Intratumoral Canine Distemper Virus Infection Inhibits Tumor Growth by Modulation of the Tumor Microenvironment in a Murine Xenograft Model of Canine Histiocytic Sarcoma.

Histiocytic sarcomas refer to highly aggressive tumors with a poor prognosis that respond poorly to conventional treatment approaches. Oncolytic viruses, which have gained significant traction as a cancer therapy in recent decades, represent a promising option for treating histiocytic sarcomas through their replication and/or by modulating the tumor microenvironment. The live attenuated canine distemper virus (CDV) vaccine strain Onderstepoort represents an attractive candidate for oncolytic viral therapy. In the present study, oncolytic virotherapy with CDV was used to investigate the impact of this virus infection on tumor cell growth through direct oncolytic effects or by virus-mediated modulation of the tumor microenvironment with special emphasis on angiogenesis, expression of selected MMPs and TIMP-1 and tumor-associated macrophages in a murine xenograft model of canine histiocytic sarcoma. Treatment of mice with xenotransplanted canine histiocytic sarcomas using CDV induced overt retardation in tumor progression accompanied by necrosis of neoplastic cells, increased numbers of intratumoral macrophages, reduced angiogenesis and modulation of the expression of MMPs and TIMP-1. The present data suggest that CDV inhibits tumor growth in a multifactorial way, including direct cell lysis and reduction of angiogenesis and modulation of MMPs and their inhibitor TIMP-1, providing further support for the concept of its role in oncolytic therapies.

In Vivo Tumorigenesis, Osteolytic Sarcomas, and Tumorigenic Cell Lines from Transgenic Mice Expressing the Human T-Lymphotropic Virus Type 1 (HTLV-1) Tax Viral Oncogene.

Human T-lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukemia, a disease commonly associated with hypercalcemia and osteolysis. There is no effective treatment for HTLV-1, and the osteolytic mechanisms are not fully understood. Mice expressing the HTLV-1 oncogene Tax, driven by the human granzyme B promoter (Tax+), develop osteolytic tumors. To investigate the progression of the bone-invasive malignancies, wild-type, Tax+, and Tax+/interferon-γ-/- mice were assessed using necropsy, histologic examination, IHC analysis, flow cytometry, and advanced imaging. Tax+ and Tax+/interferon-γ-/- malignancies of the ear, tail, and foot comprised poorly differentiated, round to spindle-shaped cells with prominent neutrophilic infiltrates. Tail tumors originated from muscle, nerve, and/or tendon sheaths, with frequent invasion into adjacent bone. F4/80+ and anti-mouse CD11b (Mac-1)+ histiocytic cells predominated within the tumors. Three Tax+/interferon-γ-/- cell lines were generated for in vivo allografts, in vitro gene expression and bone resorption assays. Two cell lines were of monocyte/macrophage origin, and tumors formed in vivo in all three. Differences in Pthrp, Il6, Il1a, Il1b, and Csf3 expression in vitro were correlated with differences in in vivo plasma calcium levels, tumor growth, metastasis, and neutrophilic inflammation. Tax+ mouse tumors were classified as bone-invasive histiocytic sarcomas. The cell lines are ideal for further examination of the role of HTLV-1 Tax in osteolytic tumor formation and the development of hypercalcemia and tumor-associated inflammation.

Mesenchymal to epithelial transition driven by canine distemper virus infection of canine histiocytic sarcoma cells contributes to a reduced cell motility in vitro.

Sarcomas especially of histiocytic origin often possess a poor prognosis and response to conventional therapies. Interestingly, tumours undergoing mesenchymal to epithelial transition (MET) are often associated with a favourable clinical outcome. This process is characterized by an increased expression of epithelial markers leading to a decreased invasion and metastatic rate. Based on the failure of conventional therapies, viral oncolysis might represent a promising alternative with canine distemper virus (CDV) as a possible candidate. This study hypothesizes that a CDV infection of canine histiocytic sarcoma cells (DH82 cells) triggers the MET process leading to a decreased cellular motility. Immunofluorescence and immunoblotting were used to investigate the expression of epithelial and mesenchymal markers followed by scratch assay and an invasion assay as functional confirmation. Furthermore, microarray data were analysed for genes associated with the MET process, invasion and angiogenesis. CDV-infected cells exhibited an increased expression of epithelial markers such as E-cadherin and cytokeratin 8 compared to controls, indicating a MET process. This was accompanied by a reduced cell motility and invasiveness. Summarized, these results suggest that CDV infection of DH82 cells triggers the MET process by an increased expression of epithelial markers resulting in a decreased cell motility in vitro.

