metastasis

Objective

To determine if the density of FOXP3⁺ lymphocytes in primary tumors and lymph nodes in pancreatic cancer correlates with the presence of lymph node metastases.

Methods

FOXP3⁺ lymphocyte density in primary pancreatic cancer tissue and draining lymph nodes was measured using immunohistochemistry. We analyzed the clinical and pathological aspects associated with the accumulation of FOXP3⁺ lymphocytes in pancreatic cancer. We also analyzed the correlation of density of FOXP3⁺ lymphocytes in lymph nodes with the nodal status and distance from the primary tumor.

Results

FOXP3⁺ lymphocyte density in pancreatic cancer was significantly higher than in paratumoral pancreatic tissue. The density of FOXP3⁺ lymphocytes in local tumor tissue correlated significantly with the histological grade and overall lymph node status. Furthermore, FOXP3⁺ lymphocyte density was significantly higher in positive lymph nodes than in negative ones, while it had no correlation with the distance of the lymph node from the primary tumor.

Conclusion

FOXP3⁺ lymphocyte density in primary tumor tissue in patients with pancreatic cancer correlates with lymph node metastasis. Lymph nodes containing metastases having higher FOXP3⁺ lymphocyte densities than do negative lymph nodes.     

INPP4B suppresses prostate cancer cell invasion

Background

INPP4B and PTEN dual specificity phosphatases are frequently lost during progression of prostate cancer to metastatic disease. We and others have previously shown that loss of INPP4B expression correlates with poor prognosis in multiple malignancies and with metastatic spread in prostate cancer.

Results

We demonstrate that de novo expression of INPP4B in highly invasive human prostate carcinoma PC-3 cells suppresses their invasion both in vitro and in vivo. Using global gene expression analysis, we found that INPP4B regulates a number of genes associated with cell adhesion, the extracellular matrix, and the cytoskeleton. Importantly, de novo expressed INPP4B suppressed the proinflammatory chemokine IL-8 and induced PAK6. These genes were regulated in a reciprocal manner following downregulation of INPP4B in the independently derived INPP4B-positive LNCaP prostate cancer cell line. Inhibition of PI3K/Akt pathway, which is highly active in both PC-3 and LNCaP cells, did not reproduce INPP4B mediated suppression of IL-8 mRNA expression in either cell type. In contrast, inhibition of PKC signaling phenocopied INPP4B-mediated inhibitory effect on IL-8 in either prostate cancer cell line. In PC-3 cells, INPP4B overexpression caused a decline in the level of metastases associated BIRC5 protein, phosphorylation of PKC, and expression of the common PKC and IL-8 downstream target, COX-2. Reciprocally, COX-2 expression was increased in LNCaP cells following depletion of endogenous INPP4B.

Conclusion

Taken together, we discovered that INPP4B is a novel suppressor of oncogenic PKC signaling, further emphasizing the role of INPP4B in maintaining normal physiology of the prostate epithelium and suppressing metastatic potential of prostate tumors.

     

The highly attenuated oncolytic recombinant vaccinia virus GLV-1h68: comparative genomic features and the contribution of <Emphasis Type="Italic">F14.5L</Emphasis> inactivation

As a new anticancer treatment option, vaccinia virus (VACV) has shown remarkable antitumor activities (oncolysis) in preclinical studies, but potential infection of other organs remains a safety concern. We present here genome comparisons between the de novo sequence of GLV-1h68, a recombinant VACV, and other VACVs. The identified differences in open reading frames (ORFs) include genes encoding host-range selection, virulence and immune modulation proteins, e.g., ankyrin-like proteins, serine proteinase inhibitor SPI-2/CrmA, tumor necrosis factor (TNF) receptor homolog CrmC, semaphorin-like and interleukin-1 receptor homolog proteins. Phylogenetic analyses indicate that GLV-1h68 is closest to Lister strains but has lost several ORFs present in its parental LIVP strain, including genes encoding CrmE and a viral Golgi anti-apoptotic protein, v-GAAP. The reduced pathogenicity of GLV-1h68 is confirmed in male mice bearing C6 rat glioma and in immunocompetent mice bearing B16-F10 murine melanoma. The contribution of foreign gene expression cassettes in the F14.5L, J2R and A56R loci is analyzed, in particular the contribution of F14.5L inactivation to the reduced virulence is demonstrated by comparing the virulence of GLV-1h68 with its F14.5L-null and revertant viruses. GLV-1h68 is a promising engineered VACV variant for anticancer therapy with tumor-specific replication, reduced pathogenicity and benign tissue tropism.     

Bone microenvironment and androgen status modulate subcellular localization of ErbB3 in prostate cancer cells

ErbB-3, an ErbB receptor tyrosine kinase, has been implicated in the pathogenesis of several malignancies, including prostate cancer. We found that ErbB-3 expression was up-regulated in prostate cancer cells within lymph node and bone metastases. Despite being a plasma membrane protein, ErbB-3 was also detected in the nuclei of the prostate cancer cells in the metastatic specimens. Because most metastatic specimens were from men who had undergone androgen ablation, we examined the primary tumors from patients who have undergone hormone deprivation therapy and found that a significant fraction of these specimens showed nuclear localization of ErbB3. We thus assessed the effect of androgens and the bone microenvironment on the nuclear translocation of ErbB-3 by using xenograft tumor models generated from bone-derived prostate cancer cell lines, MDA PCa 2b, and PC-3. In subcutaneous tumors, ErbB-3 was predominantly in the membrane/cytoplasm; however, it was present in the nuclei of the tumor cells in the femur. Castration of mice bearing subcutaneous MDA PCa 2b tumors induced a transient nuclear translocation of ErbB-3, with relocalization to the membrane/cytoplasm upon tumor recurrence. These findings suggest that the bone microenvironment and androgen status influence the subcellular localization of ErbB-3 in prostate cancer cells. We speculate that nuclear localization of ErbB-3 may aid prostate cancer cell survival during androgen ablation and progression of prostate cancer in bone.     

Pim Kinases Promote Migration and Metastatic Growth of Prostate Cancer Xenografts

Background and methods

Pim family proteins are oncogenic kinases implicated in several types of cancer and involved in regulation of cell proliferation, survival as well as motility. Here we have investigated the ability of Pim kinases to promote metastatic growth of prostate cancer cells in two xenograft models for human prostate cancer. We have also evaluated the efficacy of Pim-selective inhibitors to antagonize these effects.

Results

We show here that tumorigenic growth of both subcutaneously and orthotopically inoculated prostate cancer xenografts is enhanced by stable overexpression of either Pim-1 or Pim-3. Moreover, Pim-overexpressing orthotopic prostate tumors are highly invasive and able to migrate not only to the nearby prostate-draining lymph nodes, but also into the lungs to form metastases. When the xenografted mice are daily treated with the Pim-selective inhibitor DHPCC-9, both the volumes as well as the metastatic capacity of the tumors are drastically decreased. Interestingly, the Pim-promoted metastatic growth of the orthotopic xenografts is associated with enhanced angiogenesis and lymphangiogenesis. Furthermore, forced Pim expression also increases phosphorylation of the CXCR4 chemokine receptor, which may enable the tumor cells to migrate towards tissues such as the lungs that express the CXCL12 chemokine ligand.

Conclusions

Our results indicate that Pim overexpression enhances the invasive properties of prostate cancer cells in vivo. These effects can be reduced by the Pim-selective inhibitor DHPCC-9, which can reach tumor tissues without serious side effects. Thus, Pim-targeting therapies with DHPCC-9-like compounds may help to prevent progression of local prostate carcinomas to fatally metastatic malignancies.     

Endoplasmic reticulum stress,autophagic and apoptotic cell death,and immune activation by a natural triterpenoid in human prostate cancer cells

Though the current therapies are effective at clearing an early stage prostate cancer, they often fail to treat late-stage metastatic disease. We aimed to investigate the molecular mechanisms underlying the anticancer effects of a natural triterpenoid, ganoderic acid DM (GA-DM), on two human prostate cancer cell lines: the androgen-independent prostate carcinoma (PC-3), and androgen-sensitive prostate adenocarcinoma (LNCaP). Cell viability assay showed that GA-DM was relatively more toxic to LNCaP cells than to PC-3 cells (IC₅₀s ranged 45-55 µM for PC-3, and 20-25 µM for LNCaP), which may have occurred due to differential expression of p53. Hoechst DNA staining confirmed detectable nuclear fragmentation in both cell lines irrespective of the p53 status. GA-DM treatment decreased Bcl-2 proteins while it upregulated apoptotic Bax and autophagic Beclin-1, Atg5, and LC-3 molecules, and caused an induction of both early and late events of apoptotic cell death. Biochemical analyses of GA-DM-treated prostate cancer cells demonstrated that caspase-3 cleavage was notable in GA-DM-treated PC-3 cells. Interestingly, GA-DM treatment altered cell cycle progression in the S phase with a significant growth arrest in the G2 checkpoint and enhanced CD4 ⁺ T cell recognition of prostate tumor cells. Mechanistic study of GA-DM-treated prostate cancer cells further demonstrated that calpain activation and endoplasmic reticulum stress contributed to cell death. These findings suggest that GA-DM is a candidate for future drug design for prostate cancer as it activates multiple pathways of cell death and immune recognition.     