Leukemic histiocytic sarcoma in a captive common squirrel monkey (Saimiri sciureus) with Saimiriine Gammaherpesvirus 2 (Rhadinovirus), Saimiri sciureus lymphocryptovirus 2 (Lymphocryptovirus) and Squirrel monkey retrovirus (ß-Retrovirus) coinfection.

Hematopoietic neoplasia other than lymphoma and leukemia is uncommon among non-human primates. Herein, we provide the first evidence of occurrence of leukemic histiocytic sarcoma in a captive common squirrel monkey with Saimiriine Gammaherpesvirus 2 (Rhadinovirus), Saimiri sciureus lymphocryptovirus 2 (Lymphocryptovirus), and Squirrel monkey retrovirus (ß-Retrovirus) coinfection.

Oxidative Stress in Canine Histiocytic Sarcoma Cells Induced by an Infection with Canine Distemper Virus Led to a Dysregulation of HIF-1α Downstream Pathway Resulting in a Reduced Expression of VEGF-B in vitro.

Histiocytic sarcomas represent malignant tumors which require new treatment strategies. Canine distemper virus (CDV) is a promising candidate due to its oncolytic features reported in a canine histiocytic sarcoma cell line (DH82 cells). Interestingly, the underlying mechanism might include a dysregulation of angiogenesis. Based on these findings, the aim of the present study was to investigate the impact of a persistent CDV-infection on oxidative stress mediated changes in the expression of hypoxia-inducible factor (HIF)-1α and its angiogenic downstream pathway in DH82 cells in vitro. Microarray data analysis, immunofluorescence for 8-hydroxyguanosine, superoxide dismutase 2 and catalase, and flow cytometry for oxidative burst displayed an increased oxidative stress in persistently CDV-infected DH82 cells (DH82Ond pi) compared to controls. The HIF-1α expression in DH82Ond pi increased, as demonstrated by Western blot, and showed an unexpected, often sub-membranous distribution, as shown by immunofluorescence and immunoelectron microscopy. Furthermore, microarray data analysis and immunofluorescence confirmed a reduced expression of VEGF-B in DH82Ond pi compared to controls. In summary, these results suggest a reduced activation of the HIF-1α angiogenic downstream pathway in DH82Ond pi cells in vitro, most likely due to an excessive, unusually localized, and non-functional expression of HIF-1α triggered by a CDV-induced increased oxidative stress.

Anti-tumour activity of oncolytic reovirus against canine histiocytic sarcoma cells.

Canine histiocytic sarcoma is an aggressive, fatal neoplastic disease with a poor prognosis. Lomustine is generally accepted as the first-line systemic therapy, although this compound does not provide complete regression. Therefore, research into a novel approach against canine histiocytic sarcoma is needed. However, anti-tumour effects of oncolytic therapy using reovirus against histiocytic sarcoma are unknown. Here, we showed that reovirus has oncolytic activity in canine histiocytic sarcoma cell lines in vitro and in vivo. We found that reovirus can replicate and induce caspase-dependent apoptosis in canine histiocytic sarcoma cell lines. A single intra-tumoural injection of reovirus completely suppressed the growth of subcutaneously grafted tumours in NOD/SCID mice. Additionally, we demonstrated that susceptibility to reovirus-induced cell death was attributable to the extent of expression of type I interferons induced by reovirus infection in vitro. In conclusion, oncolytic reovirus appears to be an effective treatment option for histiocytic sarcoma, and therefore warrants further investigation in early clinical trials.

Persistent Morbillivirus Infection Leads to Altered Cortactin Distribution in Histiocytic Sarcoma Cells with Decreased Cellular Migration Capacity.

Histiocytic sarcomas represent rare but fatal neoplasms in humans. Based on the absence of a commercially available human histiocytic sarcoma cell line the frequently affected dog displays a suitable translational model. Canine distemper virus, closely related to measles virus, is a highly promising candidate for oncolytic virotherapy. Therapeutic failures in patients are mostly associated with tumour invasion and metastasis often induced by misdirected cytoskeletal protein activities. Thus, the impact of persistent canine distemper virus infection on the cytoskeletal protein cortactin, which is frequently overexpressed in human cancers with poor prognosis, was investigated in vitro in a canine histiocytic sarcoma cell line (DH82). Though phagocytic activity, proliferation and apoptotic rate were unaltered, a significantly reduced migration activity compared to controls (6 hours and 1 day after seeding) accompanied by a decreased number of cortactin mRNA transcripts (1 day) was detected. Furthermore, persistently canine distemper virus infected DH82 cells showed a predominant diffuse intracytoplasmic cortactin distribution at 6 hours and 1 day compared to controls with a prominent membranous expression pattern (p ≤ 0.05). Summarized, persistent canine distemper virus infection induces reduced tumour cell migration associated with an altered intracellular cortactin distribution, indicating cytoskeletal changes as one of the major pathways of virus-associated inhibition of tumour spread.