Prostate Cancer Heterogeneous High-Metastatic Multi-Organ-Colonizing Chemo-Resistant Variants Selected by Serial Metastatic Passage in Nude Mice Are Highly Enriched for Multinucleate Giant Cells

In order to further understand the role of tumor heterogeneity in metastasis and chemo-resistance, high metastatic PC-3 human prostate cancer variants were selected by injecting parental PC-3 cells, expressing green fluorescent protein (GFP) in the footpad of nude mice, which then metastasize to inguinal lymph nodes. The PC-3-GFP cells which metastasized to the inguinal lymph nodes were collected and were re-injected to the footpad. After 6 such cycles, the PC-3-GFP cells collected from inguinal lymph nodes (PC-3-GFP-LN) were again injected to the footpad. PC-3-GFP-LN showed 100% metastasis to major lymph nodes (popliteal, inguinal, axillary, and cervical), and 100% metastasis to bone and lung. The percent of giant cell variants was enriched in PC-3-GFP-LN-6 compared to parental cells and increased with each cycle of selection, which in turn had increased metastasis. PC-3-GFP-LN-6 cells were resistant to 5-fluorouracil, doxorubicin and cisplatinum, compared to parental PC-3. However, PC-3-GFP-LN-6 was sensitive to the traditional Chinese medicine (TCM) herbal mixture LQ, similar to the parental cells. These results suggest that PC-3 tumors are heterogenous and that subpopulations of highly metastatic, drug-resistant cells can be step-wise selected using a mouse model of tumor progression.     

Flavonoid ampelopsin inhibits the growth and metastasis of prostate cancer in vitro and in mice

The objective of this study was to evaluate the chemopreventive effect of a novel flavonoid, ampelopsin (AMP) on the growth and metastasis of prostate cancer cells. AMP showed the more potent activity in inhibiting the proliferation of androgen-sensitive LNCaP and, to less extent, androgen-independent PC-3 human prostate cancer cell lines in vitro, primarily by induction of apoptosis associated with down-regulation of bcl-2. On the other hand, AMP showed much less activity in inhibiting the proliferation of normal prostate epithelial cells than that of prostate cancer cell lines. AMP also inhibited the migration and invasion of PC-3 cells in vitro associated with down-regulation of CXCR4 expression. In the animal study using an orthotopic prostate tumor model, AMP (150 and 300 mg/kg body weight) inhibited the growth of PC-3 tumors and lymph node and lung metastases in a dose-dependent manner. Compared to the control mice, mice treated with AMP at 300 mg/kg BW had reduced final tumor weight by 49.2% (P<0.05), lymph node metastases by 54.5% (P?=?0.3) and lung metastases by 93% (P<0.05), but had no apparent alteration on food intake or body weight. The in vivo anti-growth and anti-metastasis activities of AMP were associated with induction of apoptosis and inhibition of proliferation of prostate cancer cells, reduction of prostate tumor angiogenesis, and reduction of CXCR4 expression. Our results provide supporting evidence to warrant further investigation to develop AMP as a novel efficacious and safe candidate agent against progression and metastasis of prostate cancer.     

Evaluation of a New Recombinant Oncolytic Vaccinia Virus Strain GLV-5b451 for Feline Mammary Carcinoma Therapy

Virotherapy on the basis of oncolytic vaccinia virus (VACV) infection is a promising approach for cancer therapy. In this study we describe the establishment of a new preclinical model of feline mammary carcinoma (FMC) using a recently established cancer cell line, DT09/06. In addition, we evaluated a recombinant vaccinia virus strain, GLV-5b451, expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as an oncolytic agent against FMC. Cell culture data demonstrate that GLV-5b451 virus efficiently infected, replicated in and destroyed DT09/06 cancer cells. In the selected xenografts of FMC, a single systemic administration of GLV-5b451 led to significant inhibition of tumor growth in comparison to untreated tumor-bearing mice. Furthermore, tumor-specific virus infection led to overproduction of functional scAb GLAF-2, which caused drastic reduction of intratumoral VEGF levels and inhibition of angiogenesis.In summary, here we have shown, for the first time, that the vaccinia virus strains and especially GLV-5b451 have great potential for effective treatment of FMC in animal model.     

In vivo dynamics of pulmonary lymphoid cell subpopulations generated against pulmonary metastasis: evaluation by broncho alveolar lavage fluid

The dynamics of lymphoid cell subpopulations in bronchoalveolar lavage fluid (BALF) and the systemic lymphoid organs of mice after intravenous injection of B16 melanoma cells were examined with a fluorescence-activated cell sorter. The lymphoid cell subpopulations of BALF and mediastinal lymph nodes showed significant changes in numbers and proportions, while those of other lymphoid organs including inguinal lymph nodes, thymus and spleen, showed little change. In week 1, the cells with a Thy-1.2⁺, Lyt-1⁺, L3T4⁻, Lyt-2⁻ phenotype and asialo-Gm1⁺ cells in BALF significantly increased and L3T4⁺ cells slightly increased in number. By week 3, the numbers of Lyt-2⁺ cells in BALF markedly increased in number (by about 90 times) compared with controls. The number of Thy1.2⁺ cells in mediastinal lymph nodes also increased significantly by week 3. Mice that had been pretreated with an immunosuppressive dose of cyclophosphamide were also inoculated intravenously with B16 melanoma cells. In these mice, a significantly increased number of pleural tumors developed and the number of Thy-1.2⁺ cells in BALF was markedly reduced from week 1 to 3. The results indicate that L3T4 and Lyt-2 double negative T-cells and natural killer (NK) cells may be generated and/or mobilized to the lung in an early phase of experimental metastasis of B16 melanoma cells and that at a later stage, when multiple metastases develop, T-cells with a Lyt-2⁺ phenotype markedly increase, probably as an expression of a host reaction against proliferating metastatic tumor cells.     

Comparison of Immunity in Mice Cured of Primary/Metastatic Growth of EMT6 or 4THM Breast Cancer by Chemotherapy or Immunotherapy

Purpose

We have compared cure from local/metastatic tumor growth in BALB/c mice receiving EMT6 or the poorly immunogenic, highly metastatic 4THM, breast cancer cells following manipulation of immunosuppressive CD200:CD200R interactions or conventional chemotherapy.

Methods

We reported previously that EMT6 tumors are cured in CD200R1KO mice following surgical resection and immunization with irradiated EMT6 cells and CpG oligodeoxynucleotide (CpG), while wild-type (WT) animals developed pulmonary and liver metastases within 30 days of surgery. We report growth and metastasis of both EMT6 and a highly metastatic 4THM tumor in WT mice receiving iv infusions of Fab anti-CD200R1 along with CpG/tumor cell immunization. Metastasis was followed both macroscopically (lung/liver nodules) and microscopically by cloning tumor cells at limiting dilution in vitro from draining lymph nodes (DLN) harvested at surgery. We compared these results with local/metastatic tumor growth in mice receiving 4 courses of combination treatment with anti-VEGF and paclitaxel.

Results

In WT mice receiving Fab anti-CD200R, no tumor cells are detectable following immunotherapy, and CD4+ cells produced increased TNFα/IL-2/IFNγ on stimulation with EMT6 in vitro. No long-term cure was seen following surgery/immunotherapy of 4THM, with both microscopic (tumors in DLN at limiting dilution) and macroscopic metastases present within 14 d of surgery. Chemotherapy attenuated growth/metastases in 4THM tumor-bearers and produced a decline in lung/liver metastases, with no detectable DLN metastases in EMT6 tumor-bearing mice-these latter mice nevertheless showed no significantly increased cytokine production after restimulation with EMT6 in vitro. EMT6 mice receiving immunotherapy were resistant to subsequent re-challenge with EMT6 tumor cells, but not those receiving curative chemotherapy. Anti-CD4 treatment caused tumor recurrence after immunotherapy, but produced no apparent effect in either EMT6 or 4THM tumor bearers after chemotherapy treatment.

Conclusion

Immunotherapy, but not chemotherapy, enhances CD4⁺ immunity and affords long-term control of breast cancer growth and resistance to new tumor foci.     