Successful treatment of histiocytic sarcoma and concurrent HIV infection using a combination of CHOP and antiretroviral therapy.

Histiocytic sarcoma (HS) is a rare malignancy of soft tissues with an unknown etiology. The CHOP (cyclophosphamide, vincristine sulfate, doxorubicin hydrochloride and prednisolone) regimen is often adopted as first-line chemotherapy; however, its therapeutic efficacy against HS is usually low. We herein first present the case of a patient with HS who was infected with human immunodeficiency virus-1 (HIV) in whom treatment with a combination of CHOP and antiretroviral therapy (ART) was successful. The patient has been in complete remission for 12 months following the discontinuation of chemotherapy under continuous ART. This case report may help to promote further investigation of both HS and HIV-related malignancy.

Subgroup J avian leukosis virus-induced histiocytic sarcomatosis occurs only in persistently viremic but not immunotolerized meat-type chickens.

The role of subgroup J avian leukosis virus (ALV J) infection profile in the development of histiocytic sarcomatosis (HS) in chickens was evaluated using retrospective analysis of 2 experiments involving in ovo and at-hatch inoculation of commercial meat-type and ADOL line 0 chickens with 100 or 10,000 TCID(50) of various strains ALV J. HS was observed only in persistently viremic, meat-type chickens that were inoculated at hatch, but not in immunotolerized (persistently viremic, with no antibodies), in ovo inoculated chickens. However, the immunotolerized, in ovo inoculated chickens developed a high incidence of myeloid tumors. HS appeared to arise from the splenic ellipsoids and red pulp, and metastasized to liver, kidney, and other organs. The neoplastic cells were diffusely positive for ChL5, CD45, and MHC class II with multifocal infiltration of T and B lymphocytes. Expression of viral antigen gp85 within HS was very low compared with that noted in ALV J-induced myelocytomas. The above observations suggest that the mechanisms of oncogenesis of HS might be different from that of other ALV J-induced tumors.

[Primitive study of the cell lineage and Epstein-Barr virus infection in so called malignant histiocytosis with tissue microarray technique].

OBJECTIVE: To investigate the cell lineage and Epstein-Barr virus infection in previously diagnosed cases of malignant histiocytosis (MH) with tissue microarray technique. METHODS: Using tissue-chips, immunohistochemical staining, in situ hybridization and PCR to analyze 5 autopsy cases of MH. RESULTS: (1) In all 5 cases, positive reactions of CD45RO, CD3 epsilon, TIA-1, Granzyme B were detected in the neoplastic cells, whereas negative reactions were found with CD30, CD20, CD56. (2) All cases revealed EBER1/2 positivity in neoplastic cells. CONCLUSION: Previously diagnosed malignant histiocytosis is an EBV-associated aggressive NK/T cell lymphoma.

Genetic and immunological parameters governing in vivo susceptibility/resistance to retrovirally induced murine malignant histiocytosis.

Malignant histiocytosis sarcoma virus (MHSV) arose as a recombinant of c-Harvey-ras murine sarcoma virus (Ha-MuSV) and Friend mink cell focus-forming virus (F-MCFV). It is a defective acute transforming retrovirus that, along with Friend murine leukemia helper virus (F-MuLV), induces malignant histiocytosis (MH) in susceptible adult mice. We have assessed the in vivo susceptibility to MHSV in inbred homozygous, F1 hybrid, congenic, and recombinant inbred (RI) mice. We have shown that: (1) in vivo resistance to MHSV is multigenic, regulated by MHC and non-MHC genes in a different fashion than with F-MCFV, F-MuLV, or Ha-MuSV; (2) using BXD RI mice, the resistance phenotype is linked with 95.8% probability to two linked loci, Pmv-9 and Iapls3-14, on chromosome 13 (homologous to the area of human chromosome 5 for which a chromosomal break point at position 5q35 is associated with human MH); and (3) CD4+ T cells are critical for MHSV resistance.