PRC2/EED-EZH2 Complex Is Up-Regulated in Breast Cancer Lymph Node Metastasis Compared to Primary Tumor and Correlates with Tumor Proliferation In Situ

Background

Lymph node metastasis is a key event in the progression of breast cancer. Therefore it is important to understand the underlying mechanisms which facilitate regional lymph node metastatic progression.

Methodology/Principal Findings

We performed gene expression profiling of purified tumor cells from human breast tumor and lymph node metastasis. By microarray network analysis, we found an increased expression of polycomb repression complex 2 (PRC2) core subunits EED and EZH2 in lymph node metastatic tumor cells over primary tumor cells which were validated through real-time PCR. Additionally, immunohistochemical (IHC) staining and quantitative image analysis of whole tissue sections showed a significant increase of EZH2 expressing tumor cells in lymph nodes over paired primary breast tumors, which strongly correlated with tumor cell proliferation in situ. We further explored the mechanisms of PRC2 gene up-regulation in metastatic tumor cells and found up-regulation of E2F genes, MYC targets and down-regulation of tumor suppressor gene E-cadherin targets in lymph node metastasis through GSEA analyses. Using IHC, the expression of potential EZH2 target, E-cadherin was examined in paired primary/lymph node samples and was found to be significantly decreased in lymph node metastases over paired primary tumors.

Conclusions/Significance

This study identified an over expression of the epigenetic silencing complex PRC2/EED-EZH2 in breast cancer lymph node metastasis as compared to primary tumor and its positive association with tumor cell proliferation in situ. Concurrently, PRC2 target protein E-cadherin was significant decreased in lymph node metastases, suggesting PRC2 promotes epithelial mesenchymal transition (EMT) in lymph node metastatic process through repression of E-cadherin. These results indicate that epigenetic regulation mediated by PRC2 proteins may provide additional advantage for the outgrowth of metastatic tumor cells in lymph nodes. This opens up epigenetic drug development possibilities for the treatment and prevention of lymph node metastasis in breast cancer.     

Response of the mouse lung transcriptome to welding fume: effects of stainless and mild steel fumes on lung gene expression in A/J and C57BL/6J mice

Background

Nitrogen dioxide (NO₂) is an air pollutant associated with poor respiratory health, asthma exacerbation, and an increased likelihood of inhalational allergies. NO₂ is also produced endogenously in the lung during acute inflammatory responses. NO₂ can function as an adjuvant, allowing for allergic sensitization to an innocuous inhaled antigen and the generation of an antigen-specific Th2 immune response manifesting in an allergic asthma phenotype. As CD11c⁺ antigen presenting cells are considered critical for naïve T cell activation, we investigated the role of CD11c⁺ cells in NO₂-promoted allergic sensitization.

Methods

We systemically depleted CD11c⁺ cells from transgenic mice expressing a simian diphtheria toxin (DT) receptor under of control of the CD11c promoter by administration of DT. Mice were then exposed to 15 ppm NO₂ followed by aerosolized ovalbumin to promote allergic sensitization to ovalbumin and were studied after subsequent inhaled ovalbumin challenges for manifestation of allergic airway disease. In addition, pulmonary CD11c⁺ cells from wildtype mice were studied after exposure to NO₂ and ovalbumin for cellular phenotype by flow cytometry and in vitro cytokine production.

Results

Transient depletion of CD11c⁺ cells during sensitization attenuated airway eosinophilia during allergen challenge and reduced Th2 and Th17 cytokine production. Lung CD11c⁺ cells from wildtype mice exhibited a significant increase in MHCII, CD40, and OX40L expression 2 hours following NO₂ exposure. By 48 hours, CD11c⁺MHCII⁺ DCs within the mediastinal lymph node (MLN) expressed maturation markers, including CD80, CD86, and OX40L. CD11c⁺CD11b^- and CD11c⁺CD11b⁺ pulmonary cells exposed to NO₂ in vivo increased uptake of antigen 2 hours post exposure, with increased ova-Alexa 647⁺ CD11c⁺MHCII⁺ DCs present in MLN from NO₂-exposed mice by 48 hours. Co-cultures of ova-specific CD4⁺ T cells from naïve mice and CD11c⁺ pulmonary cells from NO₂-exposed mice produced IL-1, IL-12p70, and IL-6 in vitro and augmented antigen-induced IL-5 production.

Conclusions

CD11c⁺ cells are critical for NO₂-promoted allergic sensitization. NO₂ exposure causes pulmonary CD11c⁺ cells to acquire a phenotype capable of increased antigen uptake, migration to the draining lymph node, expression of MHCII and co-stimulatory molecules required to activate naïve T cells, and secretion of polarizing cytokines to shape a Th2/Th17 response.     

Étude de faisabilité du ganglion sentinelle dans le cancer de la prostate par cœlioscopie : premiers résultats

Objectives

The standard lymphadenectomy is currently a challenge in the management of prostate cancer. The aim of this prospective study was to evaluate the performance of the sentinel lymph node (SLN) by laparoscopy in patients with localized prostate cancer, candidates for local treatment.

Patients and methods

Patients were injected transrectal ultrasound-guided with 0.3 mL/100MBq 99^mTc-Sulfur rhenium colloid in each prostatic lobe, the day before surgery. Lymphoscintigraphy was performed after 2 hours. The detection was realized intraoperatively with a laparoscopic probe (Gamma Sup Clerad^®) followed by extensive dissection. Counts of SLN were performed in vivo and confirmed ex vivo. The histological analysis was performed by HPS staining and followed by immunochemistry if SLN was free.

Results

Seventy patients with carcinoma of the prostate at intermediate or high risk of lymph node metastases (D’Amico), PSA median 9.5 ng/mL [6–130], were included in the study. The lymphoscintigraphic detection rate was 94.2% (66/70) and intraoperative of 97.0% (68/70). Fourteen patients had lymph node metastases, six only in SLN. The false negative rate was 2/14 (14.0%). The internal iliac region is the first metastatic site (40.9%). Limited or standard lymph node dissection would have ignored respectively 72.7% and 59.0% of lymph node metastases.

Conclusion

The laparoscopy is adapted to a broad identification of SLN and targeted dissection of these lymph nodes significantly limits the risk of surgical extended dissection while maintaining the accuracy of the information.     

Autochthonous primary and metastatic melanomas in Hgf-Cdk4R24C mice evade T-cell-mediated immune surveillance

Genetically engineered mouse models offer new opportunities to investigate the role of cell-mediated immunity in the natural progression of melanoma in an immunocompetent host. Here we report that Hgf-Cdk4^R24C mice spontaneously develop a spectrum of primary melanomas with high penetrance during their first year of life. Malignant transformation proceeds in a stepwise manner from multiple melanocytic nevi to single nodular melanomas and disseminated metastases in most mice. Migrating melanoma cells invade the draining lymph nodes without activating the immune system. Autochthonous primary tumors are destroyed following experimental introduction of immune surveillance using an adoptive lymphocyte transfer approach. However, some tumor cells are able to survive, evade immune cell control, and recur both locally and systemically. Immune tolerance in recurring tumors may be supported by immunosuppressive Gr1⁺ myeloid cells. Taken together, our results demonstrate that primary and metastatic melanomas developing spontaneously in Hgf-Cdk4^R24C mice effectively evade cellular immune surveillance.     

Myxoma Virus Oncolytic Efficiency Can Be Enhanced Through Chemical or Genetic Disruption of the Actin Cytoskeleton

Myxoma virus (MYXV) is one of many animal viruses that exhibit oncolytic properties in transformed human cells. Compared to orthopoxviruses like vaccinia (VACV), MYXV spreads inefficiently, which could compromise its use in treating tumors and their associated metastases. The VACV F11 protein promotes virus exit and rapid spread by inhibiting Rho signalling, which results in a disruption of cortical actin. We have previously shown that although MYXV lacks an F11 homolog, the F11L gene can be introduced into MYXV promoting the spread of this Leporipoxvirus in natural host cells. Here we show that the F11-encoding (F11L⁺) MYXV strain replicates to higher levels in a number of human cancer cells. We also show that F11L⁺ MYXV induces better tumor control and prolonged survival of mice bearing MDA-MB-231 cancer cells. Furthermore, we show that this virus also spreads more efficiently from the site of growth in one injected tumor, to a second untreated tumor.While we focused mostly on the use of a modified MYXV we were able to show that the effects of F11 on MYXV growth in cancer cells could be mimicked through the use of pharmacological inhibition or siRNA-mediated silencing of key regulators of cortical actin (RhoA, RhoC, mDia1, or LIMK2). These data suggest that it may be possible to increase the oncolytic efficacy of wild-type MYXV using chemical inhibitors of RhoA/C or their downstream targets. Furthermore, since all viruses must overcome barriers to exit posed by structures like cortical actin, these findings suggest that the oncolytic activity of other viruses may be enhanced through similar strategies.