Epstein-Barr virus (EBV)-induced CD30+ natural killer cell-type malignancy resembling malignant histiocytosis: malignant transformation in chronic active EBV infection associating hypogammaglobulinemia.

A 27-year-old male suffered from Epstein-Barr virus (EBV)-related liver dysfunction with persistent hypogammaglobulinemia. IgG titers to EBV antigens were significantly high, while other hepatitis markers were negative. Liver biopsy disclosed active intralobular inflammation. Two years later, he manifested persistent fever, leukopenia, effusions and hypoproteinemia, and his general condition worsened progressively. The peripheral blood small lymphocytes predominantly expressed natural killer (NK)-like phenotypes (CD2+, CD7+, CD16+, CD56+). Hepatosplenomegaly and marked elevation of serum lactic dehydrogenase were observed. He died of respiratory failure at the age of 29. At autopsy, the liver (2190 g), spleen (860 g), small bowel and mesenteric lymph nodes showed massive infiltration of large atypical lymphoid cells in close association with hemophagocytic histiocytes. Involvement was mildly noted also in the bone marrow, lungs, gall-bladder and kidneys. The atypical cells belonged to CD30+ activated NK-type cells expressing CD2, cytoplasmic CD3 epsilon, CD7, CD45RO, CD56, HLA-DR and HLA-DQ. T cell receptors (TCR), surface CD3, CD4, CD5 and CD8 were not expressed. Epstein-Barr virus-related small nuclear RNA (EBER1) and Epstein-Barr virus-associated nuclear antigen 1 were detected in the nuclei of a significant number of atypical cells, while EBV-related latent membrane protein-1 was negative. EBER1 was also identified in the nuclei of non-neoplastic small lymphocytes at both biopsy and autopsy. Monoclonal integration of the EBV genome into the lymphoma cells was shown by Southern blot analysis. Clonal rearrangement of TCR was undetectable. Roles of chronic active EBV infection in the development of NK cell-type malignancy resembling malignant histiocytosis are discussed.

Analysis of the Epstein-Barr viral genome in so-called malignant histiocytosis syndrome.

Malignant histiocytosis has been described as a proliferation of morphologically atypical histiocytes, but it is difficult to determine whether or not malignant proliferation is present based on morphology alone. Recently the disorder has been thought to be heterogeneous, and therefore a true histiocytic origin is considered to be rare. The Epstein-Barr virus (EBV) is thought to have the ability to transform human cells. Therefore, eight cases of malignant histiocytic (MH) syndrome and five cases of virus-associated hemophagocytic syndrome (VAHS) were analyzed using a polymerase chain reaction (PCR) and the in situ hybridization (ISH) method in order to determine their relationship to EBV infection. At the same time, the cellular origin of these syndromes was also studied. The results indicated that three of the MH cases were derived from T cells while four the MH cases were from histiocytes. The amplification of the EBV-LYDMA region, which was used to determine the monoclonality, was detected in two MH cases and one VAHS case, and all these cases showed only one band. An ISH study also demonstrated the presence of an EBV in these three cases. One of the EBV-positive cases revealed an amplification of the EBV-LYDMA region by the PCR method before showing any sign of MH clinically. In the VAHS cases, the EBV genome was detected in hemophagocytic cells. The EBV-positive cases all demonstrated a rapid clinical course. Based on these results it is possible that EBV infection causes similar rapid clinical features in some cases of both MH and VAHS by the same mechanism.

Malignant histiocytosis derived from a common histiocyte clone in a patient with chronic Epstein-Barr virus infection.

Epstein-Barr virus (EBV) has a particular propensity for B lymphocytes, but in a few cases it seems to play a role in histiocytic disorders and EVB DNA has been identified in histiocytes. To determine what kind of cell proliferate clonally, we studied a patient with malignant histiocytosis that developed after chronic EBV infection. Polymerase chain reaction (PCR) for lymphocyte-defined membrane antigen (LYDMA) of EBV, a marker of monoclonality, double stainings of cell markers (B, T lymphocytes; histiocytes), and in situ hybridization for EBV were performed in tissues obtained in 1987 and 1990 before the appearance of malignant histiocytosis and in 1991 after the disease was diagnosed. PCR for LYDMA from multiple samples during the disease showed the same single band, indicating that chronic EBV infection and malignant histiocytosis were caused by the same single virion. We also found a single terminal repeat band of EBV which supports this finding. In the studies of double stainings, EBV was present in histiocytes of the non-neoplastic early stage, and in the neoplastic cells of malignant histiocytosis. The histiocyte, infected with EBV, clonally expanded to result in malignant histiocytosis.